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

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

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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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antibody
seq id
fucosyltransferase
amino acid
dna
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PCT/JP2007/053733
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French (fr)
Japanese (ja)
Inventor
Naoyuki Taniguchi
Eiji Miyoshi
Jianguo Gu
Naoko Ohnuki
Harue Nishiya
Ryosuke Nakano
Mitsuo Satoh
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Kyowa Hakko Kogyo Co., Ltd.
Osaka University
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Priority to JP2006-052077 priority
Application filed by Kyowa Hakko Kogyo Co., Ltd., Osaka University filed Critical Kyowa Hakko Kogyo Co., Ltd.
Publication of WO2007099988A1 publication Critical patent/WO2007099988A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10Transferases (2.)
    • C12N9/1048Glycosyltransferases (2.4)
    • C12N9/1051Hexosyltransferases (2.4.1)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/26Preparation of nitrogen-containing carbohydrates

Abstract

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

Description

Specification

α -1,6- off Koshino-les-transferase variant and its applications

Technical field

[0001] The present invention, arsenic 1,6 in fucosyltransferase, Fei the enzyme Motonohi 1,6-fucosyl transferase activity are deleted or has decreased, 1,6 - fucosyl transferase peptidase mutants, and on its use.

BACKGROUND

In recent years, there has been a growing interest in the structure and function of the carbohydrate portion of the glycoconjugate contained in glycoproteins etc. from higher organisms, the research has been actively conducted.

Sugar chain of a glycoprotein by binding style with the protein moiety, 2 carbohydrate chain (0-glycosyl binding Gotokusari) that bind to sugar chains (Nyu- glycoside-linked sugar chains) that bind Asuparagin serine, threonine and It is roughly classified into types. Ν- glycoside-linked sugar chains, have a common co § structure underlying shown in but have various structures (see Non-Patent Document 1), also the following structural formulas each case (I) known is, Ru.

[Formula 1 U

Man 1 → 6

Man β l → 4GlcNAc β l → 4GlcNAc (I)

Man monument 1 → 3

[0003] Slight structural formula (I) Te, the end of the sugar chain which binds to Asuparagin called reducing end, or the opposite side is called a non-reducing end. As N- glycoside-linked sugar chains, high mannose type only mannose to the non-reducing end of the core structure binding or galactose to the non - reducing terminal side of the core structure - N- § cetyl Dano Reco Sa Min (hereinafter, the Ga preparative GlcNAc have one or a plurality of parallel branches notation for), further sialic acid to the non-reducing terminal side of the Ga DOO GlcNAc, complex type having a structure such as N- § cetyl Darco Sa Min bisecting, was or, it is known that the non-reducing end side of the core structure has a hybrid type with branches of both of the high mannose type and complex type.

[0004] N-glycoside-linked sugar chain glycoprotein with is present as heterologous glycoprotein core protein consisting of molecules with a diversity Tokusari構 granulated be identical. Further, the Tokusari構 concrete is thought to be controlled by a gene decomposing sugar degrading enzyme glycosyltransferase and a sugar chain synthesizing sugar chains.

Sugar chain of a glycoprotein, including N- glycoside-linked sugar chains, the three-dimensional structure of the protein portion

, Transport, in addition to affecting the stability and clearance, the function of the protein itself, cell-cell cells interactions, by affecting the interaction with extracellular matrix, development and immune response, such as the generation process of cancer that exhibit their important physiological role is becoming apparent (see non-patent document 2-4). Also, human congenital genetic diseases relating to sugar abnormality; Analysis force et al (CDG congenital disorders of glycosylation), the causative gene the N - it is reported a gene required for the synthesis of glycoside binding sugar chain are (see non-Patent Document 5, 6). That is, in a transporter of sugar nucleotide GDP- fucose GDP- fucose transporter one is missing Les, Ru CDG type II disease patients (OMIM § click Session Number 266 265), a sugar donor GDP- fucose cytoplasm of from for transport to the Golgi apparatus is not performed, there is almost no modification of fucose to complex carbohydrates sugar chain, mental disorders and growth delay, and, symptoms such as impaired immune function has been observed (see non-patent Document 7, 8). Under such circumstances, an enzyme involved in sugar chain modification in which 1-position of fucose is "bound to the 6-position of the N- glycoside-linked complex type sugar chain of N- § cetyl Darco Sa Min, many glycosylation enzymes Unlike, only been found a single gene in vivo so far, came to its functionality is very attention.

Enzymes 1-position of fucose to 6-position of the N- Darikoshido binding complex type sugar chain of N- § cetyl Darco Sa Min is involved in sugar chain modification to bind alpha, in animals, alpha-1,6-Fukoshinore transferase have been found (see non-Patent Document 9). Facial - 1,6 fucosyl structure sill transflector Eraze gene (EC 2.4.1,68) has been elucidated in 1996 (Non-patent document 10 11, see Patent Document 1). Facial 1,6 - enzymatic activity of fucosyltransferase has been confirmed in a number of organs, but are (see Non-Patent Document 12 13) which is reported to be relatively high enzymatic activity in inter alia brain and intestine. In the retina formed and it has been pointed out that fucose modified sugar chain plays an important physiological functions, shed 1,6 - fucosyl transflector Eraze les expression control is attention, Ru (Non-Patent Document 14 reference ). Slight blood clotting, the role of the alpha-1,6-fucosyltransferase of the platelet-derived even have attracted attention (see Non-Patent Document 15). Also, the immunoglobulin modification of fucose relative to the sugar chain structure of IgGl affects the binding of the Fc y RI Ila, the antibody-dependent cellular cytotoxicity of the antibody itself changes have also been reported (Non-Patent Document 16, 17 reference). Regarding the involvement in the pathology, in some diseases, such as liver cancer Ya sac cystic fibrosis, shed - 1,6-fucosyl transferase activity was increased, the proportion of Rukoto and enzymatic reaction product was increased Les, Rukoto is observed Les, Runode, (see non-Patent Document 18, 19) associated is assumed between these diseases and enzyme. Facial - 1,6 fucosyl trans diethyl Nick mice overexpressing fucosyltransferase is also produced, fabricated trans di We nick mouse liver and kidney Niore, degeneration of steatosis like Te is observation seen (see non-Patent Document 20). The non - 1,6-fucosyltransferase knockout mice produced Nirre, be reported les, Ru (see Patent Document 2).

Thus, shed - 1,6 fucosyltransferase which various analyzes are made as to transferase, various important physiological role in vivo of the enzyme as described above are estimated. However, there is no specific report on the amino acid variants of the α -1,6- fucosyltransferase. Cells expressing the alpha-1,6-fucosyltransferase variants, Nyu- Dali Koshido binding complex-type oligosaccharide 1-position of fucose to 6-position of Nyu- § cetyl Darco Sa Min reducing terminal sugar chain modification to bind alpha due to be less, it is assumed that produces glycoproteins bioactivity has changed. Further, assume that the direct or indirect variety of pathological symptoms develop in tissues where activity of the enzyme expressing deletion or decreased to have alpha _ 1,6-fucosyltransferase variants, It is. Relations between the true physiological roles and pathology of this enzyme is elucidated, it is clear that further usage in industry of alpha-1,6-fucosyltransferase variants les, Kukoto is expected.

Patent Document l: WO92 / 27303

Patent Document 2: US2005-0160485

Non-Patent Document 1: Reiko Takahashi, ed., "Biochemical Experimental Methods 23-glycoprotein sugar chain research methods", Society of publishing Center, 1989, p.1-4

Non-Patent Document 2: (... Curr Opin Immunol) Current 'Opinyon' in 'Imunoroji, 3, 646, 1991 Non-Patent Document 3: Glycobiology (Glycobiology), 3, 97, 1993

Non-Patent Document 4: (... Biochem Soc Trans) Biochemical 'Sosaiati' transactions, 23, 1, 1995

Non-Patent Document 5: Glycobiology (Glycobiology), 3, 423, 1993

Non-Patent Document 6: tio sip Bien 'Journal' O Bed 'Bae Deer Trick' Neurology (Eur.

J Paediatr Neurol.), 1, 61, 1997

Non-Patent Document 7: (.. Nat Genet) Neichiya one 'Jiweneteikusu, 28, 73, 2001

Non-Patent Document 8: (.. Nat Genet) Neichiya one 'Jiweneteikusu, 28, 69, 2001

Non-Patent Document 9: (.... Biochem Biophys Res Commun) Biochemical And Biophysical Research Communications's, 72, 909, 197 Ri

Non-Patent Document 10:... Journal 'O Bed' Baiorojigaru 'Chemistry CJ Biol Chem), 271, 27817, 1996

Non-Patent Document 11: (. J. Biochem) Journal 'O Bed' Biochemistry, 121, 626, 1997 Non-Patent Document 12: inter one National journal O blanking Cancer (International Jour nal of Cancer), 72, 1117 , 1997

Non-Patent Document 13: (... Biochim Biophys Acta) Baiokimi force 'E' Baiofuiji force 'Akuto, 14 73, 9, 1999

Non-Patent Document 14: Glycobiology (Glycobiology), 9, 1171, 1999

Non-Patent Document 15:.. Biochemical 'Sosaiati' transaction (Biochem Soc Trans

.), 15, 603, 1987

Non-Patent Document 16: (.. J. Biol Chem) Journal 'O Bed' Baiorojigaru 'Chemistry, 277, 26733, 2002)

Non-Patent Document 17:... The journal 'O Breakfast' Baiorojigaru 'chemistry CJ Biol Chem), 278, 3466, 2003

Non-Patent Document 18: to Patoroji (H mark atology), 13, 683, 1991)

Non-Patent Document 19: to Patoroji (H mark atology), 28, 944, 1998

Non-Patent Document 20: Glycobiology (Glycobiology), 11, 165, 2001

DISCLOSURE OF INVENTION you'll solve

[0007] The present invention, arsenic -1, 6-in-fucosyltransferase, enzyme Motonohi -1, Fei -1 6-fucosyl transferase activity are deleted or has decreased, 6 - fucosyl transferase peptidase mutant enzymes and and to provide a method of use.

Fei -1, 6-fucosyltransferase variants of the present invention are useful in elucidating the diagnosis of involvement with physiological role and conditions of the non-1,6-fucose modifying enzyme. The ratio - 1,6-fucose modifying enzyme development and sugar chain structure of drugs that target is for a Yes to the development of important sugar protein pharmaceutical.

Means for Solving the Problems

[0008] The present invention relates to the following (1) to (23).

(1) Fei - 1,6 in the amino acid sequence of fucosyltransferase, positions corresponding to the 171 th amino acid from the N-terminal amino acid sequence you express in SEQ ID NO: 7 amino acids or are deleted, or other than serine having the amino acid sequence are substituted with amino acids, shed - 1,6 full waist transferase variants.

(2) an amino acid other than serine is Asuparagin, Fei -1 according to the above (1), 6 - Fukoshiruto lance Feller peptidase mutants.

(3) Fei - 1,6-fucosyltransferase mosquito following (a) ~ the a protein of DN A code selected from the group consisting of (f) (1) or (2) Fei described -1, 6-fucosyl transformer Hue hydrolase mutant.

(A) comprising the nucleotide sequence represented by SEQ ID NO: 1 DNA;

(B) comprising the nucleotide sequence represented by SEQ ID NO: 2 DNA;

(C) comprising the nucleotide sequence represented by SEQ ID NO: 3 DNA;

(D) comprising the nucleotide sequence represented by SEQ ID NO: 4 DNA;

(E) comprising the nucleotide sequence represented by SEQ ID NO: 5 DNA;

(F) DNA comprising the nucleotide sequence represented by SEQ ID NO: 6.

(4) alpha -1, 6- fucosyltransferase mosquitoes S, a 蛋 white matter selected from the group consisting of the following (a) ~ (f), alpha -1 according to the above (1) or (2), 6 - fucosyltransferase variant (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 7;

(B) a protein comprising the amino acid sequence represented by SEQ ID NO: 8;

(C) a protein consisting of the amino acid sequence of SEQ ID NO: 9;

(D) a protein consisting of the amino acid sequence of SEQ ID NO: 10;

(E) a protein comprising the amino acid sequence represented by SEQ ID NO: 11;

(F) a protein comprising the amino acid sequence represented by SEQ ID NO: 12.

(5) SEQ ID NO 13: 17 Fukumuhi the amino acid sequence shown in any of - 1,6 Fukoshirutora Nsufuweraze variants.

(6) SEQ ID NO 13: In the amino acid sequence shown in any of 17, made of one or more amino acids are deleted, substituted, 揷入 and / or added in the amino acid sequence, Katsuhi - 1,6 full cosyl transferase activity are deleted, or SEQ ID NO: 7 or 9 lower than fly-1,6-fucosyltransferase activity in carry-1,6-fucosyltransferase consists of an amino acid sequence represented by by Tahi -1, having a 6-fucosyltransferase activity alpha-1,6-fucosyl transferase variants.

(7) SEQ ID NO 13: 17 consists either in amino acid sequence represented by 80% or more homology to have the amino acid sequence of, and alpha-1,6-fucosyltransferase activity are deleted, or, alpha with reduced alpha-1,6-fucosyl trans luciferase activity than SEQ ID NO: 7 or comprising the amino acid sequence represented by the 9 α _1,6- fucosyl trans Blow over peptidase alpha-1,6-fucosyltransferase activity 1,6-fucosyltransferase variant.

(8) above (1) to (7) Les, DNA encoding the alpha-1,6-fucosyltransferase variant according to the deviation or claim.

(9) DNA comprising the nucleotide sequence shown in any of SEQ ID NO: 18-22.

(10) and c Iburidizu the nucleotide sequence under stringent conditions represented by any of SEQ ID NO: 18-22, Katsuhi - 1,6 - fucosyltransferase activity are deleted, or in SEQ ID NO 7 or 9 Fei Fei 1,6-fucosyltransferase consists of an amino acid Hai歹 1J represented - 1,6 fucosyltransferase lower than glycosyltransferase activity Fei having Tahi 1,6 fucosyltransferase activity 1,6 - fucosyltransferase DNA encoding the variant.

(11) above (1) Les to (7), displacement or flight according to one Section 1,6 - fucosyltransferase variant variant or above (8) to (10 Les, according to the deviation or claim cells expressing alpha _1,6_ full cosyl transferases mutant DNA encodes.

(12) (8) - (10) Les, deviation or cells transfected with DNA according to one paragraph.

(13) above (1) Les to (7), Fei according to the deviation or claim 1,6 - fucosyltransferase variant variant or, wherein Re, in shift one of the above (7) to (9) DNA of that Yusuke only carry-1,6-fucosyltransferase activity of the encoded Suruhi 1,6 full waist transferase variants above (11) or (12), wherein the cell.

(14) The cell according to Les, shift one of the above in which a gene encoding the glycoprotein from (11) (13).

(15) glycoprotein is an antibody, the cell according to (14).

(16) antibodies, below), (b), (c), (d) and (an antibody selected from the group consisting of e), cells described above SL (15).

(A) human antibodies;

(B) chimeric antibody;

(C) a humanized antibody;

(D) (a) or antibody fragment comprising the Fc region of (b);

(E) (a) or a fusion protein comprising the Fc region of (b).

(17) Re above (14) to (16), the step of culturing in a medium a cell according to the deviation or claim, the glycoprotein composition comprising the culture or al glycoprotein molecule harvested and purified method for producing To蛋 white matter composition characterized.

(18) is a glycoprotein antibody, production method according to (17).

(19) The glycoprotein composition produced using the method described in (17).

(20) an antibody composition produced using the method described in the above (18).

(21) A medicament comprising as an active ingredient the glycoprotein composition according to (19).

(22) A medicament comprising as an active ingredient an antibody composition according to the above (20).

(23) cells were separated from human tissues, genomic than separated cells Tokushi collected total RNA or mRNA, from the obtained genomic, total RNA or mRNA, or were prepared using the total RNA or mRNA obtained cDNA Facial - 1,6 - fucosyltransferase isolated genes glycosyltransferases single, in the nucleotide sequence of the isolated gene, the amino acid positions corresponding from the N terminus of the amino acid sequence represented by SEQ ID NO: 7 to 171 amino acid sequence in Asuparagin diagnostic methods Azukasuru alpha _1,6- fucosyltransferase and detects whether it is substituted Seki disease.

Effect of the invention

[0009] The present invention, arsenic - 1,6 In fucosyltransferase enzyme, enzyme Motonohi - 1, 6-fucosyltransferase activity are deleted or reduced alpha -1, 6-fucosyltransferase trans Hue hydrolase variants and its use is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] [FIG. L] CHO / DG44 cells and RN6 strain Fei -1 is a diagram showing the staining by LCA is 6-fucose specific lectins. The horizontal axis logarithmic fluorescence intensity (log), shows the distribution of cell number on the vertical axis. Open box results of cells were stained by FITC-labeled streptavidin, black unplug shows the results of cells was subjected to staining with FITC-labeled LCA.

Is a diagram illustrating the FIG. 2] RN6 strain results monosaccharide composition analysis of the production antibodies of. Elution time on the horizontal axis (min), relative intensity in the vertical axis.

3 is a diagram showing the construction of a plasmid CHFUT8Comp23.

4 is a diagram showing the construction of a plasmid CHFUT8CompTA.

5 is a diagram showing the construction of a plasmid PcDNAchFUT8Comp.

Is a diagram showing the construction of FIG. 6 plasmid CHFUT8Mo3.

7 is a diagram showing the construction of a plasmid CHFUT8MoTA.

8 is a diagram showing the construction of a plasmid PcDNAchFUT8Mo.

Is a diagram showing the LCA stainability of FIG 9] FUT8 expression strain and FUT8- base substitutions expression strain. The horizontal axis logarithmic fluorescence intensity (log), shows the distribution of cell number on the vertical axis. White is the result of the cells were stained by FITC-labeled stress but-avidin, black unplug shows the results of the cells was carried out by that stained FITC-labeled LCA.

BEST MODE FOR CARRYING OUT THE INVENTION

[0011] In the present invention, arsenic -1, 6-fucosyltransferase The fucosyltransferase, N- glycoside-linked complex type sugar reducing end of N - 6-position and 1-position of fucose § cetyl Darco Sa Min is involved in the reaction to a binding if the enzyme that Les, canal enzyme are also included. Specifically, the following (a), (b), (,, (d), (e) or (protein encoded by a DNA, such as f), or the following (g), (h), (i) ( j), and the like are proteins such as (k) or (1).

(A) comprising the nucleotide sequence represented by SEQ ID NO: 1 DNA;

(B) comprising the nucleotide sequence represented by SEQ ID NO: 2 DNA;

(C) comprising the nucleotide sequence represented by SEQ ID NO: 3 DNA;

(D) comprising the nucleotide sequence represented by SEQ ID NO: 4 DNA;

(E) comprising the nucleotide sequence represented by SEQ ID NO: 5 DNA;

(F) comprising the nucleotide sequence represented by SEQ ID NO: 6 DNA;

Or,

(G) a protein comprising the amino acid sequence represented by SEQ ID NO: 7;

(H) a protein comprising the amino acid sequence represented by SEQ ID NO: 8;

(A protein comprising the amino acid sequence represented by D SEQ ID NO: 9;

(J) a protein comprising the amino acid sequence represented by SEQ ID NO: 10;

(K) a protein comprising the amino acid sequence represented by SEQ ID NO: 11;

(1) a protein comprising the amino acid sequence shown in SEQ ID NO: 12.

[0012] of the present invention, alpha -1, 6- In fucosyltransferase, positions corresponding to the 171 th amino acid from the Ν-terminus of amino acid sequence represented by SEQ ID NO: 7 occurring amino acids, alpha-1,6 - the calculated, two amino acid sequences using the homology analysis program and parameters one coater such as BLAST and FASTA described below homology with the amino acid sequence represented by amino acid sequence SEQ ID NO: 7 having the full stiffness transferase when it was aligned alpha -1, 6- fucosyltransferase trans luciferase mutants on the amino acid sequence in having the refers to the amino acid in the position corresponding the Ν-terminus of the amino acid sequence represented by SEQ ID NO: 7 to 171 amino acid.

[0013] Fei -1 of the present invention, the 6-fucosyltransferase variants, non-1,6-fucosyltransferase in the amino acid sequence of sill transflector Eraze, the amino acid sequence represented by SEQ ID NO: 7 New terminal 1 71 th amino acid at the position corresponding to amino acid has an amino acid sequence are substituted with amino acids other than the force \ or serine lacking, but may be any a-1,6-fucosyltransferase variants, for example, the following ( i), (ii), (iii), (iv), (v), (vi), (vii), (viii), (ix), (x), (xi), (xii), (xiii) , (xiv) or (xv) protein, such as, or the following (xvi), (xvii), (xviii), (xix), (xx), (xxi), (xxii), (xxiii), (xxiv) or (xxv) protein there are up to DNA, such as to code.

(Protein consisting of the amino acid sequence of 0 SEQ ID NO: 13;

(Ii) a protein comprising the amino acid sequence represented by SEQ ID NO: 14;

(Iii) a protein comprising the amino acid sequence represented by SEQ ID NO: 15;

(Iv) a protein comprising the amino acid sequence represented by SEQ ID NO: 16;

(V) a protein consisting of the amino acid sequence of SEQ ID NO: 17;

(Vi) the amino acid sequence represented by SEQ ID NO: 13, one or more amino acids are deleted, substituted, 揷 input and / or added in the amino acid sequence, Katsuhi - 1,6 - fucosyl trans Hue hydrolase activity deletions, or the flight consisting of the amino acid sequence represented by SEQ ID NO: 7 or 9 - 1, 6-fucosyltransferase Fei _1,6 - the reduced alpha-1,6-fucosyltransferase activity than fucosyltransferase activity with alpha-1,6-fucosyl transferase peptidase variants;

(Vii) the amino acid sequence represented by SEQ ID NO: 14, one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence, and alpha-1,6-fucosyltransferase transflector Eraze activity deletion or, alpha has a reduced alpha-1,6-fucosyltransferase activity than alpha-1,6-fucosyltransferase activity consisting of the amino acid sequence represented by SEQ ID NO: 7 or 9 alpha-1,6-fucosyltransferase 1,6 fucosyl transferase peptidase variants;

(Viii) an amino acid sequence represented by SEQ ID NO: 15, one or more amino acids are deleted, substituted, 揷入 and / or added in the amino acid sequence, Katsuhi - 1,6 - fucosyl transflector Eraze activity deleted loss, or, Fei 1,6 Fei-1,6-fucosyltransferase consists of an amino acid sequence represented by SEQ ID NO: 7 or 9 - having lower than fucosyltransferase activity Shitahi 1,6-fucosyltransferase activity Facial 1,6 fucosyl transferase peptidase variants;

(Ix) an amino acid sequence represented by SEQ ID NO: 16, one or more amino acids are deleted, substituted, 揷 input and / or added in the amino acid sequence, Katsuhi - 1,6 - fucosyl trans Hue hydrolase activity deletions, or consists of the amino acid sequence represented by SEQ ID NO: 7 or 9 alpha -1, 6- fucosyltransferase alpha-1,6-fucosyltransferase reduced alpha-1,6-fucosyltransferase activity than alpha-1,6-fucosyl transferase peptidase variants with;

(X) in the amino acid sequence represented by SEQ ID NO: 17, one or more amino acids are deleted, substituted, 揷 input and / or added in the amino acid sequence, Katsuhi - 1,6 - fucosyl trans Hue hydrolase activity deletions, or the flight consisting of the amino acid sequence represented by SEQ ID NO: 7 or 9 - 1, Fei 6- fucosyltransferase _1,6 - reduced Tahi 1,6-fucosyltransferase activity than fucosyltransferase activity Fei-1,6 fucosyl transferase peptidase variants with;

(Xi) consisting of an amino acid sequence having an amino acid sequence homology of 80% or more of SEQ ID NO: 13, Katsuhi - 1,6-fucosyltransferase activity are deleted or, SEQ ID NO: 7 or at 9, Fei having made amino acid sequence forces represented alpha-1,6-fucosyltransferase of alpha -1,6-7 cosyl transferases reduced alpha-1,6-fucosyltransferase activity than - 1,6 fucosyltransferase Mutant;

(Xii) consists of an amino acid sequence having an amino acid sequence homology of 80% or more represented by SEQ ID NO: 14, and alpha-1,6-fucosyltransferase activity are deleted or, SEQ ID NO: 7 or at 9, Fei having made amino acid sequence forces represented alpha-1,6-fucosyltransferase of alpha -1,6-7 cosyl transferases reduced alpha-1,6-fucosyltransferase activity than - 1,6 fucosyltransferase Mutant;

(Xiii) a protein consisting of an amino acid sequence having an amino acid sequence homology of 80% or more of SEQ ID NO: 15, Katsuhi - 1,6-fucosyltransferase activity are deleted or, SEQ ID NO: 7 or at 9, amino acid sequence force represented, Fei has reduced Tahi 1,6-fucosyltransferase activity than flight 1,6 full waist transferase activity et consisting Fei-1,6-fucosyltransferase - 1,6 - fucosyltransferase variant;

(Xiv) a protein consisting of an amino acid sequence having an amino acid sequence homology of 80% or more of SEQ ID NO: 16, Katsuhi - 1,6-fucosyltransferase activity are deleted or, SEQ ID NO: 7 or at 9, Fei having the amino acid sequence represented by force, et consisting Fei-1,6-fucosyltransferase of flight 1,6 full cosyl transferases reduced alpha-1,6-fucosyltransferase activity than - 1,6 fucosyltransferase variant;

(XV) consisting of an amino acid sequence having an amino acid sequence homology of 80% or more of SEQ ID NO: 17, Katsuhi - 1,6-fucosyltransferase activity are deleted or, SEQ ID NO: 7 or at 9, amino acid sequence force represented, Fei has reduced Tahi 1,6-fucosyltransferase activity than flight 1,6 full waist transferase activity et consisting Fei-1,6-fucosyltransferase - 1,6 - fucosyltransferase variant;

Or,

(Xvi) comprising the nucleotide sequence represented by SEQ ID NO: 18 DNA;

DNA consisting of the nucleotide sequence represented by (xvii) SEQ ID NO: 19;

(Xviii) comprising the nucleotide sequence represented by SEQ ID NO: 20 DNA;

(Xix) comprising the nucleotide sequence represented by SEQ ID NO: 21 DNA;

(XX) comprising the nucleotide sequence represented by SEQ ID NO: 22 DNA;

(Xxi) table 9 High Priestess and soybean, and alpha-1,6-fucosyltransferase activity are deleted, or was SEQ ID NO: 7 or either in the base sequence represented by stringent conditions of SEQ ID NO: 18 Fei 1,6 fucosyltransferase with reduced alpha-1,6-fucosyltransferase activity than alpha -1,6-7 waist transferase activity of the amino acid sequence power et consisting alpha-1,6-fucosyltransferase which is DNA encoding the variant;

(Xxii) table 9 hybridizes with the base sequence under stringent conditions represented by any one, and the alpha-1,6-fucosyltransferase activity deletions, or was SEQ ID NO: 7 or of SEQ ID NO: 19 Fei-1,6 having an amino acid sequence power et consisting alpha 1,6 fucosyltransferase lower than alpha -1,6-7 waist transferase activity of glycosyltransferases and Tahi 1,6-fucosyltransferase activity that is - fucosyltransferase DNA encoding the variant;

(Xxiii) hybridizes with the base sequence under stringent conditions represented by any of SEQ ID NO: 20, the table 9 Katsuhi 1,6-fucosyltransferase activity are deleted, or SEQ ID NO: 7 or amino acid sequence force, flight has reduced Tahi 1,6-fucosyltransferase activity than flight 1,6 full waist transferase activity et consisting Fei-1,6-fucosyltransferase 1,6 - fucosyl DNA encoding the transferase variant; (xxiv) and High Priestess soybean base sequence under stringent conditions represented by any of SEQ ID NO: 21, and alpha-1,6-fucosyltransferase activity are deleted or, the sequence No. 7 or is lower than alpha _1,6_ full waist transferase activity of the amino acid sequence power et consisting alpha-1,6-fucosyltransferase expressed by 9 Tahi 1,6 Fukoshi DNA encoding fucosyltransferase variants - Fei-1,6 having a transferase activity;

(XXV) table 9 and High Priestess soybean base sequence under stringent conditions represented by any one, Katsuhi 1,6-fucosyltransferase activity are deleted, or SEQ ID NO: 7 or of SEQ ID NO: 22 amino acid sequence force, flight has reduced Tahi 1,6-fucosyltransferase activity than flight 1,6 full waist transferase activity et consisting Fei-1,6-fucosyltransferase 1,6 - fucosyl DNA encoding the transferase mutant.

In the present invention, and the DNA Haiburidizu in Sutorinji Engineering cement conditions, for example, SEQ ID NO: 18, 19, 20, 21 or DNA or of a DNA having the nucleotide sequence represented by 22 pieces of part as a probe, means DNA obtained by Rukoto using colony one 'hybrida I See Chillon method, plaque • hybrida I See Chillon method or Southern blot hybrida I See Chillon method, specifically, a colony or using a filter with immobilized D NA from plaque, 0. 7: 1. under NaCl presence of 0M, after hybrida I See Chillon at 65 ° C, 0.:! ~ 2-fold concentration (composition of 1-fold concentration SSC solution, 150 mM sodium chloride, 15 mM Kuen consisting of sodium) SSC solution used, the DNA that can be identified by washing the filter with 65 ° C under Ageruko Door can be. Haiburidaise 1 ~~ Chillon ia>, Molecular cloning, a laooratory manual, Third Edition, Cold Spring Harbor Laboratory Press (2001) ( hereinafter referred to as Molecular ^ ~ Cloning, 3rd edition), Current Protocols in Molecular Biology, John Wiley & Sons, 1987-1997 (hereinafter, abbreviated as current 'Protocols' in 'Molecular' Biology), DNA Clonin g 1: Core Ί ecnmques, a Practical Approach, Second Edition, is described in Oxford University (19 95), etc. it can be carried out in accordance with the method you are. Bruno, specifically as a Iburidizu possible DNA, when calculated using BLAST or FASTA, etc., for example, SEQ ID NO: 18, 19, 20, 21 or 22 are a nucleotide sequence at least 70%, preferably 80% or more, more preferably 90. / O or more, more preferably 95% or more, particularly preferably 98% or more, most preferably be mentioned a DNA having a homology of 99% or more.

[0015] Homology of the amino acid sequence and the base sequence, for example algorithm BLAST by Karlin and Altschul [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] and FASTA [Methods Enzym ol., 183, 63 (1990)] can be determined using. And based on the algorithm BLAST, BLASTN and BLASTX a program called have been developed [J. Mol. Biol., When a nucleotide sequence is analyzed by BLASTN based on 215. 403 (1990)] BLAST is lame one coater is, for example, Score = 100, wordlength = 12. Further, when an amino acid sequence is analyzed by BL ASTX on the basis of BLAST, the parameters one has to score = 50, w ordlength = 3, for example. When BLAST and Gapped BLAST programs are used, default parameters of each program. Specific procedures for these analysis methods are known (http: //www.ncbi.nlm.nih.gov·).

[0016] In the present invention, will for example SEQ ID NO: 13, 14, 15, 16 or one or more amino acids are deleted in the amino acid sequence represented by 17, substituted, inserted and / or added in the amino acid sequence, and the protein having alpha-1,6-fucosyltransferase activity, Moreki Yura scratch. cloning, third Edition, current 'Protocols' in' Molecular ^ ~ Biology, Nucleic Acids Research, 10, 6487 (1982), Proc . Natl. Acad. Sci., USA, 79, 6409 (1 982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. A cad. Sci USA, 82, 488 using site directed mutagenesis as described in (1985) or the like, for example, SEQ ID NO: 13, 14, 15, 16 or DNA to site-specific mutagenesis that encoding a protein having an amino acid sequence represented by 17 It refers to a protein which can be obtained by introducing a. Deletion, substitution, insertion and / or number of the added amino acid is 1 or more but the number is not particularly limited, by known techniques of site-directed mutagenesis method or the like described above, deletion, substitution with or is the number enough to mosquito 卩, for example, 1 to several tens, preferably 1 to 2 0, more preferably 1 to: 10, more preferably from 1 to 5.

[0017] In addition, one or more amino acids are deleted, and substituted or added, in any position of the same sequence, it means that one or deletion of a plurality of amino acids, substitution or addition is, deletion , amino acid substitution or addition is to be Yogu substituted or added may occur simultaneously be natural or non-natural type. The natural amino acids, L- Aranin, L- Asuparagin, L Asuparagin acid, L-arginine, L-glutamine, L-glutamic acid, glycine, L-histidine, L- isoleucine, L bite leucine, L- lysine, L Mechionin, L- Hue two Ruaranin, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L heparin, such as L- cysteine, and the like.

Hereinafter, examples of the amino acids capable of mutual substitution. The amino acids in the same group are mutually substitutable.

Group A: leucine, isoleucine, norleucine, valine, Nonorebarin, Aranin, 2 Aminobu Tan acid, Mechionin, 〇_ methyl serine, t_ butyl glycine, t - Buchiruaranin, cyclohexane Kishinorearanin

Group B: Asuparagin acid, Gunoretamin acid, Isoasuparagin acid, Isogunoretamin acid, 2-Amino adipic acid, 2-Aminosu base phosphoric acid

Group C: Asuparagin, Gunoretamin

Group D: lysine, arginine, Ol two Chin, 2,4 Jiaminobutan acid, 2,3 Jiaminopuropio phosphate

Group E: proline, 3-hydroxyproline, 4-hydroxyproline

Group F: serine, threonine, homoserine

Group G: Hue two Ruaranin, tyrosine

Further, in the present invention, a protein which have the example SEQ ID NO: 13, 14, 15, 16 or has an amino acid sequence homology of 80% or more represented by 17, and alpha-1,6-fucosyltransferase activity , when calculated using analysis software such as BLAST or FASTA, SEQ ID NO: 13, 14, 15, 16 or a protein having an amino acid sequence as set forth in 17 at least 80% or more, preferably 85. / 0 or more, more preferably 90. / O or more, more preferably 95. / 0 or more, especially preferably to 97% or more, as the cells of the present invention, which means that most preferably a protein is at least 99%, expressed Fei-1,6-fucosyltransferase variants of the invention may be any cell as long as it has cells, such as yeast, animal cells, insect cells, plant cells and the like, specific examples of these cells include those described in 2 below . Specific examples of the animal cells, Chinese hamster ovary tissue - derived of CHO cells, rat myeloma cell line YB2 / 3HL.P2.G11.16Ag.20 cells, Mausumie opening Ichima cell line NS0 cell, a mouse myeloma cell line SP2 / 0-Agl4 cells, derived from a Syrian hamster kidney tissue BHK cells, hybridoma cells that produce antibodies, human leukemia cells Kabuna Malva cells, embryonic stem cells, such as fertilized egg cells. Preferably, production used in the production of a glycoprotein such as an antibody, the aforementioned myeloma cells, High Priestess dormer cells, host cells for the production of humanized antibodies or human antibodies, the transformer Jiwenikku non-human animals for producing human antibodies embryonic stem cells or fertilized egg cell is used for, and Yore, such as Ru plant cells, and the like to produce a trans-di We nick plants producing humanized and human antibodies.

The NS0 cells, Bio / Technology (BIO / TECHNOLOGY), 10, 169 (1992), Ba I O Technology 'bioengineering (Biotechnol. Bioeng.), 73, 261, have been described in the literature such as (2001) NS0 cells, and the like. In addition, RIKEN Cell Bank has been registered NS0 cell line (RCB0213), or even such as sub-cell lines were 馴 of these cell lines to various serum-free media, and the like. The SP2 / 0-Agl4 cells, journal O Bed 'Imuno Biology (J. Immunol.), 126, 317, (1981), Neichiya (Nature), 276, 269, (1978), Hiyu Man' Anti warts DIZ 'and /, Iburidomazu (Human Antibodies and Hybridomas),

3, 129, (1992) is SP2 / 0_Agl4 cells are described in documents and the like. In addition, sub-cell lines were SP2 / 0-Agl4 cells that have been registered in the AT CC (ATCC CRL-1581) or these cell lines acclimated to a variety of serum-free medium (ATCC CRL-1581.1), etc. may be mentioned. The Chiyaini hamster ovary tissue-derived CHO cells, Journal of Experimental Medicine, 10 8, 945 (1958), Proc. Natl. Acad. Sci. USA, 60, 1275 (1968), Genetics, 55, 513 (196 8) , Chromosoma, 41, 129 (1973), Methods in Cell Science, 18, 115 (1996), Radiatio n Research, 148, 260 (1997), Proc. Natl. Acad. Sci. USA, 77, 4216 (1980), proc. Na tl. Acad. Sci. 60> 1275 (1968), cell, 6, 121 (1975), Molecular cell Genetics, is CHO cells have been described in the literature such as Appen dix 1,11 (p883_900) like . Also, CHO-Kl strain registered in the ATCC (ATCC CCL-61), DUXB11 strain (ATCC CRL- 9096), Pr 0-5 strain (ATCC CRL- 1781) and the commercially available CHO- S strain (Lifetechnologies Inc. Ltd. Cat No.11619), or such as sub-cell lines obtained by naturalizing these cell lines to various serum-free medium may be mentioned. The Rattomi Eroma cell line YB2 / 3HL.P2.G11.16Ag.20 cells, Y3 / Agl.2.3 cells (ATCC C RL-1631) forces an established cell line are included. As specific examples, J. Cell. Biol., 93, 576 (1982), Methods Enzymol. 73B, 1 (1981) are described in the literature, such as Y B2 / 3HL.P2. Gil.16Ag. 20 cells, and the like. In addition, registered in the ATCC YB2 / 3H and.? Such as 2 11.16 eight 8.20 cells (eight 1 ^ Ji CRL-1662) or sub-cell lines obtained by naturalizing these cell lines to various serum-free medium may be mentioned.

[0020] can be the cell of the present invention by introducing a gene encoding the glycoprotein molecules, for producing a glycoprotein composition comprising a To蛋 white matter molecules using the cells.

Sugar chains in glycoproteins, the binding mode of the glycoprotein moiety, a sugar chain which binds to Asuparagin (N- glycoside-linked sugar chain), sugar binding such as serine or threonine (〇 - glycoside-linked sugar chain) It is roughly classified into two types of.

[0021] N-glycoside-linked sugar chains, in any case has the various structures have a common core structure shown in the above structural formula (I). In the compound of formula (I), terminal reducing end of the sugar chain which binds to Asuparagin, opposite that non-reducing end. The N- glycoside-linked sugar chain, non-reducing end only mannose high mannose type sugar chain which binds galactose to non-reducing end side of the core structure of the core structure - N- § cetyl Darco Sa Min (hereinafter, Ga preparative GlcNac and Table to. branches has one or a plurality of parallel of), further sialic acid to the non-reducing end of the Ga Bok GlcNac, complex type having a structure such as bisecting N- § cetyl Darco Sa Min (both composite referred.) sugar, such as a hybrid type sugar chain with branches of both non-reducing end HMT and complex-type core structure thereof.

[0022] 0 The glycoside-linked sugar chains, N- § cetyl Galata Tosa reducing end of Min is serine or bind shed and threonine hydroxyl groups, further galactose, N- § cetyl Darco Sa Min, N- § Sechirugaratato sugar chain the summing or sialic acid is bound, sugar xylose is bound hydroxyl group and β serine, galactose and hydroxyl groups and β linked sugar chain hydroxy lysine and the like.

[0023] sugar xylose were hydroxyl group and / 3 binding of serine, usually several sugar is attached to the 4-position of the xylose, linear polysaccharide is coupled consisting ahead disaccharide of bound sugar are doing. Cartilage Puroteodarikan thereof include substances having such a sugar chain structure. Galactose as the substance having a hydroxyl group and β bound sugar chain structure of hydroxyapatite lysine, collagen and the like.

[0024] The sugar constituting a sugar chain, Nyu- § cetyl Darco Sa Min, Nyu- § cetyl Galata acetylgalactosamine, mannose, galactose, fucose, sialic acid, xylose, has Arabinosu like included, these sugars Les, good record, be coupled with Canal order.

Accordingly, in the present invention, the glycoprotein composition, New - Les a composition comprising glycoprotein molecules with glycoside-linked sugar chain or 〇- grayed glycosidic bond sugar chains, earthenware pots.

[0025] Specific examples of the sugar protein, antibody, erythropoietin, thrombopoietin, organization-type plasminogen § Cu Chi beta, Purourokinaze, thrombomodulin, Anchitoro Nbin III, protein your blood clotting factor VII, blood coagulation factor VIII, a blood clotting factor IX, blood clotting factor X, blood coagulation factor XII, gonadotropin, thyroid stimulating hormone, epithelial increase 殖因Ko (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor, Akuchibin, bone shape formation factors, stem cell factor (SCF), interferon alpha, interferon beta, interferons port down, interleukin 2, interleukin 6, interleukin 10, interleukin 1 1, soluble interleukin-4 receptor, non-tumor necrosis factor, Dnasel, galactosidase, shed Darukoshidaze, a Darko celebrities Russia mannosidase And the like.

[0026] By having no sugar chain structure fucose modification, as a more specific example of glycoprotein that its physiological activity is significantly increased, for example, antibody. Also, the antibody composition, Le a composition comprising an antibody molecule having a N- glycoside-linked complex-type oligosaccharide in the Fc region, earthenware pots. Hereinafter, an example production of the antibody composition, showing a manufacturing method of a non-human animal and glycoprotein composition using progeny thereof of the present invention.

[0027] The antibody composition, the result of the foreign antigen stimulation, a protein that will be produced in the body by the immune response, antigen specifically as long as it has activity of binding Les, become those by any les, but animals immunized with antigens, other antibodies High Priestess dormer cells prepared from a spleen cell of the immunized animal to secrete, antibody produced by gene recombination technology, i.e., an antibody expression vector 揷入 antibody gene and such antibodies obtained by introducing into a host cell. Specifically, there may be mentioned antibody production High Priestess dormer, chimeric antibodies, humanized anti-body, and a human antibody. [0028] High Priestess dormer has a B cell obtained by immunizing antigen to a mammal other than a human, mouse, and a myeloma cell derived from a rat or the like obtained by cell fusion, a desired antigen specificity It refers to a cell to produce a monoclonal antibody having.

Chimeric antibodies, non-human animal antibody heavy chain variable region (hereinafter, the variable region referred to as HV or VH as V region) and antibody light chain variable region (hereinafter, the light chain also referred to as LV or VL as L chain) and a heavy chain constant region of a human antibody (hereinafter also referred to as CH) and the human antibody Keikusarijo constant region refers to an antibody consisting of (hereinafter also referred to as CL) and. As the animal other than human, mouse, rat, hamster, rabbit or the like, if it is possible to produce a High Priestess dormer, it is possible to use also made or Rere.

[0029] Chimeric antibodies, from a hybridoma producing a monoclonal antibody to obtain a cDNA codes the VH and VL, respectively into an expression vector for host cell having genes encoding human antibody CH and human antibody CL 揷入and to construct a human chimeric antibody expression vector, and expressed by introducing into the host cell, it can be force S to manufacture.

The CH of the chimeric antibody, a human immunoglobulin (hereinafter referred to as hlg) may be composed or not used, so long as it belongs to, but it is preferable that the hlgG class, further hlgGl belonging to hlgG class, hIgG2, hIgG3, hIgG4 any subclass such can also be used. As the CL of human chimeric antibody, any so long as it belongs to the hlg, can be used as the K-class or Ek lath.

[0030] Hitoi 匕抗 body, complementarity determining regions of the VH and VL of a non-human animal antibody (hereinafter referred to as CD R) antibody were grafted amino acid sequence of the appropriate positions of VH and VL of a human antibody the say.

Humanized antibodies, to construct a cDNA encoding the V regions grafted to the CDR sequences of VH and VL of any human antibody CDR sequences of VH and VL of a non-human animal antibody, a human antibody CH and human antibody into an expression vector for host cell having genes encoding CL their respective 揷入 to build a humanized antibody expression vector, to express humanized antibodies by introducing the expression vector into the host cell to produce be able to.

[0031] As the CH of the humanized antibody, Les I in any, so long as it belongs to the hlg, but it is preferable that the hlgG class, any subclass such further hIgGl belonging to hlgG class, hIgG2, hIgG3, hIgG4 it can be also used. As the CL of the Hitoi 匕抗 body may be composed squid used, so long as it belongs to hlg, can be used for / class or e class.

Human antibody is originally an antibody naturally existing in the human body, but genetic engineering, cell engineering, developmental engineering human antibody phage library produced by technological advances one as well as human antibody-producing trans antibodies and the like obtained from Jienikku animal or human antibody-producing transgenic click plants are also included.

[0032] antibodies present in the human body, for example, human peripheral blood lymphocytes are isolated and immortalized infected with EB virus or the like, by cloning, can be cultured lymphocytes that produce antibodies, culture it can be purified more antibodies.

The human antibody phage library one is a library Fab, antibody fragments such as single chain antibodies expressed on the phage surface by 揷入 antibody gene prepared from human B cell into a phage gene. From the library, it can Rukoto force S to recover the phage expressing an antibody fragment having the desired antigen binding activity binding activity as an indicator for an antigen-immobilized substrate. The antibody fragment can be further by genetic engineering techniques, also converted to two full H chains Oyo human antibody molecule comprising complete L chain of two beauty.

[0033] Human antibody-producing transformer diethyl nick nonhuman animal is an animal in which a human antibody gene is integrated into cells. Specifically, by introducing the mouse embryonic stem cells Hecht antibody genes after implantation of embryo stem cells into an early embryo of other mouse, making a human antibody-producing Toransuji Engineering two click animal by generating can. Further, by introducing a human antibody gene into an animal embryo, it is also possible to produce human antibody-producing transformer diethyl nick animals to generating the fertilized egg. A human antibody is prepared from the human antibody-producing trans Jie Nick animals, obtained human antibody producing High Priestess dormer by High Priestess dormer manufacturing method usually carried out in a non-human mammal, a human in culture by culturing antibodies can be a production storage.

[0034] trans-di We nicked non-human animals, © shea, Hijji, catcher formic, pigs, © Ma, mouse, rat, Niwato Li, monkey or Usagi and the like.

Further, in the present invention, the antibody, tumor-associated antigen antibody recognizing, allergies walk antibody which recognizes an antigen associated with inflammation, recognize antigens which are associated with cardiovascular disease antibody, associated with autoimmune diseases an antibody recognizing an antigen or virus, or the desirability tool antibody class that the antigens associated with bacteria infection which recognizes the antibody of IgG human antibody, are preferred.

[0035] and antibody fragments, it refers to a fragment which comprises at least a part of the Fc region of the antibody. The Fc region containing, C-terminal, the side regions of an antibody H chain means a CH2 and CH3 regions, a variant of a native Oyo benefactor. The at least part of the Fc region, preferably a fragment containing the CH2 region, and more preferably will have a region including the first Asuparagin acid present in the CH2 region. Fc region of IgG class, power bat (Kabat) et al., EU Index [Shikenshizu 'O Breakfast' Puroti lens' off 'Munoronkanore' Intaresu! (Sequences of Proteins of Immunological Inter est), o Ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1 991)] C-terminal numbering in the 226 th cysteine ​​of or 230th proline force C, It means to the end. The antibody fragment specifically, monomer H chain, such as dimer of H chains.

[0036] Examples of the fusion protein comprising the Fc region, and an antibody or antibody fragment containing the Fc region of an antibody, an enzyme, Oh those les, consisting either be a substance that combines with protein, such as site force in good.

The antibody which recognizes a tumor-related antigen includes anti-GD2 antibody (Anticancer Res., 13, 331, 19 93), anti-GD3 antibody (Cancer Immunol. Immunother "36, 260, 1993), anti-GM2 antibody (Can cer Res. , 54, 1511, 1994), anti-HER2 antibody (Proc. Natl. Acad. Sci. USA, 89, 4285, 199 2), anti-CD52 antibody (Nature, 332, 323, 1988), anti-MAGE antibody (British J. Cancer, 83, 49 3, 2000), anti-HM1.24 antibody (Molecular Immunol., 36, 387, 1999), anti-parathyroid hormone related protein (PTHrP) antibody (Cancer, 88, 2909, 2000), anti-FGF8 antibody (Proc. Natl. Aca d. Sci. USA, 86, 9911, 1989) anti-basic fibroblast growth factor antibody, anti-FGF8 receptor antibody (J. Biol. Chem., 265, 16455, 1990), anti basic fibroblast growth factor receptor antibody, anti-insulin-like growth factor antibody (J. Neurosci. Res., 40, 647, 1995), anti-insulin-like growth factor receptor antibodies (J. Neurosci. Res., 40 , 647, 1995), anti-PMSA antibody (J. Urology, 160. 2396, 1998), anti-vascular endothelial cell growth factor Antibody (Cancer Res., 57, 4593, 1997) or other anti-vascular endothelial cell growth factor receptor antibody (Oncogene, 19, 2138, 2000), an anti-CA125 antibody, anti-17-1A antibody, anti-Integurinhi v / 3 3 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), and the like, such as.

[0037] The antibody which recognizes an allergy- or inflammation-related antigen includes anti-interleukin 6 antibody (Immunol. Rev., 127, 5, 1992), anti-interleukin 6 receptor antibody (Molecul ar Immunol, 31, 371, 1994), anti-interleukin 5 antibody (Immunol. Rev., 127, 5, 1992), anti-interleukin 5 receptor antibody, anti-interleukin 4 antibody (Cytokine, 3, 562, 19 91), anti-interleukin 4 receptor antibody (J. Immunol. Meth., 217, 41, 1998), anti-tumor necrosis factor antibody (Hybridoma, 13, 183, 1994), anti-tumor necrosis factor receptor antibody (Molecula r Pharmacol., 58, 237 , 2000), anti-CCR4 antibody (Nature, 400, 776, 1999), anti-chemokine antibody (J. Immunol. Meth., 174, 249, 1994), anti-chemokine receptor antibody (J. Exp. Med., 186. 1373, 1997), anti-IgE antibodies, anti-CD23 antibody, anti-CD 11a antibody (Immunology Today, 21, 403, 2000), anti-CRTH2 antibody (J. Immunol., 162, 1278, 1999), anti-CCR8 antibody (W099 / 2 5734), anti-CCR3 antibody (US6207155), and the like.

[0038] As the recognize antigens which are associated with cardiovascular disease, anti-GpIIb / IIIa antibody (J. Immuno 1., 152, 2968, 1994), anti-platelet-derived growth factor antibody (Science, 253, 1129, 1991 ), anti-platelet-derived growth factor receptor antibodies (J. Biol. Chem., 272, 17400, 1997) or such as an anti-blood coagulation factor antibody (Circulation, 101, 1158, 2000) and the like.

(As a specific example, psoriasis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, multiple sclerosis, etc.) autoimmune diseases as the antibody recognizes an antigen associated with the anti-self DNA antibodies (Immunol. Letters, 72, 61, 2000), anti-CDl la antibodies, anti-ICAM 3 antibody, anti-CD80 antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti Integurinhi 4 j3 7 antibody, anti-CD40L antibody , such as anti-IL-2 receptor antibodies (Immunology Today, 21, 403, 2000) and the like.

[0039] The antibody which recognizes a viral or antigens associated with bacterial infection, anti-gpl20 antibody

(Structure, 8, 385, 2000), anti-CD4 antibody (J. Rheumatology, 25, 2065, 1998), anti-CCR 4 antibody, anti-verotoxin antibody (J. Clin. Microbiol., 37, 396, 1999) and can give.

Hereinafter, Fei-1,6-fucosyltransferase variants of the present invention and its use will be described in detail.

1. Preparation of DNA alpha-1,6-fucosyltransferase variants of the invention

alpha-1,6-fucosyltransferase variants of the present invention, alpha-1,6-activity of fucose modifying enzyme is released deletions or mRNA from human tissue having a disease such as reduced single, its cDNA live to prepare a slurry, it can then be prepared by obtaining the desired clones by screening the cDNA library.

[0040] The non - 1,6 fucosyltransferase activity glycosyltransferase deletion or by mutation occurs as drops,, N - glycoside bond complex type sugar chain N - of § cetyl Dano record Sa Min by using the 6-position and 1 Kuraigahi bound cultured cell lines derived from mRNA which become resistant sugar chain structure recognized by the lectin fucose, Fei 1,6 of the present invention - a fucosyl trans Hue hydrolase mutants it can be prepared.

[0041] The resistant cell lines lectin, even at an effective concentration of lectin refers to cell lines in which growth is not inhibited. Effective concentration and is described above, alpha-1,6-fucosyltransferase activity of the glycosyltransferase are deleted or previous cell lines mutations occur as reduced (hereinafter, referred to as "parent strain"), you can not normally grow Concentration or more, preferably, every time the concentration of the same concentrated to parent strain can not grow, and more preferably 2 to 5 times, more preferably 10 fold, and most preferably 20 times or more.

[0042] In the present invention, the effective concentration of lectin that does not inhibit growth, may be appropriately determined in accordance with the parent strain, normally 10 ig / ml~10mg / ml, preferably 0.5mg / ml~2.0mg / ml is there.

The lectin which recognizes a sugar chain structure in which 6-position and 1-position of fucose N- glycoside-linked sugar chain of N- § cetyl Darco Sa Min bound a, if a lectin capable of recognizing the sugar chain structure, any it can also be used in the lectin. As specific examples, the end © lectin PSA (Pisum sativum derived nea lectin) (lentil agglutinin of Lens culinaris Yukari夹) Ren Zumamerekuchin LCA, broad bean lectin VFA (agglutinin derived from Vicia faba), Hyi b Cha Wan aurantia lectin AAL (Aleuria n that from aurantia lectin), and the like

[0043] Human tissue-derived or cultured cell lines derived from mRNA, the walk following your sharpen human tissues Total RNA was prepared from cultured cell lines, Rukoto force to prepared by isolating a 該全 RNA force mRNA It can be S.

The methods for preparing total RNA from human tissues or cultured cell lines, Chioshian Thang Anijin one Torifuruoro cesium acetate method [Methods in Enzymology, 154, 3 (1987)], acidic thiocyanate guanidine 'phenolic' black hole Holm ( AGPC) method [Analytical Biochem istry, 162, 156 (1987), experimental medicine, 9, etc. 1937 (1991)] and the like. The methods for preparing mRNA as po ly (A) + RNA from total RNA, oligo (dT) immobilized cellulose force ram method (Molecular 'Cloning, Second Edition) and the like. Alternatively, Fast Track mR NA Isolation Kit (Invitrogen Corporation), can be prepared mRNA by using a kit such as Quick Prep mRNA Purification Kit (Pharmacia Co.).

[0044] Next, to prepare a cDNA library from the prepared human tissue or cultured cell line mRNA.

The cDNA library one preparation method, Molecular 'Cloning, Second Edition, Current' flop Rotokoruzu. In. Molecular. Methods described in Biology and the like or a commercially available kit, the example Superscript Plasmid system for cDNA Synthesis and Plasmid Cloning, ( Life Technologies, Inc.), such as how to use the ZAP-cDNA Synthesis Kit (STRATAGENE Co., Ltd.) and the like.

[0045] As the cloning vector for preparing the cDNA library, so long as it is autonomously replicable in Escherichia coli K12 strain, phage vectors, can be used in any plasmid vectors and the like. Specifically, ZAP Express [STRATAGENE Co., Strategies, 5, 58 (1992)], Bluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)], Lambda ZAP II (manufactured by STRATAGENE Co., Ltd.), e gtl0, λ gtl l [DNA Cloning, A Practical Approach, 丄, 49 (1 985)], lambda TRIPLEX (Clontech, Inc.), (manufactured by Pharmacia Co.) lambda ExCell, pT7T318U (Pharmacia a Inc.), PCD2 [Mol. Cell. Biol., can be cited 3, 280 (1983)] and pUC18 [Gene, 33, 103 (1985)] and the like.

[0046] As the host microorganism, a microorganism belonging to the Eshurihia genera (Escherichia), especially Eshurihi § 'stiffness (Escherichiacoli, hereinafter also referred to as "E. coli") can Rereru be force S use in any if it is a microorganism belonging to the. Specifically, EscherichiacoliXLl-Blue MRF '[STRATAGENE Co., Strategies, 5, 81 (1992)], Escherichia coliC600 [Genetics, 39, 440 (1954)], Esc herichia coliY1088 [Science, 222, 778 (1983)] , Escherichia coli Y1090 [Science, 222, 778 (1983)], Escherichia coli NM522 [J. Mol. Biol, 166, 1 (1983)], Escherichia coli K802 [J. Mol. Biol., 16, 118 (1966) 1 and Escherichiacoli JM105 [Gene, 38, 275 (19 85)] or the like is used.

[0047] Oligo this cDNA library, Yo Le be directly used in the following analysis, but lowered the ratio of incomplete length cDNA A, in order to obtain as efficiently as possible the full-length cDNA, which Kanno et al developed cap method [Gene, 138, 171 (1994), Gene, 200, 149 (1997), protein, nucleic acid enzymes, 41, 603 (1996), experimental medicine, 11, 2491 (1993), cDNA cloning, Yodo-sha ( 1996), method for preparing a gene library, Yo Le be used for the following analysis a cDNA library prepared using YODOSHA (1994)].

[0048] Each clone from a cDNA library prepared isolated and the nucleotide sequence of the cDNA from the end for each clone, nucleotide sequence analysis method generally used, for example, Sanger (Sanger) et al Jidokishi method [Proc. Natl. Acad . Sci. USA, 74, 5463 (1977)] or by analyzing using a base sequence analysis apparatus such as a BIPRISM377DNA Shikuensa one (PE Biosystems, Inc.), to determine the nucleotide sequence of the DNA.

[0049] nucleotide sequences of the cDNA is whether has a nucleotide sequence encoding an amino acid modified variant mutant enzymes of the alpha-1,6-fucosyltransferase, using a homology search program such as BLAST, GenBank by carrying out a search of nucleotide sequence databases such as EMBL and DDBJ, it affirms Shinobu by homology tone bell with a base sequence of an existing gene in the database.

[0050] obtained by the above method, the nucleotide sequence of cDNA containing the nucleotide sequence encoding the amino acid modification mutant enzyme, e.g., SEQ ID NO: 18, 19, 20, the nucleotide sequence represented by 21 or 22 and the like.

SEQ ID NO: 18, 19, 20, to ensure that does not artificially occur during molecular forces ScDNA library prepared comprising the base sequence represented by 21 or 22, use the cDNA library prepared the genomic library of human tissues or cultured cell lines had, and screened using sequence specific to SEQ ID NO: 18, 19, 20, the nucleotide sequence represented by 21 or 22, the nucleotide sequence of the obtained genomic clones it can be determined by determining.

[0051] Genomic libraries scratch, from human tissues or cultured cell lines, Molecular 'Cloning, Third Edition or Current' Protocols' in 'Molecular' be prepared using itself known methods described in Biology etc. can. Further, genomic DNA library screening system (Genome Systems, Inc.) or Universal GenomeWalker ™ Kits (CLONTEC H, Inc.) can also be prepared by the like.

[0052] The genomic library, as a method of screening using sequence specific to SEQ ID NO: 18, 19, 20, 21 or the nucleotide sequence represented by 22, SEQ ID NO: 18, 19, 20, 21 or 22 in PCR using primers specific to the nucleotide sequence represented [PCR Protocols, Acade mic Press (1990)] and, in SEQ ID NO: 18, 19, 20, the nucleotide sequence represented by 21 or 22 specific colonies hybrida I See Chillon and plaque hybrida Izeshiyon method using the oligonucleotides (Molecular 'cloning, third Edition) and the like.

[0053] In the above method in SEQ ID NO: 18, 19, 20, genomic D NA clones containing the nucleotide sequence represented by 21 or 22 is obtained. If this genomic DNA base sequence was determined SEQ ID NO 18, 19, 20, confirmed to be consistent with 2 1 or the nucleotide sequence represented by 22, the sequence was artificially generated when a cDNA library prepared it can be seen not.

SEQ ID NO: 18, 19, 20, DNA consisting of the nucleotide sequence represented by 21 or 22 is temporarily acquired, after which the nucleotide sequence was determined, the nucleotide sequence of the 5 'and 3' ends of the nucleotide sequence as 铸型 a cDNA library primers were prepared which were made using the human tissue or cultured cell lines based on, PCR method by performing the amplification of DNA using [PCR Protocols, Academic Press (1990)], it is possible to obtain the DNA of the alpha-1,6-fucosyltransferase variants of the present invention.

[0054] In addition, SEQ ID NO: 18, 19, 20, 21 or a portion total length Oh Rui DNA comprising the nucleotide sequence as a probe represented by 22, cDNA library prepared using human tissue or cultured cell lines colonies by performing hybrida I See Chillon and plaque hybrida I See sucrose emissions (Morekiyura one 'cloning 3rd edition), Fei of the invention to - obtaining a DNA 1,6 Fukoshirutora Nsufueraze variants can.

. [0055] Based on the nucleotide sequence of the determined DNA, by chemical synthesis phosphoamidite method Parkinson emissions utilizing Elma one company DNA synthesizer, such as the DNA synthesizer model 392, Fei invention - 1,6 - for the obtained DNA which can also obtain DNA fucosyltransferase variants, by expressing the protein a recombinant vector using a transformant obtained by introducing into a host cell containing the DNA, it is possible to confirm that the DNA is a DNA encoding the amino acid modified alpha-1,6-Fukoshinore transferase variants as activities of alpha-1,6-fucose modifying enzyme is deleted, or drops it can.

[0056] SEQ ID NO: 18, 19, 20, 21 or based on the nucleotide sequence or information about the sequence of the fragment represented by 22, the usual method or by using a DNA synthesizer, Fei -1 of the present invention, 6 - fucosyltransferase nucleotide sequence of the DNA of the mutants, for example SEQ ID NO: 18, 19, 2 0, 21 or 22 in the nucleotide sequence represented, contiguous 5-60 bases, preferably corresponds to 10 to 40 base sequence corresponding to a complementary sequence to the oligonucleotide or the oligonucleotides with (hereinafter, referred to as antisense O Rigo nucleotides) kill that force S is prepared.

[0057] These oligonucleotides, Fei 1,6 of the present invention - can be used to detect the fucosyltransferase variants. In particular, Origonutare Ochido capable of identifying and alpha _1,6- fucosyl transferase peptidase variants and alpha-1,6-fucosyltransferase of the present invention is the diagnosis of alpha-1,6-fucosyltransferase variants of the invention it is useful in the law.

[0058] As oligonucleotides, oligo DNA, oligonucleotide oligo RNA such, and the oligonucleotide derivatives (hereinafter, an oligonucleotide derivative and Re, U) or the like is found on.

As the oligonucleotide or antisense O Rigo nucleotides, for example, the detected record, Ore part of the base sequence of mRNA Te, 5 'Sensupu corresponds to the end side of the base sequence primers, a 3' terminal side antisense primer such that corresponding to a nucleotide sequence can be mentioned. However, bases corresponding to Urashiru in mRNA becomes thymidine in the oligonucleotide plug timer scratch.

[0059] As the sense primer and antisense primer, both the melting temperature (Tm) of the contact and the number of bases with no oligonucleotide be changed extremely, 5-60 bases, preferably mentioned those 10 to 50 bases in It is.

The oligonucleotide derivatives include an oligonucleotide derivative wherein the phosphodiester bond has been converted into phosphorothioate Chio oleate bond in an oligonucleotide, the phosphodiester bond in the oligonucleotide is converted to N3'- P5 'phosphodiester Fore Mi linkages and the oligonucleotide derivatives, ribose and an oligonucleotide derivative wherein the phosphodiester bond has been converted to a peptide-nucleic acid bond in an oligonucleotide, an oligonucleotide derivative Urashiru is substituted with C_ 5 propynyl © La sill in the oligonucleotide, in the oligonucleotide Urashiru oligonucleotide derivatives substituted with C_ 5 thiazole © La sills, Origonukureo tides derivative in which cytosine is substituted with C-5 propynyl cytosine in the O oligo nucleotides, citrate in the oligonucleotide Emissions oligonucleotide derivatives substituted with Fuenokisajin modified cytosine (phenoxazi ne-modified cytosine), ribose in the oligonucleotide is 2, an oligonucleotide derivative which is substituted by _〇 one propyl ribose, there have the ribose in the oligonucleotide 2, single-methoxyethoxy oligonucleotide derivatives substituted with ribose and the like [cell Engineering, 1 ^, 1463 (1997)].

2. Preparation of alpha-1,6-fucosyltransferase variants of the invention

(1) Preparation of a-1,6-fucosyltransferase variants

alpha-1,6-fucosyltransferase variants of the present invention, Molecular ^ ~ Cloning, 3rd Edition or Current Protocols 'in. Molecular' using whichever method described in biology and the like, for example, the following method Accordingly, the DNA of the alpha-1,6-fucosyltransferase variants of the present invention be expressed in a host cell, it can be produced.

[0060] The full-length cDNA based on, if necessary, to prepare a DNA fragment of appropriate length comprising a region encoding the alpha _1,6_ fucosyltransferase variants.

By 揷入 the DNA fragment or full-length cDNA, downstream of a promoter in an appropriate expression vector to prepare a recombinant vector.

The recombinant vector is introduced into a host cell suited for the expression base Kuta one, Fei 1,6 of the present invention - it is possible to obtain a transformant producing the fucosyltransferase variants.

[0061] As the host cell, bacteria, yeast, animal cells, insect cells, plant cells and the like, can be used, so long as it can express the gene of interest.

Expression vectors, capable of integration into the host cell autonomous replicable or chromosome in, les, shall is used at a position appropriate for the transcription of the DNA encoding the protein of the present invention contain a promoter scratch.

[0062] When using a prokaryote such as bacteria as the host cell, a recombinant vector comprising the DNA encoding the alpha-1,6-Fukoshirutora Nsufueraze variants of the present invention is a by self-replicable in prokaryotes there simultaneously, a promoter, a ribosome binding Hai歹 1 J, gene encoding 蛋 white matter of the present invention, and it is constructed vectors from transcription termination sequence good better les. It contains a gene that controls the promoter Les, even good record,.

[0063] As expression vectors, for example, pBTrp2, pBTacl, pBTac2 (all 巿販 from Boehringer Mannheim also), pKK233- 2 (Pharmacia Co.), pSE280 (Invitrogen Corp.), pGE MEX-1 (Promega Corp. ), manufactured by pQE_8 (QIAGEN Co.), PKYPIO (JP 58- 110600), pKY Ρ200 [Agricultural Biological Chemistry,, 669 (1984)], pLSAl [Agric. Biol. Chem., 53, 277 (1989)], pGELl [Proc Natl Acad Sci USA, 82, 4306 (1985)], pBluescri pt II SK.... (-) (Stratagene Co.), pTrs30 [Escherichia coli JM109 / pTrS30 (FERM BP-5 407) from preparation] , Trs32 [prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)], PGHA2 iEscherichiacoliIGHA2 prepared from (FERM B-400), JP 60-221091], from pGK A2 [Escherichia coli IGKA2 (FERM BP-6798) preparation, JP-A-60-221091], P Term2 (US4686191, US4939094, US5160735), pSupex, pUBHO, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990) ], (manufactured by Pharmacia Inc.) pGEX, pET system (Novagen Co., Ltd.), it is possible to increase the pSupex like.

[0064] The promoter may be any substance so long as it can be expressed in a host cell. For example, tro. Promoter (P), lac promoter, P promoter, P promoter trp LR

, It is possible to increase the promoter derived from such as T7 promoter, E. coli, phage and the like. The two series are not promoter and P (PX 2), £ promoter, lacT7 pro trp trp

Motor can Rukoto force S also used such as artificially designed and modified promoters like the let I promoter.

[0065] It is preferred to use a plasmid in which between adjusted to an appropriate distance (for example 6 to 18 bases) between Shine one Dalgarno (Shine-Dalgarno) sequence and the initiation codon is a ribosome binding sequence. Fei present invention 1,6 - fucosyltransferase nucleotide sequence of a portion encoding the variant, for optimum codon host expression, by replacing the base, alpha is an object -1, 6- it is possible to improve the production rate of fucosyltransferase variants.

[0066] In the recombinant vector of the present invention, but not necessarily a transcription termination sequence for the expression of the DNA of the alpha-1,6-fucosyltransferase variants of the present invention, the transcription termination sequence immediately downstream of the structural gene arrangement it is preferable to.

The host cell, Eshierihia, Serratia, Bacillus, Brevibacterium, Corynebacterium, the genus Microbacterium, microorganisms belonging to Shiyudomonasu genus, etc. For example, Escherichiacoli XLl_Blue, Escherichiacoli XL2_Blue, Escherichia coliDHl, E scherichia coli MC1000, Escherichia coli KY3276, Escherichiacoli W1485, Escheric hia coli JM109, Escherichia coliHB101, Escherichiacoli No.49, Escherichia coli W31 10, Escherichiacoli NY49, Serratia ficaria. Serratia fonticola. Serratialiguefaciens. S erratiamarcescens. Bacillus subtilis, Bacillusamvloliguefacines. Brevibacteriumimmar iophilum ATCC14068 , Brevibacteriumsaccharolvticum ATCC14066, Brevibacterium flavum ATCC14067, Brevibacteriumammoniagenes, Brevibacteriumlactofermentum ATCC13869, Corvnebacteriumglutamicum ATC and 13032, Corvnebacteriumacetoacid ophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354, Pseudomonassp . It is possible to increase the D-0110 and the like.

Introduction of the recombinant vector if Re der methods for introducing DNA into the host cell either can be used, for example, a method using calcium ion [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], the protoplast method (JP 63-248394), or Gen e, 17, 107 (1982) and Molecular & General Genetics, cliffs, can be force mentioned method described in 111 (1979).

[0067] When yeast is used as the host cell, the expression vector, for example, YEP13 (ATC C37115), YEp24 (ATCC37051), can be mentioned YCp50 (ATCC37419), and the like. The promoter can be used any one as long as it can function in yeast, for example, to promoters of genes of the glycolytic pathway such as key source kinase, PH05 promoter, PGK promoter, GAP promoter, ADH promoter , mention may be made of gal 1-flops opening motor, gal 10 promoter, heat shock protein promoter, MF monument 1 promoter one coater, the CUP 1 promoter, and the like.

[0068] The host cell, Saccharomyces, Schizosaccharomyces genus, Kuryui base mouth Mrs. genus preparative Rikosuporon genus, yeast belonging to Shuwaniomisesu genus like, for example, Saccharomvces cerevisi ae. Schizosaccharomvces pomoe. Kiuvveromvces lactis, fnchosporon pullulans. ¾ch wanniomvces alluvius can o this and force raised.

Introduction of the recombinant vector so long as it is a method for introducing DNA into yeast les, may also be used misalignment, for example, elect port Poreshiyon method [Mesozzu 'Enzaimoroji one (Me thods. Enzymol.), 194, 182 (1990)], Sufuwe opening Plast method [Proceedings 'O Breakfast' The 'National Akademi -. O Breakfast' Science (Pro Natl. Acad. Sci. USA), 84, 1929 (1 978)], the lithium acetate method [journal 'O Breakfast' Bataterioroji one (J. Bacteriology), 153, 163 (1983)], Proceedings O blanking THE Nashonanore-Akademi one 'O blanking Science (Pro c. Natl. Acad. Sci. USA ), it can be mentioned 75, 1929 (1978)] the method according to such.

[0069] When an animal cell is used as a host, an expression vector, for example, pcDNAI, pc DM8 (commercially available from Funakoshi), pAGE107 [JP-A 3-22979; site Technology (Cytotec hnology), 3, 133, ( 1990)], pAS3- 3 [JP 2- 227075], pCDM8 [Neichiya (Nature), 329, 840, (1987)], pcDNAI / Amp (Invitrogen Corporation), manufactured by pREP4 (Invitrogen Corp.), pAGE 103 [ journal 'O Bed' Biochemistry (J. Biochemistry), dishes, 1307 (1987)], can be mentioned pAGE 210 or the like.

[0070] As the promoter, so long as it can function in animal cells either can be used, for example, the cytomegalovirus (CMV) IE (immediate early) promoter one gene, early promoter of SV40, the Retorouinoresu promoter, meta opening Chioneinpuromo one coater, it is possible to increase the heat shock promoter, SR shed promoter, and the like. Further, Yo Le be used Enhansa of IE gene of human CMV together with the promoter.

[0071] The host cell is a human cell Namalwa (Namalwa) cells, C 〇_S cells which are monkey cells, Chinese 'Bruno, CHO cells are cells of Muster, HBT5637 (JP 63-29 9 ), it can be mentioned rat myeloma cells, mouse myeloma cells, Syrian hamster kidney-derived cells, embryonic stem cell, a fertilized egg cell and the like.

Introduction of the recombinant vector displacement have any one of the known methods for introducing DNA into animal cells can be used, for example, elect port Poreshiyon method Site Technology (Cytote chnology), 3, 133 (1990)], calcium phosphate law [JP-A-2-227075], Ribofuwekushiyon method [Proceedings O blanking the National Academy O blanking Science (Pro Natl • Acad. Sci. USA), 84, 7413 (1987)], the injection method [ Manipulating the mouse Embryo a Laboratory Manual, second Edition, Cold Spring Harbor Laboratory Pres s (1994) (hereinafter abbreviated as one computing 'mouse' Enburio second edition Manipiyure)], a method using a Pateiku Rugan (gene gun) [No. 2606856, No. 2517813], DEAE- dextran Hore O manual Series 4 monogenic Transfer and expression analysis (Yodo-sha) by Takashi Yokota. Arai Kenー ed. (1994)], the virus vector Chromatography method [Manipiyure one computing 'mouse' Enpuri O Second Edition] and the like.

[0072] When an insect cell is used as the host, for example current 'Protocols' in 'leakage Kyufu 1 ~' Haioron 1 ~ Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992), carbonochloridate I O / Akunoroji (Bio / Technology), by the method described in 6, 47 (1988), etc., capable of expressing the protein.

That is, after obtaining the recombinant virus in a recombinant gene transfer vector and baculovirus are cotransfected into insect cells Insect cell culture supernatant, and further infected with the recombinant virus into insect cells, that express the protein it can.

[0073] The gene transfer vector used in the method, for example, pVL1392, PVLl

393, pBlueBacIII (both manufactured by Invitorogen Co., Ltd.), etc. can be mentioned.

The baculovirus, for example, can Autogu Lafayette 'Karifuonoreni force' nuclease one 'poly to Doroshisu' Uinoresu (Autographa californica nu clear polyhedrosis virus) that use Rereru force etc. S is a virus that infects burglar Gaka insects.

[0074] As the insect cells, Sf9 ovarian Hoso朐of Soodooterafrug erda, Sf21 [Current 'pro graft 1 to Norezu' in 'Morekyufu 1 ~ - /ヽIoroshi 1 ~ Baculovirus Expression Vectors, A Labo ratory Manual, WH Freeman and Company, New York (1992)], High 5 (Invitrogen Corporation) is an ovarian cell of nchonlusiani like can be used.

For the preparation of recombinant viruses, as Cotransfection of the above recombinant gene transfer vector and the baculovirus into insect cells, for example, calcium phosphate method (JP-A-2 -227075), Ripofuekushiyon method [Proceedings' O Bed tHE 'National' Academy O Bed 'science (Proc. Natl. Acad. Sci. USA), as possible out to raise 84, 7413 (1987)], and the like.

[0075] The plant cells when used as a host cell, the expression vector, for example, Ti plasmid include a tobacco mosaic virus vector and the like.

The promoter can be used any one as long as it can function in a plant cell, for example, 35S promoter of cauliflower mosaic virus (CaMV), the Ineaku Chin 1 promoter.

[0076] As the host cell, it can be mentioned tobacco, potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat, plant cells such as Oomugi.

Introduction of the recombinant vector displacement have any one of the known methods for introducing DNA into plant cells can also be used, for example, § glow Park Teri © beam (Agrobacterium) [JP 59-140

885, JP-A-60-70080, WO94 / 00977], elect opening Poreshiyon method [JP-A-60-251887

], A method using particle gun (gene gun) [Japanese Patent No. 2606856, Japanese Patent No. 25

17813], and the like can be mentioned.

[0077] As the expression method of gene, in addition to direct expression, in accordance with the method or the like that are described in Molecular 'Cloning, Third Edition, secretory production, Ru, etc. can be performed fusion protein expression.

Yeast, when animal cells, expressed by insect cells, or plant cells can be obtained a protein to which a sugar or a sugar chain is added.

And culturing the transformant obtained as described above, alpha -Iota of the present invention in the culture, to produce and accumulate a 6-fucosyltransferase variants, Ri by the harvesting from the culture, the Fei invention 1,6 - can be produced fucosyltransferase variants. This onset Akiranohi _1,6 - method for culturing a transformant that expressed the fucosyltransferase variants the medium can be carried out according to conventional methods used for culturing a host.

[0078] As the medium for culturing a transformant eucaryotic organisms such as prokaryotes or yeast obtained as a host such as E. coli, a carbon source organism is assimilable nitrogen source, has free and inorganic salts , natural medium as long as the medium can perform culturing of the transformant efficiently may be used have shifted the synthetic medium.

The carbon source, as long as the organism can assimilate Yogu Gunorekosu, fructose, sucrose, carbohydrate molasses, starch and starch hydrolysates containing them, organic acids such as acetic acid and propionic acid, ethanol, can be used alcohols such and propanol.

The [0079] nitrogen source, ammonia, Anmoniumu chloride, Anmoniumu sulfuric acid ammonium © beam, Anmoniumu salts of organic or inorganic acids such as Anmoniumu phosphate, other nitrogen-containing compounds, as well as, peptone, meat extract, yeast extract, corn steep liquor, Kazi emissions hydrolyzate, soybean cake hydrolyzate, various fermentation bacteria and can be digested products thereof.

The [0080] inorganic salt, primary potassium phosphate, secondary potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese cancer, copper sulfate, and calcium carbonate .

Culture is carried out under aerobic conditions, for example, usually shaking culture or deep aeration stirring culture. The culturing temperature is 15 to 40 ° C Gayogu culture time is usually 16 hours to 7 days. pH during culture is maintained at 3.0 to 9.0. The pH is adjusted using inorganic or organic acid, alkaline solution, urea, calcium carbonate, ammonia and the like.

[0081] If necessary during culture, an antibiotic such as ampicillin or tetracycline may be added to the medium.

When culturing a microorganism transformed with a recombinant vector containing an inducible promoter as the promoter, an inducer may be added to the medium, if necessary. For example, indole acrylic acid in the case of a microorganism which sometimes isopropyl one beta _D_ Chio Galata galactopyranoside like culturing a microorganism transformed with a recombinant vector, transformed with a recombinant vector using a promoter with promoter or the like may be added to the medium.

[0082] The animal cells as a medium for culturing a transformant obtained as the host, commonly used RPMI1640 medium [The Journal O drive. The. American 'Medical Asoshiei Chillon (The Journal of the american Medical Association), 199, 519 (1967)], Eagle of MEM medium [Science (Science), 122, 501 (1952)], Dulbecco's modified MEM medium 邊 [Vue Uroroji (Virology), 8, 396 (1959)] , 199 medium [Proceedings Day packaging 'O Breakfast' The 'Sosaie tee' Fore 'The' Bio-Logica Norre 'Medicine (Proceeding of the Society for the Biologic al Medicine), 73, 1 (1950)], Whitten medium [Developmental Engineering experiment manual - Toransujiweni' click 'mouse recipe (Kodansha) Motoya Katsuki ed (1987)] or may be used fetal calf serum or the like medium and the like were added to these media.

[0083] The culture is usually pH6~8, 30~40 ° C, 5% CO present in conditions such as below:! Do to 7 days.

Fuedobatsuchi culture, can line TURMERIC land one day to several months cultured using the culture method such as a hollow fiber culture.

If necessary during culture, kanamycin, antibiotics such as penicillin may be added Caro to the medium.

[0084] The insect cells as a medium for culturing the transformant obtained as the host, generally employed media such as TNM-FH medium (Pharmingen Co.), Sf-900 II SFM medium (Life Technologies, Inc.), ExCell400, ExCell405 (both manufactured by JRH Biosciences, Inc.), Grace's Insect Medium [Grace, TCC, it is possible to use Nature, 195, 788 (1962)] and the like.

Culture is usually pH 6-7, under conditions such as 25 to 30 ° C, carried out 1-5 days.

If necessary during culture, an antibiotic such as gentamicin may be added to the medium

[0085] transformant plant cells obtained as a host can be used as the cell or after differentiating to cells or an organ of a plant culturing. As a medium for culturing the transformant includes generally used Murashige 'and' Sutagu (MS) medium, White (White) medium, auxin or child these media, cytokinin or the like, 添Ka卩 phytohormones the media was, or the like can and Mochiiruko.

[0086] The culturing is carried out usually pH 5 to 9, 3 to 60 days under the conditions of 20 to 40 ° C.

If necessary during culture, kanamycin, Bruno, may be added to the medium an antibiotic such Idaromaishin.

As described above, Fei 1,6 of the present invention - a microorganism carrying a fucosyl recombinant vectors incorporating DN A encoding fucosyltransferase variants, animal cells, or transformants derived from a plant cell, normal growth cultured according to the method, the "1,6-fucosyl trans luciferase mutants is produced and accumulated, by collecting the alpha-1,6-fucosyl transferase peptidase variant from the culture, the alpha-1,6 - it can be force S to produce a fucosyltransferase variants.

[0087] As the expression method of gene, in addition to direct expression, in accordance with the method or the like that are described in Molecular 'Cloning, Third Edition, secretory production, it is possible to perform the fusion protein expression and the like. Fei present invention 1,6 - as a method of producing fucosyltransferase variant, there is a method or a method in which raw Musa on the host cell membrane outer envelope, is secreted method, out of the host cell to be produced in a host cell, using and host cells, more altering the structure of the protein to be produced, it is possible to select the method.

[0088] Fei of the present invention 1,6 - if fucosyltransferase variants is produced in a host cell or on a host fine extracellular membrane, Paulson et al method [Journal 'O Bed' Biological 'Chemistry (J. Biol. Chem.), 264, 17619 (1989)], the method of Roura [Proceedings • O Breakfast 'the' National 'Academy ^ ~, O blanking Science (Proc. Natl. Acad. Sci. USA), 8 6, 8227 (1989); (. Genes Develop) Gene 'Development, 4, 1288 (1990)] or Hei 05-336963, by mutatis mutandis the methods described in JP-a-06-823021, etc., the alpha _1, , 6-fucosyltransferase variants Ru can be actively secreted out of the host cell.

[0089] That is, letting using techniques of genetic recombination, manifested originating in a form of adding a signal peptide in front of a protein containing the active site of alpha-1,6-fucosyl trans Blow over peptidase variants of the invention Accordingly, the alpha-1,6-fucosyltransferase variants of the present invention can be actively secreted out of the host cell.

Further, according to the method described in Japanese Published Unexamined Patent Application No. 227075/90, it is also possible to increase the production amount by utilizing a gene amplification system using a dihydrofolate reductase genetic child like.

Furthermore, by redifferentiation of animal or plant cells introduced genes, gene to construct a introduced animal individual (trans diethyl Nick nonhuman animal) or plant individual (transgenic click plant), these individuals Fei of the invention using - 1,6 - a fucosyl transferase peptidase variants can also be produced. [0090] When the transformant is an animal individual or plant individual, in accordance with a general method by rearing or cultivating, to produce and accumulate the alpha-1,6-fucosyltransferase variants, from the animal or plant individual by collecting the alpha _1,6- fucosyltransferase variants, 該Hi 1,6 - can be produced fucosyltransferase variants.

Fei present invention using an animal individual - As a method for producing the 1,6-fucosyltransferase variants, for example, a known method [American 'Journal' O Bed 'Clinical' Nutri Chillon (American Journal of Clinical Nutrition), 63 , 639S (1996);. American Journal Honoré 'O Bed' Clinic force Honoré 'two Yutorishiyon (American Journal of Clinical Nutrition), 63, 627 S (1996); Bio Z technology (Bio / technology), 9, 830 (1991 )] Fei present invention into an animal was constructed by introducing the gene according to - 1,6 fucosyltransferase variants how to production and the like.

[0091] In the case of an animal individual, for example, reared trans diethyl nick nonhuman animal carrying the introduced DNA encoding the fly-1,6-fucosyl transflector We hydrolase variants of the present invention, the alpha-1,6 fucosyl transferase variants is generated and accumulated in the animal, by taking 該Hi 1,6-fucosyltransferase variants than animal in, the alpha - 1,6-fucosyl trans Blow over peptidase mutants it can be produced. The generation and accumulation place the animal in, for example, milk of the animal (JP 63-309192), can be mentioned eggs like. The promoter used in this case, can be used, so long as it can function in an animal, for example, alpha casein promoter mammary gland cell-specific promoters, beta Kazi down promoter, lactoglobulin promoter, whey acid 'protein promoter and the like.

[0092] Fei of the invention using the plant individual - a process for preparing 1,6-fucosyltransferase variants, Fei example, the present invention - was introduced that encoding a 1,6-fucosyltransferase variant DNA trans-di We nick plant known method [tissue culture, (1994); tissue culture, ^ 1 (1995); trend 'in' biotechnology (trends in biotechnology), 15, 45 (1997)] cultivated in accordance with the the 該Hi 1,6-fucosyltransferase variants is generated and accumulated in the plant, 該Hi from said plant in - by Rukoto be taken 1,6-fucosyltransferase variants, 該Hi - 1,6 - how to produce fucosyltransferase variant is Ru and the like.

[0093] alpha-1,6-fucosyltransferase variant mutant produced by the transformant cell of the present invention, when expressed in a dissolve state, for example, alpha-1,6-fucosyltransferase variants strength within cells of the present invention the, after completion of the cultivation, cells were harvested by centrifugation, after suspended in an aqueous buffer, sonicator, French press, Manton Gaulin homogenizer, the cells were disrupted by da Inomiru or the like to obtain a cell-free extract . Cell-free extract was centrifuged to supernatant force obtained by Rukoto the normal isolation and purification methods of the enzyme, i.e., solvent extraction, salting out with ammonium sulfate or the like, desalting, precipitation with an organic solvent, Jefferies chill aminoethyl (DEAE) - Sepharose, DIAION ΗΡΑ-75 (Mitsubishi Chemical Industries, Ltd.) anion exchange black Matogurafi one using resins, cation using a resin such as SS mark harose FF (Pharmacia Co.) exchange chromatography, butyl Sepharose, phenylene Rusefarosu hydrophobic chromatography was use Rere resins such as gel filtration using a molecular sieve, Afi two tea chromatography grayed Rafi one, chromatofocusing, isoelectric using a technique electrophoresis such as focusing, alone or in combination, to obtain a purified preparation.

[0094] In addition, the alpha _1,6- If fucosyltransferase variants revealed issued as an inclusion body in cells, similarly the cells disrupted after collection, followed by centrifugation, the precipitate fraction and to recover the inclusion body of α -1,6- fucosyltransferase variant. The recovered alpha-1,6 - inclusion body fucosyltransferase variant is solubilized with a protein denaturing agent. By diluting or dialyzing the solvent of solution, after returning the alpha-1,6-fucosyltransferase variants normal three-dimensional structure, by the same isolation and purification methods 該Hi - 1,6 to obtain a purified preparation of Fukoshirutora transferase variants.

[0095] Fei of the present invention 1,6 - if fucosyltransferase variant or derivative conductor such as a glycosylated product is secreted extracellularly, 該Hi the culture supernatant 1,6 - fucosyl trans Blow over Ze variant or it can be recovered derivative of the sugar chain adduct. That is, the culture Tokushi preparative soluble fraction is treated by a technique such as centrifugation in the same manner as above, from the soluble fraction by using the same isolation and purification methods as described above, a purified preparation You can get that force S.

[0096] As a way proteins are obtained, for example, it may be mentioned a protein having an amino acid sequence represented by SEQ ID NO: 13, 14, 15, 16 or 17.

Further, Fei present invention - 1, 6-fucosyltransferase variants, Fmoc method (Furuoreni methyl O alkoxycarbonyl method), also be produced by tBoc method (t-butyl O alkoxycarbonyl method) chemical synthesis methods such it can. In addition, Advanced ChemTech, Inc., Perkin 'E Norema "~, Inc., Pharmacia, Inc., Protein technology Instrument earth, Synthecel Bok Vega child earth, Per S mark tive, Inc., also be chemically synthesized using a peptide synthesizer of Shimadzu Corporation, etc. it can.

(2) a-1,6-activity measurement of fucosyltransferase variants

Fei -1 of the present invention, 6-fucosyltransferase variants 及 Bihi -1, as the measuring method of 6-fucosyl transflector Eraze activity, for example, a known method [Uozumi et al., J Biochem, 120. 385-392 ( 1996)] in analogously, Fei -1 of the present invention adjusted, a method of measuring the activity of 6-fucosyl transferase peptidase variants thereof. Specific examples, the following method can be exemplified.

Reaction as follows (i) ~ (v) were mixed in a microcentrifuge tube to prepare [(i) Enzyme source: a solution containing the enzyme of 5 mL, such as purified enzyme, cell extract, or tissue extract); ( ii) pH adjustment buffer: 8 mL of 200mM MES (2- (N-Morpholino) ethanesulfonic Acid) -NaOH (H 6) (manufactured by Nacalai tester Co.); (iii) surfactant: 10% ImL Triton X-100 ( manufactured by Wako Pure Chemical Industries, Ltd.); (iv) fucose § click Scepter: 4 mL of 5mM Pirijiruamino - Puchiruamin labeled 'fucose (I) complex duplex type sugar chain solution (Takara Bio Inc., and trade name PA-sugar chain 012) ; (v) fucose donor: 2 mL of 500 mM GDP-L-fucose solution (Sigma Co.). After the adjusted reaction was allowed to react for 2 hours at 37 ° C, and 添Ka卩 sterile distilled water 80mL to the reaction solution, to inactivate the enzyme was boiled hand for 1 minute to 100 ° C. Thereafter, centrifugation for 10 minutes at 15,000 g, and separate supernatant of 90 mL, TSK-gel / ODS 80TM column HPLC equipped with (Tosoh Ltd. one company) (Shimadzu Seisakusho, Modenore SCL 6A) to to apply. 55. At C, to elute the glycans with 20 m M sodium acetate buffer containing 0.1% butanol (pH 4), the excitation wavelength 320nm of the eluate fluorometer fluorescence intensity at a wavelength of 400nm radiate (Shimadzu Corporation, Model measured by the RF535). From the measured fluorescence intensity to calculate the amount of fucosylated complex duplex-type sugar chain produced in the enzymatic reaction. Enzyme specific activity in the sample, per unit amount of protein contained in Sanpunore, per unit reaction time is expressed in moles of the fucosylated complex duplex type sugar chain molecule (unit: pmol / hour / mg ). Furthermore, protein content in the sample is measured © shea serum albumin (Pi ERCE Co.) in Yore the standard, was BCA Protein mediation Si kit (Pierce Co.).

3. part of the alpha-1,6-fucosyltransferase Fei-1,6-fucosyltransferase variants of the preparation the present invention the variant antibody recognizing, and they shed 1,6 Fukoshiruto lance Feller peptidase variants of the invention fragment polypeptide of purified specimen, or flight of the present invention - by using a 1,6-fucosyl peptide having a partial amino acid sequence of glycosyltransferase mutants as an antigen, a polyclonal antibody, monoclonal antibody, etc., Fei invention - 1,6 - fucosyltransferase variant can be produced antibodies that recognize.

(1) Preparation of polyclonal antibody

Fei-1,6-fucosyltransferase variants of the present invention, and they shed 1,6 Fukoshiruto lance Feller peptidase variants of partial fragment polypeptide of the purified preparation, or flight of the present invention - 1,6-fucosyltransferase variants using a peptide having a partial amino acid sequence of the body as an antigen, it is possible to produce polyclonal antibodies by administering to the animal.

[0098] As animals to be administered, Usagi, catcher formic, rats, mice, Ru can be used hamsters.

The dose of antigen is 50 per animal: 100 mu § are preferred.

When using a peptide to those that were covalently conjugated to a carrier protein to the peptide to keyhole Moshianin etc. (keyhole limpet haem ocyanin) Ya bovine thyroglobulin and antigen is desired. Peptide of the antigen can be synthesized by a peptide synthesizer.

[0099] of the antigen administration, after the first dose:! Do 10 times: 3 to week or every two weeks. After each administration, blood was collected from the venous plexus of the fundus of 3-7 days, enzyme immunoassay that react with antigens that serum was used for immunization Enzyme immunoassay (ELISA method): Igaku Shoin, published (1976 ), Antibodies-A La boratory Manual, confirmed by Cold Spring Harbor Laboratory (1988)] or the like, and.

Against the antigen used for immunization, it is possible that serum acquires the serum from a non-human mammal showing a sufficient antibody titer to obtain polyclonal antibodies by the serum separated and purified.

[0100] separation, as a method of purification, centrifugation, salting out by 40-50% saturated sulfuric Anmoniumu, Kapurinore acid precipitation [Antibodies, A Laboratory manual, Cold Spring Harbor Labor atory (1988)], or DEAE- Sepharose column , an anion exchange column, a chromatography using protein a or G- force ram or Genore filtration column or the like, a method of processing and the like alone or in combination.

(2) Preparation of monoclonal antibody

(A) Preparation of antibody-producing cells

Fei present invention used for immunization - 1,6 fucosyltransferase to a partial fragment polypeptide of glycosyltransferase mutants subjected to the rat whose serum shows a sufficient antibody titer as a supply source of antibody-producing cells.

3-7 days after the final administration of the antigenic substance in rats showed antibody titers, it spleens removed.

The spleen was cut to pieces in MEM medium (Nissui Pharmaceutical), loosened with forceps, 1, was centrifuged for 5 minutes at 200 rpm, the supernatant is discarded.

Precipitated fraction of spleen cells obtained tris monochloride Anmoniumu buffer solution (ρΗ7 · 65):! After removal of erythrocytes was treated to 2 minutes, washed three times with MEM medium, splenocytes obtained antibody used as production cell.

(B) Preparation of myeloma cells

The myeloma cells, using a cell line obtained from mouse or rat. For example, 8-Azaguanin resistant mouse (8 eight teeth 8 do derived) myeloma cell lines P3-X63Ag8-Ul (hereinafter, abbreviated as P 3- U1) [Curr. Topics. Microbiol. Immunol., 81, 1 (1978) , Europ. J. Immunol., 6, 511 (1976)], SP2 / 0-Agl4 (SP2) [Nature, 276, 269 (1978)], P3- X63- Ag8653 (65 3) [J. Immunol ., 123, 1548 (1979)], P3-X63-Ag8 (X63) [can be used Nature, 256, 495 (1975)] and the like. These cell lines include 8-Azaguanin medium [RPMI_ 1640 medium Gunoretamin (1. 5mmol / L), 2- mercaptoethanol (5 X 10- 5 mol / L ), gentamicin (10 x gZml) and fetal bovine serum (FCS) (CSL Ltd., 10./O) was added medium (hereinafter referred to as normal medium), and further 8 Azaguanin (15 x gZml). However passaged in mosquito 卩 example medium], cell fusion and cultured in the normal medium 3 to 4 days, using the cells 2 X 10 7 or more in the fusion.

(C) High Priestess dormer Preparation (a) MEM medium or PB S (disodium phosphate myeloma cells obtained in the acquired antibody-producing cells (b) in 1. 83 g, monopotassium phosphate 0 · 21g, salt . 7. 65 g, one liter of distilled water, pH 7 2) was washed well with, cell number, antibody-producing cells: myeloma cells = 5: 10: earthenware pots by becomes 1 were mixed, centrifuged 5 minutes at 1, 200 rpm after separation, the supernatant is discarded.

[0102] thoroughly loosened and the resulting precipitated fraction of cell groups, the said group of cells, with stirring, at 37 ° C, 10 8 antibody-producing cells per polyethylene of glycol - 1000 (PEG- 1000) 2g, MEM 2ml and dimethyl sulfoxide (DMS_〇) 0. mixed solution of 7ml was 0. 2~Lml added Caro, adding several more times MEM medium l~2ml every 1-2 minutes. After the addition, the total amount by adding MEM medium prepared such that 50 ml. 5 minutes preparation liquid at 900rpm After centrifugation, the supernatant is discarded. After the precipitated fraction of the cells obtained was gently loosened, suction by measuring pipette, gently HAT medium [normal medium hypoxanthine (10- 4 mol / L) at blowing, thymidine (1. 5 X 10 - 5 molZL) and aminopterin (4 X 10- 7 molZL) was added medium] is suspended in 100 ml.

[0103] The suspension dispensed at 100 mu 1 / well to the plate for a 96-well culture in 5% CO incubator, 7 at 37 ° C: 14 days culturing.

After culturing, a part taken anti body I's [in Antibodies in the culture supernatants, A Laboratory manual, Cold Spring Harbor Laboratory, by Chapter 14 (1988)] the enzyme immunoassay is described in such as the present invention alpha - selecting a specifically reactive High Priestess dormer the partial fragment Poribe peptide of 1,6 fucosyltransferase variants.

[0104] Specific examples of the enzyme immunoassay, it may be mentioned the following method.

Upon immunization, the parts divided pieces polypeptide of alpha-1,6-fucosyltransferase variants of the present invention using the antigen coated on an appropriate plate, obtained by High Priestess dormer culture supernatant or the rear predicate (d) the purified antibody was reacted as a primary antibody, corresponding to the further second antibody as Pio Chin, enzymatic labeling substance after an anti-rat labeled or anti-mouse sweep rate Takeno microglobulin antibody reacted with a chemiluminescent substance, a radioactive compound or the like reaction the performed, Fei present invention - 1,6-fucosyl Fei -1 of the present invention which specifically reacts with the fucosyltransferase variants is selected as high Puridoma of producing monoclonal antibodies which recognize 6-fucosyltransferase variants . [0105] Using the hybridoma, repeated twice cloned by limiting dilution method [first time, HT medium (medium prepared by removing aminopterin from HAT medium), a second time, using the normal medium], stable alpha -1 of the strong present invention antibody titer recognized what Te is selected as high Priestess dormer lines that produce monoclonal antibodies recognizing the 6-fucosyl transflector Weraze variants.

(D) the preparation of a monoclonal antibody

Pristane-treated [2, 6, 10, 14-tetramethyl pentadecane (Pristane) O. 5ml was intraperitoneally administered 2 weeks of feeding to] the 8: 10-week-old mice or nude mice with (c) Fei -1 acquired present invention, 6 - recognizes fucosyltransferase variant monoclonal antibody-producing hybridoma cells 5 to 20 X 10 6 cells / mouse are injected intraperitoneally. High Priestess Doma causes ascites tumor in 10 to 2 for 1 day.

The ascitic fluid is collected from the ascites water cancerous mice, removing the solids by centrifugation for 5 minutes at 3000 rpm.

[0106] From the resulting supernatant, purified monoclonal antibody in a manner similar to that used in the polyclonal can be obtained.

The subclass of the antibody can be determined using a mouse monoclonal antibody typing kit or a rat monoclonal antibody typing kit. Protein content is calculated Lowry method or from the absorbance at 2 80 nm.

4. Preparation of cells expressing alpha -1, 6- fucosyltransferase variants of the invention

(1) a -1, preparation of cells expressing 6-fucosyltransferase variants

alpha -1 of the present invention, cells expressing 6-fucosyltransferase variants, Fei -1 of the present invention having the above 2., 6 - fucosyltransferase that the various using the methods described for the preparation of sill transflector We hydrolase mutants it can be produced using the host cell.

[0107] In this case, arsenic -1, 6 - by using a host cell not expressing fucose modifying enzyme, Fei invention -1, from 6-fucosyltransferase variants Fei 1,6 fucosyl trans cells may be prepared with only luciferase activity.

The non - 1,6-fucose modifying enzyme gene was targeted, by use Rereru how genomic genetic modification, from Fei -1, 6-fucosyltransferase variants of the present invention Fei - 1, 6- it can be prepared cells having only fucose modifying enzyme activity. The a fucose qualified enzyme, specifically, alpha -1, 6- fucosyltransferase and the like.

[0108] As a method for genomic modification, any method is also included as long as it is a method capable of specifically modifying the genome gene of the target enzyme. Examples include a homologous recombinant method, RDO method, a method using retrovirus, a method using a transposon or the like mentioned et be. These methods are specifically described below.

By genomic genetic modification methods using (a) homologous recombination, Fei invention - Preparation of cells expressing 1,6_ Fukoshi Honoré transferase mutants

Fei -1 of the present invention, cells expressing 6-fucosyltransferase variants shed -1, 6-off the gene of course modifying enzyme which targets, have to alter use of homologous recombination of the target gene on the chromosome it can be made by.

[0109] modification of the target gene on the chromosome, Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994) (hereinafter referred to as "Ma two Publicis rating 'The' mouse 'Enburio' §. Laboratory one 'abbreviated as manual "), Gene T argeting, a Practical Approach, IRL Press at Oxford University Press (1993), Nono O manual Series 8, Gene targeting, Preparation of mutant mice using ES cells, Yodo-sha (1995) ( hereinafter referred to as "ES cells Preparation of mutant mice using") using the technique of chromosome engineering according the like, for example, as follows.

[0110] α -1, to obtain a cDNA of 6-fucose modifying enzyme.

Based on the nucleotide sequence of the obtained cDNA, alpha -1, genomic DNA is prepared of 6-fucose modifying enzyme.

Also based on the nucleotide sequence of the genomic DNA, a target gene to be modified (e.g., arsenic -1, 6 - structural gene of fucose modifying enzyme, or introns gene) to produce a data one target vector for homologous recombination .

[0111] The prepared target vector is introduced into cells by selecting the cells that have undergone homologous recombination between the target gene and target vector, Fei present invention - expressing 1,6 Fukoshirutoran Sufueraze variants cells can be prepared.

a-1,6 - a method for obtaining a cDNA or genomic DNA of fucose modifying enzyme, For example, the method described in the above 1. and the like.

[0112] The target vector for the homologous recombination of the target gene, Gene Targeting, A Pra ctical Approach, IRL Press at Oxford University Press (1993), carbonochloridate I O Mani Your Honoré Series 8, Gene Targeting, ES cells it can be prepared according to the method described in Preparation of mutant mice (Yodosha) (1995) or the like using. The target vector can be used replace main cement type, Insashiyon type, either gene trap type.

[0113] As the method for introducing the target vector into the cell, if way of introducing DNA into animal cells either can be used, for example, elect port Poreshiyon method Site Technology (Cytotechnology), 3, 133 (1990)], the calcium phosphate method [Japanese Published Unexamined Patent Application No. 227075/90], Li Pofuwekushiyon method [Proceedings Ding das O blanking-the-Nashonanore. Akademi scratch. O drive. science (Proc. Natl. Acad. Sci. USA), 84, 7413 (1987)], the injection method [Manipulati ng the mouse Embryo a Laboratory Manual, second Edition, Cold Spring Harbor La boratory Press (1994) (hereinafter abbreviated as one computing 'mouse' Enburio second edition Manipiyure)], a method using particle gun (gene gun) [No. 2,606,856, No. 2,517,813], D EAE dextran method [Biomanual Series 4 gene Transfer and expression. analysis (sheep Dosha) Takashi Yokota 'Kenichi Arai Hen (1994 )], Viral vector method [Manipiyure one computing 'mouse • Enburio Second Edition] and the like.

[0114] homologous recombinants as a method for efficiently screening, for example, Gene Targeting, A Practic al Approach, IRL Press at Oxford University Press (1993), carbonochloridate I O Mani Your Honoré Series 8, Gene Targeting, ES positive selection according to Preparation of mutant mice using cells (Yodosha) (1995), etc., promoters selected, it is possible to use a method such as negative selection, poly a selection. Specifically, in the case of the target vector containing hprt gene, after introduction into a cell lacking the hprt gene, culturing the cells in a medium containing aminopterin, hypoxanthine and thymidine, selecting a strain of aminopterin resistant it is thereby possible to perform the positive selection to select homologous recombinants containing hprt gene. The case of the target vector containing a neomycin resistance gene, cells into which the vector has been introduced is cultured in a medium containing G418, by selecting the strains of G418-resistant, positive selection to select homologous recombinants containing a neomycin resistance gene it can be carried out. If the DT gene including target vector, culturing the cells transfected with vector, randomly inserted into the chromosome recombinants except (homologous recombination of selecting strain grown in, DT gene on chromosome to express built-, can not grow by the toxicity of DT) by, it is possible to perform a negative selection to select homologous recombinants not containing DT gene. The method for selecting the homologous recombinant of interest from the selected cell lines include Southern hybrida I See Chillon Method for genomic D NA (Morekiyura one 'Cloning, Second Edition), P CR method [CPC Earl. Protocols 's (PCR Protocols), Academic Press (1990)], and the like.

By (b) RDO method, Fei 1,6 of the present invention - Preparation of cells expressing fucosyltransferase variants

Cells expressing the fly-1,6-fucosyltransferase variants of the present invention, the gene of the non-1,6-fucose modifying enzyme which targets, using RDO (RNA-DNA oligonucleotide) method, for example, the following it can be manufactured so.

[0115] To prepare a cDNA or genomic DNA of alpha-1,6-fucose modifying enzyme.

Determining the nucleotide sequence of the prepared cDNA or genomic DNA.

Based on the sequence of the determined DNA, portion encoding the alpha-1,6-fucose modifying enzyme, designed to synthesize the construct of the RDO appropriate length comprising a region or intron portion of the untranslated region.

[0116] Synthesis was RDO is introduced into cells, enzymes that target, i.e. by selecting a cell that mutation occurs in the alpha-1,6-fucose modifying enzyme, the present invention alpha-1,6-fucosyl it can be prepared cells expressing trans Blow over peptidase mutants.

The introduction of RD_〇 into cells can be used a method of introducing the target vector according to (a) above 4..

[0117] Fei - The method for preparing cDNA of the 1,6-fucose modifying enzyme, prepared by the methods for preparing a cDNA placing serial above 1. and the like.

Facial - 1,6 - a method for preparing a genomic DNA encoding the fucose modifying enzyme is prepared by the methods for preparing a genomic DNA described in the above 1. and the like.

Nucleotide sequence of the DNA after cleavage with appropriate restriction enzymes, pBluescript SK (-) was cloned into (Stratagen e Corp.) plasmid such as base sequence analysis method generally used, for example, Jidokishi Sanger (Sanger) et law [Proceedings. O drive. the. National. § mosquito Demi one 'O Bed' Science (Pro Natl. Acad. Sci., USA), 74, 5463 (1977)] was reacted, such as, automatic base sequence analyzer device, for example, it is possible to determine the nucleotide sequence of the DNA by analysis to using A. and F. DNA Shikuensa one (Pharmacia Co., Ltd.).

[0118] RDO can be prepared by known methods or by using a DNA synthesizer.

The RDO introduced into a cell, an enzyme that target, shed 1,6 -. The method for selecting the cells that mutation occurs in the gene of fucose modifying enzyme, Molecular 'Cloning, Second Edition, Current' Protocols in . Molecular. method for detecting directly a mutation in chromosomal genes described in Biology and the like.

[0119] constructs of RDO can Science (Science), 273, 1386, (1996); Neichiya one 'Medicine (Nature Medicine), 4, 285, (1998); to Patoroji (H mark atology), 25, 1462 , (1997);. Gene Therapy (Gene Therapy), 5, 1960, (1999);. Gene Therapy (Gene Therapy), 5, 1960, (1999); journal 'O Breakfast' Molecular 'Medeishin (J. Mol. . Med), 75, 829, (1997);... proceedings 'O Bed-the' National 'Akademi one' O blanking Science (Pro Natl Acad Sci USA), 96, 8774, (1999); Proceedings 'O Bed' The 'National' Academy 'O Bed' science (.... Proc Natl Acad Sci USA), 96, 8768, (1999); (.. Nu Acids Res) Nukurei click 'acid' research, 27 , 1323, (1999); Investor Institut gate Chillon 'O Bed' Damato biology, HI, 1172, (1998) (Invest Dematol..); (. Nature Biotech) Neichiya ^ ~ - biotechnology, 16, 1343, (1998); Neichiya 'Biotech Noroji, 18, 43, (2000) (Nature Bi otech.); (. Nature Biotech) Neichiya ^ ~ Biotechnology, 18, 555, can be designed as described in such (2 000).

By (c) a method using a transposon, Fei invention - Preparation of cells expressing 1,6 fucosyltransferase variants

Fei invention -1,6 - (. Nature Genet) cells expressing fucosyl transferase mutants Neichiya 'Jieneteiku, 25, 35, (2000) Les use a transposon system described in such ,, shed -1 , Ru can be produced by selecting a mutant of 6-fucose modifying enzyme. [0120] The transposon system is a system for inducing a mutation by insertion into the chromosome of a foreign gene randomly, usually, used as a vector for inducing a mutation wherein an exogenous gene interposed between transposons, this simultaneously introducing into the cell an expression vector transposase order to insert randomly into the chromosome of the gene. Transposase anything use even les so long as it is suitable for the sequence of the transposon to be used, it can Rukoto force S.

[0121] As the exogenous gene, so long as it can induce a mutation in DNA of a cell les, les use also Canal gene can Rukoto force S.

The introduction of genes into cells can be used to introduce how the target vector according to (a) above 4..

(2) a-1,6-activity measurement of fucosyltransferase variants

Fei -1 of the present invention, 6-fucosyltransferase variants 及 Bihi -1, 6-fucosyltransferase method for measuring sill transflector Eraze activity, 2 (2) According to the method described, including Murrell present adjustment cells the invention alpha -1, a method of measuring the activity of 6-fucosyltransferase variants thereof.

5. alpha -1 of the present invention, due to the 6-fucosyltransferase variants, the use of DNA or antibody (1) of the present invention alpha -1, 6-fucosyltransferase detect fucosyltransferase expression of the DNA encoding the variant 'quantifying determination method of disease

alpha -1 of the present invention, 6-fucosyltransferase Yore DNA or oligonucleotide de of glycosyltransferase mutants ,, Northern hybrida I See Chillon method (Molecular 'Cloning, Second Edition), PC R method and RT (reverse-transcribed) -PCR method [both PCR Protocols, Academic Pre ss (1990)] (or, together referred to as PCR method), such as a row-,, present invention Fei - the DNA encoding the 1,6 Fukoshirutoran Sufueraze variants by detecting the expression, retinal dysplasia, liver 臓癌, fatty liver, blood coagulation disorders, cystic fibrosis, lung tissue damage, growth failure, diseases involving impairment of insulin-like growth factors and growth hormone action, epidermal growth Fei such accompanied cormorants disease disorders of the action of factors - 1,6 - can and this for judging disease associated with fucose modifying enzyme have been reported.

[0122] RT- for the PCR method is a simple method, Ru particularly useful der as detection of expression of the DNA.

For example, the DN A having a sequence complementary to DNA or the DNA, having the SEQ ID NO: 18, 19, 20, 21 or sequentially with the same sequence as about 100 to 2000 bases were in the nucleotide sequence represented by 22 as a probe It performed Northern hybrida I See Chillon, SEQ ID NO: 18, 19, 20, 21 or is to quantify the expression levels of the DNA of 22, can S be force to determine the constant of the disease by comparing the healthy person .

[0123] As a specific method for the determination, for example, it may be mentioned the following method.

Subjects and healthy subjects of white blood cells or tissue from total RNA (10~20 μ g), or their ΠΙΚΝΑ the (1~5 Α ^), denaturing solution [50% (ν / ν) formamide, 2. 2 mol / L e Honoré Mua Honoré dehydrogenase, 20 mmol / LM_〇_PS [3 _ (N-morpholino) propanesulfonic acid] (P H7. 0), 5mmol / L sodium acetate, at lmmol / L EDTA] in, 65 ° C 5 heated minutes, denatured, electrophoresed on a 1% Agarosugeru containing 2. 2 mol / L formaldehyde.

[0124] After electrophoresis, the RNA in the gel nitrocellulose filter; blotted onto (Optimal BA-S85 manufactured by Schlei cher & Schuell, Inc.), immobilized by heating under reduced pressure for 1 hour 80 ° C. The filters hybrida I See Chillon solution [5 X SSPE (750mmol / L NaCl, 50 mmol / L NaH P_〇, 5mmol / L EDTA;. PH7 4), 5 X Denhardt's solution (0.1%

twenty four

Fuikonore, 0.1% poly Bie Honoré pyrrolidone, 0.1% © shea serum albumin), 1% SDS (de de Sil sodium sulfate), in 0. 2 mg / ml salmon sperm DNA (Pharmacia Biotech Inc.)] perform dipped prehybridized da I See Chillon.

[0125] After pre hybrida I See Chillon, the probe was added to the solution, performing Haiburidize Shiyon at 65 ° C.

The probes may for example SEQ ID NO: 18, 19, 20, 21 or multi-prime DNA labeling system The DNA fragment according to 22 use those labeled with 32 P (manufactured by Amersham).

[0126] the filter after hybrida I See Chillon and washed in the following order.

(A) 0. 2 X SSC (300mmolZL NaCl, 30mmol / L Taen acid sodium © beam) containing 1% SDS solution and washed for 15 minutes at room temperature. I repeated this several times. (B) 0. l X SSC (150mmol / L NaCl, 15mmol / L Taen acid sodium © beam) containing 1% SDS solution and washed for 15 minutes at 50-70 ° C. I repeated this several times.

(C) 50~70. C 0. 0. 1 X SSC containing 1% SDS (15mmol / L NaCl, 1. 5mmol / L sodium Kuen acid) solution, washed 15 minutes at 50-70 ° C. I repeated this several times.

[0127] After washing the filters, the expression of the DNA of the autoradiography in row-,, Ba I O sequences Imaging Analyzer BAS2000 (Fuji Photo Film Co., Ltd.) No. 18, 19, 20, 21 or 22 by using an imaging plate detected can be quantified.

Further, for example, flying of the present invention 1,6 - fucosyl use Le ,, subjects and from healthy persons white blood cells or tissue as primers specific set of oligonucleotides DN A encoding fucosyltransferase variants total RNA, their mRNA or their RNA forces, PCR was carried out with cDNA were et prepared as 铸型, the amplified fragment detected 'quantified, determines the disease by comparing the expression amount of the DNA of the subject and a healthy person can do.

[0128] Oligonucleotides can be used oligonucleotides 1 above described.

mRNA or total RNA becomes 铸型 of PCR from the separation 'acquired various leukocyte cells blood, or a tissue can be extracted from tissue or the like with a suspected disease.

As each of white blood cells, polymorphonuclear leukocytes, monocytes, lymphocytes, T cells, can this a force S to increase the B cells and the like.

[0129] polymorphonuclear leukocytes and mononuclear cells, from the peripheral blood of the subject, 'by using Polymo 卬 Hprep ™ is Pharma (Nycom ed Pharma) manufactured kit, separation' Nycomed and child acquires can.

From the obtained monocytes, J. Immunol., 130, 706 according to the method described in (1983), etc., monocytes Contact and lymphocytes, Tissue Antigen, 9, 153 (1977), J. Immunol, 11, 273 (1976), by the method described in manuals relating to the isolation method of Nycom ed's blood cells, separating the T cells and B cells' can be obtained.

[0130] T cells nylon wool method [Eur. J. Immunol., 3, 645 (1973)] can also be obtained using. Moreover, T-cells, B-cells, using monocytes / macrophages magnetic beads combined binding antibodies specific respectively (e.g., Dynabeads manufactured by Dynal, Inc.), can be force S each cell separation 'to get. The methods for preparing total RNA from leukocytes or tissues, Chioshian acid Guani Jin one Torifuruoro cesium acetate method [Methods in Enzymol., 154, 3 (1987)] and the like can be this a force S and the like.

[0131] The methods for preparing poly (A) + RNA from total RNA, oligo (dT) immobilized cellulose column method (Molecular Cloning, Second Edition) and the like.

In addition, fast 'track' mRNA 'isolation' kit [Fast Track mRNA Isolati on Kit; manufactured by Invitrogen Corp.], quick-prep 'mRNA' pin lily Fi cable Chillon 'kit (Quic k Prep mRNA Purification Kit; manufactured by Pharmacia Inc.) Chide monkey to prepare a mRNA using a kit of equal.

[0132] single-stranded cDNA from the total RNA or mRNA, using single-stranded cDNA synthesis kit Superscript preamplification system (BRL Co.), can be synthesized. The synthesis can be carried out in the kit according to the attached manual.

The above thus prepared may total RNA, mRNA or cDNA using RT- PCR method by [PC R Protocols, Academic Press (1990)], encoding a _1,6_ fucosyl trans Blow over peptidase variants of the invention it is possible to quantify the expression level of a gene.

(2) method for determining disease by detect only gene encoding alpha-1,6-fucosyltransferase variants of the invention

Oligonucleotides alpha-1,6-fucosyltransferase variants of the present invention as a probe, Southern hybrida I See Chillon method on genomic DNA (Molecular 'claw-learning Third Edition), by performing PCR method , it is possible to detect only the mutation in the gene encoding the alpha-1,6-fucosyltransferase variants of the present invention. Detection method, alpha -1, 6- fucose modification associated with the enzyme have been reported, retinal dysplasia, liver cancer, fatty liver, blood coagulation disorders, cystic fibrosis, lung tissue damage, growth failure, insulin it can be utilized like growth factors and diseases involving disorders of growth hormone action, the determination of diseases, such as diseases involving disorders of the effects of epidermal growth factor.

[0133] Specifically, for example, the coding region of the DNA encoding the fly-1,6-fucosyltransferase variants of the invention the patient's amplified by PCR, and sequenced, sequence number by comparing the nucleotide sequence of the DNA represented by 3, it is possible to check for mutations in the coding region.

cDNA as a 铸型 subjected to PCR method can be obtained by the method described in (1). Nucleotide sequence of the obtained cDNA, the Perkin Elmer one company of DNA Shikuensa one 377 and reaction kit (ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit: Appli ed Biosystems, Inc.) can be determined using.

(3) determining for diseases due to immunological detection method and quantification method

Fei present invention 1,6 - using an antibody against fucosyltransferase variants, Fei of the invention in the blood or tissue of a subject 1,6 - the presence or absence of the expression of fucosyltransferase transflector We hydrolase variant immunologically detecting or quantitatively, as possible out to determine the following disease by comparing the amino acid represented by SEQ ID NO: 9 and composition ratio 1,6-fucosyltransferase.

[0134] As a method for immunologically detecting, it can be mentioned ELISA method using a microtiter plate, fluorescent antibody method, Western blot method, the immunohistochemical staining method.

As a method of immunologically quantified, for example Epitopu of antibodies reactive alpha _1,6- Fukoshirutora transferase in variants specifically the present invention in the liquid phase were used two different monoclonal antibody sandwich ELISA method, radio I Takeno assay I method using a recognizing labeled RA-associated polypeptide and the polypeptide antibodies and the like with a radioisotope such as 125 1.

[0135] the immunological method is associated with the alpha-1,6-fucose modifying enzyme have been reported, retinal shaped formation disorders, liver cancer, fatty liver, blood coagulation disorders, cystic fibrosis, lung tissue failure can be utilized poor growth, diseases involving impairment of insulin-like growth factors and growth hormone action, the determination of the disease, such as diseases involving disorders of the effects of epidermal growth factor.

(4) Fei of the present invention 1,6 - fucosyltransferase screening method for a substance that controls the functions of the variant

Retinal dysplasia, liver cancer, fatty liver, blood coagulation disorders, cystic fibrosis, lung tissue damage, growth failure, diseases involving impairment of insulin-like growth factors and growth hormone action, a failure of the effect of epidermal growth factor in diseases such as diseases involving, shed 1,6 - associated with dysfunction of fucose modifying enzyme is suggested. [0136] Thus, also inhibit the function of the alpha _1,6- fucosyltransferase variants of the invention be enhanced is considered effective in the prevention and treatment of the disease. Further, even diseases that functions not attributable unusually direct alpha-1,6-fucose modifying enzyme, symptomatic by inhibiting or enhancing the function of alpha-1,6-full waist transferase variants of the invention manner can be used to prevent or treat the disease.

[0137] Accordingly, Fei-1,6-fucosyltransferase variants of the present invention because, when there is a patient physiological actions of cells is diminished or enhanced, Fei-1,6-fucosyl transformer luciferase of the present invention by administering a compound to control the function of the mutant can control the physiological action, Matahi 1,6 - functional changes fucose modifying enzyme, such a direct cause of the disorder without having the present invention diseases to symptomatic treatment by controlling the function of Nohi 1,6 fucosyltransferase variant can also be prevented and treated by administration of the compound.

[0138] The compounds can be obtained by the method shown below, for example.

[I] and cells expressing the alpha-1,6-fucosyltransferase variants of the present invention, the presence of [ii] test substance, the cells expressing alpha _1,6- fucosyltransferase variant of the invention, 2 above cultured for 2 hours to 1 week by the culture method described in, the alpha-1,6-fucosyltransferase trans Hue hydrolase mutants detect cellular responses caused by works, compared, the alpha-1,6 - substances selected to get with Fukoshi Honoré transferase variant activity to inhibit or enhance the function of.

[0139] As a method for detecting cells response generated by the function alpha-1,6-fucosyltransferase variant of the invention, for example, expressing the alpha-1,6-fucosyl transferase peptidase variants of the invention reactivity change to lectin of cells, insulin-like growth factor, growth hormone, dependent intracellular signaling in epidermal growth factor gene transcription, preparative sugar interrupt, known methods for detecting a change such as proliferation and the like (J. Biol Chem, 276, 1 1956, 2001;... J. Biol Chem, 275, 21988, 2000;. Molecular Medicine, 40, 1034, 2003; Nature, 263, 663, 1976; clinical science , 17, 958, 1981).

[0140] As the lectin, for example, a lectin that recognizes a 6-position and 1 Kuraigahi bound sugar chain structure of fucose N- glycoside-linked sugar chain of N- § cetyl Darco Sami emissions and the like.

N - N of the glycosidic bond sugar chain - The § cetyl Darco Sa Min 6-position and 1-position lectin recognizes a sugar chain structure bound a fucose, if a lectin capable of recognizing the sugar chain structure, any it can also be used in the lectin. As specific examples, the end © lectin PSA (Pisum sativum derived nea lectin) (lentil agglutinin derived from Lens culinaris) Ren Zumamerekuchin LCA, broad bean lectin VFA (Vicia faba derived agglutinin), Hyi b Cha Wan aurantia lectin AAL (Aleuria aurantia pharmaceutical comprising the compound obtained in the search method n which lectin derived from), and the like (5) (4)

Fei present invention 1,6 - compounds that regulate the function of fucosyltransferase variants shed - associated with dysfunction of the 1,6-fucose modifying enzyme is suggested, retinal dysplasia, liver cancer, fatty liver, blood coagulation disorders, cystic fibrosis, lung tissue damage, growth failure, diseases involving impairment of insulin-like growth factors and growth hormone action, preventive agent for diseases such as diseases involving disorders of the effects of epidermal growth factor and treatment it is useful as a medicine.

[0141] (4) The compound obtained in can be provided as a pharmaceutical preparation produced by any of the methods well known in the technical field of pharmaceutics shown below, for example.

As a method of administration of the prophylactic and therapeutic agent, for example, when the normal physiological action by dysfunction due to alpha-1,6-Fukoshiruto lance Feller peptidase variants of the present invention there are patients who can not be expected, (i) the be DNA that encodes a protein that controls the functions of the alpha-1,6-fucosyltransferase variants of the invention is administered to the patient the expression (ii) alpha-1,6-fucosyltransferase of the present invention to cells of interest after the proteins that control the functions of the variant is inserted DNA encoding expression, transplanting the cells to the patient, there Les, the (iii) alpha-1,6-fucosyltransferase variants of the present invention such as by administering the protein or compounds to control the function of the body to the patient, it is possible to change the function of the alpha-1,6-fucosyltransferase variants of the present invention in the body of a patient. Thus, shed - 1,6-fucose modifying functional diseases caused by abnormal enzyme, Aruiwahi - without directly attributable to 1,6-fucose modifying enzyme, the present invention Fei - 1,6 fucosyltransferase of fucosyltransferase variants prophylactic and therapeutic for diseases that can be symptomatic treatment by administration of proteins that control DNA, or the function of the flight-1,6-fucosyl trans Blow over peptidase variants of the present invention encodes a protein that controls the functions it is useful as a medicine.

[0142] Fei 1,6 of the present invention - if the DNA the protein to code that controls the functions of the fucosyltransferase variants used as the prophylactic and therapeutic agent, Fei invention - 1,6 fucosyltransferase variants alone or retroviral vector DNA of proteins that control the functions of the adenoviral vector, was 揷入 into an appropriate vector such as adenovirus § associations Ted Huy Noresubetata, manufactured according to a conventional method described below Zaika, may be formulated and administered.

[0143] retroviruses, the use of gene therapy vectors incorporating the viral vector or other vectors one for gene therapy, such as adenovirus as a gene therapeutic or prophylactic agent is used to the gene therapy vector and gene therapy agents can be prepared by formulating the base [Nature Genet., 8, 42 (1994)] 0

The base used in the gene therapy agent may be any one so long as it is a base to be used in the normal injection, distilled water, sodium chloride or sodium chloride salt, such as a mixture with mineral salt solutions, mannitol, Ratatosu, dextran, sugar solutions such as glucose, glycine, amino acid solution such as § arginine, a mixed solution or the like with an organic acid solution or salt solution and a glucose solution. The conventional method, osmotic agent into these groups, pH adjusting agents, sesame oil, using the aid of a surfactant such as a vegetable oil or lecithin or nonionic surfactants soybean oil, solution, suspensions, injections may be prepared as a dispersion. These injections, powdered, as possible out also be prepared as dissolution formulation before use by operating the freeze-drying. Gene therapy agents can be used as such in the case of liquid, in the case of an individual can be used to treat dissolved immediately prior to gene therapy in the above base was sterile processing necessary. As a method of administration of the gene therapy agent may be mentioned a method of administering such, locally absorbed into the treatment site in the patient.

[0144] Further, it this a force S to transport DNA into the treatment site by non-viral gene transfer method of interest.

The known non-viral gene transfer methods in the art, calcium phosphate co-precipitation. [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)], membrane fusion via Liposomes one arm - mediated transfection method [Pro Natl Acad Sci USA, 84, 7413-7417 (1987); Biochemistry, 28, 9508-9514 (1989); J ... Biol Chem, 264, 12126-12129 (1989);. Hum Gene Ther "3, 267-275, (1992); Science, 249, 1285-1288 (1990); Circulation, 83, 2007-2011 (1992 )] or direct DNA uptake and receptor - mediated DNA transfer method [Sc ience, 247, 1465-1468 (1990);..... J. Biol Chem, 266, 14338-14342 (1991); Proc Natl Acad Sci . USA, 87, 3655-3659 (1991);.. J. Biol Chem, 264, 16985-16987 (1989); BioTechniques, 11, 474-485 (1991);... Pro Natl Acad Sci USA, 87, 3410-3414 (19 90); Pro ... Natl Acad Sci USA, 88, 4255-4259 (1991);.... Proc Natl Acad Sci USA, 87, 4033-4037 (1990);... Pro Natl Acad Sci USA, 88, 8850-8854 (1991);.. Hum G ene Ther, 3, can be force S mentioned 147-154 (1991)] and the like.

[0145] membrane fusion via ribosome - by administering directly into the tissue to a ribosome preparations with interposed transfer methods targeting are possible localized gene incorporation and expression of the tissue in the study on tumor It has been reported [Hum. Gene Ther., 3, 399 (1992)].

The pharmaceutical containing the protein or compounds to control the functions of the alpha-1,6-fucosyltransferase variants of the present invention as an active ingredient, said that the active ingredient to be administered alone some also possible force usually the active ingredient mixed with one or more carriers that are pharmacologically acceptable, it is desirable to provide a pharmaceutical formulation manufactures by any method well known in the technical field of pharmaceutics. Preferably water or saline, glycine, Gunorekosu, sterile solution dissolved in an aqueous carrier such as an aqueous solution, such as human albumin use les are. Further, Unaryo buffer I human agents and isotonizing agents to approximate formulation solution to physiological conditions, pharmacologically acceptable additives, for example, sodium acetate, sodium chloride, lactic acid sodium, potassium, It may be added sodium Kuen acid. Also, stored and freeze-dried, it may be used by dissolving in an appropriate solvent when in use.

[0146] The route of administration, the most effective ones to use is desirable tool oral administration, Oh Rui oral cavity upon treatment, the airway, intrarectal, subcutaneous, parenteral administration intramuscular and intravenous etc. up it can Rukoto force S. The dosage form includes sprays, capsules, tablets, granules, syrups Qi 1 J, emulsions, suppositories, injections, ointments, tapes and the like.

Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like. For example, liquid preparations such as emulsions and syrups are water, sucrose, sorbitol, sugars fructose, etc., polyethylene glycol, glycol ethers such as propylene glycol, sesame oil, Oribu oil, oils such as soybean oil, P- preservatives such as hydroxybenzoate esters can be prepared using strawberry flavor, and the additive flavors such as peppermint. Capsules, tablets, powders, granules and the like are lactose, dextrose, sucrose, and mannitol, etc., starch, disintegrants such as sodium alginate, magnesium stearic phosphate, lubricants such as talc, poly Bulle alcohol , hydroxypropyl cellulose, binders such as gelatin, can be prepared by using surfactants such as fatty acid esters, plasticizers such as glycerin or the like as an additive.

[0147] Suitable formulations for parenteral administration include injections, suppositories, sprays and the like. For example, injections are prepared using a salt solution, a carrier or the like comprising a mixture of a glucose solution, or both. Suppositories can be prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid. Further, propellant protein or the compound itself, or does not stimulate the recipient's mouth contact and respiratory mucosa, and using a carrier or the like for facilitating the absorption by dispersing the protein, or the compound as fine particles prepared to. The carrier includes lactose, glycerine, and the like. The nature of the carrier used protein or said compound and, air Rozonore, it is possible to produce pharmaceutical preparations such as dry powders. It is also possible to add the components exemplified as additives also oral agent in the parenteral preparations.

[0148] dose or frequency of administration, the desired therapeutic effect, administration method, a treatment period, age, different force S by the body weight or the like, an adult per day 10 / ig / kg~8mg / kg.

6. production of glycoprotein pharmaceuticals using cells having alpha-1,6-fucosyltransferase variants of the invention

In cells with a flight-1,6-fucosyl transflector We hydrolase variants of the present invention, since the activity of the non-1,6-fucose modifying enzyme is deleted or is decreased, sugar is produced in the cell protein Wahi - 1,6 - glycosylated by fucose modifying enzyme is deleted or has decreased.

[0149] Thus Nahi 1,6 - phase and fucose modifying enzyme glycosylation by deletion or decreased glycoprotein, a change in the hemodynamic and distribution in vivo, proteins required for pharmacological activity expression has changed for interaction, it is useful as pharmaceuticals.

Examples include antibodies, erythropoietin, thrombopoietin, tissue-type plus plasminogen § Chi beta, Purourokinaze, thrombomodulin, antithrombin II I, protein your blood clotting factor VII, blood clotting factor VIII, blood coagulation factor IX, blood clotting factor X, gonadotropin, thyroid stimulating hormone, epidermal growth factor (EGF), hepatocyte increase 殖因Ko (HGF), keratinocyte growth factor, Akuchibin, osteogenic factors, stem cell factor (SCF), interferon shed, interferon beta, interferon gamma, interleukin 2, interleukin 6, interleukin 10, interleukin 11, soluble interleukin down 4 receptor, non-tumor necrosis factor, Dnasel, galactosidase, shed Darukoshidaze and Darko cerebrosidase the like .

[0150] because the fucose modification has a deletion or reduced sugar chain structure, as more specific examples of glycoproteins their physiological activity is significantly increased, for example, antibody. Hereinafter, an example production of the antibody composition, showing a method for manufacturing a glycoprotein composition using cells with alpha-1,6-fucosyltransferase variant mutant of the present invention.

Antibody compositions Morekiyura one 'Cloning, Third Edition, Current' Protocols 'in' mode Rekyufu * Nono Ioroshi one, in Antibodies, A Laboratory manual, and old Spring Harbor La boratory, 1988 (hereinafter referred to as anti-body's) , Monoclonal Antibodies:. principles an d practice, Third Edition, Acad Press, 1993 (hereinafter referred to as monochromatic over nano Les anti body's), Antibody Engineering, a Practical Approach, IRL Press at Oxford University Press, 1996 (hereinafter, anti-body use the method described in engineering and abbreviated), and the like ,, for example, may be obtained by expressing in cells expressing alpha-1,6-fucosyltransferase variants of the present invention as follows.

[0151] To prepare the cDNA of the antibody molecule.

It prepared a full-length cDNA of the antibody molecules based on, if necessary, to prepare a DNA fragment of appropriate length comprising a region that encoding a protein.

By 揷入 the DNA fragment or full-length cDNA, downstream of a promoter in an appropriate expression vector to prepare a recombinant vector.

[0152] The recombinant vector, Fei present invention suitable for the expression base Kuta one - by introducing the 1,6-fucosyltransferase trans luciferase expressed mutant cells, transformant 換個 which produces the antibody molecule it is possible to obtain.

alpha _1,6- The fucosyltransferase expressed fucosyltransferase variant cells, force may be used bacteria, yeast, animal cells, insect cells, plant cells, etc., also Re Baise long as it can express the gene of interest preferably, animal cells and the like.

[0153] The enzyme relating to the modification of Ν- glycoside-linked sugar chains bound to the Fc region of an antibody molecule is introduced using a gene engineering techniques, bacterial, yeast, animal cells, insect cells, cells such as plant cells It can also be used.

As cells used in the production method of an antibody composition of the present invention, prepared in the above 2 or 4.0 may Rukoto force S mentioned cells expressing fly-1,6-fucosyltransferase variants of the invention .

[0154] cDNA is Rukoto be prepared using from tissues or cells of 従Re ,, human or non-human animal to a method for the preparation of the cDNA described above: 1. The probe primer specific for the antibody molecule of interest It can force S.

The various cells Niore, as a specific method for producing the antibody composition Te, method of constructing the expression vector of the 2 wherein, the method introducing the expression vector into cells, cells culture methods, objects production it is possible to increase the purification method of the object.

[0155] introduced a gene involved in the synthesis of a sugar chain, bacteria, yeast, animal cells, antibodies insect cell or when expressed by the plant cell or the like, which is added a sugar or a sugar chain by the introduced gene it is possible to obtain a molecule.

As acquired the antibody compositions include, for example, antibodies, fragments of antibodies, fusion proteins with an Fc region of the antibody, and the like.

[0156] Hereinafter, as an example for obtaining the antibody composition, Hitoi 匕抗 AND PROCESS glycoproteins force other antibody compositions, such as referred method of manufacturing the above-described composition of the Fc fusion protein and It can also be obtained according to the method.

A. Preparation of humanized antibody composition

(1) Construction of humanized antibody expression vector

The humanized antibody expression vector, genes encoding CH and CL of a human antibody is an expression vector for embedded or animal cells, the genes encoding CH 及 beauty CL of a human antibody into an animal cell expression vector it can be constructed by cloning each

[0157] As the C region of human antibody may be CH and CL of any human antibody, e.g., the IgGl subclass of human antibody H chain C region (hereinafter referred to as "hC 7 1") and human antibody L chain / c class C region (hereinafter referred to as "hC kappa") and the like.

As the genes encoding CH and CL of a human antibody can be used chromosomal DNA consisting Ekison intron, also, cDNA and can Yore, also Rukoto.

[0158] As the expression vector for animal cells, inserted may be any as long as it can be inserted and expressed the gene encoding the C region of a human antibody. For example, PAGE 107 [Site Technology (Cytotechnology), 3, 133 (1990)], pAGE103 [Journal 'O Bed' bio chemistry (J. Biochem.), 101, 1307 (1987)], pHSG274 [Gene (Gene ), 27, 223 (19

84)], pKCR [Proceedings O blanking THE Nashonanore-Akademi one 'O blanking Science (

Proc. Natl. Acad. Sci. USA), 78, 1527 (1981)], pSG l β d2_4 [site technology (C ytotechnology), 4, 173 (1990)], and the like. The flop opening motor and Enhansa one in the expression vector for animal cell, SV40 early promoter and Enhansa of the journal 'O Bed' Biochemistry (J. Biochem.), Dishes, 1307 (1987)], Moroni one mouse leukemia LTR Lek Iokemikaru 'and' Biophysical 'research' communication sheet Yonzu disease virus (Biochem. Biophys. Res. Commun.), 149, 960 (1987)], immunoglobulin H chain promoter [cell (cell), 41 , 479 (1985)] and Enhansa cell (cell), 33, 717 (1983)

] And the like.

[0159] vector for expression of humanized antibody, the type present in the data drive or on the same vector that antibody H chain and L chain exist on separate vectors (hereinafter, referred to as tandem) using any throat fliers power Hitoi 匕抗-expressing ease of construction of the vector, easiness of introduction into animal cells, a tandem from the viewpoint of the balance between the expression amounts of antibody H and L chains in animal cells is balanced human can reduction is preferably towards the antibody expression vector [journal O Bed • Imunorojikaru 'Mesozzu (J. Immunol. Methods), 167, 271 (1994)].

[0160] humanized antibody expression vector constructed may be used for expression in animal cells of the human chimeric antibody and a human CDR-grafted antibody.

(2) Acquisition of cDNA encoding the V region of an antibody derived from a non-human animal

Non-animal antibody human, eg, cDNA encoding VH and VL of the mouse antibody can be obtained as follows.

[0161] mRNA is extracted from hybridoma cells producing the mouse antibody of interest to synthesize cDNA. The synthesized cDNA is cloned into a vector such as a phage or a plasmid to obtain a cDNA A library. From the library, the recombinant containing the cDNA encoding the recombinant phage some have a recombinant plasmid and VL having a cDNA encoding the use Le ,, VH a C region part or V region part of an existing mouse antibody as the probe phage or recombinant plasmid is isolated, respectively. Full nucleotide sequences of VH and VL of the mouse antibody of interest on the recombinant phage or recombinant plasmid, to estimate the total Amino acid sequences of VH and VL from the nucleotide sequence.

[0162] As the animals other than human, mouse, rat, hamster, Usagi like, if it is possible to produce a High Priestess dormer cells, can also be used Les made or not.

The method for preparing total RNA from High Priestess dormer cells Chioshian acid guanidine Torifuruoro cesium acetate method [Mesozzu 'in' Enzaimoroji one (Methods in Enzymol.), 154. 3 (1987)], also for preparing mRNA from total RNA as a method, an oligo (dT) immobilized cellulose column method [Molecular ^ ~ cloning: § 'Laboratory' manual. (Molecular and lonmg: a Laboratory manual), and old Spring Harbor Lab Press New York, 1989 "and the like can give. Further, examples of a kit for preparing mRNA from High Priestess dormer cells, Fast Tr ack mRNA Isolation Kit , Quick Prep mRNA Purification Kit (Pharm acia Co., Ltd.), and the like.

[0163] Examples of synthetic and cDNA library one method of producing cDNA, a conventional method [Morekiyura one 'Claw-learning: §' Laboratory 'Ma two Yuanore. (Molecular Cloning: A Laboratory Manual), Cold Sp ring Harbor Lab Press New York , 1989; the current 'proto Kono lesbian' in 'Molecular' Noioroji 1 ~ (current Protocols in MolecularBiology) , Supplement 1-34], Arure, f or巿sales of kit, f Retsue is ,, Super Script Plasmid System for such as cDNA Synthesis and Plasmid Cloning (GIBCO BRL Co., Ltd.) and ZAP-cDNA Synthesis Kit (Stratagene, Inc.) how to use, and the like.

[0164] In preparing the cDNA library, the vector into which a cDNA synthesized using mRNA extracted from a High Priestess dormer cells as 铸型, said cDNA can Rereru possible force S use be composed squid if vector incorporate. For example, ZAP Express [Strategies (Strategies), 5, 58 (1992)], pBluescript II SK (+) [Nucleic 'Ashizzu' research (Nucleic Acids Rese arch), 17, 9494 (1989)], λ zap II ( Stratagene, Inc.), gtlO, λ gtl l [Denue cloning:.. § 'Practitioner Atlantica Norre approach (DNA cloning: A Practical approach), I, 49 (1985)], Lambda BlueMid (Clontech Co., Ltd.), λ ExCell, pT7T3 18U (Pharmacia Inc.), PCD2 [Molecular 'and' cellular 'Biology (Mol. Cell. Biol.), 3, 280 (19 83)] and pUC18 [Gene (Gene), 33, 103 (1985)], etc. It is used.

[0165] Phage or introducing the cDNA library as E. coli for introducing the cDNA library constructed by a plasmid vector can be used as long as it can express and maintain record, even made whether. For example, XLl-Blue MRF '[Strategies (Strategies), 5, 81 (1992)], C600 [Genetics (Genetics), 39, 440 (1954)], Y1088, Y1090 [sciences (Science), 222, 778 (1983)], NM522 [journal 'O Breakfast' Molecular 'bio-opening Gee (J. Mol. Biol.), 166, 1 (1983)], K802 [journal' O Breakfast 'Molecular ^ ~ Bio opening Gee ( J. Mol. Biol.), 16, 118 (1966)] and JM105 [Gene (Gene), 38, 275 (1985)] or the like is used.

[0166] As a method for selecting a cDNA clone encoding the VH and VL of an antibody of an animal other than human from the cDNA library, isotope or fluorescence-labeled colonies using probes' hybrida I See Chillon method or plaque 'hybrida I See Chillon method [Morekiyura one' cloning: § 'Laboratory' Ma two Yuanore. (Molecular Cloning: A Laboratory Manual), C old Spring Harbor Lab Press NewYork, 1989] makes it possible to select. Further, a primer was prepared and cDNA synthesized or cDNA libraries from mRNA as 錡型, Polymerase Chain Reaction [hereinafter referred to as PCR method; Molecular 'Cloning: §' Fuhofutori ■ ~ 'Manyusunore (Molecular Cloning: A . Laboratory Manual), Cold Spring Har bor Lab Press New York, 1989; current 'Protocols' in 'Molecular' Biology (current Protocols in Molecular Biology), preparing cDNA encoding VH and VL by Supplement 1-34] it is also possible to.

[0167] The cDNA selected by the above method, after cutting with appropriate restriction enzymes, pBluescript SK (-) was cloned into a plasmid (Stratagene, Inc.) and the like, typically nucleotide sequences solution 析方 method used, for example, Sanger (Sanger) et al. Jidokishi method [Proceedings Ding Das 'O Breakfast' the 'National Akademi ~ · O Breakfast' Science (Pro Natl. Acad. ScL, USA), 74, 5463 (1 977)] to carry out the reaction such as , nucleotide sequence automatic analyzer, for example, it is possible to determine the nucleotide sequence of the cDNA by analyzing using ALF DNA Shikuen server (Pharmacia Co., Ltd.).

[0168] estimate the whole amino acid sequence of VH and VL from the determined nucleotide sequence, the total amino acid Hai歹 IJ of known antibody VH 及 beauty VL [Shikenshizu 'O Bed' Proteins' O Bed 'Imunorojikaru' Inn Taresuto ( sequences of Proteins of Immunologicailnterest), by comparing the Ub Dept. Health and Hum an Services, 1991], check obtained cDNA encodes the full amino acid sequences of VH and VL of the antibody comprising a secretory signal sequence can do.

(3) Analysis of the amino acid sequence of the V region of an antibody derived from a non-human animal

For a complete amino acid sequence of VH and VL of the antibody containing the secretion signal sequence, the entire amino acid Hai歹 IJ of VH and VL of the already known antibody [Shikenshizu 'O Breakfast' Proteins' O Breakfast 'I Munoron sword Norre' Interest (sequences of Proteins oflmmunological Interest), by comparing the US Dept. Health and Human Services, 1991], we can estimate the length and N-terminal amino acid sequence of the secretion signal sequence, that even know Sabugunorepu they belong it can. In addition, also for the amino acid sequence of each CDR of VH and VL, the amino acid sequence of a known antibody VH and VL [Shikenshizu, O blanking Proteins, O blanking-Imunorojikaru 'in Taresuto (Sequences of Proteins of Immunological Interest), Us Dept. Healtn and Hum an Services, can be found by comparison with 1991.

(4) Construction of human chimeric antibody expression vector

Upstream of the gene that encoding a human antibody CH and CL of the humanized antibody expression vector described in (1) of this section 2, the cDNA encoding VH and VL of a non-human animal antibody claw and Jung , it is possible to construct a human chimeric antibody expression vector. Eg, cDNA, and the distal 5 'CH and CL of the base sequence with a human antibody of the distal 3' antibody VH and VL of non-human animals nucleotide sequences encoding VH and VL of a non-human animal antibody It consists of a and suitable restriction linked recognition sequence, respectively a synthetic DNA having at both ends of the enzyme, encoding CH and CL of a human antibody of the vector for humanized antibody expression described respectively in (1) the above 2 these upstream of to that gene is cloned for expression in an appropriate form, it is possible to construct a human chimeric antibody expression vector.

(5) Construction of cDNA encoding V region of human CDR-grafted antibody

cDNA encoding VH and VL of human CDR-grafted antibody can this a force S be constructed as follows. First, select the amino acid sequence of the framework of the VH and VL of a human antibody for grafting CDR of VH and VL of a non-human animal antibody of interest (hereinafter referred to as FR). The amino acid sequence of FR of VH and VL of a human antibody, so long as they are derived from a human antibody, inserted may be any. For example, Protein Data Bank or the like is registered in the database of record, Ru of FR of VH and VL of a human antibody amino acid Hai歹 1 J, a common amino acid Hai歹 U [Shikenshizu of each subgroup of FR of VH and VL of a human antibody. O drive. proteins. O Breakfast 'I Munoron sword Norre' Interest (Sequences of proteins of Immunological Interest), US Dept. Although Health and human Services, 1991], and the like, among them, the human form with a sufficient activity to generate CDR grafted antibodies, Nozomu be selected § amino acid sequence having acid sequences having high homology of FR of VH and VL of a non-human animal antibody of interest (at least 60% or more) better Les,.

Next, grafted amino acid sequences of CDR of VH and VL of the antibody VH and VL FR animal amino acid sequence other than the target human of a human antibody selected, the amino acid sequences of VH and VL of human CDR-grafted antibody the design. The frequency of use of codons seen the amino acid sequence was designed on the nucleotide sequence of the antibody genes [Shikenshizu 'O Breakfast' Proteins' O Breakfast 'Imunorojikaru' I Ntaresu Bok (Sequences of Proteins of Immunological Interest), US Dept. Health and H considering UMAN Services, 1991] is converted into DNA sequences, to design DNA sequences encoding the amino acid sequences of VH and VL of a human CDR-grafted antibody. Based on the designed DNA sequences, one 00 base several synthetic DNA consisting length force behind the synthesis, and PCR is carried out using them. In this case, the length of the reaction efficiency and can be synthesized DNA in PCR, Shi preferred to design the H chain, L chain that six synthetic DNA les. [0170] Further, in Rukoto to introduce a recognition sequence for a suitable restriction enzyme at the 5 'end of the synthetic DNA located at both ends, readily Kuroyungu the humanized antibody expression vector constructed in the above 2 (1) It can be Rukoto force S. After the PCR, the amplified product pBluescript SK (-) was Kuroyungu the plasmid of (Stratagene Co., Ltd.), by the method described in the above 2 (2), to determine the nucleotide sequence of the desired human CDR-grafted antibody It acquires plasmid having the DNA sequence encoding the amino acid sequence of VH and VL of.

(6) Modification of amino acid sequence of V region of human CDR-grafted antibody

Human CDR-grafted antibody, the only transplanted with only CDR of the antibody VH and VL of a non-human animal of interest FR of VH and VL of human antibodies, antigen-binding activity of the original non-human animal it is known that lowered as compared with the antibody-les, Relais I O / technology (BIO / TE CHNOLOGY), 9, 266 (1991)]. This is probably because in VH and VL of an animal other than the original human antibody, not CDR only, FR Les, the amino acid residues of several have been directly or indirectly relate to antigen-binding activity, with the implantation of these amino acid residues are CDR, les it is considered varies to a different amino acid residues of FR of VH and VL of a human antibody, Ru. To solve this problem, in human CDR-grafted antibody, in amino acid sequence of FR of VH and VL of a human antibody, an amino acid residue of the amino acid residues or CDR that directly relates to binding to an antigen group or interact with, and maintaining the three-dimensional structure of an antibody, indirectly involved in binding to antigen were identified les, Ru amino acid residues, amino acids found in antibodies of their original non-human animal modified residues have been made to increase the reduced antigen binding activity [Bio / technology (BIO / TECHNOLOGY), 9, 266 (1991)].

[0171] In the production of a human CDR-grafted antibody, or to identify their antigen binding activity Amino acid residues of the FR relating to how efficiently the most is an important point, X-rays crystal analysis for the [ journal 'O Bed' Morekiyura one 'Biology (J. Mol. Biol.), 112, 535 (1977)] some Rere is computer modeling [protein. engineering (protein engineering), the antibody according to 7, 1501 (1994)], etc. construction and analysis of the three-dimensional structure have been made. Information of the three-dimensional structure of these antibodies has been a lot of useful information for the preparation of human CDR-grafted antibody, no method for producing any antibody adaptable human CDR-grafted antibody is established yet and not not, at present to produce several variants for each antibody is required various attempts, such as to consider the correlation between their respective antigen binding activity.

[0172] Modification of the amino acid sequence of FR in VH and VL of a human antibody, by performing PCR method described in using various synthetic DNA for modification according to claim 2 (5), can be achieved. By the method described in (2) in this section 2 for the amplified product obtained by the PCR, the nucleotide sequence was determined to confirm that the desired modification has been achieved.

(7) Construction of human CDR-grafted antibody expression vector

Upstream of the gene that encoding a human antibody CH and CL of the humanized antibody expression vector described in (1) of this section 2, human CDR-grafted constructed in the above 2 (5) and (6) cDNA encoding VH and VL of the antibody was Kuroyungu can this a force S to construct a human CDR-grafted antibody expression vector. For example, the above 2 (5) and of the synthetic DNA using VH and VL of a human CDR-grafted antibody when building (6), suitable limits to the 5 'end of the synthetic DNA located at both ends by introducing the recognition sequence of the enzyme, upstream of genes encoding CH and CL of the humanized antibody expression base Kuta one human antibody described in the above 2 (1) they be expressed in an appropriate form cloned into so that it is possible to construct a human CDR-grafted antibody expression vector.

(8) Stable production of humanized antibody

The above 2 (4) and (7) a humanized antibody expression human chimeric antibody by Rukoto to introduce vectors into appropriate animal cells and human CDR-grafted antibody according to (hereinafter, referred to as humanized antibodies together ) can be obtained transformant capable of stably producing.

[0173] Introduction of the humanized antibody expression vector into an animal cell, elect port Poreshiyon method [Japanese Published Unexamined Patent Application No. 257891/90; site Technology (Cytotechnology), 3,133 (1990)] or the like is Ru mentioned.

As the animal cell for introducing the Hitoi 匕抗-expressing vectors, so long as it is an animal cell which can produce the humanized antibody can be used in any cell.

Specifically, NS0 cells, a mouse myeloma cells, SP2 / 0 cells, Chinese hamster ovary cells CHO / dhfr_ cells, CHO / DG44 cell, a rat myeloma YB2 / 0 cells, IR 983F cells, derived from a Syrian hamster kidney there BHK cells, force etc. Namalwa human myeloma cell like S, preferably, CHO / DG44 cell is a Chinese hamster ovary cell, a rat myeloma YB2 / 0 cells, wherein the 2 or the onset according to 4. cells, etc. expressing Ming alpha-1,6-fucosyltransferase variants thereof.

[0174] After introduction of the humanized antibody expression vector, the transformant capable of stably producing the humanized antibody, in accordance with the method disclosed in JP-A-2-257891, G418 sulfate (hereinafter referred to as G418; SIGMA Co. It can be selected by a medium for animal cell culture containing an agent Ltd.) and the like. As a medium for animal cell culture (manufactured by Nissui Pharmaceutical Co., Ltd.) RPMI1640 medium, (manufactured by Nippon Pharmaceutical Co., Ltd.) GIT medium (manufactured by JRH Co.) EX-C ELL302 medium (manufactured by GIBCO BRL, Inc.) IMDM medium, Hybridoma-SFM medium (GI BCO BRL Co.), or fetal calf serum (hereinafter referred to as FBS) to these media can be used media obtained by adding various additives such like. The humanized antibody in the culture supernatant by the obtained transformant in a medium culture can be the production storage. Production of the humanized antibody in the culture supernatant and the antigen-binding activity is an enzyme immunosorbent assay [hereinafter, referred to as ELISA method; Ante body's: § 'Laboratory' Ma two Yuanore (Antibodies: A Laboratory Manual), Cold Spring Harbor Laboratory, Chapter 14, 1998, monochrome one Nanore 'ante body's: Purinshipunorezu' and 'practice (Monoclonal Antibodies: Principles and practice), can be measured by Academic Press Limited, 1996] and the like. Moreover, transformants were, in accordance with the method disclosed in Japanese Published Unexamined Patent Application No. 257891/90, it is possible to increase the production of human anti bodies by utilizing a DHFR gene amplification system or the like.

[0175] Hitoi 匕抗 body can be purified using a protein A column from the culture supernatant of the transformant [Ante body's: § 'laboratory ^ ~ Manual (Antibodies: A Laboratory Ma nual), Cold Spring Harbor Laboratory, Chapter 8, 1988, monoclonal Antibodies Antibo Didsbury: Principles 'and' practices (monoclonal Antibodies: Principles and Pr actice), Academic Press Limited, 1996]. In addition, purification methods generally used for the purification of proteins can be used. For example, by a combination of gel filtration, ion-exchange chromatography, ultrafiltration and the like can be purified. H chain of the purified humanized antibody, L chain or the molecular weight of the whole antibody molecule, polyacrylamide gel electrophoresis kinematic [hereinafter referred to as SDS-PAGE; Neichiya (Nature), 227, 680 (1970)] and Western blot Teingu method [ante body's: § 'Laboratory' manual (Antibodies: A Labora tory manual), and old Spring Harbor Laboratory, Chapter 12, 1988, black-and-white "~ Nanore-7 Ntibodizu: Principles 'and' practices (Monoclonal Antibodies: Principle s and Practice), can be measured by Academic Press Limited, 1996] and the like.

Production of B. Fc fusion protein composition

(1) Construction of the Fc fusion protein expression vector

The Fc fusion protein expression vector is an animal cell expression vector and protein gene encoding has been incorporated to be fused with Fc region of a human antibody, an Fc region of a human antibody into an animal cell expression downy click ter gene encoding the protein to be fused can be constructed by cloning.

[0176] The Fc region of a human antibody, Ho force ,, hinge region of the region containing the CH2 and CH3 regions, it is also encompassed intended to include part of CH1. The CH2 or at least one amino acid deletion CH3, substituted, added or inserted may be any as long as it has a binding activity to substantially Fe y receptor.

The gene encoding the protein to be fused to the Fc region of a human antibody can be used chromosomal DNA comprising Ekison and intron, also, cDNA and can Yore, also Rukoto. It found as a method for connecting a gene and Fc regions, each gene sequence as 铸型, PCR method (Les Kiyura ~ Cloning, Second Edition; Current Protocols 'in' Molecular ^ ~ & Baioroji one, Supplement 1- it may be mentioned that performs 34).

[0177] As the expression vector for animal cells, inserted may be any as long as it can be inserted and expressed the gene encoding the C region of a human antibody. For example, pAGE107 [site technology (Cytotechnology), 3, 133 (1990)], pAGE103 [journal 'O Breakfast' bio-chemistry (J. Biochem.), Dishes, 1307 (1987)], pHSG274 [Gene (Gene) , 27, 223 (1984)], pKCR [Proceedings 'O Breakfast' The 'National' Akademi one 'O Breakfast' Saien scan (Proc. Natl. Acad. Sci. USA), 78, 1527 (1981)], pSGl β d2-4 [site technology one (Cytotechnology), 4, 173 (1990)], and the like. The promoter and Enhansa one who is use in animal cell expression vector, SV40 early promoter and Enhansa the Journal 'O Bed' Biochemistry (J. Biochem.), 101, 1307 (1987)], Moroni one mouse leukemia virus the LTR Les Iokemikaru and Biophysical research community two Keshiyonzu (Biochem. Biophys. Res. Commun.), 149, 960 (1987)], immunoglobulin H chain promoter [cell (cell). 41, 479 ( 1985)] and Enhansa cell (cell), 33, 71 7 (1983)] and the like.

(2) Acquisition of DNA coding for the protein to be fused to the Fc region of a human antibody

DNA encoding the protein to be fused to the Fc region of a human antibody can S forces be obtained as follows.

[0178] mRNA is extracted from cells or tissues expressing the protein to be fused with the purpose of Fc, to synthesize cDNA A. The synthesized cDNA is cloned into a vector such as a phage or a plasmid to prepare a cDNA library. From the library, isolate recombinant phages or recombinant plasmid having a cDNA encoding the protein of use Le ,, intended gene sequence part of the protein of interest as a probe. Full nucleotide sequences of purpose of the protein on the recombinant phage or recombinant plasmid, to estimate the entire amino acid sequence from the nucleotide sequence.

[0179] As the animals other than human, mouse, rat, hamster, if it is possible to remove the Usagi like, cells or tissues can be used les, also made or.

The methods for preparing total RNA from cells or tissues, Chioshian acid guanidine - triflic O b cesium acetate method [.. (. Methods in Enzymol) Mesozzu in Enzaimoroji one, 154, 3 (1987)], also mRNA from total RNA the methods for preparing the oligo (dT) immobilized cellulose column method (Molecular 'cloning, second Edition) and the like. Further, examples of a kit for preparing m RNA from cells or tissues, (manufactured by Invitrogen Corp.) Fast Track mRNA Isolation Kit, Qui ck Prep mRNA Purification Kit (Pharmacia Co., Ltd.).

[0180] As an production method synthesis and cDNA library of cDNA, a conventional method (Morekiyura one 'claw-learning Third Edition; Current' Protocols' in 'Molecular' Biology, Supplement 1_ 34), or a commercially available kit, for example, such as Super Script ™ Plasmid System for cDNA Synthesi s and Plasmid Cloning (GIBCO BRL Co., Ltd.) and ZAP- cDNA Synthesis Kit (Stratagene, Inc.) a method using, and the like.

[0181] In preparing the cDNA library, the vector into which a cDNA synthesized using mRNA extracted from a cell or tissue as 錡型 can be used in any well of as long as a vector incorporate the cDNA. For example, ZAP Express [Strategies (Strategies), 5, 58 (1 992)], pBluescript II SK (+) [Nukureitsuku 'Ashizzu' research (Nucleic Acids Research), 17, 9494 (1989)], λ zapll (Stratagene Co. Inc.), gtl0, λ gtl l [Denue one 'black-learning: §' Practical 'approach (DNA Cloning: A Practical approach), I, 49 (1985)], lambda BlueMid (Clontech, Inc.), lambda ExCel pT7T3 18U (Pharmacia, Inc.), CD2 [Molecular and 'cellular ~ Biology (Mol. Cell. Biol.), 3, 280 (1983)] and pUC18 [Gene (Gene), 33, 103 (1985) ] or the like is used.

[0182] Phage or introducing the cDNA library as E. coli for introducing the cDNA library constructed by a plasmid vector can be used as long as it can express and maintain record, even made whether. For example, XLl-Blue MRF '[Strategies (Strategies), 5, 81 (1992)], C600 [Genetics (Genetics), 39, 440 (1954)], Y1088, Y1090 [Sa Iensu (Science), 222, 778 (1983)], NM522 [journal 'O Bed' Molecular 'by Oroji one (J. Mol. Biol), 166, 1 (1983)], K802 [journal' O Bed 'Molecular' by Oroji one (J. Mol . Biol), 16, 118 (1966)] and JM105 [Gene (Gene), 38, 275 (1985)] or the like is used.

[0183] As the method for selecting a cDNA clone encoding the protein of interest also cDNA library Ichiriki, isotope or fluorescence-labeled colonies 'Bruno, Eve lida I See Chillon method or plaque' hybrida I See Chillon method using a probe It can be force to select Ri by the (Molecular 'cloning, third Edition). Further, Chide monkey that a primer was prepared and cDNA synthesized or cDNA libraries from mRNA as 铸型, preparing cDNA encoding the protein of interest by PCR.

[0184] The protein of interest as a method of fusing the Fc region of a human antibody, PCR method.

For example, set any synthetic oligo DNA (primer I) 5 'and 3' side of the gene sequence encoding the protein of interest, to obtain a PCR product subjected to PCR method. Similarly, to set an arbitrary primer to the gene sequence of the Fc area of ​​a human antibody to be fused to obtain a PCR product. At this time, the 5 'side of the PCR product side and Fc region 3' of the protein of the PCR product to fuse setting the primers so that there is the same restriction enzyme site or the same gene sequence. If the amino acid change near the connecting portion is required, it is introducing a mutation by using primers to introduce the mutation. Two PCR fragments obtained by further performing a PCR using, for connecting both genes. Or it may be force S coupling after treatment with the same restriction enzyme even with Raigeshiyon child. [0185] The gene sequence linked by the above method, after cutting with appropriate restriction enzymes, pBluesc ript SK (-) was cloned into a plasmid (Stratagene, Inc.) or the like, usually a base sequence analysis method used, for example, Sanger (Sanger) et al. Jidokishi method [Proceedings Ding Das' O Breakfast 'the * National Akademi ~ · O Bed' science (Proc. Natl. Acad. Sci. USA), 74, 54 63 (1977)] or the ABI PRISM by analysis using 377DNA Shikuensa (manufactured by PE Biosystems Co.) single nucleotide sequence analyzer such as can be used to determine the nucleotide sequence of the DNA.

[0186] estimate the determined entire amino acid sequence of the Fc fusion protein from the nucleotide sequence, encoding the complete amino acid sequence of the Fc fusion protein comprising by comparing the amino acid distribution 歹 IJ purpose, the cDNA is secretory signal sequence obtained it is possible to check whether to have you.

(3) Stable production of Fc fusion protein composition

To obtain a transformant capable of stably producing the Fc fusion protein composition by introducing into a suitable animal cell an Fc fusion protein expression vector according to item (1) of this section 2 B

[0187] The method for introducing the Fc fusion protein expression vector into an animal cell, elect port Poresho down method [Japanese Published Unexamined Patent Application No. 257891/90; site Technology (Cytotechnology), 3, 133 (1990)] Hitoshigaa is down.

As the animal cell for introducing the Fc fusion protein expression vector, so long as it is an animal cell which can produce the Fc fusion protein, can be used in any cell.

[0188] More specifically, NS0 cells, a mouse myeloma cells, SP2 / 0 cells, Chinese hamster ovary cells CHO / dhfr- cells, CHO / DG44 cell, a rat myeloma YB2 / 0 cells, IR 983F cells, Syrian hamster BHK cells derived from kidney, preferably a force such as Na Malva cells like a human myeloma cells, CHO / DG44 cell is a Chinese Nono Muster ovary cells, rat myeloma YB2 / 0 cells, according to the 2 or 4. Fei of the present invention - such as cells expressing a 1,6-fucosyltransferase variants thereof.

[0189] After introduction of the Fc fusion protein expression vector, form transformant capable of stably producing the Fc fusion protein compositions in accordance with the method disclosed in Japanese Published Unexamined Patent Application No. 257891/90, animal cell culture containing an agent such as G418 It can be selected by use medium. As a medium for animal cell culture (manufactured by Nissui Pharmaceutical Co., Ltd.) RPMI1640 culture areas, (manufactured by Nippon Pharmaceutical Co., Ltd.) GIT medium, EX-CELL302 medium (JRH Co., Ltd.), IMD M medium (GIBCO BRL Co., Ltd.), Hybridoma- SFM medium (GIBCO BRL) or can be used fetal bovine media prepared by adding various additives such as serum or the like to these media. Transformants obtained can be produced and accumulated the Fc fusion protein composition in the culture supernatant by culturing in a medium. Production and antigen binding activity of the Fc fusion protein composition in the culture supernatant can be measured by EUSA method. Further, it transformants in accordance with method shown open in Japanese Published Unexamined Patent Application No. 257891/90, it is possible to increase the production of the Fc fusion protein composition by using a dhfr gene amplification system or the like.

[0190] Fc fusion protein composition can be purified using a protein A column or Puroti emission G column the culture supernatant of the transformant (anti body's, Chapter 8, monochrome chromatography nano les • ante body's) . It is also possible to use a use record, is a purification method other normal, purification of the protein. For example, by a combination of gel filtration, ion exchange chromatography and ultrafiltration Filtration like, it can be purified. Purified Fc fusion protein molecule overall molecular weight, SDS-PAGE [Neichiya (Nature), 227, 680 (1970)] and Western blotting method measured at (anti body's, Chapter 12, Monoclonal 'Ante body's) or the like It can be force S to.

[0191] Although the animal cell describes the preparation of antibody composition as host, as described above, can be force S also prepared bacteria, yeast, insect cells, plant cell, an animal individual or a plant individual .

Already, when the cells have the ability to express a glycoprotein composition such as an antibody molecule, said after preparation of cells using the method described in 4., culturing the cells, the cultures by purifying the antibody composition of interest from, it is possible to produce an antibody composition Ya glycoprotein compositions of the present invention.

Activity evaluation of 7. glycoprotein composition

Protein amount of the purified glycoprotein composition, affinity for the receptor, half-life in the blood, the distribution of the tissue after blood administration, or a change in protein-protein interactions required for pharmacological activity expressed as a method for measuring may, Current Protocols in Protein Science, John Wiley & Sons Inc., (1995), Japanese biochemical Society new biochemical experiment lecture 19 animal experimentation, Tokyo Kagaku Dojin (199 1), Japanese biochemical Society new biochemical experiment course 8 cells internal information and the cell response, Tokyo Kagaku Dojin (1990), Japanese biochemical Society new biochemical experiment course 9 hormone I peptide hormone, Tokyo Kagaku Dojin (1991), experimental biology Department, 3 isotope experimental methods, Maruzen Co., Ltd. (1982), Monocl onal antibodies: Principles and Applications, Wiley-Liss, Inc., (1995), enzyme immunoassay measurement method, third Edition, Igaku Shoin (1987), revised version of the enzyme antibody method, publicly known as described in interdisciplinary planning (1985), etc. the method can be used for.

[0192] As a specific example, labeled glycoprotein composition purified by compounds of such radioisotope, the binding reaction between proteins and receptors, or the interaction of the labeled glycoprotein composition strength quantitatively method of measuring the like the of. Further, by using various devices such as Biacore's BIACOR e series, it is also possible to measure the protein protein interaction (

J. Immnunol. Methods, 145, 229 (1991), Experimental Medicine separate volume Bio Manual UP Series Zutanpaku protein molecular interaction experiment method, sheep soil, Inc. shed 996)).

[0193] The labeled glycoprotein compositions to administer into the body, it is possible to know the half-life or distribution to tissue after blood administration in blood, the detection of label, a labeling substance Align the other system set specific antibody-antigen reaction to glycoprotein compositions comprising a method for detect the subject of the detection is preferred.

8. activity evaluation of the antibody compositions

Protein amount of the purified antibody composition, as a method for measuring the binding or effector function to an antigen can be used known methods described in Monoclonal Antibodies or anti body engineering, etc.,.

[0194] As a specific example, if the binding activity of the antibody with the antigen composition chimeric antibody composition or a humanized antibody compositions, binding activity to an antigen-positive cultured clone EUSA method and fluorescent 饥body method [Cancer 'Imunoroshi one' Imunoserapi (Cancer Immunol. Immunotherj, 36, 373 (1993)] can be measured by such. the cytotoxic activity against an antigen-positive cultured clone, CDC activity, by measuring the ADCC activity or the like, can evaluate [Cancer 'Imunoroji one' Imunoserapi (Cancer Immunol. Immunother.), 36, 373 (1993)].

[0195] In addition, safety, therapeutic efficacy of the antibody composition in human can be evaluated using a force two Kuizaru such relatively near have suitable animal species model to human. 9. analysis of the sugar chain of the antibody compositions

Sugar chain structure of an antibody molecule expressed in various cells can be performed in accordance with a conventional analysis of the sugar chain structure of a glycoprotein. For example, a sugar chain bound to an IgG molecule galactose, mannose, neutral sugars such as fucose, amino sugars such as N- § cetyl Darco Sa Min, and acidic sugars such as sialic acid, a sugar composition analysis and it can be carried out using the technique of the sugar chain structure analysis or the like use les was like a two-dimensional sugar chain mapping.

(1) neutral sugar 'amino sugar composition analysis

Composition analysis of the sugar chain of the antibody molecule is a Torifuruoro acetate, by a child that is an acid hydrolysis of sugar chains, liberates neutral sugars or amino sugars, it is possible to analyze the composition ratio.

[0196] As a specific method, a method using a Dionex Corp. sugar composition analyzer (BioLC) is exemplified al are. BioL Mr. HPAE - PAD (high perrormance anion-exchange chromatography-pui sed amperometric detection) method [(.. J.Liq Chromatogr) Jananore-O Bed Liquid Chromatography, 6, 1577 (1983)] sugar composition by it is a device to analyze.

Further, it is also possible to analyze the composition ratio by the fluorescence labeling method using 2-Aminobirijin. Specifically, a known method [§ glycanase Ruchu Lal and 'Biological Chemistry (Agm Biol.Chem.), 55 (1) .283 ~ 284 (1991)] 2_ Aminobirijiru the acid hydrolyzed samples according to in fluorescently labeled, it is possible to calculate the composition ratio by HPLC analysis.

(2) sugar chain structure analysis

Structural analysis of sugar chains of an antibody molecule, two-dimensional sugar chain mapping method [Analytical 'Bio Chemist Lee (Anal.Biochem.), M, 73 (1988), Biochemical Experimental Methods 23-glycoprotein sugar chain research methods ( can be carried out by the Society of publication Center) Reiko Takahashi ed. (1989)]. 2 dimensional sugar chain mapping method, for example, the retention time or elution position of reverse phase chromatography sugar chains in X-axis, the retention time or elution position of the sugar chain by normal phase chromatography as the Y axis, respectively plot and, by comparing the results of their known sugar chains, a method for estimating the sugar chain structure.

[0197] Specifically, antibodies to hydrazinolysis to release the sugar chain from the antibody, 2-aminopyridine (hereinafter referred to as PA) fluorescent labeled [Journal O Bed 'Biochemistry of the sugar chain by ( J. Biochem.), 95, 197 (1984)] after, etc. and then separated from an excess PA-treating reagent by gel filtration, the sugar chain is subjected to reversed phase chromatography. Then, perform the normal phase chromatography for each peak of the sugar chain. Based on these results, plotted on a two dimensional sugar chain mapping, (manufactured by TaKaRa Co.) sugar chain standards, Ref Anaritikanoi Biochemistry (Anal. Bi ochem.), 171, 73 (1988)] with it is possible to estimate the sugar chain structure than the comparison spot.

[0198] can further confirm the MALDI-TOF-MS structure deduced Ri by the row-,, 2 dimensional sugar chain mapping method mass spectrometry, such as for each sugar chain.

10. Use of Fei-1,6-fucosyltransferase cells glycoprotein composition produced using with variants and antibody compositions of the present invention

Fei 1,6 of the present invention - fucosyltransferase variant glycoprotein produced using the cells having the composition and antibody compositions has a sugar chain structure with no fucose modification, if example embodiment, receptor affinity improvement of the improvement of the blood half-life, improved tissue distribution after blood administration, or the effect is high can be expected physiological activity such as enhancement of the interaction between proteins required for pharmacological activity expression show. In particular, an antibody composition of this invention, a high effector function, les have ie antibody-dependent cellular cytotoxicity, Ru. High Les these physiological activities glycoprotein sets Narubutsu or Kore, antibody compositions with ADCC activity, cancer, inflammatory diseases, autoimmune diseases, immune diseases such as allergies, cardiovascular diseases or viral or bacterial, the infected useful in the prevention and treatment of various diseases and dimethyl.

[0199] cancer, i.e. cancer cells in malignant tumors are growing. Ordinary anticancer agent is characterized by inhibiting the growth of cancer cells. However, Kore, antibodies with ADCC activity, it is possible to treat cancer by damaging cancer cells by cell killing effect, it is effective as a therapeutic agent than the normal anti-cancer agents. Particularly in treatment of cancer, but at present the combination therapy with the case antitumor effect insufficient number chemotherapy antibody drugs alone have been made [Saien scan (Science), 280, 1197, 1998], the as long observed stronger antitumor effect of antibody composition alone invention, reliance is reduced to chemotherapy, leading to a reduction of side effects.

[0200] inflammatory diseases, autoimmune diseases, immune diseases such as allergies, contact Keru vivo reactions to them disease, because it is caused by the release of a mediator molecule by immunocytes, using an antibody having Kore, ADCC activity by removing the immune cells Te, it can be force obtain an allergic reaction. The cardiovascular diseases, such as arteriosclerosis and the like. Arteriosclerosis, performs the current treatment with a balloon catheter one ether, than to suppress using an antibody having the growth high ADCC activity of arterial cells in restenosis after treatment, that prevent and treat cardiovascular diseases it can

[0201] The growth of cells infected with viruses or bacteria, by suppressing using an antibody having high ADCC activity can be force S to prevent and therapy of various diseases including viral or bacterial infections.

Tumor-associated antigen antibodies that recognize an antibody that be recognized allergy- or inflammation-related antigen, an antibody that recognizes an antigen associated with cardiovascular disease, recognize antigen associated with an autoimmune disease antibody or viral or bacterial, specific examples of the recognizing antibody antigens associated with infection described below.

[0202] The tumor-associated antigen antibody recognizing the anti-CA125 antibodies (Immunology Today, 21, 4 03-410, 2000), anti-17-1A antibody (Immunology Today, 21, 403-410, 2000), anti-integrin Glynn ct v i3 3 antibodies (Immunology Today, 21, 403-410, 2000), anti-CD33 antibody (Immuno logy Today, 21, 403-410, 2000), anti-CD22 antibodies (Immunology Today, 21, 403-410, 2 000), anti-HLA antibodies (Immunology Today, 21, 403-410, 2000), anti-HLA-DR antibody (I mmunology Today, 21, 403-410, 2000), anti-CD20 antibodies (Immunology Today, 21, 403 -410 , 2000), anti-CD19 antibodies (Immunology Today, 21, 403-410, 2000), anti-EGF receptor antibody (Immunology Today, 21, 403-410, 2000), anti-CD 10 antibody (American Journal of Clinical Pathology, U3 , 374-382, 2000; Proc Natl Acad Sci USA, 79:..... 4386-43 91, 1982), anti-GD2 antibody (Anticancer Res, 13, 331-336, 1993), anti-GD3 antibody (Cance r Immunol. Immunother., 36, 260-266, 1993), anti-GM2 antibody (Cancer Res., 54, 1511 -1516, 1994), anti-HER2 antibody ( Proc. Natl. Acad. Sci. USA, 89, 4285-4289, 1992), anti-CD52 antibody (Nature, 332, 323-327, 1988), anti-MAGE antibody (British J. Cancer, 83, 493-497, 2000 ), anti-HM1. 24 antibody (Molecular Immunol "36, 387-395, 1999), anti-parathyroid hormone-related protein (PTHrP) antibody (Cancer, 88, 2909-2911, 2000), anti-FG F8 antibody (Proc. Natl . Acad. Sci. USA, 86, 9911-9915, 1989) anti-basic fibroblast increase 殖因Ko antibody, anti-FGF8 receptor antibody (J. Biol. Chem., 265, 16455-16463, 1990), anti-salt group fibroblast growth factor receptor antibody, anti-insulin-like growth factor antibody (J. Neurosci. R es., 40, 647-659, 1995), anti-insulin-like growth factor receptor antibodies (J. Neurosci. Res. , 40, 647-659, 1995), anti-PMSA antibody (J. Urology, 160, 2396-2401, 1998), anti-vascular endothelial cell growth factor antibody (Cancer Res., 57, 4593-4599, 1997) or anti-vascular endothelial cells increase 殖因Ko receptor antibody (Oncogene, 19, 2138-2146, 2000) and the like.

[0203] The antibody which recognizes an allergy- or inflammation-related antigen, anti-IgE antibodies (Im munology Today, 21, 403-410, 2000), anti-CD23 antibodies (Immunology Today, 21, 403- 410, 2000), anti CD1 la antibodies (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-inter one interleukin 5 antibody ( Immunol. Rev., 127, 5-24, 1992), anti-interleukin 5 receptor antibody, anti-interleukin 4 antibody (Cytokine, 3, 562-567, 1991), anti-interleukin 4 receptor antibody (J. Immunol. Meth., 211, 41-50, 1998), anti-S heavy 瘍壊 death factor antibody (Hybridoma, 13, 183-190, 1994), anti-tumor necrosis factor receptor antibody (Molecular Pharmacol., 58, 237 -245 , 2000), the anti-CCR4 antibody (Nature, 400 , 776-780, 1999), anti-chemokine antibody (J. Im munol. Meth., 174, 249-257, 1994) or anti-chemokine receptor antibody (J. Exp. Med., 1 86, 1373-1381, 1997 ), and the like.

[0204] As the recognize antigens which are associated with cardiovascular disease, anti-GpIIb / lIIa antibodies (J. Im solid nol., 152, 2968-2976, 1994), anti-platelet-derived growth factor antibody (Science, 253, 1129-113 2, 1991), anti-platelet-derived growth factor receptor antibodies (J. Biol. Chem., 272, 17400-17404, 1997) or anti-blood coagulation factor antibody (Circulation, 101, 1158-1164, 2000), etc. and the like.

[0205] Autoimmune diseases, such as psoriasis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, as an antibody which recognizes an antigen associated with multiple sclerosis, anti-self D NA antibodies (Immunol. Letters , 72, 61-68, 2000), anti-CD 1 la antibody (Immunology Today, 21, 403-410, 2000), anti-ICAM3 antibodies (Immunology Today, 21, 403-410, 2000), anti-C D80 antibody (Immunology Today, 21, 403-410, 2000), anti-CD2 antibodies (Immunology Toda y, 21, 403-410, 2000), anti-CD3 antibodies (Immunology Today, 21, 403-410, 2000), anti-C D4 antibodies (Immunology Today, 21, 403-410, 2000), anti-integrin a 4 7 antibody (Imm unology Today, 21, 403-410, 2000), anti-CD40L antibodies (Immunology Today, 21, 403- 410, 2000), anti IL_ 2 receptor antibody (Immunology Today, 21, 403-410, 2000), etc. can be mentioned up.

[0206] Virus or The antibody which recognizes an antigen associated with bacterial infection, anti-g P 120 antibody (Structure, 8, 385-395, 2000 ), anti-CD4 antibody (J. Rheumatology, 25, 2065-2076, 1 998), anti-CCR4 antibodies or anti-verotoxin antibody (J. Clin. Microbiol., 37, 396-399, 1999) and the like.

The antibody, ATCC (The American Type Culture Collection), RIKEN cells Development Bank, Agency of life Industrial Technology Research Institute public institutions of like, or manufactured by Dainippon drug Co., Ltd.,, R & D SYSTEMS Inc., PharMingen, Inc. , Cosmo Bio, it is possible to obtain private reagent sales companies force such as Funakoshi.

[0207] The pharmaceutical comprising the glycoprotein composition of the present invention, there is also be administered alone as therapeutic agents, but is usually mixed with one or more of the carriers pharmacologically acceptable and, it is desirable to provide as a pharmaceutical preparation produced by methods well known in the technical field of pharmaceutics.

The route of administration, the most effective ones to use is desirable tool orally, or buccal in the treatment, the airway, intrarectal, subcutaneous, Rukoto force S mentioned parenteral administration intramuscular and intravenous etc. it can, in the case of an antibody preparation, preferably can be mentioned intravenous administration.

[0208] The dosage form includes 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 are water, sucrose, sorbitol, sugars fructose, etc., polyethylene glycol, Darikoru such as propylene glycol, sesame oil, olive oil, oils such as soybean oil, p- preservatives such as hydroxybenzoate esters can be prepared using stringent port Beri one flavor, flavors such as peppermint as an additive. [0209] Capsules, tablets, powders, granules and the like are lactose, dextrose, sucrose, and mannitol, etc., starch, disintegrants such as sodium alginate, a lubricant, such as magnesium stearate, Tal click, polyvinyl alcohol, hydroxypropyl cellulose, binders such as gelatin, can be prepared by using surfactants such as fatty acid esters, plasticizers such as glycerin or the like as an additive.

[0210] Suitable formulations for parenteral administration include injections, suppositories, sprays and the like.

Injections can be prepared using a carrier such as a salt solution, glucose solution or a mixture thereof. Alternatively, the glycoprotein composition is lyophilized by a conventional method, it is also this to prepare a powder injection by adding chloride sodium © beam.

Suppositories can be prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.

[0211] Also, sprays glycoprotein composition itself, or does not stimulate the recipient's mouth and airway mucous membranes, and the carrier is to facilitate absorption by dispersing a glycoprotein composition as fine particles, etc. It is prepared using.

The carrier includes lactose, glycerol and the like. Due to the nature of the glycoprotein compositions and carrier, it is possible to prepare aerosols, dry powders, etc.. Also, as possible out also be added to the components exemplified as additives for oral preparations in these parenteral preparations.

[0212] dose or frequency of administration, a therapeutic effect, administration method, treating period, age, different forces adult per day by body weight such as 10 / ig / kg~20mg / kg of interest.

Further, a method to examine antitumor effects on various tumor cells of a glycoprotein composition, for example, in the case of antibodies, in vitro experiments, CDC (complement_dependent cytotoxic! Ty) activity assay, ADCC (antibody- d mark endent cellular cytotoxicity ) activity measuring method or the like there Gerare, as in vivo experiments, anti-tumor experiments like using tumor systems in experimental animals such as a mouse.

[0213] CDC activity, ADCC activity, antitumor experiments, the literature [Cancer 'Imunoroji one' Imunosera heat 1 ~ (and ancer Immunology Immunotherapy), όΌ, 373 (1993); Kyansa "~ 'RIQUET" ~ te (Can cer Research), it can be performed according to the method 54, 1511 (1994)] or the like, wherein.

The present invention will be described more specifically by the following examples, which examples are merely illustrative of the invention and is not intended to limit the scope of the present invention.

Example 1

[0214] acquisition of fucose modified variant RN6 shares

1. acquisition of RN6 shares

Chinese Nono Muster ovary C HO / DG44 cells deficient in dihydrofolate reductase gene (dhfr) [Somatic Cell and Moleculer Genetics, 12, 555 (1986)] and, 400 μ g / ml lentil lectin (LCA; EY Laboratory company) were cultured for 3 weeks in the presence of.

[0215] resistant strain obtained by the LCA co culture, were seeded such that 1.5 X 10 5 cells / well in adherent cells for 24-well plates (one company Guraina), 5% CO, 37 ° C conditions after 4 hours incubation under, 400 / ig / ml LCA (EY Laboratory, Inc.) and replace the medium containing 100 / i M L_ fucose (Nacalai tester, Inc.), and cultured further 5 days. After incubation, replace the medium containing 10% WST-1 (Takara Bio Inc.), was incubated for 30 minutes under the conditions of 5% CO, 37 ° C, and measuring the absorbance at a wavelength 450 s Awakening of the culture supernatant, It was used as an indicator of viable cell density. As a result, it was found resistant RN6 strain LCA even in the presence of L- fucose.

2. α -1,6- fucose addition capability analysis of RN6 shares

Dulbecco's phosphate buffer containing 1% © shea serum-derived albumin (Sigma) (Invitrogen Corp.) (hereinafter, referred to as 1% BSA_PBS) to and suspended 2 X 10 5 cells RN6 strain or CHO / DG44 cells, FITC labeled LCA (Vector Laboratories, Inc.) or 100-fold diluted FITC-labeled Sutoreputa neutravidin a (KPL Co.) was 添Ka卩. 4 ° Cells were stained with to stand for 30 minutes at C, After the cells were washed with 1% BSA-PB S, were analyzed IX 10 4 cells in FACSCalibur (BD Biosciences Inc.).

[0216] showed reactivity against LCA of each strain in FIG. While stained with LCA is a CHO / DG44 cell Wahi _1,6- fucose singular lectin was observed, the RN6 strain, it was not stained against LCA.

3. monosaccharide composition analysis of the antibody composition RN6 strain produced

First, with respect to RN6 strain, an anti-CCR4 antibody expression vector PKANTEX216 0 of WO01 / 64754, wherein, introduced by elect port Poreshiyon method [Cytotechnology, 3, 133 (1990)], were obtained stable expression strain. From the culture supernatant of the anti-CCR4 antibody expression strains were purified antibody composition using Mab Select (Amersham Pha rmacia Biotech Inc.). To obtain anti-CCR4 antibodies, methods publicly known [Journal 'O Bed' liquid 'chromatography (Journal of Liquid Chromatog raphy), 6, 1577, (1983)] was carried out monosaccharide composition analysis in accordance with. As a result, the antibody composition obtained from RN6 strain, fucose content was below the limit of quantification (Figure 2). This result is RN6 strain, N - 1-position of fucose is suggested that lack the function of a binding to the 6-position of § cetyl Darco Sa Min - linked complex type sugar chain of N.

Example 2

[0217] analysis of the mutation point of RN6 shares

1. expression level analysis of fucose modifying enzyme

From RN6 strains and CHO / DG44 cells obtained in Example 1, total RNA was extracted using the RNeasy Mini Kit (QIAGEN Inc.). Total RNA 5 beta g and 铸型, subjected to reverse transcription reaction by oligo dT primer using the Superscript First-Strand Synthe sis System for RT_PCR (Invitrogen Corp.), was synthesized single-stranded cDNA.

[0218] Expression Analysis of GDP-mannose 4.6 dehydratase (GMD) was performed as follows. Synthesizing specific forward primer (SEQ ID NO: 23) and reverse primer (SEQ ID NO: 24) into Chinese hamster GMD cDNA sequence described in patent W 002/31140, DNA polymerase Ex Taq (Takara Bio Inc.) and the cDNA 1 20 mu 1 of the reaction solution containing the μ 1 [ΕχΤ aq buffer (Takara Bio Inc.), 0.2 mmol / 1 dNTPs, 0.5 μ mol / 1 above gene-specific primers (SEQ ID NO: 23 and SEQ ID NO: 24)] was prepared , polymerase chain reaction (PCR) row ivy. PCR, after heated for 5 minutes at 94 ° C, 1 minute at 94 ° C, the reaction was conducted consisting of 2 minutes 26 cycles of steps as one cycle at 68 ° C. After the PCR, subjected to 1.2% (w / v) Agarosugeru electrophoresis after staining D NA with SYBR Green I Nucleic Acid Gel Stain (Molecular Probes, Inc.), Fluorlmager the emission intensities of the DNA fragments amplified SI (Molecular D ynamics Co.) was calculated using the.

[0219] α -1, expression analysis of the 6-fucosyltransferase (FUT8) was performed as follows. Synthesizing a patent WO02 / 31140 tea I cDNA Hai歹 lJ of hamster FUT8 (SEQ ID NO: l) the specific forward primers of (SEQ ID NO: 25) and reverse primer (SEQ ID NO: 26), DNA polymerase Ex Taq (Takara Bio) and 20 mu 1 of a reaction solution containing the cDNA 1 μ 1 [Ex Taq buffer (Takara Bio Inc.), 0.2 mmol / 1 dNTPs, 0.5 μ mol / 1 above gene-specific primers (SEQ ID NO: 25 and SEQ ID NO: 26)] was prepared and subjected to PCR. PCR, after heated for 5 minutes at 94 ° C, 1 minute at 94 ° C, the reaction was conducted consisting of 2 minutes 22 cycles of steps a 1 Saikunore at 68 ° C. After the PCR, subjected to 1.2% (w / v) Agarosugeru electrophoresis after staining DNA with SYBR Green I Nucleic Acid Gel Stain (Molecular Probes, Inc.), Fluorlmager the emission intensities of the DNA fragments amplified SI (Molecular Dynamics, Inc.) was calculated using the.

[0220] Moreover, expression analysis of FUT8 cDNA full length was performed as follows. Mouse FUT8 cDNA Hai歹 IJ

[GenBank, AB025198, SEQ ID NO: 2] was synthesized specific reverse primer (SEQ ID NO: 28) on the side 'untranslated region specific forward primer (SEQ ID NO: 27) and 3 untranslated regions 5', DNA polymerase Ex Taq (Takara Bio Inc.) and the reaction solution 25 μ ΐ containing the cDNA 1 μ 1 [Ex Taq buffer (Takara Bio Inc.), 0.2 mmol / 1 dNTPs, 4% DMSO, 0.5 β mol / 1 said gene specific primers (SEQ ID NO: 27 and SEQ ID NO: 28)] was prepared and subjected to PCR. PCR, after heating for 1 min at 94 ° C, 30 seconds at 94 ° C, 30 seconds at 55 ° C, were performed at 30 cycles of steps in which the reaction of 2 minutes as one cycle at 72 ° C. After the PCR, subjected to 1.2% (w / v) Agarosugeru electrophoresis after staining DNA with SYBR Green I Nucleic Acid Gel Stain (Molecul ar Probes, Inc.), Fluo the emission intensities of the DNA fragments amplified rlmager calculated 7 this in SI (Molecular Dynamics Shari.

[0221] GDP-expression analysis of β -L-ilicose pyrophorylase (GFPP) was performed as follows. Synthesizing a patent WO03 / 085,118 according to Chinese Nono Muster GFPP cDNA sequence specific for Fowa one Dopuraima one (SEQ ID NO: 29) and reverse primer (SEQ ID NO: 30), DN A polymerase Ex Taq (Takara Bio Inc.) and the reaction of 20 μ ΐ containing cDNA 1 μ 1 [Ex Taq buffer (Takara Bio Inc.), 0.2 mmol / 1 dNTPs, 0.5 μ mol / 1 above gene-specific primers (SEQ ID NO: 29 and SEQ ID NO: 30) prepared and it was subjected to PCR. PCR, after heated for 5 minutes at 94 ° C, 1 minute at 94 ° C, the reaction was conducted consisting of 2 minutes 24 cycles of steps as one cycle at 68 ° C. After the PCR, subjected to 1.2% (w / v) Agarosugeru electrophoresis, SYB R Green I Nucleic Acid Gel Stain (Molecular Probes, Inc.) using the after staining DNA, Fluorlmager the emission intensities of the DNA fragments amplified SI (Molecular Dynamics, Inc.) was calculated using the.

[0222] Expression Analysis of GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase (FX) was performed as follows. Synthesizing specific forward primer (SEQ ID NO: 31) and reverse primer (SEQ ID NO: 32) in Chinese hamster FX cDNA Hai歹 lj described in patent WO03 / 085118, DNA polymerase Ex Taq (Takara Bio Inc.) and the cDNA 1 the μ 1 reaction solution including 20 ^ Ding & 9 13111¾1 Takarabaio Co., Ltd.), 0.2 11111101/1 0 ^ Ding? 3, 0.5 μ mol / 1 above gene-specific primers (SEQ ID NO: 31 and SEQ ID NO: 32)] was prepared and subjected to PCR. PCR, after heated for 5 minutes at 94 ° C, 1 minute at 94 ° C, was reaction consisting of 2 minutes 24 cycles of steps as one cycle at 68 ° C. After the PCR, subjected to 1.2% (w / v) Agarosugeru electrophoresis after staining DNA with SYBR Green I Nucleic Acid Gel Stain (Molecular Probes, Inc.), Fluorlmager the emission intensities of the DNA fragments amplified SI (Molecular Dyn 纖 ics Co.) was calculated using the.

[0223] GDP-expression analysis of fucose transporter was performed as follows. The patent WO03 / 085 102 singular forward primer into Chinese hamster GDP- fucose transporter cDNA sequence set forth in (SEQ ID NO: 33) and reverse primer (SEQ ID NO: 34) and synthesis, DNA polymerase Ex Taq (Takara Bio Inc.) and the cDNA 1 μ 1 20 μ 1 of a reaction solution containing [Ex Taq buffer (Takara Bio Inc.), 0.2 mmol / 1 dNTPs, 0.5 μ mol / 1 above gene-specific primers (SEQ ID NO: 33 and SEQ ID NO: 34) It was prepared and subjected to PCR. PCR, after heated for 5 minutes at 94 ° C, 1 minute at 94 ° C, the reaction was conducted consisting of 2 minutes 24 cycles of steps a 1 Sa Ital at 68 ° C. After PCR, 1.2% were subjected to (w / v) Agarosugeru electrophoresis, SYBR Green I Nucleic Acid Gel Stain (Molecular Probes, Inc.) using the after staining DNA, the emission intensities of the DNA fragments amplified Fluorlmager SI ( calculated in Molecular Dynami cs, Inc.).

[0224] / 3 - Expression Analysis of Akuchin was performed as follows. Chiyai Nizuno ヽ Muster described in patent WO02 / 31140 beta - Akuchin specific forward primer to the cDNA sequence (SEQ ID NO: 35) and were synthesized reverse primer (SEQ ID NO: 36), DNA polymerase Ex Taq (Taka Rabaio Co.) and the the reaction solution 20 containing a cDNA 1 μ ΐ [ExTaq buffer (Takara Bio Inc.), 0.2 mmol / 1 dNTPs, 0.5 μ mol / 1 above gene-specific primers (SEQ ID NO: 35 Contact and SEQ ID NO: 36)] was prepared, PCR was carried out. PCR, after heated for 5 minutes at 94 ° C, 1 minute at 94 ° C, the reaction was conducted consisting of 2 minutes at 14 cycles of steps as one cycle at 68 ° C. After P CR, subjected to 1.2% (w / v) Agarosugeru electrophoresis after staining DNA with SYBR Green I Nucleic Acid Gel St ain (Molecular Probes, Inc.), Fluorlmager the emission intensities of the DNA fragments amplified It was calculated using the SI (Molecular Dynamics, Inc.).

More RT-PCR results, GMD of RN6 strain, FUT8, GFPP, FX, GDP-expression level of fucose transporter was comparable to CHO / DG44 cell. However, partial results of analysis using primers to amplify the full length of about 1.7 Kb of FUT8 gene, since the fragment of approximately 1.3 Kb in addition to a fragment of about 1.7 Kb from the RN6 strain is detected in one of the FUT8 allele Les, is Rukoto showed deficits have occurred.

2. Sequence analysis of the FUT8 cDNA of RN6 shares

The method described in paragraph 1 embodiment, to amplify the FUT8 cDNA full length than RN6 strain, having conducted a direct sequence analysis according to methods known [Morekiyura one 'Cloning 3rd edition. As a result, a fragment of about 1.3 Kb that was found in the above item 1 of this Example, the Ekuson human FUT8

3, Ekuson 4, revealed that lacks the region corresponding to Ekuson 5. In the determined nucleotide sequence of the RN6 strain derived FUT8 deletion SEQ ID NO: 19, and were respectively the amino acid sequence inferred from the nucleotide sequence in SEQ ID NO: 14. On the other hand, the fragments of the look out has been approximately 1.7 Kb in the above item 1 of this Example had occurred single base substitution with an amino acid mutation. In other words, 512th Guanin of F UT8 translated region is substituted with the adenine, resulting serine is 171 amino acid residues were found to be substituted into Asuparagin. In the determined nucleotide sequence of the RN6 strain derived FUT8- nucleotide substitutions SEQ ID NO: 18, and were respectively the amino acid sequence inferred from the nucleotide sequence in SEQ ID NO: 13.

3. Analysis of RN6 shares by genomic Southern blot

From RN6 strains and CHO / DG44 cells obtained in Example 1, a known method [Nucleic 'Ashi' de' Research (Nucleic Acids Research), 3, 2303, (1976)] Genomic D NA of each clone was prepared, each was dissolved in TE-RNase buffer (pH8.0) [10mmol / l Tris- HC1, lmmol / 1 EDTA, 200 μ g / ml RNase a]. Prepared genomic DNA is digested with the restriction enzyme Xbal, it was subjected to 0. 8% (w / v) Agarosugeru electrophoresis. After electrophoresis, a known method [Proceedings Day ring scan 'O Breakfast' The 'National' Academy ^ ~, O blanking 'Science (Proc. Natl. Acad. Sci. USA), 7 6, 3683, in accordance with (1979), a nylon membrane to the transfer of the genomic DNA. After completion of the transfer, the heat treatment for 1 hour at 80 ° C with respect to a nylon membrane KoTsuta.

[0226] First, among the Chinese Nono Muster FUT8, using probes specific for regions of the person from Ekuson 6 human FUT8 to Ekuson 9, having conducted a Southern blot analysis by the following procedure. Plasmid CHfFUT8_pCR2.1 described in patent WO02 / 31140 and 錡型, using specific forward primer FUT8 Ekuso emissions 6 (SEQ ID NO: 37) and specific Riva over scan primer Ekuson 9 (SEQ ID NO: 38), DNA the PCR due to the polymerase Ex Taq (Takara Bio Inc.) was carried out. PCR was 30 seconds at 94 ° C, 30 seconds at 60 ° C, was reaction consisting of 1 minute under the conditions of 25 cycles wherein one cycle at 74 ° C. After the PCR, and purified the amplified fragment of approximately 400 bp, was radiolabeled probed with [a_ 32 P] dCTP 1.75MBq and Megaprime DNA Labelling system, dCTP (Amersh am Pharmacia Biotech Co.). Hybrida I See Chillon solution of the above nylon membrane [5 X SSPE, 50 X Denhald s solution, 0.5% (w / v) SDS, 100 μ g / ml salmon sperm DNA] was immersed in 15 ml, 3 at 65 ° C were prehybridized da See Chillon of time. Next, 32 P-labeled probe DNA was introduced heat denatured and continue Botonore was allowed 65 ° C De晚加. After hybrida I See Chillon, Nairon membrane 2 33 G 0.1% ^ / ¥) was immersed in SDS 50 ml, and heated for 15 minutes at 65 ° C. After repeating twice the foregoing cleaning operations, the membrane was immersed in 0.2 X SSC- 0.1% (w / V) SDS 50ml, heated 15 minutes at 65 ° C. After washing, the nylon membrane was exposed and developed in -80 ° C to X-ray film.

[0227] From Ekuson 6 genomic Southern blot using primers specific to Ekuson 9 result detects the three fragments from RN6 strains and CHO / DG44 cell, the intensity of each fragment was similar in the two strains .

Next, of the Chinese hamster FUT8, using probes specific for regions of the person from Ekuson 3 human FUT8 to Ekuson 5, having conducted a Southern blot analysis by the following procedure. Plasmid CHfFUT8_pCR2.1 and 铸型, using FUT8 Ekuson 3-specific forward primer (SEQ ID NO: 39) and specific reverse primers in Ekuson 5 (SEQ ID NO: 40), DNA polymerase Ex Taq (Takara Bio Inc. PCR was performed by). PCR was 30 seconds at 94 ° C, 30 seconds at 60 ° C, were performed at 25 cycles of steps in which the reaction of one minute as one cycle at 74 ° C. After the PCR, and purified the amplified fragment of approximately 700 bp, was radiolabeled probed with [a- P] dCTP 1.75MBq and egapnme DNA Labelling system, dCTP (Amersham Pharmacia Biotech¾ :). The above nylon membrane hybrida I See Chillon was immersed in [5 X SSPE, 50 X Denhaldt, s solution, 0.5% (w / v) SDS, 100 μ g / ml salmon sperm DNA] 15ml, 65 ° C in was prehybridized da See Chillon of 3 hours. Next, 32 P-labeled pro one Bed DNA was heat-denatured and put into the bottle and allowed 65 ° C De晚加. After hybrida See Chillon, the Nairon film 2 53. Immersed in _0.1%) SDS 50ml, heated 15 minutes at 65 ° C. After repeating twice the foregoing cleaning operations, the membrane was immersed in 0.2 X SSC _0.1% (w / v) SDS 50ml, heated 15 minutes at 65 ° C. After washing, the nylon membrane to X-ray film - were exposed at 80 ° C development.

[0228] Ekuson 3 from genomic Southern blot using primers specific to Ekuson 5 results were detected single fragment of the same size than RN6 strains and CHO / DG44 cell. RN6 strength of KabuYukari come fragments, it was less than half of the fragment intensity derived from the CHO / DG44 cells.

These results, RN6 strain one FUT8 allele force et Ekuson 3, Ekuson 4, ethacrylic Son 5 it was confirmed lacking.

Example 3

Analysis of [0229] FUT8- base substitutions

Construction of 1. Chinese hamster FUT8 expression plasmid pcDNAchFUT8Comp

(1) Construction of plasmid CHFUT8Comp23

The plasmids were constructed CHFUT8Comp23 the following procedure (Fig. 3).

Plasmid CHiFUT8- pCR2.1 1.5 μ g of Patent WO02 / 31140, was dissolved in NEBuffer for BamHI (New England Biolabs, Inc.) 50 mu 1 containing 100 μ g / ml BS A (New England Biolabs, Inc.), limit enzyme BamHI (New England Biolabs, Inc.) 10 units and XhoI (New Engl and Biolabs, Inc.) was added to 10 units, it was carried out for 1 hour and a half of the digestion reaction at 37 ° C. The reaction solution was subjected to 0 .8% (w / v) Agarosugeru electrophoresis, a DNA fragment of about 2.0 Kb was purified by GENECLEAN Spin Kit (BIO 101 companies) and eluted with water 20 μ ΐ (hereinafter, from Agarosugeru the the DN Alpha fragments purified using this method).

[0230] On the other hand, plasmid pBlueScriptll KS (+) (Strategene Inc.) 1.0 mg, construed soluble in NEBuffer for BamHI (New England Biolabs, Inc.) 50 mu 1 containing 100 μ g / ml BSA (New England Biolabs, Inc.), restriction enzyme BamHI (New England Biolabs, Inc.) 10 units and XhoI (New England Bio labs, Inc.) 10 units mosquito 卩 Ete, was carried out for 1 hour and a half and digested at 37 ° C. The reaction solution was subjected to 0.8% (w / v) Agarosugeru electrophoresis to purify a DNA fragment of about 3.0 Kb.

[0231] BamH preparative Xhol fragment from plasmid CHiFUT8-pCR2.1 obtained above (about 2.0 Kb) 4.0 μ 1, plasmid pBlueScriptll SK (+) from the BamH preparative Xhol fragment (about 3.0 Kb) 0.5 μ 1, water 0.5 mu \, mixed Ligation High (Toyobo) 5.0 μ 1, was ligated to fragment by reaction for 30 minutes at 16 ° C. E. coli DH5a strain was transformed using the reaction solution to isolate the respective plasmid DNA in accordance with a known method from the obtained ampicillin-resistant clones. This plasmid hereinafter referred to as CHFUT8Comp23.

(2) Construction of plasmid CHFUT8CompTA

The plasmids were constructed CHFUT8CompTA the following procedure (Fig. 4).

[0232] First, a forward primer (SEQ ID NO: 41) linked Kozak sequence (KOZAK) Chinese Nono Muster FUT8 translation start site, and FUT8 reverse primer was ligated restriction enzymes Ba BamHI reaction site to the translation termination site (SEQ ID NO: 42 ) was synthesized. Next, the item (1) obtained in plasmid CHFUT8Comp23 50 ng and DNA polymerase KOD-plus (Toyo Boseki) 50 mu 1 of a reaction solution containing [KOD-plus buffer (TOYOBO), 0.2 mmol / 1 dNTPs, 1.2 m mol / 1 MgSO, 0.3 μ mol / 1 above gene-specific primers (SEQ ID NO: 41 and SEQ ID NO: 42)] was prepared and subjected to PCR. PCR, after heating for 2 minutes at 94 ° C, 10 seconds at 98 ° C, 30 seconds at 60 ° C, the reaction was conducted consisting of 1 minute 45 seconds 25 cycles of steps as one cycle at 68 ° C . After the PCR, and 添Ka卩 the Ex Taq (Takara Bio Inc.) 1 unit, and reacted for 5 minutes at 72 ° C. The reaction solution was subjected to 0.8% (w / v) Agarosugeru electrophoresis to purify a DNA fragment of about 1.7 Kb. The purified DNA fragments were 揷入 to plasmid PCR2.1 with TOPO TA cloning Kit (Invitrogen Corporation), and the Stbl2 E. coli strain was transformed. A known method from the obtained ampicillin-resistant clones, each plasmid DNA was isolated according to. This plasmid hereinafter referred to as C HFUT8CompTA.

(3) Construction of plasmid pcDNAchFUT8Comp

The plasmids were constructed pcDNAchFUT8Comp the following procedure (Fig. 5). [0233] Plasmid CHFUT8CompTA 2.0 μ g was obtained in the item (2) was dissolved in NEBuffer for EcoRI (New Engl and Biolabs, Inc.) 35 beta 1, Karoete restriction enzyme EcoRI (New England Biolabs, Inc.) 10 units, 37 ° digestion reaction 2 hours was carried out in C. After subjecting the digestion reaction was ethanol precipitated, the vector cut end was blunted with 10 mu 1 of a reaction system by Blunting High (Toyobo). Fuwenoru to smooth reaction - after the black hole Holm extraction, was dissolved in NEBuffer for BamHI (New England Biolabs, Inc.) 35 mu 1 containing 100 μ g / ml B SA (New England Biolabs, Inc.), restriction enzymes added BamHI (New England Biolabs, Inc.) 10 units, was digested reaction half for 1 hour at 37 ° C.

The reaction solution was subjected to 0.8% (w / v) Agarosugeru electrophoresis, a DNA fragment of about 1.7 Kb was made fine.

[0234] On the other hand, pcDNA3.1 (+) to (Invitrogen Corp.) 1.0 mu g was dissolved in NEBuffer2 (New England Biolabs, Inc.) 35 mu 1, by adding a restriction enzyme HindllKNew England Biolabs, Inc.) 10 units, at 37 ° C for It was half of the digestion reaction between two o'clock. After subjecting the digestion reaction was ethanol precipitated, the vector cut end was blunted with a reaction system of 10/1 by Blunting High (Toyobo). After phenol over chloroform extraction treatment to smooth reaction, and dissolved in NEBuffer for BamHI (New England Biolabs, Inc.) 35 mu 1 containing 100 β g / ml BSA (New England Biolab s Co.), restriction enzymes BamHKNew england Biolabs, Inc.) was added to 10 units, having conducted the digestion reaction of 1.5 hours at 37 ° C. The reaction solution 0.8% was subjected to (w / v) Agarosugeru electrophoresis, a DNA fragment of about 5.4 Kb; ^ the Q

[0235] EcoR preparative BamHI fragment from plasmid CHFUT8CompTA obtained above (about 1.7 Kb) 1.0 mu

1, Hindin-BamHI fragment from plasmid McKOgE2-2 (approximately 5.4 Kb) 0.5 / 1 1, water 3.5 μ 1, Li gation High (Toyobo) 5.0 mu 1 were mixed, to cause reaction for 1 hour at 16 ° C fragment was consolidated the by. The Stbl2 Escherichia coli was transformed using the reaction solution, each plasmid DNA in accordance with a known method from the obtained ampicillin-resistant clones were isolated. This plasmid hereinafter referred to as p cDNAchFUT8Comp.

2. Chinese hamster FUT8- base substitutions expression plasmid pcDNAchFUT8Mo the building

(1) to construct a plasmid CHFUT8Mo3 the following procedure Construction of plasmid CHFUT8Mo3 (Figure 6).

[0236] First, were synthesized specific forward primer sequence was substituted for 512th Guanin of FUT8 translation region to adenine (SEQ ID NO: 43) and reverse primer (SEQ ID NO: 44). Next, T4 Polynucleotide kinase (Takara Bio Inc.) 20 mu 1 of a reaction solution containing 10 units [Phosphorylation buffer (Takara Bio Inc.), 1.25 mmol / 1 ATP, 10 mmol / 1 above primer (SEQ ID NO: 43 and SEQ ID NO: by reacting for 30 minutes at 37 ° C for at 44)], it was phosphorylation of oligonucleotide terminus. Subsequently, the reaction solution 50 mu \ containing plasmids CHFUT8Comp23 50 ng and DNA polymerase KOD_plus obtained in the above item 1 of this Example (1) (Toyobo Co.) [KOD- plus buffer (TOYOBO), 0.2 mmol / 1 dNTPs, 1.2 mmol / 1 MgSO, 0.3

4 mu mol / 1 above gene-specific phosphorylation primer (SEQ ID NO: 43 and SEQ ID NO: 44)] was prepared and subjected to PCR. PCR, after heating for 2 minutes at 94 ° C, 15 seconds at 94 ° C, 3 0 seconds 60 ° C, was 5 minutes force made to react at 25 cycles of steps as one cycle at 68 ° C. The PC R reaction solution was subjected to 0.8% (w / v) Agarosugeru electrophoresis and purified DNA fragment of about 5.0 Kb multiplied diluted with water.

[0237] PCR amplification products obtained in the above (approximately 5.0) 1.0 mu 1, water 4.0 μ 1, Ligation High (Toyobo) 5.0

/ L were mixed and ligated fragments by 2 hours reaction at 16 ° C. The Stbl2 Escherichia coli was transformed using the reaction solution, the resulting ampicillin resistant Thus each plasmid DNA with a known method from clones were isolated. This plasmid hereinafter referred to as CHFUT8Mo3.

(2) Construction of plasmid CHFUT8MoTA

The plasmids were constructed CHFUT8MoTA the following procedure (Fig. 7).

[0238] First, a forward primer (SEQ ID NO: 41) linked Kozak sequence (KOZAK) Chinese Nono Muster FUT8 translation start site, and FUT8 reverse primer was ligated restriction enzymes Ba BamHI reaction site to the translation termination site (SEQ ID NO: 42 ) was synthesized. Then, 50 mu 1 of the reaction solution containing the resulting plasmid CHFUT8Mo3 50 ng and DNA polymerase KOD_plus (Toyobo) in this section (1) [KOD- plus buffer (TOYOBO), 0.2 mmol / 1 dNTPs, 1.2 mmol / 1 MgSO, 0.3 μ mol / 1 above gene-specific primers (SEQ ID NO: 41 and SEQ ID NO:

Four

42)] was prepared and subjected to PCR. PCR, after heating for 2 minutes at 94 ° C, 10 seconds at 98 ° C, 30 seconds at 60 ° C, rows 68 ° C in 1 minute 45 from consisting reactions of 25 cycles wherein one cycle second step ivy . After the PCR, the addition of Ex Taq (Takara Bio Inc.) 1 unit, and reacted for 5 minutes at 72 ° C. The reaction solution was subjected to 0.8% (w / v) Agarosugeru electrophoresis to purify a DNA fragment of about 1.7 Kb. The purified DNA fragment was 揷入 into the plasmid pC R2.1 using TOPO TA cloning Kit (Invitrogen Corporation), and the Stbl2 E. coli strain was transformed. Known methods Ri I obtained ampicillin-resistant clones, each plasmid DNA was isolated according to. This plasmid hereinafter referred to as CHFUT8 CompTA.

(3) Construction of plasmid pcDNAchFUT8Mo

The plasmids were constructed pcDNAchFUT8Mo the following procedure (Fig. 8).

[0239] Plasmid CHFUT8MoTA 2.0 μ g was obtained in the item (2) was dissolved in NEBuffer for EcoRI (New Englan d Biolabs, Inc.) 35 μ ΐ, Ete restriction enzyme EcoRI (New England Biolabs, Inc.) 10 units Caro, 37 ° digestion reaction 2 hours was carried out in C. After subjecting the digestion reaction was ethanol precipitated, the vector cut end was blunted with 10 mu 1 of a reaction system by Blunting High (Toyobo). After phenol over chloroform extraction treatment to smooth reaction, 100 M g / ml BSA (New England Biolabs, Inc.) NEBuffer for BamHI (New England Biolabs, Inc.) containing dissolved in 35 mu 1, restriction enzyme BamHI ( New England Biolabs, Inc.) was added to 10 units, it was carried out for 1 hour and a half of the digestion reaction at 37 ° C. The reaction solution was subjected to 0.8% (w / v) Agarosugeru electrophoresis to purify a DNA fragment of about 1. 7 Kb.

[0240] On the other hand, pcDNA3.1 (+) (Invitrogen Inc.) 1.0 mu g was dissolved in NEBuffer2 (New England Biolabs, Inc.) 35 mu 1, by adding a restriction enzyme HindIII (New England Biolabs, Inc.) 10 units, 37 ° half of the digestion reaction between 2:00 was carried out by C. After subjecting the digestion reaction was ethanol precipitated, the vector cut end was blunted with a reaction system of 10/1 by Blunting High (Toyobo). After phenol one black port Holm extraction process to smooth reaction, and dissolved in NEBuffer for BamHI (New England Biolabs, Inc.) 35 mu 1 containing 100 μ g / ml BSA (New England Biolab s Co.), restriction enzymes BamHKNew England Biolabs, Inc.) was added to 10 units, having conducted the digestion reaction of 1.5 hours at 37 ° C. The reaction solution was subjected to 0.8% (w / v) Agarosugeru electrophoresis to purify a DNA fragment of about 5.4 Kb.

[0241] EcoR preparative BamHI fragment from plasmid CHFUT8MoTA obtained above (about 1.7 Kb) 1.0 μ ΐ plasmid McKOgE2- 2 derived Hindin- BamHI fragment (about 5.4 Kb) 0.5 μ ΐ, water 3.5 μ 1, Ligat ion High (Toyobo Co., Ltd.) were mixed 5.0 / l, was ligated to fragment by 1 hour at 16 ° C. The reaction was transformed Stbl2 E. coli strain was used to isolate each plasmid DNA according to methods known from the ampicillin resistant clones was obtained. This plasmid hereinafter referred to as pc DNAchFUT8Mo.

3. FUT8- base substitutions introduced Fei cells - 1,6 - fucose modifying Analysis

CHO / DG44 cell-derived a FUT8 knockout cell Ms709 cells [Biotechnology and Bioengineering, 87, 614 (2004)] to the plasmid pcDNA ChFUT8Comp and pcDNAchFUT8Mo constructed in the above item 1 of this Example, elect port by the following procedure Poreshiyon law [C ytotechnology, 3, 133 (1990)] was introduced by.

[0242] First, it construed dissolved each plasmid 10 mu g in NEBuffer for SspI (New England Biolabs, Inc.) 100 mu 1, 20 units of a restriction enzyme SspI (New England Biolabs, Inc.) was added 4 hours at 37 ° C for the reaction was line was Joka. Performed Fuwenoru / black port Holm extraction and ethanol precipitation buttocks to the reaction mixture, recovered linearized plasmid was 1 / g / i l7 solution. On the other hand, the Ms709 cell K -PBS buffer [137mmol / l KC1, 2.7mmol / l NaCl, 8.1mmol / l Na HP04, 1.5mmol / l KH

PO, was suspended in 4.0 mmol / l MgCl] to 8 X 10 7 cells / ml. After the cell suspension 200 mu 1 a (1.6 X 10 6 cells) was mixed with the linearized plasmid 4/1 (4 / g) , the total amount of cellular DNA mixture solution G ene Pulser Cuvette (inter-electrode distance 2 mm) (BI_〇-RAD Co.) were transferred to, was performed using a cell fusion apparatus gene puis er (BIO_RAD Co.) pulse voltage 350 V, the gene transfer under conditions of electric capacity 250 mu F. After gene introduction, 10% cell suspension © Shi calf serum (Invitrogen) and HT s upplement (Invitrogen Corp.) were suspended in IMDM medium (Invitrogen) supplemented with adhesive culture T75 flasks (one company Guraina) They were seeded to. After culturing for 24 hours under the conditions of 5% CO, 37 ° C, culture supernatant was removed, 600 μ g / ml G418 (manufactured by Nacalai tester, Inc.), HT supplement (Invi trogen Co.) and 10% © shea fetal It was replaced to serum (Invitrogen, Inc.) IMDM medium supplemented with (Invitrogen). The medium replacement work is performed repeatedly while the 15 days of culture in 3-4 days, was to get the G418-resistant strains.

[0243] in 1% BSA-PBS, and suspended the G418 resistant clones 2 X 10 5 cells were added FITC-labeled LCA (Vector Laborat ories Co.) or 100-fold diluted FITC-labeled streptavidin (KPL, Inc.). 4 ° C at the cells were stained by standing for 30 minutes, after which the cells were washed with 1% BSA-PBS, were analyzed IX 10 4 cells in FACSCalibur (BD Biosciences Inc.).

It showed reactivity to LCA of each transgenic strain in FIG. In FUT8 expression strain (Plasmid pc DNAchFUT8Comp introduced strain), a group of cells showing high LCA reactivity of comparable CHO / DG44 cells were present. In FUT8- base substitutions expression strain (Plasmid pcDNAchFUT8Mo introduced strain), there was a group of cells showing a slightly reactive in LCA. This result from the replacement of the 512th Guanin of FUT8 translation area to adenine, Fei cell - 1,6 - was shown that fucose addition capability is greatly reduced.

Industrial Applicability

[0244] The present invention, N- glycoside-linked complex type sugar chain of N- § cetyl Darco Sa Min

In enzyme which 1-position of fucose to position 6 is involved in sugar chain modification to bind alpha, activity of the enzyme is deleted or amino acids modified variants and usage thereof to decrease, it is provide.

Sequence Listing pretend - text

[0245] SEQ ID NO: 23 - Artificial Sequence Description synthetic DNA

Description synthetic DNA of SEQ ID NO: 24 Artificial Sequence

Description synthetic DNA of SEQ ID NO: 25 Artificial Sequence

Description synthetic DNA of SEQ ID NO: 26- Artificial Sequence

Description synthetic DNA of SEQ ID NO: 27- Artificial Sequence

Description synthetic DNA of SEQ ID NO: 28- Artificial Sequence

Description synthetic DNA of SEQ ID NO: 29 - Artificial Sequence

Description synthetic DNA of SEQ ID NO: 30- Artificial Sequence

Description synthetic DNA of SEQ ID NO: 31- Artificial Sequence

Description synthetic DNA of SEQ ID NO: 32 Artificial Sequence

Description synthetic DNA of SEQ ID NO: 33-Artificial Sequence

Description synthetic DNA of SEQ ID NO: 34- Artificial Sequence

Description synthetic DNA of SEQ ID NO: 35- Artificial Sequence

Description synthetic DNA of SEQ ID NO: 36- Artificial Sequence

SEQ ID NO: 37- Description Description synthetic DNA SEQ ID NO: 38- Artificial Sequence of Artificial Sequence: Description of the synthetic DNA SEQ ID NO: 39-Artificial Sequence: Description of the synthetic DNA SEQ ID NO: 40 - Artificial Sequence: synthetic DNA SEQ ID NO: 41- Artificial Sequence description: the description of the synthetic DNA SEQ ID NO: 42 artificial sequence: description of the synthetic DNA SEQ ID NO: 43- artificial sequence: description of the synthetic DNA SEQ ID NO: 44- artificial sequence: synthetic DNA

Claims

The scope of the claims
[1] Fei - 1,6 in the amino acid sequence of fucosyltransferase force amino acid at the position corresponding to 171 amino acids from the N-terminus of § amino acid sequence represented by SEQ ID NO: 7 is that lacked \ or having the amino acid sequence are substituted with an amino acid other than serine, shed - 1,6_ Fukoshi transferase variants.
[2] an amino acid other than serine is Asuparagin, Fei of claim 1 1,6 - Fukoshirutoran Sufuweraze variants.
[3] alpha-1,6-fucosyltransferase mosquito following (a) DNA selected from the group consisting of ~ (f) is a protein encoded, alpha-1,6-fucosyl according to claim 1 or 2 transferase zero variant.
(A) comprising the nucleotide sequence represented by SEQ ID NO: 1 DNA;
(B) comprising the nucleotide sequence represented by SEQ ID NO: 2 DNA;
(C) comprising the nucleotide sequence represented by SEQ ID NO: 3 DNA;
(D) comprising the nucleotide sequence represented by SEQ ID NO: 4 DNA;
(E) comprising the nucleotide sequence represented by SEQ ID NO: 5 DNA;
(F) DNA comprising the nucleotide sequence represented by SEQ ID NO: 6.
[4] Fei - 1,6 fucosyl a protein selected from the group consisting of fucosyltransferase mosquito following (a) ~ (f), Fei-1,6-fucosyltransferase variant according to claim 1 or 2.
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 7;
(B) a protein comprising the amino acid sequence represented by SEQ ID NO: 8;
(C) a protein consisting of the amino acid sequence of SEQ ID NO: 9;
(D) a protein consisting of the amino acid sequence of SEQ ID NO: 10;
(E) a protein comprising the amino acid sequence represented by SEQ ID NO: 11;
(F) a protein comprising the amino acid sequence represented by SEQ ID NO: 12.
[5] SEQ ID NO. 13 to: alpha comprises an amino acid sequence represented by any one of the 17-1,6-fucosyl trans luciferase mutants.
[6] SEQ ID NO 13: In the amino acid sequence represented by any one of 17, one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence, and alpha-1,6-fucosyltransferase deletion glycosyltransferase activity, or SEQ ID NO: 7 or 9 comprising the amino acid sequence represented by Fei-1,6-fucosyltransferase alpha -1 was lower than alpha-1,6-fucosyltransferase activity of glycosyltransferases, having a 6-fucosyltransferase activity alpha-1,6-Fukoshirutora Nsufuweraze variants.
SEQ ID NO 13: 17 consisting of an amino acid sequence having an amino acid sequence homology of 80% or more represented by any one of, Katsuhi - 1,6-fucosyltransferase activity are deleted, or SEQ ID NO: 7 or 9 in flight consisting of the amino acid sequence 1,6 - fucosyl lower than sill transferase Zenohi 1,6-fucosyltransferase activity Fei having Tahi 1,6 fucosyl transflector Eraze active 1,6 - fucosyl transferase mutant.
Fei according to any one of claims 1 to 7 1,6 - fucosyltransferase variant code to DNA.
DNA comprising the nucleotide sequence shown in any of SEQ ID NO: 18-22.
Hybridizes with the base sequence under stringent conditions represented by any of SEQ ID NO: 18-22, and alpha-1,6-fucosyltransferase activity are deleted, or SEQ ID NO: 7 or is represented by 9 that the amino acid sequence power et consisting alpha 1,6 fucosyl Fei-1,6-fucosyltransferase variants with reduced alpha-1,6-fucosyltransferase activity than alpha _1,6_ full waist transferase activity of transferase encoding the DNA.
alpha-1,6-fucosyltransferase variant according to any one of claims 1 to 7 or, alpha-1,6-fucosyl DNA encodes according to any one of claims 8-10 cells expressing Trang Sufueraze variants.
Claim 8: 10 cells transfected with the DNA of any one of.
Fei according to any one of claims 1 to 7, 1,6 - fucosyltransferase variants or, DNA code Suruhi 1,6 fucosyl according to any one of claims 8-10 Tran Sufueraze variants Fei 1,6 - fucosyl claim 11 or 12 wherein the cell has only glycosyltransferase activity.
Claim 11 has been introduced a gene encoding a glycoprotein: 13 cells according to any one of.
Glycoprotein is an antibody, the cells of claim 14.
[16] the antibody, the following (a), (b), (, (d) and (an antibody selected from the group consisting of e), the cells of claim 1 5.
(A) human antibodies;
(B) chimeric antibody;
(C) a humanized antibody;
(D) (a) or antibody fragment comprising the Fc region of (b);
(E) (a) or a fusion protein comprising the Fc region of (b).
[17] Claim 14: 16 was cultured in a medium of cells according to any one of the glycoprotein composition comprising To蛋 white matter molecule from the culture was harvested, and wherein the step of purifying sugar method for producing a protein assembly formed products.
[18] a glycoprotein antibody, production method of claim 17.
[19] glycoprotein composition produced using the method according to claim 17.
[20] The antibody composition prepared using the method of claim 18.
[21] A medicament comprising a glycoprotein composition as an active ingredient in claim 19.
[22] A medicament comprising the antibody composition as an active ingredient in claim 20.
[23] The cells were separated from human tissues, genomic than isolated cells, it acquires the total RNA or mRNA, obtained the genome, the total RNA or mRNA, or from adjusted by using the acquired total RNA or mRNA A cDNA the gene of alpha-1,6-fucosyltransferase isolated, in the nucleotide sequence of the isolated gene, the amino acid positions corresponding to Ν-terminal power 171 amino acid sequence of the amino acid sequence represented by SEQ ID NO: 7 is the Asuparagin diagnostic methods substituted by Les disease you involved alpha-1,6-fucosyltransferase and detecting whether Luke.
PCT/JP2007/053733 2006-02-28 2007-02-28 α-1,6-FUCOSYLTRANSFERASE MUTANT AND USE THEREOF WO2007099988A1 (en)

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WO2009122667A1 (en) * 2008-04-04 2009-10-08 中外製薬株式会社 Therapeutic for hepatic cancer
WO2012175874A1 (en) 2011-06-22 2012-12-27 Lfb Biotechnologies Use of a high-adcc anti-cd20 antibody for treating waldenström's macroglobulemia
US8497355B2 (en) 2007-09-28 2013-07-30 Chugai Seiyaku Kabushiki Kaisha Anti-glypican-3 antibody having improved kinetics in plasma
WO2014096672A1 (en) 2012-12-17 2014-06-26 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Use of monoclonal antibodies for the treatment of inflammation and bacterial infections
WO2015107307A1 (en) 2014-01-17 2015-07-23 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Immunoglobulin against the anthrax toxin
WO2017006052A2 (en) 2015-07-06 2017-01-12 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Use of modified fc fragments in immunotherapy
US9975966B2 (en) 2014-09-26 2018-05-22 Chugai Seiyaku Kabushiki Kaisha Cytotoxicity-inducing theraputic agent
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WO2003085107A1 (en) * 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Cells with modified genome
WO2005035778A1 (en) * 2003-10-09 2005-04-21 Kyowa Hakko Kogyo Co., Ltd. PROCESS FOR PRODUCING ANTIBODY COMPOSITION BY USING RNA INHIBITING THE FUNCTION OF α1,6-FUCOSYLTRANSFERASE
WO2005035563A1 (en) * 2003-10-09 2005-04-21 Kyowa Hakko Kogyo Co., Ltd. Process for producing antithrombin iii composition

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WO2003085107A1 (en) * 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Cells with modified genome
WO2005035778A1 (en) * 2003-10-09 2005-04-21 Kyowa Hakko Kogyo Co., Ltd. PROCESS FOR PRODUCING ANTIBODY COMPOSITION BY USING RNA INHIBITING THE FUNCTION OF α1,6-FUCOSYLTRANSFERASE
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Cited By (11)

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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 (en) * 2008-02-13 2009-08-27 Kitasato Institute Method for fluorescent-labeling sialic acid, sialic acid-containing glucide, or sialic acid-containing glycoconjugate, and fluorescent-labeled sialic acid, sialic acid-containing glucide, or sialic acid-containing glycoconjugate obtained by the method
WO2009122667A1 (en) * 2008-04-04 2009-10-08 中外製薬株式会社 Therapeutic for hepatic cancer
JP2011068682A (en) * 2008-04-04 2011-04-07 Chugai Pharmaceut Co Ltd Therapeutic for hepatic cancer
CN102046200A (en) * 2008-04-04 2011-05-04 中外制药株式会社 Therapeutic for hepatic cancer
WO2012175874A1 (en) 2011-06-22 2012-12-27 Lfb Biotechnologies Use of a high-adcc anti-cd20 antibody for treating waldenström's macroglobulemia
WO2014096672A1 (en) 2012-12-17 2014-06-26 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Use of monoclonal antibodies for the treatment of inflammation and bacterial infections
WO2015107307A1 (en) 2014-01-17 2015-07-23 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Immunoglobulin against the anthrax toxin
US9975966B2 (en) 2014-09-26 2018-05-22 Chugai Seiyaku Kabushiki Kaisha Cytotoxicity-inducing theraputic agent
WO2017006052A2 (en) 2015-07-06 2017-01-12 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Use of modified fc fragments in immunotherapy

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