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WO2007099988A1 - α-1,6-FUCOSYLTRANSFERASE MUTANT AND USE THEREOF - Google Patents

α-1,6-FUCOSYLTRANSFERASE MUTANT AND USE THEREOF Download PDF

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Publication number
WO2007099988A1
WO2007099988A1 PCT/JP2007/053733 JP2007053733W WO2007099988A1 WO 2007099988 A1 WO2007099988 A1 WO 2007099988A1 JP 2007053733 W JP2007053733 W JP 2007053733W WO 2007099988 A1 WO2007099988 A1 WO 2007099988A1
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Prior art keywords
fucosyltransferase
antibody
seq
amino acid
dna
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PCT/JP2007/053733
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French (fr)
Japanese (ja)
Inventor
Naoyuki Taniguchi
Eiji Miyoshi
Jianguo Gu
Naoko Ohnuki
Harue Nishiya
Ryosuke Nakano
Mitsuo Satoh
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Kyowa Hakko Kogyo Co., Ltd.
Osaka University
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Publication of WO2007099988A1 publication Critical patent/WO2007099988A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates

Definitions

  • the present invention relates to a -1,6-fucosyltransferase mutant of -1,6-fucosyltransferase, wherein the -1,6-fucosyltransferase activity of the enzyme is deleted or reduced, And its use.
  • sugar chains of glycoproteins are divided into sugar chains that bind to asparagine ( ⁇ -glycoside-linked sugar chains) and sugar chains that bind to serine, threonine, etc. (0-glycosyl-linked sugar chains). Broadly divided into types. Although ⁇ -glycoside-linked sugar chains have various structures (see Non-Patent Document 1), in any case, they may have a basic common core structure represented by the following structural formula (I). It ’s known.
  • N-glycoside-linked sugar chain is a high mannose type in which only mannose binds to the non-reducing end of the core structure, or galactose—N-acetylidanorecosamine (hereinafter referred to as Gato GlcNAc) on the non-reducing end of the core structure.
  • a complex type having a structure such as sialic acid or bisecting N-acetylcylcosamine on the non-reducing end side of Gato GlcNAc or It is known that there are hybrid types having both a high mannose type and a complex type branch on the non-reducing end side of the core structure.
  • a glycoprotein having an N-glycoside-linked sugar chain has a sugar chain structure even if the core protein is the same. It exists as a heterogeneous glycoprotein composed of diverse molecules. The sugar chain structure is thought to be controlled by glycosyltransferases that synthesize sugar chains and glycolytic enzymes that degrade 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 ability of human congenital disorders of glycosylation (CDG) related to sugar chain abnormalities has been reported to be the gene required for the synthesis of N-glycosidic sugar chains. (See Non-Patent Documents 5 and 6).
  • CDG type II disease patients which lacks the GDP-fucose transporter, which is a transporter of sugar nucleotide GDP-fucose, the cytoplasm of the sugar donor, GDP-fucose.
  • Symptom such as lack of fucose modification to glycoconjugates, mental disorders and growth delays, and impaired immune function have been observed.
  • N-glycoside-linked complex-type sugar chain reducing terminal N-acetylyldarcosamine is linked to the 6th position of fucose, where the enzyme involved in the sugar chain modification is In contrast, so far only a single gene has been found in vivo, and its function has attracted much attention.
  • N-daricoside-linked glycan N-acetylyldarcosamine at the reducing end of the enzyme is involved in the sugar chain modification in which the 1-position of fucose is ⁇ -linked to the 6-position of ⁇ - 1,6-fucosinole transferase in animals has been found (see Non-Patent Document 9).
  • the structure of the -1,6-fucosyltransferase gene (EC 2.4.1,68) was clarified in 1996 (see Non-Patent Documents 10-11 and Patent Document 1).
  • Enzyme activity of -1,6-fucosyltransferase has been confirmed in many organs, but it has been reported that enzyme activity is relatively high in the brain and small intestine (see Non-Patent Documents 12-13). It has been pointed out that fucose-modified sugar chains play an important physiological function in retinal formation, and attention has been paid to the regulation of the expression of -1,6-fucosyltransferase (see Non-Patent Document 14). ). For blood clotting, The role of platelet-derived ⁇ -1,6-fucosyltransferase has attracted attention (see Non-Patent Document 15).
  • Non-Patent Document 16 discloses the modification of fucose to the sugar chain structure of immunoglobulin IgGl affects the binding to FcyRI Ila and changes the antibody-dependent cytotoxic activity of the antibody itself.
  • Non-Patent Documents 18 and 19 Regarding the relationship with the pathological condition, in some diseases such as liver cancer and cystic fibrosis, increased 1,6-fucosyltransferase activity and the ratio of the enzyme reaction product increased. Therefore, the relationship between these diseases and the enzyme is assumed (see Non-Patent Documents 18 and 19).
  • Transgenic mice overexpressing -1,6-fucosyltransferase have also been prepared, and liver and kidneys of the produced transgenic mice have been observed to have steatosis-like degeneration. (See Non-Patent Document 20).
  • a -1,6-fucosyltransferase knockout mouse is produced (see Patent Document 2).
  • Patent Literature l 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 'Opinon' Immunology (Curr. Opin. Immunol.), 3, 646, 1991
  • Non-Patent Document 3 Glycobiology, 3, 97, 1993
  • Non-Patent Document 4 Biochemical 'Society' Transaction (Biochem. Soc. Trans.), 23, 1, 1995
  • Non-Patent Document 5 Glycobiology, 3, 423, 1993
  • Non-Patent Document 6 Choi Bian 'Journal' Ob 'Pediatric' Neurology (Eur.
  • Non-Patent Document 7 Nichia's Genetys (Nat. Genet.), 28, 73, 2001
  • Non-Patent Document 8 Nichia's Genetys (Nat. Genet.), 28, 69, 2001
  • Non-Patent Document 9 Biochemical and Biophysical Research Communications (Biochem. Biophys. Res. Commun.), 72, 909, 197
  • Non-Patent Document 10 Journal 'Ob' Biologicals' Chemistry CJ. Biol. Chem.), 271, 27817, 1996
  • Non-Patent Document 11 Journal 'Ob' Biochemistry, 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' Biologic 'Chemistry CJ. Biol. Chem.), 278, 3466, 2003
  • Non-patent document 18 Hepatology (H-mark atology), 13, 683, 1991)
  • Non-Patent Document 19 Hepatology (H Mark atology), 28, 944, 1998
  • Non-Patent Document 20 Glycobiology, 11, 165, 2001
  • the present invention relates to a -1,6, -fucosyltransferase, a -1, 6-fucosyltransferase mutant enzyme in which the -1,6, -fucosyltransferase activity of the enzyme is deleted or reduced, and It is to provide a method of using the same.
  • the -1,6-fucosyltransferase mutant of the present invention is useful for elucidating and diagnosing the physiological role of -1,6-fucose modifying enzyme and its relationship with pathological conditions. It is also useful for the development of drugs targeting the 1,6-fucose modifying enzyme and for the development of glycoprotein drugs where the sugar chain structure is important.
  • the present invention relates to the following (1) to (23).
  • amino acid sequence of -1,6-fucosyltransferase the amino acid at the position corresponding to the 171st amino acid from the N-terminus of the amino acid sequence represented by SEQ ID NO: 7 has been deleted, or other than serine A mutant of 1,6-fucosyltransferase having an amino acid sequence substituted with an amino acid.
  • H-1, -6-fucosyltransferase is a protein encoded by DNA selected from the group consisting of the following (a) to (f): 6-fucosyltransferase mutant.
  • ⁇ -1, 6-fucosyltransferase S a protein selected from the group consisting of the following (a) to (f), ⁇ -1, 6 according to (1) or (2) above -Fucosyltransferase mutant (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 7;
  • the amino acid sequence represented by any one of SEQ ID NOs: 13 to 17 is composed of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and The fucosyltransferase activity is deleted, or the -1,6-fucosyltransferase having the amino acid sequence represented by SEQ ID NO: 7 or 9 has a -1,6-fucosyltransferase activity lower than -1, An ⁇ -1,6-fucosyltransferase mutant having 6-fucosyltransferase activity.
  • (7) consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by any one of SEQ ID NOs: 13 to 17 and lacking ⁇ -1,6-fucosyltransferase activity, or alpha with reduced alpha-1,6-fucosyltransferase trans luciferase activity than SEQ ID NO: 7 or comprising the amino acid sequence represented by the 9 ⁇ _1,6- fucosyl trans Blow over peptidase alpha-1,6-fucosyltransferase activity -1,6-fucosyltransferase mutant.
  • a DNA comprising the base sequence represented by any of SEQ ID Nos: 18-22.
  • a method for producing a glycoprotein composition A method for producing a glycoprotein composition.
  • a medicament comprising the glycoprotein composition according to (19) above as an active ingredient.
  • a medicament comprising the antibody composition according to (20) above as an active ingredient.
  • H-1,6-fucosyltransferase gene In the isolated nucleotide sequence of the isolated gene, it is detected whether or not the amino acid at the position corresponding to the 171st amino acid sequence from the N-terminus of the amino acid sequence represented by SEQ ID NO: 7 has been substituted with asparagine. A method for diagnosing a disease associated with the characteristic ⁇ _1,6-fucosyltransferase.
  • an ⁇ -1,6-fucosyltransferase mutant in which the -1,6-fucosyltransferase activity of the enzyme is deleted or reduced, and How to use it is provided.
  • FIG. 1 is a diagram showing staining by LCA which is a -1,6-fucose-specific lectin of CHO / DG44 cells and RN6 strain.
  • the horizontal axis shows the fluorescence intensity in logarithm (log), and the vertical axis shows the cell number distribution.
  • the white outline shows the results of cells stained with FITC-labeled streptavidin, and the black outline shows the results of cells stained with FITC-labeled LCA.
  • FIG. 2 shows the results of monosaccharide composition analysis of the antibody produced by RN6 strain.
  • the horizontal axis shows the elution time (min), and the vertical axis shows the relative intensity.
  • FIG. 3 shows the construction of plasmid CHFUT8Comp23.
  • FIG. 4 shows the construction of plasmid CHFUT8CompTA.
  • FIG. 5 shows the construction of plasmid pcDNAchFUT8Comp.
  • FIG. 6 shows the construction of plasmid CHFUT8Mo3.
  • FIG. 7 shows the construction of plasmid CHFUT8MoTA.
  • FIG. 8 shows the construction of plasmid pcDNAchFUT8Mo.
  • FIG. 9 is a graph showing LCA staining of a FUT8 expression strain and a FUT8-base substitution product expression strain.
  • the horizontal axis shows the fluorescence intensity in logarithm (log), and the vertical axis shows the cell number distribution.
  • the white outline shows the results of cells stained with FITC-labeled streptavidin, and the black outline shows the results of cells stained with FITC-labeled LCA.
  • -1,6-fucosyltransferase is an N-glycoside-linked complex type sugar chain reducing terminal N-acetylyldarcosamine 6-position and fucose 1-position a bond Any enzyme involved is also included.
  • proteins encoded by DNA such as (a), (b), (, (d), (e) or (f) below, or (g), (h), (i), ( Examples thereof include proteins such as j), (k) or (1).
  • a protein comprising the amino acid sequence represented by SEQ ID NO: 12.
  • the amino acid at the position corresponding to the 171st amino acid from the terminus of the amino acid sequence represented by SEQ ID NO: 7 is ⁇ -1,6 -Calculate the homology between the amino acid sequence of fucosyltransferase and the amino acid sequence represented by SEQ ID NO: 7 using homology analysis programs and parameters such as BLAST and FASTA, which will be described later.
  • the -1,6-fucosyltransferase mutant of the present invention refers to the 171st position from the heel of the amino acid sequence represented by SEQ ID NO: 7 in the amino acid sequence of -1, -6-fucosyltransferase. Any of the a-1,6-fucosyltransferase mutants having the ability to delete the amino acid at the position corresponding to the amino acid or an amino acid sequence substituted with an amino acid other than serine may be used.
  • the amino acid sequence represented by SEQ ID NO: 13 consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and has a 1,6-fucosyltransferase activity.
  • ⁇ -1,6-fucosyltransferase activity that is lower than the _1,6-fucosyltransferase activity of -1,6-fucosyltransferase consisting of the deletion or amino acid sequence represented by SEQ ID NO: 7 or 9.
  • An ⁇ -1,6-fucosyltransferase mutant having;
  • the amino acid sequence represented by SEQ ID NO: 14 consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and ⁇ -1,6-fucosyltransferase is deleted. Or ⁇ having a ⁇ -1,6-fucosyltransferase activity that is lower than the ⁇ -1,6-fucosyltransferase activity of ⁇ -1,6-fucosyltransferase comprising the amino acid sequence represented by SEQ ID NO: 7 or 9. -1,6-fucosyltransferase mutant;
  • the amino acid sequence represented by SEQ ID NO: 15 consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and lacks -1,6-fucosyltransferase activity. Or has a -1,6-fucosyltransferase activity lower than the -1,6-fucosyltransferase activity of the -1,6-fucosyltransferase consisting of the amino acid sequence represented by SEQ ID NO: 7 or 9. -1,6-fucosyltransferase mutant;
  • the amino acid sequence represented by SEQ ID NO: 17 consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and has a 1,6-fucosyltransferase activity. Deletion or -1,6-fucosyltransferase activity lower than the _1,6-fucosyltransferase activity of -1,6-fucosyltransferase consisting of the amino acid sequence represented by SEQ ID NO: 7 or 9.
  • a -1,6-fucosyltransferase mutant having;
  • amino acid sequence represented by ⁇ -1,6-fucosyltransferase has ⁇ -1,6-fucosyltransferase activity lower than ⁇ -1,6-fucosyltransferase activity of ⁇ -1,6-fucosyltransferase.
  • amino acid sequence represented by SEQ ID NO: 14 consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 14, and lacking ⁇ -1,6-fucosyltransferase activity, or SEQ ID NO: 7 or 9
  • the amino acid sequence represented by ⁇ -1,6-fucosyltransferase has ⁇ -1,6-fucosyltransferase activity lower than ⁇ -1,6-fucosyltransferase activity of ⁇ -1,6-fucosyltransferase.
  • XV consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 17, and lacking -1,6-fucosyltransferase activity, or SEQ ID NO: 7 or 9
  • the amino acid sequence represented by Ranarihi-1,6-fucosyltransferase has a -1,6-fucosyltransferase activity that is lower than that of 1,6-fucosyltransferase.
  • a fucosyltransferase variant
  • (XXV) Hyperhybridized under stringent conditions with the nucleotide sequence represented by any of SEQ ID NO: 22, and lacks -1,6-fucosyltransferase activity, or represented by SEQ ID NO: 7 or 9. -1,6-fucosyltransferase having lower -1,6-fucosyltransferase activity than the -1,6-fucosyltransferase activity of Ranaruhi-1,6-fucosyltransferase DNA encoding a transferase variant.
  • DNA that hybridizes under stringent conditions refers to DNA such as DNA having a base sequence represented by SEQ ID NO: 18, 19, 20, 21, or 22, or a partial fragment thereof.
  • DNA means DNA obtained by using colony hybridization method, plaque hybridization method, Southern blot hybridization method, etc., specifically, colony or Using a filter immobilized with plaque-derived DNA, 0.7 to 1.0. After hybridization at 65 ° C in the presence of 0 M sodium chloride, 0. The DNA can be identified by washing the filter under 65 ° C conditions using a SSC solution (concentrated SSC solution consisting of 150 mM sodium chloride and 15 mM sodium quenate). And can.
  • SSC solution concentrated SSC solution consisting of 150 mM sodium chloride and 15 mM sodium quenate
  • HYBRIDISE 1 ⁇ Cillon ia> Molecular cloning, a laooratory manual, Third Edition, Cold Spring Harbor Laboratory Press (2001) (hereinafter abbreviated as Molecular ⁇ ⁇ Cloning 3rd Edition), Current Protocols in Molecular Biology, John Wiley & Sons, 1987—1997 (hereinafter “abbreviated as“ protocols ”in“ molecular ”biology), DNA Clonin g 1: Core ⁇ ecnmques, A Practical Approach, Second Edition, Oxford University (1955), etc. It can be performed according to the method.
  • DNA that can be hybridized when calculated using BLAST, FASTA, etc., for example, at least 70% or more of the nucleotide sequence represented by SEQ ID NO: 18, 19, 20, 21, or 22, Preferably it is 80% or more, more preferably 90. / o or more, more preferably 95% or more, particularly preferably 98% or more, Most preferred is DNA having a homology of 99% or more.
  • the homology between the amino acid sequence and the base sequence is, for example, the algorithm BLAST [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] by Karlin and Altschul, FASTA [Methods Enzymol., 183, 63 (1990)].
  • BLAST Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)
  • FASTA Method Enzymol., 183, 63 (1990)
  • programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215. 403 (1990)]
  • the amino acid sequence is analyzed by BL ASTX based on BLAST
  • BLAST and Gapped BLAST programs use the default parameters of each program. Specific methods of these analysis methods are known (http: //www.ncbi.nlm.nih.gov ⁇
  • the amino acid sequence represented by SEQ ID NO: 13, 14, 15, 16 or 17 comprises an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and Proteins with ⁇ -1,6-fucosyltransferase activity are: Molecular Molecular Cloning, 3rd Edition, Current 'Protocols' In' Molecular ⁇ Biology, Nucleic Acids Research, 10, 6487 (1982), Proc Natl. Acad. Sci., USA, 79, 6409 (1 982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. A cad.
  • site-directed mutagenesis to DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 13, 14, 15, 16 or 17 It means a protein that can be obtained by introducing.
  • the number of amino acids to be deleted, substituted, inserted and / or added is one or more, and the number is not particularly limited. However, deletion, substitution or attachment may be performed by well-known techniques such as the above-mentioned site-directed mutagenesis. For example, the number is 1 to several tens, preferably 1 to 20, more preferably 1 to 10 and even more preferably 1 to 5.
  • one or more amino acids are deleted, substituted or added means that there is a deletion, substitution or addition of one or more amino acids at any position in the same sequence.
  • the amino acid to be substituted or added may be natural or non-natural, although substitution or addition may occur simultaneously.
  • Natural amino acids include L-alanine and 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-cystine 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, valine, nonorevaline, alanine, 2-aminobutanoic acid, methionine, _methylserine, t_butylglycine, t-butylalanine, cyclohexenolealanine
  • Group B aspartic acid, gnoretamic acid, isoaspartic acid, isognoletamic 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
  • Group G phenylalanin, tyrosine
  • Any cell may be used as long as it is a cell, for example, yeast, animal cells, insect cells, plant cells, and the like. Specific examples of these cells include those described in 2. below. . Specific examples of animal cells include Chinese hamster ovary tissue CHO cells, rat myeloma cell line YB2 / 3HL.P2.G11.16Ag.20 cell, mouse myeoma cell line NS0 cell, mouse myeloma cell line SP2 / 0-Agl4 cell, Syrian hamster kidney tissue-derived BHK cell, Examples include antibody-producing hybridoma cells, human leukemia cell line Namalba cells, embryonic stem cells, and fertilized egg cells.
  • the above-described myeloma cells, hyperpridoma cells, humanized antibodies or host cells for producing human antibodies, and non-human transgenic animals producing human antibodies, which are used for the production of glycoproteins such as antibodies, are produced.
  • examples thereof include embryonic stem cells or fertilized egg cells used for the preparation, and plant cells for use in producing a transgenic plant producing humanized antibodies and human antibodies.
  • NS0 cells are described in documents such as Bio / Technology (BIO / TECHNOLOGY), 10, 169 (1992), Biotechnology (Biotechnol. Bioeng.), 73, 261, (2001). NS0 cells. Another example is the NS0 cell line (RCB0213) registered with the RIKEN Cell Development Bank, or substrains in which these lines are conditioned to various serum-free media. SP2 / 0-Agl4 cells include Journal of Immunology (J. Immunol.), 126, 317, (1981), Nature, 276, 269, (1978), Human Anti-Ibodies. 'And /, Human Antibodies and Hybridomas,
  • SP2 / 0_Agl4 cells described in literatures such as 3, 129, (1992).
  • SP2 / 0-Agl4 cells (ATCC CRL-1581) registered in ATCC or sub-strains (ATCC CRL-1581.1) in which these strains are conditioned to various serum-free media are also included.
  • ATCC CRL-1581 registered in ATCC or sub-strains (ATCC CRL-1581.1) in which these strains are conditioned to various serum-free media are also included.
  • ATCC CRL-1581 registered in ATCC or sub-strains (ATCC CRL-1581.1) in which these strains are conditioned to various serum-free media are also included.
  • ATCC CRL-1581 registered in ATCC or sub-strains (ATCC CRL-1581.1) in which these strains are conditioned to various serum-free media are also included.
  • ATCC CRL-1581 registered in ATCC or sub-strains (ATCC CRL-1581.1) in which these strains are conditioned to
  • CHO-Kl strain ATCC CCL-61
  • DUXB11 strain ATCC CRL-9096
  • Pr 0-5 strain ATCC CRL-1781 registered in ATCC
  • commercially available CHO-S strain (Lifetechnologies) Cat No. 11619)
  • substrains obtained by acclimating these strains to various serum-free media.
  • the Eroma cell line YB2 / 3HL.P2.G11.16Ag.20 cells include cell lines in which Y3 / Agl.2.3 cells (ATCC C RL-1631) have been established. Specific examples thereof include Y B2 / 3HL.P2.Gil.16Ag. Described in documents such as J. Cell. Biol., 93, 576 (1982), Methods Enzymol. 73B, 1 (1981). There are 20 cells. Also, YB2 / 3H registered in ATCC? 2 11.16 8 .20 cells (8 1 ⁇ CRL-1662) or substrains of these strains acclimated to various serum-free media.
  • a gene encoding a glycoprotein molecule can be introduced into the cell of the present invention, and a glycoprotein composition comprising the glycoprotein molecule can be produced using the cell.
  • Glycoproteins have a sugar chain that is linked to asparagine (N-glycoside-linked sugar chain) and a sugar chain that binds to serine or threonine (o-glycoside-linked sugar chain), depending on the mode of binding to the glycoprotein moiety.
  • N-glycoside-linked sugar chain N-glycoside-linked sugar chain
  • o-glycoside-linked sugar chain a sugar chain that binds to serine or threonine
  • N-glycoside-linked sugar chains have various structures, but in any case, 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 Gato GlcNac) on the non-reducing end of the core structure.
  • the complex type has one or more branches in parallel, and further has a structure such as sialic acid or bisecting N-acetylcylcosamine at the non-reducing end of Ga ⁇ GlcNac. And glycans and hybrid glycans having both high-mannose and complex branches at the non-reducing end of the core structure.
  • N-acetylylgalatatosamine is bonded to the hydroxyl group of serine or threonine, and further, galactose, N-acetylyldarcosamine, N-acetylgalato Examples include sugar chains in which samine or 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 hydroxylysine.
  • a sugar chain in which xylose is bonded to the hydroxyl group of serine by / 3 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.
  • Galacto Examples of the substance having a sugar chain structure in which ⁇ is bonded to the hydroxyl group of hydroxylysine include collagen.
  • sugar constituting the sugar chain examples include ⁇ -acetylyl darcosamine, ⁇ -acetyl galatatosamine, mannose, galactose, fucose, sialic acid, xylose, arabinose, and the like. You can combine them in any order.
  • the glycoprotein composition refers to a composition comprising glycoprotein molecules having ⁇ -glycoside-linked sugar chains or ⁇ -glycoside-linked sugar chains.
  • glycoproteins include antibodies, erythropoietin, thrombopoietin, tissue plasminogen activator, prolokinase, thrombomodulin, antithrombin III, protein blood coagulation factor VII, blood coagulation Factor VIII, blood clotting factor IX, blood clotting factor X, blood clotting factor XII, gonadotropin, thyroid stimulating hormone, epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor, activin, bone Form factor, stem cell factor (SCF), interferon ⁇ , interferon ⁇ , interferon, interleukin 2, interleukin 6, interleukin 10, interleukin 11 1, soluble interleukin 4 receptor, tumor necrosis factor, Dnasel, Galactosidase, Dalcosidase, Dalcocerebrosidase And the like.
  • EGF epidermal growth factor
  • HGF hepatocyte growth factor
  • the antibody composition refers to a composition comprising an antibody molecule having an N-glycoside-linked complex sugar chain in the Fc region.
  • a method for producing a glycoprotein composition using the non-human animal of the present invention and its progeny will be described by taking the production of an antibody composition as an example.
  • the antibody composition is a protein produced in vivo by an immune reaction as a result of stimulation with a foreign antigen, and may be any protein that has an activity of specifically binding to an antigen.
  • the antibody expression vectors inserted with antibodies produced by genetic recombination technology, ie, antibody genes are immunized with antigens. And the like, which are obtained by introducing the protein into a host cell.
  • Specific examples include antibodies produced by Hypridoma, chimeric antibodies, humanized antibodies, human antibodies, and the like.
  • Hypridoma has a desired antigen specificity obtained by cell fusion of B cells obtained by immunizing mammals other than humans with myeloma cells derived from mice, rats and the like. It refers to a cell that produces a monoclonal antibody.
  • Chimeric antibodies are antibody heavy chain variable regions of non-human animals (hereinafter, variable regions are also referred to as HV or VH as V regions) and antibody light chain variable regions (hereinafter, light chains are also referred to as LV or VL as L chains). And a heavy chain constant region of a human antibody (hereinafter also referred to as CH) and a light chain constant region of a human antibody (hereinafter also referred to as CL).
  • variable regions are also referred to as HV or VH as V regions
  • light chains are also referred to as LV or VL as L chains.
  • CH heavy chain constant region of a human antibody
  • CL light chain constant region of a human antibody
  • animals other than humans various animals such as mice, rats, hamsters, rabbits, etc. can be used as long as it is possible to produce high pridomas.
  • cDNAs encoding VH and VL are obtained from hybridomas producing monoclonal antibodies, and inserted into expression vectors for host cells having genes encoding human antibody CH and human antibody CL, respectively.
  • a human chimeric antibody expression vector can be constructed and introduced into a host cell for expression and production.
  • the CH of the chimeric antibody may be any that belongs to human immunoglobulin (hereinafter referred to as “hlg”), but is preferably of the hlgG class, and more preferably of hlgGl, hIgG2, hIgG3, hIgG4 belonging to the hlgG class. Any of the subclasses can be used.
  • the CL of the human chimeric antibody may be any as long as it belongs to hlg, and K class or E class can be used.
  • a human rabbit antibody is an antibody in which the amino acid sequence of the VH and VL complementarity determining regions (hereinafter referred to as CDR) of an antibody of a non-human animal is transplanted to an appropriate position of the human antibody VH and VL.
  • CDR complementarity determining regions
  • Humanized antibodies are constructed by constructing cDNA encoding the V region obtained by grafting the VH and VL CDR sequences of non-human animal antibodies to the VH and VL CDR sequences of any human antibody.
  • a humanized antibody expression vector is constructed by inserting each into a host cell expression vector having a gene encoding CL, and the humanized antibody is expressed and produced by introducing the expression vector into the host cell. be able to.
  • the CH of the humanized antibody may be any as long as it belongs to the hlg, but the hlgG class is preferable, and the subclasses such as hIgGl, hIgG2, hIgG3, and hIgG4 belonging to the hlgG class are preferred. Any of these can be used.
  • the CL of human rabbit antibody may be any as long as it belongs to hlg, and those of / class or e class can be used.
  • a human antibody originally refers to an antibody that naturally exists in the human body.
  • a human antibody phage library prepared by recent advances in genetic engineering, cell engineering, and developmental engineering, as well as human antibody production trans Also included are antibodies obtained from dienic animals or human antibody-producing transgenic plants.
  • the antibody present in the human body can be cultured, for example, by isolating human peripheral blood lymphocytes, infecting an EB virus or the like, immortalizing, and cloning the lymphocytes that produce the antibody.
  • the antibody can be further purified.
  • the human antibody phage library 1 is a library in which antibody fragments such as Fab and single chain antibody are expressed on the phage surface by inserting antibody genes prepared from human B cells into the phage genes. From this library, it is possible to collect phages expressing antibody fragments having a desired antigen-binding activity using as an index the binding activity to the substrate on which the antigen is immobilized.
  • the antibody fragment can be further converted into a human antibody molecule comprising two complete heavy chains and two complete light chains by genetic engineering techniques.
  • a human antibody-producing transgenic non-human animal refers to an animal in which a human antibody gene is incorporated into cells. Specifically, a human antibody-producing transgenic animal is produced by introducing a human antibody gene into a mouse embryonic stem cell, transplanting the embryonic stem cell to an early embryo of another mouse, and generating it. Can do. It is also possible to produce a human antibody-producing transgenic animal by introducing a human antibody gene into a fertilized egg of an animal and generating the fertilized egg. The production method of human antibodies from human antibody-producing transgenic animals is obtained by cultivating and culturing human antibody-producing hyperpridoma by the conventional method of producing hyperidoma in mammals other than humans. Antibodies can be produced and accumulated.
  • transgenic non-human animals include sushi, hidge, goat, pig, horse, mouse, rat, chicken, monkey, and rabbit.
  • the antibody is an antibody recognizing a tumor-related antigen, an antibody recognizing an antigen related to allergy or inflammation, an antibody recognizing an antigen related to cardiovascular disease, or an autoimmune disease.
  • Antibody that recognizes antigen, or virus or bacterial sensation A human antibody whose antibody class is preferably an IgG that recognizes an antigen associated with a stain is preferred.
  • the antibody fragment refers to a fragment containing at least a part of the Fc region of the antibody.
  • the Fc region means a region on the C-terminal side of the H chain of an antibody, a CH2 region and a CH3 region, and includes a natural type and a mutant type thereof. At least a part of the Fc region preferably means a fragment containing the CH2 region, more preferably a region containing the first aspartic acid present in the CH2 region.
  • the Fc region of the IgG class is the EU Index [Sequences 'Ob' Proteins 'Off' Munoron Kanole'interest of Kabat et al.
  • antibody fragments include H chain monomers and H chain dimers.
  • a fusion protein having an Fc region is a substance obtained by fusing an antibody or antibody fragment containing an Fc region of an antibody with a protein such as an enzyme or a cytodynamic force. Good.
  • antibodies that recognize tumor-associated antigens include anti-GD2 antibodies (Anticancer Res., 13, 331, 1993), anti-GD3 antibodies (Cancer Immunol. Immunother "36, 260, 1993), and anti-GM2 antibodies (Cancer Res. , 54, 1511, 1994), anti-HER2 antibody (Proc. Natl. Acad. Sci. USA, 89, 4285, 199 2), anti-CD52 antibody (Nature, 332, 323, 1988), anti-MAGE antibody (British J.
  • anti-PMSA antibody J. Urology, 160. 2396, 1998)
  • anti-vascular endothelial growth factor Antibody Cancer Res., 57, 4593, 1997) or anti-vascular endothelial growth factor receptor antibody (Oncogene, 19, 2138, 2000
  • anti-CA125 antibody anti-17-1A antibody
  • anti-CD33 antibody anti-CD22 antibody
  • anti-HL A antibody anti-HLA-DR antibody
  • anti-CD20 antibody anti-CD 19 antibody
  • anti-EGF receptor antibody Immunol ogy Today, 21, 403, 2000
  • anti-CD 10 antibody American Journal of Clinical Pathology, 1 13, 374 , 2000.
  • Antibodies that recognize antigens related to allergy or inflammation include anti-interleukin 6 antibody (Immunol. Rev., 127, 5, 1992), anti-interleukin 6 receptor antibody (Molecul ar Immunol, 31, 371, 1994), anti-interleukin 5 antibody (Immunol. Rev., 127, 5, 1992), anti-interleukin 5 receptor antibody, anti-interleukin 4 antibody (Cytokine, 3, 562, 19 91), anti-interleukin 4 receptor antibody (J. Immunol.
  • anti-tumor necrosis factor antibody Hybridoma, 13, 183, 1994
  • anti-tumor necrosis factor receptor antibody Molecula r Pharmacol., 58, 237) , 2000
  • anti-CCR4 antibody Nature, 400, 776, 1999
  • anti-chemokine antibody J. Immunol. Meth., 174, 249, 1994
  • anti-chemokine receptor antibody J. Exp. Med., 186. 1373, 1997)
  • anti-IgE antibody anti-CD23 antibody, anti-CD 11a antibody (Immunology Today, 21, 403, 2000)
  • anti-CRTH2 antibody J. Immunol., 162, 1278, 1999
  • anti-CCR8 antibody W099 / 2 5734
  • anti-CCR3 antibody US6207155
  • Anti-GpIIb / IIIa antibody J. Immuno 1., 152, 2968, 1994
  • antiplatelet-derived growth factor antibody Science, 253, 1129, 1991
  • Anti-platelet-derived growth factor receptor antibody J. Biol. Chem., 272, 17400, 1997) or anti-blood clotting factor antibody (Circulation, 101, 1158, 2000).
  • Antibodies that recognize antigens associated with autoimmune diseases include anti-self DNA Antibody (Immunol. Letters, 72, 61, 2000), anti-CDlla antibody, anti-ICAM 3 antibody, anti-CD80 antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-integrin 4 j3 7 antibody, anti-CD40L antibody And anti-IL-2 receptor antibody (Immunology Today, 21, 403, 2000).
  • antibodies that recognize antigens associated with viral or bacterial infection include anti-gpl20 antibodies
  • the ⁇ -1,6-fucosyltransferase mutant of the present invention isolates mRNA derived from human tissue having a disease in which the activity of ⁇ -1,6-fucose modifying enzyme is deleted or decreased, Can be prepared by preparing a library and then screening the cDNA library to obtain the desired clone.
  • mutations that delete or reduce the activity of -1,6-fucosyltransferase result in the occurrence of N-acetylidanorecosamine at the reducing end of the N-glycoside-linked complex sugar chain.
  • the -1,6-fucosyltransferase mutant of the present invention can be isolated. Can be prepared.
  • a cell line resistant to lectin refers to a cell line in which growth is not inhibited even when an effective concentration of lectin is given.
  • the effective concentration refers to the concentration at which the above-mentioned cell line (hereinafter referred to as “parent strain”) cannot grow normally before the mutation that causes loss or decrease in the activity of ⁇ -1,6-fucosyltransferase.
  • the concentration is the same as the concentration at which the parent strain cannot grow, more preferably 2 to 5 times, still more preferably 10 times, and most preferably 20 times or more.
  • the effective concentration of a lectin whose growth is not inhibited may be appropriately determined according to the parent strain, but is usually 10 ig / ml to 10 mg / ml, preferably 0.5 mg / ml to 2.0 mg / ml. is there.
  • Any lectin that recognizes the sugar chain structure can be recognized as a lectin that recognizes the sugar chain structure in which the 6-position of N-glycidylcolacamine at the N-glycoside-linked sugar chain reducing end and the 1-position of fucose are a-linked.
  • the lectin can also be used. Specific examples of this include: Lentil lectin LCA (Lens culinaris lentil agglutinin) Endumame lectin PSA (Pisum sativum-derived nea lectin), Broad bean lectin VFA (Vicia faba-derived agglutinin), Hirochawantake lectin AAL (Aleu n that from aurantia lectin), and the like
  • mRNA derived from human tissue or cultured cell line can be prepared by preparing total RNA from human tissue or cultured cell line as follows, and isolating the total RNA mRNA. S can.
  • a method for preparing total RNA from human tissues or cultured cell lines is thiocyanate group.
  • the cesium acetate trifluoroacetate method [Methods in Enzymology, 154, 3 (1987)]
  • the guanidine thiocyanate 'Phenol' chloroform (AGPC) method [Analytical Biochem istry, 162, 156 (1987), Experimental Medicine, 9 , 1937 (1991)].
  • Examples of methods for preparing mRNA from total RNA as poly (A) + RNA include oligo (dT) -immobilized cellulose strength method (Molecular 'Cloning 2nd Edition).
  • mRNA 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 prepared human tissue or cultured cell line mRNA.
  • One method for preparing a cDNA library is the method described in Molecular 'Cloning 2nd Edition, Current' Protocols in Molecular Biology, etc., or a commercially available kit, such as the Superscript Plasmid system for cDNA Synthesis and Plasmid Cloning ( Life Technologies) and ZAP-cDNA Synthesis Kit (STRATAGENE).
  • any phage vector or plasmid vector can be used as long as it can autonomously replicate in Escherichia coli K12.
  • ZAP Express [STRATAGENE, Strategies, 5, 58 (1992)], Bluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)], Lambda ZAP II (manufactured by STRATAGENE), gtl0, ⁇ gtl l (DNA cloning, A Practical Approach, ⁇ , 49 (1985)), ⁇ TriplEx (Clontech), ⁇ ExCell (Pharmacia), pT7T318U (Pharmacia), pcD2 (Mol. Cell. Biol., 3, 280 (1983)] and pUC18 [Gene, 33, 103 (1985)].
  • any microorganism belonging to the genus Escherichia can be used.
  • Esc herichia coli Y1088 [Science, 222, 778 (1983)] Escherichia coli Y1090 [Science, 222, 778 (1983)]
  • Escherichia coli NM522 J. Mol.
  • This cDNA library can be used for the following analysis as it is, but the oligo-developed by Kanno et al. Has been developed in order to reduce the proportion of incomplete length cDNA and 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, 11, 2491 (1993), cDNA cloning, Yodosha ( 1996), cDNA library prepared using the method of gene library, Yodosha (1994)] can be used for the following analysis.
  • Each clone is isolated from the prepared cDNA library, and the cDNA base sequence of each clone is isolated from the end, and a commonly used base sequence analysis method, for example, Sanger et al.'S dideoxy method [Proc. Natl. Acad Sci. USA, 74, 5463 (1977)] or A BIPRISM377 DNA sequencer (manufactured by PE Biosystems) or the like, and then analyzing the base sequence of the DNA.
  • a commonly used base sequence analysis method for example, Sanger et al.'S dideoxy method [Proc. Natl. Acad Sci. USA, 74, 5463 (1977)] or A BIPRISM377 DNA sequencer (manufactured by PE Biosystems) or the like, and then analyzing the base sequence of the DNA.
  • each cDNA has a nucleotide sequence encoding an amino acid-mutating enzyme of ⁇ -1,6-fucosyltransferase is determined using a homology search program such as BLAST using GenBank. By searching base sequence databases such as EMBL and DDBJ, it is possible to confirm by homology with the base sequences of existing genes in the database.
  • a homology search program such as BLAST using GenBank.
  • nucleotide sequence of cDNA containing the nucleotide sequence encoding the amino acid-modified mutant enzyme obtained by the above method include the nucleotide sequence represented by SEQ ID NO: 18, 19, 20, 21, or 22.
  • the molecular force ScDNA library containing the nucleotide sequence represented by SEQ ID NO: 18, 19, 20, 21, or 22 was not artificially generated during preparation of the cDNA library, it was used for preparation of the cDNA library.
  • the genomic library of human tissue or cultured cell lines was screened using a sequence specific to the nucleotide sequence represented by SEQ ID NO: 18, 19, 20, 21 or 22, and the nucleotide sequence of the resulting genomic clone was determined. It can be judged by deciding.
  • Genomic libraries are available from human tissues or cultured cell lines as described in Molecular 'Cloning 3rd Edition' and Current 'Protocols'In'Molecular' Biology.
  • the body can be prepared using a known method. It can also be prepared by using a genomic DNA library screening system (Genome Systems) or Universal GenomeWalker TM Kits (CLONTEC H).
  • SEQ ID NO: 18, 19, 20, 21, or 22 As a method for screening a genomic library using a sequence specific to the nucleotide sequence represented by SEQ ID NO: 18, 19, 20, 21, or 22, SEQ ID NO: 18, 19, 20, 21, or 22 is used. PCR method using primers specific to the nucleotide sequence represented by (PCR Protocols, Academic Press (1990)), or specific to the nucleotide sequence represented by SEQ ID NO: 18, 19, 20, 21 or 22. Examples include colony hybridization using oligonucleotides and plaque hybridization method (Molecular 'Cloning 3rd Edition).
  • a genomic DNA clone containing the base sequence represented by SEQ ID NO: 18, 19, 20, 21 or 22 is obtained by the above method. If the base sequence of this genomic DNA was determined and confirmed to match the base sequence represented by SEQ ID NO: 18, 19, 20, 21 or 22, the sequence was generated artificially when the cDNA library was prepared. It turns out that it is not a thing.
  • a cDNA library prepared using human tissue or a cultured cell line using the full length or a part of the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 18, 19, 20, 21, or 22 as a probe.
  • SEQ ID NO: 18, 19, 20, 21, or 22 the nucleotide sequence represented by SEQ ID NO: 18, 19, 20, 21, or 22 as a probe.
  • oligonucleotides can be used for detecting the -1,6-fucosyltransferase mutant of the present invention.
  • Origonutare Ochido capable of identifying and alpha _1,6- fucosyl transferase peptidase variants and alpha-1,6-fucosyltransferase of the present invention is the diagnosis of alpha-1,6-fucosyltransferase variants of the invention Useful in law.
  • oligonucleotide examples include oligonucleotides such as oligo DNA and oligo RNA, and derivatives of the oligonucleotide (hereinafter referred to as oligonucleotide derivatives).
  • oligonucleotide or antisense oligonucleotide examples include, for example, a detected primer, a sense primer corresponding to the base sequence of the 5 ′ end, and a sense primer corresponding to the base sequence of the 5 ′ end.
  • examples include antisense primers corresponding to the base sequence.
  • the base corresponding to uracil in mRNA is thymidine in the oligonucleotide primer.
  • the sense primer and the antisense primer are oligonucleotides whose melting temperature (Tm) and the number of bases do not change drastically and are 5 to 60 bases, preferably 10 to 50 bases. It is done.
  • Oligonucleotide derivatives include phosphodiester bonds in oligonucleotides. Oligonucleotide derivatives converted to phosphorothioate bonds, oligonucleotide derivatives in which phosphodiester bonds in oligonucleotides are converted to N3'-P5 'phosphoramidate bonds, ribose and phosphodiester bonds in oligonucleotides Is an oligonucleotide derivative in which uracil in the oligonucleotide is substituted with C_5 propynyluracil, an oligonucleotide derivative in which uracil in the oligonucleotide is substituted with C_5 thiazoleuracil, Oligonucleotide derivative in which cytosine in oligonucleotide is substituted with C-5 propynylcytosine, and cytosine in oligonucleotide is substituted with phenoxa
  • the ⁇ -1,6-fucosyltransferase mutant of the present invention can be obtained by using the method described in Molecular Cloning 3rd Edition or Current Protocols “in.
  • the ⁇ -1,6-fucosyltransferase mutant DNA of the present invention can be expressed in a host cell and produced.
  • a recombinant vector is prepared by inserting the DNA fragment or full-length cDNA into the downstream of the promoter of an appropriate expression vector.
  • a transformant producing the -1,6-fucosyltransferase mutant of the present invention can be obtained.
  • any bacteria, yeast, animal cell, insect cell, plant cell and the like can be used so long as they can express the target gene.
  • an expression vector autonomous replication is possible in the above host cell or into the chromosome. And a promoter containing a promoter at a position where the DNA encoding the protein of the present invention can be transcribed is used.
  • the recombinant vector containing the DNA encoding the ⁇ -1,6-fucosyltransferase mutant of the present invention is capable of autonomous replication in prokaryotes.
  • the vector is preferably composed of a promoter, a ribosome binding ligand 1J, a gene encoding the protein of the present invention, and a transcription termination sequence. It includes genes that control the promoter.
  • Examples of expression vectors include pBTrp2, pBTacl, pBTac2 (all sold by Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGE MEX-1 (Promega) ), PQE_8 (manufactured by QIAGEN), pKYPIO (JP-A 58-110600), pKY ⁇ 200 [Agricultural Biological Chemistry,, 669 (1984)], pLSAl [Agric. Biol. Chem., 53, 277 (1989)], pGELl [Proc. Natl. Acad. Sci.
  • the promoter may be any as long as it can be expressed in the host cell.
  • tro.promoter P
  • lac promoter P promoter
  • promoters derived from E. coli and phages such as the T7 promoter.
  • P X 2 two P promoters in series (P X 2), £ promoter, lacT7 pro trp trp
  • a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (eg, 6 to 18 bases).
  • the nucleotide sequence of the portion encoding the -1,6-fucosyltransferase mutant of the present invention The production rate of the target ⁇ ⁇ 1, 6-fucosyltransferase mutant can be improved by substituting the base so as to be an optimal codon for host expression.
  • a transcription termination sequence is not necessarily required for the expression of the DNA of the ⁇ -1,6-fucosyltransferase mutant of the present invention, but a transcription termination sequence is placed directly under the structural gene. It is preferable to arrange.
  • Host cells include microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Syudomonas, etc., such as Escherichiacoli XLl_Blue, Escherichiacoli XL2_Blue, Escherichia coliDHl, Escherichia Escherichia coli KY3276, Escherichiacoli W1485, Escheric hia coli JM109, Escherichia coli HB101, Escherichiacoli No.49, Escherichia coli W31 10, Escherichiacoli NY49, Serratia ficaria. Serratia fonticola.
  • Brevibacteriumsaccharolvticum ATCC14066, Brevibacterium flavum ATCC14067, Brevibacteriumammoniagenes, Brevibacteriumlactofermentum ATCC13869, Corvnebacterium glutamicum ATC 13032, Corvnebacteriumacetoacid ophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354, Pseudomonassp D-0110 etc. can be raised.
  • any method for introducing a recombinant vector any method can be used as long as it is a method for introducing DNA into the host cell, for example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110. (1972)], the protoplast method (Japanese Patent Laid-Open No. 63-248394), or the methods described in Gene, 17, 107 (1982) and Molecular & General Genetics, Cliff, 111 (1979).
  • yeast When yeast is used as a host cell, examples of expression vectors include YEP13 (ATC C37115), YEp24 (ATCC37051), YCp50 (ATCC37419) and the like. Any promoter can be used as long as it can be expressed in yeast strains. For example, promoters of glycolytic genes such as hexose kinase, PH05 promoter, PGK promoter, GAP promoter, ADH promoter , Gal 1 probe motor, gal 10 promoter, heat shock protein promoter, MF hi 1 promo And the CUP 1 promoter.
  • promoters of glycolytic genes such as hexose kinase, PH05 promoter, PGK promoter, GAP promoter, ADH promoter , Gal 1 probe motor, gal 10 promoter, heat shock protein promoter, MF hi 1 promo And the CUP 1 promoter.
  • Examples of host cells include yeasts belonging to the genus Saccharomyces, Schizosaccharomyces, Kluybe mouth genus, Trichosporon genus, Schneomyces genus, such as Saccharomvces cerevisi ae. Schizosaccharomvces pomoe. Kiuvveromvces lactans, I can raise alluvius.
  • any method can be used as long as it is a method for introducing DNA into yeast.
  • the electoral position method [Methods. Enzymol.], 194, 182 (1990)]
  • Supo Higuchi plast method [Proceedings 'Ob' The 'National Academia ⁇ .Ob' Science (Pro Natl. Acad. Sci. USA), 84, 1929 (1 978)]
  • Lithium acetate method [Journal 'Ob' Battereriology (J. Bacteriology), 153, 163 (1983)], Proc. Natl. Acad. Sci. USA ), 75, 1929 (1978)].
  • examples of expression vectors include pcDNAI, pc DM8 (commercially available from Funakoshi), pAGE107 [Japanese Patent Laid-Open No. 3-22979; Cytotec hnology, 3, 133, ( 1990)], pAS3-3 [Japanese Patent Laid-Open No. 2-227075], pCDM8 [Nature, 329, 840, (1987)], pcDNAI / Amp (Invitrogen), pREP4 (Invitrogen), pAGE 103 [ Journal “Ob” Biochemistry (J. Biochemistry), Dish, 1307 (1987)], pAGE 210 and the like.
  • Any promoter can be used as long as it can be expressed in animal cells.
  • CMV cytomegalovirus
  • IE immediate early gene promoter 1
  • SV40 early promoter SV40 early promoter
  • retrowinores examples include promoters, meta-mouthone promoters, heat shock promoters, SR promoters, and the like.
  • Host cells include human cells such as Namalwa cells, monkey cells such as COS cells, Chinese's cells, CHO cells that are Muster cells, and HBT5637 (Japanese Patent Laid-Open No. 63-29 9). ), Rat myeloma cells, mouse myeloma cells, Syrian hamster kidney-derived cells, embryonic stem cells, fertilized egg cells, and the like.
  • Recombinant vectors can be introduced by any method that introduces DNA into animal cells. Misalignment can also be used, for example, the electopore position method [Cytote chnology, 3, 133 (1990)], the calcium phosphate method [JP-A-2-27075], the ribofusion method [Proceedings of the National Academy of Science (Pro Natl • Acad. Sci. USA), 84, 7413 (1987)], injection method [Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Pres s (1994) (Hereafter abbreviated as “Manipulating“ Mouse ”Embryo 2nd edition”)], a method using a part gun (gene gun) [Patent No.
  • the recombinant gene transfer vector and baculovirus are co-introduced into insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected into insect cells to express the protein. it can.
  • Examples of gene transfer vectors used in the method include pVL1392, pVLl.
  • autographa californica nu clear polyhedrosis virus which is a virus that infects night stealing insects
  • Autographa californica nu clear polyhedrosis virus can be used, such as Autographa californica nu clear polyhedrosis virus.
  • Insect cells include Sodooterafrug erda's ovarian cells Sf9, Sf21 [Current 'Protoco 1 ⁇ Norez' In 'Molecuff 1 ⁇ ⁇ / Yoroshi 1 ⁇ Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992)], nchonlusiani ovary cells such as High 5 (Invitrogen) can be used.
  • expression vectors include Ti plasmids and tobacco mosaic virus vectors.
  • Any promoter can be used as long as it can be expressed in plant cells, and examples thereof include the cauliflower mosaic virus (CaMV) 35S promoter and the rice 1 promoter.
  • CaMV cauliflower mosaic virus
  • host cells include tobacco, potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat, barley, and other plant cells.
  • any method can be used as long as it is a method for introducing DNA into plant cells.
  • Agrobacterium Japanese Patent Laid-Open No. 59-140
  • Japanese Patent Laid-Open No. 59-140 Japanese Patent Laid-Open No. 59-140
  • the transformant obtained as described above is cultured in a medium, and the ⁇ - ⁇ , 6-fucosyltransferase mutant of the present invention is produced and accumulated in the culture and collected from the culture.
  • the inventive 1,6-fucosyltransferase mutant can be produced.
  • the method of culturing a transformant expressing the _1,6-fucosyltransferase mutant of the present invention in a medium can be performed according to a usual method used for culturing a host.
  • the medium for culturing a transformant obtained by using a prokaryote such as E. coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the organism. Any medium that can efficiently culture transformants can be a natural or synthetic medium. A deviation may be used.
  • the carbon source if it can be assimilated by the organism, it can be gnolecose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, Alcohols such as ethanol and propanol can be used.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast Extracts, corn steep liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used.
  • inorganic salt monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, mangan sulfate, copper sulfate, calcium carbonate and the like can be used. .
  • the culture is usually carried out under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the culture temperature is 15-40 ° C, and the culture time is usually 16 hours to 7 days.
  • the pH during the culture is maintained at 3.0 to 9.0.
  • the pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.
  • an antibiotic such as ampicillin or tetracycline may be added to the medium as needed during the culture.
  • an inducer may be added to the medium as necessary.
  • an inducer may be added to the medium as necessary.
  • isopropyl-1- ⁇ _D_thiogalatatopyranoside is used.
  • indole acrylic acid is used. Etc. may be added to the medium.
  • the RPMI1640 medium [The Journal of the American American Medical Association (The Journal of the American Medical Association), 199, 519 (1967)], Eagle MEM medium [Science, 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 (1959)], 199 medium [Procedure 'Ob' The 'Society'] Fore 'the' Biologic Canore 'Medicine (Proceeding of the Society for the Biologic al Medicine), 73, 1 (1950)], Whitten Medium [Genetic Engineering Experiment Manual-Transgeneic' How to make a mouse (Kodansha) edited by Motoya Katsuki (1987)] or a medium obtained by adding fetal calf serum or the like to these mediums.
  • Cultivation is usually carried out under the conditions of pH 6-8, 30-40 ° C, 5% CO, etc .:! -7 days.
  • Cultivation can be performed for 1 day to several months using culture methods such as Fuedbacti culture and holofiber culture.
  • antibiotics such as kanamycin and penicillin may be added to the medium as needed during the culture.
  • Cultivation is usually carried out for 1 to 5 days under conditions of pH 6-7, 25-30 ° C, etc.
  • an antibiotic such as gentamicin may be added to the medium as needed during the culture.
  • a transformant obtained using a plant cell as a host can be cultured as a cell or after being differentiated into a plant cell or organ.
  • a medium for culturing the transformant commonly used Murashige 'and' Stag (MS) medium, White medium, or a plant hormone such as auxin or cytokinin is added to these mediums. Can be used.
  • Cultivation is usually carried out under conditions of pH 5-9 and 20-40 ° C for 3-60 days.
  • antibiotics such as kanamycin, no, and idaromomycin may be added to the medium as needed during culture.
  • a microorganism, animal cell, or plant cell having a recombinant vector incorporating DNA encoding the -1,6-fucosyltransferase mutant of the present invention The resulting transformant is cultured according to a normal culture method, the ⁇ -1,6-fucosyltransferase mutant is produced and accumulated, and the ⁇ -1,6-fucosyltransferase mutant is collected from the culture. By doing so, it is possible to produce the ⁇ -1,6-fucosyltransferase mutant.
  • a gene expression method in addition to direct expression, secretory production, fusion protein expression, and the like can be performed in accordance with the method described in Molecular Cloning 3rd Edition.
  • a method for producing the -1,6-fucosyltransferase mutant of the present invention there are a method in which it is produced in the host cell, a method in which it is secreted outside the host cell, and a method in which it is produced on the host cell outer membrane. This method can be selected by changing the structure of the host cell to be produced and the protein to be produced.
  • expression is performed by adding a signal peptide in front of the protein containing the active site of the ⁇ -1,6-fucosyltransferase mutant of the present invention using a genetic recombination technique.
  • the ⁇ -1,6-fucosyltransferase mutant of the present invention can be actively secreted outside the host cell.
  • the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like.
  • the transformant is an animal individual or a plant individual, it is bred or cultivated according to a normal method, and the ⁇ -1,6-fucosyltransferase mutant is produced and accumulated, from the animal individual or the plant individual.
  • the ⁇ _1,6-fucosyltransferase mutant By collecting the ⁇ _1,6-fucosyltransferase mutant, the -1,6-fucosyltransferase mutant can be produced.
  • a method for producing the -1,6-fucosyltransferase mutant of the present invention using an animal individual for example, a known method [American 'Journal' of 'Clinical' Nutrition, (American Journal of Clinical Nutrition), 63 , 639S (1996); American Journal of Clinical Nutrition, 63, 627 S (1996); Bio / Technology, 9, 830 (1991) )], A method for producing the mutant of 1,6-fucosyltransferase of the present invention in an animal constructed by introducing a gene.
  • a transgenic non-human animal introduced with a DNA encoding the -1,6-fucosyltransferase mutant of the present invention is bred, and the ⁇ -1,6- The ⁇ -1,6-fucosyltransferase mutant is obtained by generating and accumulating a fucosyltransferase mutant in the animal and collecting the -1,6-fucosyltransferase mutant from the animal.
  • Examples of the production / accumulation place in the animal include milk of the animal (Japanese Patent Laid-Open No. 63-309192), eggs and the like.
  • Any promoter can be used as long as it can be expressed in animals.
  • a sex protein promoter or the like is preferably used.
  • the ⁇ -1,6-fucosyltransferase mutant produced by the transformed cell of the present invention for example, when the ⁇ -1,6-fucosyltransferase mutant power of the present invention is expressed in a dissolved state in the cell After completion of the culture, the cells are collected by centrifugation, suspended in an aqueous buffer solution, and then disrupted by an ultrasonic crusher, French press, Manton Gaurin homogenizer, dynomill, etc. to obtain a cell-free extract. .
  • the supernatant strength obtained by centrifuging the cell-free extract the usual enzyme isolation and purification methods, that is, solvent extraction methods, salting-out methods using ammonium sulfate, desalting methods, precipitation methods using organic solvents, Anion-exchange chromatography using resin such as Jetylaminoethyl (DEAE) -Sepharose, DIAION ⁇ -75 (manufactured by Mitsubishi Kasei), Cation using resin such as SS sign harose FF (Pharmacia) Replacement chromatography method, hydrophobic chromatography method using resins such as butyl sepharose and phenyl sepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, isoelectric focusing Purified preparations can be obtained using methods such as electrophoresis, such as electrophoresis, alone or in combination.
  • solvent extraction methods salting-out methods using ammonium sulfate, desalting methods, precipitation methods using organic solvent
  • the ⁇ _1,6-fucosyltransferase mutant is expressed by forming an insoluble substance in the cells, the cells are similarly collected, disrupted, and centrifuged to obtain a precipitate fraction. As a result, an insoluble form of the ⁇ -1,6-fucosyltransferase mutant is recovered.
  • the recovered insoluble form of the ⁇ -1,6-fucosyltransferase mutant is solubilized with a protein denaturant.
  • the ⁇ -1,6-fucosyltransferase mutant is returned to its normal three-dimensional structure by diluting or dialyzing the solubilized solution, and then isolated by the same isolation and purification method as described above. A purified preparation of a fucosyltransferase mutant can be obtained.
  • the -1,6-fucosyltransferase mutant of the present invention or a modified sugar thereof is secreted outside the cell
  • the -1,6-fucosyltransferase is added to the culture supernatant. It is possible to recover derivatives such as mutase mutants or sugar chain adducts thereof. That is, a soluble fraction is obtained by treating the culture by a method such as centrifugation as described above, and a purified preparation is obtained from the soluble fraction by using the same isolation and purification method as described above. You can get power S.
  • the mutant of 1,6-fucosyltransferase of the present invention can also be produced by chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (tbutyloxycarbonyl method). it can.
  • chemical synthesis may be performed using peptide synthesizers such as Advanced ChemTech, Perkinenoma, Ltd., Pharmacia, Protein technology Instrument, Synthecel Vega, Per S India, Shimadzu, etc. it can.
  • Examples of the method for measuring the activity of the -1,6-fucosyltransferase mutant and -1,6-fucosyltransferase of the present invention include known methods [Uozumi et al., J Biochem, 120. 385-392 ( 1996)], there is a method for measuring the activity of the prepared -1,6-fucosyltransferase mutant of the present invention. Specific examples include the following methods.
  • the sugar chain was eluted with 20 mM sodium acetate buffer (pH 4) containing 0.1% butanol, and the fluorescence intensity of the eluate with an excitation wavelength of 320 nm and emission wavelength of 400 nm was measured with a fluorometer (manufactured by Shimadzu Corporation, model Measure with RF535). From the measured fluorescence intensity, the amount of fucosylated complex double-stranded sugar chain produced by the enzyme reaction is calculated. Enzyme specific activity in a sample is expressed in moles of fucosylated complex double-stranded sugar chain molecules per unit protein mass contained in the sample and per unit reaction time. (Unit: pmol / hour / mg). The amount of protein in the sample is measured with BCA protein assembly kit (Pierce) using ushi serum albumin (Pierce) as a standard product.
  • a polyclonal antibody can be prepared by administering a peptide having a partial amino acid sequence of the body as an antigen to an animal.
  • Usagi, goat, rat, mouse, hamster and the like can be used as an animal to be administered.
  • the dose of antigen is 50 per animal: 100 mu ⁇ are preferred.
  • a peptide When a peptide is used, it is desirable that the peptide is covalently bound to a carrier protein such as keyhole limpet haem ocyanin or bovine thyroglobulin.
  • a carrier protein such as keyhole limpet haem ocyanin or bovine thyroglobulin.
  • the peptide used as an antigen can be synthesized with a peptide synthesizer.
  • the administration of the antigen is carried out after the first administration:! To 3 to 10 times every 2 weeks. On the 3rd to 7th day after each administration, blood is collected from the fundus venous plexus and the serum reacts with the antigen used for immunization.
  • the enzyme immunoassay (ELISA): published by the medical school (1976) ), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)].
  • a polyclonal antibody can be obtained by obtaining serum from a non-human mammal whose serum showed a sufficient antibody titer against the antigen used for immunization, and separating and purifying the serum.
  • Separation and purification methods include centrifugation, salting out with 40-50% saturated ammonium sulfate, and caprinolic acid precipitation [Antibodies, A Laboratory manual, Cold Spring Harbor Labor atory (1988)], or chromatography using a DEAE-Sepharose column, an anion exchange column, protein A or G-force ram or Genore filtration column, etc., alone or in combination.
  • a rat whose serum showed a sufficient antibody titer against the partial fragment polypeptide of the -1,6-fucosyltransferase mutant of the present invention used for immunization is used as a source of antibody-producing cells.
  • the spleen is removed 3 to 7 days after the final administration of the antigenic substance to the rat showing the antibody titer.
  • the spleen is shredded in MEM medium (Nissui Pharmaceutical Co., Ltd.), loosened with tweezers, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded.
  • myeloma cells cell lines obtained from mice or rats are used.
  • an 8-azaguanine resistant mouse derived from 8-8) myeloma cell line P3-X63Ag8-Ul (hereinafter abbreviated as P3-U1) [Curr. Topics. Microbiol. Immunol., 81, 1 (1978) , Europ. J. Immunol., 6, 511 (1976)], SP2 / 0-Agl4 (SP-2) [Nature, 276, 269 (1978)], P3— X63— Ag8653 (65 3) [J.
  • the cell group of the obtained precipitate fraction is thoroughly loosened, and the cell group is stirred at 37 ° C, and 10 g of polyethylene glycol-1000 (PEG-1000) per 10 8 antibody-producing cells, MEM Add 0.2 ml of a solution containing 2 ml and 0.7 ml of dimethyl sulfoxide (DMS 0), and add 1-2 ml of MEM medium several times every 1-2 minutes. After the addition, add MEM medium to prepare a total volume of 50 ml. Centrifuge the preparation at 900 rpm for 5 minutes, and discard the supernatant.
  • PEG-1000 polyethylene glycol-1000
  • HAT medium normal medium hypoxanthine (10- 4 mol / L) at blowing, thymidine (1. 5 X 10 - 5 molZL) and aminopterin (4 X 10- 7 molZL) was added medium
  • HAT medium normal medium hypoxanthine (10- 4 mol / L) at blowing, thymidine (1. 5 X 10 - 5 molZL) and aminopterin (4 X 10- 7 molZL) was added medium
  • the suspension is dispensed at 100 ⁇ l / well into a 96-well culture plate, and cultured in a 5% CO incubator at 37 ° C for 7 to 14 days.
  • enzyme immunoassay include the following methods.
  • an ⁇ -1,6-fucosyltransferase mutant partial fragment polypeptide of the present invention used as an antigen is coated on an appropriate plate, and is obtained in a hyperidoma culture supernatant or as described in (d) below.
  • the purified antibody is reacted as the first antibody, and the anti-rat or anti-mouse immunoglobulin antibody labeled with piotin, enzyme, chemiluminescent substance or radiation compound as the second antibody is reacted, and then the reaction according to the labeling substance.
  • the one that specifically reacts with the -1,6-fucosyltransferase mutant of the present invention is selected as a hybridoma that produces a monoclonal antibody that recognizes the -1,6-fucosyltransferase mutant of the present invention.
  • cloning was repeated twice by the limiting dilution method (first time using HT medium (medium obtained by removing aminopterin from HAT medium), second time using normal medium), and stable.
  • having a strong antibody titer is selected as a hyperidoma strain producing a monoclonal antibody that recognizes the ⁇ -1,6-fucosyltransferase mutant of the present invention.
  • pristane 2, 6, 10, 14-tetramethylpentadecane (Pristane) O. 5ml intraperitoneally and bred for 2 weeks] 8 ⁇ :
  • Fei -1 acquired present invention 6 - recognizes fucosyltransferase variant monoclonal antibody-producing hybridoma cells 5 to 20 X 10 6 cells / mouse are injected intraperitoneally. Hypridoma becomes ascites tumor in 10-2 days.
  • Ascites fluid is collected from the ascites-bearing mouse and centrifuged at 3000 rpm for 5 minutes to remove solids.
  • a monoclonal antibody can be purified and obtained by the same method as that used for polyclonal.
  • the antibody subclass is determined using a mouse monoclonal antibody typing kit or a rat monoclonal antibody typing kit.
  • the protein mass is calculated from the Raleigh method or absorbance at 280 nm.
  • Cells expressing the ⁇ -1,6-fucosyltransferase mutant of the present invention can be used in various ways by using the method described in the production of the -1,6 -fucosyltransferase mutant of the present invention in 2. above. It can be produced using host cells.
  • the -1,6-fucosyltransferase derived from the -1,6-fucosyltransferase mutant of the present invention is used.
  • Cells having only ferrase activity can be prepared.
  • -1,6-fucose modifying enzyme by targeting the gene of -1,6-fucose modifying enzyme and using the method of genomic gene modification, -1,6-6-fucosyltransferase mutant-derived -1, -1,6-fucose transferase mutant of the present invention is used.
  • Cells having only fucose modifying enzyme activity can be produced.
  • Specific examples of the a- fucose modifying enzyme include ⁇ -1,6-fucosyltransferase.
  • any method for modifying a genomic gene any method can be used as long as it can specifically modify the genomic gene of the target enzyme. Examples thereof include homologous recombination method, RDO method, method using retrovirus, method using transposon, and the like. These will be specifically described below.
  • the cell expressing the -1,6-fucosyltransferase mutant of the present invention targets the -1,6-fucose modifying enzyme gene, and modifies the target gene on the chromosome using the homologous recombination method. Can be produced.
  • a target vector for homologous recombination of the target gene to be modified (for example, the structural gene of -1,6-fucosase modifying enzyme or intron gene) is prepared. .
  • the produced target vector is introduced into a cell, and a cell that has undergone homologous recombination between the target gene and the target vector is selected to express the -1,6-fucosyltransferase gene of the present invention.
  • Cells can be made.
  • a method for obtaining cDNA and genomic DNA of a -1,6-fucose modifying enzyme is, for example, For example, the method described in 1. above can be mentioned.
  • Target vectors for homologous recombination of target genes are: Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Nokumanyu Anoreno series 8 Gene Targeting, ES cells Production of a mutant mouse using a mouse (Yodosha) (1995) and the like.
  • the target vector may be any of a replacement type, an insertion type, or a gene trap type.
  • any method for introducing a target vector into a cell 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 [Japanese Patent Laid-Open No. 2-227075], reflection method [Procida Natl. Acad. Sci. USA], 84, 7413 (1987)], injection method [Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994) (hereinafter abbreviated as "Manipulating” Mouse “Embryo Second Edition)], Method using particle gun [Gene Gun] [Patent No. 2606856, Patent No.
  • positive selection for selecting homologous recombinants containing the hprt gene can be performed.
  • a target vector containing a neomycin resistance gene positive selection for selecting homologous recombinants containing a neomycin resistance gene by culturing cells into which the vector has been introduced in a medium containing G418 and selecting a G418 resistant strain.
  • a G418 resistant strain Can be performed.
  • Contains DT gene In the case of a target vector, the cells into which the vector has been introduced are cultured, and the growing strain is selected (in the case of recombinants randomly inserted into the chromosome other than homologous recombination, the DT gene is integrated into the chromosome and expressed.
  • Cells expressing the -1,6-fucosyltransferase mutant of the present invention target the -1,6-fucose modifying enzyme gene and use the RDO (RNA-DNA oligonucleotide) method.
  • RDO RNA-DNA oligonucleotide
  • an RDO construct of an appropriate length including the portion encoding ⁇ -1,6-fucose modifying enzyme, the untranslated region, or the intron portion is designed and synthesized.
  • the synthesized RDO By introducing the synthesized RDO into a cell and selecting the target enzyme, ie, the cell in which the ⁇ -1,6-fucose modified enzyme is mutated, the ⁇ -1,6-fucosyl of the present invention is selected. Cells expressing the transferase mutant can be generated.
  • Examples of a method for preparing cDNA for -1,6-fucose modifying enzyme include the method for preparing cDNA described in 1. above.
  • Examples of the method for preparing genomic DNA of H-1,6-fucose modifying enzyme include the method for preparing genomic DNA described in 1. above.
  • the DNA base sequence is cleaved with an appropriate restriction enzyme, and then pBluescript SK (-) (Stratagen e), etc., and a commonly used nucleotide sequence analysis method such as Sanger et al.'s dideoxy method [Proceedings.Ob.The.Naka. Pro Natl. Acad. Sci., USA), 74, 5463 (1977)], etc., and analysis using an automatic base sequence analyzer such as A. and F. DNA Sequencer (Pharmacia) By doing so, the base sequence of the DNA can be determined.
  • RDO can be prepared by a conventional method or using a DNA synthesizer.
  • RDO constructs are described in Science, 273, 1386, (1996); Nichiya's Medicine (Nature Medicine), 4, 285, (1998); Hepatology (H-signed atology), 25, 1462. , (1997); Gene Therapy, 5, 1960, (1999); Gene Therapy, 5, 1960, (1999); Journal 'Ob' Molecular 'Medicin (J. Mol. Med.), 75, 829, (1997); Proceedings 'Ob The National' Academia Sci. USA (Pro Natl. Acad. Sci. USA), 96, 8774, (1999); Proceedings 'Ob' The 'National' Academy 'Ob' Science (Proc. Natl. Acad. Sci.
  • a cell expressing the -1,6-fucosyltransferase mutant of the present invention can be obtained by using the transposon system described in Nature Genet., 25, 35, (2000), etc. Therefore, it can be prepared by selecting mutants of 6-fucose modifying enzyme.
  • the transposon system is a system that induces mutations by randomly inserting foreign genes onto chromosomes. Usually, a foreign gene inserted into a transposon is used as a vector to induce mutations. A transposase expression vector for randomly inserting the gene into the chromosome is introduced into the cell at the same time. Any transposase that is suitable for the transposon sequence to be used can be used.
  • any gene that induces a mutation in cellular DNA can be used.
  • the present method contained in cells prepared according to the method described in 2 (2). Examples include methods for measuring the activity of the ⁇ -1,6-fucosyltransferase mutant of the invention.
  • ⁇ -1,6-fucosyltransferase mutant DNA or oligonucleotide of the present invention Using the ⁇ -1,6-fucosyltransferase mutant DNA or oligonucleotide of the present invention, Northern hybridization method (Molecular 'Cloning 2nd edition), PCR method and RT (reverse-transcribed) — Perform PCR method (both PCR Protocols, Academic Press (1990)) (also referred to as PCR method above), etc., to detect the DNA encoding the mutant of 1,6-fucosyltransferase gene of the present invention.
  • Northern hybridization method Molecular 'Cloning 2nd edition
  • PCR method and RT reverse-transcribed
  • the RT-PCR method is a simple method and is particularly useful as a method for detecting DNA expression.
  • RNA 10 to 20 ⁇ g derived from white blood cells or tissues of subjects and healthy subjects, or their ⁇ (1 to 5 ⁇ ⁇ ), and denaturation solution (50% ( ⁇ / ⁇ ) formamide, 2.2 mol / L monooledehydride, 20mmol / LMoPS [3_ (N-morpholino) propanesulfonic acid] (PH 7.0), 5mmol / L sodium acetate, lmmol / L EDTA] at 65 ° C, 5 Heat for minutes, denature, and run on a 1% agarose gel containing 2.2 mol / L formaldehyde.
  • denaturation solution 50% ( ⁇ / ⁇ ) formamide, 2.2 mol / L monooledehydride, 20mmol / LMoPS [3_ (N-morpholino) propanesulfonic acid] (PH 7.0), 5mmol / L sodium acetate, lmmol / L EDTA] at 65 ° C, 5 Heat for minutes,
  • RNA in the gel is blotted on a nitrocellulose filter (Optimal BA-S85; manufactured by Schleicher & Schuell) and immobilized by heating at 80 ° C for 1 hour under reduced pressure.
  • the filter was mixed with a hybridization solution [5 X SSPE (750 mmol / L NaCl, 50 mmol / L NaHPO, 5 mmol / L EDTA; pH 7.4), 5 X Denhardt solution (0.1%
  • a DNA fragment represented by SEQ ID NO: 18, 19, 20, 21, or 22 labeled with 32 P using a multiprime DNA labeling system (Amersham) can be used.
  • a set of oligonucleotides specific for DNA encoding the -1,6-fucosyltransferase mutant of the present invention is used as a primer, derived from white blood cells or tissues of subjects and healthy subjects. Perform PCR using total RNA, their mRNA, or cDNA prepared from these RNAs, and detect and quantify the amplified fragments, and determine the above diseases by comparing the expression level of the DNA between the subject and healthy subject can do.
  • the oligonucleotide described in 1. above can be used.
  • MRNA or total RNA which is a type of PCR, can be extracted from various leukocyte cells isolated and obtained from blood, or as a tissue suspected of having a disease.
  • Each white blood cell can be exemplified by polymorphonuclear leukocytes, monocytes, lymphocytes, T cells, B cells and the like.
  • Polymorphonuclear leukocytes and mononuclear cells can be separated from the peripheral blood of the subject by using Polymo ⁇ hprep TM, a kit manufactured by Nycomed Pharma. Can do.
  • Monocytes and lymphocytes were obtained from the obtained mononuclear cells by the method described in J. Immunol., 130, 706 (1983), etc., in Tissue Antigen, 9, 153 (1977), J. Immunol, 11, 273. (1976), T cells and B cells can be separated and obtained by the method described in the manual on the isolation method of blood cells of Nycomed, Inc.
  • T cells can also be obtained using the nylon wool method [Eur. J. Immunol., 3, 645 (1973)]. Further, it is possible to separate and acquire each cell using magnetic beads (for example, Dynabeads manufactured by Dynal) in which specific antibodies are bound to T cells, B cells, and monocytes / macrophages.
  • magnetic beads for example, Dynabeads manufactured by Dynal
  • specific antibodies are bound to T cells, B cells, and monocytes / macrophages.
  • guanidine thiocyanate-trifluoroacetate method [Methods in Enzymol., 154, 3 (1987)] can be mentioned.
  • Examples of a method for preparing poly (A) + RNA from total RNA include an oligo (dT) -immobilized cellulose column method (Molecular Cloning 2nd Edition).
  • first 'track' mRNA 'isolation' kit (Fast Track mRNA Isolation Kit; manufactured by Invitrogen), Quick Prep 'mRNA' purification kit (Quic k Prep mRNA Purification Kit; manufactured by Falmacia) Prepare mRNA using a kit such as the above.
  • Single-stranded cDNA can be synthesized from total RNA or mRNA using a single-stranded cDNA synthesis kit Superscript preamplification system (manufactured by BRL). The synthesis can be performed according to the manual attached to the kit.
  • the a_1,6_fucosyltransferase mutant of the present invention is encoded by the RT-PCR method (PC R Protocols, Academic Press (1990)) using total RNA, mRNA or cDNA prepared as described above. The amount of gene expression can be quantified.
  • the translation region of DNA encoding the -1,6-fucosyltransferase mutant of the present invention possessed by a patient is amplified by PCR, the nucleotide sequence is determined, and the sequence number Mutation in the translation region by comparing with the DNA base sequence represented by 3. You can check for the presence or absence.
  • the cDNA in the form of a cage for PCR can be obtained by the method described in (1).
  • the nucleotide sequence of the obtained cDNA can be determined using a DNA sequencer 377 from Perkin Elma and a reaction kit (ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit: Applied Biosystems).
  • the presence or absence of expression of the -1,6-fucosyltransferase mutant of the present invention in the blood or tissue of a subject is immunologically determined.
  • the following diseases can be determined by detecting or quantifying and comparing with -1,6-fucosyltransferase consisting of the amino acid represented by SEQ ID NO: 9.
  • immunological detection methods include ELISA using a microtiter plate, fluorescent antibody method, Western plot method, immunohistochemical staining method, and the like.
  • a sandwich using two monoclonal antibodies having different epitopes among antibodies that specifically react with the ⁇ _1,6-fucosyltransferase mutant of the present invention in a liquid phase As a method for immunological quantification, for example, a sandwich using two monoclonal antibodies having different epitopes among antibodies that specifically react with the ⁇ _1,6-fucosyltransferase mutant of the present invention in a liquid phase.
  • ELISA method radio I Takeno assay I method using a recognizing labeled RA-associated polypeptide and the polypeptide antibodies and the like with a radioisotope such as 125 1.
  • the immunological method is reported to be associated with ⁇ -1,6-fucose modifying enzyme, retinal dysplasia, liver cancer, fatty liver, blood coagulation disorder, cystic fibrosis, lung tissue It can be used for the determination of diseases such as disorders, poor growth, diseases with impaired insulin-like growth factor and growth hormone action, and diseases with impaired epidermal growth factor action.
  • symptomatic therapy can be achieved by inhibiting or enhancing the function of the ⁇ -1,6-fucosyltransferase mutant of the present invention.
  • the above diseases can be prevented or treated.
  • the -1,6-fucosyltransferase of the present invention is used.
  • the physiological action can be controlled by administering a compound that regulates the function of the mutant, and even if the change in the function of -1,6-fucose modifying enzyme is not a direct cause of the disease, the present invention Diseases that can be symptomatically treated by controlling the function of the -1,6-fucosyltransferase mutant can also be prevented and treated by administration of the compound.
  • the compound can be obtained, for example, by the method shown below.
  • the ⁇ -1,6-fucosyltransferase mutant of the present invention is expressed.
  • Known methods for detecting changes in responsiveness of cells to lectins, insulin-like growth factors, growth hormone, epithelial growth factor-dependent intracellular signal transduction, gene transcription, sugar uptake, proliferation, etc. J. Biol. Chem., 276, 1 1956, 2001; J. Biol. Chem., 275, 21988, 2000; Molecular Medicine, 40, 1034, 2003; Nature, 263, 663, 1976; clinical science , 17, 958, 1981).
  • Examples of the lectin include a lectin that recognizes a sugar chain structure in which the N-glycoside-linked sugar chain reducing terminal N-acetylcylcosamine is linked to the 6-position of fucose and the 1-position of fucose.
  • any lectin that can recognize the sugar chain structure can be used. Specific examples of this are: Lentil lectin LCA (lentil agglutinin from lentil aculglutinin), Endo bean lectin PSA (nea lectin from Pisum sativum), Broad bean lectin VFA (agglutinin from Vicia faba), Alochawantake lectin AAL (Aleuria Pharmaceuticals containing the compound obtained by the search method of n (5) (4)
  • the compound that controls the function of the -1,6-fucosyltransferase mutant of the present invention has been suggested to be associated with abnormal function of -1,6-fucose modifying enzyme, retinal dysfunction, liver cancer, fat Prevention and treatment of diseases such as liver, blood coagulation disorder, cystic fibrosis, lung tissue disorder, growth failure, diseases with impaired insulin-like growth factor and growth hormone action, diseases with impaired epidermal growth factor action, etc. Useful as a medicine.
  • the compound obtained in (4) can be provided, for example, as a pharmaceutical preparation produced by any method well known in the technical field of pharmaceutical formulation shown below.
  • a method for administering the prophylactic and therapeutic agents for example, when there is a patient who cannot expect normal physiological action due to functional abnormality associated with the ⁇ -1,6-fucosyltransferase mutant of the present invention, (i) (Ii) administering the DNA encoding a protein that controls the function of the ⁇ -1,6-fucosyltransferase variant of the invention to the patient and expressing the DNA (ii) in the target cell, the ⁇ -1,6-fucosyltransferase of the invention After inserting and expressing a DNA encoding a protein that controls the function of the mutant, the cell is transplanted into the patient.
  • the ⁇ -1,6-fucosyltransferase mutation of the present invention The function of the ⁇ -1,6-fucosyltransferase mutant of the present invention in the body of a patient can be changed by administering a protein or compound that controls the body function to the patient. Accordingly, the -1,6-fucosyltransferase mutant of the present invention can be used even if it is not caused by a disease caused by dysfunction of -1,6-fucose modifying enzyme or directly by -1,6-fucose modifying enzyme.
  • Preventive drugs and treatments for diseases that can be symptomatically treated by administration of DNA encoding a protein that regulates the function or a protein that regulates the function of the -1,6-fucosyltransferase mutant of the present invention Useful as a medicine.
  • a protein that regulates the function of the -1,6-fucosyltransferase mutant of the present invention is identified.
  • the DNA to be loaded is used as the above-mentioned preventive or therapeutic agent
  • an appropriate vector such as a virus-associated violets betater
  • a gene therapy vector incorporated into a virus vector such as a retrovirus or adenovirus or other gene therapy vector
  • a virus vector such as a retrovirus or adenovirus or other gene therapy vector
  • a gene therapy agent such as a gene therapy drug or a preventive drug
  • a gene therapy vector and a gene therapy agent can be manufactured by blending a base [Nature Genet., 8, 42 (1994)] 0
  • the base used for the gene therapy agent may be any base as long as it is usually used for injections, such as distilled water, sodium chloride or a salt solution such as a mixture of sodium chloride and an inorganic salt, mannitol, ratatose, Examples thereof include sugar solutions such as dextran and glucose, amino acid solutions such as glycine and arginine, organic acid solutions, or mixed solutions of a salt solution and a glucose solution.
  • these bases are mixed with an osmotic pressure adjusting agent, a pH adjusting agent, a vegetable oil such as sesame oil or soybean oil, or an auxiliary agent such as a surfactant such as lecithin or a nonionic surfactant.
  • An injection may be prepared as a suspension or dispersion. These injections can be prepared as preparations for dissolution at the time of use by operations such as pulverization and freeze-drying.
  • the gene therapy agent can be used as it is in the case of a liquid, and in the case of an individual, the gene therapy agent can be dissolved in the above-mentioned base that has been sterilized if necessary before the gene therapy.
  • An example of a gene therapy agent administration method is a method of local administration so that the gene therapy agent is absorbed at a treatment site of a patient.
  • the ability to transport DNA to the target treatment site can also be achieved by non-viral gene transfer.
  • Non-viral gene transfer methods known in the art include calcium phosphate coprecipitation (Virolog y, 52, 456-467 (1973); Science, 209, 1414-1422 (1980)), microinjection [Proc. Natl. Acad. Sci. USA, 77, 5399-5403 (1980); Proc. Natl. Acad. Sci. USA, 77, 7380-7384 (1980); Cell, 27, 223-231 (1981); Nature, 294, 92 -94 (1981)], liposome-mediated membrane fusion-mediated transfer (Pro Natl. Acad. Sci. USA, 84, 7413-7417 (1987); Biochemistry, 28, 9508-9514 (1989); J. Biol.
  • ribosome preparations can be directly administered to a target tissue to allow local uptake and expression of that tissue in tumor research. It has been reported [Hum. Gene Ther., 3, 399 (1992)].
  • a drug containing a protein or compound that controls the function of the ⁇ -1,6-fucosyltransferase mutant of the present invention as an active ingredient is usually capable of administering the active ingredient alone.
  • the active ingredient is desirably mixed with one or more pharmaceutically acceptable carriers and provided as a pharmaceutical preparation prepared by any method well known in the technical field of pharmaceutics.
  • a sterile solution dissolved in water or an aqueous carrier such as an aqueous solution of salt, glycine, gnolecose, human albumin or the like is used.
  • pharmacologically acceptable additives such as buffering agents and isotonic agents for bringing the formulation solution close to physiological conditions, such as sodium acetate, sodium chloride, sodium lactate, potassium chloride, Sodium quenate and the like can also be added. It can also be lyophilized and stored, and dissolved in an appropriate solvent before use.
  • oral administration or oral administration such as buccal, intratracheal, rectal, subcutaneous, intramuscular and intravenous.
  • oral administration forms include sprays, capsules, tablets, granules, syrups 1J, emulsions, suppositories, injections, ointments, tapes, and the like.
  • Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules Agents and the like.
  • liquid preparations such as emulsions and syrups include sugars such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, P- It can be produced using preservatives such as hydroxybenzoic acid esters, and flavors such as strawberry flavor and peppermint as additives.
  • excipients such as lactose, glucose, sucrose, and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polybulu alcohol It can be produced using a binder such as hydroxypropyl cellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin as additives.
  • Suitable formulations for parenteral administration include injections, suppositories, sprays and the like.
  • an injection is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of both.
  • Suppositories are prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.
  • the propellant uses the carrier or the like which does not irritate the protein or the compound itself, or the recipient's oral cavity and airway mucosa, and is easily dispersed by dispersing the protein or the compound as fine particles.
  • Specific examples of the carrier include lactose and glycerin.
  • preparations such as aerosol or dry powder are possible.
  • the components exemplified as additives in oral preparations can also be added.
  • the dose or number of administrations is a force S that varies depending on the intended therapeutic effect, administration method, treatment period, age, body weight, etc., and is usually 10 / ig / kg to 8 mg / kg per day for an adult.
  • the activity of the -1,6-fucose modifying enzyme is deleted or decreased, so that the sugar produced in the cell Proteins lack or reduce sugar chain modification by -1,6-fucose modifying enzymes.
  • glycoproteins lacking or reducing the sugar chain modification by -1,6-fucose modifying enzyme are in phase with changes in hemodynamics and distribution in the living body and proteins necessary for the expression of pharmacological activity. The interaction has changed and it is useful as a medicine.
  • Specific examples include antibodies, erythropoietin, thrombopoietin, tissue-type plasminogen activator, prolokinase, thrombomodulin, antithrombin II I, protein blood coagulation factor VII, blood coagulation factor VIII, blood Coagulation factor IX, Blood coagulation 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 ⁇ , interferon ⁇ , interleukin 2, interleukin 6, interleukin 10, interleukin 11, soluble interleukin 4 receptor, tumor necrosis factor, Dnasel, galactosidase, dalcosidase, dalco cerebrosidase, etc. .
  • a more specific example of a glycoprotein whose physiological activity is significantly increased due to a sugar chain structure in which the fucose modification is deleted or reduced is, for example, an antibody.
  • a method for producing a glycoprotein composition using cells having the ⁇ -1,6-fucosyltransferase variant of the present invention will be described by taking the production of an antibody composition as an example.
  • the antibody composition is Moleculera 'Cloning 3rd Edition, Current' Protocols 'in' Molechu * Noroji, Antibodies, A Laboratory manual, old Spring Harbor Laboratory, 1988 (hereinafter abbreviated as Antibodies) , Monoclonal Antibodies: principles an d practice, Third Edition, Acad. Press, 1993 (hereinafter referred to as Monoclonal Nanoreantibodies), Antibody Engineering, A Practical Approach, IRL Press at Oxford University Press, 1996 (hereinafter referred to as Antibody) For example, it can be obtained by expressing in a cell expressing the ⁇ -1,6-fucosyltransferase mutant of the present invention as described below.
  • 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 recombinant vector of the present invention adapted to the expression vector A transgenic individual producing an antibody molecule can be obtained by introducing it into a cell that expresses a ferase mutant.
  • Any cell that expresses the gene of interest such as bacteria, yeast, animal cells, insect cells, or plant cells, can be used as a cell that expresses the ⁇ _1,6-fucosyltransferase mutant.
  • animal cells are used.
  • Cells such as bacteria, yeast, animal cells, insect cells, plant cells, etc. that have been introduced using genetic engineering techniques to bind the glycoside-linked sugar chain that binds to the Fc region of antibody molecules Can also be used.
  • Examples of the cells used in the method for producing the antibody composition of the present invention include the cells produced in the above 2. or 4. that express the -1,6-fucosyltransferase mutant of the present invention. .
  • cDNA should be prepared according to the cDNA preparation method described in 1. above, from human or non-human animal tissues or cells, using probe primers specific for the antibody molecule of interest. Power S can be.
  • Specific methods for preparing an antibody composition in the above various cells include the method for constructing the expression vector described in 2. above, the method for introducing the expression vector into the cell, the method for culturing the vesicle, and the target production The purification method of a thing can be mention
  • Examples of the antibody composition to be obtained include an antibody, an antibody fragment, a fusion protein having an antibody Fc region, and the like.
  • an antibody composition the power described for the method for producing the composition of the human rabbit antibody and Fc fusion protein, the glycoproteins such as other antibody compositions, etc. It can also be obtained according to the method.
  • a humanized antibody expression vector contains genes encoding CH and CL of a human antibody. It is a rare expression vector for animal cells, and can be constructed by cloning the genes encoding human antibodies CH and CL respectively into the expression vector for animal cells.
  • the C region of a human antibody can be CH and CL of any human antibody.
  • the C region of the IgGl subclass of the H chain of a human antibody hereinafter referred to as "hC 7 1”
  • the C region of the / c class hereinafter referred to as “hC ⁇ ” of the L chain of human antibodies.
  • Chromosomal DNA consisting of exons and introns can be used as the gene encoding human antibody CH and CL, and cDNA can also be used.
  • any expression vector can be used as long as it can incorporate and express a gene encoding the C region of a human antibody.
  • PAGE 107 [Cytotechnology, 3, 133 (1990)]
  • pAGE103 [Journal 'Ob' Biochemistry., 101, 1307 (1987)]
  • pHSG274 [Gene ), 27, 223 (1990)
  • the promoters and enhancers used for animal cell expression vectors include SV40 early promoter and enhancer [J. Biochem., Dish, 1307 (1987)], Moroni mouse white blood. LTR requichemical 'and' biophysical 'research' Communications (Biochem. Biophys. Res. Commun.), 149, 960 (1987)], immunoglobulin heavy chain promoter [Cell, 41 , 479 (1985)] and Enhanser [Cell, 33, 717 (1983)
  • the humanized antibody expression vector should be either the type in which the antibody H chain and L chain are present on separate vectors or the same vector (hereinafter referred to as tandem type).
  • tandem type the same vector
  • An antibody-expressing vector is preferred [Journal of Immunological Methods (J. Immunol. Methods), 167, 271 (1994)].
  • the constructed humanized antibody expression vectors are human chimeric antibodies and human CDR-grafted antibodies. It can be used for expression in animal cells.
  • Non-human animal antibodies for example, cDNAs encoding mouse antibody VH and VL can be obtained as follows.
  • mRNA is extracted from hybridoma cells producing the desired mouse antibody, and cDNA is synthesized.
  • the synthesized cDNA is cloned into a vector such as a phage or plasmid to prepare a cDNA library.
  • a vector such as a phage or plasmid
  • the C region or V region of an existing mouse antibody is used as a probe, a recombinant phage or cDNA containing VH-encoding cDNA, and a recombination containing cDNA encoding VL.
  • Isolate each phage or recombinant plasmid Determine the VH and VL base sequences of the desired mouse antibody on the recombinant phage or recombinant plasmid, and estimate the VH and VL amino acid sequences from the base sequence.
  • mice As animals other than humans, mice, rats, hamsters, rabbits, etc. can be used as long as it is possible to produce high-pridoma cells.
  • Methods for preparing total RNA from Hypridoma cells include guanidine thiocyanate, cesium trifluoroacetate method [Methods in Enzymol., 154.3 (1987)], and preparing mRNA from total RNA. Methods include oligo (dT) immobilized cellulose column method [Molecular ⁇ ⁇ Cloning: 'Laboratory' manual (Molecular and lonmg: A Laboratory Manual), old Spring Harbor Lab. Press New York, 1989], etc. can give. In addition, the Fast Track mRNA Isolation Kit is available as a kit for preparing mRNA from Hypridoma cells. Quick Prep mRNA Purification Kit (manufactured by Pharmacia).
  • any vector can be used as long as it is a vector into which cDNA synthesized by using mRNA extracted from a hyperidoma cell as a saddle type can be incorporated.
  • ZAP Express [Strategies, 5, 58 (1992)]
  • pBluescript II SK (+) [Nucleic Acids Rese arch, 17, 9494 (1989)]
  • ⁇ zap II Stratagene
  • gtlO gtl l
  • Any Escherichia coli for introducing a cDNA library constructed by a phage or plasmid vector can be used as long as it can introduce, express and maintain the cDNA library.
  • the selection of cDNA clones encoding VH and VL of non-human animal antibodies from a cDNA library includes colony hybridization methods or plaques using isotopes or fluorescently labeled probes. It can be selected by the 'Hybridization method [Molecula 1' Cloning: Laboratory ', Molecular Cloning: A Laboratory Manual, Old Spring Harbor Lab. Press NewYork, 1989]. In addition, a primer is prepared and a cDNA or cDNA library synthesized from mRNA is used as a cage.
  • PCR method Polymerase Chain Reaction
  • Molecular 'cloning A' Hofofri ⁇ ⁇ 'Manu's Nole (Molecular Cloning: A Laboratory Manual), Cold Spring Harbor Lab. Press New York, 1989; VH and VL according to Current Protocols in Molecular Biology, Supplement 1-3 CDNA encoding can also be prepared.
  • the cDNA selected by the above method is cleaved with an appropriate restriction enzyme and then cloned into a plasmid such as pBluescript SK (-) (Stratagene), and a commonly used nucleotide sequence analysis method such as Sanger. (Sanger) et al. Dideoxy [Procedidas 'Ob' The 'National Academia ⁇ ⁇ Ob' Science (Pro Natl. Acad. ScL, USA), 74, 5463 (1 977)] Then, the base sequence of the cDNA can be determined by analysis using an automatic base sequence analyzer such as an ALF DNA sequencer (Pharmacia).
  • a plasmid such as pBluescript SK (-) (Stratagene)
  • a commonly used nucleotide sequence analysis method such as Sanger. (Sanger) et al. Dideoxy [Procedidas 'Ob' The 'National Academia ⁇ ⁇ Ob' Science (Pro
  • VH and VL of the antibody including the secretion signal sequence
  • all amino acid sequences of VH and VL of the known antibody IJ [Sequences 'Ob' Proteins (Sequences of Proteins of lmmunological Interest), US Dept. Health and Human Services, 1991]
  • VH and VL CDR amino acid sequences of the known antibody VH and VL amino acid sequences [Sequences of Proteins of Immunological Interest, Us Dept. Healtn and Hum an Services, 1991].
  • the cDNA encoding the VH and VL of the non-human animal antibody is cloned upstream of the gene encoding the human antibody CH and CL of the humanized antibody expression vector described in (2) of this section 2.
  • a human chimeric antibody expression vector can be constructed.
  • cDNA encoding VH and VL of a non-human animal antibody can be obtained by using the antibodies VH and VL of a non-human animal. It is composed of a base sequence on the end side and a base sequence on the 5 'end side of CH and CL of human antibody, and is linked to a synthetic DNA having an appropriate restriction enzyme recognition sequence at both ends.
  • the humanized antibody expression vector described in (1) is cloned upstream of the gene encoding the human antibody CH and CL so that they are expressed in an appropriate form, and a human chimeric antibody expression vector is constructed. be able to.
  • CDNAs encoding human CDR-grafted antibodies VH and VL can be constructed as follows. First, the amino acid sequence of the VH and VL framework (hereinafter referred to as FR) of the human antibody to which the VH and VL CDRs of the target non-human animal antibody are transplanted is selected. Any amino acid sequence derived from a human antibody can be used as the amino acid sequence of human antibody VH and VL FR.
  • the human amino acid VH and VL FR amino acid sequences 1J, human antibody VH and VL FR subgroups, which are registered in databases such as Protein Data Bank, are common amino acid sequences U [Sequences. OB. Proteins.
  • the VH and VL CDR amino acid sequences of the target non-human animal antibody are transplanted into the VH and VL FR amino acid sequences of the selected human antibody, and the VH and VL amino acid sequences of the human CDR-grafted antibody.
  • Frequency of codon usage of the designed amino acid sequence in the base sequence of antibody genes [Sequences of Proteins of Immunological Interest], US Dept. Health and H uman Services, 1991] is converted into a DNA sequence, and a DNA sequence encoding the amino acid sequence of VH and VL of a human CDR-grafted antibody is designed. Based on the designed DNA sequence, several synthetic DNAs with a length of around 100 bases are synthesized, and PCR is performed using them.
  • the amplified product is cloned into a plasmid such as pBluescript SK (-) (Stratagene), the nucleotide sequence is determined by the method described in (2) of this section 2, and the desired human CDR-grafted antibody A plasmid having a DNA sequence encoding the amino acid sequence of VH and VL is obtained.
  • a plasmid such as pBluescript SK (-) (Stratagene)
  • the nucleotide sequence is determined by the method described in (2) of this section 2, and the desired human CDR-grafted antibody
  • the human CDR-grafted antibody can be obtained by transplanting only the VH and VL CDRs of the target non-human animal antibody to the VH and VL FRs of the human antibody. It is known that it will be lower than that of an antibody (BIO / TE CHNOLOGY), 9, 266 (1991)]. This is because, in the VH and VL of the original non-human animal antibody, not only CDR but also FR residues and some amino acid residues are directly or indirectly involved in antigen binding activity. It is thought that these amino acid residues are changed to different amino acid residues of FR of VH and VL of human antibody with CDR grafting.
  • human CDR-grafted antibodies use amino acid residues that are directly involved in antigen binding or CDR amino acid residues in the human amino acid VH and VL FR amino acid sequences.
  • Amino acids found in the antibody of the original non-human animal by identifying the amino acid residues that interact with the group or maintain the three-dimensional structure of the antibody and indirectly participate in antigen binding. It has been practiced to modify residues to increase decreased antigen binding activity [BIO / TECHNOLOGY, 9, 266 (1991)].
  • Modification of FR amino acid residues of VH and VL of a human antibody can be achieved by performing the PCR method described in (2) of this section 2 using synthetic DNA for modification. For amplified products after PCR, determine the nucleotide sequence by the method described in (2) of this section 2 and confirm that the target modification has been made.
  • the cDNA encoding the antibody VH and VL can be cloned to construct a human CDR-grafted antibody expression vector.
  • appropriate restrictions are placed on the 5 ′ ends of the synthetic DNAs located at both ends.
  • By introducing the recognition sequence of the enzyme it is expressed in an appropriate form upstream of the genes encoding the human antibody CH and CL of the humanized antibody expression vector described in (1) of this section 2.
  • a human CDR-grafted antibody expression vector can be constructed.
  • a humanized antibody expression vector described in (4) and (7) of this section 2 into an appropriate animal cell, a human chimeric antibody and a human CDR grafted antibody (hereinafter collectively referred to as a humanized antibody). ) Can be obtained stably.
  • Examples of a method for introducing a humanized antibody expression vector into animal cells include the electoral position method [Japanese Patent Laid-Open No. 2-257891; Cytotechnology, 3, 133 (1990)].
  • Any animal cell capable of producing a humanized antibody can be used as the animal cell into which the human ⁇ antibody expression vector is introduced.
  • mouse myeloma cells derived from NS0 cells, SP2 / 0 cells, Chinese hamster ovary cells CHO / dhfr_ cells, CHO / DG44 cells, rat myeloma YB2 / 0 cells, IR 983F cells, and Syrian hamster kidneys
  • Certain BHK cells, human myeloma cells Namalva cells, etc. preferably CHO / DG44 cells that are Chinese hamster ovary cells, rat myeloma YB2 / 0 cells, Examples include cells that express a bright ⁇ -1,6-fucosyltransferase mutant.
  • G418 sulfate (hereinafter referred to as G418; SIGMA) according to the method disclosed in JP-A-2-257891.
  • Selected from animal cell culture media containing drugs such as Animal cell culture media include RPMI1640 medium (Nissui Pharmaceutical), GIT medium (Nihon Pharmaceutical), EX-C ELL302 medium (JRH), IMDM medium (GIBCO BRL), Hybridoma-SFM A medium (GI BCO BRL) or a medium obtained by adding various additives such as fetal bovine serum (hereinafter referred to as FBS) to these mediums can be used.
  • the humanized antibody By culturing the obtained transformant in a medium, the humanized antibody can be produced and accumulated in the culture supernatant.
  • the production amount and antigen binding activity of the humanized antibody in the culture supernatant are the enzyme-linked immunosorbent assay (hereinafter referred to as the ELISA method; Antibodies: Laboratories: A Laboratory Manual), Cold It can be measured by Spring Harbor Laboratory, Chapter 14, 1998, Monochrome Mononole Antibodies: Principles and Practices, Academic Press Limited, 1996].
  • the transformed strain can increase the production amount of the humanized antibody using a DHFR gene amplification system or the like according to the method disclosed in JP-A-2-257891.
  • Human IgA antibody can be purified from the culture supernatant of the transformant using a protein A column [Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 8, 1988, Monoclonal Antibodies: Principles and Practices, Academic Press Limited, 1996].
  • a purification method usually used in protein purification can be used. For example, it can be purified by a combination of gel filtration, ion exchange chromatography, ultrafiltration and the like.
  • the molecular weight of the purified humanized antibody H chain, L chain, or whole antibody molecule is determined by polyacrylamide gel electrophoresis [hereinafter referred to as SDS-PAGE; Nature, 227, 680 (1970)] or Western blot.
  • the Fc fusion protein expression vector is an expression vector for animal cells in which a gene encoding a protein to be fused with the Fc region of a human antibody is incorporated, and the Fc region of the human antibody is incorporated into the expression vector for animal cells. It can be constructed by cloning a gene encoding a protein to be fused.
  • the Fc region of a human antibody includes the force of the region containing the CH2 and CH3 regions, the hinge region, and those containing a part of CH1.
  • any amino acid may be used as long as at least one amino acid of CH2 or CH3 is deleted, substituted, added, or inserted and has substantially binding activity to the Fey receptor.
  • chromosomal DNA consisting of exons and introns can be used, and cDNA can also be used.
  • cDNA can also be used.
  • each gene sequence is used as a saddle type, PCR method (Requirula ⁇ ⁇ Cloning 2nd edition; Current Protocols 'in' Molecular ⁇ ⁇ ⁇ Biology 1, Supplement 1- 34).
  • any expression vector can be used as long as it can incorporate and express a gene encoding the C region of a human antibody.
  • pAGE107 [Cytotechnology, 3, 133 (1990)]
  • pAGE103 [Journal 'Ob' by Chemistry (J. Biochem.), Dish, 1307 (1987)]
  • pHSG274 [Gene , 27, 223 (1984)]
  • pKCR Proceedings' Ob 'The' National 'Academia 1'Ob' Science (Proc. Natl. Acad. Sci. USA), 78, 1527 (1981)]
  • pSGl ⁇ d2-4 [Cytotechnology, 4, 173 (1990)].
  • the promoters and enhancers used in animal cell expression vectors include SV40 early promoters and enhancers [Journal 'Ob' Biochemistry., 101, 1307 (1987)], Moroni Mouse Leukemia Virus. LTR biochemical and biophysical research communications (Biochem. Biophys. Res. Commun.), 149, 960 (1987)], immunoglobulin heavy chain promoter [Cell. 41, 479 ( 1985)] and Enhanser [Cell, 33, 71 7 (1983)].
  • DNA encoding the protein to be fused with the Fc region of a human antibody can be obtained as follows.
  • mRNA is extracted from cells or tissues expressing the protein to be fused with the target Fc, and cDNA is synthesized.
  • the synthesized cDNA is cloned into a vector such as a phage or plasmid to prepare a cDNA library. From this library, the gene sequence portion of the target protein is used as a probe, and a recombinant phage or a recombinant plasmid having cDNA encoding the target protein is isolated.
  • the entire base sequence of the target protein on the recombinant phage or recombinant plasmid is determined, and the entire amino acid sequence is deduced from the base sequence.
  • mice As animals other than humans, mice, rats, hamsters, rabbits, etc. can be used as long as cells and tissues can be removed.
  • Methods for preparing total RNA from cells and tissues include guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymol., 154, 3 (1987)], and mRNA from total RNA.
  • Examples of the method for preparing the above include oligo (dT) -immobilized cellulose column method (Molecular 'Cloning 2nd Edition).
  • Examples of kits for preparing mRNA from cells and tissues include Fast Track mRNA Isolation Kit (manufactured by Invitrogen), Quick Prep mRNA Purification Kit (manufactured by Pharmacia), and the like.
  • Synthesis of cDNA and construction of a cDNA library include conventional methods (Molequila I Cloning 3rd Edition; Current Protocols In-Molecular Biology, Supplement 1_34), or commercially available kits, For example, a method using Super Script TM Plasmid System for cDNA Synthesis and Plasmid Cloning (GIBCO BRL) or ZAP-cDNA Synthesis Kit (Stratagene) can be mentioned.
  • any vector can be used as a vector into which cDNA synthesized by using mRNA extracted from cells or tissues as a saddle type can be incorporated.
  • ZAP Express [Strategies, 5, 58 (1 992)]
  • pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)]
  • ⁇ zapll (Stratagene )
  • Lambda BlueMid (Clontech), ⁇ ExCel pT7T3 18U (Pharmacia), cD2 (Molecular and 'Cellular Biology (Mol. Cell. Biol.), 3, 280 (1983)]
  • pUC18 [Gene, 33, 103 (1985)] and the like are used.
  • any one can be used as long as it can introduce, express and maintain the cDNA library.
  • cDNA library selection of cDNA clones encoding the protein of interest including colonies using the isotope or fluorescently labeled probe, hybridization method or plaque 'hybridization method (Molecular 'Cloning 3rd Edition) can be selected. It is also possible to prepare primers and prepare cDNA encoding the protein of interest by PCR using a cDNA or cDNA library synthesized from mRNA as a saddle.
  • Examples of a method for fusing the target protein with the Fc region of a human antibody include PCR.
  • any synthetic oligo DNA (primer) on the 5 'and 3' sides of the gene sequence of the target protein, and perform PCR to obtain a PCR product.
  • an arbitrary primer is set for the gene sequence in the Fc region of the human antibody to be fused to obtain a PCR product.
  • set primers so that the same restriction enzyme site or the same gene sequence exists on the 3 'side of the PCR product of the protein to be fused and the 5' side of the PCR product of the Fc region. If it is necessary to modify the amino acid around this ligation site, the mutation is introduced by using a primer into which the mutation has been introduced. Two genes are ligated by further PCR using the two types of PCR fragments obtained.
  • ligation can be performed after ligation after the same restriction enzyme treatment.
  • the gene sequence linked by the above method is cleaved with an appropriate restriction enzyme or the like, and then cloned into a plasmid such as pBluescript SK (-) (Stratagene). Sanger et al.'S dideoxy method [Procedinas 'Ob' The * National Academia Sci. USA (Proc. Natl. Acad. Sci. USA), 74, 54 63 (1977)] or ABI PRISM By analyzing using a base sequence analyzer such as 377 DNA Sequencer (PE Biosystems), the base sequence of the DNA can be determined.
  • a base sequence analyzer such as 377 DNA Sequencer (PE Biosystems)
  • the obtained cDNA encodes the complete amino acid sequence of the Fc fusion protein including the secretory signal sequence. You can check whether you are doing.
  • Examples of a method for introducing an Fc fusion protein expression vector into animal cells include the electoporation method [Japanese Patent Laid-Open No. 2-257891; Cytotechnology, 3, 133 (1990)].
  • Any animal cell capable of producing an Fc fusion protein can be used as an animal cell into which an Fc fusion protein expression vector is introduced.
  • Chinese nomstar ovary cells CHO / DG44 cells, rat myeloma YB2 / 0 cells, as described in 2. or 4 above.
  • cells expressing the -1,6-fucosyltransferase mutant of the present invention are examples of the present invention.
  • the transformant that stably produces the Fc fusion protein composition is an animal cell culture containing a drug such as G418 according to the method disclosed in JP-A-2-257891. It can be selected according to the culture medium.
  • RPMI1640 medium Ground manufactured by Nissui Pharmaceutical
  • GIT medium manufactured by Nippon Pharmaceutical
  • EX-CELL302 medium manufactured by JRH
  • IMD M medium manufactured by GIBCO BRL
  • Hybridoma-SFM medium manufactured by GIBCO BRL
  • a medium obtained by adding various additives such as fetal calf serum to these mediums can be used.
  • the Fc fusion protein composition can be produced and accumulated in the culture supernatant.
  • the production amount and antigen binding activity of the Fc fusion protein composition in the culture supernatant can be measured by the EUSA method or the like.
  • the transformant can increase the production amount of the Fc fusion protein composition using a dhfr gene amplification system or the like according to the method disclosed in JP-A-2-257891.
  • the Fc fusion protein composition can be purified from the culture supernatant of the transformant using a protein A column or protein G column (Antibodies, Chapter 8, Monochrome Nanore • Antibodies) .
  • purification methods generally used for protein purification can be used. For example, it can be purified by combining gel filtration, ion exchange chromatography, ultrafiltration and the like. The molecular weight of the entire purified Fc fusion protein molecule is measured by SDS-PAGE [Nature, 227, 680 (1970)] or Western blotting (Antibodies, Chapter 12, Monoclonal 'Antibodies). The power to do S.
  • the cell When the cell already has the ability to express a glycoprotein composition such as an antibody molecule, the cell is prepared using the method described in 4. above, and then the cell is cultured. From the purified antibody composition, the antibody composition of the present invention can be produced as a glycoprotein composition.
  • a purified glycoprotein composition is labeled with a compound such as a radioisotope, and the binding reaction with the receptor or interacting protein of the labeled glycoprotein composition is strong.
  • a method for quantitatively measuring the thickness It is also possible to measure protein-protein interactions using various devices such as Biacore's BIAcor e series (
  • the binding activity to the antigen and the binding activity to the antigen-positive cultured cell line are determined by the EUSA method and the fluorescent lamp. Body method [Cancer Immunol. Immunotherj, 36, 373 (1993)] etc. Cytotoxic activity against an antigen-positive cultured cell line can be measured by measuring CDC activity, ADCC activity, etc. [Cancer Immunol. Immunother., 36, 373 (1993)].
  • sugar chain structure of an antibody molecule expressed in various cells can be performed according to the analysis of the sugar chain structure of a normal glycoprotein.
  • sugar chains bound to IgG molecules are composed of neutral sugars such as galactose, mannose, and fucose, amino sugars such as N-acetyldarcosamine, and acidic sugars such as sialic acid.
  • it can be carried out by using a two-dimensional sugar chain map method or the like, or a method such as sugar chain structure analysis.
  • composition analysis of the sugar chain of the antibody molecule neutral sugar or amino sugar is liberated by acid hydrolysis of the sugar chain with trifluoroacetic acid or the like, and the composition ratio can be analyzed.
  • a specific method includes a method using a sugar composition analyzer (BioLC) manufactured by Dionex.
  • BioL is HPAE and sugar composition by PAD (high perrormance anion-exchange chromatography-puised amperometric detection) method [J. Liq. Chromatogr.], 6, 1577 (1983)] It is a device that analyzes.
  • BioLC sugar composition analyzer
  • PAD high perrormance anion-exchange chromatography-puised amperometric detection
  • the composition ratio can also be analyzed by fluorescence labeling with 2-aminoviridine. Specifically, a sample hydrolyzed according to a known method [Agricultural and Biological Chemistry (Agm Biol. Chem.), 55 (1) .283-284 (1991)] was converted to 2-aminobilidylation. The composition ratio can be calculated by fluorescent labeling and HPLC analysis.
  • the structure analysis of the glycans of antibody molecules is based on the two-dimensional glycan map method [Analytical 'Biochem. (Anal. Biochem.), M, 73 (1988), Biochemical Experimental Method 23-Glycoprotein Glycan Research Method ( It is possible to do it according to the Society Publishing Center) Takahashi Keiko (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. It is a method for estimating the sugar chain structure by comparing with the results of known sugar chains.
  • the antibody is hydrazine-degraded to release the sugar chain from the antibody, and fluorescent labeling of the sugar chain with 2-aminopyridine (hereinafter abbreviated as PA) [Journal of Biochemistry ( J. Biochem.), 95, 197 (1984)], followed by gel filtration to remove excess sugar chains, etc. And reverse phase chromatography. Next, normal phase chromatography is performed for each peak of the separated sugar chain. Based on these results, plotting on a two-dimensional glycan map, glycan standard (manufactured by TaKaRa), literature [Analytical Biochemistry (Anal. Biochem.), 171, 73 (1988)] The sugar chain structure can be estimated from spot comparison.
  • PA 2-aminopyridine
  • mass analysis such as MALDI-TOF-MS of each sugar chain can be performed, and the structure estimated by the two-dimensional sugar chain map method can be confirmed.
  • glycoprotein composition and antibody composition produced using cells having the -1,6-fucosyltransferase mutant of the present invention
  • the glycoprotein composition or antibody composition produced using the cell having the -1,6-fucosyltransferase mutant of the present invention has a sugar chain structure without fucose modification, for example, a receptor.
  • High physiological activity that can be expected to improve the affinity with the protein, improve the blood half-life, improve the tissue distribution after administration in blood, or improve the interaction with proteins required for the expression of pharmacological activity.
  • the antibody composition of the present invention has high effector function, that is, antibody-dependent cytotoxic activity.
  • An antibody composition having a high physiological activity, glycoprotein composition, or high ADCC activity can be used for cancer, inflammatory diseases, autoimmune diseases, immune diseases such as allergies, cardiovascular diseases, viruses or bacteria. It is useful in the prevention and treatment of various diseases including infection.
  • Cancer cells are proliferating in cancer, that is, malignant tumors.
  • Ordinary anticancer agents are characterized by inhibiting the growth of cancer cells.
  • an antibody having a high ADCC activity is more effective as a therapeutic agent than a conventional anticancer agent because it can treat cancer by damaging cancer cells due to a cytocidal effect.
  • the anti-tumor effects of antibody drugs alone are often insufficient at present, and combined therapy with chemotherapy [Science, 280, 1197, 1998] If a stronger antitumor effect is observed with the antibody composition of the invention alone, the dependence on chemotherapy will be reduced, and side effects will be reduced.
  • cardiovascular diseases include arteriosclerosis. Atherosclerosis is currently treated with balloon catheter, but it is possible to prevent and treat cardiovascular disease by suppressing the proliferation of arterial cells in restenosis after treatment with an antibody with high ADCC activity. it can
  • 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 or bacteria Specific examples of antibodies that recognize antigens related to infection are described below.
  • Anti-CA125 antibodies (Immunology Today, 21, 4 03-410, 2000), anti-17-1A antibodies (Immunology Today, 21, 403-410, 2000), anti-integrin antibodies are those that recognize tumor-associated antigens.
  • Grin ct v i3 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, 2) 000), 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-CD19 antibody (Immunology Today, 21, 403-410, 2000), anti-EGF receptor antibody (Immunology Today, 21, 403-410, 2000), anti-CD10 antibody (American Journal of Clinical Pathology, U3 , 374-382, 2000
  • 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)
  • Anti-basic fibroblast growth factor antibody, anti-FGF8 receptor antibody J. Biol. Chem., 265, 16455-16463, 1990
  • anti-salt Basic fibroblast growth factor receptor antibody anti-insulin-like growth factor antibody (J. Neurosci. Res., 40, 647-659, 1995), anti-insulin-like growth factor receptor antibody (J.
  • anti-PMSA antibody J. Urology, 160, 2396-2401, 1998)
  • anti-vascular endothelial growth factor antibody Cancer Res., 57, 4593-4599, 1997) or anti-vascular Examples thereof include endothelial cell growth factor receptor antibodies (Oncogene, 19, 2138-2146, 2000).
  • Anti-IgE antibodies (Immunology Today, 21, 403-410, 2000), anti-CD23 antibodies (Immunology Today, 21, 403-410, 2000), as antibodies that recognize antigens related to allergy or inflammation
  • Anti-CD1 la 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. Rev., 127, 5-24, 1992), anti-interleukin 6 receptor antibody (Molecular Immunol., 31, 371-381, 1994), anti-interleukin 5 antibody ( Immunol.
  • Anti-GpIIb / lIIa antibody J. Im solid nol., 152, 2968-2976, 1994
  • antiplatelet-derived growth factor antibody Science, 253, 1994
  • Anti-GpIIb / lIIa antibody J. Im solid nol., 152, 2968-2976, 1994
  • antiplatelet-derived growth factor antibody Science, 253, 1994
  • Antigens related to cardiovascular diseases 1129-113 2, 1991
  • antiplatelet-derived growth factor receptor antibody J. Biol. Chem., 272, 17400-17404, 1997) or anticoagulation factor antibody (Circulation, 101, 1158-1164, 2000), etc. Is mentioned.
  • Antibodies that recognize antigens associated with autoimmune diseases such as psoriasis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, and multiple sclerosis include anti-self DNA antibodies (Immunol.
  • anti-CD 1 la antibody (Immunology Today, 21, 403-410, 2000), anti-ICAM3 antibody (Immunology Today, 21, 403-410, 2000), anti-CD80 antibody (Immunology Today, 21, 403-410, 2000), anti-CD2 antibody (Immunology Toda y, 21, 403-410, 2000), anti-CD3 antibody (Immunology Today, 21, 403-410, 2000), anti-CD4 antibody (Immunology Today, 21, 403-410, 2000), anti-integrin a 47 antibody (Immunology Today, 21, 403-410, 2000), anti-CD40L antibody (Immunology Today, 21, 403-410, 2000), anti-IL_2 receptor antibody (Immunology Today, 21, 403-410, 2000), etc. Can be mentioned.
  • Virus or The antibody which recognizes an antigen associated with bacterial infection anti-g P 120 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 Co., Ltd., Funakoshi Co., Ltd., etc.
  • the medicament containing the glycoprotein composition of the present invention is usually mixed together with one or more pharmacologically acceptable carriers.
  • the route of administration is preferably oral administration, which should be the most effective for treatment, or parenteral administration such as buccal, airway, rectal, subcutaneous, intramuscular and intravenous. In the case of an antibody preparation, intravenous administration is desirable.
  • dosage 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 saccharides such as water, sucrose, sorbitol, and fructose, Daricols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, p- It can be produced using preservatives such as hydroxybenzoates and the like, flavors such as stove belly flavors 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 polyvinyl alcohol, hydroxypropyl cellulose, gelatin, a surfactant such as fatty acid ester, a plasticizer such as glycerin, and the like as additives.
  • a binder such as polyvinyl alcohol, hydroxypropyl cellulose, gelatin, a surfactant such as fatty acid ester, a plasticizer such as glycerin, and the like as additives.
  • Suitable formulations for parenteral administration include injections, suppositories, sprays and the like.
  • the injection is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of both.
  • a 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 glycoprotein composition is dispersed as fine particles to facilitate absorption, etc. 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 preparations 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 usual adult dose is 10 / ig / kg to 20 mg / kg per day.
  • the method of examining the antitumor effect of glycoprotein compositions on various tumor cells includes, for example, in the case of antibodies, in vitro experiments such as CDC (complement_dependent cytotoxicity) assay, ADCC (antibody-d marked endent cellular cytotoxicity) ) Activity measurement methods and the like, and examples of in vivo experiments include antitumor experiments using tumor systems in experimental animals such as mice.
  • Figure 1 shows the reactivity of each strain to LCA.
  • CHO / DG44 cells were stained by LCA, a _1,6-fucose specific lectin, whereas the RN6 strain was not stained by LCA.
  • the anti-CCR4 antibody expression vector pKANTEX2160 described in WO01 / 64754 was introduced into the RN6 strain by the electopore position method [Cytotechnology, 3, 133 (1990)] to obtain a stable expression strain.
  • Mab Select Anamersham Pha
  • the antibody composition was purified using rmacia Biotech).
  • the obtained anti-CCR4 antibody was subjected to monosaccharide composition analysis according to a known method [Journal of Liquid Chromatog raphy, 6, 1577, (1983)].
  • the antibody composition obtained from the RN6 strain had a fucose content below the limit of quantification (FIG. 2). From these results, it was suggested that the RN6 strain lacks the function of a-binding fucose 1-position to the 6-position of N-acetyl darcosamine at the N-linked complex sugar chain reducing terminal.
  • RNA was extracted from the RN6 strain and CHO / DG44 cells obtained in Example 1 using the RNeasy Mini Kit (QIAGEN). Single-stranded cDNA was synthesized by reverse transcription reaction with oligo dT primer using Superscript First-Strand Synthesis System for RT_PCR (Invitrogen) using 5 ⁇ g of total RNA as a cage.
  • PCR was performed in 26 cycles of a reaction consisting of heating at 94 ° C for 5 minutes, followed by a reaction consisting of 94 ° C for 1 minute and 68 ° C for 2 minutes. After PCR, it was subjected to 1.2% (w / v) agarose gel electrophoresis. After staining with DNA using SYBR Green I Nucleic Acid Gel Stain (Molecular Probes), the luminescence intensity of each amplified DNA fragment was measured using Fluorlmager SI. (Molecular Dynamics) was used for calculation.
  • reaction solution [Ex Taq buffer (Takara Bio Inc.), 0.2 mmol / 1 dNTPs, 0.5 ⁇ mol / 1 gene-specific primer (SEQ ID NO: 25 and SEQ ID NO: 26)] was prepared, and PCR was performed. PCR was performed in 22 cycles of a reaction consisting of 94 ° C for 5 minutes, 94 ° C for 1 minute, and 68 ° C for 2 minutes.
  • PCR was performed in a 30-cycle process, consisting of a reaction consisting of 94 ° C for 1 minute, 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 2 minutes. After PCR, the sample was subjected to 1.2% (w / v) agarose gel electrophoresis, and DNA was stained using SYBR Green I Nucleic Acid Gel Stain (Molecular Probes), and then the luminescence intensity of each amplified DNA fragment was measured by Fluo rlmager SI (7 calculated by Molecular Dynamics)
  • GFPP GDP- ⁇ -L-ilicose pyrophorylase
  • PCR was performed in 24 cycles, with a reaction consisting of 94 ° C for 5 minutes, 94 ° C for 1 minute, and 68 ° C for 2 minutes. After PCR, the sample was subjected to 1.2% (w / v) agarose gel electrophoresis, and DNA was stained with SYBR Green I Nucleic Acid Gel Stain (Molecular Probes). The luminescence intensity of each amplified DNA fragment was then measured using Fluorlmager SI. (Molecular Dynamics) Calculated.
  • PCR was performed in a 24 cycle process, consisting of a reaction consisting of 94 ° C for 5 minutes, 94 ° C for 1 minute, and 68 ° C for 2 minutes. After PCR, the sample was subjected to 1.2% (w / v) agarose gel electrophoresis, and DNA was stained with SYBR Green I Nucleic Acid Gel Stain (Molecular Probes). The luminescence intensity of each amplified DNA fragment was then measured using Fluorlmager SI ( Molecular Dynamics).
  • a forward primer (SEQ ID NO: 35) and reverse primer (SEQ ID NO: 36) specific for the Chinese nomstar ⁇ -actin cDNA sequence described in patent WO02 / 31140 were synthesized, and DNA polymerase Ex Taq (Takara Bio) and the above 20 reaction solutions containing 1 ⁇ cDNA [ExTaq buffer (Takara Bio), 0.2 mmol / 1 dNTPs, 0.5 ⁇ mol / 1 gene-specific primer (SEQ ID NO: 35) And SEQ ID NO: 36)] were prepared and PCR was performed.
  • PCR was performed in 14 cycles of a reaction consisting of 94 ° C for 5 minutes, 94 ° C for 1 minute, and 68 ° C for 2 minutes. After PCR, 1.2% (w / v) agarose gel electrophoresis, SYBR Green I Nucleic Acid Gel Stain (Molecular Probes) was used to stain the DNA, and then the luminescence intensity of each amplified DNA fragment was measured using Fluorlmager Calculation was performed using SI (Molecular Dynamics).
  • the full length FUT8 cDNA was amplified from the RN6 strain by the method described in Section 1 of this Example, and direct sequence analysis was performed according to a known method [Molequila 'Cloning 3rd Edition]. As a result, the fragment of about 1.3 Kb found in the first item of this example was found to be an exon of human FUT8.
  • the determined nucleotide sequence of the RN6 strain-derived FUT8 deletion product is shown in SEQ ID NO: 19, and the amino acid sequence deduced from the nucleotide sequence is shown in SEQ ID NO: 14, respectively.
  • a single base substitution accompanied by a single amino acid mutation occurred in the fragment of about 1.7 Kb found in the first section of this example. That is, it was found that the 512th guanine in the FUT8 translation region was substituted with adenine, and as a result, serine, the 171st amino acid residue, was substituted with asparagine.
  • the determined nucleotide sequence of the RN6 strain-derived FUT8-base substitution product is shown in SEQ ID NO: 18, and the amino acid sequence deduced from the base sequence is shown in SEQ ID NO: 13, respectively.
  • a genomic DNA of each clone was prepared from the RN6 strain obtained in Example 1 and CHO / DG44 cells according to a known method [Nucleic Acids Research, 3, 2303, (1976)] Each was dissolved in TE-RNase buffer (pH 8.0) [10 mmol / l Tris-HC1, lmmol / 1 EDTA, 200 ⁇ g / ml RNase A]. The prepared genomic DNA was digested with the restriction enzyme Xbal and subjected to 0.8% (w / v) agarose gel electrophoresis.
  • the amplified fragment of about 400 bp was purified and radiolabeled using [a — 32 P] dCTP 1.75 MBq and Megaprime DNA Labeling system, dCTP (Amersham Pharmacia Biotech) to obtain a probe.
  • dCTP Megaprime DNA Labeling system
  • the 32 P-labeled probe DNA was heat denatured, put into a bottle and heated at 65 ° C.
  • the Nylon membrane was immersed in 50 ml of 233 ° -0.1% ⁇ / ⁇ ) SDS and heated at 65 ° C. for 15 minutes. After the above washing operation was repeated twice, the membrane was immersed in 50 ml of 0.2 X SSC—0.1% (w / V) SDS and heated at 65 ° C. for 15 minutes. After washing, the nylon membrane was exposed to X-ray film at -80 ° C and developed.
  • Southern plot analysis was performed by the following procedure using a probe specific to the region corresponding to exon 3 to exon 5 of human FUT8 in Chinese hamster FUT8.
  • DNA polymerase Ex Taq (Takara Bio Inc.) using plasmid CHfFUT8_pCR2.1 as a saddle and using a forward primer specific for FUT8 exon 3 (SEQ ID NO: 39) and a reverse primer specific for exon 5 (SEQ ID NO: 40) ) PCR was performed.
  • PCR consists of 25 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 74 ° C for 1 minute. The process was performed.
  • Plasmid CHFUT8Comp23 was constructed according to the following procedure (Fig. 3).
  • the reaction solution was subjected to 0.8% (w / v) agarose gel electrophoresis, and a DNA fragment of about 2.0 Kb was purified by GENECLEAN Spin Kit (BIO 101) and eluted with 20 ⁇ of water (hereinafter referred to as agarose gel). This method was used to purify the DN ⁇ fragment.
  • plasmid pBlueScriptll KS (+) (Strategene) 1.0 mg, 100 ⁇ g / ml BSA (New Dissolve in NEBuffer for BamHI (New England Biolabs) 50 ⁇ 1 containing England Biolabs) and add 10 units of restriction enzymes BamHI (New England Biolabs) and 10 units of XhoI (New England Biolabs) The digestion reaction was carried out at 37 ° C for 1.5 hours. The reaction solution was subjected to 0.8% (w / v) agarose gel electrophoresis, and a DNA fragment of about 3.0 Kb was purified.
  • BamH to Xhol fragment (about 2.0 Kb) derived from plasmid CHiFUT8-pCR2.1 obtained above (about 2.0 Kb) 4.0 ⁇ 1, BamH to Xhol fragment (about 3.0 Kb) derived from plasmid pBlueScriptll SK (+) 0.5 ⁇ 1, water
  • the fragments were ligated by mixing 0.5 ⁇ ⁇ and Ligation High (Toyobo Co., Ltd.) 5.0 ⁇ 1 and reacting at 16 ° C. for 30 minutes.
  • the reaction solution was used to transform Escherichia coli DH5a strain, and plasmid DNAs were isolated from the resulting ampicillin resistant clones according to a known method. This plasmid is hereinafter referred to as CHFUT8Comp23.
  • Plasmid CHFUT8CompTA was constructed by the following procedure (Fig. 4).
  • Plasmid pcDNAchFUT8Comp was constructed according to the following procedure (FIG. 5). [0233] 2.0 ⁇ g of the plasmid CHFUT8CompTA obtained in this section (2) was dissolved in NEBuffer for EcoRI (New Engl and Biolabs) 35 ⁇ 1, and 10 units of the restriction enzyme EcoRI (New England Biolabs) were prepared. The digestion reaction was performed at ° C for 2.5 hours. After subjecting the digestion reaction solution to ethanol precipitation, the vector cut ends were smoothed in a 10 ⁇ l reaction system by Blunting High (Toyobo Co., Ltd.).
  • the smooth reaction mixture was extracted with phenol-chloroform, and then dissolved in 35 ⁇ 1 of NEBuffer for BamHI (New England Biolabs) containing 100 ⁇ g / ml BSA (New England Biolabs). 10 units of BamHI (New England Biolabs) was added, and the digestion reaction was performed at 37 ° C for 1.5 hours.
  • the reaction solution was subjected to 0.8% (w / v) agarose gel electrophoresis, and a DNA fragment of about 1.7 Kb was purified.
  • Plasmid CHFUT8Mo3 was constructed by the following procedure (Fig. 6).
  • a forward primer (SEQ ID NO: 43) and reverse primer (SEQ ID NO: 44) specific to the sequence in which the 512th guanine in the FUT8 translation region was replaced with adenine were synthesized.
  • the fragments were ligated by mixing / l and reacting at 16 ° C for 2.5 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 CHFUT8Mo3.
  • Plasmid CHFUT8MoTA was constructed by the following procedure (FIG. 7).
  • PCR was performed in 25 cycles, consisting of heating for 2 minutes at 94 ° C, 10 seconds at 98 ° C, 30 seconds at 60 ° C, and 1 minute 45 seconds at 68 ° C. I got it.
  • 1 unit of Ex Taq (Takara Bio Inc.) was added and reacted at 72 ° C for 5 minutes.
  • the reaction solution was subjected to 0.8% (w / v) agarose gel electrophoresis, and a DNA fragment of about 1.7 Kb was purified.
  • the purified DNA fragment was inserted into plasmid pCR2.1 using TOPO TA cloning Kit (Invitrogen) to transform Escherichia coli Stbl2 strain. Plasmid DNAs were isolated from the resulting ampicillin resistant clones according to a known method. This plasmid is hereinafter referred to as CHFUT8 CompTA.
  • Plasmid pcDNAchFUT8Mo was constructed according to the following procedure (FIG. 8).
  • the smooth reaction mixture was extracted with phenol-chloroform and then dissolved in 35 ⁇ 1 of NEBuffer for BamHI (New England Biolabs) containing 100 mg / ml BSA (New England Biolabs), and the restriction enzyme BamHI ( (New England Biolabs) 10 units were added and digestion reaction was performed at 37 ° C for 1.5 hours.
  • the reaction solution was subjected to 0.8% (w / v) agarose gel electrophoresis, and a DNA fragment of about 1.7 Kb was purified.
  • the plasmid pcDNA chFUT8Comp and pcDNAchFUT8Mo constructed in section 1 of this example were prepared by the following procedure. It was introduced by the method [Cytotechnology, 3, 133 (1990)].
  • the cell suspension is suspended in IMDM medium (Invitrogen) supplemented with 10% urine fetal serum (Invitrogen) and HT supplement (Invitrogen), and T75 flask for adhesion culture (Grainer) Sowing. After culturing for 24 hours at 5% CO and 37 ° C, the culture supernatant is removed, and 600 ⁇ g / ml G418 (Nacalai Testa), HT supplement (Invitrogen) and 10% fetus The medium was replaced with IMDM medium (Invitrogen) supplemented with serum (Invitrogen). This medium exchange operation was repeated every 3 to 4 days, and culturing was performed for 15 days to obtain a G418 resistant strain.
  • Figure 9 shows the reactivity of each transgenic strain to LCA.
  • FUT8 expression strain plasmid pc DNAchFUT8Comp-introduced strain
  • FUT8-base substitution expression strain plasmid pcDNAchFUT8Mo-introduced strain
  • substitution of the 512th guanine in the FUT8 translation region with adenine greatly reduces the ability of cells to add 1,6-fucose.
  • the N-glycidyl darcosamine of the N-glycoside-linked complex type sugar chain reducing end the N-glycidyl darcosamine of the N-glycoside-linked complex type sugar chain reducing end
  • SEQ ID NO: 37--Description of Artificial Sequence Synthetic DNA SEQ ID NO: 38-description of artificial sequence: synthetic DNA SEQ ID NO: 39-description of artificial sequence: synthetic DNA SEQ ID NO: 40-description of artificial sequence: synthetic DNA SEQ ID NO: 41-description of artificial sequence: synthetic DNA SEQ ID NO: 42-artificial sequence Description: Synthetic DNA SEQ ID NO: 43-Description of artificial sequence: Synthetic DNA SEQ ID NO: 44-Description of artificial sequence: Synthetic DNA

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Abstract

Disclosed is a mutant of an enzyme involved in such a sugar chain modification that an α-bonding is formed between position-6 of N-acetylglycosamine at the reducing terminus of a N-glycoside-bound composite sugar chain and position-1 of fucose, wherein the mutant has such an amino acid modification that the enzymatic activity is deleted or reduced. Also disclosed is use of the mutant.

Description

明 細 書  Specification
α -1,6-フコシノレトランスフェラーゼ変異体とその用途  α-1,6-fucosinoretransferase mutants and uses thereof
技術分野  Technical field
[0001] 本発明は、 ひ -1,6-フコシルトランスフェラーゼにおいて、該酵素のひ -1,6-フコシル トランスフェラーゼ活性が欠失、または低下しているひ -1,6 -フコシルトランスフェラー ゼ変異体、及びその利用に関する。  [0001] The present invention relates to a -1,6-fucosyltransferase mutant of -1,6-fucosyltransferase, wherein the -1,6-fucosyltransferase activity of the enzyme is deleted or reduced, And its use.
背景技術  Background art
[0002] 近年、高等生物由来の糖蛋白質等に含まれる複合糖質のうち糖鎖部分の構造と機 能に関する関心が高まっており、その研究が盛んに行われている。  [0002] In recent years, there has been an increasing interest in the structure and function of the sugar chain portion of glycoconjugates contained in glycoproteins derived from higher organisms, and their research has been actively conducted.
糖蛋白質の糖鎖は、蛋白質部分との結合様式により、ァスパラギンと結合する糖鎖 (Ν—グリコシド結合糖鎖）とセリン、スレオニンなどと結合する糖鎖（0—グリコシル結 合糖鎖）の 2種類に大別される。 Ν-グリコシド結合糖鎖は、様々な構造を有している が (非特許文献 1参照)、いずれの場合も以下の構造式 (I)に示す基本となる共通のコ ァ構造を有することが知られてレ、る。  The sugar chains of glycoproteins are divided into sugar chains that bind to asparagine (Ν-glycoside-linked sugar chains) and sugar chains that bind to serine, threonine, etc. (0-glycosyl-linked sugar chains). Broadly divided into types. Although Ν-glycoside-linked sugar chains have various structures (see Non-Patent Document 1), in any case, they may have a basic common core structure represented by the following structural formula (I). It ’s known.
[化 1コ  [Chemical 1
Man 1→6  Man 1 → 6
Man β l→4GlcNAc β l→4GlcNAc (I)  Man β l → 4GlcNAc β l → 4GlcNAc (I)
Manひ 1→3  Man hi 1 → 3
[0003] 構造式 (I)におレ、て、ァスパラギンと結合する糖鎖の末端は還元末端と呼ばれ、ま た、反対側は非還元末端と呼ばれている。 N-グリコシド結合糖鎖として、コア構造の 非還元末端にマンノースのみが結合するハイマンノース型または、コア構造の非還 元末端側にガラクトース— N-ァセチルダノレコサミン（以下、 Gaト GlcNAcと表記する） の枝を並行して 1ないしは複数本有し、更に Gaト GlcNAcの非還元末端側にシアル酸 、バイセクティングの N-ァセチルダルコサミンなどの構造を有するコンプレックス型、ま たは、コア構造の非還元末端側にハイマンノース型とコンプレックス型の両方の枝を 持つハイブリッド型などがあることが知られている。  [0003] In 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. N-glycoside-linked sugar chain is a high mannose type in which only mannose binds to the non-reducing end of the core structure, or galactose—N-acetylidanorecosamine (hereinafter referred to as Gato GlcNAc) on the non-reducing end of the core structure. Or a complex type having a structure such as sialic acid or bisecting N-acetylcylcosamine on the non-reducing end side of Gato GlcNAc, or It is known that there are hybrid types having both a high mannose type and a complex type branch on the non-reducing end side of the core structure.
[0004] N-グリコシド結合糖鎖を有する糖蛋白質は、コア蛋白質が同一であっても糖鎖構 造に多様性のある分子からなるヘテロな糖蛋白質として存在する。また、その糖鎖構 造は、糖鎖を合成する糖転移酵素と糖鎖を分解する糖分解酵素の遺伝子によって 制御されていると考えられている。 [0004] A glycoprotein having an N-glycoside-linked sugar chain has a sugar chain structure even if the core protein is the same. It exists as a heterogeneous glycoprotein composed of diverse molecules. The sugar chain structure is thought to be controlled by glycosyltransferases that synthesize sugar chains and glycolytic enzymes that degrade sugar chains.
N-グリコシド結合糖鎖をはじめとする糖蛋白質の糖鎖は、蛋白質部分の立体構造 Glycoprotein sugar chains, including N-glycoside-linked sugar chains, are the three-dimensional structure of the protein part.
、輸送、安定性及びクリアランスに影響を与えるほか、蛋白質自身の機能、細胞間細 胞相互作用、細胞外マトリックスとの相互作用に影響を与えることにより、発生や免疫 応答、癌の発生過程などに重要な生理的役目を発揮していることが明らかとなりつつ ある（非特許文献 2-4参照）。また、糖鎖異常に係わるヒトの先天性遺伝疾患（CDG; congenital disorders of glycosylation)の解析力ら、その原因遺伝子が N -グリコシド結 合糖鎖の合成に必要な遺伝子であることが報告されている（非特許文献 5, 6参照）。 すなわち、糖ヌクレオチド GDP-フコースの輸送体である GDP-フコーストランスポータ 一が欠損してレ、る CDGタイプ II疾患患者（OMIMァクセッションナンバー 266265)では 、糖供与体である GDP-フコースの細胞質からゴルジ体への輸送が行われないため、 複合糖質糖鎖へのフコースの修飾がほとんどないこと、精神的な障害や成長の遅れ 、及び、免疫機能の不全などの症状が観察されている（非特許文献 7, 8参照）。 このような中、 N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6 位にフコースの 1位が《結合する糖鎖修飾に関与する酵素は、多くの糖鎖修飾酵素 とは異なり、これまでのところ生体内で単一の遺伝子しか見出されておらず、その機 能が大変注目されるようになった。 Affects the transport, stability, and clearance of the protein, affects the function of the protein itself, the interaction between cells, and the interaction with the extracellular matrix. It is becoming clear that it plays an important physiological role (see Non-Patent Document 2-4). In addition, the analysis ability of human congenital disorders of glycosylation (CDG) related to sugar chain abnormalities has been reported to be the gene required for the synthesis of N-glycosidic sugar chains. (See Non-Patent Documents 5 and 6). In other words, in the case of CDG type II disease patients (OMIM accession number 266265), which lacks the GDP-fucose transporter, which is a transporter of sugar nucleotide GDP-fucose, the cytoplasm of the sugar donor, GDP-fucose. Symptom such as lack of fucose modification to glycoconjugates, mental disorders and growth delays, and impaired immune function have been observed. (See Non-Patent Documents 7 and 8.) Under these circumstances, N-glycoside-linked complex-type sugar chain reducing terminal N-acetylyldarcosamine is linked to the 6th position of fucose, where the enzyme involved in the sugar chain modification is In contrast, so far only a single gene has been found in vivo, and its function has attracted much attention.
N-ダリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコース の 1位が α結合する糖鎖修飾に関与する酵素としては、動物では、 α -1,6-フコシノレ トランスフェラーゼが見出されている（非特許文献 9参照）。 ひ- 1,6-フコシルトランスフ エラーゼの遺伝子（EC 2.4.1,68)の構造は 1996年に明らかにされている（非特許文献 10— 11、特許文献 1参照）。 ひ -1,6 -フコシルトランスフェラーゼの酵素活性は多くの 臓器で確認されているが、なかでも脳や小腸において酵素活性が比較的高いことが 報告されている（非特許文献 12— 13参照）。網膜形成においてフコース修飾糖鎖が 重要な生理的な機能を担っていることが指摘されており、 ひ -1,6 -フコシルトランスフ エラーゼの発現調節が注目されてレ、る（非特許文献 14参照）。血液凝固におレ、ても 血小板由来の α -1,6-フコシルトランスフェラーゼの役割が注目されている（非特許文 献 15参照）。また、免疫グロブリン IgGlの糖鎖構造に対するフコースの修飾が Fc y RI Ilaとの結合に影響を及ぼし、抗体自身の抗体依存性細胞障害活性が変化することも 報告されている (非特許文献 16、 17参照)。病態との関わりに関しては、肝臓癌ゃ嚢 胞性繊維症などいくつかの疾病において、 ひ- 1，6-フコシルトランスフェラーゼ活性が 上昇してレ、ること及び該酵素反応生成物の割合が増加してレ、ることが観察されてレ、 るので、これら疾患と該酵素との関連が想定されている（非特許文献 18, 19参照）。 ひ- 1，6-フコシルトランスフェラーゼを過剰に発現したトランスジエニックマウスも作製さ れ、作製されたトランスジヱニックマウスの肝臓や腎臓にぉレ、て脂肪症様の変性が観 察されている（非特許文献 20参照）。また、 ひ- 1，6-フコシルトランスフェラーゼノック アウトマウスの作製にっレ、ても報告されてレ、る（特許文献 2参照）。 N-daricoside-linked glycan N-acetylyldarcosamine at the reducing end of the enzyme is involved in the sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of α- 1,6-fucosinole transferase in animals Has been found (see Non-Patent Document 9). The structure of the -1,6-fucosyltransferase gene (EC 2.4.1,68) was clarified in 1996 (see Non-Patent Documents 10-11 and Patent Document 1). Enzyme activity of -1,6-fucosyltransferase has been confirmed in many organs, but it has been reported that enzyme activity is relatively high in the brain and small intestine (see Non-Patent Documents 12-13). It has been pointed out that fucose-modified sugar chains play an important physiological function in retinal formation, and attention has been paid to the regulation of the expression of -1,6-fucosyltransferase (see Non-Patent Document 14). ). For blood clotting, The role of platelet-derived α-1,6-fucosyltransferase has attracted attention (see Non-Patent Document 15). It has also been reported that the modification of fucose to the sugar chain structure of immunoglobulin IgGl affects the binding to FcyRI Ila and changes the antibody-dependent cytotoxic activity of the antibody itself (Non-Patent Document 16, 17). Regarding the relationship with the pathological condition, in some diseases such as liver cancer and cystic fibrosis, increased 1,6-fucosyltransferase activity and the ratio of the enzyme reaction product increased. Therefore, the relationship between these diseases and the enzyme is assumed (see Non-Patent Documents 18 and 19). Transgenic mice overexpressing -1,6-fucosyltransferase have also been prepared, and liver and kidneys of the produced transgenic mice have been observed to have steatosis-like degeneration. (See Non-Patent Document 20). In addition, it has been reported that a -1,6-fucosyltransferase knockout mouse is produced (see Patent Document 2).
このように、 ひ- 1，6-フコシルトランスフェラーゼに関する様々な解析が行われており 、上述のように本酵素の生体における生理的役割の多様な重要性が推定されている 。しかしながら、 α -1,6-フコシルトランスフェラーゼのアミノ酸変異体に関する具体的 な報告はない。 α -1,6-フコシルトランスフェラーゼ変異体を発現する細胞は、 Ν-ダリ コシド結合複合型糖鎖還元末端の Ν-ァセチルダルコサミンの 6位にフコースの 1位が α結合する糖鎖修飾が少なくなることに起因して、生理活性が変化した糖蛋白質を 生産していることが想定される。また、本酵素の活性が欠失、または低下している α _ 1,6-フコシルトランスフェラーゼ変異体を発現する組織においては直接的もしくは間 接的に様々な病的な症状が発現することが想定される。本酵素の真の生理的役割 や病態との関わりが解明され、 α -1,6-フコシルトランスフェラーゼ変異体の産業上の 更なる利用方法が明らかにされてレ、くことが期待されている。  As described above, various analyzes relating to -1,6-fucosyltransferase have been performed, and as described above, various importance of the physiological role of the enzyme in the living body is estimated. However, there are no specific reports on amino acid variants of α-1,6-fucosyltransferase. Cells expressing the α-1,6-fucosyltransferase mutant have a glycan modification in which the 1-position of fucose is α-linked to the 6-position of 還 元 -acetylcolcamine at the reducing end of the Ν-daricoside-linked complex. It is assumed that glycoproteins with altered physiological activity are produced due to the decrease. In addition, it is assumed that various pathological symptoms are expressed directly or indirectly in tissues expressing α_1,6-fucosyltransferase mutants in which the activity of this enzyme is deficient or decreased. Is done. It is expected that the true physiological role and pathophysiological role of this enzyme will be elucidated, and further industrial utilization of α-1,6-fucosyltransferase mutants will be clarified.
特許文献 l : WO92/27303 Patent Literature l: WO92 / 27303
特許文献 2： US2005-0160485 Patent Document 2: US2005-0160485
非特許文献 1 :高橋禮子編、「生物化学実験法 23—糖蛋白質糖鎖研究法」、学会出 版センター、 1989年、 p.1-4 Non-Patent Document 1: Tetsuko Takahashi, “Biochemical Experimental Method 23—Glycoprotein Glycan Research Method”, Academic Press Center, 1989, p.1-4
非特許文献 2 :カレント'ォピニョン 'イン 'ィムノロジー（Curr. Opin. Immunol.) , 3, 646, 1991 非特許文献 3:グリコバイオロジー（Glycobiology), 3， 97, 1993 Non-patent document 2: Current 'Opinon' Immunology (Curr. Opin. Immunol.), 3, 646, 1991 Non-Patent Document 3: Glycobiology, 3, 97, 1993
非特許文献 4:バイオケミカル'ソサイァティー'トランザクション（Biochem. Soc. Trans. )， 23, 1, 1995 Non-Patent Document 4: Biochemical 'Society' Transaction (Biochem. Soc. Trans.), 23, 1, 1995
非特許文献 5:グリコバイオロジー（Glycobiology), 3， 423, 1993 Non-Patent Document 5: Glycobiology, 3, 423, 1993
非特許文献 6:ョ一口ビアン 'ジャーナル'ォブ'ぺディアトリック'ニューロロジー（Eur.Non-Patent Document 6: Choi Bian 'Journal' Ob 'Pediatric' Neurology (Eur.
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非特許文献 7:ネィチヤ一'ジヱネテイクス（Nat. Genet.), 28, 73， 2001 Non-Patent Document 7: Nichia's Genetys (Nat. Genet.), 28, 73, 2001
非特許文献 8:ネィチヤ一'ジヱネテイクス (Nat. Genet.), 28, 69， 2001 Non-Patent Document 8: Nichia's Genetys (Nat. Genet.), 28, 69, 2001
非特許文献 9：バイオケミカル ·アンド ·バイオフィジカル ·リサーチ ·コミュニケーション ズ (Biochem. Biophys. Res. Commun.) , 72, 909， 197り Non-Patent Document 9: Biochemical and Biophysical Research Communications (Biochem. Biophys. Res. Commun.), 72, 909, 197
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非特許文献 14:グリコバイオロジー（Glycobiology), 9， 1171, 1999 Non-Patent Document 14: Glycobiology, 9, 1171, 1999
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非特許文献 18:へパトロジー（H印 atology), 13， 683， 1991) Non-patent document 18: Hepatology (H-mark atology), 13, 683, 1991)
非特許文献 19:へパトロジー（H印 atology), 28， 944, 1998 Non-Patent Document 19: Hepatology (H Mark atology), 28, 944, 1998
非特許文献 20:グリコバイオロジー（Glycobiology), 11, 165， 2001 Non-Patent Document 20: Glycobiology, 11, 165, 2001
発明の開示 発明が解決しょうとする課題 Disclosure of the invention Problems to be solved by the invention
[0007] 本発明は、 ひ -1 ,6-フコシルトランスフェラーゼにおいて、該酵素のひ -1 ,6-フコシル トランスフェラーゼ活性が欠失、または低下しているひ -1 ,6 -フコシルトランスフェラー ゼ変異酵素及びその利用方法を提供することにある。  [0007] The present invention relates to a -1,6, -fucosyltransferase, a -1, 6-fucosyltransferase mutant enzyme in which the -1,6, -fucosyltransferase activity of the enzyme is deleted or reduced, and It is to provide a method of using the same.
本発明のひ -1 ,6-フコシルトランスフェラーゼ変異体は、 ひ -1，6-フコース修飾酵素 の生理的役割及び病態との関わりの解明や診断に有用である。また、 ひ- 1，6-フコー ス修飾酵素を標的とした薬の開発や糖鎖構造が重要な糖蛋白質医薬の開発にも有 用である。  The -1,6-fucosyltransferase mutant of the present invention is useful for elucidating and diagnosing the physiological role of -1,6-fucose modifying enzyme and its relationship with pathological conditions. It is also useful for the development of drugs targeting the 1,6-fucose modifying enzyme and for the development of glycoprotein drugs where the sugar chain structure is important.
課題を解決するための手段  Means for solving the problem
[0008] 本発明は、以下の (1)〜(23)に関する。 [0008] The present invention relates to the following (1) to (23).
( 1 ) ひ- 1，6-フコシルトランスフェラーゼのアミノ酸配列において、配列番号 7で表され るアミノ酸配列の N末端から 171番目のアミノ酸に相応する位置のアミノ酸が欠失し ているか、またはセリン以外のアミノ酸に置換されたアミノ酸配列を有する、 ひ- 1，6-フ コシルトランスフェラーゼ変異体。  (1) In the amino acid sequence of -1,6-fucosyltransferase, the amino acid at the position corresponding to the 171st amino acid from the N-terminus of the amino acid sequence represented by SEQ ID NO: 7 has been deleted, or other than serine A mutant of 1,6-fucosyltransferase having an amino acid sequence substituted with an amino acid.
(2)セリン以外のアミノ酸がァスパラギンである、上記（1 )に記載のひ -1 ,6 -フコシルト ランスフェラーゼ変異体。  (2) The -1,6-fucosyltransferase mutant according to (1) above, wherein the amino acid other than serine is asparagine.
(3) ひ- 1，6-フコシルトランスフェラーゼカ 以下の（a)〜(f)からなる群から選ばれる DN Aがコードする蛋白質である上記（1 )または（2)に記載のひ -1 ,6-フコシルトランスフエ ラーゼ変異体。  (3) H-1, -6-fucosyltransferase is a protein encoded by DNA selected from the group consisting of the following (a) to (f): 6-fucosyltransferase mutant.
(a)配列番号 1で表される塩基配列からなる DNA ;  (a) DNA consisting of the base sequence represented by SEQ ID NO: 1;
(b)配列番号 2で表される塩基配列からなる DNA ;  (b) DNA consisting of the base sequence represented by SEQ ID NO: 2;
(c)配列番号 3で表される塩基配列からなる DNA ;  (c) DNA consisting of the base sequence represented by SEQ ID NO: 3;
(d)配列番号 4で表される塩基配列からなる DNA ;  (d) DNA consisting of the base sequence represented by SEQ ID NO: 4;
(e)配列番号 5で表される塩基配列からなる DNA ;  (e) DNA consisting of the base sequence represented by SEQ ID NO: 5;
(f)配列番号 6で表される塩基配列からなる DNA。  (f) DNA consisting of the base sequence represented by SEQ ID NO: 6.
(4) α -1 ,6-フコシルトランスフェラーゼカ S、以下の (a)〜(f)からなる群から選ばれる蛋 白質である、上記（1 )または（2)に記載の α -1 ,6-フコシルトランスフェラーゼ変異体 (a)配列番号 7で表されるアミノ酸配列からなる蛋白質； (4) α -1, 6-fucosyltransferase S, a protein selected from the group consisting of the following (a) to (f), α -1, 6 according to (1) or (2) above -Fucosyltransferase mutant (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 7;
(b)配列番号 8で表されるアミノ酸配列からなる蛋白質；  (b) a protein comprising the amino acid sequence represented by SEQ ID NO: 8;
(c)配列番号 9で表されるアミノ酸配列からなる蛋白質；  (c) a protein comprising the amino acid sequence represented by SEQ ID NO: 9;
(d)配列番号 10で表されるアミノ酸配列からなる蛋白質；  (d) a protein comprising the amino acid sequence represented by SEQ ID NO: 10;
(e)配列番号 11で表されるアミノ酸配列からなる蛋白質；  (e) a protein comprising the amino acid sequence represented by SEQ ID NO: 11;
(f)配列番号 12で表されるアミノ酸配列からなる蛋白質。  (f) a protein comprising the amino acid sequence represented by SEQ ID NO: 12.
(5)配列番号 13〜： 17のいずれかで表されるアミノ酸配列を含むひ- 1，6-フコシルトラ ンスフヱラーゼ変異体。  (5) A mutant of 1,6-fucosyltransferase comprising the amino acid sequence represented by any of SEQ ID NOS: 13 to 17.
(6)配列番号 13〜： 17のいずれかで表されるアミノ酸配列において、 1以上のアミノ酸 が欠失、置換、揷入および/または付加されたアミノ酸配列からなり、かつひ- 1，6-フ コシルトランスフェラーゼ活性が欠失、または、配列番号 7または 9で表されるアミノ酸 配列からなるひ -1，6-フコシルトランスフェラーゼのひ -1,6-フコシルトランスフェラーゼ 活性よりも低下したひ -1,6-フコシルトランスフェラーゼ活性を有する α -1,6-フコシル トランスフェラーゼ変異体。  (6) In the amino acid sequence represented by any one of SEQ ID NOs: 13 to 17, the amino acid sequence is composed of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and The fucosyltransferase activity is deleted, or the -1,6-fucosyltransferase having the amino acid sequence represented by SEQ ID NO: 7 or 9 has a -1,6-fucosyltransferase activity lower than -1, An α-1,6-fucosyltransferase mutant having 6-fucosyltransferase activity.
(7)配列番号 13〜： 17のいずれかで表されるアミノ酸配列と 80%以上の相同性を有 するアミノ酸配列からなり、かつ α -1,6-フコシルトランスフェラーゼ活性が欠失、また は、配列番号 7または 9で表されるアミノ酸配列からなる α _1，6-フコシルトランスフェラ ーゼの α -1,6-フコシルトランスフェラーゼ活性よりも低下した α -1,6-フコシルトランス フェラーゼ活性を有する α -1,6-フコシルトランスフェラーゼ変異体。 (7) consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by any one of SEQ ID NOs: 13 to 17 and lacking α-1,6-fucosyltransferase activity, or alpha with reduced alpha-1,6-fucosyltransferase trans luciferase activity than SEQ ID NO: 7 or comprising the amino acid sequence represented by the 9 α _1,6- fucosyl trans Blow over peptidase alpha-1,6-fucosyltransferase activity -1,6-fucosyltransferase mutant.
(8)上記（1)〜（7)のレ、ずれか一項に記載の α -1,6-フコシルトランスフェラーゼ変異 体をコードする DNA。  (8) A DNA encoding the α-1,6-fucosyltransferase mutant according to any one of (1) to (7) above.
(9)配列番号 18〜22のいずれかで表される塩基配列を含む DNA。  (9) A DNA comprising the base sequence represented by any of SEQ ID NOs: 18-22.
(10)配列番号 18〜22のいずれかで表される塩基配列とストリンジェントな条件でハ イブリダィズし、かつひ- 1，6 -フコシルトランスフェラーゼ活性が欠失、または、配列番 号 7または 9で表されるアミノ酸配歹 1Jからなるひ -1,6-フコシルトランスフェラーゼのひ - 1，6-フコシルトランスフェラーゼ活性よりも低下したひ -1，6-フコシルトランスフェラーゼ 活性を有するひ -1，6 -フコシルトランスフェラーゼ変異体をコードする DNA。  (10) Hybridizes under stringent conditions with the nucleotide sequence represented by any of SEQ ID NOs: 18 to 22 and lacks -1,6-fucosyltransferase activity, or is SEQ ID NO: 7 or 9. A -1,6-fucosyltransferase having 1-6-fucosyltransferase activity which is lower than the 1,6-fucosyltransferase activity of the -1,6-fucosyltransferase comprising 1J DNA encoding the mutant.
(11)上記（1)〜（7)のレ、ずれか一項に記載のひ -1,6 -フコシルトランスフェラーゼ変 異体または、上記（8)〜（10のレ、ずれか一項に記載の DNAがコードする α _1，6_フ コシルトランスフェラーゼ変異体を発現する細胞。 (11) The -1,6-fucosyltransferase modification described in (1) to (7) above, A cell that expresses a mutant or an α_1,6_fucosyltransferase mutant encoded by the DNA according to any one of (8) to (10) above.
(12)上記（8)〜（10)のレ、ずれか一項に記載の DNAを導入した細胞。  (12) A cell into which the DNA according to (8) to (10) above is introduced.
(13)上記（1)〜（7)のレ、ずれか一項に記載のひ -1,6 -フコシルトランスフェラーゼ変 異体または、上記（7)〜（9)のレ、ずれか一項に記載の DNAがコードするひ -1，6-フ コシルトランスフェラーゼ変異体のひ -1，6-フコシルトランスフェラーゼ活性のみ有す る上記（11)または（12)記載の細胞。  (13) The -1,6-fucosyltransferase variant described in (1) to (7) above or any one of the above, or the above (7) to (9). The cell according to the above (11) or (12), which has only the -1,6-fucosyltransferase activity of a -1,6-fucosyltransferase mutant encoded by the DNA.
(14)糖蛋白質をコードする遺伝子を導入した上記（11)から（13)のレ、ずれか一項に 記載の細胞。  (14) The cell according to any one of (11) to (13) above, into which a gene encoding a glycoprotein has been introduced.
(15)糖蛋白質が抗体である、上記（14)に記載の細胞。  (15) The cell according to (14) above, wherein the glycoprotein is an antibody.
(16)抗体が、以下の )、（b)、（c)、（d)及び (e)からなる群から選ばれる抗体である、上 記（15)に記載の細胞。  (16) The cell according to (15) above, wherein the antibody is an antibody selected from the group consisting of: (b), (c), (d) and (e) below.
(a)ヒト抗体；  (a) a human antibody;
(b)キメラ抗体；  (b) a chimeric antibody;
(c)ヒト化抗体；  (c) a humanized antibody;
(d) (a)または (b)の Fc領域を含む抗体断片；  (d) an antibody fragment comprising the Fc region of (a) or (b);
(e) (a)または (b)の Fc領域を有する融合蛋白質。  (e) A fusion protein having the Fc region of (a) or (b).
(17)上記（14)〜（16)のレ、ずれか一項に記載の細胞を培地に培養し、該培養物か ら糖蛋白質分子からなる糖蛋白質組成物を採取し、精製する工程を特徴とする糖蛋 白質組成物の製造方法。  (17) A step of culturing the cell according to any one of the above (14) to (16) in a medium, collecting a glycoprotein composition composed of glycoprotein molecules from the culture, and purifying it. A method for producing a glycoprotein composition.
( 18)糖蛋白質が抗体である、上記（ 17)に記載の製造方法。  (18) The production method according to the above (17), wherein the glycoprotein is an antibody.
( 19)上記（ 17)に記載の方法を用いて製造される糖蛋白質組成物。  (19) A glycoprotein composition produced using the method described in (17) above.
(20)上記（18)に記載の方法を用いて製造される抗体組成物。  (20) An antibody composition produced using the method according to (18) above.
( 21 )上記（ 19)に記載の糖蛋白質組成物を有効成分として含有する医薬。  (21) A medicament comprising the glycoprotein composition according to (19) above as an active ingredient.
(22)上記（20)に記載の抗体組成物を有効成分として含有する医薬。  (22) A medicament comprising the antibody composition according to (20) above as an active ingredient.
(23)ヒト組織より細胞を分離し、分離した細胞よりゲノム、全 RNAまたは mRNAを取 得し、取得したゲノム、全 RNAまたは mRNAから、あるいは取得した全 RNAまたは mRNAを用いて調整した cDNAからひ- 1，6 -フコシルトランスフェラーゼの遺伝子を 単離し、単離した遺伝子の塩基配列において、配列番号 7で表されるアミノ酸配列の N末端から 171番目のアミノ酸配列に相応する位置のアミノ酸がァスパラギンに置換 されているか否かを検出することを特徴とする α _1，6-フコシルトランスフェラーゼが関 与する疾患の診断方法。 (23) Isolate cells from human tissue, obtain genome, total RNA or mRNA from the separated cells, and use the obtained genome, total RNA or mRNA, or cDNA prepared using the obtained total RNA or mRNA. H-1,6-fucosyltransferase gene In the isolated nucleotide sequence of the isolated gene, it is detected whether or not the amino acid at the position corresponding to the 171st amino acid sequence from the N-terminus of the amino acid sequence represented by SEQ ID NO: 7 has been substituted with asparagine. A method for diagnosing a disease associated with the characteristic α_1,6-fucosyltransferase.
発明の効果  The invention's effect
[0009] 本発明により、 ひ- 1，6-フコシルトランスフェラーゼ酵素において、該酵素のひ- 1 ,6- フコシルトランスフェラーゼ活性が欠失、または低下した α -1 ,6-フコシルトランスフエ ラーゼ変異体及びその利用方法が提供される。 [0009] According to the present invention, in a -1,6-fucosyltransferase enzyme, an α -1,6-fucosyltransferase mutant in which the -1,6-fucosyltransferase activity of the enzyme is deleted or reduced, and How to use it is provided.
図面の簡単な説明  Brief Description of Drawings
[0010] [図 l]CHO/DG44細胞および RN6株のひ -1 ,6-フコース特異的レクチンである LCAに よる染色性を示した図である。横軸に蛍光強度を対数表示 (log)、縦軸に細胞数の 分布を示す。 白抜きは FITC標識ストレプトアビジンによる染色を行った細胞の結果を 、黒抜きは FITC標識 LCAによる染色を行なった細胞の結果を示す。  [0010] FIG. 1 is a diagram showing staining by LCA which is a -1,6-fucose-specific lectin of CHO / DG44 cells and RN6 strain. The horizontal axis shows the fluorescence intensity in logarithm (log), and the vertical axis shows the cell number distribution. The white outline shows the results of cells stained with FITC-labeled streptavidin, and the black outline shows the results of cells stained with FITC-labeled LCA.
[図 2]RN6株の産生した抗体の単糖組成分析の結果を示した図である。横軸に溶出 時間（min)、縦軸に相対強度を示す。  FIG. 2 shows the results of monosaccharide composition analysis of the antibody produced by RN6 strain. The horizontal axis shows the elution time (min), and the vertical axis shows the relative intensity.
[図 3]プラスミド CHFUT8Comp23の構築を示した図である。  FIG. 3 shows the construction of plasmid CHFUT8Comp23.
[図 4]プラスミド CHFUT8CompTAの構築を示した図である。  FIG. 4 shows the construction of plasmid CHFUT8CompTA.
[図 5]プラスミド pcDNAchFUT8Compの構築を示した図である。  FIG. 5 shows the construction of plasmid pcDNAchFUT8Comp.
[図 6]プラスミド CHFUT8Mo3の構築を示した図である。  FIG. 6 shows the construction of plasmid CHFUT8Mo3.
[図 7]プラスミド CHFUT8MoTAの構築を示した図である。  FIG. 7 shows the construction of plasmid CHFUT8MoTA.
[図 8]プラスミド pcDNAchFUT8Moの構築を示した図である。  FIG. 8 shows the construction of plasmid pcDNAchFUT8Mo.
[図 9]FUT8発現株および FUT8—塩基置換体発現株の LCA染色性を示した図である 。横軸に蛍光強度を対数表示 (log)、縦軸に細胞数の分布を示す。 白抜きは FITC標 識ストレブトアビジンによる染色を行った細胞の結果を、黒抜きは FITC標識 LCAによ る染色を行なった細胞の結果を示す。  FIG. 9 is a graph showing LCA staining of a FUT8 expression strain and a FUT8-base substitution product expression strain. The horizontal axis shows the fluorescence intensity in logarithm (log), and the vertical axis shows the cell number distribution. The white outline shows the results of cells stained with FITC-labeled streptavidin, and the black outline shows the results of cells stained with FITC-labeled LCA.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0011] 本発明において、 ひ -1 ,6-フコシルトランスフェラーゼとは、 N-グリコシド結合複合型 糖鎖還元末端の N -ァセチルダルコサミンの 6位とフコースの 1位が a結合する反応に 関与する酵素であればレ、かなる酵素も包含される。具体的には、下記 (a)、（b)、（ 、 (d )、（e)または (f)などの DNAがコードする蛋白質、または下記 (g)、（h)、（i)、（j)、（k)または (1 )などの蛋白質があげられる。 [0011] In the present invention, -1,6-fucosyltransferase is an N-glycoside-linked complex type sugar chain reducing terminal N-acetylyldarcosamine 6-position and fucose 1-position a bond Any enzyme involved is also included. Specifically, proteins encoded by DNA such as (a), (b), (, (d), (e) or (f) below, or (g), (h), (i), ( Examples thereof include proteins such as j), (k) or (1).
(a)配列番号 1で表される塩基配列からなる DNA ;  (a) DNA consisting of the base sequence represented by SEQ ID NO: 1;
(b)配列番号 2で表される塩基配列からなる DNA ;  (b) DNA comprising the base sequence represented by SEQ ID NO: 2;
(c)配列番号 3で表される塩基配列からなる DNA ;  (c) DNA consisting of the base sequence represented by SEQ ID NO: 3;
(d)配列番号 4で表される塩基配列からなる DNA ;  (d) DNA consisting of the base sequence represented by SEQ ID NO: 4;
(e)配列番号 5で表される塩基配列からなる DNA ;  (e) DNA consisting of the base sequence represented by SEQ ID NO: 5;
(f)配列番号 6で表される塩基配列からなる DNA ;  (f) DNA comprising the base sequence represented by SEQ ID NO: 6;
または、  Or
(g)配列番号 7で表されるアミノ酸配列からなる蛋白質；  (g) a protein comprising the amino acid sequence represented by SEQ ID NO: 7;
(h)配列番号 8で表されるアミノ酸配列からなる蛋白質；  (h) a protein comprising the amino acid sequence represented by SEQ ID NO: 8;
(D配列番号 9で表されるアミノ酸配列からなる蛋白質；  (D protein having the amino acid sequence represented by SEQ ID NO: 9;
(j)配列番号 10で表されるアミノ酸配列からなる蛋白質；  (j) a protein comprising the amino acid sequence represented by SEQ ID NO: 10;
(k)配列番号 11で表されるアミノ酸配列からなる蛋白質；  (k) a protein comprising the amino acid sequence represented by SEQ ID NO: 11;
(1)配列番号 12で表されるアミノ酸配列からなる蛋白質。  (1) A protein comprising the amino acid sequence represented by SEQ ID NO: 12.
[0012] 本発明の、 α -1 ,6-フコシルトランスフェラーゼにおいて、配列番号 7で表されるアミ ノ酸配列の Ν末端から 171番目のアミノ酸に相応する位置のアミノ酸とは、 α -1，6-フ コシルトランスフェラーゼが有するアミノ酸配列と配列番号 7で表されるアミノ酸配列と の相同性を後述する BLASTおよび FASTAなどの相同性解析プログラムおよびパラメ 一ターを用いて計算し、 2つのアミノ酸配列を整列させたとき α -1 ,6-フコシルトランス フェラーゼ変異体が有するアミノ酸配列上で、配列番号 7で表されるアミノ酸配列の Ν末端から 171番目のアミノ酸に対応する位置のアミノ酸をいう。  In the α -1,6-fucosyltransferase of the present invention, the amino acid at the position corresponding to the 171st amino acid from the terminus of the amino acid sequence represented by SEQ ID NO: 7 is α -1,6 -Calculate the homology between the amino acid sequence of fucosyltransferase and the amino acid sequence represented by SEQ ID NO: 7 using homology analysis programs and parameters such as BLAST and FASTA, which will be described later. An amino acid at a position corresponding to the 171st amino acid from the terminus of the amino acid sequence represented by SEQ ID NO: 7 on the amino acid sequence of the α -1,6-fucosyltransferase mutant when aligned.
[0013] 本発明のひ -1 ,6-フコシルトランスフェラーゼ変異体とは、 ひ -1，6-フコシルトランスフ エラーゼのアミノ酸配列において、配列番号 7で表されるアミノ酸配列の Ν末端から 1 71番目のアミノ酸に相応する位置のアミノ酸が欠失している力 \またはセリン以外の アミノ酸に置換されたアミノ酸配列を有する、 a -1，6-フコシルトランスフェラーゼ変異 体であればいずれでもよいが、例えば下記 (i)、 (ii)、 (iii)、 (iv)、 (v)、 (vi)、 (vii)、 (viii)、 (ix) 、（x)、（xi)、（xii)、 (xiii)、 (xiv)または (xv)などの蛋白質、または下記 (xvi)、 (xvii)、 (xviii)、 (xix)、 （xx)、（xxi)、 （xxii)、（xxiii)、 （xxiv)または (xxv)などの DNAがコードする蛋白質があ げられる。 [0013] The -1,6-fucosyltransferase mutant of the present invention refers to the 171st position from the heel of the amino acid sequence represented by SEQ ID NO: 7 in the amino acid sequence of -1, -6-fucosyltransferase. Any of the a-1,6-fucosyltransferase mutants having the ability to delete the amino acid at the position corresponding to the amino acid or an amino acid sequence substituted with an amino acid other than serine may be used. i), (ii), (iii), (iv), (v), (vi), (vii), (viii), (ix) , (X), (xi), (xii), (xiii), (xiv) or (xv) or a protein such as (xvi), (xvii), (xviii), (xix), (xx), Examples include (xxi), (xxii), (xxiii), (xxiv), or (xxv) proteins encoded by DNA.
(0配列番号 13で表されるアミノ酸配列からなる蛋白質；  (0 a protein consisting of the amino acid sequence represented by SEQ ID NO: 13;
(ii)配列番号 14で表されるアミノ酸配列からなる蛋白質；  (ii) a protein comprising the amino acid sequence represented by SEQ ID NO: 14;
(iii)配列番号 15で表されるアミノ酸配列からなる蛋白質；  (iii) a protein comprising the amino acid sequence represented by SEQ ID NO: 15;
(iv)配列番号 16で表されるアミノ酸配列からなる蛋白質；  (iv) a protein comprising the amino acid sequence represented by SEQ ID NO: 16;
(V)配列番号 17で表されるアミノ酸配列からなる蛋白質； (V) a protein comprising the amino acid sequence represented by SEQ ID NO: 17;
(vi)配列番号 13で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、揷 入および/または付加されたアミノ酸配列からなり、かつひ- 1，6 -フコシルトランスフエ ラーゼ活性が欠失、または、配列番号 7または 9で表されるアミノ酸配列からなるひ- 1 ，6-フコシルトランスフェラーゼのひ _1，6 -フコシルトランスフェラーゼ活性よりも低下し た α -1,6-フコシルトランスフェラーゼ活性を有する α -1,6-フコシルトランスフェラー ゼ変異体；  (vi) The amino acid sequence represented by SEQ ID NO: 13 consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and has a 1,6-fucosyltransferase activity. Α-1,6-fucosyltransferase activity that is lower than the _1,6-fucosyltransferase activity of -1,6-fucosyltransferase consisting of the deletion or amino acid sequence represented by SEQ ID NO: 7 or 9. An α-1,6-fucosyltransferase mutant having;
(vii)配列番号 14で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、 挿入および/または付加されたアミノ酸配列からなり、かつ α -1,6-フコシルトランスフ エラーゼ活性が欠失、または、配列番号 7または 9で表されるアミノ酸配列からなる α -1,6-フコシルトランスフェラーゼの α -1,6-フコシルトランスフェラーゼ活性よりも低下 した α -1,6-フコシルトランスフェラーゼ活性を有する α -1,6-フコシルトランスフェラー ゼ変異体；  (vii) The amino acid sequence represented by SEQ ID NO: 14 consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and α-1,6-fucosyltransferase is deleted. Or α having a α-1,6-fucosyltransferase activity that is lower than the α-1,6-fucosyltransferase activity of α-1,6-fucosyltransferase comprising the amino acid sequence represented by SEQ ID NO: 7 or 9. -1,6-fucosyltransferase mutant;
(viii)配列番号 15で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、 揷入および/または付加されたアミノ酸配列からなり、かつひ- 1，6 -フコシルトランスフ エラーゼ活性が欠失、または、配列番号 7または 9で表されるアミノ酸配列からなるひ -1，6-フコシルトランスフェラーゼのひ -1，6 -フコシルトランスフェラーゼ活性よりも低下 したひ -1，6-フコシルトランスフェラーゼ活性を有するひ -1,6-フコシルトランスフェラー ゼ変異体；  (viii) The amino acid sequence represented by SEQ ID NO: 15 consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and lacks -1,6-fucosyltransferase activity. Or has a -1,6-fucosyltransferase activity lower than the -1,6-fucosyltransferase activity of the -1,6-fucosyltransferase consisting of the amino acid sequence represented by SEQ ID NO: 7 or 9. -1,6-fucosyltransferase mutant;
(ix)配列番号 16で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、揷 入および/または付加されたアミノ酸配列からなり、かつひ- 1，6 -フコシルトランスフエ ラーゼ活性が欠失、または、配列番号 7または 9で表されるアミノ酸配列からなる α -1 ，6-フコシルトランスフェラーゼの α -1,6-フコシルトランスフェラーゼ活性よりも低下し た α -1,6-フコシルトランスフェラーゼ活性を有する α -1,6-フコシルトランスフェラー ゼ変異体； (ix) The amino acid sequence represented by SEQ ID NO: 16, consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added, and -1,6-fucosyltransferase The α-1,6-fucosyltransferase activity of the α-1,6-fucosyltransferase of the α-1,6-fucosyltransferase consisting of the amino acid sequence represented by SEQ ID NO: 7 or 9 is reduced. An α-1,6-fucosyltransferase mutant having fucosyltransferase activity;
(X)配列番号 17で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、揷 入および/または付加されたアミノ酸配列からなり、かつひ- 1，6 -フコシルトランスフエ ラーゼ活性が欠失、または、配列番号 7または 9で表されるアミノ酸配列からなるひ- 1 ，6-フコシルトランスフェラーゼのひ _1，6 -フコシルトランスフェラーゼ活性よりも低下し たひ -1，6-フコシルトランスフェラーゼ活性を有するひ -1,6-フコシルトランスフェラー ゼ変異体；  (X) The amino acid sequence represented by SEQ ID NO: 17 consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and has a 1,6-fucosyltransferase activity. Deletion or -1,6-fucosyltransferase activity lower than the _1,6-fucosyltransferase activity of -1,6-fucosyltransferase consisting of the amino acid sequence represented by SEQ ID NO: 7 or 9. A -1,6-fucosyltransferase mutant having;
(xi)配列番号 13で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列 からなり、かつひ- 1，6-フコシルトランスフェラーゼ活性が欠失、または、配列番号 7ま たは 9で表されるアミノ酸配列力 なる α -1,6-フコシルトランスフェラーゼの α -1,6-7 コシルトランスフェラーゼ活性よりも低下した α -1,6-フコシルトランスフェラーゼ活性 を有するひ- 1,6-フコシルトランスフェラーゼ変異体；  (xi) consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 13, and lacking the 1,6-fucosyltransferase activity, or having SEQ ID NO: 7 or 9 The amino acid sequence represented by α-1,6-fucosyltransferase has α-1,6-fucosyltransferase activity lower than α-1,6-fucosyltransferase activity of α-1,6-fucosyltransferase. Mutant;
(xii)配列番号 14で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列 からなり、かつ α -1,6-フコシルトランスフェラーゼ活性が欠失、または、配列番号 7ま たは 9で表されるアミノ酸配列力 なる α -1,6-フコシルトランスフェラーゼの α -1,6-7 コシルトランスフェラーゼ活性よりも低下した α -1,6-フコシルトランスフェラーゼ活性 を有するひ- 1,6-フコシルトランスフェラーゼ変異体；  (xii) consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 14, and lacking α-1,6-fucosyltransferase activity, or SEQ ID NO: 7 or 9 The amino acid sequence represented by α-1,6-fucosyltransferase has α-1,6-fucosyltransferase activity lower than α-1,6-fucosyltransferase activity of α-1,6-fucosyltransferase. Mutant;
(xiii)配列番号 15で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列 からなり、かつひ- 1，6-フコシルトランスフェラーゼ活性が欠失、または、配列番号 7ま たは 9で表されるアミノ酸配列力、らなるひ -1，6-フコシルトランスフェラーゼのひ -1,6-フ コシルトランスフェラーゼ活性よりも低下したひ -1,6-フコシルトランスフェラーゼ活性 を有するひ- 1，6 -フコシルトランスフェラーゼ変異体；  (xiii) consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 15, and lacking -1,6-fucosyltransferase activity, or SEQ ID NO: 7 or 9 The amino acid sequence represented by Ranarihi-1,6-fucosyltransferase has a -1,6-fucosyltransferase activity that is lower than that of 1,6-fucosyltransferase. A fucosyltransferase variant;
(xiv)配列番号 16で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列 からなり、かつひ- 1，6-フコシルトランスフェラーゼ活性が欠失、または、配列番号 7ま たは 9で表されるアミノ酸配列力、らなるひ -1，6-フコシルトランスフェラーゼのひ -1,6-フ コシルトランスフェラーゼ活性よりも低下した α -1,6-フコシルトランスフェラーゼ活性 を有するひ- 1,6-フコシルトランスフェラーゼ変異体； (xiv) consisting of an amino acid sequence having at least 80% homology with the amino acid sequence represented by SEQ ID NO: 16, and lacking the 1,6-fucosyltransferase activity, or having SEQ ID NO: 7 or 9 Amino acid sequence represented by Ranaruhi-1,6-fucosyltransferase A -1,6-fucosyltransferase mutant having α-1,6-fucosyltransferase activity that is lower than the cosyltransferase activity;
(XV)配列番号 17で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列 からなり、かつひ- 1，6-フコシルトランスフェラーゼ活性が欠失、または、配列番号 7ま たは 9で表されるアミノ酸配列力、らなるひ -1，6-フコシルトランスフェラーゼのひ -1,6-フ コシルトランスフェラーゼ活性よりも低下したひ -1,6-フコシルトランスフェラーゼ活性 を有するひ- 1，6 -フコシルトランスフェラーゼ変異体；  (XV) consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 17, and lacking -1,6-fucosyltransferase activity, or SEQ ID NO: 7 or 9 The amino acid sequence represented by Ranarihi-1,6-fucosyltransferase has a -1,6-fucosyltransferase activity that is lower than that of 1,6-fucosyltransferase. A fucosyltransferase variant;
または、 Or
(xvi)配列番号 18で表される塩基配列からなる DNA;  (xvi) DNA consisting of the base sequence represented by SEQ ID NO: 18;
(xvii)配列番号 19で表される塩基配列からなる DNA;  (xvii) DNA consisting of the base sequence represented by SEQ ID NO: 19;
(xviii)配列番号 20で表される塩基配列からなる DNA;  (xviii) DNA consisting of the base sequence represented by SEQ ID NO: 20;
(xix)配列番号 21で表される塩基配列からなる DNA;  (xix) DNA consisting of the base sequence represented by SEQ ID NO: 21;
(XX)配列番号 22で表される塩基配列からなる DNA； (XX) DNA consisting of the base sequence represented by SEQ ID NO: 22;
(xxi)配列番号 18のいずれかで表される塩基配列とストリンジェントな条件でハイプリ ダイズし、かつ α -1,6-フコシルトランスフェラーゼ活性が欠失、または、配列番号 7ま たは 9で表されるアミノ酸配列力らなる α -1,6-フコシルトランスフェラーゼの α -1,6-7 コシルトランスフェラーゼ活性よりも低下した α -1,6-フコシルトランスフェラーゼ活性 を有するひ -1,6-フコシルトランスフェラーゼ変異体をコードする DNA;  (xxi) Hyperhybridized under stringent conditions with the nucleotide sequence represented by any of SEQ ID NO: 18, and lacks α-1,6-fucosyltransferase activity, or represented by SEQ ID NO: 7 or 9. -1,6-fucosyltransferase having α-1,6-fucosyltransferase activity lower than that of α-1,6-fucosyltransferase of α-1,6-fucosyltransferase DNA encoding the variant;
(xxii)配列番号 19のいずれかで表される塩基配列とストリンジェントな条件でハイブリ ダイズし、かつ α -1,6-フコシルトランスフェラーゼ活性が欠失、または、配列番号 7ま たは 9で表されるアミノ酸配列力らなる α -1,6-フコシルトランスフェラーゼの α -1,6-7 コシルトランスフェラーゼ活性よりも低下したひ -1,6-フコシルトランスフェラーゼ活性 を有するひ -1，6 -フコシルトランスフェラーゼ変異体をコードする DNA;  (xxii) hybridizes with the nucleotide sequence represented by SEQ ID NO: 19 under stringent conditions and lacks α-1,6-fucosyltransferase activity, or is represented by SEQ ID NO: 7 or 9. -1,6-fucosyltransferase having -1,6-fucosyltransferase activity lower than that of α-1,6-fucosyltransferase of α-1,6-fucosyltransferase DNA encoding the variant;
(xxiii)配列番号 20のいずれかで表される塩基配列とストリンジェントな条件でハイブリ ダイズし、かつひ -1,6-フコシルトランスフェラーゼ活性が欠失、または、配列番号 7ま たは 9で表されるアミノ酸配列力、らなるひ -1，6-フコシルトランスフェラーゼのひ -1,6-フ コシルトランスフェラーゼ活性よりも低下したひ -1,6-フコシルトランスフェラーゼ活性 を有するひ -1，6 -フコシルトランスフェラーゼ変異体をコードする DNA; (xxiv)配列番号 21のいずれかで表される塩基配列とストリンジェントな条件でハイプリ ダイズし、かつ α -1,6-フコシルトランスフェラーゼ活性が欠失、または、配列番号 7ま たは 9で表されるアミノ酸配列力らなる α -1,6-フコシルトランスフェラーゼの α _1,6_フ コシルトランスフェラーゼ活性よりも低下したひ -1,6-フコシルトランスフェラーゼ活性 を有するひ -1，6 -フコシルトランスフェラーゼ変異体をコードする DNA; (xxiii) hybridizes with the nucleotide sequence represented by any of SEQ ID NO: 20 under stringent conditions and lacks -1,6-fucosyltransferase activity, or is represented by SEQ ID NO: 7 or 9 -1,6-fucosyltransferase having lower -1,6-fucosyltransferase activity than the -1,6-fucosyltransferase activity of Ranaruhi-1,6-fucosyltransferase DNA encoding a transferase variant; (xxiv) Hyperhybridized under stringent conditions with the nucleotide sequence represented by any of SEQ ID NO: 21, and lacks α-1,6-fucosyltransferase activity, or represented by SEQ ID NO: 7 or 9. -1,6-fucosyltransferase mutations with -1,6-fucosyltransferase activity lower than α_1,6_fucosyltransferase activity of α-1,6-fucosyltransferase DNA encoding the body;
(XXV)配列番号 22のいずれかで表される塩基配列とストリンジェントな条件でハイプリ ダイズし、かつひ -1,6-フコシルトランスフェラーゼ活性が欠失、または、配列番号 7ま たは 9で表されるアミノ酸配列力、らなるひ -1，6-フコシルトランスフェラーゼのひ -1,6-フ コシルトランスフェラーゼ活性よりも低下したひ -1,6-フコシルトランスフェラーゼ活性 を有するひ -1，6 -フコシルトランスフェラーゼ変異体をコードする DNA。 (XXV) Hyperhybridized under stringent conditions with the nucleotide sequence represented by any of SEQ ID NO: 22, and lacks -1,6-fucosyltransferase activity, or represented by SEQ ID NO: 7 or 9. -1,6-fucosyltransferase having lower -1,6-fucosyltransferase activity than the -1,6-fucosyltransferase activity of Ranaruhi-1,6-fucosyltransferase DNA encoding a transferase variant.
本発明において、ストリンジ工ントな条件下でハイブリダィズする DNAとは、例えば 配列番号 18、 19、 20、 21または 22で表される塩基配列を有する DNAなどの DNAま たはその一部の断片をプローブとして、コロニ一'ハイブリダィゼーシヨン法、プラーク •ハイブリダィゼーシヨン法あるいはサザンブロットハイブリダィゼーシヨン法等を用い ることにより得られる DNAを意味し、具体的には、コロニーあるいはプラーク由来の D NAを固定化したフィルターを用いて、 0. 7〜： 1. 0Mの塩化ナトリウム存在下、 65°C でハイブリダィゼーシヨンを行った後、 0. ：!〜 2倍濃度の SSC溶液（1倍濃度の SSC 溶液の組成は、 150mM塩化ナトリウム、 15mMクェン酸ナトリウムよりなる）を用い、 65°C条件下でフィルターを洗浄することにより同定できる DNAをあげることができる 。ハイブリダイセ1 ~~シヨン ia>、 Molecular cloning, a laooratory manual, Third Edition, Cold Spring Harbor Laboratory Press (2001) (以下、モレキュラ^ ~ ·クローニング第 3 版と略す）、 Current Protocols in Molecular Biology, John Wiley & Sons, 1987—1997 ( 以下、カレント'プロトコールズ'イン'モレキュラー 'バイオロジーと略す）、 DNA Clonin g 1 : Core Ί ecnmques, A Practical Approach, Second Edition, Oxford University (19 95)等に記載されている方法に準じて行うことができる。ノ、イブリダィズ可能な DNAと して具体的には、 BLASTや FASTA等を用いて計算したときに、例えば配列番号 18 、 19、 20、 21または 22で表される塩基配列と少なくとも 70%以上、好ましくは 80%以 上、より好ましくは 90。/o以上、さらに好ましくは 95%以上、特に好ましくは 98%以上、 最も好ましくは 99%以上の相同性を有する DNAをあげることができる。 In the present invention, DNA that hybridizes under stringent conditions refers to DNA such as DNA having a base sequence represented by SEQ ID NO: 18, 19, 20, 21, or 22, or a partial fragment thereof. As a probe, it means DNA obtained by using colony hybridization method, plaque hybridization method, Southern blot hybridization method, etc., specifically, colony or Using a filter immobilized with plaque-derived DNA, 0.7 to 1.0. After hybridization at 65 ° C in the presence of 0 M sodium chloride, 0. The DNA can be identified by washing the filter under 65 ° C conditions using a SSC solution (concentrated SSC solution consisting of 150 mM sodium chloride and 15 mM sodium quenate). And can. HYBRIDISE 1 ~~ Cillon ia>, Molecular cloning, a laooratory manual, Third Edition, Cold Spring Harbor Laboratory Press (2001) (hereinafter abbreviated as Molecular ^ ~ Cloning 3rd Edition), Current Protocols in Molecular Biology, John Wiley & Sons, 1987—1997 (hereinafter “abbreviated as“ protocols ”in“ molecular ”biology), DNA Clonin g 1: Core Ί ecnmques, A Practical Approach, Second Edition, Oxford University (1955), etc. It can be performed according to the method. More specifically, as DNA that can be hybridized, when calculated using BLAST, FASTA, etc., for example, at least 70% or more of the nucleotide sequence represented by SEQ ID NO: 18, 19, 20, 21, or 22, Preferably it is 80% or more, more preferably 90. / o or more, more preferably 95% or more, particularly preferably 98% or more, Most preferred is DNA having a homology of 99% or more.
[0015] アミノ酸配列および塩基配列の相同性は、例えば Karlin and Altschulによるアルゴリ ズム BLAST〔Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)〕や FASTA [Methods Enzym ol.， 183, 63 (1990)〕を用いて決定することができる。このアルゴリズム BLASTに基づ いて、 BLASTNや BLASTXとよばれるプログラムが開発されている〔J. Mol. Biol., 215. 403(1990)] BLASTに基づいて BLASTNによって塩基配列を解析する場合には、 ラメ一ターは例えば Score = 100、 wordlength= 12とする。また、 BLASTに基づいて BL ASTXによってアミノ酸配列を解析する場合には、パラメータ一は例えば score = 50、 w ordlength = 3とする。 BLASTと Gapped BLASTプログラムを用いる場合には、各プログ ラムのデフォルトパラメーターを用いる。これらの解析方法の具体的な手法は公知で ある (http://www.ncbi.nlm.nih.gov·)。 [0015] The homology between the amino acid sequence and the base sequence is, for example, the algorithm BLAST [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] by Karlin and Altschul, FASTA [Methods Enzymol., 183, 63 (1990)]. Based on this algorithm BLAST, programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215. 403 (1990)] When analyzing a base sequence by BLASTN based on BLAST, For example, the parameter is Score = 100 and wordlength = 12. In addition, when the amino acid sequence is analyzed by BL ASTX based on BLAST, the parameter 1 is, for example, score = 50 and word length = 3. When using BLAST and Gapped BLAST programs, use the default parameters of each program. Specific methods of these analysis methods are known (http: //www.ncbi.nlm.nih.gov·).
[0016] 本発明において、例えば配列番号 13、 14、 15、 16または 17で表されるアミノ酸配列 において 1以上のアミノ酸が欠失、置換、挿入および/または付加されたアミノ酸配 列からなり、かつ α -1,6-フコシルトランスフェラーゼ活性を有する蛋白質とは、モレキ ユラ一.クローニング第 3版、カレント'プロトコールズ 'イン'モレキュラ^ ~ ·バイオロジー 、 Nucleic Acids Research, 10, 6487 (1982)、 Proc. Natl. Acad. Sci., USA, 79, 6409(1 982)、 Gene, 34, 315 (1985)、 Nucleic Acids Research, 13， 4431 (1985)、 Proc. Natl. A cad. Sci USA,82, 488 (1985)等に記載の部位特異的変異導入法を用いて、例えば、 配列番号 13、 14、 15、 16または 17で表されるアミノ酸配列を有する蛋白質をコードす る DNAに部位特異的変異を導入することにより取得することができる蛋白質を意味 する。欠失、置換、挿入および/または付加されるアミノ酸の数は 1個以上でありその 数は特に限定されないが、上記の部位特異的変異導入法等の周知の技術により、 欠失、置換もしくは付カ卩できる程度の数であり、例えば、 1〜数十個、好ましくは 1〜2 0個、より好ましくは 1〜： 10個、さらに好ましくは 1〜5個である。 In the present invention, for example, the amino acid sequence represented by SEQ ID NO: 13, 14, 15, 16 or 17 comprises an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and Proteins with α-1,6-fucosyltransferase activity are: Molecular Molecular Cloning, 3rd Edition, Current 'Protocols' In' Molecular ^ Biology, Nucleic Acids Research, 10, 6487 (1982), Proc Natl. Acad. Sci., USA, 79, 6409 (1 982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. A cad. Sci USA, 82, Using site-directed mutagenesis described in 488 (1985), for example, site-directed mutagenesis to DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 13, 14, 15, 16 or 17 It means a protein that can be obtained by introducing. The number of amino acids to be deleted, substituted, inserted and / or added is one or more, and the number is not particularly limited. However, deletion, substitution or attachment may be performed by well-known techniques such as the above-mentioned site-directed mutagenesis. For example, the number is 1 to several tens, preferably 1 to 20, more preferably 1 to 10 and even more preferably 1 to 5.
[0017] また、 1以上のアミノ酸が欠失、置換または付加されたとは、同一配列中の任意の 位置において、 1または複数のアミノ酸の欠失、置換または付加があることを意味し、 欠失、置換または付加が同時に生じてもよぐ置換または付加されるアミノ酸は天然 型と非天然型とを問わない。天然型アミノ酸としては、 L—ァラニン、 L—ァスパラギン 、 L ァスパラギン酸、 L アルギニン、 L グルタミン、 L グルタミン酸、グリシン、 L ヒスチジン、 L—イソロイシン、 L一口イシン、 L—リジン、 L メチォニン、 L—フエ二 ルァラニン、 L プロリン、 L セリン、 L スレオニン、 L トリプトファン、 L チロシン 、 L パリン、 L—システィンなどがあげられる。 [0017] Further, that one or more amino acids are deleted, substituted or added means that there is a deletion, substitution or addition of one or more amino acids at any position in the same sequence. The amino acid to be substituted or added may be natural or non-natural, although substitution or addition may occur simultaneously. Natural amino acids include L-alanine and 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-cystine and the like.
以下に、相互に置換可能なアミノ酸の例を示す。同一群に含まれるアミノ酸は相互 に置換可能である。  Examples of 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.
A群：ロイシン、イソロイシン、ノルロイシン、バリン、ノノレバリン、ァラニン、 2-アミノブ タン酸、メチォニン、〇_メチルセリン、 t_ブチルグリシン、 t -ブチルァラニン、シクロへ キシノレァラニン  Group A: leucine, isoleucine, norleucine, valine, nonorevaline, alanine, 2-aminobutanoic acid, methionine, _methylserine, t_butylglycine, t-butylalanine, cyclohexenolealanine
B群：ァスパラギン酸、グノレタミン酸、イソァスパラギン酸、イソグノレタミン酸、 2-ァミノ アジピン酸、 2-アミノスべリン酸  Group B: aspartic acid, gnoretamic acid, isoaspartic acid, isognoletamic acid, 2-amino adipic acid, 2-aminosuberic acid
C群：ァスパラギン、グノレタミン  Group C: Asparagine, Gnoretamine
D群：リジン、アルギニン、オル二チン、 2,4-ジァミノブタン酸、 2,3-ジァミノプロピオ ン酸  Group D: lysine, arginine, ornithine, 2,4-dianaminobutanoic acid, 2,3-dianaminopropionic acid
E群：プロリン、 3-ヒドロキシプロリン、 4-ヒドロキシプロリン  Group E: proline, 3-hydroxyproline, 4-hydroxyproline
F群：セリン、スレオニン、ホモセリン  Group F: serine, threonine, homoserine
G群：フエ二ルァラニン、チロシン  Group G: phenylalanin, tyrosine
また、本発明において、例えば配列番号 13、 14、 15、 16または 17で表されるアミノ酸 配列と 80%以上の相同性を有し、かつ α -1,6-フコシルトランスフェラーゼ活性を有 する蛋白質とは、 BLASTや FASTA等の解析ソフトを用いて計算したときに、配列 番号 13、 14、 15、 16または 17に記載のアミノ酸配列を有する蛋白質と少なくとも 80% 以上、好ましくは 85。/0以上、より好ましくは 90。/o以上、さらに好ましくは 95。/0以上、特 に好ましくは 97%以上、最も好ましくは 99%以上である蛋白質であることを意味する 本発明の細胞としては、本発明のひ -1,6-フコシルトランスフェラーゼ変異体を発現 している細胞であればいかなる細胞でもよいが、例えば酵母、動物細胞、昆虫細胞、 植物細胞などがあげられ、これらの細胞の具体的な例としては、後述の 2.に記載の ものがあげられる。動物細胞の具体例としては、チャイニーズハムスター卵巣組織由 来の CHO細胞、ラットミエローマ細胞株 YB2/3HL.P2.G11.16Ag.20細胞、マウスミエ口 一マ細胞株 NS0細胞、マウスミエローマ細胞株 SP2/0-Agl4細胞、シリアンハムスター 腎臓組織由来 BHK細胞、抗体を産生するハイブリドーマ細胞、ヒト白血病細胞株ナ マルバ細胞、胚性幹細胞、受精卵細胞などがあげられる。好ましくは、抗体などの糖 蛋白質の製造に用いられる、上述のミエローマ細胞、ハイプリドーマ細胞、ヒト化抗体 あるいはヒト抗体を製造するための宿主細胞、ヒト抗体を生産するヒト以外のトランス ジヱニック動物を製造するために用いる胚性幹細胞または受精卵細胞、ならびにヒト 化抗体およびヒト抗体を生産するトランスジヱニック植物を製造するために用レ、る植 物細胞などがあげられる。 In the present invention, for example, a protein having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 13, 14, 15, 16, or 17 and having α-1,6-fucosyltransferase activity. Is at least 80% or more, preferably 85, with a protein having the amino acid sequence set forth in SEQ ID NO: 13, 14, 15, 16 or 17 when calculated using analysis software such as BLAST or FASTA. / 0 or more, more preferably 90. / o or more, more preferably 95. / 0 or more, particularly preferably 97% or more, most preferably 99% or more of the protein of the present invention means that the -1,6-fucosyltransferase mutant of the present invention is expressed. Any cell may be used as long as it is a cell, for example, yeast, animal cells, insect cells, plant cells, and the like. Specific examples of these cells include those described in 2. below. . Specific examples of animal cells include Chinese hamster ovary tissue CHO cells, rat myeloma cell line YB2 / 3HL.P2.G11.16Ag.20 cell, mouse myeoma cell line NS0 cell, mouse myeloma cell line SP2 / 0-Agl4 cell, Syrian hamster kidney tissue-derived BHK cell, Examples include antibody-producing hybridoma cells, human leukemia cell line Namalba cells, embryonic stem cells, and fertilized egg cells. Preferably, the above-described myeloma cells, hyperpridoma cells, humanized antibodies or host cells for producing human antibodies, and non-human transgenic animals producing human antibodies, which are used for the production of glycoproteins such as antibodies, are produced. Examples thereof include embryonic stem cells or fertilized egg cells used for the preparation, and plant cells for use in producing a transgenic plant producing humanized antibodies and human antibodies.
NS0細胞としては、バイオ/テクノロジー (BIO/TECHNOLOGY), 10, 169 (1992)、バ ィォテクノロジー 'バイオエンジニアリング (Biotechnol. Bioeng.), 73, 261, (2001)等の 文献に記載されている NS0細胞があげられる。また、理化学研究所細胞開発銀行に 登録されている NS0細胞株（RCB0213)、あるいはこれら株を様々な無血清培地に馴 化させた亜株などもあげられる。 SP2/0-Agl4細胞としては、ジャーナル ·ォブ'ィムノ ロジー (J. Immunol.), 126, 317，（1981)、ネイチヤー (Nature), 276, 269， (1978)、ヒユー マン'アンチイボディズ 'アンド · /、イブリドーマズ (Human Antibodies and Hybridomas), NS0 cells are described in documents such as Bio / Technology (BIO / TECHNOLOGY), 10, 169 (1992), Biotechnology (Biotechnol. Bioeng.), 73, 261, (2001). NS0 cells. Another example is the NS0 cell line (RCB0213) registered with the RIKEN Cell Development Bank, or substrains in which these lines are conditioned to various serum-free media. SP2 / 0-Agl4 cells include Journal of Immunology (J. Immunol.), 126, 317, (1981), Nature, 276, 269, (1978), Human Anti-Ibodies. 'And /, Human Antibodies and Hybridomas,
3, 129, (1992)等の文献に記載されている SP2/0_Agl4細胞があげられる。また、 AT CCに登録されている SP2/0-Agl4細胞（ATCC CRL-1581)あるいはこれら株を様々 な無血清培地に馴化させた亜株 (ATCC CRL-1581.1)などもあげられる。チヤィニー ズハムスター卵巣組織由来 CHO細胞としては、 Journal of Experimental Medicine, 10 8, 945 (1958)、 Proc. Natl. Acad. Sci. USA, 60, 1275 (1968)、 Genetics, 55, 513 (196 8)、 Chromosoma, 41, 129 (1973)、 Methods in Cell Science, 18, 115 (1996)、 Radiatio n Research, 148, 260 (1997)、 Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)、 Proc. Na tl. Acad. Sci. 60> 1275 (1968)、 Cell, 6, 121 (1975)、 Molecular Cell Genetics, Appen dix 1,11 (p883_900)等の文献に記載されている CHO細胞があげられる。また、 ATCC に登録されている CHO-Kl株（ATCC CCL-61)、 DUXB11株（ATCC CRL- 9096)、 Pr 0-5株（ATCC CRL- 1781)や、市販の CHO- S株（Lifetechnologies社製 Cat No.11619 )、あるいはこれら株を様々な無血清培地に馴化させた亜株などもあげられる。ラットミ エローマ細胞株 YB2/3HL.P2.G11.16Ag.20細胞としては、 Y3/Agl.2.3細胞（ATCC C RL-1631)力 樹立された株化細胞が包含される。その具体的な例としては、 J. Cell. Biol., 93, 576 (1982)、 Methods Enzymol. 73B, 1 (1981)等の文献に記載されている Y B2/3HL.P2. Gil.16Ag.20細胞があげられる。また、 ATCCに登録されている YB2/3H し.？2 11.16八8.20細胞（八1^じ CRL-1662)あるいはこれら株を様々な無血清培地に 馴化させた亜株などもあげられる。 SP2 / 0_Agl4 cells described in literatures such as 3, 129, (1992). In addition, SP2 / 0-Agl4 cells (ATCC CRL-1581) registered in ATCC or sub-strains (ATCC CRL-1581.1) in which these strains are conditioned to various serum-free media are also included. For CHO cells derived from Chinese hamster ovary tissue, Journal of Experimental Medicine, 10 8, 945 (1958), Proc. Natl. Acad. Sci. USA, 60, 1275 (1968), Genetics, 55, 513 (196 8) Chromosoma, 41, 129 (1973), Methods in Cell Science, 18, 115 (1996), Radiation Research, 148, 260 (1997), Proc. Natl. Acad. Sci. USA, 77, 4216 (1980), Proc. Natl. Acad. Sci. 60> 1275 (1968), Cell, 6, 121 (1975), Molecular Cell Genetics, Appen dix 1,11 (p883_900), etc. . In addition, CHO-Kl strain (ATCC CCL-61), DUXB11 strain (ATCC CRL-9096), Pr 0-5 strain (ATCC CRL-1781) registered in ATCC, commercially available CHO-S strain (Lifetechnologies) Cat No. 11619), or substrains obtained by acclimating these strains to various serum-free media. Ratmi The Eroma cell line YB2 / 3HL.P2.G11.16Ag.20 cells include cell lines in which Y3 / Agl.2.3 cells (ATCC C RL-1631) have been established. Specific examples thereof include Y B2 / 3HL.P2.Gil.16Ag. Described in documents such as J. Cell. Biol., 93, 576 (1982), Methods Enzymol. 73B, 1 (1981). There are 20 cells. Also, YB2 / 3H registered in ATCC? 2 11.16 8 .20 cells (8 1 ^ CRL-1662) or substrains of these strains acclimated to various serum-free media.
[0020] 本発明の細胞に糖蛋白質分子をコードする遺伝子を導入し、該細胞を用いて糖蛋 白質分子からなる糖蛋白質組成物を製造することができる。 [0020] A gene encoding a glycoprotein molecule can be introduced into the cell of the present invention, and a glycoprotein composition comprising the glycoprotein molecule can be produced using the cell.
糖蛋白質に糖鎖は、糖蛋白質部分との結合様式により、ァスパラギンと結合する糖 鎖 (N-グリコシド結合糖鎖）と、セリンまたはスレオニンなどと結合する糖鎖（〇 -グリコ シド結合糖鎖）の 2種類に大別される。  Glycoproteins have a sugar chain that is linked to asparagine (N-glycoside-linked sugar chain) and a sugar chain that binds to serine or threonine (o-glycoside-linked sugar chain), depending on the mode of binding to the glycoprotein moiety. There are two main types.
[0021] N-グリコシド結合糖鎖は、様々な構造を有しているがいずれの場合も上述の構造 式 (I)に示す共通のコア構造を有する。構造式 (I)において、ァスパラギンと結合する 糖鎖の末端が還元末端、反対側が非還元末端という。 N-グリコシド結合糖鎖としては 、コア構造の非還元末端にマンノースのみが結合するハイマンノース型糖鎖、コア構 造の非還元末端側にガラクトース- N-ァセチルダルコサミン（以下、 Gaト GlcNacと表 す。）の枝を並行して 1ないしは複数本有し、更に Ga卜 GlcNacの非還元末端にシアル 酸、バイセクティングの N-ァセチルダルコサミンなどの構造を有するコンプレックス型（ 複合型とも称す。）糖鎖、コア構造の非還元末端にハイマンノース型とコンプレックス 型の両方の枝を持つハイブリッド型糖鎖などがあげられる。  [0021] N-glycoside-linked sugar chains have various structures, but in any case, they have a common core structure represented by the above structural formula (I). In 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 Gato GlcNac) on the non-reducing end of the core structure. The complex type has one or more branches in parallel, and further has a structure such as sialic acid or bisecting N-acetylcylcosamine at the non-reducing end of Ga 卜 GlcNac. And glycans and hybrid glycans having both high-mannose and complex branches at the non-reducing end of the core structure.
[0022] 0-グリコシド結合糖鎖としては、 N-ァセチルガラタトサミンの還元末端がセリンまた はスレオニンの水酸基とひ結合し、更にガラクトース、 N-ァセチルダルコサミン、 N-ァ セチルガラタトサミンあるいはシアル酸が結合した糖鎖、キシロースがセリンの水酸基 と β結合した糖鎖、ガラクトースがハイドロキシリジンの水酸基と β結合した糖鎖など があげられる。  [0022] 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 Examples include sugar chains in which samine or sialic acid is bonded, sugar chains in which xylose is β-bonded to the hydroxyl group of serine, and sugar chains in which galactose is β-bonded to the hydroxyl group of hydroxylysine.
[0023] キシロースがセリンの水酸基と /3結合した糖鎖は、通常、当該キシロースの 4位に複 数の糖が結合し、結合した糖の先に 2糖からなる直鎖状の多糖が結合している。この ような糖鎖構造を有する物質としては軟骨プロテオダリカンがあげられる。ガラクトー スがハイドロキシリジンの水酸基と β結合した糖鎖構造を有する物質としては、コラー ゲン等があげられる。 [0023] A sugar chain in which xylose is bonded to the hydroxyl group of serine by / 3 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. Galacto Examples of the substance having a sugar chain structure in which β is bonded to the hydroxyl group of hydroxylysine include collagen.
[0024] 糖鎖を構成する糖としては、 Ν-ァセチルダルコサミン、 Ν-ァセチルガラタトサミン、 マンノース、ガラクトース、フコース、シアル酸、キシロース、ァラビノース等が含まれ、 これらの糖はレ、かなる順序で結合してもよレ、。  [0024] Examples of the sugar constituting the sugar chain include ァ -acetylyl darcosamine, Ν-acetyl galatatosamine, mannose, galactose, fucose, sialic acid, xylose, arabinose, and the like. You can combine them in any order.
従って、本発明において、糖蛋白質組成物とは、 Ν -グリコシド結合糖鎖または〇-グ リコシド結合糖鎖を有する糖蛋白質分子からなる組成物をレ、う。  Therefore, in the present invention, the glycoprotein composition refers to a composition comprising glycoprotein molecules having Ν-glycoside-linked sugar chains or ◯ -glycoside-linked sugar chains.
[0025] 糖蛋白質の具体的な例としては、抗体、エリスロポイエチン、トロンボポイエチン、組 織型プラスミノーゲンァクチベータ、プロゥロキナーゼ、トロンボモジュリン、アンチトロ ンビン III、プロテインお血液凝固因子 VII、血液凝固因子 VIII、血液凝固因子 IX、血 液凝固因子 X、血液凝固因子 XII、性腺刺激ホルモン、甲状腺刺激ホルモン、上皮増 殖因子 (EGF)、肝細胞増殖因子 (HGF)、ケラチノサイト増殖因子、ァクチビン、骨形 成因子、幹細胞因子（SCF)、インターフェロン α、インターフェロン β、インターフエ口 ン 、インターロイキン 2、インターロイキン 6、インターロイキン 10、インターロイキン 1 1、可溶性インターロイキン 4受容体、腫瘍壊死因子ひ、 Dnasel,ガラクトシダーゼ、 ひ ダルコシダーゼ、ダルコセレブロシダーゼなどがあげられる。  [0025] Specific examples of glycoproteins include antibodies, erythropoietin, thrombopoietin, tissue plasminogen activator, prolokinase, thrombomodulin, antithrombin III, protein blood coagulation factor VII, blood coagulation Factor VIII, blood clotting factor IX, blood clotting factor X, blood clotting factor XII, gonadotropin, thyroid stimulating hormone, epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor, activin, bone Form factor, stem cell factor (SCF), interferon α, interferon β, interferon, interleukin 2, interleukin 6, interleukin 10, interleukin 11 1, soluble interleukin 4 receptor, tumor necrosis factor, Dnasel, Galactosidase, Dalcosidase, Dalcocerebrosidase And the like.
[0026] フコース修飾を有しない糖鎖構造を有することで、その生理活性が大幅に上昇する 糖蛋白質のより具体的な例としては、例えば、抗体があげられる。また、抗体組成物 とは、 N-グリコシド結合複合型糖鎖を Fc領域に有する抗体分子からなる組成物をレ、う 。以下に、抗体組成物の製造を例に、本発明の非ヒト動物及びその子孫を用いた糖 蛋白質組成物の製造方法を示す。  [0026] More specific examples of glycoproteins that have a sugar chain structure that does not have fucose modification and that significantly increase their physiological activity include, for example, antibodies. The antibody composition refers to a composition comprising an antibody molecule having an N-glycoside-linked complex sugar chain in the Fc region. In the following, a method for producing a glycoprotein composition using the non-human animal of the present invention and its progeny will be described by taking the production of an antibody composition as an example.
[0027] 抗体組成物としては、外来抗原刺激の結果、免疫反応によって生体内に生産され る蛋白質で、抗原と特異的に結合する活性を有するものであればレ、かなるものでもよ レ、が、動物に抗原を免疫し、免疫動物の脾臓細胞より作製したハイプリドーマ細胞が 分泌する抗体のほか、遺伝子組換え技術により作製された抗体、すなわち、抗体遺 伝子を揷入した抗体発現ベクターを、宿主細胞へ導入することにより取得された抗体 などがあげられる。具体的には、ハイプリドーマが生産する抗体、キメラ抗体、ヒト化抗 体、ヒト抗体などをあげることができる。 [0028] ハイプリドーマは、ヒト以外の哺乳動物に抗原を免疫して取得された B細胞と、マウ ス、ラット等に由来するミエローマ細胞とを細胞融合させて得られる、所望の抗原特異 性を有したモノクローナル抗体を生産する細胞をいう。 [0027] The antibody composition is a protein produced in vivo by an immune reaction as a result of stimulation with a foreign antigen, and may be any protein that has an activity of specifically binding to an antigen. However, in addition to antibodies secreted by hyperprideoma cells prepared from spleen cells of immunized animals, the antibody expression vectors inserted with antibodies produced by genetic recombination technology, ie, antibody genes, are immunized with antigens. And the like, which are obtained by introducing the protein into a host cell. Specific examples include antibodies produced by Hypridoma, chimeric antibodies, humanized antibodies, human antibodies, and the like. [0028] Hypridoma has a desired antigen specificity obtained by cell fusion of B cells obtained by immunizing mammals other than humans with myeloma cells derived from mice, rats and the like. It refers to a cell that produces a monoclonal antibody.
キメラ抗体は、ヒト以外の動物の抗体重鎖可変領域 (以下、可変領域は V領域として HVまたは VHとも称す）および抗体軽鎖可変領域（以下、軽鎖は L鎖として LVまたは V Lとも称す）とヒト抗体の重鎖定常領域 (以下、 CHとも称す）およびヒト抗体の軽鎖定 常領域 (以下、 CLとも称す）とからなる抗体を意味する。ヒト以外の動物としては、マウ ス、ラット、ハムスター、ラビット等、ハイプリドーマを作製することが可能であれば、レヽ かなるものも用いることができる。  Chimeric antibodies are antibody heavy chain variable regions of non-human animals (hereinafter, variable regions are also referred to as HV or VH as V regions) and antibody light chain variable regions (hereinafter, light chains are also referred to as LV or VL as L chains). And a heavy chain constant region of a human antibody (hereinafter also referred to as CH) and a light chain constant region of a human antibody (hereinafter also referred to as CL). As animals other than humans, various animals such as mice, rats, hamsters, rabbits, etc. can be used as long as it is possible to produce high pridomas.
[0029] キメラ抗体は、モノクローナル抗体を生産するハイブリドーマより、 VHおよび VLをコ ードする cDNAを取得し、ヒト抗体 CHおよびヒト抗体 CLをコードする遺伝子を有する 宿主細胞用発現ベクターにそれぞれ揷入してヒト型キメラ抗体発現ベクターを構築し 、宿主細胞へ導入することにより発現させ、製造すること力 Sできる。  [0029] For chimeric antibodies, cDNAs encoding VH and VL are obtained from hybridomas producing monoclonal antibodies, and inserted into expression vectors for host cells having genes encoding human antibody CH and human antibody CL, respectively. Thus, a human chimeric antibody expression vector can be constructed and introduced into a host cell for expression and production.
キメラ抗体の CHとしては、ヒトイムノグロブリン（以下、 hlgと表記する）に属すればい かなるものでもよいが、 hlgGクラスのものが好適であり、更に hlgGクラスに属する hlgGl 、 hIgG2、 hIgG3、 hIgG4といったサブクラスのいずれも用いることができる。また、ヒト型 キメラ抗体の CLとしては、 hlgに属すればいかなるものでもよく、 Kクラスあるいはえク ラスのものを用いることができる。  The CH of the chimeric antibody may be any that belongs to human immunoglobulin (hereinafter referred to as “hlg”), but is preferably of the hlgG class, and more preferably of hlgGl, hIgG2, hIgG3, hIgG4 belonging to the hlgG class. Any of the subclasses can be used. In addition, the CL of the human chimeric antibody may be any as long as it belongs to hlg, and K class or E class can be used.
[0030] ヒトイ匕抗体は、ヒト以外の動物の抗体の VHおよび VLの相補性決定領域（以下、 CD Rと表記する）のアミノ酸配列をヒト抗体の VHおよび VLの適切な位置に移植した抗体 をいう。  [0030] A human rabbit antibody is an antibody in which the amino acid sequence of the VH and VL complementarity determining regions (hereinafter referred to as CDR) of an antibody of a non-human animal is transplanted to an appropriate position of the human antibody VH and VL. Say.
ヒト化抗体は、ヒト以外の動物の抗体の VHおよび VLの CDR配列を任意のヒト抗体 の VHおよび VLの CDR配列に移植した V領域をコードする cDNAを構築し、ヒト抗体の CHおよびヒト抗体の CLをコードする遺伝子を有する宿主細胞用発現ベクターにそれ ぞれ揷入してヒト化抗体発現ベクターを構築し、該発現ベクターを宿主細胞へ導入 することによりヒト化抗体を発現させ、製造することができる。  Humanized antibodies are constructed by constructing cDNA encoding the V region obtained by grafting the VH and VL CDR sequences of non-human animal antibodies to the VH and VL CDR sequences of any human antibody. A humanized antibody expression vector is constructed by inserting each into a host cell expression vector having a gene encoding CL, and the humanized antibody is expressed and produced by introducing the expression vector into the host cell. be able to.
[0031] ヒト化抗体の CHとしては、 hlgに属すればいかなるものでもよレ、が、 hlgGクラスのもの が好適であり、更に hlgGクラスに属する hIgGl、 hIgG2、 hIgG3、 hIgG4といったサブクラ スのいずれも用いることができる。また、ヒトイ匕抗体の CLとしては、 hlgに属すればいか なるものでもよく、 / クラスあるいはえクラスのものを用いることができる。 [0031] The CH of the humanized antibody may be any as long as it belongs to the hlg, but the hlgG class is preferable, and the subclasses such as hIgGl, hIgG2, hIgG3, and hIgG4 belonging to the hlgG class are preferred. Any of these can be used. In addition, the CL of human rabbit antibody may be any as long as it belongs to hlg, and those of / class or e class can be used.
ヒト抗体は、元来、ヒト体内に天然に存在する抗体をいうが、最近の遺伝子工学的、 細胞工学的、発生工学的な技術の進歩により作製されたヒト抗体ファージライブラリ 一ならびにヒト抗体生産トランスジエニック動物あるいはヒト抗体生産トランスジェニッ ク植物から得られる抗体等も含まれる。  A human antibody originally refers to an antibody that naturally exists in the human body. However, a human antibody phage library prepared by recent advances in genetic engineering, cell engineering, and developmental engineering, as well as human antibody production trans Also included are antibodies obtained from dienic animals or human antibody-producing transgenic plants.
[0032] ヒト体内に存在する抗体は、例えば、ヒト末梢血リンパ球を単離し、 EBウィルス等を 感染させ不死化、クローニングすることにより、該抗体を生産するリンパ球を培養でき 、培養物中より該抗体を精製することができる。  [0032] The antibody present in the human body can be cultured, for example, by isolating human peripheral blood lymphocytes, infecting an EB virus or the like, immortalizing, and cloning the lymphocytes that produce the antibody. The antibody can be further purified.
ヒト抗体ファージライブラリ一は、ヒト B細胞から調製した抗体遺伝子をファージ遺伝 子に揷入することにより Fab、一本鎖抗体等の抗体断片をファージ表面に発現させた ライブラリーである。該ライブラリーより、抗原を固定化した基質に対する結合活性を 指標として所望の抗原結合活性を有する抗体断片を発現しているファージを回収す ること力 Sできる。該抗体断片は、更に遺伝子工学的手法により、 2本の完全な H鎖およ び 2本の完全な L鎖からなるヒト抗体分子へも変換することができる。  The human antibody phage library 1 is a library in which antibody fragments such as Fab and single chain antibody are expressed on the phage surface by inserting antibody genes prepared from human B cells into the phage genes. From this library, it is possible to collect phages expressing antibody fragments having a desired antigen-binding activity using as an index the binding activity to the substrate on which the antigen is immobilized. The antibody fragment can be further converted into a human antibody molecule comprising two complete heavy chains and two complete light chains by genetic engineering techniques.
[0033] ヒト抗体生産トランスジエニック非ヒト動物は、ヒト抗体遺伝子が細胞内に組込まれた 動物をいう。具体的には、マウス胚性幹細胞ヘヒト抗体遺伝子を導入し、該胚性幹細 胞を他のマウスの初期胚へ移植後、発生させることによりヒト抗体生産トランスジ工ニ ック動物を作製することができる。また、動物の受精卵にヒト抗体遺伝子を導入し、該 受精卵を発生させることにヒト抗体生産トランスジエニック動物を作製することもできる 。ヒト抗体生産トランスジエニック動物からのヒト抗体の作製方法は、通常のヒト以外の 哺乳動物で行われているハイプリドーマ作製方法によりヒト抗体生産ハイプリドーマを 得、培養することで培養物中にヒト抗体を生産蓄積させることができる。  [0033] A human antibody-producing transgenic non-human animal refers to an animal in which a human antibody gene is incorporated into cells. Specifically, a human antibody-producing transgenic animal is produced by introducing a human antibody gene into a mouse embryonic stem cell, transplanting the embryonic stem cell to an early embryo of another mouse, and generating it. Can do. It is also possible to produce a human antibody-producing transgenic animal by introducing a human antibody gene into a fertilized egg of an animal and generating the fertilized egg. The production method of human antibodies from human antibody-producing transgenic animals is obtained by cultivating and culturing human antibody-producing hyperpridoma by the conventional method of producing hyperidoma in mammals other than humans. Antibodies can be produced and accumulated.
[0034] トランスジヱニック非ヒト動物は、ゥシ、ヒッジ、ャギ、ブタ、ゥマ、マウス、ラット、ニヮト リ、サル又はゥサギ等があげられる。  [0034] Examples of transgenic non-human animals include sushi, hidge, goat, pig, horse, mouse, rat, chicken, monkey, and rabbit.
また、本発明において、抗体が、腫瘍関連抗原を認識する抗体、アレルギーあるい は炎症に関連する抗原を認識する抗体、循環器疾患に関連する抗原を認識する抗 体、自己免疫疾患に関連する抗原を認識する抗体、またはウィルスあるいは細菌感 染に関連する抗原を認識する抗体であることが好ましぐ抗体のクラスが IgGのヒト抗 体が好ましい。 In the present invention, the antibody is an antibody recognizing a tumor-related antigen, an antibody recognizing an antigen related to allergy or inflammation, an antibody recognizing an antigen related to cardiovascular disease, or an autoimmune disease. Antibody that recognizes antigen, or virus or bacterial sensation A human antibody whose antibody class is preferably an IgG that recognizes an antigen associated with a stain is preferred.
[0035] 抗体断片とは、上記抗体の少なくとも Fc領域の一部を含んだ断片をいう。 Fc領域と は、抗体の H鎖の C末端側の領域、 CH2領域および CH3領域を意味し、天然型およ びその変異型を包含する。少なくとも Fc領域の一部とは、好ましくは CH2領域を含む 断片、より好ましくは CH2領域内に存在する 1番目のァスパラギン酸を含む領域をい う。 IgGクラスの Fc領域は、力バット（Kabat)らの EU Index [シーケンシズ'ォブ'プロティ ンズ 'オフ ' ムノロンカノレ'インタレス！ (Sequences of Proteins of Immunological Inter est) , o Ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1 991)]のナンバリングで 226番目のシスティンから C末端、あるいは 230番目のプロリン 力 C末端までを意味する。抗体の断片としては、具体的には、 H鎖の単量体、 H鎖 の 2量体などがあげられる。  [0035] The antibody fragment refers to a fragment containing at least a part of the Fc region of the antibody. The Fc region means a region on the C-terminal side of the H chain of an antibody, a CH2 region and a CH3 region, and includes a natural type and a mutant type thereof. At least a part of the Fc region preferably means a fragment containing the CH2 region, more preferably a region containing the first aspartic acid present in the CH2 region. The Fc region of the IgG class is the EU Index [Sequences 'Ob' Proteins 'Off' Munoron Kanole'interest of Kabat et al. (Sequences of Proteins of Immunological Interest), o Ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1 991)] numbering from 226th cysteine to C-terminal or 230th proline force C It means to the end. Specific examples of antibody fragments include H chain monomers and H chain dimers.
[0036] Fc領域を有する融合蛋白質としては、抗体の Fc領域を含んだ抗体あるいは抗体の 断片と、酵素、サイト力インなどの蛋白質とを融合させた物質であればレ、かなるもので あよい。  [0036] A fusion protein having an Fc region is a substance obtained by fusing an antibody or antibody fragment containing an Fc region of an antibody with a protein such as an enzyme or a cytodynamic force. Good.
腫瘍関連抗原を認識する抗体としては、抗 GD2抗体 (Anticancer Res., 13, 331, 19 93)、抗 GD3抗体（Cancer Immunol. Immunother" 36, 260, 1993)、抗 GM2抗体（Can cer Res., 54, 1511, 1994)、抗 HER2抗体（Proc. Natl. Acad. Sci. USA, 89, 4285, 199 2)、抗 CD52抗体（Nature, 332, 323, 1988)、抗 MAGE抗体（British J. Cancer, 83, 49 3， 2000)、抗 HM1.24抗体（Molecular Immunol., 36, 387, 1999)、抗副甲状腺ホルモ ン関連蛋白（PTHrP)抗体（Cancer, 88, 2909， 2000)、抗 FGF8抗体（Proc. Natl. Aca d. Sci. USA, 86, 9911， 1989)抗塩基性繊維芽細胞増殖因子抗体、抗 FGF8受容体 抗体 (J. Biol. Chem., 265, 16455， 1990)、抗塩基性繊維芽細胞増殖因子受容体抗 体、抗インスリン様増殖因子抗体（J. Neurosci. Res. , 40, 647， 1995)、抗インスリン様 増殖因子受容体抗体 (J. Neurosci. Res., 40, 647, 1995)、抗 PMSA抗体（J. Urology, 160. 2396, 1998)、抗血管内皮細胞増殖因子抗体（Cancer Res. , 57, 4593, 1997)ま たは抗血管内皮細胞増殖因子受容体抗体（Oncogene, 19, 2138, 2000)、抗 CA125 抗体、抗 17-1A抗体、抗インテグリンひ v /3 3抗体、抗 CD33抗体、抗 CD22抗体、抗 HL A抗体、抗 HLA-DR抗体、抗 CD20抗体、抗 CD 19抗体、抗 EGF受容体抗体（Immunol ogy Today, 21, 403, 2000)、抗 CD 10抗体（American Journal of Clinical Pathology, 1 13, 374, 2000)などがあげられる。 Examples of antibodies that recognize tumor-associated antigens include anti-GD2 antibodies (Anticancer Res., 13, 331, 1993), anti-GD3 antibodies (Cancer Immunol. Immunother "36, 260, 1993), and anti-GM2 antibodies (Cancer Res. , 54, 1511, 1994), anti-HER2 antibody (Proc. Natl. Acad. Sci. USA, 89, 4285, 199 2), anti-CD52 antibody (Nature, 332, 323, 1988), anti-MAGE antibody (British J. Cancer, 83, 49 3, 2000), anti-HM1.24 antibody (Molecular Immunol., 36, 387, 1999), anti-parathyroid hormone related protein (PTHrP) antibody (Cancer, 88, 2909, 2000), anti-FGF8 Antibody (Proc. Natl. Aca d. Sci. USA, 86, 9911, 1989) Anti-basic fibroblast growth factor antibody, anti-FGF8 receptor antibody (J. Biol. Chem., 265, 16455, 1990), anti Basic fibroblast growth factor receptor antibody, anti-insulin-like growth factor antibody (J. Neurosci. Res., 40, 647, 1995), anti-insulin-like growth factor receptor antibody (J. Neurosci. Res., 40 , 647, 1995), anti-PMSA antibody (J. Urology, 160. 2396, 1998), anti-vascular endothelial growth factor Antibody (Cancer Res., 57, 4593, 1997) or anti-vascular endothelial growth factor receptor antibody (Oncogene, 19, 2138, 2000), anti-CA125 antibody, anti-17-1A antibody, anti-integrin antibody v / 3 3 antibodies, anti-CD33 antibody, anti-CD22 antibody, anti-HL A antibody, anti-HLA-DR antibody, anti-CD20 antibody, anti-CD 19 antibody, anti-EGF receptor antibody (Immunol ogy Today, 21, 403, 2000), anti-CD 10 antibody (American Journal of Clinical Pathology, 1 13, 374 , 2000).
[0037] アレルギーあるいは炎症に関連する抗原を認識する抗体としては、抗インターロイ キン 6抗体（Immunol. Rev. , 127, 5， 1992)、抗インターロイキン 6受容体抗体（Molecul ar Immunol, 31, 371， 1994)、抗インターロイキン 5抗体（Immunol. Rev., 127, 5， 1992 )、抗インターロイキン 5受容体抗体、抗インターロイキン 4抗体（Cytokine, 3, 562, 19 91)、抗インターロイキン 4受容体抗体 (J. Immunol. Meth., 217, 41， 1998)、抗腫瘍 壊死因子抗体（Hybridoma, 13, 183， 1994)、抗腫瘍壊死因子受容体抗体（Molecula r Pharmacol., 58, 237, 2000)、抗 CCR4抗体（Nature, 400, 776, 1999)、抗ケモカイン 抗体（J. Immunol. Meth., 174, 249, 1994)、抗ケモカイン受容体抗体（J. Exp. Med., 186. 1373, 1997)、抗 IgE抗体、抗 CD23抗体、抗 CD 11a抗体（Immunology Today, 21, 403, 2000)、抗 CRTH2抗体 (J. Immunol., 162, 1278, 1999)、抗 CCR8抗体 (W099/ 25734)、抗 CCR3抗体（US6207155)などがあげられる。  [0037] Antibodies that recognize antigens related to allergy or inflammation include anti-interleukin 6 antibody (Immunol. Rev., 127, 5, 1992), anti-interleukin 6 receptor antibody (Molecul ar Immunol, 31, 371, 1994), anti-interleukin 5 antibody (Immunol. Rev., 127, 5, 1992), anti-interleukin 5 receptor antibody, anti-interleukin 4 antibody (Cytokine, 3, 562, 19 91), anti-interleukin 4 receptor antibody (J. Immunol. Meth., 217, 41, 1998), anti-tumor necrosis factor antibody (Hybridoma, 13, 183, 1994), anti-tumor necrosis factor receptor antibody (Molecula r Pharmacol., 58, 237) , 2000), anti-CCR4 antibody (Nature, 400, 776, 1999), anti-chemokine antibody (J. Immunol. Meth., 174, 249, 1994), anti-chemokine receptor antibody (J. Exp. Med., 186. 1373, 1997), anti-IgE antibody, anti-CD23 antibody, anti-CD 11a antibody (Immunology Today, 21, 403, 2000), anti-CRTH2 antibody (J. Immunol., 162, 1278, 1999), anti-CCR8 antibody (W099 / 2 5734), anti-CCR3 antibody (US6207155) and the like.
[0038] 循環器疾患に関連する抗原を認識する抗体としては、抗 GpIIb/IIIa抗体 (J. Immuno 1.， 152, 2968, 1994)、抗血小板由来増殖因子抗体（Science, 253, 1129， 1991)、抗 血小板由来増殖因子受容体抗体 (J. Biol. Chem., 272, 17400, 1997)または抗血液 凝固因子抗体（Circulation, 101, 1158, 2000)などがあげられる。  [0038] Anti-GpIIb / IIIa antibody (J. Immuno 1., 152, 2968, 1994), antiplatelet-derived growth factor antibody (Science, 253, 1129, 1991) are recognized as antibodies that recognize antigens related to cardiovascular diseases. ), Anti-platelet-derived growth factor receptor antibody (J. Biol. Chem., 272, 17400, 1997) or anti-blood clotting factor antibody (Circulation, 101, 1158, 2000).
自己免疫疾患 (具体的な例としては、乾癬、関節リウマチ、クローン病、潰瘍性大腸 炎、全身性エリテマトーデス、多発性硬化症など）に関連する抗原を認識する抗体と しては、抗自己 DNA抗体（Immunol. Letters, 72, 61， 2000)、抗 CDl la抗体、抗 ICAM 3抗体、抗 CD80抗体、抗 CD2抗体、抗 CD3抗体、抗 CD4抗体、抗インテグリンひ 4 j3 7 抗体、抗 CD40L抗体、抗 IL-2受容体抗体（Immunology Today, 21, 403, 2000)などが あげられる。  Antibodies that recognize antigens associated with autoimmune diseases (specific examples include psoriasis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, multiple sclerosis) include anti-self DNA Antibody (Immunol. Letters, 72, 61, 2000), anti-CDlla antibody, anti-ICAM 3 antibody, anti-CD80 antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-integrin 4 j3 7 antibody, anti-CD40L antibody And anti-IL-2 receptor antibody (Immunology Today, 21, 403, 2000).
[0039] ウィルスあるいは細菌感染に関連する抗原を認識する抗体としては、抗 gpl20抗体  [0039] Examples of antibodies that recognize antigens associated with viral or bacterial infection include anti-gpl20 antibodies
(Structure, 8, 385, 2000)、抗 CD4抗体（J. Rheumatology, 25, 2065, 1998)、抗 CCR 4抗体、抗ベロ毒素抗体（J. Clin. Microbiol., 37, 396, 1999)などがあげられる。  (Structure, 8, 385, 2000), anti-CD4 antibody (J. Rheumatology, 25, 2065, 1998), anti-CCR 4 antibody, anti-verotoxin antibody (J. Clin. Microbiol., 37, 396, 1999), etc. can give.
以下、本発明のひ -1,6-フコシルトランスフェラーゼ変異体とその利用について詳細 に説明する。 Hereinafter, the -1,6-fucosyltransferase mutant of the present invention and its use are described in detail. Explained.
1.本発明の α -1,6-フコシルトランスフェラーゼ変異体の DNAの調製  1. Preparation of α-1,6-fucosyltransferase mutant DNA of the present invention
本発明の α -1,6-フコシルトランスフェラーゼ変異体は、 α -1,6-フコース修飾酵素 の活性が欠失、または低下するような疾患を有するヒト組織由来の mRNAを単離し、 その cDNAライブラリーを作製し、次いで該 cDNAライブラリーをスクリーニングして 目的のクローンを得ることにより調製することができる。  The α-1,6-fucosyltransferase mutant of the present invention isolates mRNA derived from human tissue having a disease in which the activity of α-1,6-fucose modifying enzyme is deleted or decreased, Can be prepared by preparing a library and then screening the cDNA library to obtain the desired clone.
[0040] また、 ひ- 1，6-フコシルトランスフェラーゼの活性が欠失、または低下するような変異 が生じることによって、 N -グリコシド結合複合型糖鎖還元末端の N -ァセチルダノレコサ ミンの 6位とフコースの 1位がひ結合した糖鎖構造を認識するレクチンに耐性となった 培養細胞株由来の mRNAを用いることにより、本発明のひ -1,6 -フコシルトランスフエ ラーゼ変異体を調製することができる。  [0040] In addition, mutations that delete or reduce the activity of -1,6-fucosyltransferase result in the occurrence of N-acetylidanorecosamine at the reducing end of the N-glycoside-linked complex sugar chain. By using mRNA derived from a cultured cell line that has become resistant to a lectin that recognizes a sugar chain structure in which 6-position and 1-position of fucose are linked, the -1,6-fucosyltransferase mutant of the present invention can be isolated. Can be prepared.
[0041] レクチンに耐性な細胞株とは、レクチンを有効濃度与えたときにも、生育が阻害され ない細胞株をいう。有効濃度とは、上述の、 α -1,6-フコシルトランスフェラーゼの活 性が欠失、または低下するような変異が生じる以前の細胞株 (以下、「親株」と称す） が正常に生育できない濃度以上であり、好ましくは、親株が生育できない濃度と同濃 度、より好ましくは 2〜5倍、さらに好ましくは 10倍、最も好ましくは 20倍以上である。  [0041] A cell line resistant to lectin refers to a cell line in which growth is not inhibited even when an effective concentration of lectin is given. The effective concentration refers to the concentration at which the above-mentioned cell line (hereinafter referred to as “parent strain”) cannot grow normally before the mutation that causes loss or decrease in the activity of α-1,6-fucosyltransferase. Preferably, the concentration is the same as the concentration at which the parent strain cannot grow, more preferably 2 to 5 times, still more preferably 10 times, and most preferably 20 times or more.
[0042] 本発明において、生育が阻害されないレクチンの有効濃度は、親株に応じて適宜 定めればよいが、通常 10 i g/ml〜10mg/ml、好ましくは 0.5mg/ml〜2.0mg/mlである。  [0042] In the present invention, the effective concentration of a lectin whose growth is not inhibited may be appropriately determined according to the parent strain, but is usually 10 ig / ml to 10 mg / ml, preferably 0.5 mg / ml to 2.0 mg / ml. is there.
N-グリコシド結合糖鎖還元末端の N-ァセチルダルコサミンの 6位とフコースの 1位が a結合した糖鎖構造を認識するレクチンとしては、該糖鎖構造を認識できるレクチン であれば、いずれのレクチンでも用いることができる。その具体的な例としては、レン ズマメレクチン LCA (Lens culinaris由夹の lentil agglutinin)エンドゥマメレクチン PSA (Pisum sativum由来の nea lectin) ,ソラマメレクチン VFA (Vicia faba由来の agglutinin )、ヒィロチャワンタケレクチン AAL (Aleuria aurantia由来の lectin)などがあげられる n Any lectin that recognizes the sugar chain structure can be recognized as a lectin that recognizes the sugar chain structure in which the 6-position of N-glycidylcolacamine at the N-glycoside-linked sugar chain reducing end and the 1-position of fucose are a-linked. The lectin can also be used. Specific examples of this include: Lentil lectin LCA (Lens culinaris lentil agglutinin) Endumame lectin PSA (Pisum sativum-derived nea lectin), Broad bean lectin VFA (Vicia faba-derived agglutinin), Hirochawantake lectin AAL (Aleu n that from aurantia lectin), and the like
[0043] ヒト組織由来あるいは培養細胞株由来の mRNAは、以下のごとぐヒト組織あるい は培養細胞株から全 RNAを調製し、該全 RNA力 mRNAを単離することで調製す ること力 Sできる。  [0043] mRNA derived from human tissue or cultured cell line can be prepared by preparing total RNA from human tissue or cultured cell line as follows, and isolating the total RNA mRNA. S can.
ヒト組織あるいは培養細胞株から全 RNAを調製する方法としては、チォシアン酸グ ァニジン一トリフルォロ酢酸セシウム法 [Methods in Enzymology, 154, 3 (1987)〕、酸 性チオシアン酸グァニジン'フエノール 'クロ口ホルム（AGPC)法 [Analytical Biochem istry, 162, 156 (1987)、実験医学， 9, 1937 (1991)〕などがあげられる。全 RNAから po ly (A) + RNAとして mRNAを調製する方法としては、オリゴ（dT)固定化セルロース力 ラム法（モレキュラー 'クローニング第 2版）等があげられる。あるいは、 Fast Track mR NA Isolation Kit (Invitrogen社製)、 Quick Prep mRNA Purification Kit (Pharmacia社 製）などのキットを用いることにより mRNAを調製することができる。 A method for preparing total RNA from human tissues or cultured cell lines is thiocyanate group. The cesium acetate trifluoroacetate method [Methods in Enzymology, 154, 3 (1987)], the guanidine thiocyanate 'Phenol' chloroform (AGPC) method [Analytical Biochem istry, 162, 156 (1987), Experimental Medicine, 9 , 1937 (1991)]. Examples of methods for preparing mRNA from total RNA as poly (A) + RNA include oligo (dT) -immobilized cellulose strength method (Molecular 'Cloning 2nd Edition). Alternatively, mRNA can be prepared by using a kit such as Fast Track mRNA Isolation Kit (Invitrogen) or Quick Prep mRNA Purification Kit (Pharmacia).
[0044] 次に、調製したヒト組織あるいは培養細胞株 mRNAから cDNAライブラリーを作製 する。 [0044] Next, a cDNA library is prepared from the prepared human tissue or cultured cell line mRNA.
cDNAライブラリ一作製法としては、モレキュラー 'クローニング第 2版、カレント'プ ロトコールズ.イン.モレキュラー.バイオロジー等に記載された方法、あるいは市販の キット、例えは Superscript Plasmid system for cDNA Synthesis and Plasmid Cloning ( Life Technologies社製）、 ZAP-cDNA Synthesis Kit (STRATAGENE社製）を用いる方 法などがあげられる。  One method for preparing a cDNA library is the method described in Molecular 'Cloning 2nd Edition, Current' Protocols in Molecular Biology, etc., or a commercially available kit, such as the Superscript Plasmid system for cDNA Synthesis and Plasmid Cloning ( Life Technologies) and ZAP-cDNA Synthesis Kit (STRATAGENE).
[0045] cDNAライブラリーを作製するためのクローニングベクターとしては、大腸菌 K12株 中で自立複製できるものであれば、ファージベクター、プラスミドベクター等いずれで も使用できる。具体的には、 ZAP Express [STRATAGENE社、 Strategies, 5, 58 (1992 )〕、 Bluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)〕、 Lambda ZAP II ( STRATAGENE社製）、 え gtl0、 λ gtl l〔DNA cloning, A Practical Approach,丄， 49 (1 985)〕、 λ TriplEx (Clontech社製）、 λ ExCell (Pharmacia社製）、 pT7T318U (Pharmaci a社製）、 pcD2〔Mol. Cell. Biol., 3, 280 (1983)〕および pUC18〔Gene, 33, 103 (1985)〕 等をあげることができる。  [0045] As a cloning vector for preparing a cDNA library, any phage vector or plasmid vector can be used as long as it can autonomously replicate in Escherichia coli K12. Specifically, ZAP Express [STRATAGENE, Strategies, 5, 58 (1992)], Bluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)], Lambda ZAP II (manufactured by STRATAGENE), gtl0, λ gtl l (DNA cloning, A Practical Approach, 丄, 49 (1985)), λ TriplEx (Clontech), λ ExCell (Pharmacia), pT7T318U (Pharmacia), pcD2 (Mol. Cell. Biol., 3, 280 (1983)] and pUC18 [Gene, 33, 103 (1985)].
[0046] 宿主微生物としては、エシュリヒア属（Escherichia)に属する微生物、特にエシュリヒ ァ'コリ（Escherichiacoli、以下、「大腸菌」ともいう）に属する微生物であればいずれで も用レヽること力 Sできる。具体的には、 EscherichiacoliXLl-Blue MRF' [STRATAGENE 社、 Strategies, 5, 81 (1992)]、 Escherichia coliC600 [Genetics, 39, 440 (1954)]、 Esc herichia coliY1088 [Science, 222, 778 (1983)〕、 Escherichia coli Y1090 [Science, 222, 778 (1983)〕、 Escherichia coli NM522 [J. Mol. Biol, 166, 1 (1983)〕、 Escherichia coli K802 [J. Mol. Biol., 16, 118 (1966)1および Escherichiacoli JM105 [Gene, 38, 275 (19 85)〕等が用いられる。 [0046] As the host microorganism, any microorganism belonging to the genus Escherichia, particularly a microorganism belonging to Escherichia coli (hereinafter also referred to as "Escherichia coli") can be used. Specifically, EscherichiacoliXLl-Blue MRF '[STRATAGENE, Strategies, 5, 81 (1992)], Escherichia coli C600 [Genetics, 39, 440 (1954)], Esc herichia coli Y1088 [Science, 222, 778 (1983)] Escherichia coli Y1090 [Science, 222, 778 (1983)], Escherichia coli NM522 [J. Mol. Biol, 166, 1 (1983)], Escherichia coli K802 [J. Mol. Biol., 16, 118 (1966) 1 and Escherichiacoli JM105 [Gene, 38, 275 (1985)] etc. are used.
[0047] この cDNAライブラリーを、そのまま以下の解析に用いてもょレ、が、不完全長 cDN Aの割合を下げ、完全長 cDNAをできるだけ効率よく取得するために、菅野らが開発 したオリゴキャップ法〔Gene， 138, 171 (1994)、 Gene, 200, 149 (1997)、蛋白質核酸酵 素， 41, 603 (1996)、実験医学， 11, 2491 (1993)、 cDNAクローニング,羊土社 (1996) 、遺伝子ライブラリーの作製法，羊土社（1994)〕を用いて調製した cDNAライブラリー を以下の解析に用いてもょレ、。  [0047] This cDNA library can be used for the following analysis as it is, but the oligo-developed by Kanno et al. Has been developed in order to reduce the proportion of incomplete length cDNA and 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, 11, 2491 (1993), cDNA cloning, Yodosha ( 1996), cDNA library prepared using the method of gene library, Yodosha (1994)], can be used for the following analysis.
[0048] 作製した cDNAライブラリーから各クローンを単離し、それぞれのクローンについて cDNAの塩基配列を末端から、通常用いられる塩基配列解析方法、例えばサンガー (Sanger)らのジデォキシ法〔Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)〕あるいは A BIPRISM377DNAシークェンサ一（PE Biosystems社製）等の塩基配列分析装置 を用いて分析することにより、該 DNAの塩基配列を決定する。  [0048] Each clone is isolated from the prepared cDNA library, and the cDNA base sequence of each clone is isolated from the end, and a commonly used base sequence analysis method, for example, Sanger et al.'S dideoxy method [Proc. Natl. Acad Sci. USA, 74, 5463 (1977)] or A BIPRISM377 DNA sequencer (manufactured by PE Biosystems) or the like, and then analyzing the base sequence of the DNA.
[0049] それぞれの cDNAの塩基配列が、 α -1,6-フコシルトランスフェラーゼのアミノ酸改 変変異酵素をコードする塩基配列を有しているかどうかは、 BLAST等の相同性検索 プログラムを用いて、 GenBank, EMBLおよび DDBJなどの塩基配列データベース を検索することにより、データベース中の既存の遺伝子の塩基配列との相同性を調 ベることにより確言忍できる。  [0049] Whether each cDNA has a nucleotide sequence encoding an amino acid-mutating enzyme of α-1,6-fucosyltransferase is determined using a homology search program such as BLAST using GenBank. By searching base sequence databases such as EMBL and DDBJ, it is possible to confirm by homology with the base sequences of existing genes in the database.
[0050] 上記の方法で得られる、アミノ酸改変変異酵素をコードする塩基配列を含む cDNA の塩基配列として、例えば、配列番号 18、 19、 20、 21または 22で表される塩基配列が あげられる。  [0050] Examples of the nucleotide sequence of cDNA containing the nucleotide sequence encoding the amino acid-modified mutant enzyme obtained by the above method include the nucleotide sequence represented by SEQ ID NO: 18, 19, 20, 21, or 22.
配列番号 18、 19、 20、 21または 22で表される塩基配列を含む分子力 ScDNAライブラ リー作製の際に人工的に生じたものではないことを確認するためには、 cDNAライブ ラリー作製に用いたヒト組織あるいは培養細胞株のゲノムライブラリーを、配列番号 18 、 19、 20、 21または 22で表される塩基配列に特異的な配列を用いてスクリーニングし 、得られたゲノムクローンの塩基配列を決定することで判断することができる。  In order to confirm that the molecular force ScDNA library containing the nucleotide sequence represented by SEQ ID NO: 18, 19, 20, 21, or 22 was not artificially generated during preparation of the cDNA library, it was used for preparation of the cDNA library. The genomic library of human tissue or cultured cell lines was screened using a sequence specific to the nucleotide sequence represented by SEQ ID NO: 18, 19, 20, 21 or 22, and the nucleotide sequence of the resulting genomic clone was determined. It can be judged by deciding.
[0051] ゲノムライブラリ一は、ヒト組織あるいは培養細胞株から、モレキュラー 'クローニング 第 3版やカレント'プロトコールズ 'イン'モレキュラー 'バイオロジー等に記載された自 体公知の方法を用いて調製することができる。また、ゲノム DNAライブラリースクリー ニングシステム（Genome Systems社）や Universal GenomeWalker™ Kits (CLONTEC H社)などを用いることにより調製することもできる。 [0051] Genomic libraries are available from human tissues or cultured cell lines as described in Molecular 'Cloning 3rd Edition' and Current 'Protocols'In'Molecular' Biology. The body can be prepared using a known method. It can also be prepared by using a genomic DNA library screening system (Genome Systems) or Universal GenomeWalker ™ Kits (CLONTEC H).
[0052] ゲノムライブラリーを、配列番号 18、 19、 20、 21または 22で表される塩基配列に特異 的な配列を用いてスクリーニングする方法としては、配列番号 18、 19、 20、 21または 22 で表される塩基配列に特異的なプライマーを用いた PCR法〔PCR Protocols, Acade mic Press (1990)〕や、配列番号 18、 19、 20、 21または 22で表される塩基配列に特異 的なオリゴヌクレオチドを用いたコロニーハイブリダィゼーシヨンやプラークハイブリダ ィゼーシヨン法 (モレキュラー 'クローニング第 3版)などがあげられる。  [0052] As a method for screening a genomic library using a sequence specific to the nucleotide sequence represented by SEQ ID NO: 18, 19, 20, 21, or 22, SEQ ID NO: 18, 19, 20, 21, or 22 is used. PCR method using primers specific to the nucleotide sequence represented by (PCR Protocols, Academic Press (1990)), or specific to the nucleotide sequence represented by SEQ ID NO: 18, 19, 20, 21 or 22. Examples include colony hybridization using oligonucleotides and plaque hybridization method (Molecular 'Cloning 3rd Edition).
[0053] 上記の方法で配列番号 18、 19、 20、 21または 22で表される塩基配列を含むゲノム D NAクローンが得られる。このゲノム DNAの塩基配列を決定し配列番号 18、 19、 20、 2 1または 22で表される塩基配列と一致することが確認できれば、該配列は cDNAライ ブラリー作製の際に人工的に生じたものではないことが分かる。  [0053] A genomic DNA clone containing the base sequence represented by SEQ ID NO: 18, 19, 20, 21 or 22 is obtained by the above method. If the base sequence of this genomic DNA was determined and confirmed to match the base sequence represented by SEQ ID NO: 18, 19, 20, 21 or 22, the sequence was generated artificially when the cDNA library was prepared. It turns out that it is not a thing.
配列番号 18、 19、 20、 21または 22で表される塩基配列からなる DNAが一旦取得さ れ、その塩基配列が決定された後は、該塩基配列の 5'端および 3'端の塩基配列に 基づいたプライマーを調製し、ヒト組織あるいは培養細胞株を用いて作製した cDNA ライブラリーを铸型として、 PCR法〔PCR Protocols, Academic Press (1990)〕を用いて DNAの増幅を行うことにより、本発明の α -1,6-フコシルトランスフェラーゼ変異体の DNAを取得することができる。  After DNA having the base sequence represented by SEQ ID NO: 18, 19, 20, 21, or 22 is once obtained and its base sequence has been determined, the base sequences at the 5 ′ end and 3 ′ end of the base sequence By preparing a primer based on the above and using a PCR library [PCR Protocols, Academic Press (1990)] using a cDNA library prepared using human tissue or a cultured cell line as a saddle, The DNA of the α-1,6-fucosyltransferase mutant of the present invention can be obtained.
[0054] また、配列番号 18、 19、 20、 21または 22で表される塩基配列からなる DNAの全長あ るいは一部をプローブとして、ヒト組織あるいは培養細胞株を用いて作製した cDNA ライブラリーに対してコロニーハイブリダィゼーシヨンやプラークハイブリダィゼーショ ン（モレキユラ一'クローニング第 3版）を行うことにより、本発明のひ- 1，6-フコシルトラ ンスフエラーゼ変異体の DNAを取得することができる。  [0054] In addition, a cDNA library prepared using human tissue or a cultured cell line using the full length or a part of the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 18, 19, 20, 21, or 22 as a probe. To obtain the DNA of the mutant of 1,6-fucosyl transferase of the present invention by performing colony hybridization or plaque hybridization (Molecular 1 'Cloning 3rd edition) on Can do.
[0055] 決定された DNAの塩基配列に基づいて、フォスフォアミダイト法を利用したパーキ ン.エルマ一社の DNA合成機 model 392等の DNA合成機で化学合成することにより 、本発明のひ -1,6 -フコシルトランスフェラーゼ変異体の DNAを取得することもできる 取得した DNAについて、該 DNAを含む組換えベクターを宿主細胞に導入して得 られる形質転換体を用いて蛋白質を発現させることにより、該 DNAが α -1,6-フコー ス修飾酵素の活性が欠失、または低下するようにアミノ酸改変された α -1,6-フコシノレ トランスフェラーゼ変異体をコードする DNAであることを確認することができる。 [0055] Based on the determined DNA base sequence, Parkin using the phosphoramidite method. By chemically synthesizing with a DNA synthesizer such as Elma's DNA synthesizer model 392, the It is also possible to obtain DNA for 1,6-fucosyltransferase mutants With respect to the obtained DNA, by expressing a protein using a transformant obtained by introducing a recombinant vector containing the DNA into a host cell, the DNA has the activity of an α-1,6-fusose modifying enzyme. It can be confirmed that the DNA encodes an α-1,6-fucosinole transferase mutant that has been amino acid modified so as to be deleted or reduced.
[0056] 配列番号 18、 19、 20、 21または 22で表される塩基配列またはその断片の塩基配列 に関する情報に基づき、常法または DNA合成機を用いることにより、本発明のひ -1, 6-フコシルトランスフェラーゼ変異体の DNAの塩基配列、例えば配列番号 18、 19、 2 0、 21または 22表される塩基配列のうち、連続した 5〜60塩基、好ましくは 10〜40塩 基に相当する配列を有するオリゴヌクレオチドまたは該オリゴヌクレオチドと相補的な 配列に相当するオリゴヌクレオチド（以下、アンチセンス ·ォリゴヌクレオチドという）を 調製すること力 Sきる。 [0056] Based on the information on the base sequence represented by SEQ ID NO: 18, 19, 20, 21 or 22, or the base sequence of the fragment thereof, by using a conventional method or a DNA synthesizer, -1, 6 -Fucosyltransferase mutant DNA base sequence, for example, a sequence corresponding to a continuous 5 to 60 bases, preferably 10 to 40 bases of the base sequence represented by SEQ ID NO: 18, 19, 20, 21, or 22 Or an oligonucleotide corresponding to a sequence complementary to the oligonucleotide (hereinafter referred to as an antisense oligonucleotide).
[0057] これらオリゴヌクレオチドは、本発明のひ -1,6 -フコシルトランスフェラーゼ変異体を 検出するために用いることができる。特に、本発明の α _1，6-フコシルトランスフェラー ゼ変異体と α -1,6-フコシルトランスフェラーゼとを識別することが可能なオリゴヌタレ ォチドは本発明の α -1,6-フコシルトランスフェラーゼ変異体の診断法において有用 である。 [0057] These oligonucleotides can be used for detecting the -1,6-fucosyltransferase mutant of the present invention. In particular, Origonutare Ochido capable of identifying and alpha _1,6- fucosyl transferase peptidase variants and alpha-1,6-fucosyltransferase of the present invention is the diagnosis of alpha-1,6-fucosyltransferase variants of the invention Useful in law.
[0058] オリゴヌクレオチドとしては、オリゴ DNA、オリゴ RNA等のオリゴヌクレオチド、およ び該オリゴヌクレオチドの誘導体（以下、オリゴヌクレオチド誘導体とレ、う）等があげら れる。  [0058] Examples of the oligonucleotide include oligonucleotides such as oligo DNA and oligo RNA, and derivatives of the oligonucleotide (hereinafter referred to as oligonucleotide derivatives).
該オリゴヌクレオチドまたはアンチセンス ·ォリゴヌクレオチドとして、例えば、検出し たレ、mRNAの一部の塩基配列にぉレ、て、 5'末端側の塩基配列に相当するセンスプ ライマー、 3'末端側の塩基配列に相当するアンチセンスプライマー等をあげることが できる。ただし、 mRNAにおいてゥラシルに相当する塩基は、オリゴヌクレオチドプラ イマ一においてはチミジンとなる。  Examples of the oligonucleotide or antisense oligonucleotide include, for example, a detected primer, a sense primer corresponding to the base sequence of the 5 ′ end, and a sense primer corresponding to the base sequence of the 5 ′ end. Examples include antisense primers corresponding to the base sequence. However, the base corresponding to uracil in mRNA is thymidine in the oligonucleotide primer.
[0059] センスプライマーおよびアンチセンスプライマーとしては、両者の融解温度（Tm)お よび塩基数が極端に変わることのないオリゴヌクレオチドで、 5〜60塩基、好ましくは 10〜50塩基数のものがあげられる。 [0059] The sense primer and the antisense primer are oligonucleotides whose melting temperature (Tm) and the number of bases do not change drastically and are 5 to 60 bases, preferably 10 to 50 bases. It is done.
オリゴヌクレオチド誘導体としては、オリゴヌクレオチド中のリン酸ジエステル結合が ホスフォロチォエート結合に変換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド 中のリン酸ジエステル結合が N3'— P5'ホスフォアミデート結合に変換されたオリゴヌ クレオチド誘導体、オリゴヌクレオチド中のリボースとリン酸ジエステル結合がペプチド 核酸結合に変換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド中のゥラシルが C_ 5プロピニルゥラシルで置換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド 中のゥラシルが C_ 5チアゾールゥラシルで置換されたオリゴヌクレオチド誘導体、ォ リゴヌクレオチド中のシトシンが C— 5プロピニルシトシンで置換されたオリゴヌクレオ チド誘導体、オリゴヌクレオチド中のシトシンがフエノキサジン修飾シトシン（phenoxazi ne-modified cytosine)で置換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド中 のリボースが 2， _〇一プロピルリボースで置換されたオリゴヌクレオチド誘導体、ある いはオリゴヌクレオチド中のリボースが 2，一メトキシエトキシリボースで置換されたオリ ゴヌクレオチド誘導体等があげられる〔細胞工学， 1^, 1463 (1997)〕。 Oligonucleotide derivatives include phosphodiester bonds in oligonucleotides. Oligonucleotide derivatives converted to phosphorothioate bonds, oligonucleotide derivatives in which phosphodiester bonds in oligonucleotides are converted to N3'-P5 'phosphoramidate bonds, ribose and phosphodiester bonds in oligonucleotides Is an oligonucleotide derivative in which uracil in the oligonucleotide is substituted with C_5 propynyluracil, an oligonucleotide derivative in which uracil in the oligonucleotide is substituted with C_5 thiazoleuracil, Oligonucleotide derivative in which cytosine in oligonucleotide is substituted with C-5 propynylcytosine, and cytosine in oligonucleotide is substituted with phenoxazine-modified cytosine Tide derivatives, oligonucleotide derivatives in which the ribose in the oligonucleotide is substituted with 2, _0-propylpropylose, or oligonucleotide derivatives in which the ribose in the oligonucleotide is substituted with 2,1 methoxyethoxyribose [Cell engineering, 1 ^, 1463 (1997)].
2.本発明の α -1,6-フコシルトランスフェラーゼ変異体の調製  2. Preparation of α-1,6-fucosyltransferase mutant of the present invention
(1) a -1,6-フコシルトランスフェラーゼ変異体の製造  (1) Production of a-1,6-fucosyltransferase mutant
本発明の α -1,6-フコシルトランスフェラーゼ変異体は、モレキュラ^ ~ ·クローニング 第 3版やカレント ·プロトコールズ 'イン.モレキュラー 'バイオロジー等に記載された方 法等を用い、例えば以下の方法により、本発明の α -1,6-フコシルトランスフェラーゼ 変異体の DNAを宿主細胞中で発現させて、製造することができる。  The α-1,6-fucosyltransferase mutant of the present invention can be obtained by using the method described in Molecular Cloning 3rd Edition or Current Protocols “in. Thus, the α-1,6-fucosyltransferase mutant DNA of the present invention can be expressed in a host cell and produced.
[0060] 全長 cDNAをもとにして、必要に応じて、該 α _1，6_フコシルトランスフェラーゼ変異 体をコードする部分を含む適当な長さの DNA断片を調製する。 [0060] Based on the full-length cDNA, if necessary, a DNA fragment of an appropriate length containing a portion encoding the α_1,6_fucosyltransferase mutant is prepared.
該 DNA断片、または全長 cDNAを適当な発現ベクターのプロモーターの下流に 揷入することにより、組換えベクターを作製する。  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.
該組換えベクターを、該発現べクタ一に適合した宿主細胞に導入することにより、 本発明のひ -1,6 -フコシルトランスフェラーゼ変異体を生産する形質転換体を得ること ができる。  By introducing the recombinant vector into a host cell suitable for the expression vector, a transformant producing the -1,6-fucosyltransferase mutant of the present invention can be obtained.
[0061] 宿主細胞としては、細菌、酵母、動物細胞、昆虫細胞、植物細胞等、 目的とする遺 伝子を発現できるものであればいずれも用いることができる。  [0061] As the host cell, any bacteria, yeast, animal cell, insect cell, plant cell and the like can be used so long as they can express the target gene.
発現ベクターとしては、上記宿主細胞において自立複製可能ないしは染色体中へ の組込が可能で、本発明の蛋白質をコードする DNAを転写できる位置にプロモータ 一を含有してレ、るものが用いられる。 As an expression vector, autonomous replication is possible in the above host cell or into the chromosome. And a promoter containing a promoter at a position where the DNA encoding the protein of the present invention can be transcribed is used.
[0062] 細菌等の原核生物を宿主細胞として用いる場合は、本発明の α -1,6-フコシルトラ ンスフエラーゼ変異体をコードする DNAを含有してなる組換えベクターは原核生物 中で自立複製可能であると同時に、プロモーター、リボソーム結合配歹 1J、本発明の蛋 白質をコードする遺伝子、及び転写終結配列より構成されたベクターであることが好 ましレ、。プロモーターを制御する遺伝子が含まれてレ、てもよレ、。  [0062] When a prokaryotic organism such as a bacterium is used as a host cell, the recombinant vector containing the DNA encoding the α-1,6-fucosyltransferase mutant of the present invention is capable of autonomous replication in prokaryotes. At the same time, the vector is preferably composed of a promoter, a ribosome binding ligand 1J, a gene encoding the protein of the present invention, and a transcription termination sequence. It includes genes that control the promoter.
[0063] 発現ベクターとしては、例えば、 pBTrp2, pBTacl、 pBTac2 (いずれもベーリンガー マンハイム社より巿販）、 pKK233- 2 (Pharmacia社製）、 pSE280 (Invitrogen社製）、 pGE MEX-1 (Promega社製）、 pQE_8 (QIAGEN社製）、 pKYPIO (特開昭 58- 110600)、 pKY Ρ200 [Agricultural Biological Chemistry, ， 669 (1984)] , pLSAl [Agric. Biol. Chem. ， 53, 277 (1989)〕、 pGELl [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)〕、 pBluescri pt II SK (-) (Stratagene社製）、 pTrs30 [Escherichia coli JM109/pTrS30 (FERM BP-5 407)より調製〕、 Trs32 [Escherichia coli JM109/pTrS32 (FERM BP-5408)より調製〕 、 PGHA2 iEscherichiacoliIGHA2 (FERM B-400)より調製、特開昭 60-221091〕、 pGK A2 [Escherichia coli IGKA2 (FERM BP-6798)より調製、特開昭 60-221091〕、 PTerm2 (US4686191、 US4939094、 US5160735)、 pSupex、 pUBHO, pTP5、 pC194、 pEG400 [ J. Bacteriol.,172, 2392 (1990)〕、 pGEX (Pharmacia社製）、 pETシステム（Novagen社 製）、 pSupex等をあげることができる。 [0063] Examples of expression vectors include pBTrp2, pBTacl, pBTac2 (all sold by Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGE MEX-1 (Promega) ), PQE_8 (manufactured by QIAGEN), pKYPIO (JP-A 58-110600), pKY Ρ200 [Agricultural Biological Chemistry,, 669 (1984)], pLSAl [Agric. Biol. Chem., 53, 277 (1989)], pGELl [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescri pt II SK (-) (Stratagene), pTrs30 (prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5 407)) , Trs32 (prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)), PGHA2 prepared from iEscherichiacoliIGHA2 (FERM B-400) Preparation, JP-A-60-221091], P Term2 (US4686191, US4939094, US5160735), pSupex, pUBHO, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX (Pharmacia), pET system (Novagen Co., Ltd.), it is possible to increase the pSupex like.
[0064] プロモーターとしては、宿主細胞中で発現できるものであればいかなるものでもよい 。例えば、 tro.プロモーター（P )、 lacプロモーター、 Pプロモーター、 Pプロモーター trp L R  [0064] The promoter may be any as long as it can be expressed in the host cell. For example, tro.promoter (P), lac promoter, P promoter, P promoter trp L R
、 T7プロモーター等の、大腸菌やファージ等に由来するプロモーターをあげることが できる。また P を 2つ直列させたプロモーター（P X 2)、 £プロモーター、 lacT7プロ trp trp  And promoters derived from E. coli and phages, such as the T7 promoter. Also, two P promoters in series (P X 2), £ promoter, lacT7 pro trp trp
モーター、 let Iプロモーターのように人為的に設計改変されたプロモーター等も用い ること力 Sできる。  It is also possible to use artificially designed and modified promoters such as motors and let I promoters.
[0065] リボソーム結合配列であるシャイン一ダルガノ（Shine-Dalgarno)配列と開始コドンと の間を適当な距離 (例えば 6〜18塩基）に調節したプラスミドを用いることが好ましい。 本発明のひ -1,6 -フコシルトランスフェラーゼ変異体をコードする部分の塩基配列を 、宿主の発現に最適なコドンとなるように、塩基を置換することにより、 目的とする α -1 ，6-フコシルトランスフェラーゼ変異体の生産率を向上させることができる。 [0065] It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (eg, 6 to 18 bases). The nucleotide sequence of the portion encoding the -1,6-fucosyltransferase mutant of the present invention The production rate of the target α −1, 6-fucosyltransferase mutant can be improved by substituting the base so as to be an optimal codon for host expression.
[0066] 本発明の組換えベクターにおいては、本発明の α -1,6-フコシルトランスフェラーゼ 変異体の DNAの発現には転写終結配列は必ずしも必要ではないが、構造遺伝子 の直下に転写終結配列を配置することが好ましい。 [0066] In the recombinant vector of the present invention, a transcription termination sequence is not necessarily required for the expression of the DNA of the α-1,6-fucosyltransferase mutant of the present invention, but a transcription termination sequence is placed directly under the structural gene. It is preferable to arrange.
宿主細胞としては、ェシエリヒア属、セラチア属、バチルス属、ブレビバクテリウム属、 コリネバクテリウム属、ミクロバクテリウム属、シユードモナス属等に属する微生物、例 ば、 Escherichiacoli XLl_Blue、 Escherichiacoli XL2_Blue、 Escherichia coliDHl、 E scherichia coli MC1000、 Escherichia coli KY3276、 Escherichiacoli W1485、 Escheric hia coli JM109、 Escherichia coliHB101、 Escherichiacoli No.49、 Escherichia coli W31 10、 Escherichiacoli NY49、 Serratia ficaria. Serratia fonticola. Serratialiguefaciens. S erratiamarcescens. Bacillus subtilis、 Bacillusamvloliguefacines. Brevibacteriumimmar iophilum ATCC14068、 Brevibacteriumsaccharolvticum ATCC14066、 Brevibacterium flavum ATCC14067、 Brevibacteriumammoniagenes、 Brevibacteriumlactofermentum ATCC13869、 Corvnebacteriumglutamicum ATCし 13032、 Corvnebacteriumacetoacid ophilum ATCC13870、 Microbacterium ammoniaphilum ATCC15354、 Pseudomonassp . D-0110等をあげることができる。  Host cells include microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Syudomonas, etc., such as Escherichiacoli XLl_Blue, Escherichiacoli XL2_Blue, Escherichia coliDHl, Escherichia Escherichia coli KY3276, Escherichiacoli W1485, Escheric hia coli JM109, Escherichia coli HB101, Escherichiacoli No.49, Escherichia coli W31 10, Escherichiacoli NY49, Serratia ficaria. Serratia fonticola. , Brevibacteriumsaccharolvticum ATCC14066, Brevibacterium flavum ATCC14067, Brevibacteriumammoniagenes, Brevibacteriumlactofermentum ATCC13869, Corvnebacterium glutamicum ATC 13032, Corvnebacteriumacetoacid ophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354, Pseudomonassp D-0110 etc. can be raised.
組換えベクターの導入方法としては、上記宿主細胞へ DNAを導入する方法であ ればいずれも用いることができ、例えば、カルシウムイオンを用いる方法〔Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)〕、プロトプラスト法（特開昭 63-248394)、または Gen e, 17, 107 (1982)や Molecular & General Genetics,崖， 111 (1979)に記載の方法等 をあげること力できる。  As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into the host cell, for example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110. (1972)], the protoplast method (Japanese Patent Laid-Open No. 63-248394), or the methods described in Gene, 17, 107 (1982) and Molecular & General Genetics, Cliff, 111 (1979).
[0067] 酵母を宿主細胞として用いる場合には、発現ベクターとして、例えば、 YEP13 (ATC C37115)、 YEp24 (ATCC37051)、 YCp50 (ATCC37419)等をあげることができる。 プロモーターとしては、酵母菌株中で発現できるものであればいずれのものを用い てもよく、例えば、へキソースキナーゼ等の解糖系の遺伝子のプロモーター、 PH05 プロモーター、 PGKプロモーター、 GAPプロモーター、 ADHプロモーター、 gal 1プ 口モーター、 gal 10プロモーター、ヒートショック蛋白質プロモーター、 MFひ 1プロモ 一ター、 CUP 1プロモーター等をあげることができる。 [0067] When yeast is used as a host cell, examples of expression vectors include YEP13 (ATC C37115), YEp24 (ATCC37051), YCp50 (ATCC37419) and the like. Any promoter can be used as long as it can be expressed in yeast strains. For example, promoters of glycolytic genes such as hexose kinase, PH05 promoter, PGK promoter, GAP promoter, ADH promoter , Gal 1 probe motor, gal 10 promoter, heat shock protein promoter, MF hi 1 promo And the CUP 1 promoter.
[0068] 宿主細胞としては、サッカロミセス属、シゾサッカロミセス属、クリュイべ口ミセス属、ト リコスポロン属、シュヮニォミセス属等に属する酵母、例えば、 Saccharomvces cerevisi ae. Schizosaccharomvces pomoe. Kiuvveromvces lactis、 fnchosporon pullulans. ¾ch wanniomvces alluvius をあげ oこと力できる。  [0068] Examples of host cells include yeasts belonging to the genus Saccharomyces, Schizosaccharomyces, Kluybe mouth genus, Trichosporon genus, Schneomyces genus, such as Saccharomvces cerevisi ae. Schizosaccharomvces pomoe. Kiuvveromvces lactans, I can raise alluvius.
組換えベクターの導入方法としては、酵母に DNAを導入する方法であればレ、ずれ も用いることができ、例えば、エレクト口ポレーシヨン法 [メソッズ 'ェンザィモロジ一 (Me thods. Enzymol.), 194, 182 (1990)]、スフヱ口プラスト法 [プロシーディングス 'ォブ 'ザ 'ナショナル ·ァカデミ ~ .ォブ 'サイエンス (Pro Natl. Acad. Sci. U.S.A), 84, 1929 (1 978)]、酢酸リチウム法 [ジャーナル'ォブ'バタテリォロジ一 (J. Bacteriology), 153, 163 (1983)]、プロシーディングス ·ォブ ·ザ ·ナショナノレ ·ァカデミ一'ォブ ·サイエンス (Pro c. Natl. Acad. Sci. U.S.A), 75, 1929 (1978)]に記載の方法等をあげることができる。  As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into yeast. For example, the electoral position method [Methods. Enzymol.], 194, 182 (1990)], Supo Higuchi plast method [Proceedings 'Ob' The 'National Academia ~ .Ob' Science (Pro Natl. Acad. Sci. USA), 84, 1929 (1 978)], Lithium acetate method [Journal 'Ob' Battereriology (J. Bacteriology), 153, 163 (1983)], Proc. Natl. Acad. Sci. USA ), 75, 1929 (1978)].
[0069] 動物細胞を宿主として用いる場合には、発現ベクターとして、例えば、 pcDNAI, pc DM8 (フナコシ社より市販）、 pAGE107 [特開平 3-22979 ;サイトテクノロジー (Cytotec hnology), 3, 133, (1990)]、 pAS3- 3 [特開平 2- 227075]、 pCDM8 [ネイチヤー (Nature), 329, 840, (1987)]、 pcDNAI/Amp (Invitrogen社製）、 pREP4 (Invitrogen社製）、 pAGE 103 [ジャーナル'ォブ'バイオケミストリー (J. Biochemistry)，皿， 1307 (1987)]、 pAGE 210等をあげることができる。  [0069] When animal cells are used as hosts, examples of expression vectors include pcDNAI, pc DM8 (commercially available from Funakoshi), pAGE107 [Japanese Patent Laid-Open No. 3-22979; Cytotec hnology, 3, 133, ( 1990)], pAS3-3 [Japanese Patent Laid-Open No. 2-227075], pCDM8 [Nature, 329, 840, (1987)], pcDNAI / Amp (Invitrogen), pREP4 (Invitrogen), pAGE 103 [ Journal "Ob" Biochemistry (J. Biochemistry), Dish, 1307 (1987)], pAGE 210 and the like.
[0070] プロモーターとしては、動物細胞中で発現できるものであればいずれも用いることが でき、例えば、サイトメガロウィルス（CMV)の IE (immediate early)遺伝子のプロモータ 一、 SV40の初期プロモーター、レトロゥイノレスのプロモーター、メタ口チォネインプロモ 一ター、ヒートショックプロモーター、 SRひプロモーター等をあげることができる。また 、ヒト CMVの IE遺伝子のェンハンサーをプロモーターと共に用いてもょレ、。  [0070] Any promoter can be used as long as it can be expressed in animal cells. For example, cytomegalovirus (CMV) IE (immediate early) gene promoter 1, SV40 early promoter, retrowinores Examples include promoters, meta-mouthone promoters, heat shock promoters, SR promoters, and the like. You can also use the enhancer of the human CMV IE gene with a promoter.
[0071] 宿主細胞としては、ヒトの細胞であるナマルバ（Namalwa)細胞、サルの細胞である C 〇S細胞、チャイニーズ'ノ、ムスターの細胞である CHO細胞、 HBT5637 (特開昭 63-29 9)、ラットミエローマ細胞、マウスミエローマ細胞、シリアンハムスター腎臓由来細胞、 胚性幹細胞、受精卵細胞等をあげることができる。  [0071] Host cells include human cells such as Namalwa cells, monkey cells such as COS cells, Chinese's cells, CHO cells that are Muster cells, and HBT5637 (Japanese Patent Laid-Open No. 63-29 9). ), Rat myeloma cells, mouse myeloma cells, Syrian hamster kidney-derived cells, embryonic stem cells, fertilized egg cells, and the like.
組換えベクターの導入方法としては、動物細胞に DNAを導入する方法であればい ずれも用いることができ、例えば、エレクト口ポレーシヨン法 [サイトテクノロジー (Cytote chnology), 3, 133 (1990)]、リン酸カルシウム法 [特開平 2-227075]、リボフヱクシヨン 法 [プロシーディングス ·ォブ ·ザ ·ナショナル ·アカデミー ·ォブ ·サイエンス (Pro Natl • Acad. Sci. U.S.A.), 84, 7413 (1987)]、インジェクション法 [Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Pres s (1994) (以下、マニピユレ一ティング'マウス'ェンブリオ第 2版と略す) ]、パーテイク ルガン (遺伝子銃）を用いる方法 [特許第 2606856、特許第 2517813]、 DEAE—デキ ストラン法レ ォマニュアルシリーズ 4一遺伝子導入と発現 ·解析法（羊土社)横田 崇.新井賢ー編 (1994)]、ウィルスベクター法 [マニピユレ一ティング'マウス'ェンプリ ォ第 2版]等をあげることができる。 Recombinant vectors can be introduced by any method that introduces DNA into animal cells. Misalignment can also be used, for example, the electopore position method [Cytote chnology, 3, 133 (1990)], the calcium phosphate method [JP-A-2-27075], the ribofusion method [Proceedings of the National Academy of Science (Pro Natl • Acad. Sci. USA), 84, 7413 (1987)], injection method [Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Pres s (1994) (Hereafter abbreviated as “Manipulating“ Mouse ”Embryo 2nd edition”)], a method using a part gun (gene gun) [Patent No. 2606856, Patent No. 2517813], DEAE—dextran method manual series 4 single gene introduction And expression / analysis method (Yodosha) Takashi Yokota. Ken Arai (1994)], virus vector method [Manipulating "Mouse" Emprio 2nd edition], etc. .
[0072] 昆虫細胞を宿主として用いる場合には、例えばカレント 'プロトコールズ 'イン 'モレ キュフ1 ~ 'ハイォロン1 ~ Baculovirus Expression Vectors, A Laboratory Manual, W. H. Freeman and Company, New York (1992)、 ノくィォ /ァクノロジー (Bio/Technology), 6, 47 (1988)等に記載された方法によって、タンパク質を発現することができる。 [0072] When insect cells are used as hosts, for example, Current 'Protocols' in 'Molecuff 1 ~' Hyoron 1 ~ Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992), Proteins can be expressed by the method described in Bio / Technology, 6, 47 (1988).
即ち、組換え遺伝子導入ベクターおよびバキュロウィルスを昆虫細胞に共導入して 昆虫細胞培養上清中に組換えウィルスを得た後、さらに組換えウィルスを昆虫細胞 に感染させ、タンパク質を発現させることができる。  That is, the recombinant gene transfer vector and baculovirus are co-introduced into insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected into insect cells to express the protein. it can.
[0073] 該方法において用いられる遺伝子導入ベクターとしては、例えば、 pVL1392、 pVLl[0073] Examples of gene transfer vectors used in the method include pVL1392, pVLl.
393、 pBlueBacIII (ともに Invitorogen社製）等をあげることができる。 393, pBlueBacIII (both manufactured by Invitorogen) and the like.
バキュロウィルスとしては、例えば、夜盗蛾科昆虫に感染するウィルスであるアウトグ ラファ 'カリフオノレニ力'ヌクレア一'ポリへドロシス'ウイノレス (Autographa californica nu clear polyhedrosis virus)等を用レヽること力 Sできる。  As a baculovirus, for example, autographa californica nu clear polyhedrosis virus, which is a virus that infects night stealing insects, can be used, such as Autographa californica nu clear polyhedrosis virus.
[0074] 昆虫細胞としては、 Soodooterafrug erdaの卵巣細朐である Sf9、 Sf21 [カレント 'プロ トコ1 ~ノレズ'イン'モレキュフ 1 ~ · /ヽィォロシ1 ~ Baculovirus Expression Vectors, A Labo ratory Manual, W. H. Freeman and Company, New York (1992)]、 nchonlusianiの 卵巣細胞である High 5 (Invitrogen社製）等を用いることができる。 [0074] Insect cells include Sodooterafrug erda's ovarian cells Sf9, Sf21 [Current 'Protoco 1 ~ Norez' In 'Molecuff 1 ~ · / Yoroshi 1 ~ Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992)], nchonlusiani ovary cells such as High 5 (Invitrogen) can be used.
組換えウィルスを調製するための、昆虫細胞への上記組換え遺伝子導入ベクター と上記バキュロウィルスの共導入方法としては、例えば、リン酸カルシウム法（特開平 2 -227075)、リポフエクシヨン法 [プロシーディングス 'ォブ ·ザ'ナショナル 'アカデミー · ォブ'サイエンス (Proc. Natl. Acad. Sci. U.S.A.), 84, 7413 (1987)]等をあげることがで きる。 As a method for co-introducing the above recombinant gene transfer vector and the above baculovirus into insect cells for preparing a recombinant virus, for example, the calcium phosphate method (Japanese Patent Laid-Open No. Hei 2). -227075), the lipofusion method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].
[0075] 植物細胞を宿主細胞として用いる場合には、発現ベクターとして、例えば、 Tiプラス ミド、タバコモザイクウィルスベクター等をあげることができる。  [0075] When plant cells are used as host cells, examples of expression vectors include Ti plasmids and tobacco mosaic virus vectors.
プロモーターとしては、植物細胞中で発現できるものであればいずれのものを用い てもよく、例えば、カリフラワーモザイクウィルス（CaMV)の 35Sプロモーター、イネァク チン 1プロモーター等をあげることができる。  Any promoter can be used as long as it can be expressed in plant cells, and examples thereof include the cauliflower mosaic virus (CaMV) 35S promoter and the rice 1 promoter.
[0076] 宿主細胞としては、タバコ、ジャガイモ、トマト、ニンジン、ダイズ、アブラナ、アルファ ルファ、イネ、コムギ、ォォムギ等の植物細胞等をあげることができる。 [0076] Examples of host cells include tobacco, potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat, barley, and other plant cells.
組換えベクターの導入方法としては、植物細胞に DNAを導入する方法であればい ずれも用いることができ、例えば、ァグロパクテリゥム（Agrobacterium) [特開昭 59-140 As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into plant cells. For example, Agrobacterium [Japanese Patent Laid-Open No. 59-140] is available.
885、特開昭 60-70080、 WO94/00977]、エレクト口ポレーシヨン法 [特開昭 60-251887885, Japanese Patent Laid-Open No. 60-70080, WO94 / 00977], Elect Mouth Position Method [Japanese Patent Laid-Open No. 60-251887]
]、パーティクルガン (遺伝子銃）を用いる方法 [日本特許第 2606856、 日本特許第 25], Method using particle gun (gene gun) [Japanese Patent No. 2606856, Japanese Patent No. 25
17813]等をあげることができる。 17813] and the like.
[0077] 遺伝子の発現方法としては、直接発現以外に、モレキュラー 'クローニング第 3版に 記載されている方法等に準じて、分泌生産、融合タンパク質発現等を行うことができ る。 [0077] As a gene expression method, in addition to direct expression, secretory production, fusion protein expression, etc. can be performed according to the method described in Molecular Cloning 3rd Edition.
酵母、動物細胞、昆虫細胞または植物細胞により発現させた場合には、糖あるいは 糖鎖が付加された蛋白質を得ることができる。  When expressed in yeast, animal cells, insect cells or plant cells, a protein to which a sugar or sugar chain has been added can be obtained.
以上のようにして得られる形質転換体を培地に培養し、培養物中に本発明の α-ι， 6-フコシルトランスフェラーゼ変異体を生成蓄積させ、該培養物から採取することによ り、本発明のひ -1,6 -フコシルトランスフェラーゼ変異体を製造することができる。本発 明のひ _1,6 -フコシルトランスフェラーゼ変異体を発現した形質転換体を培地に培養 する方法は、宿主の培養に用いられる通常の方法に従って行うことができる。 The transformant obtained as described above is cultured in a medium, and the α- ι, 6-fucosyltransferase mutant of the present invention is produced and accumulated in the culture and collected from the culture. The inventive 1,6-fucosyltransferase mutant can be produced. The method of culturing a transformant expressing the _1,6-fucosyltransferase mutant of the present invention in a medium can be performed according to a usual method used for culturing a host.
[0078] 大腸菌等の原核生物あるいは酵母等の真核生物を宿主として得られた形質転換 体を培養する培地としては、該生物が資化し得る炭素源、窒素源、無機塩類等を含 有し、形質転換体の培養を効率的に行える培地であれば天然培地、合成培地のい ずれを用いてもよい。 [0078] The medium for culturing a transformant obtained by using a prokaryote such as E. coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the organism. Any medium that can efficiently culture transformants can be a natural or synthetic medium. A deviation may be used.
炭素源としては、該生物が資化し得るものであればよぐグノレコース、フラクトース、 スクロース、これらを含有する糖蜜、デンプンあるいはデンプン加水分解物等の炭水 化物、酢酸、プロピオン酸等の有機酸、エタノール、プロパノールなどのアルコール 類等を用いることができる。  As the carbon source, if it can be assimilated by the organism, it can be gnolecose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, Alcohols such as ethanol and propanol can be used.
[0079] 窒素源としては、アンモニア、塩化アンモニゥム、硫酸アンモニゥム、酢酸アンモニ ゥム、リン酸アンモニゥム等の無機酸もしくは有機酸のアンモニゥム塩、その他の含窒 素化合物、ならびに、ペプトン、肉エキス、酵母エキス、コーンスチープリカー、カゼィ ン加水分解物、大豆粕および大豆粕加水分解物、各種発酵菌体およびその消化物 等を用いることができる。  [0079] Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast Extracts, corn steep liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used.
[0080] 無機塩としては、リン酸第一カリウム、リン酸第二カリウム、リン酸マグネシウム、硫酸 マグネシウム、塩化ナトリウム、硫酸第一鉄、硫酸マン癌、硫酸銅、炭酸カルシウム等 を用いることができる。  [0080] As the inorganic salt, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, mangan sulfate, copper sulfate, calcium carbonate and the like can be used. .
培養は、通常振盪培養または深部通気攪拌培養などの好気的条件下で行う。培養 温度は 15〜40°Cがよぐ培養時間は、通常 16時間〜 7日間である。培養中の pHは 3. 0〜9. 0に保持する。 pHの調整は、無機または有機の酸、アルカリ溶液、尿素、 炭酸カルシウム、アンモニアなどを用いて行う。  The culture is usually carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is 15-40 ° C, and the culture time is usually 16 hours to 7 days. The pH during the culture is maintained at 3.0 to 9.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.
[0081] また、培養中必要に応じて、アンピシリンやテトラサイクリン等の抗生物質を培地に 添加してもよい。  [0081] Further, an antibiotic such as ampicillin or tetracycline may be added to the medium as needed during the culture.
プロモーターとして誘導性のプロモーターを用いた組換えベクターで形質転換した 微生物を培養するときには、必要に応じてインデューサーを培地に添加してもよい。 例えば、 プロモーターを用いた組換えベクターで形質転換した微生物を培養する ときにはイソプロピル一 β _D_チォガラタトピラノシド等を、 プロモーターを用い た組換えベクターで形質転換した微生物を培養するときにはインドールアクリル酸等 を培地に添加してもよい。  When culturing a microorganism transformed with a recombinant vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with a recombinant vector using a promoter, isopropyl-1-β_D_thiogalatatopyranoside is used. When cultivating a microorganism transformed with a recombinant vector using a promoter, indole acrylic acid is used. Etc. may be added to the medium.
[0082] 動物細胞を宿主として得られた形質転換体を培養する培地としては、一般に使用さ れている RPMI1640培地 [ザ ·ジャーナル ·ォブ .ザ.アメリカン'メディカル ·ァソシエイ シヨン (The Journal of the American Medical Association), 199, 519 (1967)]、 Eagleの MEM培地 [サイエンス (Science),122， 501 (1952)]、ダルベッコ改変 MEM培地邊 [ヴュ ゥロロジー (Virology), 8, 396 (1959)]、 199培地 [プロシーデイング'ォブ 'ザ 'ソサイエ ティ 'フォア'ザ 'バイオロジカノレ'メディスン (Proceeding of the Society for the Biologic al Medicine), 73, 1 (1950)]、 Whitten培地 [発生工学実験マニュアル-トランスジヱニッ ク 'マウスの作り方 (講談社)勝木元也編（ 1987) ]またはこれら培地に牛胎児血清等を 添加した培地等を用いることができる。 [0082] As a medium for culturing transformants obtained using animal cells as a host, the RPMI1640 medium [The Journal of the American American Medical Association (The Journal of the American Medical Association), 199, 519 (1967)], Eagle MEM medium [Science, 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 (1959)], 199 medium [Procedure 'Ob' The 'Society'] Fore 'the' Biologic Canore 'Medicine (Proceeding of the Society for the Biologic al Medicine), 73, 1 (1950)], Whitten Medium [Genetic Engineering Experiment Manual-Transgeneic' How to make a mouse (Kodansha) edited by Motoya Katsuki (1987)] or a medium obtained by adding fetal calf serum or the like to these mediums.
[0083] 培養は、通常 pH6〜8、 30〜40°C、 5%CO存在下等の条件下で:!〜 7日間行う。 [0083] Cultivation is usually carried out under the conditions of pH 6-8, 30-40 ° C, 5% CO, etc .:! -7 days.
フエドバツチ培養、ホロファイバー培養などの培養法を用いて 1日〜数ケ月培養を行 うことちできる。  Cultivation can be performed for 1 day to several months using culture methods such as Fuedbacti culture and holofiber culture.
また、培養中必要に応じて、カナマイシン、ペニシリン等の抗生物質を培地に添カロ してもよい。  Further, antibiotics such as kanamycin and penicillin may be added to the medium as needed during the culture.
[0084] 昆虫細胞を宿主として得られた形質転換体を培養する培地としては、一般に使用さ れている TNM-FH培地（Pharmingen社製）、 Sf-900 II SFM培地（Life Technologies社 製）、 ExCell400、 ExCell405 (いずれも JRH Biosciences社製）、 Grace's Insect Medium [Grace,T.C.C, Nature, 195, 788 (1962)〕等を用いることができる。  [0084] As a medium for culturing a transformant obtained using insect cells as a host, commonly used TNM-FH medium (Pharmingen), Sf-900 II SFM medium (Life Technologies), ExCell400, ExCell405 (both manufactured by JRH Biosciences), Grace's Insect Medium [Grace, TCC, Nature, 195, 788 (1962)] and the like can be used.
培養は、通常 pH6〜7、 25〜30°C等の条件下で、 1〜5日間行う。  Cultivation is usually carried out for 1 to 5 days under conditions of pH 6-7, 25-30 ° C, etc.
また、培養中必要に応じて、ゲンタマイシン等の抗生物質を培地に添加してもよい  In addition, an antibiotic such as gentamicin may be added to the medium as needed during the culture.
[0085] 植物細胞を宿主として得られた形質転換体は、細胞として、または植物の細胞や器 官に分化させて培養することができる。該形質転換体を培養する培地としては、一般 に使用されているムラシゲ'アンド'スターグ (MS)培地、ホワイト (White)培地、またはこ れら培地にオーキシン、サイトカイニン等、植物ホルモンを添カ卩した培地等を用いるこ とができる。 [0085] A transformant obtained using a plant cell as a host can be cultured as a cell or after being differentiated into a plant cell or organ. As a medium for culturing the transformant, commonly used Murashige 'and' Stag (MS) medium, White medium, or a plant hormone such as auxin or cytokinin is added to these mediums. Can be used.
[0086] 培養は、通常 pH5〜9、 20〜40°Cの条件下で 3〜60日間行う。  [0086] Cultivation is usually carried out under conditions of pH 5-9 and 20-40 ° C for 3-60 days.
また、培養中必要に応じて、カナマイシン、ノ、イダロマイシン等の抗生物質を培地 に添加してもよい。  In addition, antibiotics such as kanamycin, no, and idaromomycin may be added to the medium as needed during culture.
上記のとおり、本発明のひ -1,6 -フコシルトランスフェラーゼ変異体をコードする DN Aを組み込んだ組換え体ベクターを保有する微生物、動物細胞、あるいは植物細胞 由来の形質転換体を、通常の培養方法に従って培養し、該《 -1,6-フコシルトランス フェラーゼ変異体を生成蓄積させ、該培養物より該 α -1,6-フコシルトランスフェラー ゼ変異体を採取することにより、該 α -1,6-フコシルトランスフェラーゼ変異体を製造 すること力 Sできる。 As described above, a microorganism, animal cell, or plant cell having a recombinant vector incorporating DNA encoding the -1,6-fucosyltransferase mutant of the present invention The resulting transformant is cultured according to a normal culture method, the <<-1,6-fucosyltransferase mutant is produced and accumulated, and the α-1,6-fucosyltransferase mutant is collected from the culture. By doing so, it is possible to produce the α -1,6-fucosyltransferase mutant.
[0087] 遺伝子の発現方法としては、直接発現以外に、モレキュラー 'クローニング第 3版に 記載されている方法等に準じて、分泌生産、融合蛋白質発現等を行うことができる。 本発明のひ -1,6 -フコシルトランスフェラーゼ変異体の生産方法としては、宿主細胞 内に生産させる方法、宿主細胞外に分泌させる方法、あるいは宿主細胞外膜上に生 産させる方法があり、使用する宿主細胞や、生産させる蛋白質の構造を変えることに より、該方法を選択することができる。  [0087] As a gene expression method, in addition to direct expression, secretory production, fusion protein expression, and the like can be performed in accordance with the method described in Molecular Cloning 3rd Edition. As a method for producing the -1,6-fucosyltransferase mutant of the present invention, there are a method in which it is produced in the host cell, a method in which it is secreted outside the host cell, and a method in which it is produced on the host cell outer membrane. This method can be selected by changing the structure of the host cell to be produced and the protein to be produced.
[0088] 本発明のひ -1,6 -フコシルトランスフェラーゼ変異体が宿主細胞内あるいは宿主細 胞外膜上に生産される場合、ポールソンらの方法 [ジャーナル'ォブ 'バイオロジカル 'ケミストリー (J. Biol. Chem.), 264, 17619 (1989)]、ロウらの方法 [プロシーディングス •ォブ'ザ 'ナショナル 'アカデミ^ ~ ·ォブ ·サイエンス (Proc. Natl. Acad. Sci. U.S.A.), 8 6, 8227 (1989);ジーン'デベロップメント (Genes Develop.), 4, 1288 (1990) ]、または 特開平 05-336963、特開平 06-823021等に記載の方法を準用することにより、該 α _1 ，6-フコシルトランスフェラーゼ変異体を宿主細胞外に積極的に分泌させることができ る。  [0088] When the -1,6-fucosyltransferase mutant of the present invention is produced in the host cell or on the host cell outer membrane, the method of Paulson et al. [Journal 'Ob' Biological 'Chemistry (J. Biol. Chem.), 264, 17619 (1989)], Law et al. [Proc. Natl. Acad. Sci. USA], 8 6, 8227 (1989); Gene's Development (Genes Develop.), 4, 1288 (1990)], or Japanese Patent Application Laid-Open No. 05-336963, Japanese Patent Application Laid-Open No. 06-823021, etc. , 6-fucosyltransferase mutants can be actively secreted outside the host cell.
[0089] すなわち、遺伝子組換えの手法を用いて、本発明の α -1,6-フコシルトランスフェラ ーゼ変異体の活性部位を含む蛋白質の手前にシグナルペプチドを付加した形で発 現させることにより、本発明の α -1,6-フコシルトランスフェラーゼ変異体を宿主細胞 外に積極的に分泌させることができる。  [0089] That is, expression is performed by adding a signal peptide in front of the protein containing the active site of the α-1,6-fucosyltransferase mutant of the present invention using a genetic recombination technique. Thus, the α-1,6-fucosyltransferase mutant of the present invention can be actively secreted outside the host cell.
また、特開平 2-227075に記載されている方法に準じて、ジヒドロ葉酸還元酵素遺伝 子等を用いた遺伝子増幅系を利用して生産量を上昇させることもできる。  Further, according to the method described in JP-A-2-27075, the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like.
さらに、遺伝子導入した動物または植物の細胞を再分化させることにより、遺伝子 が導入された動物個体（トランスジエニック非ヒト動物）または植物個体（トランスジェニ ック植物）を造成し、これらの個体を用いて本発明のひ- 1，6 -フコシルトランスフェラー ゼ変異体を製造することもできる。 [0090] 形質転換体が動物個体または植物個体の場合は、通常の方法に従って、飼育また は栽培し、該 α -1,6-フコシルトランスフェラーゼ変異体を生成蓄積させ、該動物個体 または植物個体より該 α _1，6-フコシルトランスフェラーゼ変異体を採取することにより 、該ひ -1，6 -フコシルトランスフェラーゼ変異体を製造することができる。 Furthermore, by redifferentiating the cells of the animal or plant into which the gene has been introduced, animal individuals (transgenic non-human animals) or plant individuals (transgenic plants) into which the gene has been introduced are created, and these individuals are It can also be used to produce the 1,6-fucosyltransferase mutant of the present invention. [0090] When the transformant is an animal individual or a plant individual, it is bred or cultivated according to a normal method, and the α-1,6-fucosyltransferase mutant is produced and accumulated, from the animal individual or the plant individual. By collecting the α_1,6-fucosyltransferase mutant, the -1,6-fucosyltransferase mutant can be produced.
動物個体を用いて本発明のひ- 1，6-フコシルトランスフェラーゼ変異体を製造する 方法としては、例えば公知の方法 [アメリカン 'ジャーナル'ォブ 'クリニカル'ニュートリ シヨン (American Journal of Clinical Nutrition), 63, 639S (1996);アメリカン.ジャーナ ノレ'ォブ 'クリニ力ノレ'二ユートリシヨン (American Journal of Clinical Nutrition), 63, 627 S (1996);バイオ Zテクノロジー (Bio/Technology), 9, 830 (1991)]に準じて遺伝子を 導入して造成した動物中に本発明のひ- 1，6-フコシルトランスフェラーゼ変異体を生 産する方法があげられる。  As a method for producing the -1,6-fucosyltransferase mutant of the present invention using an animal individual, for example, a known method [American 'Journal' of 'Clinical' Nutrition, (American Journal of Clinical Nutrition), 63 , 639S (1996); American Journal of Clinical Nutrition, 63, 627 S (1996); Bio / Technology, 9, 830 (1991) )], A method for producing the mutant of 1,6-fucosyltransferase of the present invention in an animal constructed by introducing a gene.
[0091] 動物個体の場合は、例えば、本発明のひ -1,6-フコシルトランスフヱラーゼ変異体を コードする DNAを導入したトランスジエニック非ヒト動物を飼育し、該 α -1,6-フコシル トランスフェラーゼ変異体を該動物中に生成 ·蓄積させ、該動物中より該ひ-1，6-フコ シルトランスフェラーゼ変異体を採取することにより、該 α - 1,6-フコシルトランスフェラ ーゼ変異体を製造することができる。該動物中の生成 ·蓄積場所としては、例えば、 該動物のミルク（特開昭 63-309192)、卵等をあげることができる。この際に用いられる プロモーターとしては、動物で発現できるものであればいずれも用いることができるが 、例えば、乳腺細胞特異的なプロモーターである αカゼインプロモーター、 βカゼィ ンプロモーター、 ラクトグロブリンプロモーター、ホエー酸'性プロテインプロモーター 等が好適に用いられる。  [0091] In the case of an animal individual, for example, a transgenic non-human animal introduced with a DNA encoding the -1,6-fucosyltransferase mutant of the present invention is bred, and the α-1,6- The α-1,6-fucosyltransferase mutant is obtained by generating and accumulating a fucosyltransferase mutant in the animal and collecting the -1,6-fucosyltransferase mutant from the animal. Can be manufactured. Examples of the production / accumulation place in the animal include milk of the animal (Japanese Patent Laid-Open No. 63-309192), eggs and the like. Any promoter can be used as long as it can be expressed in animals. For example, α-casein promoter, β-casein promoter, lactoglobulin promoter, whey acid, which are mammary cell-specific promoters. A sex protein promoter or the like is preferably used.
[0092] 植物個体を用いて本発明のひ- 1，6-フコシルトランスフェラーゼ変異体を製造する 方法としては、例えば本発明のひ- 1，6-フコシルトランスフェラーゼ変異体をコードす る DNAを導入したトランスジヱニック植物を公知の方法 [組織培養， (1994);組織 培養， ^1 (1995);トレンド'イン'バイオテクノロジー (Trends in Biotechnology), 15, 45 (1997)]に準じて栽培し、該ひ -1,6-フコシルトランスフェラーゼ変異体を該植物中に 生成 ·蓄積させ、該植物中より該ひ- 1，6-フコシルトランスフェラーゼ変異体を採取す ることにより、該ひ- 1，6 -フコシルトランスフェラーゼ変異体を生産する方法があげられ る。 [0092] As a method for producing a mutant of -1,6-fucosyltransferase of the present invention using an individual plant, for example, DNA encoding the mutant of -1,6-fucosyltransferase of the present invention was introduced. Transgenic plants were cultivated according to known methods [tissue culture, (1994); tissue culture, ^ 1 (1995); Trends in Biotechnology, 15, 45 (1997)]. The -1,6-fucosyltransferase mutant is produced and accumulated in the plant, and the -1,6-fucosyltransferase mutant is collected from the plant to obtain the 1,6-fucosyltransferase mutant. -How to produce fucosyltransferase mutants The
[0093] 本発明の形質転換した細胞により製造された α -1,6-フコシルトランスフェラーゼ変 異体は、例えば本発明の α -1,6-フコシルトランスフェラーゼ変異体力 細胞内に溶 解状態で発現した場合には、培養終了後、細胞を遠心分離により回収し、水系緩衝 液にけん濁後、超音波破砕機、フレンチプレス、マントンガウリンホモゲナイザー、ダ イノミル等により細胞を破砕し、無細胞抽出液を得る。該無細胞抽出液を遠心分離す ることにより得られる上清力 、通常の酵素の単離精製法、即ち、溶媒抽出法、硫安 等による塩析法、脱塩法、有機溶媒による沈殿法、ジェチルアミノエチル (DEAE) - セファロース、 DIAION ΗΡΑ-75 (三菱化成社製）等レジンを用いた陰イオン交換クロ マトグラフィ一法、 S-S印 harose FF (Pharmacia社製）等のレジンを用いた陽イオン交 換クロマトグラフィー法、ブチルセファロース、フエ二ルセファロース等のレジンを用レヽ た疎水性クロマトグラフィー法、分子篩を用いたゲルろ過法、ァフィ二ティークロマトグ ラフィ一法、クロマトフォーカシング法、等電点電気泳動等の電気泳動法等の手法を 単独あるいは組み合わせて用い、精製標品を得ることができる。  [0093] The α-1,6-fucosyltransferase mutant produced by the transformed cell of the present invention, for example, when the α-1,6-fucosyltransferase mutant power of the present invention is expressed in a dissolved state in the cell After completion of the culture, the cells are collected by centrifugation, suspended in an aqueous buffer solution, and then disrupted by an ultrasonic crusher, French press, Manton Gaurin homogenizer, dynomill, etc. to obtain a cell-free extract. . The supernatant strength obtained by centrifuging the cell-free extract, the usual enzyme isolation and purification methods, that is, solvent extraction methods, salting-out methods using ammonium sulfate, desalting methods, precipitation methods using organic solvents, Anion-exchange chromatography using resin such as Jetylaminoethyl (DEAE) -Sepharose, DIAION ΗΡΑ-75 (manufactured by Mitsubishi Kasei), Cation using resin such as SS sign harose FF (Pharmacia) Replacement chromatography method, hydrophobic chromatography method using resins such as butyl sepharose and phenyl sepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, isoelectric focusing Purified preparations can be obtained using methods such as electrophoresis, such as electrophoresis, alone or in combination.
[0094] また、該 α _1，6-フコシルトランスフェラーゼ変異体が細胞内に不溶体を形成して発 現した場合は、同様に細胞を回収後破砕し、遠心分離を行うことにより、沈殿画分と して α -1,6-フコシルトランスフェラーゼ変異体の不溶体を回収する。回収した α -1,6 -フコシルトランスフェラーゼ変異体の不溶体を蛋白質変性剤で可溶化する。該可溶 化液を希釈または透析することにより、該 α -1,6-フコシルトランスフェラーゼ変異体を 正常な立体構造に戻した後、上記と同様の単離精製法により該ひ- 1,6-フコシルトラ ンスフェラーゼ変異体の精製標品を得ることができる。  [0094] If the α_1,6-fucosyltransferase mutant is expressed by forming an insoluble substance in the cells, the cells are similarly collected, disrupted, and centrifuged to obtain a precipitate fraction. As a result, an insoluble form of the α-1,6-fucosyltransferase mutant is recovered. The recovered insoluble form of the α-1,6-fucosyltransferase mutant is solubilized with a protein denaturant. The α-1,6-fucosyltransferase mutant is returned to its normal three-dimensional structure by diluting or dialyzing the solubilized solution, and then isolated by the same isolation and purification method as described above. A purified preparation of a fucosyltransferase mutant can be obtained.
[0095] 本発明のひ -1,6 -フコシルトランスフェラーゼ変異体あるいはその糖修飾体等の誘 導体が細胞外に分泌された場合には、培養上清に該ひ -1,6 -フコシルトランスフェラ ーゼ変異体あるいはその糖鎖付加体等の誘導体を回収することができる。即ち、該 培養物を上記と同様の遠心分離等の手法により処理することにより可溶性画分を取 得し、該可溶性画分から、上記と同様の単離精製法を用いることにより、精製標品を 得ること力 Sできる。  [0095] When a derivative of the -1,6-fucosyltransferase mutant of the present invention or a modified sugar thereof is secreted outside the cell, the -1,6-fucosyltransferase is added to the culture supernatant. It is possible to recover derivatives such as mutase mutants or sugar chain adducts thereof. That is, a soluble fraction is obtained by treating the culture by a method such as centrifugation as described above, and a purified preparation is obtained from the soluble fraction by using the same isolation and purification method as described above. You can get power S.
[0096] このようにして取得される蛋白質として、例えば、配列番号 13、 14、 15、 16または 17 で表されるアミノ酸配列を有する蛋白質をあげることができる。 [0096] As the protein thus obtained, for example, SEQ ID NO: 13, 14, 15, 16 or 17 And a protein having an amino acid sequence represented by
また、本発明のひ- 1 ,6-フコシルトランスフェラーゼ変異体は、 Fmoc法（フルォレニ ルメチルォキシカルボニル法）、 tBoc法（t ブチルォキシカルボニル法）等の化学 合成法によっても製造することができる。また、 Advanced ChemTech社、パーキン'ェ ノレマ" ~社、 Pharmacia社、 Protein technology Instrument土、 Synthecel卜 Vega子土、 Per S印 tive社、島津製作所等のペプチド合成機を利用して化学合成することもできる。  The mutant of 1,6-fucosyltransferase of the present invention can also be produced by chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (tbutyloxycarbonyl method). it can. In addition, chemical synthesis may be performed using peptide synthesizers such as Advanced ChemTech, Perkinenoma, Ltd., Pharmacia, Protein technology Instrument, Synthecel Vega, Per S India, Shimadzu, etc. it can.
(2) a -1，6-フコシルトランスフェラーゼ変異体の活性測定 (2) Activity measurement of a-1,6-fucosyltransferase mutant
本発明のひ -1 ,6-フコシルトランスフェラーゼ変異体及びひ -1 ,6-フコシルトランスフ エラーゼの活性の測定方法としては、例えば公知の方法 [Uozumi et al.， J Biochem, 120. 385-392 (1996)]に準じて、調整した本発明のひ -1 ,6-フコシルトランスフェラー ゼ変異体の活性を測定する方法があげられる。具体的例として、以下の方法があげ られる。  Examples of the method for measuring the activity of the -1,6-fucosyltransferase mutant and -1,6-fucosyltransferase of the present invention include known methods [Uozumi et al., J Biochem, 120. 385-392 ( 1996)], there is a method for measuring the activity of the prepared -1,6-fucosyltransferase mutant of the present invention. Specific examples include the following methods.
反応液として下記 (i)〜(v)をマイクロ遠心管中で混合し調製する [(i)酵素源： 5mLの 酵素を含む溶液、例えば精製酵素、細胞抽出液、もしくは組織抽出液）； (ii)pH調整 バッファー： 8mLの 200mM MES (2-(N-Morpholino)ethanesulfonic Acid)-NaOH ( H 6 ) (ナカライテスタ社製）； (iii)界面活性剤： ImLの 10% Triton X-100 (和光純薬社製） ; ( iv)フコースァクセプター： 4mLの 5mMピリジルァミノ-プチルァミン標識 'フコース（一） 複合二本鎖型糖鎖溶液 (タカラバイオ社製、商品名 PA-Sugar Chain 012など）； (v)フ コースドナー： 2mLの 500mM GDP-L-フコース溶液（Sigma社製）]。調整した該反応液 を 37°Cにて 2時間反応させた後、該反応液に 80mLの滅菌蒸留水を添カ卩し、 100°Cに て 1分間ボイルし酵素を失活させる。その後、 15,000gにて 10分間遠心を行い、 90mL の上清を分取し、 TSK-ゲル/ ODS- 80TMカラム（東ソ一社製）を装着した HPLC (島津 製作所製、モデノレ SCL 6A)にアプライする。 55。Cにて、 0.1 %のブタノールを含む 20m M酢酸ナトリウムバッファー (pH 4)で糖鎖を溶出させ、溶出液の励起波長 320nm、放 射波長 400nmの蛍光強度を蛍光光度計（島津製作所製、モデル RF535)で測定する 。測定した蛍光強度から、酵素反応で生成したフコシル化複合二本鎖型糖鎖の量を 算出する。サンプル中の酵素比活性は、サンプノレ中に含まれる単位蛋白質量あたり 、単位反応時間あたりの、フコシル化された複合二本鎖型糖鎖分子のモル数で表記 する（単位： pmol/hour/mg)。また、サンプル中の蛋白質量は、ゥシ血清アルブミン（Pi erce社製)を標準品に用レ、た BCAプロテインアツセィキット (Pierce社製)で測定する。 Prepare the following (i) to (v) as reaction solutions in a microcentrifuge tube [(i) Enzyme source: solution containing 5 mL of enzyme, such as purified enzyme, cell extract, or tissue extract); ii) pH adjustment buffer: 8 mL of 200 mM MES (2- (N-Morpholino) ethanesulfonic acid) -NaOH (H 6) (manufactured by Nacalai Testa); (iii) Surfactant: ImL of 10% Triton X-100 ( (Iv) Fucose acceptor: 4 mL of 5 mM pyridylamino-ptylamamine labeled 'fucose (one) complex double chain type sugar chain solution (trade name PA-Sugar Chain 012, etc., manufactured by Takara Bio Inc.) (V) Fucose donor: 2 mL of 500 mM GDP-L-fucose solution (Sigma)]. After the prepared reaction solution is reacted at 37 ° C for 2 hours, 80 mL of sterilized distilled water is added to the reaction solution and boiled at 100 ° C for 1 minute to deactivate the enzyme. Then, centrifuge at 15,000g for 10 minutes, collect 90mL of the supernatant, and apply to HPLC (Shimadzu Corporation, Modenore SCL 6A) equipped with TSK-gel / ODS-80TM column (manufactured by Tosohichi Co., Ltd.). Apply. 55. In C, the sugar chain was eluted with 20 mM sodium acetate buffer (pH 4) containing 0.1% butanol, and the fluorescence intensity of the eluate with an excitation wavelength of 320 nm and emission wavelength of 400 nm was measured with a fluorometer (manufactured by Shimadzu Corporation, model Measure with RF535). From the measured fluorescence intensity, the amount of fucosylated complex double-stranded sugar chain produced by the enzyme reaction is calculated. Enzyme specific activity in a sample is expressed in moles of fucosylated complex double-stranded sugar chain molecules per unit protein mass contained in the sample and per unit reaction time. (Unit: pmol / hour / mg). The amount of protein in the sample is measured with BCA protein assembly kit (Pierce) using ushi serum albumin (Pierce) as a standard product.
3.本発明の α -1,6-フコシルトランスフェラーゼ変異体を認識する抗体の調製 本発明のひ -1,6-フコシルトランスフェラーゼ変異体、またそれらひ -1,6-フコシルト ランスフェラーゼ変異体の部分断片ポリペプチドの精製標品、あるいは本発明のひ- 1，6-フコシルトランスフェラーゼ変異体の一部のアミノ酸配列を有するペプチドを抗原 として用いることにより、ポリクローナル抗体、モノクローナル抗体等、本発明のひ -1,6 -フコシルトランスフェラーゼ変異体を認識する抗体を作製することができる。  3. Preparation of antibodies recognizing α-1,6-fucosyltransferase mutants of the present invention The -1,6-fucosyltransferase mutants of the present invention, and parts of these -1,6-fucosyltransferase mutants By using a purified product of a fragment polypeptide or a peptide having a partial amino acid sequence of the -1,6-fucosyltransferase variant of the present invention as an antigen, polyclonal antibodies, monoclonal antibodies, etc. Antibodies that recognize 1,6-fucosyltransferase mutants can be generated.
(1)ポリクローナル抗体の作製  (1) Production of polyclonal antibodies
本発明のひ -1,6-フコシルトランスフェラーゼ変異体、またそれらひ -1,6-フコシルト ランスフェラーゼ変異体の部分断片ポリペプチドの精製標品、あるいは本発明のひ- 1，6-フコシルトランスフェラーゼ変異体の一部のアミノ酸配列を有するペプチドを抗原 として用い、動物に投与することによりポリクローナル抗体を作製することができる。  A purified preparation of the -1,6-fucosyltransferase mutant of the present invention, a partial fragment polypeptide of the -1,6-fucosyltransferase mutant, or the -1,6-fucosyltransferase mutant of the present invention A polyclonal antibody can be prepared by administering a peptide having a partial amino acid sequence of the body as an antigen to an animal.
[0098] 投与する動物として、ゥサギ、ャギ、ラット、マウス、ハムスター等を用いることができ る。 [0098] Usagi, goat, rat, mouse, hamster and the like can be used as an animal to be administered.
該抗原の投与量は動物 1匹当たり 50〜： 100 μ §が好ましい。 The dose of antigen is 50 per animal: 100 mu § are preferred.
ペプチドを用いる場合は、ペプチドをスカシガイへモシァニン（keyhole limpet haem ocyanin)ゃ牛チログロブリンなどのキャリア蛋白に共有結合させたものを抗原とするの が望ましい。抗原とするペプチドは、ペプチド合成機で合成することができる。  When a peptide is used, it is desirable that the peptide is covalently bound to a carrier protein such as keyhole limpet haem ocyanin or bovine thyroglobulin. The peptide used as an antigen can be synthesized with a peptide synthesizer.
[0099] 該抗原の投与は、 1回目の投与の後:!〜 2週間おきに 3〜： 10回行う。各投与後、 3 〜7日目に眼底静脈叢より採血し、該血清が免疫に用いた抗原と反応することを酵 素免疫測定法〔酵素免疫測定法 (ELISA法）：医学書院刊（1976)、 Antibodies-A La boratory Manual, Cold Spring Harbor Laboratory (1988)〕等で確認、する。 [0099] The administration of the antigen is carried out after the first administration:! To 3 to 10 times every 2 weeks. On the 3rd to 7th day after each administration, blood is collected from the fundus venous plexus and the serum reacts with the antigen used for immunization. The enzyme immunoassay (ELISA): published by the medical school (1976) ), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)].
免疫に用いた抗原に対し、その血清が充分な抗体価を示した非ヒト哺乳動物より血 清を取得し、該血清を分離、精製することによりポリクローナル抗体を取得することが できる。  A polyclonal antibody can be obtained by obtaining serum from a non-human mammal whose serum showed a sufficient antibody titer against the antigen used for immunization, and separating and purifying the serum.
[0100] 分離、精製する方法としては、遠心分離、 40〜50%飽和硫酸アンモニゥムによる 塩析、カプリノレ酸沈殿 [Antibodies, A Laboratory manual, Cold Spring Harbor Labor atory (1988)〕、または DEAE—セファロースカラム、陰イオン交換カラム、プロテイン Aまたは G—力ラムあるいはゲノレ濾過カラム等を用いるクロマトグラフィー等を、単独 または組み合わせて処理する方法があげられる。 [0100] Separation and purification methods include centrifugation, salting out with 40-50% saturated ammonium sulfate, and caprinolic acid precipitation [Antibodies, A Laboratory manual, Cold Spring Harbor Labor atory (1988)], or chromatography using a DEAE-Sepharose column, an anion exchange column, protein A or G-force ram or Genore filtration column, etc., alone or in combination.
(2)モノクローナル抗体の作製 (2) Production of monoclonal antibodies
(a)抗体産性細胞の調製  (a) Preparation of antibody-producing cells
免疫に用いた本発明のひ- 1，6-フコシルトランスフェラーゼ変異体の部分断片ポリ ペプチドに対し、その血清が十分な抗体価を示したラットを抗体産生細胞の供給源と して供する。  A rat whose serum showed a sufficient antibody titer against the partial fragment polypeptide of the -1,6-fucosyltransferase mutant of the present invention used for immunization is used as a source of antibody-producing cells.
該抗体価を示したラットに抗原物質を最終投与した後 3〜7日目に、脾臓を摘出す る。  The spleen is removed 3 to 7 days after the final administration of the antigenic substance to the rat showing the antibody titer.
該脾臓を MEM培地（日水製薬社製）中で細断し、ピンセットでほぐし、 1 , 200rpm で 5分間遠心分離した後、上清を捨てる。  The spleen is shredded in MEM medium (Nissui Pharmaceutical Co., Ltd.), loosened with tweezers, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded.
得られた沈殿画分の脾細胞をトリス一塩化アンモニゥム緩衝液 (ρΗ7· 65)で:!〜 2 分間処理し赤血球を除去した後、 MEM培地で 3回洗浄し、得られた脾細胞を抗体 産生細胞として用いる。  Treat the spleen cells of the resulting precipitate fraction with Tris ammonium monochloride buffer (ρΗ7 · 65):! ~ 2 minutes to remove erythrocytes, and then wash with MEM medium 3 times. Used as production cells.
(b)骨髄腫細胞の調製  (b) Preparation of myeloma cells
骨髄腫細胞としては、マウスまたはラットから取得した株化細胞を使用する。例えば 、 8—ァザグァニン耐性マウス（8八し8ん由来）骨髄腫細胞株 P3-X63Ag8-Ul (以下、 P 3— U1と略す）〔Curr. Topics. Microbiol. Immunol., 81, 1 (1978)、 Europ. J. Immunol. ， 6, 511 (1976)] , SP2/0-Agl4(SP-2) [Nature, 276, 269 (1978)〕、 P3— X63— Ag8653(65 3) [J. Immunol., 123, 1548 (1979)〕、 P3-X63-Ag8(X63) [Nature, 256, 495 (1975)〕等を 用いることができる。これらの細胞株は、 8—ァザグァニン培地〔RPMI_ 1640培地 にグノレタミン（1. 5mmol/L) , 2—メルカプトエタノール（5 X 10— 5mol/L)、ジェンタ マイシン（10 x gZml)および牛胎児血清（FCS) (CSL社製、 10。/o)を加えた培地（ 以下、正常培地という）に、さらに 8—ァザグァニン（15 x gZml)をカ卩えた培地〕で継 代するが、細胞融合の 3〜4日前に正常培地で培養し、融合には該細胞を 2 X 107個 以上用いる。 As myeloma cells, cell lines obtained from mice or rats are used. For example, an 8-azaguanine resistant mouse (derived from 8-8) myeloma cell line P3-X63Ag8-Ul (hereinafter abbreviated as P3-U1) [Curr. Topics. Microbiol. Immunol., 81, 1 (1978) , Europ. J. Immunol., 6, 511 (1976)], SP2 / 0-Agl4 (SP-2) [Nature, 276, 269 (1978)], P3— X63— Ag8653 (65 3) [J. Immunol , 123, 1548 (1979)], P3-X63-Ag8 (X63) [Nature, 256, 495 (1975)], and the like. These cell lines include 8-Azaguanin medium [RPMI_ 1640 medium Gunoretamin (1. 5mmol / L), 2- mercaptoethanol (5 X 10- 5 mol / L ), gentamicin (10 x gZml) and fetal bovine serum (FCS) (CSL, 10./o) added medium (hereinafter referred to as normal medium), further subcultured with 8-azaguanine (15 x gZml). Cultivate in normal medium 3-4 days ago and use at least 2 X 10 7 cells for fusion.
(c)ハイプリドーマの作製 (a)で取得した抗体産生細胞と（b)で取得した骨髄腫細胞を MEM培地または PB S (リン酸ニナトリウム 1. 83g、リン酸一カリウム 0· 21g、食塩 7. 65g、蒸留水 1リットル 、 pH7. 2)でよく洗浄し、細胞数が、抗体産生細胞：骨髄腫細胞 = 5〜： 10 : 1になるよ う混合し、 1 , 200rpmで 5分間遠心分離した後、上清を捨てる。 (c) Production of High Pridoma The antibody-producing cells obtained in (a) and the myeloma cells obtained in (b) are mixed with MEM medium or PBS (disodium phosphate 1.83 g, monopotassium phosphate 0.21 g, sodium chloride 7.65 g, distilled water 1 Wash well with 1 liter, pH 7.2), mix so that the number of cells is antibody-producing cells: myeloma cells = 5 to: 10: 1, and centrifuge at 1,200 rpm for 5 minutes, then remove the supernatant. throw away.
[0102] 得られた沈澱画分の細胞群をよくほぐし、該細胞群に、攪拌しながら、 37°Cで、 108 抗体産生細胞あたり、ポリエチレングライコール— 1000 (PEG— 1000) 2g、 MEM 2mlおよびジメチルスルホキシド（DMS〇）0. 7mlを混合した溶液を 0. 2〜lml添カロ し、さらに 1〜2分間毎に MEM培地 l〜2mlを数回添加する。添加後、 MEM培地を 加えて全量が 50mlになるように調製する。該調製液を 900rpmで 5分間遠心分離後 、上清を捨てる。得られた沈殿画分の細胞を、ゆるやかにほぐした後、メスピペットに よる吸込み、吹出しでゆるやかに HAT培地〔正常培地にヒポキサンチン（10— 4mol/ L)、チミジン（1. 5 X 10— 5molZL)およびアミノプテリン（4 X 10— 7molZL)を加えた 培地〕 100ml中に懸濁する。 [0102] The cell group of the obtained precipitate fraction is thoroughly loosened, and the cell group is stirred at 37 ° C, and 10 g of polyethylene glycol-1000 (PEG-1000) per 10 8 antibody-producing cells, MEM Add 0.2 ml of a solution containing 2 ml and 0.7 ml of dimethyl sulfoxide (DMS 0), and add 1-2 ml of MEM medium several times every 1-2 minutes. After the addition, add MEM medium to prepare a total volume of 50 ml. Centrifuge the preparation at 900 rpm for 5 minutes, and discard the supernatant. After the precipitated fraction of the cells obtained was gently loosened, suction by measuring pipette, gently HAT medium [normal medium hypoxanthine (10- 4 mol / L) at blowing, thymidine (1. 5 X 10 - 5 molZL) and aminopterin (4 X 10- 7 molZL) was added medium] is suspended in 100 ml.
[0103] 該懸濁液を 96穴培養用プレートに 100 μ 1/穴ずつ分注し、 5% COインキュベー ター中、 37°Cで 7〜： 14日間培養する。  [0103] The suspension is dispensed at 100 µl / well into a 96-well culture plate, and cultured in a 5% CO incubator at 37 ° C for 7 to 14 days.
培養後、培養上清の一部をとりアンチボディィズ [Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory, Chapter 14 (1988)〕等に述べられている酵素免疫 測定法により、本発明の α -1,6-フコシルトランスフェラーゼ変異体の部分断片ポリべ プチドに特異的に反応するハイプリドーマを選択する。  After culturing, a part of the culture supernatant is removed and subjected to the enzyme immunoassay described in Antibodies [Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory, Chapter 14 (1988)], etc. Select a hyperidoma that specifically reacts with a partial fragment polypeptide of a 1,6-fucosyltransferase mutant.
[0104] 酵素免疫測定法の具体的例として、以下の方法をあげることができる。 [0104] Specific examples of the enzyme immunoassay include the following methods.
免疫の際、抗原に用いた本発明の α -1,6-フコシルトランスフェラーゼ変異体の部 分断片ポリペプチドを適当なプレートにコートし、ハイプリドーマ培養上清もしくは後 述の（d)で得られる精製抗体を第一抗体として反応させ、さらに第二抗体としてピオ チン、酵素、化学発光物質あるいは放射線化合物等で標識した抗ラットまたは抗マウ スィムノグロブリン抗体を反応させた後に標識物質に応じた反応を行ない、本発明の ひ- 1，6-フコシルトランスフェラーゼ変異体に特異的に反応するものを本発明のひ -1, 6-フコシルトランスフェラーゼ変異体を認識するモノクローナル抗体を生産するハイ プリドーマとして選択する。 [0105] 該ハイブリドーマを用いて、限界希釈法によりクローニングを 2回繰り返し〔1回目は 、 HT培地（HAT培地からアミノプテリンを除いた培地）、 2回目は、正常培地を使用 する〕、安定して強い抗体価の認められたものを本発明の α -1 ,6-フコシルトランスフ ヱラーゼ変異体を認識するモノクローナル抗体を産生するハイプリドーマ株として選 択する。 In immunization, an α-1,6-fucosyltransferase mutant partial fragment polypeptide of the present invention used as an antigen is coated on an appropriate plate, and is obtained in a hyperidoma culture supernatant or as described in (d) below. The purified antibody is reacted as the first antibody, and the anti-rat or anti-mouse immunoglobulin antibody labeled with piotin, enzyme, chemiluminescent substance or radiation compound as the second antibody is reacted, and then the reaction according to the labeling substance. And the one that specifically reacts with the -1,6-fucosyltransferase mutant of the present invention is selected as a hybridoma that produces a monoclonal antibody that recognizes the -1,6-fucosyltransferase mutant of the present invention. . [0105] Using the hybridoma, cloning was repeated twice by the limiting dilution method (first time using HT medium (medium obtained by removing aminopterin from HAT medium), second time using normal medium), and stable. And having a strong antibody titer is selected as a hyperidoma strain producing a monoclonal antibody that recognizes the α-1,6-fucosyltransferase mutant of the present invention.
(d)モノクローナル抗体の調製  (d) Preparation of monoclonal antibody
プリスタン処理〔2, 6， 10, 14—テトラメチルペンタデカン（Pristane) O. 5mlを腹 腔内投与し、 2週間飼育する〕した 8〜： 10週令のマウスまたはヌードマウスに、 (c)で 取得した本発明のひ -1 ,6 -フコシルトランスフェラーゼ変異体を認識するモノクローナ ル抗体産生ハイブリドーマ細胞 5〜20 X 106細胞/匹を腹腔内に注射する。 10〜2 1日間でハイプリドーマは腹水癌化する。 Treated with pristane [2, 6, 10, 14-tetramethylpentadecane (Pristane) O. 5ml intraperitoneally and bred for 2 weeks] 8 ~: For 10-week-old mice or nude mice, (c) Fei -1 acquired present invention, 6 - recognizes fucosyltransferase variant monoclonal antibody-producing hybridoma cells 5 to 20 X 10 6 cells / mouse are injected intraperitoneally. Hypridoma becomes ascites tumor in 10-2 days.
該腹水癌化したマウスから腹水を採取し、 3000rpmで 5分間遠心分離して固形分 を除去する。  Ascites fluid is collected from the ascites-bearing mouse and centrifuged at 3000 rpm for 5 minutes to remove solids.
[0106] 得られた上清より、ポリクローナルで用いた方法と同様の方法でモノクローナル抗体 を精製、取得することができる。  [0106] From the obtained supernatant, a monoclonal antibody can be purified and obtained by the same method as that used for polyclonal.
抗体のサブクラスの決定は、マウスモノクローナル抗体タイピングキットまたはラット モノクローナル抗体タイピングキットを用いて行う。蛋白質量は、ローリー法あるいは 2 80nmでの吸光度より算出する。  The antibody subclass is determined using a mouse monoclonal antibody typing kit or a rat monoclonal antibody typing kit. The protein mass is calculated from the Raleigh method or absorbance at 280 nm.
4.本発明の α -1 ,6-フコシルトランスフェラーゼ変異体を発現する細胞の調製  4. Preparation of cells expressing the α -1,6-fucosyltransferase mutant of the present invention
( 1 ) a -1 ,6-フコシルトランスフェラーゼ変異体を発現する細胞の調製  (1) Preparation of a cell expressing a -1,6-fucosyltransferase mutant
本発明の α -1 ,6-フコシルトランスフェラーゼ変異体を発現する細胞は、上記 2.の 本発明のひ -1 ,6 -フコシルトランスフヱラーゼ変異体の製造に記載した方法を用いる ことで様々な宿主細胞を用いて作製することができる。  Cells expressing the α -1,6-fucosyltransferase mutant of the present invention can be used in various ways by using the method described in the production of the -1,6 -fucosyltransferase mutant of the present invention in 2. above. It can be produced using host cells.
[0107] この場合、 ひ -1 ,6 -フコース修飾酵素を発現していない宿主細胞を用いることにより 、本発明のひ -1 ,6-フコシルトランスフェラーゼ変異体由来のひ -1，6-フコシルトランス フェラーゼ活性のみ有する細胞を調製することができる。 [0107] In this case, by using a host cell that does not express the -1,6-fucose modifying enzyme, the -1,6-fucosyltransferase derived from the -1,6-fucosyltransferase mutant of the present invention is used. Cells having only ferrase activity can be prepared.
また、 ひ- 1，6-フコース修飾酵素の遺伝子を標的とし、ゲノム遺伝子改変の方法を 用レヽることにより、本発明のひ -1 ,6-フコシルトランスフェラーゼ変異体由来のひ- 1 ,6- フコース修飾酵素活性のみ有する細胞を作製することができる。 a フコース修 飾酵素としては、具体的には、 α -1 ,6-フコシルトランスフェラーゼがあげられる。 In addition, by targeting the gene of -1,6-fucose modifying enzyme and using the method of genomic gene modification, -1,6-6-fucosyltransferase mutant-derived -1, -1,6-fucose transferase mutant of the present invention is used. Cells having only fucose modifying enzyme activity can be produced. Specific examples of the a- fucose modifying enzyme include α -1,6-fucosyltransferase.
[0108] ゲノム遺伝子改変の方法としては、標的とする酵素のゲノム遺伝子を特異的に改変 することができる方法であればいかなる方法も包含される。その例としては、相同組換 え法、 RDO法、レトロウイルスを用いた方法、トランスポゾンを用いた方法等があげら れる。以下これらを具体的に説明する。 [0108] As a method for modifying a genomic gene, any method can be used as long as it can specifically modify the genomic gene of the target enzyme. Examples thereof include homologous recombination method, RDO method, method using retrovirus, method using transposon, and the like. These will be specifically described below.
(a)相同組換え法を用いたゲノム遺伝子改変の方法による、本発明のひ- 1，6_フコシ ノレトランスフェラーゼ変異体を発現する細胞の作製  (a) Preparation of cells expressing the -1,6_fucosinoretransferase mutant of the present invention by a method of genomic gene modification using homologous recombination method
本発明のひ -1 ,6-フコシルトランスフェラーゼ変異体を発現する細胞は、 ひ -1 ,6-フ コース修飾酵素の遺伝子を標的とし、染色体上の標的遺伝子を相同組換え法を用 い改変することによって作製することができる。  The cell expressing the -1,6-fucosyltransferase mutant of the present invention targets the -1,6-fucose modifying enzyme gene, and modifies the target gene on the chromosome using the homologous recombination method. Can be produced.
[0109] 染色体上の標的遺伝子の改変は、 Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994) (以下、「マ二 ピュレイティング'ザ'マウス'ェンブリオ 'ァ.ラボラトリ一'マニュアル」と略す）、 Gene T argeting, A Practical Approach, IRL Press at Oxford University Press (1993)、 ノヽ ォマニュアルシリーズ 8ジーンターゲッティング， ES細胞を用いた変異マウスの作製， 羊土社（1995) (以下、「ES細胞を用いた変異マウスの作製」と略す)等に記載の染色 体工学の手法を用い、例えば以下のように行うことができる。 [0109] The modification of target genes on chromosomes is described in Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994) (hereinafter referred to as "Mani Pureating 'The' Mouse 'Embryo' A. Abbreviated 'manual'), Gene Taringing, A Practical Approach, IRL Press at Oxford University Press (1993), Non-Manual Series 8 Gene Targeting, Production of Mutant Mice Using ES Cells, Yodosha (1995) ( Hereinafter, the method of chromosome engineering described in “abbreviation of mutant mouse using ES cells”) can be used, for example, as follows.
[0110] α -1 ,6-フコース修飾酵素の cDNAを取得する。  [0110] Obtain cDNA of α -1,6-fucose modifying enzyme.
取得した cDNAの塩基配列に基づき、 α -1 ,6-フコース修飾酵素のゲノム DNAを 調製する。  Prepare genomic DNA of α-1,6-fucose modifying enzyme based on the obtained cDNA base sequence.
該ゲノム DNAの塩基配列にも基づき、改変する標的遺伝子（例えば、 ひ -1 ,6 -フコ ース修飾酵素の構造遺伝子、あるいはイントロン遺伝子）を相同組換えするためのタ 一ゲットベクターを作製する。  Based on the base sequence of the genomic DNA, a target vector for homologous recombination of the target gene to be modified (for example, the structural gene of -1,6-fucosase modifying enzyme or intron gene) is prepared. .
[0111] 作製したターゲットベクターを細胞に導入し、標的遺伝子とターゲットベクターの間 で相同組換えを起こした細胞を選択することにより、本発明のひ- 1，6-フコシルトラン スフエラーゼ変異体を発現する細胞を作製することができる。 [0111] The produced target vector is introduced into a cell, and a cell that has undergone homologous recombination between the target gene and the target vector is selected to express the -1,6-fucosyltransferase gene of the present invention. Cells can be made.
a -1，6 -フコース修飾酵素の cDNA及びゲノム DNAを取得する方法としては、例え ば、上記 1. に記載の方法があげられる。 A method for obtaining cDNA and genomic DNA of a -1,6-fucose modifying enzyme is, for example, For example, the method described in 1. above can be mentioned.
[0112] 標的遺伝子を相同組換えするためのターゲットベクターは、 Gene Targeting, A Pra ctical Approach, IRL Press at Oxford University Press (1993)、 ノくィォマニユアノレシリ ーズ 8ジーンターゲッティング， ES細胞を用いた変異マウスの作製 (羊土社) (1995)等 に記載の方法にしたがって作製することができる。ターゲットベクターは、リプレースメ ント型、インサーシヨン型、ジーントラップ型いずれでも用いることができる。  [0112] Target vectors for homologous recombination of target genes are: Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Nokumanyu Anoreno series 8 Gene Targeting, ES cells Production of a mutant mouse using a mouse (Yodosha) (1995) and the like. The target vector may be any of a replacement type, an insertion type, or a gene trap type.
[0113] 細胞へのターゲットベクターの導入方法としては、動物細胞に DNAを導入する方 法であればいずれも用いることができ、例えば、エレクト口ポレーシヨン法 [サイトテクノ ロジー (Cytotechnology), 3, 133 (1990)]、リン酸カルシウム法 [特開平 2-227075]、リ ポフヱクシヨン法 [プロシーディンダス ·ォブ ·ザ ·ナショナノレ .ァカデミ一.ォブ .サイエ ンス (Proc. Natl. Acad. Sci. U.S.A.), 84, 7413 (1987)]、インジェクション法 [Manipulati ng the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor La boratory Press (1994) (以下、マニピユレ一ティング'マウス'ェンブリオ第 2版と略す）] 、パーティクルガン (遺伝子銃）を用いる方法 [特許第 2606856、特許第 2517813]、 D EAE デキストラン法 [バイオマニュアルシリーズ 4 遺伝子導入と発現.解析法（羊 土社)横田崇'新井賢一編 (1994)]、ウィルスベクター法 [マニピユレ一ティング 'マウス •ェンブリオ第 2版]等をあげることができる。  [0113] As a method for introducing a target vector into a cell, any method can be used as long as it introduces DNA into animal cells. For example, the electoral position method [Cytotechnology, 3, 133]. (1990)], calcium phosphate method [Japanese Patent Laid-Open No. 2-227075], reflection method [Procida Natl. Acad. Sci. USA], 84, 7413 (1987)], injection method [Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994) (hereinafter abbreviated as "Manipulating" Mouse "Embryo Second Edition)], Method using particle gun [Gene Gun] [Patent No. 2606856, Patent No. 2517813], D EAE Dextran Method [Biomanual Series 4 Gene Introduction and Expression. Analytical Method (Sheepsoil) Takashi Yokota, Kenichi Arai (1 994)], the virus vector method [Manipulating “Mouse • Embryo 2nd edition”] and the like.
[0114] 相同組換え体を効率的に選別する方法として、例えば、 Gene Targeting, A Practic al Approach, IRL Press at Oxford University Press (1993)、 ノくィォマニユアノレシリー ズ 8ジーンターゲッティング， ES細胞を用いた変異マウスの作製 (羊土社) (1995)等に 記載のポジティブ選択、プロモーター選択、ネガティブ選択、ポリ A選択などの方法 を用いることができる。具体的には、 hprt遺伝子を含むターゲットベクターの場合は、 hprt遺伝子を欠損した細胞に導入後、該細胞をアミノプテリン、ヒポキサンチンおよび チミジンを含む培地で培養し、アミノプテリン耐性の株を選別することにより、 hprt遺伝 子を含む相同組換え体を選別するポジティブ選択を行なうことができる。ネオマイシ ン耐性遺伝子を含むターゲットベクターの場合は、ベクターを導入した細胞を G418を 含む培地で培養し、 G418耐性の株を選別することにより、ネオマイシン耐性遺伝子を 含む相同組換え体を選別するポジティブ選択を行なうことができる。 DT遺伝子を含 むターゲットベクターの場合は、ベクターを導入した細胞を培養し、生育してきた株を 選別する（相同組換え以外のランダムに染色体に挿入された組換え体は、 DT遺伝子 が染色体に組み込まれて発現するため、 DTの毒性により生育できない）ことにより、 D T遺伝子を含まない相同組換え体を選別するネガティブ選択を行なうことができる。 選別した細胞株の中から目的とする相同組換え体を選択する方法としては、ゲノム D NAに対するサザンハイブリダィゼーシヨン法（モレキユラ一'クローニング第 2版）や P CR法 [ピーシーアール.プロトコールズ (PCR Protocols), Academic Press (1990)]等 があげられる。 [0114] As a method for efficiently selecting homologous recombinants, for example, Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Nokumanyu Announceries 8 Gene Targeting, ES Preparation of mutant mice using cells (Yodosha) (1995) and other methods such as positive selection, promoter selection, negative selection, and poly A selection can be used. Specifically, in the case of a target vector containing the hprt gene, after introduction into a cell lacking the hprt gene, the cell is cultured in a medium containing aminopterin, hypoxanthine and thymidine, and an aminopterin resistant strain is selected. Thus, positive selection for selecting homologous recombinants containing the hprt gene can be performed. In the case of a target vector containing a neomycin resistance gene, positive selection for selecting homologous recombinants containing a neomycin resistance gene by culturing cells into which the vector has been introduced in a medium containing G418 and selecting a G418 resistant strain. Can be performed. Contains DT gene In the case of a target vector, the cells into which the vector has been introduced are cultured, and the growing strain is selected (in the case of recombinants randomly inserted into the chromosome other than homologous recombination, the DT gene is integrated into the chromosome and expressed. Therefore, negative selection for selecting homologous recombinants that do not contain the DT gene can be performed. Methods for selecting the desired homologous recombinants from the selected cell lines include the Southern hybridization method (Molequila's Cloning 2nd edition) for genomic DNA and the PCR method [PC protocol. (PCR Protocols, Academic Press (1990)].
(b)RDO方法による、本発明のひ -1,6 -フコシルトランスフェラーゼ変異体を発現する 細胞の作製  (b) Preparation of cells expressing the -1,6-fucosyltransferase mutant of the present invention by the RDO method
本発明のひ -1,6-フコシルトランスフェラーゼ変異体を発現する細胞は、 ひ -1,6-フ コース修飾酵素の遺伝子を標的とし、 RDO (RNA-DNA oligonucleotide)法を用い、 例えば、以下のように作製することができる。  Cells expressing the -1,6-fucosyltransferase mutant of the present invention target the -1,6-fucose modifying enzyme gene and use the RDO (RNA-DNA oligonucleotide) method. Can be produced as follows.
[0115] α -1,6-フコース修飾酵素の cDNAあるいはゲノム DNAを調製する。 [0115] Prepare cDNA or genomic DNA of α-1,6-fucose modifying enzyme.
調製した cDNAあるいはゲノム DNAの塩基配列を決定する。  Determine the base sequence of the prepared cDNA or genomic DNA.
決定した DNAの配列に基づき、 α -1,6-フコース修飾酵素をコードする部分、非翻 訳領域の部分あるいはイントロン部分を含む適当な長さの RDOのコンストラクトを設 計し合成する。  Based on the determined DNA sequence, an RDO construct of an appropriate length including the portion encoding α-1,6-fucose modifying enzyme, the untranslated region, or the intron portion is designed and synthesized.
[0116] 合成した RDOを細胞に導入し、標的とした酵素、すなわち α -1,6-フコース修飾酵 素に変異が生じた細胞を選択することにより、本発明の α -1,6-フコシルトランスフェラ ーゼ変異体を発現する細胞を作製することができる。 [0116] By introducing the synthesized RDO into a cell and selecting the target enzyme, ie, the cell in which the α -1,6-fucose modified enzyme is mutated, the α -1,6-fucosyl of the present invention is selected. Cells expressing the transferase mutant can be generated.
細胞への RD〇の導入には、上記 4.の（a)に記載のターゲットベクターの導入方法 を用いることができる。  For the introduction of RD0 into cells, the method for introducing a target vector described in 4. (a) above can be used.
[0117] ひ- 1，6-フコース修飾酵素の cDNAを調製する方法としては、例えば、上記 1.に記 載の cDNAの調製方法などがあげられる。  [0117] Examples of a method for preparing cDNA for -1,6-fucose modifying enzyme include the method for preparing cDNA described in 1. above.
ひ- 1，6 -フコース修飾酵素のゲノム DNAを調製する方法としては、例えば、上記 1. に記載のゲノム DNAの調製方法などがあげられる。  Examples of the method for preparing genomic DNA of H-1,6-fucose modifying enzyme include the method for preparing genomic DNA described in 1. above.
DNAの塩基配列は、適当な制限酵素などで切断後、 pBluescript SK (-) (Stratagen e社製)等のプラスミドにクローニングし、通常用いられる塩基配列解析方法、例えば 、サンガー（Sanger)らのジデォキシ法 [プロシーディングス.ォブ.ザ.ナショナル.ァカ デミ一'ォブ'サイエンス (Pro Natl. Acad. Sci., U.S.A.), 74, 5463 (1977)]等の反応 を行い、塩基配列自動分析装置、例えば、 A.し F. DNAシークェンサ一（Pharmacia 社製）等を用いて解析することで該 DNAの塩基配列を決定することができる。 The DNA base sequence is cleaved with an appropriate restriction enzyme, and then pBluescript SK (-) (Stratagen e), etc., and a commonly used nucleotide sequence analysis method such as Sanger et al.'s dideoxy method [Proceedings.Ob.The.Naka. Pro Natl. Acad. Sci., USA), 74, 5463 (1977)], etc., and analysis using an automatic base sequence analyzer such as A. and F. DNA Sequencer (Pharmacia) By doing so, the base sequence of the DNA can be determined.
[0118] RDOは、常法または DNA合成機を用いることにより調製することができる。  [0118] RDO can be prepared by a conventional method or using a DNA synthesizer.
RDOを細胞に導入し、標的とした酵素、 ひ -1,6 -フコース修飾酵素の遺伝子に変 異が生じた細胞を選択する方法としては、モレキュラー 'クローニング第 2版、カレント 'プロトコールズ.イン.モレキュラー.バイオロジー等に記載された染色体上の遺伝子 の変異を直接検出する方法があげられる。  As a method of introducing RDO into cells and selecting cells in which the target enzyme, -1,6-fucose modifying enzyme gene, has changed, Molecular 'Cloning 2nd Edition, Current' Protocols. A method for directly detecting gene mutations on chromosomes described in molecular biology and the like can be mentioned.
[0119] RDOのコンストラクトは、サイエンス (Science), 273, 1386, (1996);ネィチヤ一'メディ シン (Nature Medicine), 4, 285, (1998);へパトロジー (H印 atology), 25, 1462, (1997); ジーン.セラピー (Gene Therapy), 5, 1960, (1999);ジーン.セラピー (Gene Therapy), 5, 1960, (1999);ジャーナル'ォブ 'モレキュラー'メデイシン (J. Mol. Med.), 75, 829， ( 1997);プロシーディングス 'ォブ ·ザ'ナショナル'ァカデミ一'ォブ ·サイエンス (Pro Natl. Acad. Sci. USA), 96, 8774， (1999);プロシーディングス 'ォブ 'ザ'ナショナル' アカデミー'ォブ 'サイエンス (Proc. Natl. Acad. Sci. USA), 96, 8768， (1999);ヌクレイ ック'アシッド'リサーチ (Nu Acids. Res.), 27, 1323， (1999);インべスティゲーシヨン' ォブ'ダーマトロジー (Invest. Dematol.), HI, 1172, (1998);ネイチヤ^ ~ ·バイオテクノ ロジー (Nature Biotech.), 16, 1343, (1998);ネイチヤー'バイオテクノロジー (Nature Bi otech.), 18, 43, (2000);ネイチヤ^ ~ ·バイオテクノロジー (Nature Biotech.), 18, 555， (2 000)等の記載に従って設計することができる。  [0119] RDO constructs are described in Science, 273, 1386, (1996); Nichiya's Medicine (Nature Medicine), 4, 285, (1998); Hepatology (H-signed atology), 25, 1462. , (1997); Gene Therapy, 5, 1960, (1999); Gene Therapy, 5, 1960, (1999); Journal 'Ob' Molecular 'Medicin (J. Mol. Med.), 75, 829, (1997); Proceedings 'Ob The National' Academia Sci. USA (Pro Natl. Acad. Sci. USA), 96, 8774, (1999); Proceedings 'Ob' The 'National' Academy 'Ob' Science (Proc. Natl. Acad. Sci. USA), 96, 8768, (1999); Nucleic 'Acid' Research (Nu Acids. Res.), 27 , 1323, (1999); Investigation 'Ob' Dermatology (Invest. Dematol.), HI, 1172, (1998); Nature Biotech., 16, 1343, (1998); Nature's biotech It can be designed according to the description of Nature Biotech., 18, 43, (2000); Nature Biotech., 18, 555, (2 000), etc.
(c)トランスポゾンを用いた方法による、本発明のひ- 1，6-フコシルトランスフェラーゼ 変異体を発現する細胞の作製  (c) Production of a cell expressing the -1,6-fucosyltransferase mutant of the present invention by a method using a transposon
本発明のひ -1,6 -フコシルトランスフェラーゼ変異体を発現する細胞は、ネイチヤー 'ジエネテイク (Nature Genet.), 25, 35，（2000)等に記載のトランスポゾンのシステムを 用レ、、 ひ -1,6-フコース修飾酵素の突然変異体を選択することで作製することができ る。 [0120] トランスポゾンのシステムとは、外来遺伝子をランダムに染色体上に挿入させること で突然変異を誘発させるシステムであり、通常、トランスポゾンに挿まれた外来遺伝子 を突然変異を誘発させるベクターとして用い、この遺伝子を染色体上にランダムに挿 入させるためのトランスポゼースの発現ベクターを同時に細胞の中に導入する。 トランスポゼースは、用いるトランスポゾンの配列に適したものであればいかなるもの も用レ、ること力 Sできる。 A cell expressing the -1,6-fucosyltransferase mutant of the present invention can be obtained by using the transposon system described in Nature Genet., 25, 35, (2000), etc. Therefore, it can be prepared by selecting mutants of 6-fucose modifying enzyme. [0120] The transposon system is a system that induces mutations by randomly inserting foreign genes onto chromosomes. Usually, a foreign gene inserted into a transposon is used as a vector to induce mutations. A transposase expression vector for randomly inserting the gene into the chromosome is introduced into the cell at the same time. Any transposase that is suitable for the transposon sequence to be used can be used.
[0121] 外来遺伝子としては、細胞の DNAに変異を誘起するものであればレ、かなる遺伝子 も用レ、ること力 Sできる。  [0121] As a foreign gene, any gene that induces a mutation in cellular DNA can be used.
細胞への遺伝子の導入には、上記 4.の（a)に記載のターゲットベクターの導入方 法を用いることができる。  For introduction of a gene into a cell, the method for introducing a target vector described in (a) of 4. above can be used.
(2) a -1，6-フコシルトランスフェラーゼ変異体の活性測定  (2) Activity measurement of a-1,6-fucosyltransferase mutant
本発明のひ -1 ,6-フコシルトランスフェラーゼ変異体及びひ -1 ,6-フコシルトランスフ エラーゼの活性の測定方法としては、 2(2)記載の方法に準じて、調整した細胞に含 まれる本発明の α -1 ,6-フコシルトランスフェラーゼ変異体の活性を測定する方法が あげられる。  As a method for measuring the activity of the -1,6-fucosyltransferase mutant and -1,6-fucosyltransferase of the present invention, the present method contained in cells prepared according to the method described in 2 (2). Examples include methods for measuring the activity of the α -1,6-fucosyltransferase mutant of the invention.
5.本発明の α -1 ,6-フコシルトランスフェラーゼ変異体、 DNAまたは抗体の利用 ( 1 )本発明の α -1 ,6-フコシルトランスフェラーゼ変異体をコードする DNAの発現を 検出'定量することによる疾患の判定方法  5. Use of α -1,6-fucosyltransferase mutant, DNA or antibody of the present invention (1) By detecting and quantifying the expression of DNA encoding the α -1,6-fucosyltransferase mutant of the present invention Disease determination method
本発明の α -1 ,6-フコシルトランスフェラーゼ変異体の DNAまたはオリゴヌクレオチ ドを用レ、、ノーザンハイブリダィゼーシヨン法（モレキュラー 'クローニング第 2版）、 PC R法および RT (reverse-transcribed)—PCR法〔ともに PCR Protocols, Academic Pre ss (1990)〕（以上、併せて PCR法ともいう）等を行レ、、本発明のひ- 1，6-フコシルトラン スフエラーゼ変異体をコードする DNAの発現を検出することで、網膜形成障害、肝 臓癌、脂肪肝、血液凝固障害、嚢胞性繊維症、肺組織障害、成長不良、インスリン様 増殖因子や成長ホルモン作用の障害を伴う疾患、上皮増殖因子の作用の障害を伴 う疾患等のひ- 1，6 -フコース修飾酵素との関連が報告されている疾患の判定を行うこ とができる。  Using the α -1,6-fucosyltransferase mutant DNA or oligonucleotide of the present invention, Northern hybridization method (Molecular 'Cloning 2nd edition), PCR method and RT (reverse-transcribed) — Perform PCR method (both PCR Protocols, Academic Press (1990)) (also referred to as PCR method above), etc., to detect the DNA encoding the mutant of 1,6-fucosyltransferase gene of the present invention. By detecting expression, retinal dysfunction, liver cancer, fatty liver, blood coagulation disorder, cystic fibrosis, lung tissue disorder, growth failure, diseases with impaired insulin-like growth factor and growth hormone action, epithelial proliferation It is possible to determine a disease that has been reported to be associated with a 1,6-fucose-modifying enzyme, such as a disease that is accompanied by impaired factor action.
[0122] RT— PCR法は簡便な方法であるため、 DNAの発現の検出法として特に有用であ る。 [0122] The RT-PCR method is a simple method and is particularly useful as a method for detecting DNA expression. The
例えば、配列番号 18、 19、 20、 21または 22で表される塩基配列中の連続した約 100 〜2000塩基と同じ配列を有する DNA、または該 DNAと相補的な配列を有する DN Aをプローブとしてノーザンハイブリダィゼーシヨンを行い、配列番号 18、 19、 20、 21ま たは 22に記載の DNAの発現量を定量し、健常者と比較することにより上記疾患を判 定すること力 Sできる。  For example, DNA having the same sequence as about 100 to 2000 bases in the base sequence represented by SEQ ID NO: 18, 19, 20, 21, or 22 or DNA having a sequence complementary to the DNA as a probe. The ability to determine the above-mentioned disease by performing Northern hybridization, quantifying the expression level of the DNA represented by SEQ ID NO: 18, 19, 20, 21, or 22 and comparing it with a healthy person .
[0123] 該判定の具体的方法として、例えば、以下の方法をあげることができる。  [0123] Specific examples of the determination include the following methods.
被検者および健常者の白血球細胞または組織由来全 RNA(10〜20 μ g)、または それらの ΠΙΚΝΑ (1〜5 Α^)を、変性溶液〔50% (ν/ν)ホルムアミド、 2. 2mol/Lホ ノレムァノレデヒド、 20mmol/L M〇PS [3 _ (N—モルホリノ）プロパンスルホン酸] (P H7. 0)、 5mmol/L酢酸ナトリウム、 lmmol/L EDTA]中、 65°Cで 5分間加熱し 、変性させ、 2. 2mol/Lホルムアルデヒドを含む 1 %ァガロースゲルで電気泳動す る。  Total RNA (10 to 20 μg) derived from white blood cells or tissues of subjects and healthy subjects, or their ΠΙΚΝΑ (1 to 5 Α ^), and denaturation solution (50% (ν / ν) formamide, 2.2 mol / L monooledehydride, 20mmol / LMºPS [3_ (N-morpholino) propanesulfonic acid] (PH 7.0), 5mmol / L sodium acetate, lmmol / L EDTA] at 65 ° C, 5 Heat for minutes, denature, and run on a 1% agarose gel containing 2.2 mol / L formaldehyde.
[0124] 電気泳動後、ゲル中の RNAをニトロセルロースフィルター（Optimal BA-S85; Schlei cher & Schuell社製)上にブロッテイングし、減圧下 80°Cで 1時間加熱し固定化する。 該フィルターをハイブリダィゼーシヨン溶液〔5 X SSPE (750mmol/L NaCl、 50 mmol/L NaH P〇、 5mmol/L EDTA; pH7. 4)、 5 Xデンハルト溶液（0. 1 %  [0124] After electrophoresis, the RNA in the gel is blotted on a nitrocellulose filter (Optimal BA-S85; manufactured by Schleicher & Schuell) and immobilized by heating at 80 ° C for 1 hour under reduced pressure. The filter was mixed with a hybridization solution [5 X SSPE (750 mmol / L NaCl, 50 mmol / L NaHPO, 5 mmol / L EDTA; pH 7.4), 5 X Denhardt solution (0.1%
2 4  twenty four
フイコーノレ、 0. 1%ポリビエノレピロリドン、 0. 1 %ゥシ血清アルブミン）、 1%SDS (ドデ シル硫酸ナトリウム）、 0. 2mg/mlのサケ精子 DNA(Pharmacia Biotech社製)〕中に 浸漬しプレハイブリダィゼーシヨンを行う。  (Fuconore, 0.1% polybienopyrrolidone, 0.1% ushi serum albumin), 1% SDS (sodium dodecyl sulfate), 0.2 mg / ml salmon sperm DNA (Pharmacia Biotech)) Immerse and perform prehybridization.
[0125] プレハイブリダィゼーシヨン後、該溶液にプローブを添加し、 65°Cでハイブリダィゼ ーシヨンを行う。 [0125] After prehybridization, a probe is added to the solution, and hybridization is performed at 65 ° C.
プローブとしては、例えば配列番号 18、 19、 20、 21または 22に記載の DNA断片を マルチプライム DNA標識システム（アマシャム社製）を用いて32 Pで標識したものを使 用できる。 As the probe, for example, a DNA fragment represented by SEQ ID NO: 18, 19, 20, 21, or 22 labeled with 32 P using a multiprime DNA labeling system (Amersham) can be used.
[0126] ハイブリダィゼーシヨン後のフィルターを、以下の順で洗浄する。  [0126] The filter after the hybridization is washed in the following order.
(a) 0. 1%SDSを含む 2 X SSC (300mmolZL NaCl、 30mmol/Lタエン酸ナトリ ゥム)溶液中、室温で 15分間洗浄する。これを数回繰り返す。 (b) 0. 1 %SDSを含む l X SSC (150mmol/L NaCl、 15mmol/Lタエン酸ナトリ ゥム)溶液中、 50〜70°Cで 15分間洗浄する。これを数回繰り返す。 (a) Wash in a solution of 2X SSC (300 mmol ZL NaCl, 30 mmol / L sodium taenoate) containing 0.1% SDS at room temperature for 15 minutes. Repeat this several times. (b) Wash in lX SSC (150 mmol / L NaCl, 15 mmol / L sodium taenoate) solution containing 0.1% SDS for 15 minutes at 50-70 ° C. Repeat this several times.
(c) 50〜70。C 0. 1%SDSを含む 0. 1 X SSC (15mmol/L NaCl、 1. 5mmol/ Lクェン酸ナトリウム)溶液中、 50〜70°Cで 15分間洗浄する。これを数回繰り返す。  (c) 50-70. Wash in 0.1X SSC (15 mmol / L NaCl, 1.5 mmol / L sodium quenate) solution containing 0.1% SDS for 15 minutes at 50-70 ° C. Repeat this several times.
[0127] フィルター洗浄後、イメージングプレートを用いてオートラジオグラフィーを行レ、、バ ィォイメージングアナライザー BAS2000 (富士写真フィルム）で配列番号 18、 19、 20 、 21または 22に記載の DNAの発現を検出し定量することができる。  [0127] After the filter was washed, autoradiography was performed using an imaging plate, and the DNA of SEQ ID NO: 18, 19, 20, 21 or 22 was expressed using a bioimaging analyzer BAS2000 (Fuji Photo Film). Can be detected and quantified.
また、例えば、本発明のひ -1,6 -フコシルトランスフェラーゼ変異体をコードする DN Aに特異的な 1組のオリゴヌクレオチドをプライマーとして用レ、、被検者および健常者 の白血球細胞または組織由来全 RNA、それらの mRNAまたはこれら RNA力、ら調製 した cDNAを铸型として PCRを行い、増幅断片を検出'定量し、被験者と健常者の 該 DNAの発現量を比較することにより上記疾患を判定することができる。  Further, for example, a set of oligonucleotides specific for DNA encoding the -1,6-fucosyltransferase mutant of the present invention is used as a primer, derived from white blood cells or tissues of subjects and healthy subjects. Perform PCR using total RNA, their mRNA, or cDNA prepared from these RNAs, and detect and quantify the amplified fragments, and determine the above diseases by comparing the expression level of the DNA between the subject and healthy subject can do.
[0128] オリゴヌクレオチドとしては、上記 1.記載のオリゴヌクレオチドを用いることができる。 [0128] As the oligonucleotide, the oligonucleotide described in 1. above can be used.
PCRの铸型となる mRNAまたは全 RNAは、血液より分離'取得した各種白血球細 胞から、または組織としては疾患の疑いがある組織等から抽出できる。  MRNA or total RNA, which is a type of PCR, can be extracted from various leukocyte cells isolated and obtained from blood, or as a tissue suspected of having a disease.
各白血球細胞としては、多形核白血球、単球、リンパ球、 T細胞、 B細胞等をあげる こと力 Sできる。  Each white blood cell can be exemplified by polymorphonuclear leukocytes, monocytes, lymphocytes, T cells, B cells and the like.
[0129] 多形核白血球および単核球は、被検者の末梢血より、ナイコメッド'ファーマ (Nycom ed Pharma)社製のキットである Polymo卬 hprep™を用いることにより、分離'取得するこ とができる。  [0129] Polymorphonuclear leukocytes and mononuclear cells can be separated from the peripheral blood of the subject by using Polymo 卬 hprep ™, a kit manufactured by Nycomed Pharma. Can do.
取得した単核球より、 J. Immunol., 130, 706 (1983)等に記載の方法により、単球お よびリンパ球を、 Tissue Antigen, 9, 153 (1977)、 J. Immunol, 11, 273 (1976)、 Nycom ed社の血球細胞の単離法に関するマニュアル等に記載の方法により、 T細胞や B細 胞を分離 '取得することができる。  Monocytes and lymphocytes were obtained from the obtained mononuclear cells by the method described in J. Immunol., 130, 706 (1983), etc., in Tissue Antigen, 9, 153 (1977), J. Immunol, 11, 273. (1976), T cells and B cells can be separated and obtained by the method described in the manual on the isolation method of blood cells of Nycomed, Inc.
[0130] T細胞はナイロンウール法〔Eur. J. Immunol., 3, 645 (1973)〕を用いて取得すること もできる。また、 T細胞、 B細胞、単球/マクロファージにそれぞれ特異的な抗体を結 合した磁気ビーズ (例えば、 Dynal社製の Dynabeads)を用いて、各細胞を分離'取得 すること力 Sできる。 白血球細胞または組織から全 RNAを調製する方法としては、チォシアン酸グァニ ジン一トリフルォロ酢酸セシウム法 [Methods in Enzymol., 154, 3 (1987)〕等をあげる こと力 Sできる。 [0130] T cells can also be obtained using the nylon wool method [Eur. J. Immunol., 3, 645 (1973)]. Further, it is possible to separate and acquire each cell using magnetic beads (for example, Dynabeads manufactured by Dynal) in which specific antibodies are bound to T cells, B cells, and monocytes / macrophages. As a method for preparing total RNA from white blood cells or tissues, guanidine thiocyanate-trifluoroacetate method [Methods in Enzymol., 154, 3 (1987)] can be mentioned.
[0131] 全 RNAからポリ（A) +RNAを調製する方法としては、オリゴ（dT)固定化セルロース カラム法（モレキュラー ·クローニング第 2版)等をあげることができる。 [0131] Examples of a method for preparing poly (A) + RNA from total RNA include an oligo (dT) -immobilized cellulose column method (Molecular Cloning 2nd Edition).
また、ファースト 'トラック 'mRNA'アイソレーション 'キット〔Fast Track mRNA Isolati on Kit； Invitrogen社製〕、クイック ·プレップ 'mRNA'ピユリフィケーシヨン'キット（Quic k Prep mRNA Purification Kit ;フアルマシア社製）等のキットを用いて mRNAを調製 することちでさる。  In addition, first 'track' mRNA 'isolation' kit (Fast Track mRNA Isolation Kit; manufactured by Invitrogen), Quick Prep 'mRNA' purification kit (Quic k Prep mRNA Purification Kit; manufactured by Falmacia) Prepare mRNA using a kit such as the above.
[0132] 一本鎖 cDNAは、全 RNAまたは mRNAから、一本鎖 cDNA合成キット Superscript preamplification system (BRL社製）を用いて、合成することができる。合成は、キット に添付のマニュアルに従って行うことができる。  [0132] Single-stranded cDNA can be synthesized from total RNA or mRNA using a single-stranded cDNA synthesis kit Superscript preamplification system (manufactured by BRL). The synthesis can be performed according to the manual attached to the kit.
上記のように調製できる全 RNA、 mRNAまたは cDNAを用いた RT— PCR法〔PC R Protocols, Academic Press (1990)〕により、本発明の a _1，6_フコシルトランスフェラ ーゼ変異体をコードする遺伝子の発現量を定量することができる。  The a_1,6_fucosyltransferase mutant of the present invention is encoded by the RT-PCR method (PC R Protocols, Academic Press (1990)) using total RNA, mRNA or cDNA prepared as described above. The amount of gene expression can be quantified.
(2)本発明の α -1,6-フコシルトランスフェラーゼ変異体をコードする遺伝子のみを検 出することによる疾患の判定方法  (2) A method for determining a disease by detecting only the gene encoding the α-1,6-fucosyltransferase mutant of the present invention
本発明の α -1,6-フコシルトランスフェラーゼ変異体のオリゴヌクレオチドをプローブ として、ゲノム DNAに対してサザンハイブリダィゼーシヨン法（モレキュラー 'クロー二 ング第 3版）、 PCR法等を行うことにより、本発明の α -1,6-フコシルトランスフェラーゼ 変異体をコードする遺伝子の変異のみを検出することができる。該検出方法は、 α -1 ，6-フコース修飾酵素との関連が報告されている、網膜形成障害、肝臓癌、脂肪肝、 血液凝固障害、嚢胞性繊維症、肺組織障害、成長不良、インスリン様増殖因子や成 長ホルモン作用の障害を伴う疾患、上皮増殖因子の作用の障害を伴う疾患等の疾 患の判定に利用することができる。 By performing the Southern hybridization method (Molecular Cloning 3rd edition), PCR method, etc. on genomic DNA using the α-1,6-fucosyltransferase mutant oligonucleotide of the present invention as a probe Only the mutation of the gene encoding the α -1,6-fucosyltransferase mutant of the present invention can be detected. The detection method has been reported to be associated with α -1,6-fucose modifying enzyme, retinal dysfunction, liver cancer, fatty liver, blood coagulation disorder, cystic fibrosis, lung tissue disorder, growth failure, insulin It can be used to determine diseases such as diseases accompanied by impaired growth factor and growth hormone action and diseases accompanied by impaired epidermal growth factor action.
[0133] 具体的には、例えば、患者が保有する本発明のひ -1,6-フコシルトランスフェラーゼ 変異体をコードする DNAの翻訳領域を PCR法で増幅させ、塩基配列を決定し、配 列番号 3で表される DNAの塩基配列と比較することにより、翻訳領域における変異 の有無を調べることができる。 [0133] Specifically, for example, the translation region of DNA encoding the -1,6-fucosyltransferase mutant of the present invention possessed by a patient is amplified by PCR, the nucleotide sequence is determined, and the sequence number Mutation in the translation region by comparing with the DNA base sequence represented by 3. You can check for the presence or absence.
PCR法に供する铸型となる cDNAは（1)に記載した方法により取得できる。取得さ れた cDNAの塩基配列は、パーキンエルマ一社の DNAシークェンサ一 377と反応 キット (ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit:Appli ed Biosystems社製）を用いて決定できる。  The cDNA in the form of a cage for PCR can be obtained by the method described in (1). The nucleotide sequence of the obtained cDNA can be determined using a DNA sequencer 377 from Perkin Elma and a reaction kit (ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit: Applied Biosystems).
(3)免疫学的検出法 ·定量法による疾患の判定法  (3) Immunological detection methodDetermination method of disease by quantitative method
本発明のひ -1,6 -フコシルトランスフェラーゼ変異体に対する抗体を用いて、被験者 の血液中または組織における本発明のひ -1,6 -フコシルトランスフヱラーゼ変異体の 発現の有無を免疫学的に検出し、または定量し、配列番号 9で表されるアミノ酸から なるひ -1,6-フコシルトランスフェラーゼと比較することで下記疾患を判定することがで きる。  Using an antibody against the -1,6-fucosyltransferase mutant of the present invention, the presence or absence of expression of the -1,6-fucosyltransferase mutant of the present invention in the blood or tissue of a subject is immunologically determined. The following diseases can be determined by detecting or quantifying and comparing with -1,6-fucosyltransferase consisting of the amino acid represented by SEQ ID NO: 9.
[0134] 免疫学的に検出する方法としては、マイクロタイタープレートを用いる ELISA法、蛍 光抗体法、ウェスタンプロット法、免疫組織染色法等をあげることができる。  [0134] Examples of immunological detection methods include ELISA using a microtiter plate, fluorescent antibody method, Western plot method, immunohistochemical staining method, and the like.
免疫学的に定量する方法としては、例えば液相中で本発明の α _1，6-フコシルトラ ンスフェラーゼ変異体と特異的に反応する抗体のうちェピトープが異なる 2種類のモ ノクローナル抗体を用いたサンドイッチ ELISA法、 1251等の放射性同位体で標識した RA関連ポリペプチドと該ポリペプチドを認識する抗体を用いるラジオィムノアッセィ 法等があげられる。 As a method for immunological quantification, for example, a sandwich using two monoclonal antibodies having different epitopes among antibodies that specifically react with the α_1,6-fucosyltransferase mutant of the present invention in a liquid phase. ELISA method, radio I Takeno assay I method using a recognizing labeled RA-associated polypeptide and the polypeptide antibodies and the like with a radioisotope such as 125 1.
[0135] 該免疫学的方法は、 α -1,6-フコース修飾酵素との関連が報告されている、網膜形 成障害、肝臓癌、脂肪肝、血液凝固障害、嚢胞性繊維症、肺組織障害、成長不良、 インスリン様増殖因子や成長ホルモン作用の障害を伴う疾患、上皮増殖因子の作用 の障害を伴う疾患等の疾患の判定に利用することができる。  [0135] The immunological method is reported to be associated with α-1,6-fucose modifying enzyme, retinal dysplasia, liver cancer, fatty liver, blood coagulation disorder, cystic fibrosis, lung tissue It can be used for the determination of diseases such as disorders, poor growth, diseases with impaired insulin-like growth factor and growth hormone action, and diseases with impaired epidermal growth factor action.
(4)本発明のひ -1,6 -フコシルトランスフェラーゼ変異体の機能を制御する物質のスク リーニング方法  (4) A method for screening a substance that controls the function of the -1,6-fucosyltransferase mutant of the present invention
網膜形成障害、肝臓癌、脂肪肝、血液凝固障害、嚢胞性繊維症、肺組織障害、成 長不良、インスリン様増殖因子や成長ホルモン作用の障害を伴う疾患、上皮増殖因 子の作用の障害を伴う疾患等の疾患では、 ひ -1,6 -フコース修飾酵素の機能異常と の関連が示唆されている。 [0136] したがって、本発明の α _1，6-フコシルトランスフェラーゼ変異体の機能を阻害また は亢進させることが上記疾患の予防および治療に有効と考えられる。また α -1,6-フ コース修飾酵素の機能異常に直接起因しない疾患であっても、本発明の α -1,6-フ コシルトランスフェラーゼ変異体の機能を阻害または亢進させることにより対症療法的 に上記疾患の予防または治療を行うことができる。 Retinal dysfunction, liver cancer, fatty liver, blood coagulation disorder, cystic fibrosis, lung tissue disorder, poor growth, diseases with impaired insulin-like growth factor and growth hormone action, impaired epithelial growth factor action In diseases such as accompanying diseases, it has been suggested that it is associated with abnormal function of -1,6-fucose modifying enzyme. [0136] Therefore, it is considered effective to prevent or treat the above-mentioned diseases by inhibiting or enhancing the function of the α_1,6-fucosyltransferase mutant of the present invention. In addition, even for diseases that are not directly caused by abnormal function of α-1,6-fucose modifying enzyme, symptomatic therapy can be achieved by inhibiting or enhancing the function of the α-1,6-fucosyltransferase mutant of the present invention. In particular, the above diseases can be prevented or treated.
[0137] 従って、本発明のひ -1,6-フコシルトランスフェラーゼ変異体が原因で、細胞の生理 作用が減退または亢進している患者がいる場合、本発明のひ -1,6-フコシルトランス フェラーゼ変異体の機能を制御する化合物を投与することにより該生理作用を制御 することができ、またひ -1，6 -フコース修飾酵素の機能変化が疾患の直接の原因でな くても、本発明のひ -1,6-フコシルトランスフェラーゼ変異体の機能を制御することで 対症療法が可能な疾患もまた、該化合物の投与により予防および治療できる。  [0137] Therefore, when there is a patient whose cellular physiological activity is reduced or increased due to the -1,6-fucosyltransferase mutant of the present invention, the -1,6-fucosyltransferase of the present invention is used. The physiological action can be controlled by administering a compound that regulates the function of the mutant, and even if the change in the function of -1,6-fucose modifying enzyme is not a direct cause of the disease, the present invention Diseases that can be symptomatically treated by controlling the function of the -1,6-fucosyltransferase mutant can also be prevented and treated by administration of the compound.
[0138] 該化合物は、例えば以下に示す方法により取得できる。  [0138] The compound can be obtained, for example, by the method shown below.
[i]本発明の α -1,6-フコシルトランスフェラーゼ変異体を発現する細胞と、 [ii]被験物 質の存在下、本発明の α _1，6-フコシルトランスフェラーゼ変異体を発現する細胞を、 上記 2.に記載の培養法で 2時間から 1週間培養後、該 α -1,6-フコシルトランスフエ ラーゼ変異体が機能することにより生じる細胞応答を検出、比較し、該 α -1,6-フコシ ノレトランスフェラーゼ変異体の機能を阻害または亢進させる活性を有する物質を選択 し取得する。 [i] a cell expressing the α-1,6-fucosyltransferase variant of the present invention; and [ii] a cell expressing the α_1,6-fucosyltransferase variant of the present invention in the presence of the test substance, After culturing for 2 hours to 1 week according to the culture method described in 2. above, the cellular response produced by the function of the α-1,6-fucosyltransferase mutant is detected and compared, and the α- 1,6 -Select and obtain substances that have the activity of inhibiting or enhancing the function of fucosinotransferase mutants.
[0139] 本発明の α -1,6-フコシルトランスフェラーゼ変異体が機能することにより生じる細 胞応答を検出する方法としては、例えば、本発明の α -1,6-フコシルトランスフェラー ゼ変異体を発現する細胞のレクチンに対する反応性の変化、インスリン様増殖因子、 成長ホルモン、上皮増殖因子に依存的な細胞内情報伝達、遺伝子の転写、糖の取 り込み、増殖などの変化を検出する公知の方法が挙げられる (J. Biol. Chem., 276, 1 1956， 2001; J. Biol. Chem., 275, 21988, 2000; Molecular Medicine, 40, 1034, 2003; Nature, 263, 663， 1976;臨床科学， 17, 958， 1981)。  [0139] As a method for detecting a cell response generated by the function of the α-1,6-fucosyltransferase mutant of the present invention, for example, the α-1,6-fucosyltransferase mutant of the present invention is expressed. Known methods for detecting changes in responsiveness of cells to lectins, insulin-like growth factors, growth hormone, epithelial growth factor-dependent intracellular signal transduction, gene transcription, sugar uptake, proliferation, etc. (J. Biol. Chem., 276, 1 1956, 2001; J. Biol. Chem., 275, 21988, 2000; Molecular Medicine, 40, 1034, 2003; Nature, 263, 663, 1976; clinical science , 17, 958, 1981).
[0140] レクチンとしては、例えば、 N-グリコシド結合糖鎖還元末端の N-ァセチルダルコサミ ンの 6位とフコースの 1位がひ結合した糖鎖構造を認識するレクチンがあげられる。 [0140] Examples of the lectin include a lectin that recognizes a sugar chain structure in which the N-glycoside-linked sugar chain reducing terminal N-acetylcylcosamine is linked to the 6-position of fucose and the 1-position of fucose.
N -グリコシド結合糖鎖還元末端の N -ァセチルダルコサミンの 6位とフコースの 1位が a結合した糖鎖構造を認識するレクチンとしては、該糖鎖構造を認識できるレクチン であれば、いずれのレクチンでも用いることができる。その具体的な例としては、レン ズマメレクチン LCA (Lens culinaris由来の lentil agglutinin)エンドゥマメレクチン PSA (Pisum sativum由来の nea lectin) ,ソラマメレクチン VFA (Vicia faba由来の agglutinin )、ヒィロチャワンタケレクチン AAL (Aleuria aurantia由来の lectin)などがあげられる n (5) (4)の探索法で得られた化合物を含有する医薬 N-glycoside-linked sugar chain reducing terminal N-acetylyldarcosamine position 6 and fucose position 1 As the lectin that recognizes the a-linked sugar chain structure, any lectin that can recognize the sugar chain structure can be used. Specific examples of this are: Lentil lectin LCA (lentil agglutinin from lentil aculglutinin), Endo bean lectin PSA (nea lectin from Pisum sativum), Broad bean lectin VFA (agglutinin from Vicia faba), Alochawantake lectin AAL (Aleuria Pharmaceuticals containing the compound obtained by the search method of n (5) (4)
本発明のひ -1,6 -フコシルトランスフェラーゼ変異体の機能を制御する化合物は、 ひ- 1，6-フコース修飾酵素の機能異常との関連が示唆されている、網膜形成障害、 肝臓癌、脂肪肝、血液凝固障害、嚢胞性繊維症、肺組織障害、成長不良、インスリン 様増殖因子や成長ホルモン作用の障害を伴う疾患、上皮増殖因子の作用の障害を 伴う疾患等の疾患の予防薬および治療薬として有用である。  The compound that controls the function of the -1,6-fucosyltransferase mutant of the present invention has been suggested to be associated with abnormal function of -1,6-fucose modifying enzyme, retinal dysfunction, liver cancer, fat Prevention and treatment of diseases such as liver, blood coagulation disorder, cystic fibrosis, lung tissue disorder, growth failure, diseases with impaired insulin-like growth factor and growth hormone action, diseases with impaired epidermal growth factor action, etc. Useful as a medicine.
[0141] (4)で得られた化合物は、例えば以下に示す製剤学の技術分野においてよく知ら れる任意の方法により製造した医薬製剤として提供することができる。 [0141] The compound obtained in (4) can be provided, for example, as a pharmaceutical preparation produced by any method well known in the technical field of pharmaceutical formulation shown below.
該予防薬および治療薬の投与方法としては、例えば、本発明の α -1,6-フコシルト ランスフェラーゼ変異体に伴う機能異常により正常な生理作用が期待できない患者 がいる場合に、（i)本発明の α -1,6-フコシルトランスフェラーゼ変異体の機能を制御 する蛋白質をコードする DNAを該患者に投与し発現させること（ii)対象となる細胞に 本発明の α -1,6-フコシルトランスフェラーゼ変異体の機能を制御する蛋白質をコー ドする DNAを挿入し発現させた後に、該細胞を該患者に移植すること、あるレ、は (iii) 本発明の α -1,6-フコシルトランスフェラーゼ変異体の機能を制御する蛋白質や化合 物を該患者に投与することなどによって、患者の体内における本発明の α -1,6-フコ シルトランスフェラーゼ変異体の機能を変化させることができる。したがって、 ひ- 1,6- フコース修飾酵素の機能異常に起因する疾患、あるいはひ- 1，6-フコース修飾酵素 に直接起因しなくとも、本発明のひ- 1，6-フコシルトランスフェラーゼ変異体の機能を 制御する蛋白質をコードする DNA、あるいは本発明のひ -1,6-フコシルトランスフェラ ーゼ変異体の機能を制御する蛋白質の投与により対症療法的な治療が可能な疾患 に対する予防薬および治療薬として有用である。 As a method for administering the prophylactic and therapeutic agents, for example, when there is a patient who cannot expect normal physiological action due to functional abnormality associated with the α -1,6-fucosyltransferase mutant of the present invention, (i) (Ii) administering the DNA encoding a protein that controls the function of the α-1,6-fucosyltransferase variant of the invention to the patient and expressing the DNA (ii) in the target cell, the α-1,6-fucosyltransferase of the invention After inserting and expressing a DNA encoding a protein that controls the function of the mutant, the cell is transplanted into the patient. (Iii) The α-1,6-fucosyltransferase mutation of the present invention The function of the α-1,6-fucosyltransferase mutant of the present invention in the body of a patient can be changed by administering a protein or compound that controls the body function to the patient. Accordingly, the -1,6-fucosyltransferase mutant of the present invention can be used even if it is not caused by a disease caused by dysfunction of -1,6-fucose modifying enzyme or directly by -1,6-fucose modifying enzyme. Preventive drugs and treatments for diseases that can be symptomatically treated by administration of DNA encoding a protein that regulates the function or a protein that regulates the function of the -1,6-fucosyltransferase mutant of the present invention Useful as a medicine.
[0142] 本発明のひ -1,6 -フコシルトランスフェラーゼ変異体の機能を制御する蛋白質をコ ードする DNAを上記予防薬および治療薬として使用する場合は、本発明のひ- 1,6- フコシルトランスフェラーゼ変異体の機能を制御する蛋白質の DNAを単独あるいは レトロウイルスベクター、アデノウイルスベクター、アデノウイルスァソシエーテッドウイ ノレスベタターなどの適当なベクターに揷入した後、下記に記載した常法に従って製 剤化、処方および投与することができる。 [0142] A protein that regulates the function of the -1,6-fucosyltransferase mutant of the present invention is identified. When the DNA to be loaded is used as the above-mentioned preventive or therapeutic agent, the DNA of the protein that controls the function of the -1,6-fucosyltransferase mutant of the present invention alone or retrovirus vector, adenovirus vector, adeno After being inserted into an appropriate vector such as a virus-associated violets betater, it can be formulated, formulated and administered according to a conventional method described below.
[0143] レトロウイルス、アデノウイルス等のウィルスベクターやその他遺伝子治療用のベクタ 一に組み込んだ遺伝子治療用ベクターを遺伝子治療薬または予防薬として用いる には、該遺伝子治療用ベクターと遺伝子治療剤に用いる基剤を調合することにより 製造することができる [Nature Genet., 8, 42 (1994)] 0 [0143] In order to use a gene therapy vector incorporated into a virus vector such as a retrovirus or adenovirus or other gene therapy vector as a gene therapy drug or a preventive drug, use it as a gene therapy vector and a gene therapy agent. Can be manufactured by blending a base [Nature Genet., 8, 42 (1994)] 0
遺伝子治療剤に用いる基剤としては、通常注射剤に用いる基剤であればどのよう なものでもよく、蒸留水、塩化ナトリウム又は塩化ナトリウムと無機塩との混合物等の 塩溶液、マンニトール、ラタトース、デキストラン、グルコース等の糖溶液、グリシン、ァ ルギニン等のアミノ酸溶液、有機酸溶液又は塩溶液とグルコース溶液との混合溶液 等があげられる。また常法に従い、これらの基剤に浸透圧調整剤、 pH調整剤、ゴマ 油、ダイズ油等の植物油又はレシチンもしくは非イオン界面活性剤等の界面活性剤 等の助剤を用いて、溶液、懸濁液、分散液として注射剤を調製してもよい。これらの 注射剤を、粉末化、凍結乾燥等の操作により用時溶解用製剤として調製することもで きる。遺伝子治療剤は、液体の場合はそのままで、個体の場合は必要により滅菌処 理をした上記の基剤に遺伝子治療の直前に溶解して治療に使用することができる。 遺伝子治療剤の投与方法としては、患者の治療部位に吸収されるように、局所的に 投与する方法をあげることができる。  The base used for the gene therapy agent may be any base as long as it is usually used for injections, such as distilled water, sodium chloride or a salt solution such as a mixture of sodium chloride and an inorganic salt, mannitol, ratatose, Examples thereof include sugar solutions such as dextran and glucose, amino acid solutions such as glycine and arginine, organic acid solutions, or mixed solutions of a salt solution and a glucose solution. In addition, according to a conventional method, these bases are mixed with an osmotic pressure adjusting agent, a pH adjusting agent, a vegetable oil such as sesame oil or soybean oil, or an auxiliary agent such as a surfactant such as lecithin or a nonionic surfactant. An injection may be prepared as a suspension or dispersion. These injections can be prepared as preparations for dissolution at the time of use by operations such as pulverization and freeze-drying. The gene therapy agent can be used as it is in the case of a liquid, and in the case of an individual, the gene therapy agent can be dissolved in the above-mentioned base that has been sterilized if necessary before the gene therapy. An example of a gene therapy agent administration method is a method of local administration so that the gene therapy agent is absorbed at a treatment site of a patient.
[0144] また、非ウィルス遺伝子移入法によっても目的とする治療部位に DNAを輸送する こと力 Sできる。  [0144] The ability to transport DNA to the target treatment site can also be achieved by non-viral gene transfer.
当該分野で公知の非ウィルス遺伝子移入法には、リン酸カルシウム共沈法〔Virolog y, 52, 456-467 (1973)； Science, 209, 1414-1422 (1980)〕、マイクロインジェクション法 [Proc. Natl. Acad. Sci. USA, 77, 5399-5403 (1980) ; Proc. Natl. Acad. Sci. USA, 77 ， 7380-7384 (1980) ; Cell, 27, 223-231 (1981) ; Nature, 294, 92-94 (1981)〕、リポソ一 ムを介した膜融合-介在移入法〔Pro Natl. Acad. Sci. USA, 84, 7413-7417 (1987) ; Biochemistry, 28, 9508-9514 (1989) ; J. Biol. Chem., 264, 12126-12129 (1989) ; Hum . Gene Ther" 3, 267-275, (1992) ; Science, 249, 1285-1288 (1990) ; Circulation, 83, 2007-2011 (1992)〕あるいは直接 DNA取り込みおよび受容体-媒介 DNA移入法〔Sc ience, 247, 1465-1468 (1990) ; J. Biol. Chem., 266, 14338-14342 (1991) ; Proc. Natl. Acad. Sci. USA, 87, 3655-3659 (1991) ; J. Biol. Chem., 264, 16985-16987 (1989) ; BioTechniques, 11, 474-485 (1991) ; Pro Natl. Acad. Sci. USA, 87, 3410-3414 (19 90) ; Pro Natl. Acad. Sci. USA, 88, 4255-4259 (1991) ; Proc. Natl. Acad. Sci. USA , 87, 4033-4037 (1990) ; Pro Natl. Acad. Sci. USA, 88, 8850-8854 (1991) ; Hum. G ene Ther., 3, 147-154 (1991)〕等をあげること力 Sできる。 Non-viral gene transfer methods known in the art include calcium phosphate coprecipitation (Virolog y, 52, 456-467 (1973); Science, 209, 1414-1422 (1980)), microinjection [Proc. Natl. Acad. Sci. USA, 77, 5399-5403 (1980); Proc. Natl. Acad. Sci. USA, 77, 7380-7384 (1980); Cell, 27, 223-231 (1981); Nature, 294, 92 -94 (1981)], liposome-mediated membrane fusion-mediated transfer (Pro Natl. Acad. Sci. USA, 84, 7413-7417 (1987); Biochemistry, 28, 9508-9514 (1989); J. Biol. Chem., 264, 12126-12129 (1989); Hum. Gene Ther "3, 267-275, (1992); Science, 249, 1285-1288 ( 1990); Circulation, 83, 2007-2011 (1992)] or direct DNA uptake and receptor-mediated DNA transfer (Science, 247, 1465-1468 (1990); J. Biol. Chem., 266, 14338- 14342 (1991); Proc. Natl. Acad. Sci. USA, 87, 3655-3659 (1991); J. Biol. Chem., 264, 16985-16987 (1989); BioTechniques, 11, 474-485 (1991) Pro Natl. Acad. Sci. USA, 87, 3410-3414 (19 90); Pro Natl. Acad. Sci. USA, 88, 4255-4259 (1991); Proc. Natl. Acad. Sci. USA, 87, 4033-4037 (1990); Pro Natl. Acad. Sci. USA, 88, 8850-8854 (1991); Hum. Gene Ther., 3, 147-154 (1991)].
[0145] リボソームを介した膜融合-介在移入法ではリボソーム調製物を標的とする組織に 直接投与することにより、当該組織の局所的な遺伝子の取り込みおよび発現が可能 であることが腫瘍に関する研究において報告されている〔Hum. Gene Ther., 3, 399 ( 1992)〕。 [0145] In ribosome-mediated membrane fusion-mediated transfer methods, ribosome preparations can be directly administered to a target tissue to allow local uptake and expression of that tissue in tumor research. It has been reported [Hum. Gene Ther., 3, 399 (1992)].
また、本発明の α -1,6-フコシルトランスフェラーゼ変異体の機能を制御する蛋白質 や化合物を有効成分として含有する医薬は、該有効成分を単独で投与することも可 能ではある力 通常は該有効成分を薬理学的に許容される一つあるいはそれ以上の 担体と一緒に混合し、製剤学の技術分野においてよく知られる任意の方法により製 造した医薬製剤として提供するのが望ましい。好ましくは水、あるいは食塩、グリシン 、グノレコース、ヒトアルブミン等の水溶液等の水性担体に溶解した無菌的な溶液が用 レ、られる。また、製剤溶液を生理的条件に近づけるための緩衝ィヒ剤や等張化剤のよ うな、薬理学的に許容される添加剤、例えば、酢酸ナトリウム、塩化ナトリウム、乳酸ナ トリウム、塩化カリウム、クェン酸ナトリウム等を添加することもできる。また、凍結乾燥し て貯蔵し、使用時に適当な溶媒に溶解させて用いることもできる。  In addition, a drug containing a protein or compound that controls the function of the α-1,6-fucosyltransferase mutant of the present invention as an active ingredient is usually capable of administering the active ingredient alone. The active ingredient is desirably mixed with one or more pharmaceutically acceptable carriers and provided as a pharmaceutical preparation prepared by any method well known in the technical field of pharmaceutics. Preferably, a sterile solution dissolved in water or an aqueous carrier such as an aqueous solution of salt, glycine, gnolecose, human albumin or the like is used. Also, pharmacologically acceptable additives such as buffering agents and isotonic agents for bringing the formulation solution close to physiological conditions, such as sodium acetate, sodium chloride, sodium lactate, potassium chloride, Sodium quenate and the like can also be added. It can also be lyophilized and stored, and dissolved in an appropriate solvent before use.
[0146] 投与経路は、治療に際し最も効果的なものを使用するのが望ましぐ経口投与、あ るいは口腔内、気道内、直腸内、皮下、筋肉内および静脈内等の非経口投与をあげ ること力 Sできる。投与形態としては、噴霧剤、カプセル剤、錠剤、顆粒剤、シロップ斉 1J、 乳剤、座剤、注射剤、軟膏、テープ剤等があげられる。 [0146] It is desirable to use the most effective route of treatment for oral administration, or oral administration such as buccal, intratracheal, rectal, subcutaneous, intramuscular and intravenous. The ability to raise S. Examples of administration forms include sprays, capsules, tablets, granules, syrups 1J, emulsions, suppositories, injections, ointments, tapes, and the like.
経口投与に適当な製剤としては、乳剤、シロップ剤、カプセル剤、錠剤、散剤、顆粒 剤等があげられる。例えば乳剤およびシロップ剤のような液体調製物は、水、ショ糖、 ソルビトール、果糖等の糖類、ポリエチレングリコール、プロピレングリコール等のグリ コール類、ごま油、ォリーブ油、大豆油などの油類、 P—ヒドロキシ安息香酸エステル 類等の防腐剤、ストロベリーフレーバー、ペパーミント等のフレーバー類等を添加剤と して用いて製造できる。カプセル剤、錠剤、散剤、顆粒剤等は、乳糖、ブドウ糖、ショ 糖、マンニトール等の賦形剤、デンプン、アルギン酸ナトリウム等の崩壊剤、ステアリ ン酸マグネシウム、タルク等の滑沢剤、ポリビュルアルコール、ヒドロキシプロピルセル ロース、ゼラチン等の結合剤、脂肪酸エステル等の界面活性剤、グリセリン等の可塑 剤等を添加剤として用いて製造できる。 Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules Agents and the like. For example, liquid preparations such as emulsions and syrups include sugars such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, P- It can be produced using preservatives such as hydroxybenzoic acid esters, and flavors such as strawberry flavor and peppermint as additives. Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polybulu alcohol It can be produced using a binder such as hydroxypropyl cellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin as additives.
[0147] 非経口投与に適当な製剤としては、注射剤、座剤、噴霧剤等があげられる。例えば 、注射剤は、塩溶液、ブドウ糖溶液、あるいは両者の混合物からなる担体等を用いて 調製する。座剤はカカオ脂、水素化脂肪またはカルボン酸等の担体を用いて調製さ れる。また、噴霧剤は該蛋白質または該化合物そのもの、ないしは受容者の口腔お よび気道粘膜を刺激せず、かつ該蛋白質または該化合物を微細な粒子として分散さ せ吸収を容易にさせる担体等を用いて調製する。担体として具体的には乳糖、グリセ リン等が例示される。該蛋白質または該化合物および用いる担体の性質により、エア ロゾノレ、ドライパウダー等の製剤が可能である。また、これらの非経口剤においても経 口剤で添加剤として例示した成分を添加することもできる。  [0147] Suitable formulations for parenteral administration include injections, suppositories, sprays and the like. For example, an injection is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of both. Suppositories are prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid. In addition, the propellant uses the carrier or the like which does not irritate the protein or the compound itself, or the recipient's oral cavity and airway mucosa, and is easily dispersed by dispersing the protein or the compound as fine particles. Prepare. Specific examples of the carrier include lactose and glycerin. Depending on the properties of the protein or the compound and the carrier used, preparations such as aerosol or dry powder are possible. In these parenteral preparations, the components exemplified as additives in oral preparations can also be added.
[0148] 投与量または投与回数は、 目的とする治療効果、投与方法、治療期間、年齢、体 重等により異なる力 S、通常成人 1日当たり 10 /i g/kg〜8mg/kgである。 [0148] The dose or number of administrations is a force S that varies depending on the intended therapeutic effect, administration method, treatment period, age, body weight, etc., and is usually 10 / ig / kg to 8 mg / kg per day for an adult.
6.本発明の α -1,6-フコシルトランスフェラーゼ変異体を有する細胞を用いた糖蛋白 医薬品の製造  6. Production of glycoprotein pharmaceuticals using cells having the α-1,6-fucosyltransferase mutant of the present invention
本発明のひ -1,6-フコシルトランスフヱラーゼ変異体を有する細胞では、 ひ -1,6-フ コース修飾酵素の活性が欠失、または低下しているため、その細胞において産生さ れる糖蛋白質はひ- 1，6 -フコース修飾酵素による糖鎖修飾が欠失、または低下して いる。  In the cell having the -1,6-fucosyltransferase mutant of the present invention, the activity of the -1,6-fucose modifying enzyme is deleted or decreased, so that the sugar produced in the cell Proteins lack or reduce sugar chain modification by -1,6-fucose modifying enzymes.
[0149] このようなひ -1,6 -フコース修飾酵素による糖鎖修飾が欠失、または低下した糖蛋白 質は、生体における血液動態や分布の変化、薬理活性発現に必要な蛋白質との相 互作用が変化しており、医薬品として有用である。 [0149] Such glycoproteins lacking or reducing the sugar chain modification by -1,6-fucose modifying enzyme are in phase with changes in hemodynamics and distribution in the living body and proteins necessary for the expression of pharmacological activity. The interaction has changed and it is useful as a medicine.
その具体的な例としては、抗体、エリスロポイエチン、トロンボポイエチン、組織型プ ラスミノーゲンァクチベータ、プロゥロキナーゼ、トロンボモジュリン、アンチトロンビン II I、プロテインお血液凝固因子 VII、血液凝固因子 VIII、血液凝固因子 IX、血液凝固 因子 X、性腺刺激ホルモン、甲状腺刺激ホルモン、上皮増殖因子 (EGF)、肝細胞増 殖因子 (HGF)、ケラチノサイト増殖因子、ァクチビン、骨形成因子、幹細胞因子 (SCF )、インターフェロンひ、インターフェロン β、インターフェロン γ、インターロイキン 2、 インターロイキン 6、インターロイキン 10、インターロイキン 11、可溶性インターロイキ ン 4受容体、腫瘍壊死因子ひ、 Dnasel,ガラクトシダーゼ、 ひダルコシダーゼ、ダルコ セレブロシダーゼなどがあげられる。  Specific examples include antibodies, erythropoietin, thrombopoietin, tissue-type plasminogen activator, prolokinase, thrombomodulin, antithrombin II I, protein blood coagulation factor VII, blood coagulation factor VIII, blood Coagulation factor IX, Blood coagulation 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 β, interferon γ, interleukin 2, interleukin 6, interleukin 10, interleukin 11, soluble interleukin 4 receptor, tumor necrosis factor, Dnasel, galactosidase, dalcosidase, dalco cerebrosidase, etc. .
[0150] フコース修飾が欠失、または低下した糖鎖構造を有することが原因で、その生理活 性が大幅に上昇する糖蛋白質のより具体的な例としては、例えば、抗体があげられる 。以下に、抗体組成物の製造を例に、本発明の α -1,6-フコシルトランスフェラーゼ変 異体を有する細胞を用いた糖蛋白質組成物の製造方法を示す。  [0150] A more specific example of a glycoprotein whose physiological activity is significantly increased due to a sugar chain structure in which the fucose modification is deleted or reduced is, for example, an antibody. In the following, a method for producing a glycoprotein composition using cells having the α-1,6-fucosyltransferase variant of the present invention will be described by taking the production of an antibody composition as an example.
抗体組成物は、モレキユラ一'クローニング第 3版、カレント'プロトコールズ'イン'モ レキュフー *ノヽィォロシ一、 Antibodies, A Laboratory manual, し old Spring Harbor La boratory, 1988 (以下、アンチボディズと略す)、 Monoclonal Antibodies: principles an d practice, Third Edition, Acad. Press, 1993 (以下、モノクローナノレアンチボディズと 略す)、 Antibody Engineering, A Practical Approach, IRL Press at Oxford University Press, 1996 (以下、アンチボディエンジニアリングと略す)等に記載された方法を用 レ、、例えば、以下のように本発明の α -1,6-フコシルトランスフェラーゼ変異体を発現 した細胞で発現させて取得することができる。  The antibody composition is Moleculera 'Cloning 3rd Edition, Current' Protocols 'in' Molechu * Noroji, Antibodies, A Laboratory manual, old Spring Harbor Laboratory, 1988 (hereinafter abbreviated as Antibodies) , Monoclonal Antibodies: principles an d practice, Third Edition, Acad. Press, 1993 (hereinafter referred to as Monoclonal Nanoreantibodies), Antibody Engineering, A Practical Approach, IRL Press at Oxford University Press, 1996 (hereinafter referred to as Antibody) For example, it can be obtained by expressing in a cell expressing the α-1,6-fucosyltransferase mutant of the present invention as described below.
[0151] 抗体分子の cDNAを調製する。  [0151] Prepare cDNA of the antibody molecule.
調製した抗体分子の全長 cDNAをもとにして、必要に応じて、該蛋白質をコードす る部分を含む適当な長さの DNA断片を調製する。  Based on the prepared full-length cDNA of the antibody molecule, if necessary, a DNA fragment having an appropriate length containing a portion encoding the protein is prepared.
該 DNA断片、または全長 cDNAを適当な発現ベクターのプロモーターの下流に 揷入することにより、組換えベクターを作製する。  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.
[0152] 該組換えベクターを、該発現べクタ一に適合した本発明のひ- 1，6-フコシルトランス フェラーゼ変異体を発現した細胞に導入することにより、抗体分子を生産する形質転 換個体を得ることができる。 [0152] The recombinant vector of the present invention adapted to the expression vector A transgenic individual producing an antibody molecule can be obtained by introducing it into a cell that expresses a ferase mutant.
α _1，6-フコシルトランスフェラーゼ変異体を発現した細胞としては、細菌、酵母、動 物細胞、昆虫細胞、植物細胞等、 目的とする遺伝子を発現できるものであればいず れも用いることができる力 好ましくは動物細胞があげられる。  Any cell that expresses the gene of interest, such as bacteria, yeast, animal cells, insect cells, or plant cells, can be used as a cell that expresses the α_1,6-fucosyltransferase mutant. Preferably, animal cells are used.
[0153] 抗体分子の Fc領域に結合する Ν—グリコシド結合糖鎖の修飾に係わる酵素を遺伝 子工学的な手法を用いて導入した、細菌、酵母、動物細胞、昆虫細胞、植物細胞等 の細胞を用いることもできる。 [0153] Cells, such as bacteria, yeast, animal cells, insect cells, plant cells, etc. that have been introduced using genetic engineering techniques to bind the glycoside-linked sugar chain that binds to the Fc region of antibody molecules Can also be used.
本発明の抗体組成物の製造方法に用いられる細胞としては、上記 2.または 4.で 作製した、本発明のひ -1,6-フコシルトランスフェラーゼ変異体を発現する細胞をあげ ること力 Sできる。  Examples of the cells used in the method for producing the antibody composition of the present invention include the cells produced in the above 2. or 4. that express the -1,6-fucosyltransferase mutant of the present invention. .
[0154] cDNAは、上記 1.に記載の cDNAの調製方法に従レ、、ヒト又は非ヒト動物の組織 又は細胞より、 目的とする抗体分子に特異的なプローブプライマーを用いて調製す ること力 Sできる。  [0154] cDNA should be prepared according to the cDNA preparation method described in 1. above, from human or non-human animal tissues or cells, using probe primers specific for the antibody molecule of interest. Power S can be.
上記各種細胞にぉレ、て抗体組成物を製造するための具体的な方法としては、上記 2.記載の発現ベクターの構築方法、細胞への発現ベクターの導入方法、胞の培養 方法、 目的生産物の精製方法をあげることができる。  Specific methods for preparing an antibody composition in the above various cells include the method for constructing the expression vector described in 2. above, the method for introducing the expression vector into the cell, the method for culturing the vesicle, and the target production The purification method of a thing can be mention | raise | lifted.
[0155] 糖鎖の合成に関与する遺伝子を導入した、細菌、酵母、動物細胞、昆虫細胞また は植物細胞等により発現させた場合には、導入した遺伝子によって糖あるいは糖鎖 が付加された抗体分子を得ることができる。 [0155] When a gene involved in the synthesis of a sugar chain is introduced and expressed in bacteria, yeast, animal cells, insect cells, or plant cells, an antibody to which a sugar or sugar chain is added by the introduced gene A molecule can be obtained.
取得される抗体組成物として、例えば、抗体、抗体の断片、抗体の Fc領域を有する 融合蛋白質などを挙げることができる。  Examples of the antibody composition to be obtained include an antibody, an antibody fragment, a fusion protein having an antibody Fc region, and the like.
[0156] 以下に、抗体組成物の取得のより具体的な例として、ヒトイ匕抗体および Fc融合蛋白 質の組成物の製造方法について記す力 他の抗体組成物等の糖蛋白質を上述の 方法および当該方法に準じて取得することもできる。 [0156] In the following, as a more specific example of obtaining an antibody composition, the power described for the method for producing the composition of the human rabbit antibody and Fc fusion protein, the glycoproteins such as other antibody compositions, etc. It can also be obtained according to the method.
A.ヒト化抗体組成物の製造  A. Manufacture of humanized antibody composition
(1)ヒト化抗体発現用ベクターの構築  (1) Construction of humanized antibody expression vector
ヒト化抗体発現用ベクターとは、ヒト抗体の CH及び CLをコードする遺伝子が組み込 まれた動物細胞用発現ベクターであり、動物細胞用発現ベクターにヒト抗体の CH及 び CLをコードする遺伝子をそれぞれクローニングすることにより構築することができる A humanized antibody expression vector contains genes encoding CH and CL of a human antibody. It is a rare expression vector for animal cells, and can be constructed by cloning the genes encoding human antibodies CH and CL respectively into the expression vector for animal cells.
[0157] ヒト抗体の C領域としては、任意のヒト抗体の CH及び CLであることができ、例えば、 ヒト抗体の H鎖の IgGlサブクラスの C領域（以下、「hC 7 1」と表記する）及びヒト抗体の L鎖の /cクラスの C領域（以下、「hC κ」と表記する）等があげられる。 [0157] The C region of a human antibody can be CH and CL of any human antibody. For example, the C region of the IgGl subclass of the H chain of a human antibody (hereinafter referred to as "hC 7 1") And the C region of the / c class (hereinafter referred to as “hC κ”) of the L chain of human antibodies.
ヒト抗体の CH及び CLをコードする遺伝子としてはェキソンとイントロンから成る染色 体 DNAを用いることができ、また、 cDNAを用レ、ることもできる。  Chromosomal DNA consisting of exons and introns can be used as the gene encoding human antibody CH and CL, and cDNA can also be used.
[0158] 動物細胞用発現ベクターとしては、ヒト抗体の C領域をコードする遺伝子を組込み 発現できるものであればいかなるものでも用いることができる。例えば、 PAGE 107 [サ イトテクノロジー (Cytotechnology), 3, 133 (1990)]、 pAGE103 [ジャーナル'ォブ 'バイ オケミストリー (J. Biochem.), 101 , 1307 (1987)]、 pHSG274 [ジーン (Gene), 27, 223 (19 [0158] As an expression vector for animal cells, any expression vector can be used as long as it can incorporate and express a gene encoding the C region of a human antibody. For example, PAGE 107 [Cytotechnology, 3, 133 (1990)], pAGE103 [Journal 'Ob' Biochemistry., 101, 1307 (1987)], pHSG274 [Gene ), 27, 223 (19
84)]、 pKCR [プロシーディングス ·ォブ ·ザ ·ナショナノレ ·ァカデミ一'ォブ ·サイエンス（84)], pKCR [Proceedings · Ob · The · Nashonanore · Academia 1 · Ob · Science (
Proc. Natl. Acad. Sci. U. S.A.), 78, 1527 (1981)]、 pSG l β d2_4 [サイトテクノロジー (C ytotechnology), 4, 173 (1990)]等があげられる。動物細胞用発現ベクターに用いるプ 口モーターとェンハンサ一としては、 SV40の初期プロモーターとェンハンサー [ジャー ナル'ォブ'バイオケミストリー (J. Biochem.),皿， 1307 (1987)]、モロニ一マウス白血 病ウィルスの LTRレくィォケミカル'アンド'バイオフィジカル 'リサーチ 'コミュニケーシ ヨンズ (Biochem. Biophys. Res. Commun.), 149, 960 (1987)]、免疫グロブリン H鎖の プロモーター [セル (Cell), 41 , 479 (1985)]とェンハンサー [セル (Cell), 33, 717 (1983)Proc. Natl. Acad. Sci. USA), 78, 1527 (1981)], pSGlβ d2_4 [Cytotechnology, 4, 173 (1990)] and the like. The promoters and enhancers used for animal cell expression vectors include SV40 early promoter and enhancer [J. Biochem., Dish, 1307 (1987)], Moroni mouse white blood. LTR requichemical 'and' biophysical 'research' Communications (Biochem. Biophys. Res. Commun.), 149, 960 (1987)], immunoglobulin heavy chain promoter [Cell, 41 , 479 (1985)] and Enhanser [Cell, 33, 717 (1983)
]等があげられる。 ].
[0159] ヒト化抗体発現用ベクターは、抗体 H鎖及び L鎖が別々のベクター上に存在するタ イブあるいは同一のベクター上に存在するタイプ (以下、タンデム型と表記する）のど ちらでも用いることができる力 ヒトイ匕抗体発現ベクターの構築の容易さ、動物細胞へ の導入の容易さ、動物細胞内での抗体 H鎖及び L鎖の発現量のバランスが均衡する 等の点からタンデム型のヒト化抗体発現用ベクターの方が好ましい [ジャーナル ·ォブ •ィムノロジカル'メソッズ (J. Immunol. Methods), 167, 271 (1994)]。  [0159] The humanized antibody expression vector should be either the type in which the antibody H chain and L chain are present on separate vectors or the same vector (hereinafter referred to as tandem type). The ability to construct a human 型 antibody expression vector, ease of introduction into animal cells, balance of expression levels of antibody H and L chains in animal cells, etc. An antibody-expressing vector is preferred [Journal of Immunological Methods (J. Immunol. Methods), 167, 271 (1994)].
[0160] 構築したヒト化抗体発現用ベクターは、ヒト型キメラ抗体及びヒト型 CDR移植抗体の 動物細胞での発現に使用できる。 [0160] The constructed humanized antibody expression vectors are human chimeric antibodies and human CDR-grafted antibodies. It can be used for expression in animal cells.
(2)ヒト以外の動物の抗体の V領域をコードする cDNAの取得  (2) Acquisition of cDNA encoding the V region of non-human animal antibodies
ヒト以外の動物の抗体、例えば、マウス抗体の VH及び VLをコードする cDNAは以下 のようにして取得することができる。  Non-human animal antibodies, for example, cDNAs encoding mouse antibody VH and VL can be obtained as follows.
[0161] 目的のマウス抗体を産生するハイブリドーマ細胞より mRNAを抽出し、 cDNAを合成 する。合成した cDNAをファージ或いはプラスミド等のベクターにクローニングして cDN Aライブラリーを作製する。該ライブラリーより、既存のマウス抗体の C領域部分或いは V領域部分をプローブとして用レ、、 VHをコードする cDNAを有する組換えファージ或 いは組換えプラスミド及び VLをコードする cDNAを有する組換えファージ或いは組換 えプラスミドをそれぞれ単離する。組換えファージ或いは組換えプラスミド上の目的の マウス抗体の VH及び VLの全塩基配列を決定し、塩基配列より VH及び VLの全ァミノ 酸配列を推定する。 [0161] mRNA is extracted from hybridoma cells producing the desired mouse antibody, and cDNA is synthesized. The synthesized cDNA is cloned into a vector such as a phage or plasmid to prepare a cDNA library. From this library, the C region or V region of an existing mouse antibody is used as a probe, a recombinant phage or cDNA containing VH-encoding cDNA, and a recombination containing cDNA encoding VL. Isolate each phage or recombinant plasmid. Determine the VH and VL base sequences of the desired mouse antibody on the recombinant phage or recombinant plasmid, and estimate the VH and VL amino acid sequences from the base sequence.
[0162] ヒト以外の動物としては、マウス、ラット、ハムスター、ゥサギ等、ハイプリドーマ細胞 を作製することが可能であれば、レ、かなるものも用いることができる。  [0162] As animals other than humans, mice, rats, hamsters, rabbits, etc. can be used as long as it is possible to produce high-pridoma cells.
ハイプリドーマ細胞から全 RNAを調製する方法としては、チォシアン酸グァニジン トリフルォロ酢酸セシウム法 [メソッズ'イン'ェンザィモロジ一 (Methods in Enzymol.), 154. 3 (1987)]、また全 RNAから mRNAを調製する方法としては、オリゴ (dT)固定化セ ルロースカラム法 [モレキュラ^ ~ ·クローニング：ァ 'ラボラトリー 'マニュアル (Molecular し lonmg: A Laboratory Manual), し old Spring Harbor Lab. Press New York, 1989」等 があげられる。また、ハイプリドーマ細胞から mRNAを調製するキットとしては、 Fast Tr ack mRNA Isolation Kit
Figure imgf000062_0001
、 Quick Prep mRNA Purification Kit (Pharm acia社製）等があげられる。 Methods for preparing total RNA from Hypridoma cells include guanidine thiocyanate, cesium trifluoroacetate method [Methods in Enzymol., 154.3 (1987)], and preparing mRNA from total RNA. Methods include oligo (dT) immobilized cellulose column method [Molecular ^ ~ Cloning: 'Laboratory' manual (Molecular and lonmg: A Laboratory Manual), old Spring Harbor Lab. Press New York, 1989], etc. can give. In addition, the Fast Track mRNA Isolation Kit is available as a kit for preparing mRNA from Hypridoma cells.
Figure imgf000062_0001
Quick Prep mRNA Purification Kit (manufactured by Pharmacia).
[0163] cDNAの合成及び cDNAライブラリ一作製法としては、常法 [モレキユラ一'クロー二 ング：ァ 'ラボラトリー 'マ二ユアノレ (Molecular Cloning: A Laboratory Manual), Cold Sp ring Harbor Lab. Press New York, 1989 ;カレント 'プロトコーノレズ'イン'モレキュラー' ノィォロジ1 ~ (Current Protocols in MolecularBiology), Supplement 1-34]、或レ、 fま巿 販のキット、 f列えは、、 Super Script Plasmid System for cDNA Synthesis and Plasmid Cloning (GIBCO BRL社製）や ZAP-cDNA Synthesis Kit (Stratagene社製）を用いる方 法などがあげられる。 [0163] For cDNA synthesis and cDNA library preparation, conventional methods [Molecular Y'cloning: Molecular Laboratory: A Laboratory Manual), Cold Spring Harbor Lab. Press New York , 1989; Current 'Protocorenoles' in 'Molecular' Neurology 1 ~ (Current Protocols in Molecular Biology), Supplement 1-34], or a kit for sale, f lineup, Super Script Plasmid System for Those who use cDNA Synthesis and Plasmid Cloning (GIBCO BRL) or ZAP-cDNA Synthesis Kit (Stratagene) Law.
[0164] cDNAライブラリーの作製の際、ハイプリドーマ細胞から抽出した mRNAを铸型として 合成した cDNAを組み込むベクターは、該 cDNAを組み込めるベクターであればいか なるものでも用レヽること力 Sできる。例えば、 ZAP Express [ストラテジーズ (Strategies), 5, 58 (1992)]、 pBluescript II SK(+) [ヌクレイック 'ァシッズ'リサーチ (Nucleic Acids Rese arch), 17, 9494 (1989)]、 λ zap II (Stratagene社製）、 gtlO、 λ gtl l [ディーェヌエー .クローニング：ァ'プラクティカノレ.アプローチ (DNA Cloning: A Practical Approach), I ， 49 (1985)]、 Lambda BlueMid (Clontech社製）、 λ ExCell, pT7T3 18U (Pharmacia社 製）、 pcD2 [モレキュラー'アンド'セルラー'バイオロジー (Mol. Cell. Biol.), 3, 280 (19 83)]及び pUC18 [ジーン (Gene), 33, 103 (1985)]等が用いられる。  [0164] When a cDNA library is prepared, any vector can be used as long as it is a vector into which cDNA synthesized by using mRNA extracted from a hyperidoma cell as a saddle type can be incorporated. For example, ZAP Express [Strategies, 5, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Rese arch, 17, 9494 (1989)], λ zap II ( Stratagene), gtlO, λ gtl l [D.N. Cloning: A Practical Approach, I, 49 (1985)], Lambda BlueMid (Clontech), λ ExCell, pT7T3 18U (manufactured by Pharmacia), pcD2 [Molecular & Cellular Biology (Mol. Cell. Biol.), 3, 280 (19 83)] and pUC18 [Gene, 33, 103 (1985)], etc. Is used.
[0165] ファージ或いはプラスミドベクターにより構築される cDNAライブラリーを導入する大 腸菌としては該 cDNAライブラリーを導入、発現及び維持できるものであればレ、かなる ものでも用いることができる。例えば、 XLl-Blue MRF' [ストラテジーズ (Strategies), 5, 81 (1992)]、 C600 [ジェネティックス (Genetics), 39, 440 (1954)]、 Y1088、 Y1090 [サイ エンス (Science), 222, 778 (1983)]、 NM522 [ジャーナル'ォブ 'モレキュラー 'バイオ口 ジー (J. Mol. Biol.), 166, 1 (1983)]、 K802 [ジャーナル'ォブ 'モレキュラ^ ~ ·バイオ口 ジー (J. Mol. Biol.), 16, 118 (1966)]及び JM105 [ジーン (Gene), 38, 275 (1985)]等が 用いられる。  [0165] Any Escherichia coli for introducing a cDNA library constructed by a phage or plasmid vector can be used as long as it can introduce, express and maintain the cDNA library. For example, XLl-Blue MRF '[Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088, Y1090 [Science, 222, 778 (1983)], NM522 [Journal 'Ob' Molecular 'Bio Mouth (J. Mol. Biol.), 166, 1 (1983)], K802 [Journal' Ob 'Molecular ^ ~ · Bio Mouth Gee ( J. Mol. Biol.), 16, 118 (1966)] and JM105 [Gene, 38, 275 (1985)] and the like are used.
[0166] cDNAライブラリーからのヒト以外の動物の抗体の VH及び VLをコードする cDNAクロ ーンの選択法としては、アイソトープ或いは蛍光標識したプローブを用いたコロニー' ハイブリダィゼーシヨン法或いはプラーク 'ハイブリダィゼーシヨン法 [モレキユラ一'ク ローニング：ァ'ラボラトリー 'マ二ユアノレ (Molecular Cloning: A Laboratory Manual), C old Spring Harbor Lab. Press NewYork, 1989]により選択することができる。また、プ ライマーを調製し、 mRNAから合成した cDNA或いは cDNAライブラリーを錡型として、 Polymerase Chain Reaction [以下、 PCR法と表記する；モレキュラー 'クローニング：ァ 'フホフトリ■ ~ 'マニュスノレ (Molecular Cloning: A Laboratory Manual), Cold Spring Har bor Lab. Press New York, 1989 ;カレント'プロトコールズ'イン'モレキュラー 'バイオ ロジー (Current Protocols in Molecular Biology), Supplement 1— 34]により VH及び VL をコードする cDNAを調製することもできる。 [0166] The selection of cDNA clones encoding VH and VL of non-human animal antibodies from a cDNA library includes colony hybridization methods or plaques using isotopes or fluorescently labeled probes. It can be selected by the 'Hybridization method [Molecula 1' Cloning: Laboratory ', Molecular Cloning: A Laboratory Manual, Old Spring Harbor Lab. Press NewYork, 1989]. In addition, a primer is prepared and a cDNA or cDNA library synthesized from mRNA is used as a cage. Polymerase Chain Reaction [Hereinafter referred to as PCR method; Molecular 'cloning: A' Hofofri ■ ~ 'Manu's Nole (Molecular Cloning: A Laboratory Manual), Cold Spring Harbor Lab. Press New York, 1989; VH and VL according to Current Protocols in Molecular Biology, Supplement 1-3 CDNA encoding can also be prepared.
[0167] 上記方法により選択された cDNAを、適当な制限酵素などで切断後、 pBluescript S K (-) (Stratagene社製)等のプラスミドにクローニングし、通常用いられる塩基配列解 析方法、例えば、サンガー（Sanger)らのジデォキシ法 [プロシーディンダス 'ォブ 'ザ' ナショナル ·ァカデミ ~ ·ォブ 'サイエンス (Pro Natl. Acad. ScL, U.S.A.), 74, 5463 (1 977)]等の反応を行い、塩基配列自動分析装置、例えば、 A. L. F. DNAシークェン サー（Pharmacia社製）等を用いて解析することで該 cDNAの塩基配列を決定すること ができる。 [0167] The cDNA selected by the above method is cleaved with an appropriate restriction enzyme and then cloned into a plasmid such as pBluescript SK (-) (Stratagene), and a commonly used nucleotide sequence analysis method such as Sanger. (Sanger) et al. Dideoxy [Procedidas 'Ob' The 'National Academia ~ · Ob' Science (Pro Natl. Acad. ScL, USA), 74, 5463 (1 977)] Then, the base sequence of the cDNA can be determined by analysis using an automatic base sequence analyzer such as an ALF DNA sequencer (Pharmacia).
[0168] 決定した塩基配列から VH及び VLの全アミノ酸配列を推定し、既知の抗体の VH及 び VLの全アミノ酸配歹 IJ [シーケンシズ'ォブ'プロテインズ'ォブ'ィムノロジカル 'イン タレスト (Sequences of Proteins of Immunologicailnterest), Ub Dept. Health and Hum an Services, 1991]と比較することにより、取得した cDNAが分泌シグナル配列を含む 抗体の VH及び VLの完全なアミノ酸配列をコードしているかを確認することができる。  [0168] From the determined nucleotide sequence, all amino acid sequences of VH and VL are deduced, and all amino acid sequences of VH and VL of known antibodies IJ [Sequences'Ob'Proteins'Ob'Immunological'Interest ( Sequences of Proteins of Immunologicailnterest), Ub Dept. Health and Human Services, 1991] to confirm that the cDNA obtained encodes the complete amino acid sequence of the antibody VH and VL, including the secretory signal sequence can do.
(3)ヒト以外の動物の抗体の V領域のアミノ酸配列の解析  (3) Analysis of the amino acid sequence of the V region of non-human animal antibodies
分泌シグナル配列を含む抗体の VH及び VLの完全なアミノ酸配列に関しては、既 知の抗体の VH及び VLの全アミノ酸配歹 IJ [シーケンシズ 'ォブ 'プロテインズ 'ォブ 'ィ ムノロン刀ノレ'インタレスト (Sequences of Proteins oflmmunological Interest), US Dept. Health and Human Services, 1991]と比較することにより、分泌シグナル配列の長さ 及び N末端アミノ酸配列を推定でき、更にはそれらが属するサブグノレープを知ること ができる。また、 VH及び VLの各 CDRのアミノ酸配列についても、既知の抗体の VH 及び VLのアミノ酸配列 [シーケンシズ ·ォブ ·プロテインズ ·ォブ ·ィムノロジカル 'イン タレスト (Sequences of Proteins of Immunological Interest), Us Dept. Healtn and Hum an Services, 1991]と比較することによって見出すことができる。  For the complete amino acid sequence of VH and VL of the antibody including the secretion signal sequence, all amino acid sequences of VH and VL of the known antibody IJ [Sequences 'Ob' Proteins (Sequences of Proteins of lmmunological Interest), US Dept. Health and Human Services, 1991], it is possible to estimate the length of the secretory signal sequence and the N-terminal amino acid sequence, and to know the subgnolep to which they belong. it can. In addition, the VH and VL CDR amino acid sequences of the known antibody VH and VL amino acid sequences [Sequences of Proteins of Immunological Interest, Us Dept. Healtn and Hum an Services, 1991].
(4)ヒト型キメラ抗体発現ベクターの構築  (4) Construction of human chimeric antibody expression vector
本項 2の（1)に記載のヒト化抗体発現用ベクターのヒト抗体の CH及び CLをコードす る遺伝子の上流に、ヒト以外の動物の抗体の VH及び VLをコードする cDNAをクロー ユングし、ヒト型キメラ抗体発現ベクターを構築することができる。例えば、ヒト以外の 動物の抗体の VH及び VLをコードする cDNAを、ヒト以外の動物の抗体 VH及び VLの 3 '末端側の塩基配列とヒト抗体の CH及び CLの 5'末端側の塩基配列とから成り、かつ 適当な制限酵素の認識配列を両端に有する合成 DNAとそれぞれ連結し、それぞれ を本項 2の（1)に記載のヒト化抗体発現用ベクターのヒト抗体の CH及び CLをコードす る遺伝子の上流にそれらが適切な形で発現するようにクローニングし、ヒト型キメラ抗 体発現ベクターを構築することができる。 The cDNA encoding the VH and VL of the non-human animal antibody is cloned upstream of the gene encoding the human antibody CH and CL of the humanized antibody expression vector described in (2) of this section 2. A human chimeric antibody expression vector can be constructed. For example, cDNA encoding VH and VL of a non-human animal antibody can be obtained by using the antibodies VH and VL of a non-human animal. It is composed of a base sequence on the end side and a base sequence on the 5 'end side of CH and CL of human antibody, and is linked to a synthetic DNA having an appropriate restriction enzyme recognition sequence at both ends. The humanized antibody expression vector described in (1) is cloned upstream of the gene encoding the human antibody CH and CL so that they are expressed in an appropriate form, and a human chimeric antibody expression vector is constructed. be able to.
(5)ヒト型 CDR移植抗体の V領域をコードする cDNAの構築 (5) Construction of cDNA encoding human CDR-grafted antibody V region
ヒト型 CDR移植抗体の VH及び VLをコードする cDNAは、以下のようにして構築する こと力 Sできる。まず、 目的のヒト以外の動物の抗体の VH及び VLの CDRを移植するヒト 抗体の VH及び VLのフレームワーク（以下、 FRと表記する）のアミノ酸配列を選択する 。ヒト抗体の VH及び VLの FRのアミノ酸配列としては、ヒト抗体由来のものであれば、 いかなるものでも用いることができる。例えば、 Protein Data Bank等のデータベース に登録されてレ、るヒト抗体の VH及び VLの FRのアミノ酸配歹 1J、ヒト抗体の VH及び VLの FRの各サブグループの共通アミノ酸配歹 U [シーケンシズ.ォブ.プロテインズ.ォブ 'ィ ムノロン刀ノレ'インタレスト (Sequences of Proteins of Immunological Interest), US Dept . Health and Human Services, 1991]等があげられるが、その中でも、十分な活性を 有するヒト型 CDR移植抗体を作製するためには、 目的のヒト以外の動物の抗体の VH 及び VLの FRのアミノ酸配列とできるだけ高い相同性 (少なくとも 60%以上）を有するァ ミノ酸配列を選択することが望ましレ、。  CDNAs encoding human CDR-grafted antibodies VH and VL can be constructed as follows. First, the amino acid sequence of the VH and VL framework (hereinafter referred to as FR) of the human antibody to which the VH and VL CDRs of the target non-human animal antibody are transplanted is selected. Any amino acid sequence derived from a human antibody can be used as the amino acid sequence of human antibody VH and VL FR. For example, the human amino acid VH and VL FR amino acid sequences 1J, human antibody VH and VL FR subgroups, which are registered in databases such as Protein Data Bank, are common amino acid sequences U [Sequences. OB. Proteins. OB 'I Munolon Katana' Interest (Sequences of Proteins of Immunological Interest), US Dept. Health and Human Services, 1991]. Among them, human type with sufficient activity In order to produce CDR-grafted antibodies, it is desirable to select amino acid sequences that have as high a homology as possible (at least 60% or more) with the VH and VL FR amino acid sequences of the non-human animal antibodies of interest. Masle.
次に、選択したヒト抗体の VH及び VLの FRのアミノ酸配列に目的のヒト以外の動物 の抗体の VH及び VLの CDRのアミノ酸配列を移植し、ヒト型 CDR移植抗体の VH及び VLのアミノ酸配列を設計する。設計したアミノ酸配列を抗体の遺伝子の塩基配列に 見られるコドンの使用頻度 [シーケンシズ'ォブ'プロテインズ'ォブ'ィムノロジカル'ィ ンタレス卜 (Sequences of Proteins of Immunological Interest), US Dept. Health and H uman Services, 1991]を考慮して DNA配列に変換し、ヒト型 CDR移植抗体の VH及び VLのアミノ酸配列をコードする DNA配列を設計する。設計した DNA配列に基づき、 1 00塩基前後の長さ力 成る数本の合成 DNAを合成し、それらを用いて PCR法を行う。 この場合、 PCRでの反応効率及び合成可能な DNAの長さから、 H鎖、 L鎖とも 6本の 合成 DNAを設計することが好ましレ、。 [0170] また、両端に位置する合成 DNAの 5'末端に適当な制限酵素の認識配列を導入す ることで、本項 2の（1)で構築したヒト化抗体発現用ベクターに容易にクローユングす ること力 Sできる。 PCR後、増幅産物を pBluescript SK (-) (Stratagene社製）等のプラスミ ドにクローユングし、本項 2の（2)に記載の方法により、塩基配列を決定し、所望のヒト 型 CDR移植抗体の VH及び VLのアミノ酸配列をコードする DNA配列を有するプラスミ ドを取得する。 Next, the VH and VL CDR amino acid sequences of the target non-human animal antibody are transplanted into the VH and VL FR amino acid sequences of the selected human antibody, and the VH and VL amino acid sequences of the human CDR-grafted antibody. To design. Frequency of codon usage of the designed amino acid sequence in the base sequence of antibody genes [Sequences of Proteins of Immunological Interest], US Dept. Health and H uman Services, 1991] is converted into a DNA sequence, and a DNA sequence encoding the amino acid sequence of VH and VL of a human CDR-grafted antibody is designed. Based on the designed DNA sequence, several synthetic DNAs with a length of around 100 bases are synthesized, and PCR is performed using them. In this case, it is preferable to design 6 synthetic DNAs for both the H and L chains, based on the PCR reaction efficiency and the length of DNA that can be synthesized. [0170] In addition, by introducing an appropriate restriction enzyme recognition sequence into the 5 'end of the synthetic DNA located at both ends, it can be easily cloned into the humanized antibody expression vector constructed in (2) of this section 2. Sliding power S After PCR, the amplified product is cloned into a plasmid such as pBluescript SK (-) (Stratagene), the nucleotide sequence is determined by the method described in (2) of this section 2, and the desired human CDR-grafted antibody A plasmid having a DNA sequence encoding the amino acid sequence of VH and VL is obtained.
(6)ヒト型 CDR移植抗体の V領域のアミノ酸配列の改変  (6) Modification of amino acid sequence of V region of human CDR-grafted antibody
ヒト型 CDR移植抗体は、 目的のヒト以外の動物の抗体の VH及び VLの CDRのみをヒ ト抗体の VH及び VLの FRに移植しただけでは、その抗原結合活性は元のヒト以外の 動物の抗体に比べて低下してしまうことが知られてレ、るレ ィォ /テクノロジー (BIO/TE CHNOLOGY), 9, 266 (1991)]。この原因としては、元のヒト以外の動物の抗体の VH 及び VLでは、 CDRのみならず、 FRのレ、くつかのアミノ酸残基が直接的或いは間接的 に抗原結合活性に関与しており、それらアミノ酸残基が CDRの移植に伴い、ヒト抗体 の VH及び VLの FRの異なるアミノ酸残基へと変化してしまうことが考えられてレ、る。こ の問題を解決するため、ヒト型 CDR移植抗体では、ヒト抗体の VH及び VLの FRのアミ ノ酸配列の中で、直接抗原との結合に関与しているアミノ酸残基や CDRのアミノ酸残 基と相互作用したり、抗体の立体構造を維持し、間接的に抗原との結合に関与して レ、るアミノ酸残基を同定し、それらを元のヒト以外の動物の抗体に見出されるアミノ酸 残基に改変し、低下した抗原結合活性を上昇させることが行われている [バイオ/テク ノロジー (BIO/TECHNOLOGY)， 9, 266 (1991)]。  The human CDR-grafted antibody can be obtained by transplanting only the VH and VL CDRs of the target non-human animal antibody to the VH and VL FRs of the human antibody. It is known that it will be lower than that of an antibody (BIO / TE CHNOLOGY), 9, 266 (1991)]. This is because, in the VH and VL of the original non-human animal antibody, not only CDR but also FR residues and some amino acid residues are directly or indirectly involved in antigen binding activity. It is thought that these amino acid residues are changed to different amino acid residues of FR of VH and VL of human antibody with CDR grafting. To solve this problem, human CDR-grafted antibodies use amino acid residues that are directly involved in antigen binding or CDR amino acid residues in the human amino acid VH and VL FR amino acid sequences. Amino acids found in the antibody of the original non-human animal by identifying the amino acid residues that interact with the group or maintain the three-dimensional structure of the antibody and indirectly participate in antigen binding. It has been practiced to modify residues to increase decreased antigen binding activity [BIO / TECHNOLOGY, 9, 266 (1991)].
[0171] ヒト型 CDR移植抗体の作製においては、それら抗原結合活性に関わる FRのァミノ 酸残基を如何に効率よく同定するかが、最も重要な点であり、そのために X線結晶解 析 [ジャーナル'ォブ'モレキユラ一'バイオロジー (J. Mol. Biol.), 112, 535 (1977)]或 レヽはコンピューターモデリング [プロテイン.エンジニアリング (Protein Engineering), 7, 1501 (1994)]等による抗体の立体構造の構築及び解析が行われている。これら抗体 の立体構造の情報は、ヒト型 CDR移植抗体の作製に多くの有益な情報をもたらして 来たが、その一方、あらゆる抗体に適応可能なヒト型 CDR移植抗体の作製法は未だ 確立されておらず、現状ではそれぞれの抗体について数種の改変体を作製し、それ ぞれの抗原結合活性との相関を検討する等の種々の試行錯誤が必要である。 [0171] In the production of human CDR-grafted antibodies, the most important point is how to identify the FR amino acid residues involved in these antigen-binding activities. For this reason, X-ray crystallography [ J. Mol. Biol., 112, 535 (1977)] or the label is an antibody from computer modeling [Protein Engineering, 7, 1501 (1994)] etc. The three-dimensional structure is being constructed and analyzed. The information on the three-dimensional structure of these antibodies has provided a lot of useful information for the production of human CDR-grafted antibodies, but on the other hand, methods for producing human-type CDR-grafted antibodies that can be applied to all antibodies are still established. Currently, we have created several variants for each antibody, Various trials and errors such as examining the correlation with each antigen binding activity are required.
[0172] ヒト抗体の VH及び VLの FRのアミノ酸残基の改変は、改変用合成 DNAを用いて本 項 2の（5)に記載の PCR法を行うことにより、達成できる。 PCR後の増幅産物について 本項 2の（2)に記載の方法により、塩基配列を決定し、 目的の改変が施されたことを 確認する。 [0172] Modification of FR amino acid residues of VH and VL of a human antibody can be achieved by performing the PCR method described in (2) of this section 2 using synthetic DNA for modification. For amplified products after PCR, determine the nucleotide sequence by the method described in (2) of this section 2 and confirm that the target modification has been made.
(7)ヒト型 CDR移植抗体発現ベクターの構築  (7) Construction of human CDR-grafted antibody expression vector
本項 2の（1)に記載のヒト化抗体発現用ベクターのヒト抗体の CH及び CLをコードす る遺伝子の上流に、本項 2の（5)及び（6)で構築したヒト型 CDR移植抗体の VH及び VLをコードする cDNAをクローユングし、ヒト型 CDR移植抗体発現ベクターを構築する こと力 Sできる。例えば、本項 2の（5)及び（6)でヒト型 CDR移植抗体の VH及び VLを構 築する際に用いる合成 DNAのうち、両端に位置する合成 DNAの 5'末端に適当な制 限酵素の認識配列を導入することで、本項 2の（1)に記載のヒト化抗体発現用べクタ 一のヒト抗体の CH及び CLをコードする遺伝子の上流にそれらが適切な形で発現す るようにクローニングし、ヒト型 CDR移植抗体発現ベクターを構築することができる。 The human CDR graft constructed in (5) and (6) of this section 2 upstream of the gene encoding the human antibody CH and CL of the humanized antibody expression vector of (2) of this section 2 The cDNA encoding the antibody VH and VL can be cloned to construct a human CDR-grafted antibody expression vector. For example, among the synthetic DNAs used in constructing human CDR-grafted antibody VH and VL in (2) and (6) of this section 2, appropriate restrictions are placed on the 5 ′ ends of the synthetic DNAs located at both ends. By introducing the recognition sequence of the enzyme, it is expressed in an appropriate form upstream of the genes encoding the human antibody CH and CL of the humanized antibody expression vector described in (1) of this section 2. And a human CDR-grafted antibody expression vector can be constructed.
(8)ヒト化抗体の安定的生産 (8) Stable production of humanized antibodies
本項 2の（4)及び（7)に記載のヒト化抗体発現ベクターを適当な動物細胞に導入す ることによりヒト型キメラ抗体及びヒト型 CDR移植抗体（以下、併せてヒト化抗体と称す )を安定に生産する形質転換株を得ることができる。  By introducing the humanized antibody expression vector described in (4) and (7) of this section 2 into an appropriate animal cell, a human chimeric antibody and a human CDR grafted antibody (hereinafter collectively referred to as a humanized antibody). ) Can be obtained stably.
[0173] 動物細胞へのヒト化抗体発現ベクターの導入法としては、エレクト口ポレーシヨン法 [ 特開平 2-257891 ;サイトテクノロジー (Cytotechnology), 3，133 (1990)]等があげられ る。 [0173] Examples of a method for introducing a humanized antibody expression vector into animal cells include the electoral position method [Japanese Patent Laid-Open No. 2-257891; Cytotechnology, 3, 133 (1990)].
ヒトイ匕抗体発現ベクターを導入する動物細胞としては、ヒト化抗体を生産させること ができる動物細胞であれば、いかなる細胞でも用いることができる。  Any animal cell capable of producing a humanized antibody can be used as the animal cell into which the human 匕 antibody expression vector is introduced.
具体的には、マウスミエローマ細胞である NS0細胞、 SP2/0細胞、チャイニーズハム スター卵巣細胞 CHO/dhfr_細胞、 CHO/DG44細胞、ラットミエローマ YB2/0細胞、 IR 983F細胞、シリアンハムスター腎臓由来である BHK細胞、ヒトミエローマ細胞である ナマルバ細胞などがあげられる力 S、好ましくは、チャイニーズハムスター卵巣細胞で ある CHO/DG44細胞、ラットミエローマ YB2/0細胞、前記 2.または 4.に記載の本発 明の α -1,6-フコシルトランスフェラーゼ変異体を発現する細胞等があげられる。 Specifically, mouse myeloma cells derived from NS0 cells, SP2 / 0 cells, Chinese hamster ovary cells CHO / dhfr_ cells, CHO / DG44 cells, rat myeloma YB2 / 0 cells, IR 983F cells, and Syrian hamster kidneys Certain BHK cells, human myeloma cells Namalva cells, etc., preferably CHO / DG44 cells that are Chinese hamster ovary cells, rat myeloma YB2 / 0 cells, Examples include cells that express a bright α-1,6-fucosyltransferase mutant.
[0174] ヒト化抗体発現ベクターの導入後、ヒト化抗体を安定に生産する形質転換株は、特 開平 2-257891に開示されている方法に従い、 G418 sulfate (以下、 G418と表記する； SIGMA社製）等の薬剤を含む動物細胞培養用培地により選択できる。動物細胞培養 用培地としては、 RPMI1640培地（日水製薬社製）、 GIT培地（日本製薬社製）、 EX-C ELL302培地（JRH社製）、 IMDM培地（GIBCO BRL社製）、 Hybridoma-SFM培地（GI BCO BRL社製）、またはこれら培地に牛胎児血清 (以下、 FBSと表記する）等の各種 添加物を添加した培地等を用いることができる。得られた形質転換株を培地中で培 養することで培養上清中にヒト化抗体を生産蓄積させることができる。培養上清中の ヒト化抗体の生産量及び抗原結合活性は酵素免疫抗体法 [以下、 ELISA法と表記す る；アンティボディズ：ァ 'ラボラトリー 'マ二ユアノレ (Antibodies: A Laboratory Manual), Cold Spring Harbor Laboratory, Chapter 14, 1998、モノクロ一ナノレ'アンティボディズ ：プリンシプノレズ 'アンド'プラクティス (Monoclonal Antibodies: Principles and Practice )， Academic Press Limited, 1996]等により測定できる。また、形質転換株は、特開平 2-257891に開示されている方法に従い、 DHFR遺伝子増幅系等を利用してヒト化抗 体の生産量を上昇させることができる。 [0174] After the introduction of the humanized antibody expression vector, a transformant that stably produces a humanized antibody is G418 sulfate (hereinafter referred to as G418; SIGMA) according to the method disclosed in JP-A-2-257891. Selected from animal cell culture media containing drugs such as Animal cell culture media include RPMI1640 medium (Nissui Pharmaceutical), GIT medium (Nihon Pharmaceutical), EX-C ELL302 medium (JRH), IMDM medium (GIBCO BRL), Hybridoma-SFM A medium (GI BCO BRL) or a medium obtained by adding various additives such as fetal bovine serum (hereinafter referred to as FBS) to these mediums can be used. By culturing the obtained transformant in a medium, the humanized antibody can be produced and accumulated in the culture supernatant. The production amount and antigen binding activity of the humanized antibody in the culture supernatant are the enzyme-linked immunosorbent assay (hereinafter referred to as the ELISA method; Antibodies: Laboratories: A Laboratory Manual), Cold It can be measured by Spring Harbor Laboratory, Chapter 14, 1998, Monochrome Mononole Antibodies: Principles and Practices, Academic Press Limited, 1996]. In addition, the transformed strain can increase the production amount of the humanized antibody using a DHFR gene amplification system or the like according to the method disclosed in JP-A-2-257891.
[0175] ヒトイ匕抗体は、形質転換株の培養上清よりプロテイン Aカラムを用いて精製すること ができる [アンティボディズ：ァ'ラボラトリ^ ~ ·マニュアル (Antibodies: A Laboratory Ma nual), Cold Spring Harbor Laboratory, Chapter 8， 1988、モノクローナル ·アンティボ ディズ：プリンシプルズ'アンド'プラクティス (Monoclonal Antibodies: Principles and Pr actice), Academic Press Limited, 1996]。また、その他に通常、タンパク質の精製で 用いられる精製方法を使用することができる。例えば、ゲル濾過、イオン交換クロマト グラフィー及び限外濾過等を組み合わせて行い、精製することができる。精製したヒト 化抗体の H鎖、 L鎖或いは抗体分子全体の分子量は、ポリアクリルアミドゲル電気泳 動 [以下、 SDS-PAGEと表記する；ネイチヤー (Nature), 227, 680 (1970)]やウェスタン ブロッテイング法 [アンティボディズ：ァ 'ラボラトリー 'マニュアル (Antibodies: A Labora tory Manual),し old Spring Harbor Laboratory, Chapter 12, 1988、モノクロ" ~ナノレ · 7 ンティボディズ：プリンシプルズ'アンド'プラクティス (Monoclonal Antibodies: Principle s and Practice), Academic Press Limited, 1996]等で測定することができる。 [0175] Human IgA antibody can be purified from the culture supernatant of the transformant using a protein A column [Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 8, 1988, Monoclonal Antibodies: Principles and Practices, Academic Press Limited, 1996]. In addition, a purification method usually used in protein purification can be used. For example, it can be purified by a combination of gel filtration, ion exchange chromatography, ultrafiltration and the like. The molecular weight of the purified humanized antibody H chain, L chain, or whole antibody molecule is determined by polyacrylamide gel electrophoresis [hereinafter referred to as SDS-PAGE; Nature, 227, 680 (1970)] or Western blot. Antibodies: A Labora tory Manual, Old Spring Harbor Laboratory, Chapter 12, 1988, Monochrome "~ Nanore 7 Antibodies: Principles and Practice (Monoclonal Antibodies: Principle s and Practice), Academic Press Limited, 1996].
B. Fc融合蛋白質組成物の製造  B. Production of Fc fusion protein composition
(1) Fc融合蛋白質発現用ベクターの構築  (1) Construction of Fc fusion protein expression vector
Fc融合蛋白質発現用ベクターとは、ヒト抗体の Fc領域と融合させる蛋白質とをコー ドする遺伝子が組み込まれた動物細胞用発現ベクターであり、動物細胞用発現べク ターにヒト抗体の Fc領域と融合させる蛋白質とをコードする遺伝子をクローニングする ことにより構築することができる。  The Fc fusion protein expression vector is an expression vector for animal cells in which a gene encoding a protein to be fused with the Fc region of a human antibody is incorporated, and the Fc region of the human antibody is incorporated into the expression vector for animal cells. It can be constructed by cloning a gene encoding a protein to be fused.
[0176] ヒト抗体の Fc領域としては、 CH2と CH3領域を含む領域のほ力、、ヒンジ領域、 CH1の 一部が含まれるものも包含される。また CH2または CH3の少なくとも 1つのアミノ酸が 欠失、置換、付加または挿入され、実質的に Fe y受容体への結合活性を有するもの であればいかなるものでもよい。  [0176] The Fc region of a human antibody includes the force of the region containing the CH2 and CH3 regions, the hinge region, and those containing a part of CH1. In addition, any amino acid may be used as long as at least one amino acid of CH2 or CH3 is deleted, substituted, added, or inserted and has substantially binding activity to the Fey receptor.
ヒト抗体の Fc領域と融合させる蛋白質とをコードする遺伝子としてはェキソンとイント ロンから成る染色体 DNAを用いることができ、また、 cDNAを用レ、ることもできる。それ ら遺伝子と Fc領域を連結する方法としては、各遺伝子配列を铸型として、 PCR法（レ キユラ ~ ·クローニング第 2版；カレント ·プロトコールズ'イン'モレキュラ^ ~ ·バイオロジ 一, Supplement 1-34)を行うことがあげられる。  As a gene encoding a protein to be fused with the Fc region of a human antibody, chromosomal DNA consisting of exons and introns can be used, and cDNA can also be used. As a method of linking these genes and the Fc region, each gene sequence is used as a saddle type, PCR method (Requirula ~ · Cloning 2nd edition; Current Protocols 'in' Molecular ^ ~ · Biology 1, Supplement 1- 34).
[0177] 動物細胞用発現ベクターとしては、ヒト抗体の C領域をコードする遺伝子を組込み 発現できるものであればいかなるものでも用いることができる。例えば、 pAGE107 [サ イトテクノロジー（Cytotechnology), 3, 133 (1990)]、 pAGE103 [ジャーナル'ォブ'バイ オケミストリー (J. Biochem.),皿, 1307 (1987)]、 pHSG274 [ジーン (Gene), 27, 223 ( 1984)]、 pKCR [プロシーディングス 'ォブ 'ザ'ナショナル'ァカデミ一'ォブ'サイェン ス (Proc. Natl. Acad. Sci. U.S.A.), 78, 1527 (1981)]、 pSGl β d2-4 [サイトテクノロジ 一 (Cytotechnology), 4, 173 (1990)]等があげられる。動物細胞用発現ベクターに用 いるプロモーターとェンハンサ一としては、 SV40の初期プロモーターとェンハンサー [ ジャーナル'ォブ 'バイオケミストリー (J. Biochem.), 101, 1307 (1987)]、モロニ一マウ ス白血病ウィルスの LTRレ ィォケミカル ·アンド ·バイオフィジカル ·リサーチ ·コミュ二 ケーシヨンズ (Biochem. Biophys. Res. Commun.), 149, 960 (1987)]、免疫グロブリン H鎖のプロモーター [セル（Cell). 41, 479 (1985)]とェンハンサー [セル（Cell), 33, 71 7 (1983)]等があげられる。 [0177] As an expression vector for animal cells, any expression vector can be used as long as it can incorporate and express a gene encoding the C region of a human antibody. For example, pAGE107 [Cytotechnology, 3, 133 (1990)], pAGE103 [Journal 'Ob' by Chemistry (J. Biochem.), Dish, 1307 (1987)], pHSG274 [Gene , 27, 223 (1984)], pKCR [Proceedings' Ob 'The' National 'Academia 1'Ob' Science (Proc. Natl. Acad. Sci. USA), 78, 1527 (1981)], pSGl β d2-4 [Cytotechnology, 4, 173 (1990)]. The promoters and enhancers used in animal cell expression vectors include SV40 early promoters and enhancers [Journal 'Ob' Biochemistry., 101, 1307 (1987)], Moroni Mouse Leukemia Virus. LTR biochemical and biophysical research communications (Biochem. Biophys. Res. Commun.), 149, 960 (1987)], immunoglobulin heavy chain promoter [Cell. 41, 479 ( 1985)] and Enhanser [Cell, 33, 71 7 (1983)].
(2)ヒト抗体の Fc領域と融合させる蛋白質とをコードする DNAの取得  (2) Acquisition of DNA encoding a protein to be fused with the Fc region of a human antibody
ヒト抗体の Fc領域と融合させる蛋白質とをコードする DNAは以下のようにして取得 すること力 Sできる。  DNA encoding the protein to be fused with the Fc region of a human antibody can be obtained as follows.
[0178] 目的の Fcと融合させる蛋白質を発現している細胞や組織より mRNAを抽出し、 cDN Aを合成する。合成した cDNAをファージ或いはプラスミド等のベクターにクローニング して cDNAライブラリーを作製する。該ライブラリーより、 目的の蛋白質の遺伝子配列 部分をプローブとして用レ、、 目的の蛋白質をコードする cDNAを有する組換えファー ジ或いは組換えプラスミドを単離する。組換えファージ或いは組換えプラスミド上の目 的の蛋白質の全塩基配列を決定し、塩基配列より全アミノ酸配列を推定する。  [0178] mRNA is extracted from cells or tissues expressing the protein to be fused with the target Fc, and cDNA is synthesized. The synthesized cDNA is cloned into a vector such as a phage or plasmid to prepare a cDNA library. From this library, the gene sequence portion of the target protein is used as a probe, and a recombinant phage or a recombinant plasmid having cDNA encoding the target protein is isolated. The entire base sequence of the target protein on the recombinant phage or recombinant plasmid is determined, and the entire amino acid sequence is deduced from the base sequence.
[0179] ヒト以外の動物としては、マウス、ラット、ハムスター、ゥサギ等、細胞や組織を摘出 することが可能であれば、レ、かなるものも用いることができる。  [0179] As animals other than humans, mice, rats, hamsters, rabbits, etc. can be used as long as cells and tissues can be removed.
細胞や組織から全 RNAを調製する方法としては、チォシアン酸グァニジン-トリフル ォロ酢酸セシウム法 [メソッズ.イン.ェンザィモロジ一（Methods in Enzymol.), 154, 3 (1987)]、また全 RNAから mRNAを調製する方法としては、オリゴ (dT)固定化セルロース カラム法（モレキュラー 'クローニング第 2版）等があげられる。また、細胞や組織から m RNAを調製するキットとしては、 Fast Track mRNA Isolation Kit (Invitrogen社製)、 Qui ck Prep mRNA Purification Kit (Pharmacia社製）等があげられる。  Methods for preparing total RNA from cells and tissues include guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymol., 154, 3 (1987)], and mRNA from total RNA. Examples of the method for preparing the above include oligo (dT) -immobilized cellulose column method (Molecular 'Cloning 2nd Edition). Examples of kits for preparing mRNA from cells and tissues include Fast Track mRNA Isolation Kit (manufactured by Invitrogen), Quick Prep mRNA Purification Kit (manufactured by Pharmacia), and the like.
[0180] cDNAの合成及び cDNAライブラリ一作製法としては、常法（モレキユラ一'クロー二 ング第 3版；カレント'プロトコールズ 'イン'モレキュラー 'バイオロジー， Supplement 1_ 34)、或いは市販のキット、例えば、 Super Script™ Plasmid System for cDNA Synthesi s and Plasmid Cloning (GIBCO BRL社製）や ZAP— cDNA Synthesis Kit (Stratagene 社製)を用いる方法などがあげられる。  [0180] Synthesis of cDNA and construction of a cDNA library include conventional methods (Molequila I Cloning 3rd Edition; Current Protocols In-Molecular Biology, Supplement 1_34), or commercially available kits, For example, a method using Super Script ™ Plasmid System for cDNA Synthesis and Plasmid Cloning (GIBCO BRL) or ZAP-cDNA Synthesis Kit (Stratagene) can be mentioned.
[0181] cDNAライブラリーの作製の際、細胞や組織から抽出した mRNAを錡型として合成し た cDNAを組み込むベクターは、該 cDNAを組み込めるベクターであればいかなるも のでも用いることができる。例えば、 ZAP Express [ストラテジーズ (Strategies), 5, 58 (1 992)]、 pBluescript II SK(+) [ヌクレイツク'ァシッズ 'リサーチ (Nucleic Acids Research), 17， 9494 (1989)]、 λ zapll (Stratagene社製)、 gtl0、 λ gtl l [ディーェヌェ一'クロ 一二ング：ァ 'プラクティカル 'アプローチ（DNA Cloning: A Practical Approach), I, 49 (1985)]、 Lambda BlueMid (Clontech社製)、 λ ExCel pT7T3 18U (Pharmacia社製） 、 cD2 [モレキュラー ·アンド 'セルラ ~ ·バイオロジー (Mol. Cell. Biol.), 3, 280 (1983 )]及び pUC18 [ジーン (Gene), 33, 103 (1985)]等が用いられる。 [0181] When preparing a cDNA library, any vector can be used as a vector into which cDNA synthesized by using mRNA extracted from cells or tissues as a saddle type can be incorporated. For example, ZAP Express [Strategies, 5, 58 (1 992)], pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)], λ zapll (Stratagene ), Gtl0, λ gtl l 12: Practical approach (DNA Cloning: A Practical Approach), I, 49 (1985)], Lambda BlueMid (Clontech), λ ExCel pT7T3 18U (Pharmacia), cD2 (Molecular and 'Cellular Biology (Mol. Cell. Biol.), 3, 280 (1983)], pUC18 [Gene, 33, 103 (1985)] and the like are used.
[0182] ファージ或いはプラスミドベクターにより構築される cDNAライブラリーを導入する大 腸菌としては該 cDNAライブラリーを導入、発現及び維持できるものであればレ、かなる ものでも用いることができる。例えば、 XLl-Blue MRF' [ストラテジーズ（Strategies), 5, 81 (1992)]、 C600 [ジェネティックス (Genetics), 39, 440 (1954)]、 Y1088、 Y1090 [サ ィエンス（Science), 222, 778 (1983)]、 NM522 [ジャーナル'ォブ'モレキュラー 'バイ ォロジ一 (J. Mol. Biol), 166, 1 (1983)]、 K802 [ジャーナル'ォブ 'モレキュラー 'バイ ォロジ一 (J. Mol. Biol), 16, 118 (1966)]及び JM105 [ジーン (Gene), 38, 275 (1985)] 等が用いられる。 [0182] As the Escherichia coli for introducing a cDNA library constructed by a phage or a plasmid vector, any one can be used as long as it can introduce, express and maintain the cDNA library. For example, XLl-Blue MRF '[Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088, Y1090 [Science, 222, 778 (1983)], NM522 [Journal 'Ob' Molecular 'Biol (J. Mol. Biol), 166, 1 (1983)], K802 [Journal' Ob 'Molecular' Biological (J. Mol) Biol), 16, 118 (1966)] and JM105 [Gene, 38, 275 (1985)] and the like are used.
[0183] cDNAライブラリ一力もの目的の蛋白質をコードする cDNAクローンの選択法としては 、アイソトープ或いは蛍光標識したプローブを用いたコロニー'ノ、イブリダィゼーシヨン 法或いはプラーク 'ハイブリダィゼーシヨン法（モレキュラー 'クローニング第 3版）によ り選択すること力できる。また、プライマーを調製し、 mRNAから合成した cDNA或いは cDNAライブラリーを铸型として、 PCR法により目的の蛋白質をコードする cDNAを調製 することちでさる。  [0183] cDNA library selection of cDNA clones encoding the protein of interest, including colonies using the isotope or fluorescently labeled probe, hybridization method or plaque 'hybridization method (Molecular 'Cloning 3rd Edition) can be selected. It is also possible to prepare primers and prepare cDNA encoding the protein of interest by PCR using a cDNA or cDNA library synthesized from mRNA as a saddle.
[0184] 目的の蛋白質をヒト抗体の Fc領域と融合させる方法としては、 PCR法があげられる。  [0184] Examples of a method for fusing the target protein with the Fc region of a human antibody include PCR.
例えば、 目的の蛋白質の遺伝子配列の 5'側と 3'側に任意の合成オリゴ DNA (プライマ 一)を設定し、 PCR法を行い PCR産物を取得する。同様に、融合させるヒト抗体の Fc領 域の遺伝子配列に対しても任意のプライマーを設定し、 PCR産物を得る。このとき、 融合させる蛋白質の PCR産物の 3'側と Fc領域の PCR産物の 5'側には同じ制限酵素 部位もしくは同じ遺伝子配列が存在するようにプライマーを設定する。この連結部分 周辺のアミノ酸改変が必要である場合には、その変異を導入したプライマーを用いる ことで変異を導入する。得られた 2種類の PCR断片を用いてさらに PCRを行うことで、 両遺伝子を連結する。もしくは、同一の制限酵素処理をした後にライゲーシヨンするこ とでも連結すること力 Sできる。 [0185] 上記方法により連結された遺伝子配列を、適当な制限酵素などで切断後、 pBluesc ript SK (-) (Stratagene社製）等のプラスミドにクローニングし、通常用いられる塩基配 列解析方法、例えばサンガー（Sanger)らのジデォキシ法 [プロシーディンダス 'ォブ 'ザ*ナショナル ·ァカデミ ~ ·ォブ 'サイエンス (Proc. Natl. Acad. Sci. U.S.A.), 74, 54 63 (1977)]あるいは ABI PRISM 377DNAシークェンサ一（PE Biosystems社製）等の 塩基配列分析装置を用いて分析することにより、該 DNAの塩基配列を決定すること ができる。 For example, place any synthetic oligo DNA (primer) on the 5 'and 3' sides of the gene sequence of the target protein, and perform PCR to obtain a PCR product. Similarly, an arbitrary primer is set for the gene sequence in the Fc region of the human antibody to be fused to obtain a PCR product. At this time, set primers so that the same restriction enzyme site or the same gene sequence exists on the 3 'side of the PCR product of the protein to be fused and the 5' side of the PCR product of the Fc region. If it is necessary to modify the amino acid around this ligation site, the mutation is introduced by using a primer into which the mutation has been introduced. Two genes are ligated by further PCR using the two types of PCR fragments obtained. Alternatively, ligation can be performed after ligation after the same restriction enzyme treatment. [0185] The gene sequence linked by the above method is cleaved with an appropriate restriction enzyme or the like, and then cloned into a plasmid such as pBluescript SK (-) (Stratagene). Sanger et al.'S dideoxy method [Procedinas 'Ob' The * National Academia Sci. USA (Proc. Natl. Acad. Sci. USA), 74, 54 63 (1977)] or ABI PRISM By analyzing using a base sequence analyzer such as 377 DNA Sequencer (PE Biosystems), the base sequence of the DNA can be determined.
[0186] 決定した塩基配列から Fc融合蛋白質の全アミノ酸配列を推定し、 目的のアミノ酸配 歹 IJと比較することにより、取得した cDNAが分泌シグナル配列を含む Fc融合蛋白質の 完全なアミノ酸配列をコードしているかを確認することができる。  [0186] By deducing the total amino acid sequence of the Fc fusion protein from the determined nucleotide sequence and comparing it with the target amino acid sequence IJ, the obtained cDNA encodes the complete amino acid sequence of the Fc fusion protein including the secretory signal sequence. You can check whether you are doing.
(3) Fc融合蛋白質組成物の安定的生産  (3) Stable production of Fc fusion protein composition
本項 2の Bの（1)項に記載の Fc融合蛋白質発現ベクターを適当な動物細胞に導入 することにより Fc融合蛋白質組成物を安定に生産する形質転換株を得ることができる  By introducing the Fc fusion protein expression vector described in paragraph (1) of B in this section 2 into an appropriate animal cell, a transformant that stably produces the Fc fusion protein composition can be obtained.
[0187] 動物細胞への Fc融合蛋白質発現ベクターの導入法としては、エレクト口ポレーショ ン法 [特開平 2-257891 ;サイトテクノロジー（Cytotechnology), 3, 133 (1990)]等があ げられる。 [0187] Examples of a method for introducing an Fc fusion protein expression vector into animal cells include the electoporation method [Japanese Patent Laid-Open No. 2-257891; Cytotechnology, 3, 133 (1990)].
Fc融合蛋白質発現ベクターを導入する動物細胞としては、 Fc融合蛋白質を生産さ せることができる動物細胞であれば、いかなる細胞でも用いることができる。  Any animal cell capable of producing an Fc fusion protein can be used as an animal cell into which an Fc fusion protein expression vector is introduced.
[0188] 具体的には、マウスミエローマ細胞である NS0細胞、 SP2/0細胞、チャイニーズハム スター卵巣細胞 CHO/dhfr-細胞、 CHO/DG44細胞、ラットミエローマ YB2/0細胞、 IR 983F細胞、シリアンハムスター腎臓由来である BHK細胞、ヒトミエローマ細胞であるナ マルバ細胞などがあげられる力 好ましくは、チャイニーズノヽムスター卵巣細胞である CHO/DG44細胞、ラットミエローマ YB2/0細胞、前記 2.または 4.に記載の本発明の ひ - 1，6-フコシルトランスフェラーゼ変異体を発現する細胞等があげられる。  [0188] Specifically, mouse myeloma cells NS0 cells, SP2 / 0 cells, Chinese hamster ovary cells CHO / dhfr- cells, CHO / DG44 cells, rat myeloma YB2 / 0 cells, IR 983F cells, Syrian hamsters Ability to increase kidney-derived BHK cells, human myeloma cells such as Namalba cells, etc. Preferably, Chinese nomstar ovary cells CHO / DG44 cells, rat myeloma YB2 / 0 cells, as described in 2. or 4 above. And cells expressing the -1,6-fucosyltransferase mutant of the present invention.
[0189] Fc融合蛋白質発現ベクターの導入後、 Fc融合蛋白質組成物を安定に生産する形 質転換株は、特開平 2-257891に開示されている方法に従い、 G418等の薬剤を含む 動物細胞培養用培地により選択できる。動物細胞培養用培地としては、 RPMI1640培 地（日水製薬社製)、 GIT培地（日本製薬社製)、 EX-CELL302培地（JRH社製)、 IMD M培地（GIBCO BRL社製)、 Hybridoma-SFM培地（GIBCO BRL社製)、またはこれら 培地に牛胎児血清等の各種添加物を添加した培地等を用いることができる。得られ た形質転換株を培地中で培養することで培養上清中に Fc融合蛋白質組成物を生産 蓄積させることができる。培養上清中の Fc融合蛋白質組成物の生産量及び抗原結 合活性は EUSA法等により測定できる。また、形質転換株は、特開平 2-257891に開 示されている方法に従い、 dhfr遺伝子増幅系等を利用して Fc融合蛋白質組成物の 生産量を上昇させることができる。 [0189] After the introduction of the Fc fusion protein expression vector, the transformant that stably produces the Fc fusion protein composition is an animal cell culture containing a drug such as G418 according to the method disclosed in JP-A-2-257891. It can be selected according to the culture medium. As an animal cell culture medium, RPMI1640 medium Ground (manufactured by Nissui Pharmaceutical), GIT medium (manufactured by Nippon Pharmaceutical), EX-CELL302 medium (manufactured by JRH), IMD M medium (manufactured by GIBCO BRL), Hybridoma-SFM medium (manufactured by GIBCO BRL), or A medium obtained by adding various additives such as fetal calf serum to these mediums can be used. By culturing the obtained transformant in a medium, the Fc fusion protein composition can be produced and accumulated in the culture supernatant. The production amount and antigen binding activity of the Fc fusion protein composition in the culture supernatant can be measured by the EUSA method or the like. In addition, the transformant can increase the production amount of the Fc fusion protein composition using a dhfr gene amplification system or the like according to the method disclosed in JP-A-2-257891.
[0190] Fc融合蛋白質組成物は、形質転換株の培養上清よりプロテイン Aカラムやプロティ ン Gカラムを用いて精製することができる（アンチボディズ， Chapter 8、モノクローナノレ •アンティボディズ)。また、その他に通常、タンパク質の精製で用レ、られる精製方法を 使用することができる。例えば、ゲル濾過、イオン交換クロマトグラフィー及び限外濾 過等を組み合わせて行い、精製することができる。精製した Fc融合蛋白質分子全体 の分子量は、 SDS-PAGE [ネイチヤー（Nature), 227, 680 (1970)]やウェスタンブロッ ティング法（アンチボディズ， Chapter 12、モノクローナル 'アンティボディズ）等で測 定すること力 Sできる。 [0190] The Fc fusion protein composition can be purified from the culture supernatant of the transformant using a protein A column or protein G column (Antibodies, Chapter 8, Monochrome Nanore • Antibodies) . In addition, purification methods generally used for protein purification can be used. For example, it can be purified by combining gel filtration, ion exchange chromatography, ultrafiltration and the like. The molecular weight of the entire purified Fc fusion protein molecule is measured by SDS-PAGE [Nature, 227, 680 (1970)] or Western blotting (Antibodies, Chapter 12, Monoclonal 'Antibodies). The power to do S.
[0191] 以上、動物細胞を宿主とした抗体組成物の製造方法を示したが、上述したように、 細菌、酵母、昆虫細胞、植物細胞または動物個体あるいは植物個体においても製造 すること力 Sできる。  [0191] Although the method for producing an antibody composition using animal cells as a host has been described above, as described above, it is possible to produce bacteria, yeast, insect cells, plant cells, or animal individuals or plant individuals. .
既に、細胞が抗体分子等の糖蛋白質組成物を発現する能力を有している場合に は、前記 4.に記載の方法を用いて細胞を調製した後に、該細胞を培養し、該培養 物から目的とする抗体組成物を精製することにより、本発明の抗体組成物ゃ糖蛋白 質組成物を製造することができる。  When the cell already has the ability to express a glycoprotein composition such as an antibody molecule, the cell is prepared using the method described in 4. above, and then the cell is cultured. From the purified antibody composition, the antibody composition of the present invention can be produced as a glycoprotein composition.
7.糖蛋白質組成物の活性評価  7. Activity evaluation of glycoprotein composition
精製した糖蛋白質組成物の蛋白量、受容体との親和性、血液中での半減期、血液 投与後の組織への分布、あるいは薬理活性発現に必要な蛋白質相互作用の変化を 測 する方法としては、 Current Protocols In Protein Science, John Wiley & Sons Inc .，（1995)、 日本生化学会編新生化学実験講座 19動物実験法，東京化学同人（199 1)、 日本生化学会編新生化学実験講座 8細胞内情報と細胞応答，東京化学同人（ 1990)、 日本生化学会編新生化学実験講座 9ホルモン Iペプチドホルモン，東京化学 同人（1991)、実験生物学講座 3アイソトープ実験法，丸善株式会社（1982)、 Monocl onal Antibodies: Principles and Applications, Wiley-Liss, Inc., (1995)、酵素免疫測 定法第 3版，医学書院（1987)、改訂版酵素抗体法，学際企画（1985)等に記載の公 知の方法を用いることができる。 As a method to measure the amount of protein in the purified glycoprotein composition, affinity with receptors, half-life in blood, distribution to tissues after blood administration, or changes in protein interaction necessary for expression of pharmacological activity Current Protocols In Protein Science, John Wiley & Sons Inc., (1995), Japan Biochemical Society, Shinsei Chemistry Laboratory 19 Animal Experiment Method, Tokyo Chemical Doujin (199 1), Japan Biochemical Society edited new chemistry experiment course 8 Intracellular information and cell response, Tokyo Chemical Doujin (1990), Japan Biochemical Society edited new biochemistry experiment 9 hormone I peptide hormone, Tokyo Chemical Doujin (1991), experimental biology Lecture 3 Isotope Experiment, Maruzen Co., Ltd. (1982), Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., (1995), Enzyme Immunoassay 3rd Edition, Medical School (1987), Revised Enzyme Antibody The publicly known methods described in Law, Interdisciplinary Planning (1985), etc. can be used.
[0192] その具体的な例としては、精製した糖蛋白質組成物をラジオアイソトープなどの化 合物で標識し、標識した糖蛋白質組成物の受容体あるいは相互作用をする蛋白質と の結合反応の強さを定量的に測定する方法があげられる。また、 Biacore社の BIAcor eシリーズなどの各種装置を用いて、蛋白質蛋白質相互作用を測定することもできる（[0192] As a specific example, a purified glycoprotein composition is labeled with a compound such as a radioisotope, and the binding reaction with the receptor or interacting protein of the labeled glycoprotein composition is strong. There is a method for quantitatively measuring the thickness. It is also possible to measure protein-protein interactions using various devices such as Biacore's BIAcor e series (
J. Immnunol. Methods, 145, 229 (1991)、実験医学別冊バイオマニュアル UPシリー ズタンパク質の分子間相互作用実験法，羊土社ひ 996))。 J. Immnunol. Methods, 145, 229 (1991), Biomedical Biomedical Supplement, UP Series Protein Intermolecular Interaction Experiment, Yodosha 996)) .
[0193] 標識した糖蛋白質組成物を体内に投与することで、血液中での半減期あるいは血 液投与後の組織への分布を知ることができるが、標識体の検出には、標識物質を検 出する方法と検出の対象となる糖蛋白質組成物に特異的な抗体抗原反応を組み合 わせた系が好ましい。  [0193] By administering the labeled glycoprotein composition into the body, it is possible to know the half-life in blood or the distribution to the tissue after blood administration. A system in which a detection method and a specific antibody-antigen reaction are combined with the glycoprotein composition to be detected is preferable.
8.抗体組成物の活性評価  8. Activity evaluation of antibody composition
精製した抗体組成物の蛋白量、抗原との結合性あるいはエフェクター機能を測定 する方法としては、モノクローナルアンチボディズ、あるいはアンチボディエンジニアリ ング等に記載の公知の方法を用いることができる。  As a method for measuring the protein amount, antigen binding property or effector function of the purified antibody composition, a known method described in Monoclonal Antibodies, Antibody Engineering, or the like can be used.
[0194] その具体的な例としては、抗体組成物がキメラ抗体組成物やヒト化抗体組成物の場 合、抗原との結合活性、抗原陽性培養細胞株に対する結合活性は EUSA法及び蛍 光饥体法 [キャンサー'ィムノロシ一'ィムノセラピー (Cancer Immunol. Immunotherj, 36, 373 (1993)]等により測定できる。抗原陽性培養細胞株に対する細胞障害活性は 、 CDC活性、 ADCC活性等を測定することにより、評価することができる [キャンサー' ィムノロジ一'ィムノセラピー (Cancer Immunol. Immunother.), 36, 373 (1993)]。  [0194] As specific examples, when the antibody composition is a chimeric antibody composition or a humanized antibody composition, the binding activity to the antigen and the binding activity to the antigen-positive cultured cell line are determined by the EUSA method and the fluorescent lamp. Body method [Cancer Immunol. Immunotherj, 36, 373 (1993)] etc. Cytotoxic activity against an antigen-positive cultured cell line can be measured by measuring CDC activity, ADCC activity, etc. [Cancer Immunol. Immunother., 36, 373 (1993)].
[0195] また、抗体組成物のヒトでの安全性、治療効果は、力二クイザル等のヒトに比較的近 い動物種の適当なモデルを用いて評価することができる。 9.抗体組成物の糖鎖の分析 [0195] Further, the safety and therapeutic effect of an antibody composition in humans can be evaluated using an appropriate model of an animal species that is relatively close to humans, such as power quizzes. 9. Analysis of sugar chain of antibody composition
各種細胞で発現させた抗体分子の糖鎖構造は、通常の糖蛋白質の糖鎖構造の解 析に準じて行うことができる。例えば、 IgG分子に結合している糖鎖はガラクトース、マ ンノース、フコースなどの中性糖、 N-ァセチルダルコサミンなどのアミノ糖、シアル酸 などの酸性糖から構成されており、糖組成分析および二次元糖鎖マップ法などを用 レ、た糖鎖構造解析等の手法を用いて行うことができる。  The sugar chain structure of an antibody molecule expressed in various cells can be performed according to the analysis of the sugar chain structure of a normal glycoprotein. For example, sugar chains bound to IgG molecules are composed of neutral sugars such as galactose, mannose, and fucose, amino sugars such as N-acetyldarcosamine, and acidic sugars such as sialic acid. Alternatively, it can be carried out by using a two-dimensional sugar chain map method or the like, or a method such as sugar chain structure analysis.
(1)中性糖'アミノ糖組成分析  (1) Neutral sugar 'amino sugar composition analysis
抗体分子の糖鎖の組成分析は、トリフルォロ酢酸等で、糖鎖の酸加水分解を行うこ とにより、中性糖またはアミノ糖を遊離し、その組成比を分析することができる。  In the composition analysis of the sugar chain of the antibody molecule, neutral sugar or amino sugar is liberated by acid hydrolysis of the sugar chain with trifluoroacetic acid or the like, and the composition ratio can be analyzed.
[0196] 具体的な方法として、 Dionex社製糖組成分析装置（BioLC)を用いる方法が挙げら れる。 BioLしは HPAEし- PAD (high perrormance anion-exchange chromatography-pui sed amperometric detection)法 [ジャーナノレ ·ォブ ·リキッド ·クロマトグラフィー (J.Liq. Chromatogr.) , 6, 1577 (1983)]によって糖組成を分析する装置である。 [0196] A specific method includes a method using a sugar composition analyzer (BioLC) manufactured by Dionex. BioL is HPAE and sugar composition by PAD (high perrormance anion-exchange chromatography-puised amperometric detection) method [J. Liq. Chromatogr.], 6, 1577 (1983)] It is a device that analyzes.
また、 2-アミノビリジンによる蛍光標識化法でも組成比を分析することができる。具体 的には、公知の方法 [ァグリカルチュラル ·アンド'バイオロジカル ·ケミストリー (Agm Biol.Chem.), 55(1).283~284 (1991)]に従って酸加水分解した試料を 2_アミノビリジル 化で蛍光ラベル化し、 HPLC分析して組成比を算出することができる。  The composition ratio can also be analyzed by fluorescence labeling with 2-aminoviridine. Specifically, a sample hydrolyzed according to a known method [Agricultural and Biological Chemistry (Agm Biol. Chem.), 55 (1) .283-284 (1991)] was converted to 2-aminobilidylation. The composition ratio can be calculated by fluorescent labeling and HPLC analysis.
(2)糖鎖構造解析  (2) Sugar chain structure analysis
抗体分子の糖鎖の構造解析は、 2次元糖鎖マップ法 [アナリティカル 'バイオケミスト リー (Anal.Biochem.) , m，73 (1988)、生物化学実験法 23—糖蛋白質糖鎖研究法（ 学会出版センター）高橋禮子編（1989年） ]により行うことができる。 2次元糖鎖マップ 法は、例えば、 X軸には逆相クロマトグラフィー糖鎖の保持時間または溶出位置を、 Y 軸には順相クロマトグラフィーによる糖鎖の保持時間または溶出位置を、それぞれプ ロットし、既知糖鎖のそれらの結果と比較することにより、糖鎖構造を推定する方法で ある。  The structure analysis of the glycans of antibody molecules is based on the two-dimensional glycan map method [Analytical 'Biochem. (Anal. Biochem.), M, 73 (1988), Biochemical Experimental Method 23-Glycoprotein Glycan Research Method ( It is possible to do it according to the Society Publishing Center) Takahashi Keiko (1989)]. In 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. It is a method for estimating the sugar chain structure by comparing with the results of known sugar chains.
[0197] 具体的には、抗体をヒドラジン分解して、抗体から糖鎖を遊離し、 2-アミノピリジン（ 以下、 PAと略記する）による糖鎖の蛍光標識 [ジャーナル ·ォブ'バイオケミストリー (J. Biochem.) , 95, 197 (1984)]を行った後、ゲルろ過により糖鎖を過剰の PA化試薬など と分離し、逆相クロマトグラフィーを行う。次いで、分取した糖鎖の各ピークについて 順相クロマトグラフィーを行う。これらの結果をもとに、 2次元糖鎖マップ上にプロットし 、糖鎖スタンダード（TaKaRa社製）、文献 [アナリティカノいバイオケミストリー (Anal. Bi ochem.) , 171, 73 (1988)]とのスポットの比較より糖鎖構造を推定することができる。 [0197] Specifically, the antibody is hydrazine-degraded to release the sugar chain from the antibody, and fluorescent labeling of the sugar chain with 2-aminopyridine (hereinafter abbreviated as PA) [Journal of Biochemistry ( J. Biochem.), 95, 197 (1984)], followed by gel filtration to remove excess sugar chains, etc. And reverse phase chromatography. Next, normal phase chromatography is performed for each peak of the separated sugar chain. Based on these results, plotting on a two-dimensional glycan map, glycan standard (manufactured by TaKaRa), literature [Analytical Biochemistry (Anal. Biochem.), 171, 73 (1988)] The sugar chain structure can be estimated from spot comparison.
[0198] さらに各糖鎖の MALDI-TOF-MSなどの質量分析を行レ、、 2次元糖鎖マップ法によ り推定される構造を確認することができる。 [0198] Furthermore, mass analysis such as MALDI-TOF-MS of each sugar chain can be performed, and the structure estimated by the two-dimensional sugar chain map method can be confirmed.
10.本発明のひ -1,6-フコシルトランスフェラーゼ変異体を有する細胞を用いて製造 された糖蛋白質組成物および抗体組成物の利用  10. Use of glycoprotein composition and antibody composition produced using cells having the -1,6-fucosyltransferase mutant of the present invention
本発明のひ -1,6 -フコシルトランスフェラーゼ変異体を有する細胞を用いて製造され た糖蛋白質組成物や抗体組成物は、フコース修飾のない糖鎖構造を有しており、例 えば、受容体との親和性の向上、血中半減期の向上、血中投与後の組織分布の改 善、または薬理活性発現に必要な蛋白質との相互作用の向上などの効果が期待で き高い生理活性を示す。特に、本発明の抗体組成物は、高いエフェクター機能、す なわち抗体依存性細胞障害活性を有してレ、る。これら生理活性の高レ、糖蛋白質組 成物、あるいは高レ、 ADCC活性を有する抗体組成物は、癌、炎症疾患、 自己免疫疾 患、アレルギーなどの免疫疾患、循環器疾患、またはウィルスあるいは細菌感染をは じめとする各種疾患の予防および治療において有用である。  The glycoprotein composition or antibody composition produced using the cell having the -1,6-fucosyltransferase mutant of the present invention has a sugar chain structure without fucose modification, for example, a receptor. High physiological activity that can be expected to improve the affinity with the protein, improve the blood half-life, improve the tissue distribution after administration in blood, or improve the interaction with proteins required for the expression of pharmacological activity. Show. In particular, the antibody composition of the present invention has high effector function, that is, antibody-dependent cytotoxic activity. An antibody composition having a high physiological activity, glycoprotein composition, or high ADCC activity can be used for cancer, inflammatory diseases, autoimmune diseases, immune diseases such as allergies, cardiovascular diseases, viruses or bacteria. It is useful in the prevention and treatment of various diseases including infection.
[0199] 癌、すなわち悪性腫瘍では癌細胞が増殖している。通常の抗癌剤は癌細胞の増殖 を抑制することを特徴とする。しかし、高レ、 ADCC活性を有する抗体は、殺細胞効果 により癌細胞を障害することにより癌を治療することができるため、通常の抗癌剤より も治療薬として有効である。特に癌の治療薬において、現状では抗体医薬単独の抗 腫瘍効果は不充分な場合が多く化学療法との併用療法が行われているが [サイェン ス (Science), 280, 1197， 1998]、本発明の抗体組成物単独でのより強い抗腫瘍効果 が認められれば、化学療法に対する依存度が低くなり、副作用の低減にもつながる。  [0199] Cancer cells are proliferating in cancer, that is, malignant tumors. Ordinary anticancer agents are characterized by inhibiting the growth of cancer cells. However, an antibody having a high ADCC activity is more effective as a therapeutic agent than a conventional anticancer agent because it can treat cancer by damaging cancer cells due to a cytocidal effect. Particularly in the case of cancer therapeutics, the anti-tumor effects of antibody drugs alone are often insufficient at present, and combined therapy with chemotherapy [Science, 280, 1197, 1998] If a stronger antitumor effect is observed with the antibody composition of the invention alone, the dependence on chemotherapy will be reduced, and side effects will be reduced.
[0200] 炎症疾患、 自己免疫疾患、アレルギーなどの免疫疾患において、それら疾患にお ける生体内反応は、免疫細胞によるメディエータ分子の放出により惹起されるため、 高レ、 ADCC活性を有する抗体を用いて免疫細胞を除去することにより、アレルギー反 応を えること力できる。 循環器疾患としては、動脈硬化などがあげられる。動脈硬化は、現在バルーンカテ 一テルによる治療を行うが、治療後の再狭窄での動脈細胞の増殖を高い ADCC活性 を有する抗体を用いて抑えることより、循環器疾患を予防および治療することができる [0200] In immune diseases such as inflammatory diseases, autoimmune diseases, and allergies, in vivo reactions in these diseases are triggered by the release of mediator molecules by immune cells, so antibodies with high levels and ADCC activity are used. By removing immune cells, allergic reactions can be enhanced. Examples of cardiovascular diseases include arteriosclerosis. Atherosclerosis is currently treated with balloon catheter, but it is possible to prevent and treat cardiovascular disease by suppressing the proliferation of arterial cells in restenosis after treatment with an antibody with high ADCC activity. it can
[0201] ウィルスまたは細菌に感染した細胞の増殖を、高い ADCC活性を有する抗体を用い て抑えることにより、ウィルスまたは細菌感染をはじめとする各種疾患を予防および治 療すること力 Sできる。 [0201] By suppressing the growth of cells infected with a virus or bacteria using an antibody having high ADCC activity, it is possible to prevent and treat various diseases including virus or bacteria infection.
腫瘍関連抗原を認識する抗体、アレルギーあるいは炎症に関連する抗原を認識す る抗体、循環器疾患に関連する抗原を認識する抗体、自己免疫疾患に関連する抗 原を認識する抗体、またはウィルスあるいは細菌感染に関連する抗原を認識する抗 体の具体例を以下に述べる。  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 or bacteria Specific examples of antibodies that recognize antigens related to infection are described below.
[0202] 腫瘍関連抗原を認識する抗体としては、抗 CA125抗体（Immunology Today, 21, 4 03-410, 2000)、抗 17— 1A抗体（Immunology Today, 21, 403-410, 2000)、抗インテ グリン ct v i3 3抗体（Immunology Today, 21, 403-410, 2000)、抗 CD33抗体（Immuno logy Today, 21, 403-410， 2000)、抗 CD22抗体（Immunology Today, 21, 403-410, 2 000)、抗 HLA抗体（Immunology Today, 21, 403-410， 2000)、抗 HLA— DR抗体（I mmunology Today, 21, 403-410, 2000)、抗 CD20抗体（Immunology Today, 21, 403 -410, 2000)、抗 CD19抗体（Immunology Today, 21, 403-410， 2000)、抗 EGF受容 体抗体（Immunology Today, 21, 403-410， 2000)、抗 CD 10抗体（American Journal of Clinical Pathology, U3, 374-382, 2000; Proc. Natl. Acad. Sci. USA, 79:4386-43 91， 1982)、抗 GD2抗体 (Anticancer Res., 13, 331-336, 1993)、抗 GD3抗体（Cance r Immunol. Immunother., 36, 260-266， 1993)、抗 GM2抗体（Cancer Res., 54, 1511 -1516， 1994)、抗 HER2抗体（Proc. Natl. Acad. Sci. USA, 89, 4285-4289, 1992)、 抗 CD52抗体（Nature, 332, 323-327, 1988)、抗 MAGE抗体（British J. Cancer, 83, 493-497, 2000)、抗 HM1. 24抗体（Molecular Immunol" 36, 387-395， 1999)、抗 副甲状腺ホルモン関連蛋白（PTHrP)抗体（Cancer, 88, 2909-2911, 2000)、抗 FG F8抗体（Proc. Natl. Acad. Sci. USA, 86, 9911-9915， 1989)抗塩基性繊維芽細胞増 殖因子抗体、抗 FGF8受容体抗体 (J. Biol. Chem., 265, 16455-16463, 1990)、抗塩 基性繊維芽細胞増殖因子受容体抗体、抗インスリン様増殖因子抗体 (J. Neurosci. R es.， 40, 647-659, 1995)、抗インスリン様増殖因子受容体抗体（J. Neurosci. Res., 40 ， 647-659, 1995)、抗 PMSA抗体（J. Urology, 160, 2396-2401， 1998)、抗血管内皮 細胞増殖因子抗体（Cancer Res. , 57, 4593-4599， 1997)または抗血管内皮細胞増 殖因子受容体抗体（Oncogene, 19, 2138-2146, 2000)などが挙げられる。 [0202] Anti-CA125 antibodies (Immunology Today, 21, 4 03-410, 2000), anti-17-1A antibodies (Immunology Today, 21, 403-410, 2000), anti-integrin antibodies are those that recognize tumor-associated antigens. Grin ct v i3 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, 2) 000), 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-CD19 antibody (Immunology Today, 21, 403-410, 2000), anti-EGF receptor antibody (Immunology Today, 21, 403-410, 2000), anti-CD10 antibody (American Journal of Clinical Pathology, U3 , 374-382, 2000; Proc. Natl. Acad. Sci. USA, 79: 4386-43 91, 1982), anti-GD2 antibody (Anticancer Res., 13, 331-336, 1993), anti-GD3 antibody (Cancer) Immunol. Immunother., 36, 260-266, 1993), anti-GM2 antibody (Cancer Res., 54, 1511-1516, 1994), anti-HER2 antibody ( Proc. Natl. Acad. Sci. USA, 89, 4285-4289, 1992), anti-CD52 antibody (Nature, 332, 323-327, 1988), anti-MAGE antibody (British J. Cancer, 83, 493-497, 2000) ), 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) Anti-basic fibroblast growth factor antibody, anti-FGF8 receptor antibody (J. Biol. Chem., 265, 16455-16463, 1990), anti-salt Basic fibroblast growth factor receptor antibody, anti-insulin-like growth factor antibody (J. Neurosci. Res., 40, 647-659, 1995), anti-insulin-like growth factor receptor antibody (J. Neurosci. Res. , 40, 647-659, 1995), anti-PMSA antibody (J. Urology, 160, 2396-2401, 1998), anti-vascular endothelial growth factor antibody (Cancer Res., 57, 4593-4599, 1997) or anti-vascular Examples thereof include endothelial cell growth factor receptor antibodies (Oncogene, 19, 2138-2146, 2000).
[0203] アレルギーあるいは炎症に関連する抗原を認識する抗体としては、抗 IgE抗体 (Im munology Today, 21, 403-410, 2000)、抗 CD23抗体（Immunology Today, 21, 403- 410, 2000)、抗 CD1 la抗体（Immunology Today, 21, 403-410, 2000)、抗 CRTH2 抗体 (J. Immunol., 162, 1278-1286, 1999)、抗 CCR8抗体（W099/25734)、抗 CCR 3抗体（US6207155)、抗インターロイキン 6抗体（Immunol. Rev., 127, 5-24, 1992)、 抗インターロイキン 6受容体抗体（Molecular Immunol., 31, 371-381, 1994)、抗インタ 一ロイキン 5抗体（Immunol. Rev. , 127, 5-24, 1992)、抗インターロイキン 5受容体抗 体、抗インターロイキン 4抗体（Cytokine, 3, 562-567, 1991)、抗インターロイキン 4受 容体抗体（J. Immunol. Meth., 211, 41-50, 1998)、抗 S重瘍壊死因子抗体（Hybridoma ， 13, 183-190, 1994)、抗腫瘍壊死因子受容体抗体（Molecular Pharmacol., 58, 237 -245, 2000)、抗 CCR4抗体（Nature, 400, 776-780， 1999)、抗ケモカイン抗体（J. Im munol. Meth., 174, 249-257, 1994)または抗ケモカイン受容体抗体（J. Exp. Med., 1 86, 1373-1381, 1997)などが挙げられる。  [0203] Anti-IgE antibodies (Immunology Today, 21, 403-410, 2000), anti-CD23 antibodies (Immunology Today, 21, 403-410, 2000), as antibodies that recognize antigens related to allergy or inflammation Anti-CD1 la 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. Rev., 127, 5-24, 1992), anti-interleukin 6 receptor antibody (Molecular Immunol., 31, 371-381, 1994), anti-interleukin 5 antibody ( Immunol. Rev., 127, 5-24, 1992), anti-interleukin 5 receptor antibody, anti-interleukin 4 antibody (Cytokine, 3, 562-567, 1991), anti-interleukin 4 receptor antibody (J. Immunol. Meth., 211, 41-50, 1998), anti-S-hepatic necrosis factor antibody (Hybridoma, 13, 183-190, 1994), anti-tumor necrosis factor receptor antibody (Molecular Pharmacol., 58, 237 -245) , 2000), anti-CCR4 antibody (Nature, 400) , 776-780, 1999), anti-chemokine antibody (J. Im munol. Meth., 174, 249-257, 1994) or anti-chemokine receptor antibody (J. Exp. Med., 1 86, 1373-1381, 1997) ) And the like.
[0204] 循環器疾患に関連する抗原を認識する抗体としては、抗 GpIIb/lIIa抗体 (J. Im固 nol.， 152, 2968-2976, 1994)、抗血小板由来増殖因子抗体（Science, 253, 1129-113 2， 1991)、抗血小板由来増殖因子受容体抗体（J. Biol. Chem., 272, 17400-17404, 1997)または抗血液凝固因子抗体（Circulation, 101, 1158-1164, 2000)などが挙げ られる。  [0204] Anti-GpIIb / lIIa antibody (J. Im solid nol., 152, 2968-2976, 1994), antiplatelet-derived growth factor antibody (Science, 253, 1994) are recognized as antibodies that recognize antigens related to cardiovascular diseases. 1129-113 2, 1991), antiplatelet-derived growth factor receptor antibody (J. Biol. Chem., 272, 17400-17404, 1997) or anticoagulation factor antibody (Circulation, 101, 1158-1164, 2000), etc. Is mentioned.
[0205] 自己免疫疾患、例えば、乾癬、関節リウマチ、クローン病、潰瘍性大腸炎、全身性 エリテマトーデス、多発性硬化症に関連する抗原を認識する抗体としては、抗自己 D NA抗体（Immunol. Letters, 72, 61-68, 2000)、抗 CD 1 la抗体（Immunology Today, 21, 403-410, 2000)、抗 ICAM3抗体（Immunology Today, 21, 403-410, 2000)、抗 C D80抗体（Immunology Today, 21, 403-410, 2000)、抗 CD2抗体（Immunology Toda y, 21, 403-410, 2000)、抗 CD3抗体（Immunology Today, 21, 403-410, 2000)、抗 C D4抗体（Immunology Today, 21, 403-410, 2000)、抗インテグリン a 4 7抗体（Imm unology Today, 21, 403-410， 2000)、抗 CD40L抗体（Immunology Today, 21, 403- 410, 2000)、抗 IL_ 2受容体抗体（Immunology Today, 21, 403-410, 2000)などが挙 げられる。 Antibodies that recognize antigens associated with autoimmune diseases such as psoriasis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, and multiple sclerosis include anti-self DNA antibodies (Immunol. Letters , 72, 61-68, 2000), anti-CD 1 la antibody (Immunology Today, 21, 403-410, 2000), anti-ICAM3 antibody (Immunology Today, 21, 403-410, 2000), anti-CD80 antibody (Immunology Today, 21, 403-410, 2000), anti-CD2 antibody (Immunology Toda y, 21, 403-410, 2000), anti-CD3 antibody (Immunology Today, 21, 403-410, 2000), anti-CD4 antibody (Immunology Today, 21, 403-410, 2000), anti-integrin a 47 antibody (Immunology Today, 21, 403-410, 2000), anti-CD40L antibody (Immunology Today, 21, 403-410, 2000), anti-IL_2 receptor antibody (Immunology Today, 21, 403-410, 2000), etc. Can be mentioned.
[0206] ウィルスあるいは細菌感染に関連する抗原を認識する抗体としては、抗 gP120抗 体（Structure, 8, 385-395, 2000)、抗 CD4抗体（J. Rheumatology, 25, 2065-2076, 1 998)、抗 CCR4抗体または抗ベロ毒素抗体（J. Clin. Microbiol., 37, 396-399, 1999) などが挙げられる。 [0206] Virus or The antibody which recognizes an antigen associated with bacterial infection, anti-g P 120 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).
上記抗体は、 ATCC (The American Type Culture Collection)、理化学研究所細 胞開発銀行、工業技術院生命工業技術研究所等の公的な機関、あるいは大日本製 薬株式会社、 R&D SYSTEMS社、 PharMingen社、コスモバイオ社、フナコシ株式会社 等の民間試薬販売会社力 入手することができる。  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 Co., Ltd., Funakoshi Co., Ltd., etc.
[0207] 本発明の糖蛋白質組成物を含有する医薬を、治療薬として単独で投与することも 可能ではあるが、通常は薬理学的に許容される一つあるいはそれ以上の担体と一緒 に混合し、製剤学の技術分野においてよく知られる任意の方法により製造した医薬 製剤として提供するのが望ましい。  [0207] Although it is possible to administer the medicament containing the glycoprotein composition of the present invention alone as a therapeutic agent, it is usually mixed together with one or more pharmacologically acceptable carriers. However, it is desirable to provide it as a pharmaceutical preparation produced by any method well known in the technical field of pharmaceutical science.
投与経路は、治療に際して最も効果的なものを使用するのが望ましぐ経口投与、 または口腔内、気道内、直腸内、皮下、筋肉内および静脈内等の非経口投与をあげ ること力 Sでき、抗体製剤の場合、望ましくは静脈内投与をあげることができる。  The route of administration is preferably oral administration, which should be the most effective for treatment, or parenteral administration such as buccal, airway, rectal, subcutaneous, intramuscular and intravenous. In the case of an antibody preparation, intravenous administration is desirable.
[0208] 投与形態としては、噴霧剤、カプセル剤、錠剤、顆粒剤、シロップ剤、乳剤、座剤、 注射剤、軟膏、テープ剤等があげられる。  [0208] Examples of dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.
経口投与に適当な製剤としては、乳剤、シロップ剤、カプセル剤、錠剤、散剤、顆粒 剤等があげられる。  Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
乳剤およびシロップ剤のような液体調製物は、水、ショ糖、ソルビトール、果糖等の 糖類、ポリエチレングリコール、プロピレングリコール等のダリコール類、ごま油、オリ ーブ油、大豆油等の油類、 p—ヒドロキシ安息香酸エステル類等の防腐剤、スト口ベリ 一フレーバー、ペパーミント等のフレーバー類等を添加剤として用いて製造できる。 [0209] カプセル剤、錠剤、散剤、顆粒剤等は、乳糖、ブドウ糖、ショ糖、マンニトール等の 賦形剤、デンプン、アルギン酸ナトリウム等の崩壊剤、ステアリン酸マグネシウム、タル ク等の滑沢剤、ポリビニルアルコール、ヒドロキシプロピルセルロース、ゼラチン等の 結合剤、脂肪酸エステル等の界面活性剤、グリセリン等の可塑剤等を添加剤として 用いて製造できる。 Liquid preparations such as emulsions and syrups include saccharides such as water, sucrose, sorbitol, and fructose, Daricols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, p- It can be produced using preservatives such as hydroxybenzoates and the like, flavors such as stove belly flavors and peppermint as additives. [0209] 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 polyvinyl alcohol, hydroxypropyl cellulose, gelatin, a surfactant such as fatty acid ester, a plasticizer such as glycerin, and the like as additives.
[0210] 非経口投与に適当な製剤としては、注射剤、座剤、噴霧剤等があげられる。  [0210] Suitable formulations for parenteral administration include injections, suppositories, sprays and the like.
注射剤は、塩溶液、ブドウ糖溶液、あるいは両者の混合物からなる担体等を用いて 調製される。または、糖蛋白質組成物を常法に従って凍結乾燥し、これに塩化ナトリ ゥムを加えることによって粉末注射剤を調製することもできる。  The injection is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of both. Alternatively, a powder injection can be prepared by freeze-drying a glycoprotein composition according to a conventional method and adding sodium chloride thereto.
座剤はカカオ脂、水素化脂肪またはカルボン酸等の担体を用いて調製される。  Suppositories are prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.
[0211] また、噴霧剤は該糖蛋白質組成物そのもの、ないしは受容者の口腔および気道粘 膜を刺激せず、かつ該糖蛋白質組成物を微細な粒子として分散させ吸収を容易にさ せる担体等を用いて調製される。 [0211] In addition, the propellant does not irritate the glycoprotein composition itself or the recipient's oral cavity and airway mucous membrane, and the glycoprotein composition is dispersed as fine particles to facilitate absorption, etc. It is prepared using.
担体として具体的には乳糖、グリセリン等が例示される。該糖蛋白質組成物および 用いる担体の性質により、エアロゾル、ドライパウダー等の製剤が可能である。また、 これらの非経口剤においても経口剤で添加剤として例示した成分を添加することもで きる。  Specific examples of the carrier include lactose and glycerin. Depending on the properties of the glycoprotein composition and the carrier used, preparations such as aerosols and dry powders are possible. In these parenteral preparations, the components exemplified as additives for oral preparations can also be added.
[0212] 投与量または投与回数は、 目的とする治療効果、投与方法、治療期間、年齢、体 重等により異なる力 通常成人 1日当たり 10 /i g/kg〜20mg/kgである。  [0212] The dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, body weight, etc. The usual adult dose is 10 / ig / kg to 20 mg / kg per day.
また、糖蛋白質組成物の各種腫瘍細胞に対する抗腫瘍効果を検討する方法は、 例えば抗体の場合、インビトロ実験としては、 CDC(complement_dependent cytotoxic! ty)活性測定法、 ADCC(antibody- d印 endent cellular cytotoxicity)活性測定法等があ げられ、インビボ実験としては、マウス等の実験動物での腫瘍系を用いた抗腫瘍実験 等があげられる。  In addition, the method of examining the antitumor effect of glycoprotein compositions on various tumor cells includes, for example, in the case of antibodies, in vitro experiments such as CDC (complement_dependent cytotoxicity) assay, ADCC (antibody-d marked endent cellular cytotoxicity) ) Activity measurement methods and the like, and examples of in vivo experiments include antitumor experiments using tumor systems in experimental animals such as mice.
[0213] CDC活性、 ADCC活性、抗腫瘍実験は、文献 [キャンサー'ィムノロジ一'ィムノセラ ヒ1 ~ (し ancer Immunology Immunotherapy), όΌ, 373 (1993) ;キャンサ" ~ 'リケ" ~テ (Can cer Research), 54, 1511 (1994)]等記載の方法に従って行うことができる。 [0213] CDC activity, ADCC activity, antitumor experiments, the literature [Cancer 'Imunoroji one' Imunosera heat 1 ~ (and ancer Immunology Immunotherapy), όΌ, 373 (1993); Kyansa "~ 'RIQUET" ~ te (Can cer Research), 54, 1511 (1994)] and the like.
以下の実施例により本発明をより具体的に説明するが、実施例は本発明の単なる 例示にすぎず、本発明の範囲を限定するものではない。 The present invention will be described more specifically with reference to the following examples. It is only an example and does not limit the scope of the present invention.
実施例 1  Example 1
[0214] フコース修飾変異体 RN6株の取得  [0214] Acquisition of fucose-modified mutant RN6 strain
1. RN6株の取得  1. Acquisition of RN6 shares
ジヒドロ葉酸還元酵素遺伝子（dhfr)を欠損したチャイニーズノヽムスター卵巣由来 C HO/DG44細胞 [Somatic Cell and Moleculer Genetics, 12, 555 (1986)]を、 400 μ g/mlレンズマメ由来レクチン（LCA; EY Laboratory社）存在下で 3週間培養した。  Chinese hamster ovary-derived CHO / DG44 cells deficient in the dihydrofolate reductase gene (dhfr) [Somatic Cell and Moleculer Genetics, 12, 555 (1986)] were transformed into 400 μg / ml lentil-derived lectin (LCA; EY Laboratory For 3 weeks.
[0215] 上記 LCA共存下培養により取得した耐性株を、接着細胞用 24穴プレート (グライナ 一社）に 1.5 X 105個/穴となるように播種し、 5% CO、 37°Cの条件下で 4時間培養した 後、 400 /i g/ml LCA (EY Laboratory社）および 100 /i M L_フコース（ナカライテスタ 社)を含む培地へ交換し、さらに 5日間培養した。培養後、 10% WST-1 (タカラバイオ 社)を含む培地へ交換し、 5% CO、 37°Cの条件下で 30分間培養した後、培養上清の 波長 450醒の吸光度を測定し、生細胞密度の指標とした。 その結果、 L-フコース 存在下でも LCAに耐性な RN6株を見出した。 [0215] The above-mentioned resistant strain obtained by the culture in the presence of LCA was seeded in a 24-well plate for adherent cells (Greiner) at 1.5 × 10 5 cells / hole, and the conditions were 5% CO and 37 ° C. After culturing for 4 hours, the medium was replaced with a medium containing 400 / ig / ml LCA (EY Laboratory) and 100 / i ML_fucose (Nacalai Testa), and further cultured for 5 days. After culturing, the medium is replaced with a medium containing 10% WST-1 (Takara Bio Inc.), and cultured for 30 minutes under conditions of 5% CO and 37 ° C. Then, the absorbance of the culture supernatant at wavelength 450 awakening is measured, It was used as an index of viable cell density. As a result, an RN6 strain resistant to LCA even in the presence of L-fucose was found.
2. RN6株の α -1,6-フコース付加能解析  2. Analysis of α-1,6-fucose addition ability of RN6 strain
1%ゥシ血清由来アルブミン（シグマ社）を含むダルベッコリン酸緩衝液（Invitrogen 社）（以下、 1%BSA_PBSと称す）に、 RN6株または CHO/DG44細胞 2 X 105個を懸濁し 、 FITC標識 LCA (Vector Laboratories社）または 100倍希釈した FITC標識ストレプトァ ビジン (KPL社）を添カ卩した。 4°Cにて 30分間静置することで細胞を染色し、 1%BSA-PB Sを用いて細胞を洗浄した後、 I X 104個の細胞を FACSCalibur (BD Biosciences社） で解析した。 Suspend 2 x 10 5 RN6 strains or CHO / DG44 cells in Dulbecco's phosphate buffer (Invitrogen) (hereinafter referred to as 1% BSA_PBS) containing albumin (Sigma) from 1% ushi serum. Labeled LCA (Vector Laboratories) or FITC-labeled streptavidin (KPL) diluted 100-fold was added. 4 ° Cells were stained with to stand for 30 minutes at C, After the cells were washed with 1% BSA-PB S, were analyzed IX 10 4 cells in FACSCalibur (BD Biosciences Inc.).
[0216] 図 1に各株の LCAに対する反応性を示した。 CHO/DG44細胞はひ _1，6-フコース特 異的レクチンである LCAにより染色が認められたのに対し、 RN6株は、 LCAに対して 染色性されなかった。  [0216] Figure 1 shows the reactivity of each strain to LCA. CHO / DG44 cells were stained by LCA, a _1,6-fucose specific lectin, whereas the RN6 strain was not stained by LCA.
3. RN6株が産生する抗体組成物の単糖組成分析  3. Monosaccharide composition analysis of antibody composition produced by RN6 strain
まず、 RN6株に対し、 WO01/64754記載の抗 CCR4抗体発現ベクター pKANTEX216 0を、エレクト口ポレーシヨン法 [Cytotechnology, 3, 133 (1990)]により導入し、安定的 発現株を取得した。抗 CCR4抗体発現株の培養上清より、 Mab Select(Amersham Pha rmacia Biotech社)を用いて抗体組成物を精製した。取得した抗 CCR4抗体に対し、公 知の方法 [ジャーナル'ォブ'リキッド 'クロマトグラフィー (Journal of Liquid Chromatog raphy), 6, 1577, (1983)]に従って単糖組成分析を行った。その結果、 RN6株より得た 抗体組成物は、フコース含量が定量限界以下であった（図 2)。この結果より、 RN6株 は、 N -結合型複合型糖鎖還元末端の N -ァセチルダルコサミンの 6位にフコースの 1 位が a結合する機能を欠失することが示唆された。 First, the anti-CCR4 antibody expression vector pKANTEX2160 described in WO01 / 64754 was introduced into the RN6 strain by the electopore position method [Cytotechnology, 3, 133 (1990)] to obtain a stable expression strain. From the culture supernatant of the anti-CCR4 antibody expression strain, Mab Select (Amersham Pha The antibody composition was purified using rmacia Biotech). The obtained anti-CCR4 antibody was subjected to monosaccharide composition analysis according to a known method [Journal of Liquid Chromatog raphy, 6, 1577, (1983)]. As a result, the antibody composition obtained from the RN6 strain had a fucose content below the limit of quantification (FIG. 2). From these results, it was suggested that the RN6 strain lacks the function of a-binding fucose 1-position to the 6-position of N-acetyl darcosamine at the N-linked complex sugar chain reducing terminal.
実施例 2  Example 2
[0217] RN6株の変異点の解析  [0217] Mutation point analysis of RN6 strain
1.フコース修飾酵素の発現量解析  1. Expression analysis of fucose modifying enzyme
実施例 1で得た RN6株および CHO/DG44細胞より、 RNeasy Mini Kit(QIAGEN社)を 用いて全 RNAを抽出した。全 RNA 5 β gを铸型とし、 Superscript First-Strand Synthe sis System for RT_PCR (Invitrogen社）を用いてオリゴ dTプライマーにより逆転写反応 を行い、一本鎖 cDNAを合成した。  Total RNA was extracted from the RN6 strain and CHO / DG44 cells obtained in Example 1 using the RNeasy Mini Kit (QIAGEN). Single-stranded cDNA was synthesized by reverse transcription reaction with oligo dT primer using Superscript First-Strand Synthesis System for RT_PCR (Invitrogen) using 5β g of total RNA as a cage.
[0218] GDP-mannose 4.6 dehydratase (GMD)の発現解析は以下のように行った。特許 W 002/31140に記載のチャイニーズハムスター GMD cDNA配列に特異的なフォワード プライマー（配列番号 23)およびリバースプライマー（配列番号 24)を合成し、 DNAポ リメラーゼ Ex Taq (タカラバイオ社)および上記 cDNA 1 μ 1を含む 20 μ 1の反応液 [ΕχΤ aq buffer (タカラバイオ社)、 0.2 mmol/1 dNTPs、 0.5 μ mol/1上記遺伝子特異的プライ マー（配列番号 23および配列番号 24) ]を調製し、ポリメラーゼ連鎖反応 (PCR)を行 つた。 PCRは、 94°Cで 5分間加熱した後、 94°Cで 1分間、 68°Cで 2分間から成る反応 を 1サイクルとした 26サイクルの工程で行った。 PCR後、 1.2%(w/v)ァガロースゲル電気 泳動に供し、 SYBR Green I Nucleic Acid Gel Stain (Molecular Probes社）を用いて D NAを染色した後、増幅された各 DNA断片の発光強度を Fluorlmager SI (Molecular D ynamics社）を用いて算出した。  [0218] Expression analysis of GDP-mannose 4.6 dehydratase (GMD) was performed as follows. A forward primer (SEQ ID NO: 23) and reverse primer (SEQ ID NO: 24) specific to the Chinese hamster GMD cDNA sequence described in Patent W 002/31140 were synthesized, and DNA polymerase Ex Taq (Takara Bio Inc.) and the above cDNA 1 Prepare a 20 μ 1 reaction solution [ΕχΤ aq buffer (Takara Bio Inc.), 0.2 mmol / 1 dNTPs, 0.5 μmol / 1 above gene-specific primers (SEQ ID NO: 23 and SEQ ID NO: 24)] containing μ 1 The polymerase chain reaction (PCR) was performed. PCR was performed in 26 cycles of a reaction consisting of heating at 94 ° C for 5 minutes, followed by a reaction consisting of 94 ° C for 1 minute and 68 ° C for 2 minutes. After PCR, it was subjected to 1.2% (w / v) agarose gel electrophoresis. After staining with DNA using SYBR Green I Nucleic Acid Gel Stain (Molecular Probes), the luminescence intensity of each amplified DNA fragment was measured using Fluorlmager SI. (Molecular Dynamics) was used for calculation.
[0219] α -1 ,6-フコース転移酵素（FUT8)の発現解析は以下のように行った。特許 WO02/ 31140に記載のチャィニーズハムスターFUT8のcDNA配歹lJ (配列番号l)に特異的な フォワードプライマー（配列番号 25)およびリバースプライマー（配列番号 26)を合成 し、 DNAポリメラーゼ Ex Taq (タカラバイオ社)および上記 cDNA 1 μ 1を含む 20 μ 1の 反応液 [Ex Taq buffer (タカラバイオ社)、 0.2 mmol/1 dNTPs、 0.5 μ mol/1上記遺伝子 特異的プライマー（配列番号 25および配列番号 26) ]を調製し、 PCRを行った。 PCR は、 94°Cで 5分間加熱した後、 94°Cで 1分間、 68°Cで 2分間から成る反応を 1サイクノレ とした 22サイクルの工程で行った。 PCR後、 1.2%(w/v)ァガロースゲル電気泳動に供し 、 SYBR Green I Nucleic Acid Gel Stain (Molecular Probes社）を用いて DNAを染色し た後、増幅された各 DNA断片の発光強度を Fluorlmager SI (Molecular Dynamics社） を用いて算出した。 [0219] Expression analysis of α-1,6-fucose transferase (FUT8) was performed as follows. A forward primer (SEQ ID NO: 25) and reverse primer (SEQ ID NO: 26) specific for the Chinese hamster FUT8 cDNA array IJ (SEQ ID NO: 1) described in patent WO02 / 31140 were synthesized, and DNA polymerase Ex Taq (Takara) was synthesized. Bio) and 20 μ1 of the above cDNA 1 μ1 A reaction solution [Ex Taq buffer (Takara Bio Inc.), 0.2 mmol / 1 dNTPs, 0.5 μmol / 1 gene-specific primer (SEQ ID NO: 25 and SEQ ID NO: 26)] was prepared, and PCR was performed. PCR was performed in 22 cycles of a reaction consisting of 94 ° C for 5 minutes, 94 ° C for 1 minute, and 68 ° C for 2 minutes. After PCR, the sample was subjected to 1.2% (w / v) agarose gel electrophoresis, and DNA was stained using SYBR Green I Nucleic Acid Gel Stain (Molecular Probes), and the luminescence intensity of each amplified DNA fragment was measured using Fluorlmager SI. (Molecular Dynamics).
[0220] また、 FUT8 cDNA全長の発現解析は以下のように行った。マウス FUT8 cDNA配歹 IJ  [0220] Expression analysis of the full length FUT8 cDNA was performed as follows. Mouse FUT8 cDNA IJ
[GenBank,AB025198、配列番号 2]の 5'側非翻訳領域に特異的なフォワードプライ マー（配列番号 27)および 3'側非翻訳領域に特異的なリバースプライマー (配列番号 28)を合成し、 DNAポリメラーゼ Ex Taq (タカラバイオ社)および上記 cDNA 1 μ 1を含む 25 μ ΐの反応液 [Ex Taq buffer (タカラバイオ社)、 0.2 mmol/1 dNTPs、 4% DMSO、 0.5 β mol/1上記遺伝子特異的プライマー（配列番号 27および配列番号 28) ]を調製し、 PCRを行った。 PCRは、 94°Cで 1分間加熱した後、 94°Cで 30秒間、 55°Cで 30秒間、 72 °Cで 2分間から成る反応を 1サイクルとした 30サイクルの工程で行った。 PCR後、 1.2%( w/v)ァガロースゲル電気泳動に供し、 SYBR Green I Nucleic Acid Gel Stain (Molecul ar Probes社）を用いて DNAを染色した後、増幅された各 DNA断片の発光強度を Fluo rlmager SI (Molecular Dynamics社リで算出し 7こ。  A forward primer (SEQ ID NO: 27) specific for the 5 ′ untranslated region and a reverse primer (SEQ ID NO: 28) specific for the 3 ′ untranslated region of [GenBank, AB025198, SEQ ID NO: 2] 25 μΐ reaction solution containing DNA polymerase Ex Taq (Takara Bio Inc.) and the above cDNA 1 μ 1 (Ex Taq buffer (Takara Bio Inc.), 0.2 mmol / 1 dNTPs, 4% DMSO, 0.5 β mol / 1 above gene Specific primers (SEQ ID NO: 27 and SEQ ID NO: 28)] were prepared and PCR was performed. PCR was performed in a 30-cycle process, consisting of a reaction consisting of 94 ° C for 1 minute, 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 2 minutes. After PCR, the sample was subjected to 1.2% (w / v) agarose gel electrophoresis, and DNA was stained using SYBR Green I Nucleic Acid Gel Stain (Molecular Probes), and then the luminescence intensity of each amplified DNA fragment was measured by Fluo rlmager SI (7 calculated by Molecular Dynamics)
[0221] GDP- β -L-ilicose pyrophorylase (GFPP)の発現解析は以下のように行った。特許 WO03/085118に記載のチャイニーズノヽムスター GFPP cDNA配列に特異的なフォヮ 一ドプライマ一（配列番号 29)およびリバースプライマー（配列番号 30)を合成し、 DN Aポリメラーゼ Ex Taq (タカラバイオ社)および上記 cDNA 1 μ 1を含む 20 μ ΐの反応液 [ Ex Taq buffer (タカラバイオ社)、 0.2 mmol/1 dNTPs、 0.5 μ mol/1上記遺伝子特異的 プライマー（配列番号 29および配列番号 30) ]を調製し、 PCRを行った。 PCRは、 94 °Cで 5分間加熱した後、 94°Cで 1分間、 68°Cで 2分間から成る反応を 1サイクルとした 24サイクルの工程で行った。 PCR後、 1.2%(w/v)ァガロースゲル電気泳動に供し、 SYB R Green I Nucleic Acid Gel Stain (Molecular Probes社）を用いて DNAを染色した後、 増幅された各 DNA断片の発光強度を Fluorlmager SI (Molecular Dynamics社）を用い て算出した。 [0221] Expression analysis of GDP-β-L-ilicose pyrophorylase (GFPP) was performed as follows. A first primer (SEQ ID NO: 29) and a reverse primer (SEQ ID NO: 30) specific to the Chinese nomstar GFPP cDNA sequence described in patent WO03 / 085118 were synthesized, DNA polymerase Ex Taq (Takara Bio Inc.) and the above Prepare 20 μΐ reaction solution [Ex Taq buffer (Takara Bio), 0.2 mmol / 1 dNTPs, 0.5 μmol / 1 above gene-specific primers (SEQ ID NO: 29 and SEQ ID NO: 30)] containing 1 μ1 cDNA PCR was performed. PCR was performed in 24 cycles, with a reaction consisting of 94 ° C for 5 minutes, 94 ° C for 1 minute, and 68 ° C for 2 minutes. After PCR, the sample was subjected to 1.2% (w / v) agarose gel electrophoresis, and DNA was stained with SYBR Green I Nucleic Acid Gel Stain (Molecular Probes). The luminescence intensity of each amplified DNA fragment was then measured using Fluorlmager SI. (Molecular Dynamics) Calculated.
[0222] GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase (FX)の発現解析は以下 のように行った。特許 WO03/085118に記載のチャイニーズハムスター FX cDNA配歹 lj に特異的なフォワードプライマー（配列番号 31)およびリバースプライマー（配列番号 32)を合成し、 DNAポリメラーゼ Ex Taq (タカラバイオ社)および上記 cDNA 1 μ 1を含 む 20 の反応液 ^ 丁&9 13111¾1タカラバィォ社)、0.2 11111101/1 0^丁？3、 0.5 μ mol/1 上記遺伝子特異的プライマー（配列番号 31および配列番号 32) ]を調製し、 PCRを 行った。 PCRは、 94°Cで 5分間加熱した後、 94°Cで 1分間、 68°Cで 2分間から成る反 応を 1サイクルとした 24サイクルの工程で行った。 PCR後、 1.2%(w/v)ァガロースゲル電 気泳動に供し、 SYBR Green I Nucleic Acid Gel Stain (Molecular Probes社）を用いて DNAを染色した後、増幅された各 DNA断片の発光強度を Fluorlmager SI (Molecular Dyn纖 ics社）を用いて算出した。  [0222] Expression analysis of GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase (FX) was performed as follows. A forward primer (SEQ ID NO: 31) and reverse primer (SEQ ID NO: 32) specific to Chinese hamster FX cDNA array lj described in patent WO03 / 085118 were synthesized, and DNA polymerase Ex Taq (Takara Bio Inc.) and the above cDNA 1 20 reaction solutions containing μ 1 ^ Ding & 9 13111¾1 Takarabio), 0.2 11111101/1 0 ^ Ding? 3, 0.5 μmol / 1 The above gene-specific primers (SEQ ID NO: 31 and SEQ ID NO: 32)] were prepared and PCR was performed. PCR was performed in 24 cycles, with a reaction consisting of 94 ° C for 5 minutes, 94 ° C for 1 minute, and 68 ° C for 2 minutes. After PCR, the sample was subjected to 1.2% (w / v) agarose gel electrophoresis, and DNA was stained with SYBR Green I Nucleic Acid Gel Stain (Molecular Probes), and the luminescence intensity of each amplified DNA fragment was measured using Fluorlmager SI. (Molecular Dynamics) was used for calculation.
[0223] GDP-フコーストランスポーターの発現解析は以下のように行った。特許 WO03/085 102に記載のチャイニーズハムスター GDP-フコーストランスポーター cDNA配列に特 異的なフォワードプライマー（配列番号 33)およびリバースプライマー（配列番号 34) を合成し、 DNAポリメラーゼ Ex Taq (タカラバイオ社)および上記 cDNA 1 μ 1を含む 20 μ 1の反応液 [Ex Taq buffer (タカラバイオ社)、 0.2 mmol/1 dNTPs、 0.5 μ mol/1上記 遺伝子特異的プライマー（配列番号 33および配列番号 34) ]を調製し、 PCRを行った 。 PCRは、 94°Cで 5分間加熱した後、 94°Cで 1分間、 68°Cで 2分間から成る反応を 1サ イタルとした 24サイクルの工程で行った。 PCR後、 1.2%(w/v)ァガロースゲル電気泳動 に供し、 SYBR Green I Nucleic Acid Gel Stain (Molecular Probes社）を用いて DNAを 染色した後、増幅された各 DNA断片の発光強度を Fluorlmager SI (Molecular Dynami cs社)で算出した。  [0223] Expression analysis of the GDP-fucose transporter was performed as follows. Patent WO03 / 085 Chinese hamster GDP-fucose transporter described in 102 A special forward primer (SEQ ID NO: 33) and reverse primer (SEQ ID NO: 34) were synthesized into the DNA sequence, and DNA polymerase Ex Taq (Takara Bio Inc.) And 20 μ 1 reaction solution containing 1 μ 1 of the above cDNA [Ex Taq buffer (Takara Bio Inc.), 0.2 mmol / 1 dNTPs, 0.5 μmol / 1 gene specific primer (SEQ ID NO: 33 and SEQ ID NO: 34)] Was prepared and PCR was performed. PCR was performed in a 24 cycle process, consisting of a reaction consisting of 94 ° C for 5 minutes, 94 ° C for 1 minute, and 68 ° C for 2 minutes. After PCR, the sample was subjected to 1.2% (w / v) agarose gel electrophoresis, and DNA was stained with SYBR Green I Nucleic Acid Gel Stain (Molecular Probes). The luminescence intensity of each amplified DNA fragment was then measured using Fluorlmager SI ( Molecular Dynamics).
[0224] /3 -ァクチンの発現解析は以下のように行った。特許 WO02/31140に記載のチヤィ ニーズノヽムスター β -ァクチン cDNA配列に特異的なフォワードプライマー（配列番号 35)およびリバースプライマー（配列番号 36)を合成し、 DNAポリメラーゼ Ex Taq (タカ ラバイオ社)および上記 cDNA 1 μ ΐを含む 20 の反応液 [ExTaq buffer (タカラバイオ 社)、 0.2 mmol/1 dNTPs、 0.5 μ mol/1上記遺伝子特異的プライマー（配列番号 35お よび配列番号 36) ]を調製し、 PCRを行った。 PCRは、 94°Cで 5分間加熱した後、 94°C で 1分間、 68°Cで 2分間から成る反応を 1サイクルとした 14サイクルの工程で行った。 P CR後、 1.2%(w/v)ァガロースゲル電気泳動に供し、 SYBR Green I Nucleic Acid Gel St ain (Molecular Probes社）を用いて DNAを染色した後、増幅された各 DNA断片の発光 強度を Fluorlmager SI (Molecular Dynamics社）を用いて算出した。 [0224] The expression analysis of / 3-actin was performed as follows. A forward primer (SEQ ID NO: 35) and reverse primer (SEQ ID NO: 36) specific for the Chinese nomstar β-actin cDNA sequence described in patent WO02 / 31140 were synthesized, and DNA polymerase Ex Taq (Takara Bio) and the above 20 reaction solutions containing 1 μΐ cDNA [ExTaq buffer (Takara Bio), 0.2 mmol / 1 dNTPs, 0.5 μmol / 1 gene-specific primer (SEQ ID NO: 35) And SEQ ID NO: 36)] were prepared and PCR was performed. PCR was performed in 14 cycles of a reaction consisting of 94 ° C for 5 minutes, 94 ° C for 1 minute, and 68 ° C for 2 minutes. After PCR, 1.2% (w / v) agarose gel electrophoresis, SYBR Green I Nucleic Acid Gel Stain (Molecular Probes) was used to stain the DNA, and then the luminescence intensity of each amplified DNA fragment was measured using Fluorlmager Calculation was performed using SI (Molecular Dynamics).
以上の RT-PCRの結果、 RN6株の GMD、 FUT8、 GFPP、 FX、 GDP-フコーストランス ポーターの発現量は、 CHO/DG44細胞と同等であった。しかし、 FUT8遺伝子の全長 約 1.7 Kbを増幅させるプライマーを用いて解析した結果、 RN6株より約 1.7 Kbの断片 に加えて約 1.3 Kbの断片が検出されたことから、一方の FUT8対立遺伝子に部分的 な欠損が生じてレ、ることが示された。  As a result of the above RT-PCR, the expression levels of GMD, FUT8, GFPP, FX, and GDP-fucose transporter of the RN6 strain were equivalent to those of CHO / DG44 cells. However, as a result of analysis using a primer that amplifies the full length of the FUT8 gene of about 1.7 Kb, a fragment of about 1.3 Kb was detected in addition to the fragment of about 1.7 Kb from the RN6 strain. It was shown that there was a target deficiency.
2. RN6株の FUT8 cDNAの配列解析  2. Sequence analysis of FUT8 cDNA of RN6 strain
本実施例第 1項に記述した方法により、 RN6株より FUT8 cDNA全長を増幅し、公知 の方法 [モレキユラ一'クローニング第 3版]に従ってダイレクトシーケンス解析を行つ た。その結果、本実施例第 1項で見出された約 1.3 Kbの断片は、ヒト FUT8のェクソン The full length FUT8 cDNA was amplified from the RN6 strain by the method described in Section 1 of this Example, and direct sequence analysis was performed according to a known method [Molequila 'Cloning 3rd Edition]. As a result, the fragment of about 1.3 Kb found in the first item of this example was found to be an exon of human FUT8.
3、ェクソン 4、ェクソン 5に該当する領域を欠損していることが明らかになった。決定 した RN6株由来 FUT8欠失体の塩基配列を配列番号 19に、および、塩基配列から推 測されるアミノ酸配列を配列番号 14にそれぞれ示した。一方、本実施例第 1項で見出 された約 1.7 Kbの断片には、一アミノ酸変異を伴う一塩基置換が生じていた。即ち、 F UT8翻訳領域の 512番目のグァニンがアデニンへ置換しており、その結果、 171番目 のアミノ酸残基であるセリンがァスパラギンへ置換することが判明した。決定した RN6 株由来 FUT8—塩基置換体の塩基配列を配列番号 18に、および、塩基配列から推 測されるアミノ酸配列を配列番号 13にそれぞれ示した。 It was revealed that the areas corresponding to 3, Exon 4 and Exon 5 were missing. The determined nucleotide sequence of the RN6 strain-derived FUT8 deletion product is shown in SEQ ID NO: 19, and the amino acid sequence deduced from the nucleotide sequence is shown in SEQ ID NO: 14, respectively. On the other hand, a single base substitution accompanied by a single amino acid mutation occurred in the fragment of about 1.7 Kb found in the first section of this example. That is, it was found that the 512th guanine in the FUT8 translation region was substituted with adenine, and as a result, serine, the 171st amino acid residue, was substituted with asparagine. The determined nucleotide sequence of the RN6 strain-derived FUT8-base substitution product is shown in SEQ ID NO: 18, and the amino acid sequence deduced from the base sequence is shown in SEQ ID NO: 13, respectively.
3.ゲノムサザンブロットによる RN6株の解析  3. Analysis of RN6 strain by genomic Southern blot
実施例 1で得た RN6株および CHO/DG44細胞より、公知の方法 [ヌクレイック'ァシッ ド 'リサーチ (Nucleic Acids Research), 3, 2303, (1976)]に従って各クローンのゲノム D NAを調製し、各々 TE-RNase緩衝液 (pH8.0) [10mmol/l Tris- HC1、 lmmol/1 EDTA、 200 μ g/ml RNase A]に溶解した。調製したゲノム DNAを制限酵素 Xbalで消化し、 0. 8%(w/v)ァガロースゲル電気泳動に供した。泳動後、公知の方法 [プロシーデイング ス 'ォブ 'ザ'ナショナル 'アカデミ^ ~ ·ォブ 'サイエンス (Proc. Natl. Acad. Sci. USA), 7 6, 3683, (1979)に従い、ナイロン膜へゲノム DNAを転写した。転写終了後、ナイロン 膜に対し 80°Cで 1時間の熱処理を行つた。 A genomic DNA of each clone was prepared from the RN6 strain obtained in Example 1 and CHO / DG44 cells according to a known method [Nucleic Acids Research, 3, 2303, (1976)] Each was dissolved in TE-RNase buffer (pH 8.0) [10 mmol / l Tris-HC1, lmmol / 1 EDTA, 200 μg / ml RNase A]. The prepared genomic DNA was digested with the restriction enzyme Xbal and subjected to 0.8% (w / v) agarose gel electrophoresis. After electrophoresis, a known method [Procedure Genomic DNA was transcribed onto nylon membranes according to 'Ob' The 'National' Academi ^ ~ Ob 'Science (Proc. Natl. Acad. Sci. USA), 7 6, 3683, (1979). After the transfer, the nylon membrane was heat treated at 80 ° C for 1 hour.
[0226] まず、チャイニーズノヽムスター FUT8のうち、ヒト FUT8のェクソン 6からェクソン 9に該 当する領域に特異的なプローブを用いて、以下の手順でサザンプロット解析を行つ た。特許 WO02/31140に記載のプラスミド CHfFUT8_pCR2.1を錡型とし、 FUT8ェクソ ン 6に特異的なフォワードプライマー（配列番号 37)およびェクソン 9に特異的なリバ ースプライマー（配列番号 38)を用いて、 DNAポリメラーゼ Ex Taq (タカラバイオ社)に よる PCRを行った。 PCRは、 94°Cで 30秒間、 60°Cで 30秒間、 74°Cで 1分間から成る反 応を 1サイクルとした 25サイクルの条件で行った。 PCR後、約 400bpの増幅断片を精製 し、 [a_32P] dCTP 1.75MBqおよび Megaprime DNA Labelling system, dCTP (Amersh am Pharmacia Biotech社）を用いて放射標識しプローブとした。上記のナイロン膜を ハイブリダィゼーシヨン液 [5 X SSPE、 50 X Denhald s液、 0.5%(w/v) SDS、 100 μ g/ml サケ精子 DNA] 15mlに浸漬し、 65°Cで 3時間のプレハイブリダィゼーシヨンを行った。 次に、 32P標識したプローブ DNAを熱変性してボトノレへ投入し、 65°Cでー晚加温した。 ハイブリダィゼーシヨン後、ナィロン膜を2 33じー0.1%^/¥) SDS 50mlに浸漬し、 65 °Cで 15分間加温した。上記の洗浄操作を 2回繰り返した後、膜を 0.2 X SSC— 0.1%(w/ V) SDS 50mlに浸漬し、 65°Cで 15分間加温した。洗浄後、ナイロン膜を X線フィルムへ -80°Cで暴露し現像した。 [0226] First, Southern plot analysis was performed by the following procedure using a probe specific to the region corresponding to exon 6 to exon 9 of human FUT8 in Chinese Nomenster FUT8. Using the plasmid CHfFUT8_pCR2.1 described in patent WO02 / 31140 as a saddle and using a forward primer specific for FUT8 exon 6 (SEQ ID NO: 37) and a reverse primer specific for exon 9 (SEQ ID NO: 38), DNA PCR was performed with polymerase Ex Taq (Takara Bio Inc.). PCR was performed under 25 cycles of a reaction consisting of 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 74 ° C for 1 minute. After PCR, the amplified fragment of about 400 bp was purified and radiolabeled using [a — 32 P] dCTP 1.75 MBq and Megaprime DNA Labeling system, dCTP (Amersham Pharmacia Biotech) to obtain a probe. Immerse the above nylon membrane in 15 ml of a hybridization solution [5 X SSPE, 50 X Denhald s solution, 0.5% (w / v) SDS, 100 μg / ml salmon sperm DNA], 3 at 65 ° C A prehybridization of time was performed. Next, the 32 P-labeled probe DNA was heat denatured, put into a bottle and heated at 65 ° C. After hybridization, the Nylon membrane was immersed in 50 ml of 233 ° -0.1% ^ / ¥) SDS and heated at 65 ° C. for 15 minutes. After the above washing operation was repeated twice, the membrane was immersed in 50 ml of 0.2 X SSC—0.1% (w / V) SDS and heated at 65 ° C. for 15 minutes. After washing, the nylon membrane was exposed to X-ray film at -80 ° C and developed.
[0227] ェクソン 6からェクソン 9に特異的なプライマーを用いたゲノムサザンブロットの結果 、 RN6株および CHO/DG44細胞より 3本の断片を検出し、各断片の強度は両株間で 同等であった。  [0227] As a result of genomic Southern blot using primers specific to exon 6 to exon 9, three fragments were detected from RN6 and CHO / DG44 cells, and the strength of each fragment was the same between the two strains. .
次に、チャイニーズハムスター FUT8のうち、ヒト FUT8のェクソン 3からェクソン 5に該 当する領域に特異的なプローブを用いて、以下の手順でサザンプロット解析を行つ た。プラスミド CHfFUT8_pCR2.1を铸型とし、 FUT8ェクソン 3に特異的なフォワードプ ライマー（配列番号 39)およびェクソン 5に特異的なリバースプライマー（配列番号 40 )を用いて、 DNAポリメラーゼ Ex Taq (タカラバイオ社)による PCRを行った。 PCRは、 94 °Cで 30秒間、 60°Cで 30秒間、 74°Cで 1分間から成る反応を 1サイクルとした 25サイク ルの工程で行った。 PCR後、約 700bpの増幅断片を精製し、 [a- P] dCTP 1.75MBq よび egapnme DNA Labelling system, dCTP (Amersham Pharmacia Biotech¾：) を用いて放射標識しプローブとした。上記のナイロン膜をハイブリダィゼーシヨン液 [5 X SSPE、 50 X Denhaldt，s液、 0.5%(w/v) SDS、 100 μ g/mlサケ精子 DNA] 15mlに浸 漬し、 65°Cで 3時間のプレハイブリダィゼーシヨンを行った。次に、 32P標識したプロ一 ブ DNAを熱変性してボトルへ投入し、 65°Cでー晚加温した。ハイブリダィゼーシヨン 後、ナィロン膜を2 53。_0.1% ） SDS 50mlに浸漬し、 65°Cで 15分間加温した。 上記の洗浄操作を 2回繰り返した後、膜を 0.2 X SSC _0.1%(w/v) SDS 50mlに浸漬し、 65°Cで 15分間加温した。洗浄後、ナイロン膜を X線フィルムへ- 80°Cで暴露し現像し た。 Next, Southern plot analysis was performed by the following procedure using a probe specific to the region corresponding to exon 3 to exon 5 of human FUT8 in Chinese hamster FUT8. DNA polymerase Ex Taq (Takara Bio Inc.) using plasmid CHfFUT8_pCR2.1 as a saddle and using a forward primer specific for FUT8 exon 3 (SEQ ID NO: 39) and a reverse primer specific for exon 5 (SEQ ID NO: 40) ) PCR was performed. PCR consists of 25 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 74 ° C for 1 minute. The process was performed. After PCR, an amplified fragment of about 700 bp was purified and radiolabeled using [a-P] dCTP 1.75 MBq and egapnme DNA Labeling system, dCTP (Amersham Pharmacia Biotech¾ :) to obtain a probe. Immerse the above nylon membrane in hybridization solution [5 X SSPE, 50 X Denhaldt, s solution, 0.5% (w / v) SDS, 100 μg / ml salmon sperm DNA], 65 ° C A 3 hour prehybridization was performed. Next, the 32 P-labeled probe DNA was heat denatured, put into a bottle, and heated at 65 ° C. After hybridization, 253 Nylon membrane. _0.1%) It was immersed in SDS 50ml and heated at 65 ° C for 15 minutes. After the above washing operation was repeated twice, the membrane was immersed in 50 ml of 0.2 X SSC_0.1% (w / v) SDS and heated at 65 ° C. for 15 minutes. After washing, the nylon membrane was exposed to X-ray film at -80 ° C and developed.
[0228] ェクソン 3からェクソン 5に特異的なプライマーを用いたゲノムサザンブロットの結果 、 RN6株および CHO/DG44細胞より同じサイズの単一の断片を検出した。 RN6株由 来の断片の強度は、 CHO/DG44細胞由来の断片強度の 1/2以下であった。  [0228] As a result of genomic Southern blotting using primers specific to exon 3 to exon 5, a single fragment of the same size was detected from RN6 strain and CHO / DG44 cells. The strength of the fragment derived from the RN6 strain was less than half of the strength of the fragment derived from CHO / DG44 cells.
以上の結果から、 RN6株の一方の FUT8対立遺伝子力らェクソン 3、ェクソン 4、エタ ソン 5が欠損していることが確認された。  From the above results, it was confirmed that one FUT8 allele strength of RN6 strain lacked exon 3, exon 4 and etason 5.
実施例 3  Example 3
[0229] FUT8—塩基置換体の解析 [0229] FUT8—Analysis of base substitutions
1.チャイニーズハムスター FUT8発現プラスミド pcDNAchFUT8Compの構築  1. Construction of Chinese hamster FUT8 expression plasmid pcDNAchFUT8Comp
(1)プラスミド CHFUT8Comp23の構築  (1) Construction of plasmid CHFUT8Comp23
以下の手順でプラスミド CHFUT8Comp23を構築した（図 3)。  Plasmid CHFUT8Comp23 was constructed according to the following procedure (Fig. 3).
特許 WO02/31140に記載のプラスミド CHiFUT8- pCR2.1 1.5 μ gを、 100 μ g/ml BS A(New England Biolabs社）を含む NEBuffer for BamHI(New England Biolabs社） 50 μ 1に溶解し、制限酵素 BamHI(New England Biolabs社） 10単位および XhoI(New Engl and Biolabs社） 10単位を加えて、 37°Cで 1時間半の消化反応を行った。該反応液を 0 .8%(w/v)ァガロースゲル電気泳動に供し、約 2.0 Kbの DNA断片を GENECLEAN Spin Kit(BIO 101社)により精製して水 20 μ ΐで溶出した（以下、ァガロースゲルからの DN Α断片の精製にはこの方法を用いた)。  Dissolve 1.5 µg of the plasmid CHiFUT8-pCR2.1 described in patent WO02 / 31140 in 50 µ1 of NEBuffer for BamHI (New England Biolabs) containing 100 µg / ml BS A (New England Biolabs) Enzyme BamHI (New England Biolabs) 10 units and XhoI (New Engl and Biolabs) 10 units were added, and the digestion reaction was performed at 37 ° C for 1.5 hours. The reaction solution was subjected to 0.8% (w / v) agarose gel electrophoresis, and a DNA fragment of about 2.0 Kb was purified by GENECLEAN Spin Kit (BIO 101) and eluted with 20 μΐ of water (hereinafter referred to as agarose gel). This method was used to purify the DN Α fragment.
[0230] 一方、プラスミド pBlueScriptll KS (+) (Strategene社） 1.0 mgを、 100 μ g/ml BSA(New England Biolabs社）を含む NEBuffer for BamHI(New England Biolabs社） 50 μ 1に溶 解し、制限酵素 BamHI(New England Biolabs社） 10単位および XhoI(New England Bio labs社） 10単位をカ卩えて、 37°Cで 1時間半の消化反応を行った。該反応液を 0.8%(w/v )ァガロースゲル電気泳動に供し、約 3.0 Kbの DNA断片を精製した。 [0230] On the other hand, plasmid pBlueScriptll KS (+) (Strategene) 1.0 mg, 100 μg / ml BSA (New Dissolve in NEBuffer for BamHI (New England Biolabs) 50 μ1 containing England Biolabs) and add 10 units of restriction enzymes BamHI (New England Biolabs) and 10 units of XhoI (New England Biolabs) The digestion reaction was carried out at 37 ° C for 1.5 hours. The reaction solution was subjected to 0.8% (w / v) agarose gel electrophoresis, and a DNA fragment of about 3.0 Kb was purified.
[0231] 上記で得たプラスミド CHiFUT8-pCR2.1由来の BamHト Xhol断片 (約 2.0 Kb) 4.0 μ 1 、プラスミド pBlueScriptll SK(+)由来の BamHト Xhol断片 (約 3.0 Kb) 0.5 μ 1、水 0.5 μ \ 、 Ligation High (東洋紡社） 5.0 μ 1を混合し、 16°Cで 30分間反応させることにより断片 を連結した。該反応液を用いて大腸菌 DH5a株を形質転換し、得られたアンピシリン 耐性クローンより公知の方法に従って各々プラスミド DNAを単離した。本プラスミドを 以下、 CHFUT8Comp23と称す。 [0231] BamH to Xhol fragment (about 2.0 Kb) derived from plasmid CHiFUT8-pCR2.1 obtained above (about 2.0 Kb) 4.0 μ1, BamH to Xhol fragment (about 3.0 Kb) derived from plasmid pBlueScriptll SK (+) 0.5 μ1, water The fragments were ligated by mixing 0.5 μ \ and Ligation High (Toyobo Co., Ltd.) 5.0 μ 1 and reacting at 16 ° C. for 30 minutes. The reaction solution was used to transform Escherichia coli DH5a strain, and plasmid DNAs were isolated from the resulting ampicillin resistant clones according to a known method. This plasmid is hereinafter referred to as CHFUT8Comp23.
(2)プラスミド CHFUT8CompTAの構築  (2) Construction of plasmid CHFUT8CompTA
以下の手順でプラスミド CHFUT8CompTAを構築した（図 4)。  Plasmid CHFUT8CompTA was constructed by the following procedure (Fig. 4).
[0232] まず、コザック配列（KOZAK)とチャイニーズノヽムスター FUT8翻訳開始部位を連結 したフォワードプライマー（配列番号 41)、および FUT8翻訳終止部位に制限酵素 Ba mHI反応部位を連結したリバースプライマー（配列番号 42)を合成した。次に、本項（ 1)で得たプラスミド CHFUT8Comp23 50 ngおよび DNAポリメラーゼ KOD-plus (東洋 紡社）を含む 50 μ 1の反応液 [KOD-plus buffer (東洋紡社)、 0.2 mmol/1 dNTPs、 1.2 m mol/1 MgSO、 0.3 μ mol/1上記遺伝子特異的プライマー（配列番号 41および配列 番号 42) ]を調製し、 PCRを行った。 PCRは、 94°Cで 2分間加熱した後、 98°Cで 10秒間 、 60°Cで 30秒間、 68°Cで 1分 45秒から成る反応を 1サイクルとした 25サイクルの工程 で行った。 PCR後、 Ex Taq (タカラバイオ社） 1単位を添カ卩し、 72°Cで 5分間反応させ た。該反応液を 0.8%(w/v)ァガロースゲル電気泳動に供し、約 1.7 Kbの DNA断片を 精製した。精製した DNA断片を、 TOPO TA cloning Kit (Invitrogen社）を用いてプラ スミド PCR2.1へ揷入し、大腸菌 Stbl2株を形質転換した。得られたアンピシリン耐性ク ローンより公知の方法に従って各々プラスミド DNAを単離した。本プラスミドを以下、 C HFUT8CompTAと称す。 [0232] First, a Kosak sequence (KOZAK) and a Chinese nomstar FUT8 translation start site linked forward primer (SEQ ID NO: 41), and a FUT8 translation stop site linked to a restriction enzyme Ba mHI reaction site (SEQ ID NO: 42). ) Was synthesized. Next, a reaction solution of 50 μ1 containing 50 ng of the plasmid CHFUT8Comp23 obtained in this section (1) and DNA polymerase KOD-plus (Toyobo) [KOD-plus buffer (Toyobo), 0.2 mmol / 1 dNTPs, 1.2 mMol / 1 MgSO, 0.3 μmol / 1 gene-specific primers (SEQ ID NO: 41 and SEQ ID NO: 42)] were prepared and PCR was performed. PCR was performed in 25 cycles of a reaction consisting of heating at 94 ° C for 2 minutes, followed by 98 ° C for 10 seconds, 60 ° C for 30 seconds, and 68 ° C for 1 minute 45 seconds. . After PCR, 1 unit of Ex Taq (Takara Bio Inc.) was added and reacted at 72 ° C for 5 minutes. The reaction solution was subjected to 0.8% (w / v) agarose gel electrophoresis, and a DNA fragment of about 1.7 Kb was purified. The purified DNA fragment was inserted into plasmid PCR2.1 using TOPO TA cloning Kit (Invitrogen) to transform E. coli Stbl2. Each plasmid DNA was isolated from the obtained ampicillin resistant clone according to a known method. This plasmid is hereinafter referred to as C HFUT8CompTA.
(3)プラスミド pcDNAchFUT8Compの構築  (3) Construction of plasmid pcDNAchFUT8Comp
以下の手順でプラスミド pcDNAchFUT8Compを構築した（図 5)。 [0233] 本項（2)で得たプラスミド CHFUT8CompTA 2.0 μ gを NEBuffer for EcoRI(New Engl and Biolabs社） 35 β 1に溶解し、制限酵素 EcoRI(New England Biolabs社） 10単位を カロえて、 37°Cで 2時間半の消化反応を行った。消化反応液をエタノール沈殿に付し た後、 Blunting High (東洋紡社）により 10 μ 1の反応系でベクター切断端を平滑化し た。平滑反応液に対しフヱノール—クロ口ホルム抽出処理を行った後、 100 μ g/ml B SA(New England Biolabs社）を含む NEBuffer for BamHI(New England Biolabs社） 35 μ 1に溶解し、制限酵素 BamHI(New England Biolabs社） 10単位を加えて、 37°Cで 1時 間半の消化反応を行った。 Plasmid pcDNAchFUT8Comp was constructed according to the following procedure (FIG. 5). [0233] 2.0 μg of the plasmid CHFUT8CompTA obtained in this section (2) was dissolved in NEBuffer for EcoRI (New Engl and Biolabs) 35 β1, and 10 units of the restriction enzyme EcoRI (New England Biolabs) were prepared. The digestion reaction was performed at ° C for 2.5 hours. After subjecting the digestion reaction solution to ethanol precipitation, the vector cut ends were smoothed in a 10 μl reaction system by Blunting High (Toyobo Co., Ltd.). The smooth reaction mixture was extracted with phenol-chloroform, and then dissolved in 35 μ 1 of NEBuffer for BamHI (New England Biolabs) containing 100 μg / ml BSA (New England Biolabs). 10 units of BamHI (New England Biolabs) was added, and the digestion reaction was performed at 37 ° C for 1.5 hours.
該反応液を 0.8%(w/v)ァガロースゲル電気泳動に供し、約 1.7 Kbの DNA断片を精 製した。  The reaction solution was subjected to 0.8% (w / v) agarose gel electrophoresis, and a DNA fragment of about 1.7 Kb was purified.
[0234] 一方、 pcDNA3.1(+) (Invitrogen社） 1.0 μ gを NEBuffer2(New England Biolabs社） 35 μ 1に溶解し、制限酵素 HindllKNew England Biolabs社） 10単位を加えて、 37°Cで 2時 間半の消化反応を行った。消化反応液をエタノール沈殿に付した後、 Blunting High ( 東洋紡社）により 10 / 1の反応系でベクター切断端を平滑化した。平滑反応液に対し フエノールークロロホルム抽出処理を行った後、 100 β g/ml BSA(New England Biolab s社）を含む NEBuffer for BamHI(New England Biolabs社） 35 μ 1に溶解し、制限酵素 BamHKNew England Biolabs社） 10単位を加えて、 37°Cで 1時間半の消化反応を行つ た。該反応液を 0.8%(w/v)ァガロースゲル電気泳動に供し、約 5.4 Kbの DNA断片を ；^した Q [0234] On the other hand, 1.0 μg of pcDNA3.1 (+) (Invitrogen) was dissolved in NEBuffer2 (New England Biolabs) 35 μ1, and 10 units of restriction enzyme HindllKNew England Biolabs) were added at 37 ° C. The digestion reaction was performed for 2.5 hours. After subjecting the digestion reaction solution to ethanol precipitation, the vector cut ends were smoothed by Blunting High (Toyobo Co., Ltd.) in a 10/1 reaction system. The smooth reaction mixture was extracted with phenol-chloroform and then dissolved in 35 µ 1 of NEBuffer for BamHI (New England Biolabs) containing 100 β g / ml BSA (New England Biolabs). England Biolabs) 10 units were added, and the digestion reaction was performed at 37 ° C for 1.5 hours. The reaction solution 0.8% was subjected to (w / v) Agarosugeru electrophoresis, a DNA fragment of about 5.4 Kb; ^ the Q
[0235] 上記で得たプラスミド CHFUT8CompTA由来の EcoRト BamHI断片 (約 1.7 Kb) 1.0 μ [0235] EcoR to BamHI fragment (about 1.7 Kb) derived from plasmid CHFUT8CompTA obtained above 1.0 μm
1、プラスミド mcKOgE2-2由来の Hindin-BamHI断片 (約 5.4 Kb) 0.5 /1 1、水3.5 μ 1、 Li gation High (東洋紡社） 5.0 μ 1を混合し、 16°Cで 1時間反応させることにより断片を連 結した。該反応液を用いて大腸菌 Stbl2株を形質転換し、得られたアンピシリン耐性ク ローンより公知の方法に従って各々プラスミド DNAを単離した。本プラスミドを以下、 p cDNAchFUT8Compと称す。 1. Mix Hind-BamHI fragment derived from plasmid mcKOgE2-2 (approx. 5.4 Kb) 0.5 / 1, water 3.5 μ1, Ligation High (Toyobo) 5.0 μ1, and react at 16 ° C for 1 hour The fragments were linked together. Escherichia coli Stbl2 strain was transformed with the reaction solution, and plasmid DNA was isolated from the resulting ampicillin resistant clone according to a known method. This plasmid is hereinafter referred to as p cDNAchFUT8Comp.
2.チャイニーズハムスター FUT8—塩基置換体発現プラスミド pcDNAchFUT8Moの構 築  2. Construction of Chinese hamster FUT8—base substitution plasmid pcDNAchFUT8Mo
(1)プラスミド CHFUT8Mo3の構築 以下の手順でプラスミド CHFUT8Mo3を構築した（図 6)。 (1) Construction of plasmid CHFUT8Mo3 Plasmid CHFUT8Mo3 was constructed by the following procedure (Fig. 6).
[0236] まず、 FUT8翻訳領域の 512番目のグァニンをアデニンへ置換した配列に特異的な フォワードプライマー（配列番号 43)およびリバースプライマー（配列番号 44)を合成 した。次に、 T4 polynucleotide kinase (タカラバイオ社） 10単位を含む 20 μ 1の反応液 [Phosphorylation buffer (タカラバイオ社）、 1.25 mmol/1 ATP、 10 mmol/1上記プライ マー（配列番号 43および配列番号 44) ]にて 37°Cで 30分間反応することにより、オリ ゴヌクレオチド末端のリン酸化を行った。続いて、本実施例第 1項（1)で得たプラスミ ド CHFUT8Comp23 50 ngおよび DNAポリメラーゼ KOD_plus (東洋紡社）を含む 50 μ \ の反応液 [KOD- plus buffer (東洋紡社)、 0.2 mmol/1 dNTPs、 1.2 mmol/1 MgSO、 0.3 [0236] First, a forward primer (SEQ ID NO: 43) and reverse primer (SEQ ID NO: 44) specific to the sequence in which the 512th guanine in the FUT8 translation region was replaced with adenine were synthesized. Next, 20 μ 1 reaction solution containing 10 units of T4 polynucleotide kinase (Takara Bio) [Phosphorylation buffer (Takara Bio), 1.25 mmol / 1 ATP, 10 mmol / 1 above primer (SEQ ID NO: 43 and SEQ ID NO: 44)] was reacted at 37 ° C for 30 minutes to phosphorylate the oligonucleotide terminal. Subsequently, a reaction solution (KOD-plus buffer (Toyobo Co., Ltd.), 0.2 mmol / 1) containing 50 ng of the plasmid CHFUT8Comp23 obtained in the first item (1) of this example and DNA polymerase KOD_plus (Toyobo Co., Ltd.) dNTPs, 1.2 mmol / 1 MgSO, 0.3
4 μ mol/1上記遺伝子特異的リン酸化プライマー（配列番号 43および配列番号 44) ] を調製し、 PCRを行った。 PCRは、 94°Cで 2分間加熱した後、 94°Cで 15秒間、 60°Cで 3 0秒間、 68°Cで 5分間力 成る反応を 1サイクルとした 25サイクルの工程で行った。 PC R反応液を 0.8%(w/v)ァガロースゲル電気泳動に供し、約 5.0 Kbの DNA断片を精製し て水で倍希釈した。  4 μmol / 1 of the above gene-specific phosphorylated primers (SEQ ID NO: 43 and SEQ ID NO: 44)] were prepared and subjected to PCR. PCR was performed in 25 cycles of heating at 94 ° C for 2 minutes, followed by a reaction consisting of 94 ° C for 15 seconds, 60 ° C for 30 seconds, and 68 ° C for 5 minutes. The PCR reaction solution was subjected to 0.8% (w / v) agarose gel electrophoresis, and a DNA fragment of about 5.0 Kb was purified and diluted twice with water.
[0237] 上記で得た PCR増幅産物（約 5.0) 1.0 μ 1、水 4.0 μ 1、 Ligation High (東洋紡社） 5.0  [0237] PCR amplification product obtained above (approximately 5.0) 1.0 μ1, water 4.0 μ1, Ligation High (Toyobo) 5.0
/ lを混合し、 16°Cで 2時間半反応させることにより断片を連結した。該反応液を用い て大腸菌 Stbl2株を形質転換し、得られたアンピシリン耐性クローンより公知の方法に 従って各々プラスミド DNAを単離した。本プラスミドを以下、 CHFUT8Mo3と称す。  The fragments were ligated by mixing / l and reacting at 16 ° C for 2.5 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 CHFUT8Mo3.
(2)プラスミド CHFUT8MoTAの構築  (2) Construction of plasmid CHFUT8MoTA
以下の手順でプラスミド CHFUT8MoTAを構築した（図 7)。  Plasmid CHFUT8MoTA was constructed by the following procedure (FIG. 7).
[0238] まず、コザック配列（KOZAK)とチャイニーズノヽムスター FUT8翻訳開始部位を連結 したフォワードプライマー（配列番号 41)、および FUT8翻訳終止部位に制限酵素 Ba mHI反応部位を連結したリバースプライマー（配列番号 42)を合成した。次に、本項（ 1)で得たプラスミド CHFUT8Mo3 50 ngおよび DNAポリメラーゼ KOD_plus (東洋紡社 )を含む 50 μ 1の反応液 [KOD- plus buffer (東洋紡社)、 0.2 mmol/1 dNTPs、 1.2 mmol /1 MgSO、 0.3 μ mol/1上記遺伝子特異的プライマー（配列番号 41および配列番号 [0238] First, a forward primer (SEQ ID NO: 41) in which a Kozak sequence (KOZAK) and a Chinese nomstar FUT8 translation start site were linked, and a reverse primer (SEQ ID NO: 42) in which a restriction enzyme Ba mHI reaction site was linked to the FUT8 translation termination site. ) Was synthesized. Next, 50 μl of the reaction solution containing 50 ng of the plasmid CHFUT8Mo3 obtained in this section (1) and DNA polymerase KOD_plus (Toyobo) [KOD-plus buffer (Toyobo), 0.2 mmol / 1 dNTPs, 1.2 mmol / 1 MgSO, 0.3 μmol / 1 gene-specific primer (SEQ ID NO: 41 and SEQ ID NO:
4 Four
42) ]を調製し、 PCRを行った。 PCRは、 94°Cで 2分間加熱した後、 98°Cで 10秒間、 60 °Cで 30秒間、 68°Cで 1分 45秒から成る反応を 1サイクルとした 25サイクルの工程で行 つた。 PCR後、 Ex Taq (タカラバイオ社） 1単位を添加し、 72°Cで 5分間反応させた。該 反応液を 0.8%(w/v)ァガロースゲル電気泳動に供し、約 1.7 Kbの DNA断片を精製し た。精製した DNA断片を、 TOPO TA cloning Kit (Invitrogen社）を用いてプラスミド pC R2.1へ揷入し、大腸菌 Stbl2株を形質転換した。得られたアンピシリン耐性クローンよ り公知の方法に従って各々プラスミド DNAを単離した。本プラスミドを以下、 CHFUT8 CompTAと称す。 42)] was prepared and PCR was performed. PCR is performed in 25 cycles, consisting of heating for 2 minutes at 94 ° C, 10 seconds at 98 ° C, 30 seconds at 60 ° C, and 1 minute 45 seconds at 68 ° C. I got it. After PCR, 1 unit of Ex Taq (Takara Bio Inc.) was added and reacted at 72 ° C for 5 minutes. The reaction solution was subjected to 0.8% (w / v) agarose gel electrophoresis, and a DNA fragment of about 1.7 Kb was purified. The purified DNA fragment was inserted into plasmid pCR2.1 using TOPO TA cloning Kit (Invitrogen) to transform Escherichia coli Stbl2 strain. Plasmid DNAs were isolated from the resulting ampicillin resistant clones according to a known method. This plasmid is hereinafter referred to as CHFUT8 CompTA.
(3)プラスミド pcDNAchFUT8Moの構築  (3) Construction of plasmid pcDNAchFUT8Mo
以下の手順でプラスミド pcDNAchFUT8Moを構築した（図 8)。  Plasmid pcDNAchFUT8Mo was constructed according to the following procedure (FIG. 8).
[0239] 本項（2)で得たプラスミド CHFUT8MoTA 2.0 μ gを NEBuffer for EcoRI(New Englan d Biolabs社） 35 μ ΐに溶解し、制限酵素 EcoRI(New England Biolabs社） 10単位をカロ えて、 37°Cで 2時間半の消化反応を行った。消化反応液をエタノール沈殿に付した 後、 Blunting High (東洋紡社）により 10 μ 1の反応系でベクター切断端を平滑化した。 平滑反応液に対しフエノールークロロホルム抽出処理を行った後、 100 M g/ml BSA( New England Biolabs社）を含む NEBuffer for BamHI(New England Biolabs社） 35 μ 1 に溶解し、制限酵素 BamHI(New England Biolabs社） 10単位を加えて、 37°Cで 1時間 半の消化反応を行った。該反応液を 0.8%(w/v)ァガロースゲル電気泳動に供し、約 1. 7 Kbの DNA断片を精製した。  [0239] 2.0 μg of the plasmid CHFUT8MoTA obtained in (2) above was dissolved in 35 μΐ of NEBuffer for EcoRI (New England Biolabs), and 10 units of the restriction enzyme EcoRI (New England Biolabs) was added. The digestion reaction was performed at ° C for 2.5 hours. The digestion reaction solution was subjected to ethanol precipitation, and then the vector cut end was smoothed in a 10 μ1 reaction system by Blunting High (Toyobo Co., Ltd.). The smooth reaction mixture was extracted with phenol-chloroform and then dissolved in 35 μ 1 of NEBuffer for BamHI (New England Biolabs) containing 100 mg / ml BSA (New England Biolabs), and the restriction enzyme BamHI ( (New England Biolabs) 10 units were added and digestion reaction was performed at 37 ° C for 1.5 hours. The reaction solution was subjected to 0.8% (w / v) agarose gel electrophoresis, and a DNA fragment of about 1.7 Kb was purified.
[0240] 一方、 pcDNA3.1(+) (Invitrogen社） 1.0 μ gを NEBuffer2(New England Biolabs社） 35 μ 1に溶解し、制限酵素 HindIII(New England Biolabs社） 10単位を加えて、 37°Cで 2時 間半の消化反応を行った。消化反応液をエタノール沈殿に付した後、 Blunting High ( 東洋紡社）により 10 / 1の反応系でベクター切断端を平滑化した。平滑反応液に対し フエノール一クロ口ホルム抽出処理を行った後、 100 μ g/ml BSA(New England Biolab s社）を含む NEBuffer for BamHI(New England Biolabs社） 35 μ 1に溶解し、制限酵素 BamHKNew England Biolabs社） 10単位を加えて、 37°Cで 1時間半の消化反応を行つ た。該反応液を 0.8%(w/v)ァガロースゲル電気泳動に供し、約 5.4 Kbの DNA断片を 精製した。  [0240] On the other hand, 1.0 μg of pcDNA3.1 (+) (Invitrogen) was dissolved in NEBuffer2 (New England Biolabs) 35 μ1, and 10 units of restriction enzyme HindIII (New England Biolabs) was added at 37 °. The digestion reaction was performed for 2.5 hours with C. After subjecting the digestion reaction solution to ethanol precipitation, the vector cut ends were smoothed by Blunting High (Toyobo Co., Ltd.) in a 10/1 reaction system. The smooth reaction mixture was subjected to phenol-chloroform extraction and then dissolved in 35 μ 1 of NEBuffer for BamHI (New England Biolabs) containing 100 μg / ml BSA (New England Biolabs). BamHKNew England Biolabs) 10 units were added, and the digestion reaction was performed at 37 ° C for 1.5 hours. The reaction solution was subjected to 0.8% (w / v) agarose gel electrophoresis, and a DNA fragment of about 5.4 Kb was purified.
[0241] 上記で得たプラスミド CHFUT8MoTA由来の EcoRト BamHI断片 (約 1.7 Kb) 1.0 μ ΐ プラスミド mcKOgE2- 2由来の Hindin- BamHI断片 (約 5.4 Kb) 0.5 μ ΐ、水 3.5 μ 1、 Ligat ion High (東洋紡社） 5.0 / lを混合し、 16°Cで 1時間反応させることにより断片を連結 した。該反応液を用いて大腸菌 Stbl2株を形質転換し、得られたアンピシリン耐性クロ ーンより公知の方法に従って各々プラスミド DNAを単離した。本プラスミドを以下、 pc DNAchFUT8Moと称す。 [0241] EcoR to BamHI fragment derived from plasmid CHFUT8MoTA obtained above (approximately 1.7 Kb) 1.0 μΐ Hindin-BamHI fragment derived from plasmid mcKOgE2-2 (approximately 5.4 Kb) 0.5 μΐ, water 3.5 μ1, Ligat Ion High (Toyobo Co., Ltd.) 5.0 / l was mixed and the fragments were ligated by reacting at 16 ° C for 1 hour. Escherichia coli Stbl2 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 pc DNAchFUT8Mo.
3. FUT8—塩基置換体導入細胞のひ- 1，6 -フコース修飾解析  3. FUT8—Hydrogen 1,6-fucose modification analysis of base-substituted cells
CHO/DG44細胞由来 FUT8ノックアウト細胞である Ms709細胞 [Biotechnology and Bioengineering, 87, 614 (2004)]に対し、本実施例第 1項で構築したプラスミド pcDNA chFUT8Compおよび pcDNAchFUT8Moを、以下の手順でエレクト口ポレーシヨン法 [C ytotechnology, 3, 133 (1990)]により導入した。  For the Ms709 cells [Biotechnology and Bioengineering, 87, 614 (2004)], which are FUT8 knockout cells derived from CHO / DG44 cells, the plasmid pcDNA chFUT8Comp and pcDNAchFUT8Mo constructed in section 1 of this example were prepared by the following procedure. It was introduced by the method [Cytotechnology, 3, 133 (1990)].
[0242] まず、各プラスミド 10 μ gを NEBuffer for SspI(New England Biolabs社） 100 μ 1に溶 解し、 20単位の制限酵素 SspI(New England Biolabs社)を加えて 37°Cで 4時間半反応 し線状化した。該反応液に対しフヱノール/クロ口ホルム抽出処理およびエタノール沈 殿を行い、回収した線状化プラスミドを 1 / g/ i l7 溶液とした。一方、 Ms709細胞を K -PBS緩衝液 [137mmol/l KC1、 2.7mmol/l NaCl、 8.1mmol/l Na HP04、 1.5mmol/l KH[0242] First, 10 µg of each plasmid was dissolved in 100 µ 1 of NEBuffer for SspI (New England Biolabs), and 20 units of restriction enzyme SspI (New England Biolabs) was added, followed by 4 hours and a half at 37 ° C. It reacted and became linear. The reaction solution was subjected to phenol / chloroform extraction treatment and ethanol precipitation, and the recovered linearized plasmid was used as a 1 / g / il 7 solution. Meanwhile, Ms709 cells were mixed with K-PBS buffer [137mmol / l KC1, 2.7mmol / l NaCl, 8.1mmol / l Na HP04, 1.5mmol / l KH.
PO、 4.0mmol/l MgCl ]に懸濁して 8 X 107個/ mlとした。細胞懸濁液 200 μ 1 (1.6 X 106 個）を上記線状化プラスミド 4 / 1 (4 / g)と混和した後、細胞 DNA混和液の全量を G ene Pulser Cuvette (電極間距離 2mm)(BI〇-RAD社)へ移し、細胞融合装置 Gene Puis er(BIO_RAD社)を用いてパルス電圧 350V、電気容量 250 μ Fの条件で遺伝子導入を 行った。遺伝子導入後、細胞懸濁液を 10%ゥシ胎児血清（Invitrogen社）および HT s upplement (Invitrogen社）を添加した IMDM培地（Invitrogen社）に懸濁し、接着培養 用 T75フラスコ（グライナ一社)へ播種した。 5% CO、 37°Cの条件下で 24時間培養し た後、培養上清を除去し、 600 μ g/ml G418 (ナカライテスタ社）、 HT supplement (Invi trogen社）および 10%ゥシ胎児血清（Invitrogen社）を添加した IMDM培地 (Invitrogen 社)へ交換した。この培地交換作業を 3〜4日毎に繰り返しながら 15日間の培養を行い 、 G418耐性株を取得した。 It was suspended in PO, 4.0 mmol / l MgCl] to give 8 × 10 7 cells / ml. After mixing 200 μ 1 of cell suspension (1.6 × 10 6 cells) with the above linearized plasmid 4/1 (4 / g), the total volume of the cell DNA mixture is Gene Pulser Cuvette (distance between electrodes 2 mm) (BI〇-RAD) was transferred, and gene transfer was carried out using a cell fusion device Gene Puiser (BIO_RAD) under conditions of a pulse voltage of 350 V and an electric capacity of 250 μF. After gene transfer, the cell suspension is suspended in IMDM medium (Invitrogen) supplemented with 10% urine fetal serum (Invitrogen) and HT supplement (Invitrogen), and T75 flask for adhesion culture (Grainer) Sowing. After culturing for 24 hours at 5% CO and 37 ° C, the culture supernatant is removed, and 600 μg / ml G418 (Nacalai Testa), HT supplement (Invitrogen) and 10% fetus The medium was replaced with IMDM medium (Invitrogen) supplemented with serum (Invitrogen). This medium exchange operation was repeated every 3 to 4 days, and culturing was performed for 15 days to obtain a G418 resistant strain.
[0243] 1%BSA- PBSに、各 G418耐性株 2 X 105個を懸濁し、 FITC標識 LCA (Vector Laborat ories社）または 100倍希釈した FITC標識ストレプトアビジン（KPL社）を添加した。 4°C にて 30分間静置することで細胞を染色し、 1%BSA-PBSを用いて細胞を洗浄した後、 I X 104個の細胞を FACSCalibur (BD Biosciences社）で解析した。 [0243] 2 X 10 5 strains of each G418 resistant strain were suspended in 1% BSA-PBS, and FITC-labeled LCA (Vector Laboratories) or 100-fold diluted FITC-labeled streptavidin (KPL) was added. After staining the cells by standing at 4 ° C for 30 minutes, washing the cells with 1% BSA-PBS, IX 10 Four cells were analyzed with FACSCalibur (BD Biosciences).
図 9に各遺伝子導入株の LCAに対する反応性を示した。 FUT8発現株（プラスミド pc DNAchFUT8Comp導入株）では、 CHO/DG44細胞と同等の高い LCA反応性を示す 細胞群が存在した。 FUT8—塩基置換体発現株（プラスミド pcDNAchFUT8Mo導入株 )では、 LCAに僅かに反応性を示す細胞群が存在した。この結果から、 FUT8翻訳領 域の 512番目のグァニンをアデニンへ置換することより、細胞のひ- 1，6 -フコース付加 能が大きく低減されることが示された。  Figure 9 shows the reactivity of each transgenic strain to LCA. In the FUT8 expression strain (plasmid pc DNAchFUT8Comp-introduced strain), there was a group of cells showing high LCA reactivity equivalent to CHO / DG44 cells. In the FUT8-base substitution expression strain (plasmid pcDNAchFUT8Mo-introduced strain), there was a group of cells slightly reactive to LCA. These results indicate that the substitution of the 512th guanine in the FUT8 translation region with adenine greatly reduces the ability of cells to add 1,6-fucose.
産業上の利用可能性  Industrial applicability
[0244] 本発明により、 N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの [0244] According to the present invention, the N-glycidyl darcosamine of the N-glycoside-linked complex type sugar chain reducing end
6位にフコースの 1位が α結合する糖鎖修飾に関与する酵素において、該酵素の活 性が欠失、または低下するようにアミノ酸改変された変異体及びその利用方法が提 供される。  In the enzyme involved in the sugar chain modification in which the 1-position of fucose is α-bonded at the 6-position, a variant in which the amino acid is modified so that the activity of the enzyme is deleted or decreased, and a method for using the same are provided.
配列表フリ -テキスト  Sequence Listing Free-Text
[0245] 配列番号 23-人工配列の説明 合成 DNA  [0245] SEQ ID NO: 23-Description of Artificial Sequence Synthetic DNA
配列番号 24-人工配列の説明 合成 DNA  SEQ ID NO: 24-Description of Artificial Sequence Synthetic DNA
配列番号 25-人工配列の説明 合成 DNA  SEQ ID NO: 25--Description of Artificial Sequence Synthetic DNA
配列番号 26-人工配列の説明 合成 DNA  SEQ ID NO: 26--Description of Artificial Sequence Synthetic DNA
配列番号 27-人工配列の説明 合成 DNA  SEQ ID NO: 27--Description of Artificial Sequence Synthetic DNA
配列番号 28-人工配列の説明 合成 DNA  SEQ ID NO: 28--Description of Artificial Sequence Synthetic DNA
配列番号 29-人工配列の説明 合成 DNA  SEQ ID NO: 29--Description of Artificial Sequence Synthetic DNA
配列番号 30-人工配列の説明 合成 DNA  SEQ ID NO: 30--Description of Artificial Sequence Synthetic DNA
配列番号 31-人工配列の説明 合成 DNA  SEQ ID NO: 31--Description of Artificial Sequence Synthetic DNA
配列番号 32-人工配列の説明 合成 DNA  SEQ ID NO: 32-Description of Artificial Sequence Synthetic DNA
配列番号 33-人工配列の説明 合成 DNA  SEQ ID NO: 33--Description of Artificial Sequence Synthetic DNA
配列番号 34-人工配列の説明 合成 DNA  SEQ ID NO: 34--Description of Artificial Sequence Synthetic DNA
配列番号 35-人工配列の説明 合成 DNA  SEQ ID NO: 35--Description of Artificial Sequence Synthetic DNA
配列番号 36-人工配列の説明 合成 DNA  SEQ ID NO: 36--Description of Artificial Sequence Synthetic DNA
配列番号 37-人工配列の説明 合成 DNA 配列番号 38-人工配列の説明：合成 DNA 配列番号 39-人工配列の説明 ：合成 DNA 配列番号 40-人工配列の説明 ：合成 DNA 配列番号 41-人工配列の説明 ：合成 DNA 配列番号 42-人工配列の説明 ：合成 DNA 配列番号 43-人工配列の説明 ：合成 DNA 配列番号 44-人工配列の説明 ：合成 DNA SEQ ID NO: 37--Description of Artificial Sequence Synthetic DNA SEQ ID NO: 38-description of artificial sequence: synthetic DNA SEQ ID NO: 39-description of artificial sequence: synthetic DNA SEQ ID NO: 40-description of artificial sequence: synthetic DNA SEQ ID NO: 41-description of artificial sequence: synthetic DNA SEQ ID NO: 42-artificial sequence Description: Synthetic DNA SEQ ID NO: 43-Description of artificial sequence: Synthetic DNA SEQ ID NO: 44-Description of artificial sequence: Synthetic DNA

Claims

請求の範囲 The scope of the claims
[1] ひ- 1，6-フコシルトランスフェラーゼのアミノ酸配列において、配列番号 7で表されるァ ミノ酸配列の N末端から 171番目のアミノ酸に相応する位置のアミノ酸が欠失してい る力 \またはセリン以外のアミノ酸に置換されたアミノ酸配列を有する、 ひ- 1，6_フコシ ルトランスフェラーゼ変異体。  [1] The ability to delete the amino acid at the position corresponding to the 171st amino acid from the N-terminus of the amino acid sequence represented by SEQ ID NO: 7 in the amino acid sequence of -1,6-fucosyltransferase \ or A mutant of 1,6_fucosyltransferase having an amino acid sequence substituted with an amino acid other than serine.
[2] セリン以外のアミノ酸がァスパラギンである、請求項 1に記載のひ -1,6 -フコシルトラン スフヱラーゼ変異体。  [2] The -1,6-fucosyltransferase variant according to claim 1, wherein the amino acid other than serine is asparagine.
[3] α -1,6-フコシルトランスフェラーゼカ 以下の（a)〜(f)からなる群から選ばれる DNAが コードする蛋白質である、請求項 1または 2に記載の α -1,6-フコシルトランスフェラー ゼ変異体。  [3] The α-1,6-fucosyltransferase according to claim 1 or 2, wherein the α-1,6-fucosyltransferase is a protein encoded by a DNA selected from the group consisting of the following (a) to (f): Transferase mutant.
(a)配列番号 1で表される塩基配列からなる DNA ;  (a) DNA consisting of the base sequence represented by SEQ ID NO: 1;
(b)配列番号 2で表される塩基配列からなる DNA ;  (b) DNA consisting of the base sequence represented by SEQ ID NO: 2;
(c)配列番号 3で表される塩基配列からなる DNA ;  (c) DNA consisting of the base sequence represented by SEQ ID NO: 3;
(d)配列番号 4で表される塩基配列からなる DNA ;  (d) DNA consisting of the base sequence represented by SEQ ID NO: 4;
(e)配列番号 5で表される塩基配列からなる DNA ;  (e) DNA consisting of the base sequence represented by SEQ ID NO: 5;
(f)配列番号 6で表される塩基配列からなる DNA。  (f) DNA consisting of the base sequence represented by SEQ ID NO: 6.
[4] ひ- 1，6-フコシルトランスフェラーゼカ 以下の（a)〜(f)からなる群から選ばれる蛋白質 である、請求項 1または 2に記載のひ -1,6-フコシルトランスフェラーゼ変異体。  [4] A -1,6-fucosyltransferase mutant according to claim 1 or 2, which is a protein selected from the group consisting of the following (a) to (f):
(a)配列番号 7で表されるアミノ酸配列からなる蛋白質；  (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 7;
(b)配列番号 8で表されるアミノ酸配列からなる蛋白質；  (b) a protein comprising the amino acid sequence represented by SEQ ID NO: 8;
(c)配列番号 9で表されるアミノ酸配列からなる蛋白質；  (c) a protein comprising the amino acid sequence represented by SEQ ID NO: 9;
(d)配列番号 10で表されるアミノ酸配列からなる蛋白質；  (d) a protein comprising the amino acid sequence represented by SEQ ID NO: 10;
(e)配列番号 11で表されるアミノ酸配列からなる蛋白質；  (e) a protein comprising the amino acid sequence represented by SEQ ID NO: 11;
(f)配列番号 12で表されるアミノ酸配列からなる蛋白質。  (f) a protein comprising the amino acid sequence represented by SEQ ID NO: 12.
[5] 配列番号 13〜： 17のいずれかで表されるアミノ酸配列を含む α -1,6-フコシルトランス フェラーゼ変異体。  [5] An α-1,6-fucosyltransferase mutant comprising the amino acid sequence represented by any one of SEQ ID NOs: 13 to 17:
[6] 配列番号 13〜： 17のいずれかで表されるアミノ酸配列において、 1以上のアミノ酸が 欠失、置換、挿入および/または付加されたアミノ酸配列からなり、かつ α -1,6-フコ シルトランスフェラーゼ活性が欠失、または、配列番号 7または 9で表されるアミノ酸配 列からなるひ -1,6-フコシルトランスフェラーゼの α -1,6-フコシルトランスフェラーゼ活 性よりも低下した α -1,6-フコシルトランスフェラーゼ活性を有する α -1,6-フコシルトラ ンスフヱラーゼ変異体。 [6] In the amino acid sequence represented by any one of SEQ ID NOs: 13 to 17, the amino acid sequence consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and α-1,6-fuco Α -1, which has a reduced syltransferase activity or is less than the α-1,6-fucosyltransferase activity of -1,6-fucosyltransferase comprising the amino acid sequence represented by SEQ ID NO: 7 or 9. Α-1,6-fucosyltransferase mutant having 6-fucosyltransferase activity.
配列番号 13〜： 17のいずれかで表されるアミノ酸配列と 80%以上の相同性を有する アミノ酸配列からなり、かつひ- 1，6-フコシルトランスフェラーゼ活性が欠失、または、 配列番号 7または 9で表されるアミノ酸配列からなるひ -1,6 -フコシルトランスフェラー ゼのひ -1，6-フコシルトランスフェラーゼ活性よりも低下したひ -1，6-フコシルトランスフ エラーゼ活性を有するひ -1，6 -フコシルトランスフェラーゼ変異体。 It consists of an amino acid sequence having 80% or more homology with the amino acid sequence represented by any one of SEQ ID NOs: 13 to 17 and lacks -1,6-fucosyltransferase activity, or SEQ ID NO: 7 or 9 A -1,6-fucosyltransferase having a -1,6-fucosyltransferase activity lower than the -1,6-fucosyltransferase activity of -1,6-fucosyltransferase having the amino acid sequence represented by A transferase mutant.
請求項 1〜7のいずれか一項に記載のひ -1,6 -フコシルトランスフェラーゼ変異体をコ ードする DNA。 DNA encoding the mutant of -1,6-fucosyltransferase according to any one of claims 1 to 7.
配列番号 18〜22のいずれかで表される塩基配列を含む DNA。 DNA containing the base sequence represented by any of SEQ ID NOs: 18-22.
配列番号 18〜22のいずれかで表される塩基配列とストリンジェントな条件でハイブリ ダイズし、かつ α -1,6-フコシルトランスフェラーゼ活性が欠失、または、配列番号 7ま たは 9で表されるアミノ酸配列力らなる α -1,6-フコシルトランスフェラーゼの α _1,6_フ コシルトランスフェラーゼ活性よりも低下した α -1,6-フコシルトランスフェラーゼ活性 を有するひ -1,6-フコシルトランスフェラーゼ変異体をコードする DNA。 It hybridizes with the nucleotide sequence represented by any of SEQ ID NOs: 18 to 22 under stringent conditions and lacks α-1,6-fucosyltransferase activity, or is represented by SEQ ID NO: 7 or 9. A -1,6-fucosyltransferase mutant having an α-1,6-fucosyltransferase activity that is lower than the α_1,6_fucosyltransferase activity of α-1,6-fucosyltransferase DNA encoding.
請求項 1〜7のいずれか一項に記載の α -1,6-フコシルトランスフェラーゼ変異体また は、請求項 8〜10のいずれか一項に記載の DNAがコードする α -1,6-フコシルトラン スフエラーゼ変異体を発現する細胞。 The α-1,6-fucosyltransferase mutant according to any one of claims 1 to 7, or the α-1,6-fucosyl encoded by the DNA according to any one of claims 8 to 10. A cell that expresses a transphrase mutant.
請求項 8〜： 10のいずれか一項に記載の DNAを導入した細胞。 A cell into which the DNA according to any one of claims 8 to 10 is introduced.
請求項 1〜7のいずれか一項に記載のひ -1,6 -フコシルトランスフェラーゼ変異体また は、請求項 8〜10のいずれか一項に記載の DNAがコードするひ -1,6-フコシルトラン スフエラーゼ変異体のひ -1,6 -フコシルトランスフェラーゼ活性のみ有する請求項 11 または 12記載の細胞。 A -1,6-fucosyltransferase mutant according to any one of claims 1 to 7, or a -1,6-fucosyl encoded by the DNA according to any one of claims 8 to 10. 13. The cell according to claim 11 or 12, which has only the -1,6-fucosyltransferase activity of a transphrease mutant.
糖蛋白質をコードする遺伝子を導入した請求項 11〜： 13のいずれか一項に記載の細 胞。 14. The cell according to any one of claims 11 to 13, wherein a gene encoding a glycoprotein is introduced.
糖蛋白質が抗体である、請求項 14に記載の細胞。 15. The cell according to claim 14, wherein the glycoprotein is an antibody.
[16] 抗体が、以下の (a)、（b)、（ 、（d)及び (e)からなる群から選ばれる抗体である、請求項 1 5に記載の細胞。 [16] The cell according to claim 15, wherein the antibody is an antibody selected from the group consisting of the following (a), (b), (, (d) and (e).
(a)ヒト抗体；  (a) a human antibody;
(b)キメラ抗体；  (b) a chimeric antibody;
(c)ヒト化抗体；  (c) a humanized antibody;
(d) (a)または (b)の Fc領域を含む抗体断片；  (d) an antibody fragment comprising the Fc region of (a) or (b);
(e) (a)または (b)の Fc領域を有する融合蛋白質。  (e) A fusion protein having the Fc region of (a) or (b).
[17] 請求項 14〜： 16のいずれか一項に記載の細胞を培地に培養し、該培養物から糖蛋 白質分子からなる糖蛋白質組成物を採取し、精製する工程を特徴とする糖蛋白質組 成物の製造方法。  [17] A saccharide characterized by culturing the cell according to any one of claims 14 to 16 in a medium, collecting a glycoprotein composition comprising glycoprotein molecules from the culture, and purifying the composition. A method for producing a protein composition.
[18] 糖蛋白質が抗体である、請求項 17に記載の製造方法。 18. The production method according to claim 17, wherein the glycoprotein is an antibody.
[19] 請求項 17に記載の方法を用いて製造される糖蛋白質組成物。 [19] A glycoprotein composition produced using the method according to claim 17.
[20] 請求項 18に記載の方法を用いて製造される抗体組成物。 [20] An antibody composition produced using the method according to claim 18.
[21] 請求項 19に記載の糖蛋白質組成物を有効成分として含有する医薬。 [21] A medicament comprising the glycoprotein composition according to claim 19 as an active ingredient.
[22] 請求項 20に記載の抗体組成物を有効成分として含有する医薬。 [22] A medicament comprising the antibody composition according to claim 20 as an active ingredient.
[23] ヒト組織より細胞を分離し、分離した細胞よりゲノム、全 RNAまたは mRNAを取得し、 取得したゲノム、全 RNAまたは mRNAから、あるいは取得した全 RNAまたは mRN Aを用いて調整した cDNAから α -1,6-フコシルトランスフェラーゼの遺伝子を単離し 、単離した遺伝子の塩基配列において、配列番号 7で表されるアミノ酸配列の Ν末端 力 171番目のアミノ酸配列に相応する位置のアミノ酸がァスパラギンに置換されて レ、るか否かを検出することを特徴とする α -1,6-フコシルトランスフェラーゼが関与す る疾患の診断方法。 [23] Cells are isolated from human tissue, and genomic, total RNA, or mRNA is obtained from the separated cells. From the obtained genomic, total RNA, or mRNA, or from cDNA prepared using the obtained total RNA or mRNA. The α-1,6-fucosyltransferase gene was isolated, and in the base sequence of the isolated gene, the amino acid at the position corresponding to the 171st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 7 was changed to asparagine. A method for diagnosing a disease involving α-1,6-fucosyltransferase, which comprises detecting whether or not the substance is substituted.
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