JP2002291478A - Method for improving accumulating tendency of glycoprotein in liver - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for improving the accumulating tendency of glycoprotein in the liver by modifying the structure of the sugar chain added to the glycoprotein. SOLUTION: This method comprises increasing the number of galactose residues exposed at the unreduced terminals in the sugar chain added to a glycoprotein by increasing the branching number thereof and, thereby, increasing the accumulating tendency of the glycoprotein in the liver.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for improving the hepatic accumulation of a glycoprotein, and improves the hepatic accumulation of a glycoprotein by modifying a sugar chain added to the glycoprotein. About things.

[0002]

2. Description of the Related Art In recent years, with the advance of biotechnology, many bioactive proteins have been mass-produced by techniques such as gene recombination. As a result, various bioactive proteins have been used as pharmaceuticals. However, such a bioactive protein shows high activity in a very small amount and also has various biological activities. Therefore, when put into practical use as a pharmaceutical, side effects become a serious problem.

For example, one of the cytokines, interferon, has antiviral activity, cell growth inhibitory activity, etc.
It is a protein that shows various biological activities. Utilizing this pharmacological effect, it is used for the treatment of chronic active hepatitis B and C, renal cancer and the like. However, when interferon is administered to a living body, it is rapidly excreted from the body, so it is difficult to maintain the interferon in the target organ at an effective concentration, and in order to enhance the therapeutic effect of interferon, It requires doses and prolonged treatment, and carries the risk of side effects. The side effects of interferon include influenza-like symptoms accompanied by fever, headache, and general malaise and decreased white blood cells and platelets as early symptoms, continuation of low-grade fever, anorexia, insomnia, and depression as medium-term symptoms, and hair loss and thyroid gland as late symptoms. There is a decline in function. When these side effects occur, it is not possible to administer the amount of interferon necessary for the onset of the medicinal effect, and it may be necessary to discontinue or reduce the dosage.

As described above, when a physiologically active protein is administered to a living body, the problem of side effects is serious. Therefore, it is required to control the behavior of the protein in the body to enhance the therapeutic effect and reduce the side effects. As a means for solving this problem, development of a system for specifically delivering a protein to a target tissue has been desired.

[0005] In vivo, there are proteins having affinity for sugar, and these are called lectins. There are various types of lectins, each of which recognizes and binds a sugar having a specific structure. Therefore, the lectin present on the cell surface can be considered as a receptor molecule for a ligand substance having a sugar at the terminal. Since each organ has its own unique lectin, lectins can be used as a clue for recognition of a specific organ in drug delivery technology. From this, it is expected that a protein having a sugar chain is delivered to a specific organ by utilizing the affinity between the sugar chain and lectin.

[0006] Affinity between the liver and galactose has been reported in relation to accumulation in the liver as a target organ (Ashwell, G. and Morell, AG (1977) Trends Bioche).
m.Sci.Vol.2, 76-78, Ashwell, G. and Harford, J. (198
2) Ann. Rev. Biochem. Vol. 51, 531-554). Based on this finding, by incorporating a sugar having a galactose terminal to a protein having a physiological activity effective for liver diseases such as liver cancer, cirrhosis, and hepatitis, the uptake of the protein into the liver can be efficiently increased. It is thought that the treatment effect can be enhanced. Examples of the protein modified with galactose for such a purpose include a bioactive protein modified with D-galactopyranosyl polyethylene glycol (JP-A-63
-152393). There is also a finding that clustering galactose increases the affinity for the liver (Lee, YC et al. (1983) J. Bi
ol.Chem. Vol.258, 199-202, Nishikawa, M. et al. (19
95) Am. J. Physiol. Vol. 268, G849-G856). From the same viewpoint, it is known that a glycoprotein containing a large amount of a sugar chain having a galactose-galactose bond at a terminal also has an accumulation property in the liver (Japanese Patent Laid-Open No. 1-102099).
However, the conventional technology requires reaction conditions that may denature the bioactive protein, the added sugar chain is of a non-natural type, or shows antigenicity to the human body. There are inconveniences such as slipping. In addition, it has not been put to practical use because the degree of protein modification cannot be controlled or the organ accumulation is not sufficient.

In recent years, attempts have been made to modify the sugar chains of glycoproteins in order to improve the functions of the glycoproteins. Means for modifying the sugar chain of glycoprotein include N-acetylglucosaminyltransferase I in glycoprotein-producing cells.
There is known a method in which a gene for V or N-acetylglucosaminyltransferase V is introduced and a glycoprotein is produced by the cells to increase the number of branched sugar chains (JP-A-11-346770). However, JP-A-11-346770 does not show that increasing the number of branched sugar chains has the effect of accumulating glycoproteins in specific organs.

[0008] Liver cells show an affinity for a glycoprotein that exposes galactose, but a hepatic affinity of a glycoprotein whose galactose end is protected by sialic acid is low (More
ll, AG et al. (1971) J. Biol. Chem. Vol. 246, 1461-1
467). All the above findings on the affinity between galactose and the liver relate to the affinity between galactose to which sialic acid is not added and the liver. It is necessary to artificially remove the sialic acid at the terminal of the sugar chain in order to increase the hepatic accumulation of the glycoprotein to which the sialic acid is added. An example of enhancing hepatic accumulation of glycoproteins by removing sialic acid at the sugar chain terminal is asialo-interferon-
β (Kasama, K. et al. (1995) J. Interferon cytokine
Res. Vol. 15, 407-415).

[0009] The sugar chain is usually composed of monosaccharides such as N-acetylglucosamine, mannose, galactose, fucose and sialic acid. As the N-linked sugar chain naturally produced in the human body, a branched type as shown in FIG. 1 is known (Takeuchi, M. et al. (1988) J. Biol. Che.
m. Vol. 263, 3657-3663, Tsuda, E. et al. (1988) Bioc
hemistry Vol. 27, 5646-5654), increasing the number of sugar chains increases the number of galactose contained. However, sialic acid is usually added to the end of galactose, and the sugar chain ends are capped with sialic acid (Varki A. et al. (1999) Essentials of Glycobiolo
gy, pp.195-209, Cold Spring Harbor Laboratory Pres
s, Cold Spring Harbor, NY, Kornfeld, R. and Kornfel
d, S. (1985) Ann. Rev. Biochem. Vol. 54, 631-664). Therefore, although an increase in the number of branched sugar chains increases the number of galactose contained, it does not mean an increase in exposed galactose recognized in the liver.

As described above, it has been known that increasing the number of exposed galactoses of glycoprotein sugar chains can enhance the hepatic accumulation of glycoproteins. The increase is not considered to be directly related, and if sialic acid is not removed artificially,
It was unexpected that increasing the number of branched sugar chains would lead to an increase in the accumulation of glycoproteins in the liver.

[0011]

SUMMARY OF THE INVENTION An object of the present invention is to control the sugar chain synthase gene in a sugar chain added to a glycoprotein to modify the sugar chain structure so that the glycoprotein is not reduced. An object of the present invention is to provide a method for improving hepatic accumulation by increasing the number of galactose residues exposed at the ends.

[0012]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems. As a result, the addition of sialic acid is often not complete at the sugar chain terminal of a glycoprotein, It was found that increasing the number of branches increased the number of galactose partially exposed at the sugar chain end. Further, based on this finding, it is possible to improve the affinity between glycoproteins and the liver by using the increase in exposed galactose accompanying the increase in the number of sugar chains, and to improve the liver accumulation of the glycoproteins. The present inventors have found that the present invention is possible, and completed the present invention based on the findings.

That is, the present invention is as follows. [1] Hepatic accumulation of glycoprotein, characterized by increasing the number of galactose residues exposed at the non-reducing end of the sugar chain by increasing the number of branches of the sugar chain added to the glycoprotein. How to improve. [2] The number of sugar chains of a glycoprotein is increased by the gene expression of N-acetylglucosaminyltransferase IV and / or N-acetylglucosaminyltransferase V in glycoprotein-producing cells [1] ]. [3] removing the sialic acid at the terminal of the sugar chain or modifying the addition of sialic acid so as to reduce the addition of sialic acid, thereby further increasing the number of galactose residues exposed at the non-reducing terminal; The method according to [1] or [2]. [4] Glycoprotein is produced using glycoprotein-producing cells that lack sialic acid addition ability or have extremely low sialic acid addition ability, and the number of galactose residues exposed at the non-reducing end is increased. The method according to [1] or [2], wherein the number is increased. [5] The method according to any one of [1] to [4], wherein the glycoprotein-producing cell is a Chinese hamster ovary cell. [6] A glycoprotein having improved liver accumulation obtained by the method according to any one of [1] to [5]. [7] An interferon with improved liver accumulation obtained by the method according to any one of [1] to [5]. [8] A fibroblast growth factor having improved liver accumulation obtained by the method according to any one of [1] to [5]. [9] A hepatocyte growth factor with improved liver accumulation obtained by the method according to [1] to [5]. [10] A pharmaceutical composition comprising a glycoprotein obtained by the method according to any one of [6] to [9].

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The glycoprotein according to the present invention is a protein in which a sugar chain is bound to at least one site of the main protein. Examples of such are interferon (IFN) group, fibroblast growth factor (FGF), hepatocyte growth factor (HG
F), tissue plasminogen activator (t-PA),
Granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), immunoglobulin (Ig) M and G, interleukin (IL) group, thrombopoietin (TP
O) and the like, and variants thereof. The glycoprotein of the present invention may be derived from various animals including humans (including those obtained by cell culture), derived from recombinant organisms, or synthesized. Also, the present invention
The present invention can also be applied to a protein modified so as to add a sugar chain by introducing a sugar chain addition sequence into an amino acid sequence having no sugar chain addition site. For example, the interferon-α group often has no sugar chain added thereto, but the amino acid sequence is mutated so that the sugar chain is added to such interferon-α, and then the sugar chain is added. By increasing the number of branched sugar chains according to the present invention,
It is thought that the ability of interferon-α to accumulate in the liver can be improved, and such interferon-α having high liver accumulation is considered to be effective as a therapeutic agent for liver disease.

The sugar chain according to the present invention is composed of monosaccharides such as N-acetylglucosamine, mannose, galactose, fucose and sialic acid. Usually, sialic acid is often added to the terminal of the sugar chain, but the addition rate of sialic acid is not always perfect. As a result, the partially exposed galactose residues can play a role in liver accumulation. In addition, a sugar chain from which galactose is exposed by intentionally removing sialic acid is more preferable for liver accumulation. The sugar chain according to the present invention may be natural or chemically synthesized.

In the present invention, increasing the number of branched sugar chains means increasing the proportion of sugar chains having a large number of branched chains in the composition of sugar chains of glycoprotein. The range of increase in the number of branches is
In consideration of the safety as a pharmaceutical, it is desirable to increase the number of branches within the range of the sugar chain structure naturally found, and usually the number of branches is two, three or four (FIG. 1). However, if safety is ignored and only the effect of accumulation in the liver is considered, if the branch structure is modified beyond the range of the structure found in nature and the number of branches is increased, the accumulation in the liver will increase. Is better. In the present invention, it is also possible to control the accumulation in the liver by controlling the number of branched sugar chains.

In the present invention, the addition of a sugar chain to a protein
It can be performed using the sugar chain biosynthesis function of the cell. That is, a sugar chain is added by producing a target protein by cells having a sugar chain biosynthesis mechanism. The glycoprotein thus obtained can be treated with sialidase to remove sialic acid, thereby exposing galactose residues artificially. In this case, accumulation in the liver is further improved. . On the other hand, by producing a protein in a form in which a sugar chain is not added by genetic recombination in a cell having no sugar chain biosynthesis mechanism, such as Escherichia coli, the sugar chain is enzymatically or chemically added to the obtained protein. Is also good. In this case, the accumulation in the liver is improved by preparing a sugar chain having a large number of branches and a large number of exposed galactose residues at the non-reducing terminal in advance and adding the sugar chain.

In the present invention, when producing glycoproteins using cells, the cells that can be used for producing glycoproteins are not particularly limited as long as they can produce glycoproteins. Plants, eukaryotic microbial cells and the like can be mentioned. As the animal cells, both adherent cells and planktonic cells can be used, and animal cells that produce and accumulate glycoproteins in the cells or animal cells that secrete and produce glycoproteins outside the cells may be used. These cells may be natural cells that produce the desired glycoprotein, or may be recombinant cells into which a gene encoding the desired protein has been introduced. Host cells used for recombinant production include CHO cells (Chinese hamster ovary cells), monkey Vero cells, mouse L cells, and B cells.
HK cells, φ2 (NIH3T3) cells, mouse C127
Cells, monkey COS cells, Hela cells, mouse myelo-
And human B cells. As a recombinant cell into which the human interferon-γ gene has been introduced, the CHO cell line HIIF-D is an American type culture.
Deposited in the collection as ATCC CRL-8200. In the present invention, the use of cells having a low ability to add sialic acid to the glycoprotein to be produced is more effective in accumulating the produced glycoprotein in the liver. Cells lacking the ability to add sialic acid include CHO cell L
ec2 cells are known (Deutscher, SL et al. (1
984) CellVol.39, 295-299).

When producing glycoproteins using cells, as a method of increasing the number of sugar chains, a cell that biosynthesizes a sugar chain having a high number of branches is selected, and the desired glycoprotein is produced by the cells. Production. Cells that biosynthesize a sugar chain with a high number of branches are transferred to a glycoprotein-producing cell by N-acetylglucosaminyltransferase IV (hereinafter referred to as Gn).
It can also be obtained by introducing a gene for T-IV) or N-acetylglucosaminyltransferase V (hereinafter abbreviated as GnT-V) and expressing it at a high level. GnT-
IV and GnT-V are N-branch or 3-branch structures
-Transfer of an N-acetylglucosamine residue to a linked sugar chain to generate a sugar chain having a three- or four-branch structure.

As described above, the number of galactose residues at the terminal of the sugar chain increases as the number of branches of the sugar chain increases. Sialic acid is often added to the end of galactose, but the addition of sialic acid is often not complete. As a result, galactose is partially exposed at the terminal of the sugar chain, and such exposed galactose is recognized in the liver. If the addition rate of sialic acid is the same, increasing the number of sugar chains to increase the number of galactose residues results in an increase in the number of exposed galactose. On the other hand, reducing the addition of sialic acid is effective in increasing the accumulation in the liver. If the addition of sialic acid is suppressed together with the increase in the number of branched sugar chains, the accumulation in the liver is further increased due to the synergistic effect. Such a glycoprotein can be obtained by producing a glycoprotein having a large number of branches in the form of a large amount of sialic acid addition and then enzymatically or chemically removing the sialic acid. It can also be obtained by applying cells having a low ability to add sialic acid to the present invention.

As a vector for introducing a gene encoding a protein to be produced or a gene for increasing the number of sugar chains into a glycoprotein-producing cell, any vector can be used so long as the gene of interest can be expressed in the cell. Although it is possible, specifically, SV40, BPV (bovine papilloma virus), adenovirus, and retrovirus are used as those utilizing animal viruses. Animal viruses generally use promoters, RNA,
Since it has a self-replicating ability in addition to splicing signals, poly-A addition signals, and signals necessary for gene expression such as enhancers that increase the activity of the promoter, the use of such animal virus-based And increase gene expression. In addition, it is preferable to use one that has the function of a selection marker such as a neomycin resistance gene or a hygromycin resistance gene, thereby providing a means for selecting transformed cells and facilitating isolation of the desired transformed cells. As a promoter or enhancer which is a control site for expressing a gene, a promoter or an enhancer which can obtain a desired effect in a cell can be used. For example, LTR (retrovirus long terminal repeata) can be used.
t), SV40, CMV (cytomegalovirus), M
Examples include promoters such as T (meta-mouth thionein) and actin, and enhancer sequences such as LTR, SV40 and CMV. The expression vector for animal cells that can be used in the present invention specifically enables high expression of a foreign gene by replacing a part of the nucleotide sequence of the chicken β-actin gene promoter with a gene derived from rabbit β-globin. PCXN-based expression vectors, especially pCXN2 (Niwa, H., Yamamur
a, K. and Miyazaki, J., Gene vol.108, p.193-200, 199
1, JP-A-03-168087), but not particularly limited thereto.

The method of introducing the prepared expression vector into cells includes the most common calcium phosphate method, electroporation method, microinjection method, protoplast fusion method, liposome fusion method, erythrocyte ghost fusion method and the like. Is used.

The cultivation of the cells can be carried out using various known methods, and there is no particular limitation as long as the growth of the cells and the production of glycoprotein are not inhibited. For example, suspension culture in a tank, adhesion culture in which cells are adhered to the surface of styrene microbeads or the inner wall of a roller bottle, stationary culture using a flask, and the like can be appropriately selected according to the cell strain. When culturing by a batch method, the culturing time may be such that cells are sufficiently grown and glycoprotein is sufficiently produced, and is usually about one week to six months. When performing continuous culture while aseptically replacing a part of the culture medium during the culture, the culture time is about one week to six months.
In culturing, after inoculating a cell line that produces a glycoprotein, the cell line is cultured while maintaining an appropriate temperature, aeration, and pH of the medium.

As a medium that can be used for culturing cells, a medium obtained by adding an additive such as serum to a basic medium can be used. As the basic medium, commercially available medium for cell culture can be used. For example, Eagle's minimum essential medium, RPMI-1640 medium, Ham F12 medium, Dulbecco's modified Eagle medium, CHO-S-SFMII medium (GI
BCO BRL), Opti-MEM medium (GIBCO BRL), etc.
They may be used alone or in a suitable mixture. In addition, cell culture can be performed in two stages, a growth culture for increasing the number of cells and a production culture for producing a glycoprotein, and two different types of media can be used. In this case, for example, a nutrient medium obtained by adding fetal bovine serum (FCS) at a concentration of 1 to 30% to the above basal medium is used as a growth medium, and the above basal medium containing no fetal calf serum (FCS) is used as a production medium. Thus, the load in the purification step of the produced glycoprotein can be reduced.

Purification of the glycoprotein from the cell culture can be performed by a conventional method. It can be separated and purified from the cell culture by various known purification methods utilizing, for example, ion exchange, biological affinity, adsorption or hydrophobicity, hydrophilicity, molecular size, ultrafiltration and the like. When a sugar chain is enzymatically or chemically added to a protein to which no sugar chain is added, after the sugar chain addition reaction, the reaction solution is dialyzed, salted out, ultrafiltered, ion-exchanged chromatography, Gel filtration, H
The desired glycoprotein can be obtained by purification by a conventional protein purification method such as PLC or electrophoresis.

When sialic acid is acted on the purified glycoprotein to remove sialic acid enzymatically or chemically, sialic acid is effectively removed from the liver.

The sugar chain structure of the glycoprotein of the present invention can be analyzed by various known methods. That is, by using a chemical method such as hydrazinolysis or an enzyme such as N-glycanase, O-glycanase, and glycopeptidase, sugar chains are released from the protein itself, and the free sugar chains are subjected to various known column chromatography. The structure can be analyzed by analyzing the obtained sugar chain with a device such as HPLC, NMR, and mass spectrometry. It is also possible to label a sugar chain to improve detection sensitivity. Labeling methods include 2-aminopyridine, 2-aminobenzamide (2-AB), 1,2-diamino-4,5-
There are a method of labeling with a compound such as methylenedioxydibenzene (DMB) and a method of labeling with a radioactive compound such as ³ H and ¹³ C. In the case of N-linked glycans, the free glycans are fluorescently labeled by pyridyl amination, and HPLC analysis is performed using reversed-phase chromatography and normal-phase chromatography, and the structure is analyzed in combination with various exoglycosidase treatments. it can.

In the method of the present invention, in order to increase the number of exposed galactose residues at the sugar chain terminals by increasing the number of branched sugar chains, the characteristics of hepatocytes having affinity for galactose are utilized. The glycoprotein can selectively and efficiently reach the liver tissue. Therefore, the glycoprotein of the present invention is considered to be particularly effective for treating or preventing liver diseases such as liver cancer, cirrhosis and hepatitis. In addition, since the glycoprotein of the present invention is efficiently transported to the liver, it is expected that a therapeutic effect can be obtained at a low dose. Furthermore, as the sugar chain of the glycoprotein of the present invention, a sugar chain modified within the range of a naturally occurring sugar chain structure can be used, and there is no problem of antigenicity and high safety. In addition, when producing the glycoprotein of the present invention, it is possible to control the accumulation in the liver by controlling the number of branched sugar chains.

The glycoprotein of the present invention can be administered orally or parenterally to a mammal (eg, human, monkey, dog, pig, rabbit, mouse, rat, etc.) as a pharmaceutical composition. It can be used in the form of general medical preparations. Examples of such drugs include preparations containing interferon-α, β, γ, or fibroblast growth factor, hepatocyte growth factor, and the like. Pharmaceutical preparations containing such a glycoprotein of the present invention as an active ingredient include commonly used fillers, extenders, binders, humectants,
It can be prepared using carriers such as disintegrants, surfactants and lubricants, diluents or excipients. Various forms of the pharmaceutical preparation of the present invention can be selected according to the purpose of treatment, and typical examples are tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, injections (solutions) , Suspension preparations). Further, if necessary, coloring agents, preservatives, flavoring agents, sweeteners and other pharmaceuticals can be contained.

[0030]

EXAMPLES Hereinafter, the present invention will be described in more detail by way of examples, but these examples do not limit the scope of the present invention at all.

Example 1 Production of human interferon-γ having a biantennary sugar chain Human interferon-γ (hereinafter abbreviated as hIFN-γ) producing Chinese hamster ovary (hereinafter abbreviated as CHO) cells, HIIF-D strain ( Purchased from ATTC, ATC
C NO. CRL-8200) in 10% dialyzed fetal calf serum (dFCS), 250 nM methotrexate (M
TX) containing CHO-S-SFMII medium (Life
Technologies, Inc. Cultivation in a T-flask at 37 ° C. under 5% CO ₂ conditions to grow the cells, then remove the medium, wash the cells with PBS, and add L
-Serum-free CD-CHO medium supplemented with glutamine (Life
eTechnologies, Inc. Made)
HIFN-γ was secreted into the medium. The medium was collected every 24 hours and replaced with fresh medium each time. The medium supernatant was filtered through a 0.22 micron filter, and then anti-hIFN
HIFN-γ was purified by affinity chromatography using an antibody column to which -γ antibody was bound. The sugar chain structure of the obtained hIFN-γ was analyzed as follows. After digesting the purified hIFN-γ with trypsin, gel filtration was performed with Sephadex G-25 (manufactured by Amersham Pharmacia Biotech) to collect a glycopeptide fraction. Almond-derived glycopeptidase A (manufactured by Seikagaku Corporation) was added to the obtained glycopeptide and reacted at 37 ° C. for 20 hours. Separate the released sugar chains
Purification was carried out using a -pak C18 cartridge (manufactured by Waters) and a cellulose cartridge (manufactured by Takara Shuzo).
Arthrobacter ure is added to the purified free sugar chains.
By reacting sialidase (manufactured by Nacalai Tesque) derived from Afaciens, sialic acid was first released, and then β-galactosidase (produced by Seikagaku) derived from common bean was allowed to act to release galactose.
The released sialic acid and galactose are separated by CarboPak
PA1 guard column (inner diameter 4 x length 50 mm, DIO
NEX) and CarboPak PA1 (4x inside diameter)
It is quantified by a sugar analysis system Bio-LC (manufactured by DIONEX) using a 250 mm length, manufactured by DIONEX,
The sialic acid addition rate was calculated by determining the ratio between galactose and sialic acid. Further, the purified sugar chain was fluorescently labeled by pyridyl amination (PA conversion). Artifact PA sugar chain
digested with sialidase (manufactured by Nacalai Tesque) derived from H. ureafaciens, and the desialic acid fraction was subjected to an anion exchange column MonoQ HR5 /
5 (inner diameter 5 mm × length 50 mm, manufactured by Amersham Pharmacia Biotech) by HPLC. The sugar chain from which sialic acid has been removed (asialo sugar chain) is supplied to a reverse phase column (Shim-pack CLC-ODS, inner diameter 6 × length 150 mm, Shimadzu Corporation) and an amide adsorption column (T
SKgel Amide-80, inner diameter 4.6 x length 250
mm, Tosoh Corporation). HPLC
In the analysis, the sugar chain was digested with various glycosidases, and the behavior of peaks of the sample before and after the digestion was analyzed to determine the sugar chain structure. The glycosidase used was α-L-fucosidase from bovine kidney (Boehringer).
Mannheim), bean-derived β-D-galactosidase (manufactured by Seikagaku Corporation), and bean-derived β-
N-acetylhexosaminidase (manufactured by Seikagaku Corporation),
Alternatively, it is endo-β-galactosidase from Escherichia Freundii (manufactured by Seikagaku Corporation). As a result of sugar chain analysis, hI produced by the HIIF-D strain
The sugar chain structure of FN-γ is mostly 2 as shown in column B of Table 1.
It was found to be a branched structure. The sialic acid addition rate was 51.6%. The number of galactose residues exposed at the non-reducing end by not being capped with sialic acid was calculated as follows per hIFN-γ polypeptide molecule.
2.01 were calculated. In addition, human natural hIFN-
When the sugar chain structure of γ (purchased from Hayashibara Biochemical Laboratory) was analyzed in the same manner, most of the sugar chain structures were biantennary structures (Table 1).
-Column A). The sialic acid addition rate was 92.6%. The number of galactose residues exposed at the non-reducing end in the sugar chain of human native hIFN-γ was calculated to be 0.20 per hIFN-γ polypeptide molecule.

[0032]

[Table 1]

Example 2 hIFN having a three-branched sugar chain
Production of -γ The bovine N-acetylglucosamimyltransferase IV (hereinafter abbreviated as GnT-IV) gene (SEQ ID NO: 1) was transformed into a vector pCXN2 (Niwa, H. et al. (1991) Ge
ne Vol.108, 193-200)
An -IV expression vector (pCXN2-bGnT-IV) was constructed (FIG. 2). This pCXN2-bGnT-IV was purified by electroporation with the HI described in Example 1.
It was introduced into IF-D cells. Neomycin analog G418
Select transformed cells by resistance to G4
A clone IM4 / IV4 strain having high GnT-IV activity was selected from the 18 resistant clones. GnT-IV activity is determined by performing an enzymatic reaction using a cell lysate as an enzyme source, UDP-N-acetylglucosamine (UDP-GlcNAc) and PA-galactosyl biantennary sugar chain as substrates, and quantifying the product by HPLC. Asked by.
The reaction for this was 10 mM HEPES (pH 7.
2), 80 mM UDP-GlcNAc, 10 mM M
nCl ₂ , 33 mM NaCl, 3 mM KCl, 200
mM GlcNAc, 0.2% Triton X-1
The reaction was carried out at 37 ° C. in 25 μl of a reaction solution containing 00, 800 μMPA agaractosyl biantennary sugar chain. After the reaction, the reaction was stopped by boiling for 3 minutes, and the product in the reaction solution was quantified by HPLC analysis using a Shim-pack CLC-ODS (inner diameter 6 × length 150 mm, Shimadzu Corporation). 300 μg / ml G418 in medium
(GENETICIN, Life Technology
ies, Inc. IM4 / IV4 cells were cultured in the same manner as in Example 1 except that
FN-γ was produced. HIFN-γ was purified from the medium thus obtained in the same manner as in Example 1.
The sugar chain structure was analyzed by the same method as in Example 1. As a result, the main sugar chain structure was a three-branched sugar chain (Table 1-D column). Sialic acid addition rate is 41.2%
Met. The number of galactose residues exposed at the non-reducing end by not being capped with sialic acid was
It was calculated to be 3.24 per molecule of N-γ polypeptide.

Example 3 hIFN having a triantennary sugar chain
Production of -γ N-acetylglucosamimyltransferase V (hereinafter abbreviated as GnT-V) gene derived from human (SEQ ID NO: 2)
Was transformed into the vector pCXN2 (Niwa, H. et al. (1991) Gene
Vol. 108, 193-200), and by incorporating it into the vector pCXH1 constructed by the method of FIG.
An expression vector (pCXH1-hGnT-V) was constructed (FIG. 4). This pCXH1-hGnT-V was introduced into the HIIF-D cells described in Example 1 by electroporation. Transformed cells were selected by hygromycin resistance, and a clone IM4 / V26 strain having high GnT-V activity was selected from hygromycin-resistant clones. GnT-V activity was measured using a cell lysate as an enzyme source and using UDP-N-acetylglucosamine (UDP-Gl).
An enzymatic reaction was carried out using cNAc) and PA-galactosyl biantennary sugar chain as substrates, and the product was determined by quantification by HPLC. The reaction for this is 10m
M HEPES (pH 7.2), 80 mM UDP-G
lcNAc, 10 mM EDTA, 33 mM NaCl,
3 mM KCl, 200 mM GlcNAc, 0.2%
In a 25 μl reaction solution containing Triton X-100, 800 μM PA-galactosylated biantennary sugar chain,
Performed at 37 ° C. After the reaction, the reaction was stopped by boiling for 3 minutes, and the product in the reaction solution was subjected to Shim-pack CL.
HPLC analysis was performed using C-ODS (inner diameter 6 x length 150 mm, Shimadzu Corporation) to quantify. 200 in the medium
IM4 / V26 cells were cultured in the same manner as in Example 1 except for containing μg / ml of hygromycin B (manufactured by Wako Pure Chemical Industries), and hIFN-γ was produced in the medium. HIFN-γ was purified from the medium thus obtained in the same manner as in Example 1. The sugar chain structure was analyzed by the same method as in Example 1. As a result, the sugar chain structure becomes 3
The branched sugar chain was the main structure (Table 1-E). The sialic acid addition rate was 61.2%. The number of galactose residues exposed at the non-reducing end due to not being capped with sialic acid was calculated to be 2.23 per hIFN-γ polypeptide molecule.

Example 4 hIFN having 4-branched sugar chain
Preparation of -γ The pCXN2-bGnT-IV described in Example 2 was electroporated with IM4 / described in Example 3.
Introduced into V26 strain. Transformed cells were selected by resistance to the neomycin analog G418,
Among the resistant clones, GnT-IV activity and GnT-V
A highly active clone IM4 / V26 / IV5 strain was selected. GnT-IV activity and GnT-V activity were measured in the same manner as in Examples 2 and 3. 200 μg / ml hygromycin B (manufactured by Wako Pure Chemical Industries) and 300 μg / ml in the medium
G418 (GENETICIN, Life Tech
nologies, Inc. IM4 / V26 / IV5 cells were cultured in the same manner as in Example 1 except for containing hIFN-γ in the medium. From the medium thus obtained, hIF was prepared in the same manner as in Example 1.
N-γ was purified. The sugar chain structure was analyzed by the same method as in Example 1. As a result, the main sugar chain structure was a 4-branched sugar chain (Table 1-F). The addition rate of sialic acid was 36.9%. The number of galactose residues exposed at the non-reducing end due to not being capped with sialic acid was calculated to be 4.45 per hIFN-γ polypeptide molecule.

Example 5 High sialic acid added hIFN-γ
Production of mouse α2,3-sialyltransferase (α2,3-SiaT) gene (SEQ ID NO: 3) was carried out using the vector pCXN2 (Niwa, H. et al. (1991) Gene Vol. 108,
193-200) by the method shown in FIG.
By incorporating into XN2Zb, α2,3-SiaT
An expression vector pCXN2Zb-mα2,3-SiaT was constructed (FIG. 6). In addition, pCXN2Zb-rα2,6-SiaT was constructed by incorporating the rat-derived α2,6-sialyltransferase (α2,6-SiaT) gene (SEQ ID NO: 4) into pCXN2Zb (FIG. 7). The neomycin resistance gene (SalI / SspI fragment) was removed from pCXN2Zb-rα2,6-SiaT, the ends were blunted, and cyclized to form α2,6-SiA.
aT expression vector pCXN2Zb (-Neo) -rα
2,6-SiaT was constructed. First, pCXN2Zb-
mα2,3-SiaT was converted to IM4 /
V26 cells were introduced by electroporation. Transformed cells were selected by resistance to the neomycin analog G418, and clones S3-2 having high α2,3-SiaT activity were selected from G418-resistant clones.
One strain was selected. Subsequently, α2,6 was added to this S3-21 strain.
-SiaT expression vector pCXN2Zb (-Neo)-
rα2,6-SiaT was introduced by electroporation. SSA (Sambucus sie), a lectin that specifically binds to α2,6-linked sialic acid
(FIT Boldiana) labeled with FITC (FIT
C-SSA) (manufactured by Seikagaku Corporation) using a flow cytometer (EPICS ELITE, Coulter).
Was performed, and several types of clones which expressed a large amount of α2,6-linked sialic acid on the cell surface were obtained. Among them, α2,3-SiaT activity and α
Clone S3-21 / S6 with high 2,6-SiaT activity
-5 strains were selected. α2,3-SiaT and α2,6
-SiaT activity was determined by using cell lysate as an enzyme source, CMP-
Enzymatic reaction was performed using N-acetylneuraminic acid (CMP-NeuAC) and PA-lacto-N-neotetraose as substrates, and the product was determined by quantification by HPLC. The reaction for this was 10 mM HEPES
(PH 7.2), 80 mM CMP-NeuAC, 10
mM MnCl ₂ , 33 mM NaCl, 3 mM KC
1, 20 μM 2,3-dehydro-2-deox
y-NeuAc, 5.6 mM γ-galactono
lactone, 0.2% Triton X-100,
The reaction was performed at 37 ° C. in a 25 μl reaction solution containing 800 μM PA-lacto-N-neotetraose. After the reaction, 3
The reaction in the reaction solution is stopped by boiling for 10 minutes.
him-pack CLC-ODS (inner diameter 6 x length 15
(0 mm, Shimadzu Corporation), and quantified. 200 μg / ml hygromycin B (manufactured by Wako Pure Chemical Industries) and 300 μg / ml G418 (GE
NETICIN, Life Technology
s, Inc. S3-21 / S6-5 cells were cultured in the same manner as in Example 1 except that
IFN-γ was produced. HIFN-γ was purified from the medium in the same manner as in Example 1. The sugar chain structure was analyzed by the same method as in Example 1. As a result, the main sugar chain structure was a three-branch type sugar chain as in the case of hIFN-γ of the IM4 / V26 strain (Table 1-H). The addition rate of sialic acid increased to 86.4%. The number of galactose residues exposed at the non-reducing end by not being capped with sialic acid was determined by the polypeptide 1 of hIFN-γ.
It was calculated to be 0.79 per molecule.

Example 6 Production of asialo-type hIFN-γ Arthrr was added to the hIFN-γ obtained in Examples 1 and 4.
The sialic acid at the sugar chain end was removed by the action of sialidase (manufactured by Nacalai Tesque) derived from O. ureafaciens. HIFN-γ was repurified by applying the sialidase reaction solution to an antibody column to obtain asialo hIFN-γ. Each hIFN-γ having asialo 2-branched sugar chains and asialo 4-branched sugar chains thus obtained
As a result of analyzing sugar chains by the same method as in Example 1, no sialic acid was added (Table 1-CG columns). The number of galactose residues exposed at the non-reducing end was 4.16 (asialo form derived from the HIIF-D strain) and 7.05 (derived from the IM4 / V26 / IV5 strain) per hIFN-γ polypeptide molecule. Asialo).

Example 7 hIF having various sugar chain structures obtained in Examples 1 to 5
N-γ was labeled with ¹²⁵ I, and ¹²⁵ I-hIFN-γ was administered from the tail vein of a 5-week-old male ddY mouse. In addition, 1
Five mice were administered as a group per sample. Mice were sacrificed over time after administration, and radioactivity of blood and each organ was measured to examine pharmacokinetics. When the accumulation of hIFN-γ in the liver 5 minutes after administration was compared, the accumulation in the liver increased as the number of exposed galactose residues per hIFN-γ molecule increased (FIG. 8). As shown in Examples 1 to 3, the greater the number of branches in the sugar chain of hIFN-γ, the greater the number of exposed galactose residues per hIFN-γ molecule, so the number of branches in the sugar chain is increased. It can be seen that the hepatic accumulation was increased by performing the treatment. In addition, those in which the sugar chain is asialized have a higher accumulation in the liver, and among the asialo bodies, the h of the two-branched sugar chain is higher.
Liver accumulation was higher in hIFN-γ, a four-branched sugar chain, than in IFN-γ. On the other hand, in the case where the degree of addition of sialic acid was increased, the liver accumulation was decreased, and it was found that the liver accumulation could be controlled by controlling the degree of addition of sialic acid together with the degree of branching of the sugar chain.

[0039]

According to the method of the present invention, the ability of glycoproteins to accumulate in the liver can be easily improved, and the degree of glycoprotein accumulation in the liver can be controlled by controlling the number of branched sugar chains. It is also possible to control. The method of the present invention comprises:
It is characterized in that the number of sugar chains is increased, and the degree of sugar chain modification can be kept within the range of naturally occurring sugar chain structures. Therefore, the method of the present invention is highly practical as a method for improving the accumulation of glycoproteins in the liver. Glycoproteins with improved liver accumulation of the present invention have high accumulation in the liver, and can be modified within the range of naturally-occurring sugar chain structures. It is expected to be extremely effective in treating liver diseases such as liver cancer, cirrhosis and hepatitis.

[0040]

[Sequence List] SEQUENCE LISTING <110> MITSUI CHEMICALS INC. <120> A glycoprotein which apts to accumulate in iver <160> 4 <210> 1 <211> 1608 <212> DNA <213> Bos taurus <223> GnT- IV gene <400> 1 atg agg ctc cga aat gga act gta gcc act gtt tta gca ttt atc acc 48 Met Arg Leu Arg Asn Gly Thr Val Ala Thr Val Leu Ala Phe Ile Thr 5 10 15 tcg ttc ctc act tta tct tgg tat aca aca tgg caa aat ggg aaa gaa 96 Ser Phe Leu Thr Leu Ser Trp Tyr Thr Thr Trp Gln Asn Gly Lys Glu 20 25 30 aaa gtg att gct tat caa cga gaa ttt ctt gct ctg aaa gaa cgt ctc 144 Lys Val Ile Ala Tyr Gln Arg Glu Phe Leu Ala Leu Lys Glu Arg Leu 35 40 45 cga ata gct gaa cat cga atc tct cag cgc tct tct gag ctc agt gcc 192 Arg Ile Ala Glu His Arg Ile Ser Gln Arg Ser Ser Glu Leu Ser Ala 50 55 60 att gta cag caa ttc aag cgt gta gaa gca gaa aca aac agg agt aag 240 Ile Val Gln Gln Phe Lys Arg Val Glu Ala Glu Thr Asn Arg Ser Lys 65 70 75 80 gat cca gtg aat aaa ttt tca gat gat acc cta aag ata cta aag gag 288 Asp Pro Val Asn Lys Phe Ser Asp Asp Thr Leu Lys Ile Leu Lys Glu 85 90 95 tta aca agc aaa aag tct ctt caa gtg cca agt att tat tat cat ttg 336 Leu Thr Ser Lys Lys Ser Leu Gln Val Pro Ser Ile Tyr Tyr His Leu 100 105 110 cct cat tta ttg caa aat gaa gga agc ctt caa cct gcc gtg cag atc 384 Pro His Leu Leu Gln Asn Glu Gly Ser Leu Gln Pro Ala Val Gln Ile 115 120 125 gga aat gga cga aca gga gtt tca ata gta atg gga att cct aca gtg 432 Gly Asn Gly Arg Thr Gly Val Ser Ile Val Met Gly Ile Pro Thr Val 130 135 140 aag aga gaa gtt aaa tct tac ctc ata gaa act ctt cat tcc ctt att 480 Lys Arg Glu Val Lys Ser Tyr Leu Ile Glu Thr Leu His Ser Leu Ile 145 150 155 160 gat aat ctg tat cct gaa gag aag ttg gac tgt gtt ata gta gtc ttc 528 Asp Asn Leu Tyr Pro Glu Glu Lys Leu Asp Cys Val Ile Val Val Phe 165 170 175 ata gga gag aca gat act gat tat gta aat ggt gtt gta gcc aac ctg 576 Ile Gly Glu Thr Asp Thr Asp Tyr Val Asn Gly Val Val Ala Asn Leu 180 185 190 gag aaa gaa ttt tct aaa gaa atc agt tct ggc ttg gtg gaa ata ata 624 Glu Lys Glu Plu Lys Glu Ile Ser Ser Gly Leu Val Glu Ile Ile 195 200 205 tca cct cct gaa agc tat tat cct gac ctg acg aac tta aag gag aca 672 Ser Pro Pro Glu Ser Tyr Tyr Pro Asp Leu Thr Asn Leu Lys Glu Thr 210 215 220 ttt gga gat tct aaa gaa aga gta aga tgg aga aca aag caa aac cta 720 Phe Gly Asp Ser Lys Glu Arg Val Arg Trp Arg Thr Lys Gln Asn Leu 225 230 235 240 gat tat tgt ttt cta atg atg tat gct cag gaa aaa ggc aca tac tac 768 Asp Tyr Cys Phe Leu Met Met Tyr Ala Gln Glu Lys Gly Thr Tyr Tyr 245 250 255 atc cag ctt gaa gat gat att att gtc aaa cag aat tac ttt aac acc 816 Ile Gln Leu Glu Asp Asp Ile Ile Val Lys Gln Asn Tyr Phe Asn Thr 260 265 270 ata aag aat ttt gca ctt caa ctt tct tct gag gaa tgg atg ata ctt 864 Ile Lys Asn Phe Ala Leu Gln Leu Ser Ser Glu Glu Trp Met Ile Leu 275 280 285 gag ttc tcc cag ctg ttc att ggt aaa atg ttt caa gca cct gac 912 Glu Phe Ser Gln Leu Gly Phe Ile Gly Lys Met Phe Gln Ala Pro Asp 290 295 300 ctc act ctg att gtg gag ttc ata ttt atg ttc tat aag gag Leag Thrcc 960 Ile Val Glu Phe Ile Phe Met Phe Tyr Lys Glu Lys Pro 305 310 315 320 atc gac tgg ctc ttg gac cat att ctg tgg gtc aaa gtc tgc aac ccg 1008 Ile Asp Trp Leu Leu Asp His Ile Leu Trp Val Lys Val Cys Asn 325 330 335 gaa aaa gat gca aaa cac tgt gat cga cag aag gca aat ctg cga att 1056 Glu Lys Asp Ala Lys His Cys Asp Arg Gln Lys Ala Asn Leu Arg Ile 340 345 350 cgt ttc aga ccg tcc ctt ttc cacac ctg cat tct tca ctc 1104 Arg Phe Arg Pro Ser Leu Phe Gln His Val Gly Leu His Ser Ser Leu 355 360 365 aca gga aaa att cag aaa ctc acg gat aaa gat tac atg aaa cca tta 1152 Thr Gly Lys Ile Gln Lys Leu Thr Asp Lys Asp Tyr Met Lys Pro Leu 370 375 380 ctg ctc aaa atc cat gta aac ccc cct gca gag gta tct act tct ttg 1200 Leu Leu Lys Ile His Val Asn Pro Pro Ala Glu Val Ser Thr Ser Leu 385 390 395 395 400 aag gtc tac caa ggt cat aca ctg gag aaa act tac atg ggt gag gac 1248 Lys Val Tyr Gln Gly His Thr Leu Glu Lys Thr Tyr Met Gly Glu Asp 405 410 415 ttc ttc tgg gct ata acc cca gta gct gga gac tac atc cta t tt aaa 1296 Phe Phe Trp Ala Ile Thr Pro Val Ala Gly Asp Tyr Ile Leu Phe Lys 420 425 430 ttc gac aag cca gtc aat gtg gaa agt tat ttg ttc cat agt ggc aac 1344 Phe Asp Lys Pro Val Asn Val Glu Ser Tyru Phe His Ser Gly Asn 435 440 445 cag gat cat cca ggg gat att ctg ctc aac aca acg gtg gaa gtt ctg 1392 Gln Asp His Pro Gly Asp Ile Leu Leu Asn Thr Thr Val Glu Val Leu 450 455 460 cct ttg aag agt gagat ttg gac atc agc aaa gaa acc aaa gac aaa 1440 Pro Leu Lys Ser Glu Gly Leu Asp Ile Ser Lys Glu Thr Lys Asp Lys 465 470 475 480 cga tta gaa gat ggc tat ttc aga ata ggg aaa ttt gaa aac ggt gtt 1488 Glu Asp Gly Tyr Phe Arg Ile Gly Lys Phe Glu Asn Gly Val 485 490 495 gcg gaa ggg atg gtg gat ccc agc cta aac ccc att tcg gcc ttc cga 1536 Ala Glu Gly Met Val Asp Pro Ser Leu Asn Pro Ile Ser Ala Pla 500 505 510 ctt tca gtt att cag aat tct gct gtt tgg gcc att ctt aat gag atc 1584 Leu Ser Val Ile Gln Asn Ser Ala Val Trp Ala Ile Leu Asn Glu Ile 515 520 525 cat att aaa aaa gtc aca aac tga 1608 H is Ile Lys Lys Val Thr Asn *** 530 535 <210> 2 <211> 2226 <212> DNA <213> Homo sapiens sapiens <223> GnT-V gene <400> 2 atg gct ctc ttc act ccg tgg aag ttg tcc tct cag aag ctg ggc ttt 48 Met Ala Leu Phe Thr Pro Trp Lys Leu Ser Ser Gln Lys Leu Gly Phe 5 10 15 ttc ctg gtg act ttt ggc ttc att tgg ggt atg atg ctt ctg cac ttt 96 Phe Leu Val Thr Phe Gly Phe Ile Trp Gly Met Met Leu Leu His Phe 20 25 30 acc atc cag cag cga act cag cct gaa agc agc tcc atg ctg cgc gag 144 Thr Ile Gln Gln Arg Thr Gln Pro Glu Ser Ser Ser Met Leu Arg Glu 35 40 45 cag atc ctg gac ctc agc aaa agg tac atc aag gca ctg gca gaa gaa 192 Gln Ile Leu Asp Leu Ser Lys Arg Tyr Ile Lys Ala Leu Ala Glu Glu 50 55 60 aac agg aat gtg gtg gat ggg cca tc gct gga gt tat 240 Asn Arg Asn Val Val Asp Gly Pro Tyr Ala Gly Val Met Thr Ala Tyr 65 70 75 80 gat ctg aag aaa acc ctt gct gtg tta tta gat aac att ttg cag cgc 288 Asp Leu Lys Lys Thr Leu Ala Val Leu Leu Asp Asn Ile Leu Gln Arg 85 90 95 att ggc aag ttg gag tcg aag gtg ga c aat ctt gtt gtc aat ggc acc 336 Ile Gly Lys Leu Glu Ser Lys Val Asp Asn Leu Val Val Asn Gly Thr 100 105 110 gga aca aac tca acc aac tcc act aca gct gtt ccc agc ttg gtt gca 384 Gly Thr Asn Ser Thr Asn Ser Thr Thr Ala Val Pro Ser Leu Val Ala 115 120 125 ctt gag aaa att aat gtg gca gat atc att aac gga gct caa gaa aaa 432 Leu Glu Lys Ile Asn Val Ala Asp Ile Ile Asn Gly Ala Gln Glu Lys 130 135 140 tgt gta ttg cct cct atg gac ggc tac cct cac tgt gag gga aag atc 480 Cys Val Leu Pro Pro Met Asp Gly Tyr Pro His Cys Glu Gly Lys Ile 145 150 155 160 aag tgg atg aaa gac atg tgg cgt tca gat ccc tgc tac gca gac tat 528 Lys Trp Met Lys Asp Met Trp Arg Ser Asp Pro Cys Tyr Ala Asp Tyr 165 170 175 gga gtg gat gga tcc acc tgc tct ttt ttt att tac ctc agt gag gtt 576 Gly Val Asp Gly Ser Thr Cys Ser Phe Phe Ile Tyr Leu Ser Glu Val 180 185 190 gaa aat tgg tgt cct cat tta cct tgg aga gca aaa aat ccc tac gaa 624 Glu Asn Trp Cys Pro His Leu Pro Trp Arg Ala Lys Asn Pro Tyr Glu 195 200 205 gaa gct gat cat aat tc a ttg gcg gaa att cgt aca gat ttt aat att 672 Glu Ala Asp His Asn Ser Leu Ala Glu Ile Arg Thr Asp Phe Asn Ile 210 215 220 ctc tac agt atg atg aaa aag cat gaa gaa ttc cgg tgg atg Tyr aga cta 720 Le Ser Met Met Lys Lys His Glu Glu Phe Arg Trp Met Arg Leu 225 230 235 240 cgg atc cgg cga atg gct gac gca tgg atc caa gca atc aag tcc ctg 768 Arg Ile Arg Arg Met Ala Asp Ala Trp Ile Gln Ala Ile Lys Ser Leu 245 250 255 gca gaa aag cag aac ctt gaa aag aga aag cgg aag aaa gtc ctc gtt 816 Ala Glu Lys Gln Asn Leu Glu Lys Arg Lys Arg Lys Lys Val Leu Val 260 265 270 cac ctg gga ctc ctg acc aagga ttt aag att gca gag aca 864 His Leu Gly Leu Leu Thr Lys Glu Ser Gly Phe Lys Ile Ala Glu Thr 275 280 285 gct ttc agt ggt ggc cct ctt ggt gaa tta gtt caa tgg agt gat tta 912 Ala Phe Ser Gly Gly Pro Leu Gly Glu Leu Val Gln Trp Ser Asp Leu 290 295 300 att aca tct ctg tac tta ctg ggc cat gac att agg att tca gct tca 960 Ile Thr Ser Leu Tyr Leu Leu Gly His Asp Ile Arg Ile Ser Ala Ser 305 310 315 320 ctg gc t gag ctc aag gaa atc atg aag aag gtt gta gga aac cga tct 1008 Leu Ala Glu Leu Lys Glu Ile Met Lys Lys Val Val Gly Asn Arg Ser 325 330 335 ggc tgc cca act gta gga gac aga att gtt gag ctc attac gat 1056 Gly Cys Pro Thr Val Gly Asp Arg Ile Val Glu Leu Ile Tyr Ile Asp 340 345 350 att gta gga ctt gct caa ttc aag aaa act ctt gga cca tcc tgg gtt 1104 Ile Val Gly Leu Ala Gln Phe Lys Lys Thr Leu Gly Pro Ser Trp Val 355 360 365 cat tac cag tgc atg ctc cga gtc ctt gat tca ttt ggt act gaa ccc 1152 His Tyr Gln Cys Met Leu Arg Val Leu Asp Ser Phe Gly Thr Glu Pro 370 375 380 gaa ttt aat cat gca aat tat gcc caa tcg aaa ggc cac aag acc cct 1200 Glu Phe Asn His Ala Asn Tyr Ala Gln Ser Lys Gly His Lys Thr Pro 385 390 395 400 tgg gga aaa tgg aat ctg aac cct cag cag ttt tat acc atg ttc cct 1248 Trp Gly Lys Trp Asn Leu Asn Pro Gln Gln Phe Tyr Thr Met Phe Pro 405 410 415 cat acc cca gac aac agc ttt ctg ggg ttt gtg gtt gag cag cac ctg 1296 His Thr Pro Asp Asn Ser Phe Leu Gly Phe Val Val Glu Gln His Leu 420 425 430 aac tcc agt gat atc cac cac att aat gaa atc aaa agg cag aac cag 1344 Asn Ser Ser Asp Ile His His Ile Asn Glu Ile Lys Arg Gln Asn Gln 435 440 445 tcc ctt gtg tat ggc aaa gtg gat agc ttg aag aat aag aag atc 1392 Ser Leu Val Tyr Gly Lys Val Asp Ser Phe Trp Lys Asn Lys Lys Ile 450 455 460 tac ttg gac att att cac aca tac atg gaa gtg cat gca act gtt tat 1440 Tyr Leu Asp Ile Ile His Thr Tyr Met Glu Val His Ala Thr Val Tyr 465 470 475 480 ggc tcc agc aca aag aat att ccc agt tac gtg aaa aac cat ggt atc 1488 Gly Ser Ser Thr Lys Asn Ile Pro Ser Tyr Val Lys Asn His Gly Ile 485 490 495 495 ctc agt gga cgg gac ctg cag ttc ctt ctt cga gaa acc aag ttg ttt 1536 Leu Ser Gly Arg Asp Leu Gln Phe Leu Leu Arg Glu Thr Lys Leu Phe 500 505 510 gtt gga ctt ggg ttc cct tac gag ggc cca gct ccc ctg 1584 Val Gly Leu Gly Phe Pro Tyr Glu Gly Pro Ala Pro Leu Glu Ala Ile 515 520 525 gca aat gga tgt gct ttt ctg aat ccc aag ttc aac cca ccc aaa agc 1632 Ala Asn Gly Cys Ala Phe Leu Asn Pro Lys Phe Asn Pro Pro Lys Ser 530 535 540 agc aaa aac aca gac ttt ttc att ggc aag cca act ctg aga gag ctg 1680 Ser Lys Asn Thr Asp Phe Phe Ile Gly Lys Pro Thr Leu Arg Glu Leu 545 550 555 555 560 aca tcc cag cat cct tac gct gaa gtt ttc atc ggg cgg cca cat gtg 1728 Thr Ser Gln His Pro Tyr Ala Glu Val Phe Ile Gly Arg Pro His Val 565 570 575 tgg act gtt gac ctc aac aat cag gag gaa gta gag gat gca gtg aaa 1776 Trp Thr Val Asp Leu Asn Asn Gln Glu Glu Val Glu Asp Ala Val Lys 580 585 590 gca att tta aat cag aag att gag cca tac atg cca tat gaa ttt acg 1824 Ala Ile Leu Asn Gln Lys Ile Glu Pro Tyr Met Pro Tyr Glu Phe Thr 595 600 605 tgc gag ggg atg cta cag aga atc aat gct ttc att gaa aaa cag gac 1872 Cys Glu Gly Met Leu Gln Arg Ile Asn Ala Phe Ile Glu Lys Gln Asp 610 615 620 ttc tgc cat ggg caa gtg atg tcc agc gcc cta cag gtc 1920 Phe Cys His Gly Gln Val Met Trp Pro Pro Leu Ser Ala Leu Gln Val 625 630 635 640 aag ctt gct gag ccc ggg cag tcc tgc aag cag gtg tgc cag gag agc 1968 Lys Leu Ala Glu Pro G ly Gln Ser Cys Lys Gln Val Cys Gln Glu Ser 645 650 655 cag ctc atc tgc gag cct tct ttc ttc cag cac ctc aac aag gac aag 2016 Gln Leu Ile Cys Glu Pro Ser Phe Phe Gln His Leu Asn Lys Asp Lys 660 665 gac atg ctg aag tac aag gtg acc tgc caa agc tca gag ctg gcc aag 2064 Asp Met Leu Lys Tyr Lys Val Thr Cys Gln Ser Ser Glu Leu Ala Lys 675 680 685 gac atc ctg gtg ccc tcc ttt gac cct aag aatag gtg ttt 2112 Asp Ile Leu Val Pro Ser Phe Asp Pro Lys Asn Lys His Cys Val Phe 690 695 700 caa ggt gac ctc ctg ctc ttc agc tgt gca ggc gcc cac ccc agg cac 2160 Gln Gly Asp Leu Leu Leu Phe Ser Cys Ala Gla Ala His Pro Arg His 705 710 715 715 720 cag agg gtc tgc ccc tgc cgg gac ttc atc aag ggc cag gtg gct ctc 2208 Gln Arg Val Cys Pro Cys Arg Asp Phe Ile Lys Gly Gln Val Ala Leu 725 730 735 tgc c gac tac tag 2226 Cys Lys Asp Cys Leu *** <210> 3 <211> 1002 <212> DNA <213> Mus musculus domesticus <223> α2,3-SiaT gene <400> 3 atg acc agc aaa tct cac tgg aag ctc ctg gcc ctg gct ctg gtc ctt 48 Met Thr Ser Lys Ser His Trp Lys Leu Leu Ala Leu Ala Leu Val Leu 5 10 15 gtt gtt gtc atg gtg tgg tat tcc atc tcc cga gaa gat agg tac att 96 Val Val Val Met Val Trp Tyr Ser Ile Ser Arg Glu Asp Arg Tyr Ile 20 25 30 gag ttc ttt tat ttt ccc atc tca gag aag aaa gag cca tgc ttc cag 144 Glu Phe Phe Tyr Phe Pro Ile Ser Glu Lys Lys Glu Pro Cys Phe Gln 35 40 45 ggt gag gca gag aga cag gcc tct aag att ttt ggc aac cgt tct agg 192 Gly Glu Ala Glu Arg Gln Ala Ser Lys Ile Phe Gly Asn Arg Ser Arg 50 55 60 gac cag ccc atc ttt ctg cag ctt aag gat tat ttc tgg gta aag acg 240 Asp Gln Pro Ile Phe Leu Gln Leu Lys Asp Tyr Phe Trp Val Lys Thr 35 70 75 80 cca tcc acc tat gag ctg ccc ttt ggg act aaa gga agt gaa gac ctt 288 Pro Ser Thr Tyr Glu Leu Pro Phe Gly Thr Lys Gly Ser Glu Asp Leu 85 90 95 ctt ctc cgg gtg ctg gcc atc act agc tat tct ata cct gag agc ata 336 Leu Leu Arg Val Leu Ala Ile Thr Ser Tyr Ser Ile Pro Glu Ser Ile 100 105 110 aag agc ctg gag tgt cgt cgc tgt gtt gtg gtg gga aat ggg cac 384 Lys Ser Leu Glu Cys Arg Arg Cys Val Val Val Gly Asn Gly His Arg 115 120 125 ttg cgg aac agc tcg ctg ggc ggt gtc atc aac aag tac gac gtg gtc 432 Leu Arg Asn Ser Ser Leu Gly Gly Val Ile Asn Lys Tyr Asp Val Val 130 135 140 atc aga ttg aac aat gct cct gtg gct ggc tac gag gga gat gtg ggc 480 Ile Arg Leu Asn Asn Ala Pro Val Ala Gly Tyr Glu Gly Asp Val Gly 145 150 155 160 tcc aag acc acc ata cgt ctc ttc tat cct gag gcc cac ttt gac 528 Ser Lys Thr Thr Ile Arg Leu Phe Tyr Pro Glu Ser Ala His Phe Asp 165 170 175 cct aaa ata gaa aac aac cca gac acg ctc ttg gtc ctg gta gct ttc 576 Pro Lys Ile Glu Asn Asn Pro Asp Thr Leu Leu Val Leu Val Ala Phe 180 185 190 aag gcg atg gac ttc cac tgg att gag acc atc ttg agt gat aag aag 624 Lys Ala Met Asp Phe His Trp Ile Glu Thr Ile Leu Ser Asp Lys Lys 195 200 205 cgg gtg cga aaa ggc ttc tgg aaa cag cct ccc ctc atc tgg gat gtc 672 Arg Val Arg Lys Gly Phe Trp Lys Gln Pro Pro Leu Ile Trp Asp Val 210 215 220 aac ccc aaa cag gtc cgg att cta aac ccc ttc ttt atg gag att gca 720Pro Lys Gln Val Arg Ile Leu Asn Pro Phe Phe Met Glu Ile Ala 225 230 235 240 gca gac aag ctc ctg agc ctg ccc ata caa cag cct cga aag atc aag 768 Ala Asp Lys Leu Leu Ser Leu Pro Ile Gln Gln Pro Arg Lys Ile Lys 245 250 255 cag aag cca acc acg ggt ctg cta gcc atc acc ttg gct cta cac ctc 816 Gln Lys Pro Thr Thr Gly Leu Leu Ala Ile Thr Leu Ala Leu His Leu 260 265 270 270 tgc gac tta gtg cac att gct ggc ttt ggc tat cca gat gcc tcc aac 864 Cys Asp Leu Val His Ile Ala Gly Phe Gly Tyr Pro Asp Ala Ser Asn 275 280 285 aag aag cag acc atc cac tac tat gaa cag atc aca ctt aag tct atg 912 Lys Lys Gln Thr Ile His Tyr Tyr Glu Gln Ile Thr Leu Lys Ser Met 290 295 300 gcg gga tca ggc cat aat gtc tcc caa gag gct atc gcc atc aag cgg 960 Ala Gly Ser Gly His Asn Val Ser Gln Glu Ala Ile Ala Ile Lys Arg 305 310 315 320 atg cta gag atg gga gct gtc aag aac ctc aca tac ttc tga 1002 Met Leu Glu Met Gly Ala Val Lys Asn Leu Thr Tyr Phe *** 325 330 <210> 4 <211> 1212 <212> DNA <213> Rattus norvegicus <223> α2,6-SiaT gene <400> 4 atg att cat acc aac ttg aag aaa aag ttc agc ctc ttc atc ctg gtc 48 Met Ile His Thr Asn Leu Lys Lys Lys Phe Ser Leu Phe Ile Leu Val 5 10 15 ttt ctc ctg ttc gca gtc atc tgt gtt tgg aag aaa ggg agc gac tat 96 Phe Leu Leu Phe Ala Val Ile Cys Val Trp Lys Lys Gly Ser Asp Tyr 20 25 30 gag gcc ctt aca ctg caa gcc aag gaa ttc cag atg ccc aag agc cag 144 Glu Ala Leu Thr Leu Gln Ala Lys Glu Phe Gln Met Pro Lys Ser Gln 35 40 45 gag aaa gtg gcc atg ggg tct gct tcc cag gtt gtg ttc tca aac agc 192 Glu Lys Val Ala Met Gly Ser Ala Ser Gln Val Val Phe Ser Asn Ser 50 55 60 aag caa gac cct aag gaa gac att cca atc ctc agt tac cac agg gtc 240 Lys Gln Asp Pro Lys Glu Asp Ile Pro Ile Leu Ser Tyr His Arg Val 35 70 75 80 aca gcc aag gtc aaa cca cag cct tcc ttc cag gtg tgg gac aag gac 288 Thr Ala Lys Val Lys Pro Gln Pro Ser Phe Gln Val Trp Asp Lys Asp 85 90 95 tcc aca tac tca aaa ctt aac ccc agg ctg ctg aag atc tgg aga aac 336 Ser Thr Tyr Ser Lys Leu Asn Pro Arg Leu Leu Lys Ile Trp Arg Asn 100 105 110 ta t ctg aac atg aac aaa tat aaa gta tcc tac aag gga ccg ggg cca 384 Tyr Leu Asn Met Asn Lys Tyr Lys Val Ser Tyr Lys Gly Pro Gly Pro 115 120 125 gga gtc aag ttc agc gta gaa gca ctg cgt tgc cac gac cat 432 Gly Val Lys Phe Ser Val Glu Ala Leu Arg Cys His Leu Arg Asp His 130 135 140 gtg aac gtg tct atg ata gag gcc aca gat ttt ccc ttc aac acc act 480 Val Asn Val Ser Met Ile Glu Ala Thr Asp Phe Pro Phe Asn Thr Thr 145 150 155 160 gag tgg gag ggt tac ctg ccc aag gag aac ttt aga acc aag gtt ggg 528 Glu Trp Glu Gly Tyr Leu Pro Lys Glu Asn Phe Arg Thr Lys Val Gly 165 170 175 cct tgg caa agg tgt gcc gtc gtc tct tct gca gga tct ctg aaa aac 576 Pro Trp Gln Arg Cys Ala Val Val Ser Ser Ala Gly Ser Leu Lys Asn 180 185 190 TCC CAG CTT GGT CGA GAG ATT GAT AAT CAT GAT GCA GTT CTG AGG TTT 624 Ser Gln Leu Gly Arg Glu Ile Asp Asn His Asp Ala Val Leu Arg Phe 195 200 205 aat ggg gcc cct acc gac aac ttc caa cag gat gtg ggc tca aaa act 672 Asn Gly Ala Pro Thr Asp Asn Phe Gln Gln Asp Val Gly Ser Lys Thr twenty one 0 215 220 acc att cgc cta atg aac tct cag tta gtc acc aca gaa aag cgc ttc 720 Thr Ile Arg Leu Met Asn Ser Gln Leu Val Thr Thr Glu Lys Arg Phe 225 230 235 240 ctc aag gac agt ttg tac acc gaa gga atc cta att gta tgg gac cca 768 Leu Lys Asp Ser Leu Tyr Thr Glu Gly Ile Leu Ile Val Trp Asp Pro 245 250 255 tcc gtg tat cat gca gat atc cca aag tgg tat cag aaa cca gac tac 816 Ser Val Tyr His Ala Asp Ile Pro Lys Trp Tyr Gln Lys Pro Asp Tyr 260 265 270 270 aat ttc ttc gaa acc tat aag agt tac cga agg ctg aac ccc agc cag 864 Asn Phe Phe Glu Thr Tyr Lys Ser Tyr Arg Arg Leu Asn Pro Ser Gln 275 280 285 ccatt tat atc ctc aag ccc cag atg cca tgg gaa ctg tgg gac atc 912 Pro Phe Tyr Ile Leu Lys Pro Gln Met Pro Trp Glu Leu Trp Asp Ile 290 295 300 att cag gaa atc tct gca gat ctg att cag cca aat cccca 960 Ile Gln Glu Ile Ser Ala Asp Leu Ile Gln Pro Asn Pro Pro Ser Ser 305 310 315 320 ggc atg ctg ggt atc atc atc atg atg acg ctg tgt gac cag gta gat 1008 Gly Met Leu Gly Ile Ile Ile Met Met Thr Leu Cys A sp Gln Val Asp 325 330 335 att tac gag ttc ctc cca tcc aag cgc aag acg gac gtg tgc tat tat 1056 Ile Tyr Glu Phe Leu Pro Ser Lys Arg Lys Thr Asp Val Cys Tyr Tyr 340 345 350 cac caa aag ttc ttt gac gct tgc acg atg ggt gcc tac cac ccg 1104 His Gln Lys Phe Phe Asp Ser Ala Cys Thr Met Gly Ala Tyr His Pro 345 350 355 ctc ctc ttc gag aag aat atg gtg aag cat ctc aat gag gga aca gat 1152 Leu Leu Phe Lys Asn Met Val Lys His Leu Asn Glu Gly Thr Asp 360 365 370 gaa gac att tat ttg ttt ggg aaa gcc acc ctt tct ggc ttc cgg aac 1200 Glu Asp Ile Tyr Leu Phe Gly Lys Ala Thr Leu Ser Gly Phe Arg Asn 375 380 380 385 390 att cgt tgt tga 1212 Ile Arg Cys ***

[Brief description of the drawings]

FIG. 1 is a diagram showing a branched type of a sugar chain.

FIG. 2: GnT-IV expression vector pCXN2-bGn
It is a figure showing the manufacturing method of T-IV.

FIG. 3 is a diagram showing a method for producing a vector pCXH1.

FIG. 4. GnT-V expression vector pCXH1-hGnT
It is a figure which shows the manufacturing method of -V.

FIG. 5 is a diagram showing a method for producing a vector pCXN2Zb.

FIG. 6: α2,3-SiaT expression vector pCXN2Z
It is a figure which shows the manufacturing method of b-m (alpha) 2,3-SiaT.

FIG. 7. Vector pCXN2Zb-rα2,6-Sia
FIG. 4 is a diagram illustrating a method for manufacturing T.

FIG. 8 is a graph showing the degree of liver accumulation of each hIFN-γ.
In the figure, Bi has h having a biantennary type sugar chain as a main sugar chain.
IFN-γ (derived from strain HIIF-D), Tri is hIFN-γ (derived from IM4 / IV4 strain) having a three-branched (β1,4-branched) sugar chain as a main sugar chain, and Tri ′ is 3 HIFN-γ (derived from the IM4 / V26 strain) having a branched (β1,6-branched) sugar chain as a main sugar chain was obtained from Te
tra is a hIF having a 4-branched sugar chain as a main sugar chain
9 shows N-γ (derived from IM4 / V26 / IV5 strain). Bi
-Asialo indicates that the hIFN-γ (derived from the HIIF-D strain) having a biantennary sugar chain as a main sugar chain is asialized. Tetra-asialo has a hIFN-γ (IM4
/ V26 / IV5 strain). Tri'-high sialo is a three-branch type (β
HIFN-γ (S3-21 / S6-5) having a 1,6-branched) sugar chain as a main sugar chain and having a high sialic acid addition rate.
(From strain).

[Explanation of symbols]

 Gal: D-galactose GlcNAc: N-acetyl-D-glucosamine Man: D-mannose Fuc: L-fucose

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 43/00 117 C07K 14/555 C07K 14/46 C12P 21/02 C 14/50 C12R 1:91 14 / 555 C12N 15/00 ZNAA C12P 21/02 A61K 37/02 // (C12P 21/02 37/66 C C12R 1:91) (72) Inventor Minako Tanigawa 1144 Togo, Mobara-shi, Chiba Mitsui Chemicals, Inc. (72 Inventor Reiko Abe 1144 Togo, Mobara-shi, Chiba Prefecture Mitsui Chemicals, Inc. (72) Inventor Tomoko Kubota 1144 Togo, Mobara-shi, Chiba Mitsui Chemicals Inc. (72) Inventor Tadashi Makino 1144 Togo, Mobara-shi, Chiba Mitsui Chemicals Co., Ltd. Inside the company (72) Inventor Yasuhisa Fujibayashi 23-2 Shimogotsuki, Matsuoka-cho, Yoshida-gun, Fukui Prefecture Inside the Research Center for High Energy Medicine, Fukui Medical University (72) Inventor High Shinji Matsu 23-2 Shimogotsuki, Matsuoka-machi, Yoshida-gun, Fukui Prefecture F-term in Fukui Medical University High Energy Medical Research Center 4B024 AA01 BA10 BA21 BA22 CA04 DA02 EA04 GA14 HA01 4B064 AG02 AG12 CA10 CA19 CA21 CC24 DA01 DA16 4C084 AA02 BA44 DA21 DB54 DB62 MA01 NA05 NA06 ZA752 ZB222 ZB262 ZB332 4H045 AA10 BA10 CA40 DA01 DA15 DA20 EA22 EA28 FA74

Claims (10)

[Claims] 1. A liver for a glycoprotein, characterized by increasing the number of galactose residues exposed at the non-reducing end of the sugar chain by increasing the number of branches of the sugar chain added to the glycoprotein. A method to improve integration. 2. The method according to claim 1, wherein the number of sugar chains of the glycoprotein is increased by expressing the gene for N-acetylglucosaminyltransferase IV and / or N-acetylglucosaminyltransferase V in the glycoprotein-producing cell. The method of claim 1. 3. The method according to claim 1, wherein the number of galactose residues exposed at the non-reducing terminal is further increased by removing sialic acid at the terminal of the sugar chain or modifying the sialic acid to reduce the addition of sialic acid. The method according to claim 1 or 2, wherein 4. The number of galactose residues exposed at the non-reducing end, when a glycoprotein is produced using glycoprotein-producing cells that lack sialic acid addition ability or have extremely low sialic acid addition ability. 3. The method according to claim 1, further comprising: 5. The method according to claim 1, wherein the glycoprotein-producing cell is a Chinese hamster ovary cell. 6. A glycoprotein having improved liver accumulation obtained by the method according to any one of claims 1 to 5. 7. An interferon having improved liver accumulation obtained by the method according to any one of claims 1 to 5. 8. A fibroblast growth factor having improved liver accumulation obtained by the method according to any one of claims 1 to 5. 9. A hepatocyte growth factor obtained by the method according to claim 1 and having improved liver accumulation. 10. A pharmaceutical composition comprising a glycoprotein obtained by the method according to any one of claims 6 to 9.