WO2007099988A1 - mutant de α-1,6-FUCOSYLTRANSFERASE et son utilisation - Google Patents

mutant de α-1,6-FUCOSYLTRANSFERASE et son utilisation Download PDF

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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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fucosyltransferase
antibody
seq
amino acid
dna
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PCT/JP2007/053733
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Japanese (ja)
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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/fr

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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

Abstract

L'invention concerne un mutant d'une enzyme participant à la modification d'une chaîne glucidique telle qu'une liaison α se forme entre la position 6 de N-acétylglycosamine à la terminaison de réduction d'une chaîne glucidique composite à liaison N-glycoside et la position 1 du fucose, ledit mutant présentant une modification de l'acide aminé telle que l'activité enzymatique est supprimée ou réduite. L'invention concerne également l'emploi dudit mutant.
PCT/JP2007/053733 2006-02-28 2007-02-28 mutant de α-1,6-FUCOSYLTRANSFERASE et son utilisation WO2007099988A1 (fr)

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JP2009190993A (ja) * 2008-02-13 2009-08-27 Kitasato Institute シアル酸、シアル酸含有糖質、乃至シアル酸含有複合糖質の蛍光標識化方法、及び、前記方法により得られた蛍光標識化されたシアル酸、シアル酸含有糖質、乃至シアル酸含有複合糖質
WO2009122667A1 (fr) * 2008-04-04 2009-10-08 中外製薬株式会社 Thérapie pour cancer hépatique
WO2012175874A1 (fr) 2011-06-22 2012-12-27 Lfb Biotechnologies Utilisation d'un anticorps anti-cd20 a haute adcc pour le traitement de la maladie de waldenstrom
US8497355B2 (en) 2007-09-28 2013-07-30 Chugai Seiyaku Kabushiki Kaisha Anti-glypican-3 antibody having improved kinetics in plasma
WO2014096672A1 (fr) 2012-12-17 2014-06-26 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Utilisation d'anticorps monoclonaux pour le traitement de l'inflammation et d'infections bacteriennes
WO2015107307A1 (fr) 2014-01-17 2015-07-23 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Immunoglobuline anti-toxine du charbon
WO2017006052A2 (fr) 2015-07-06 2017-01-12 Laboratoire Francais Du Fractionnement Et Des Biotechnologies UTILISATION DE FRAGMENTS Fc MODIFIÉS EN IMMUNOTHÉRAPIE
US9975966B2 (en) 2014-09-26 2018-05-22 Chugai Seiyaku Kabushiki Kaisha Cytotoxicity-inducing theraputic agent
US10118959B2 (en) 2005-10-14 2018-11-06 Chugai Seiyaku Kabushiki Kaisha Anti-glypican-3 antibody

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WO2005035563A1 (fr) * 2003-10-09 2005-04-21 Kyowa Hakko Kogyo Co., Ltd. Procede de production d'une composition d'antithrombine iii
WO2005035778A1 (fr) * 2003-10-09 2005-04-21 Kyowa Hakko Kogyo Co., Ltd. Procede permettant de produire une composition d'anticorps par inhibition par l'arn de la fonction de $g(a)1,6-fucosyltransferase

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WO2003085107A1 (fr) * 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Cellules à génome modifié
WO2005035563A1 (fr) * 2003-10-09 2005-04-21 Kyowa Hakko Kogyo Co., Ltd. Procede de production d'une composition d'antithrombine iii
WO2005035778A1 (fr) * 2003-10-09 2005-04-21 Kyowa Hakko Kogyo Co., Ltd. Procede permettant de produire une composition d'anticorps par inhibition par l'arn de la fonction de $g(a)1,6-fucosyltransferase

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10118959B2 (en) 2005-10-14 2018-11-06 Chugai Seiyaku Kabushiki Kaisha Anti-glypican-3 antibody
US8497355B2 (en) 2007-09-28 2013-07-30 Chugai Seiyaku Kabushiki Kaisha Anti-glypican-3 antibody having improved kinetics in plasma
JP2009190993A (ja) * 2008-02-13 2009-08-27 Kitasato Institute シアル酸、シアル酸含有糖質、乃至シアル酸含有複合糖質の蛍光標識化方法、及び、前記方法により得られた蛍光標識化されたシアル酸、シアル酸含有糖質、乃至シアル酸含有複合糖質
WO2009122667A1 (fr) * 2008-04-04 2009-10-08 中外製薬株式会社 Thérapie pour cancer hépatique
JP2011068682A (ja) * 2008-04-04 2011-04-07 Chugai Pharmaceut Co Ltd 肝癌治療剤
CN102046200A (zh) * 2008-04-04 2011-05-04 中外制药株式会社 肝癌治疗剂
WO2012175874A1 (fr) 2011-06-22 2012-12-27 Lfb Biotechnologies Utilisation d'un anticorps anti-cd20 a haute adcc pour le traitement de la maladie de waldenstrom
WO2014096672A1 (fr) 2012-12-17 2014-06-26 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Utilisation d'anticorps monoclonaux pour le traitement de l'inflammation et d'infections bacteriennes
EP3514175A1 (fr) 2012-12-17 2019-07-24 Laboratoire Français du Fractionnement et des Biotechnologies Utilisation d'anticorps monoclonaux pour le traitement de l'inflammation et d'infections bacteriennes
WO2015107307A1 (fr) 2014-01-17 2015-07-23 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Immunoglobuline anti-toxine du charbon
US9975966B2 (en) 2014-09-26 2018-05-22 Chugai Seiyaku Kabushiki Kaisha Cytotoxicity-inducing theraputic agent
US11001643B2 (en) 2014-09-26 2021-05-11 Chugai Seiyaku Kabushiki Kaisha Cytotoxicity-inducing therapeutic agent
WO2017006052A2 (fr) 2015-07-06 2017-01-12 Laboratoire Francais Du Fractionnement Et Des Biotechnologies UTILISATION DE FRAGMENTS Fc MODIFIÉS EN IMMUNOTHÉRAPIE

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