JPH0586099A - Interleukin 6 modification and therapeutic agent for thrombocytopenia with the same as active ingredient - Google Patents

Abstract

PURPOSE:To provide the title modification improved in stability, having blood platelet-increasing activity, thus useful for the title agents, having plural specific amino acid sequences containing Asn, rich in sugar chain addition sites, thus having more sugar chains than those in natural type interleukin 6. CONSTITUTION:The entire RNA is prepared by guanidium thiocyanate process from human thyroid-derived cell strain NIM-1 (FERM P-11103), mRNA is purified using an oligo dT cellulose column and cDNA is synthesized therefrom; the interleukin 6 cDNA is amplified through PCR process using, as primer, a synthesized oligomer coding the N- and C-terminals of interleukin 6, followed by site-specific mutation to prepare a DNA with part of the amino acids converted to asparagine, and this DNA is bonded to a vector and manifested in a host, thus giving the objective interleukin 6 modification having at least three sequences of formula Asn-X-Thr (X is amino acid other than Pro) or Asn-X-Ser.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to interleukin-6.
The present invention relates to a modified product and a therapeutic agent for thrombocytopenia containing the modified product.

[0002]

BACKGROUND OF THE INVENTION Interleukin-6 is being revealed to be an important factor for biological defense that controls proliferation / differentiation and expression of functions of the immune system, blood system, liver and nervous system (Toshio Hirano ( 1988) History of Medicine 146,301-304
), The development as a medicine has started. As a method for obtaining this interleukin-6, a method using a cultured cell or (Shimizu et al. (1985) J. Immunol. 134, 1728-1733) is used.
), Methods utilizing genetically modified cells or microorganisms have already been developed (Hirano et al. (1986) Nature 324,7).
3-76). By using these methods, interleukin-6 having a sugar chain added thereto or interleukin-6 protein having no sugar chain can be obtained. Although the molecular weight of interleukin-6 as a simple protein is 21.8 kDa, it has been reported that a sugar chain-added molecular weight of 22.5, 23.3, 27.5 kDa can be obtained from cultured cells (Gross et al. 1989) FEBS Lett. 247, 32
3-326). These are produced by addition of N-glycoside type sugar chain to Asn-X-Ser sequence existing in interleukin-6 amino acid sequence or addition of 0-glycoside type sugar chain to Thr or Ser. It is said that.

Recently, it has become clear that sugar chains added to such natural proteins may play an active role in protein stabilization and expression of physiological functions (Hayakawa et al. (1989) Pharmaceutical Research 20,735-759). Furthermore, in urokinase and G-CSF, protease resistance can be imparted by introducing a sugar chain into a site where no sugar chain was added in the native protein, by using a genetic engineering method. It has been reported (Sasaki et al., Yasumura et al. (1990), Annual Meeting of the Japanese Society of Agricultural Chemistry).

[0004]

The interleukin-6 obtained at present is limited to existing ones having a sugar chain at the sugar chain addition site or to a simple protein having no sugar chain. There are no examples in which a sugar chain is positively added to a site other than that seen to stabilize the molecule or modify the physiological activity.

The present invention introduces a glycosylation site into interleukin-6, which can be used as a medicine or a diagnostic agent, by a gene manipulation technique, and is modified with more sugar chains than natural products. It is intended to obtain interleukin-6 and provide interleukin-6 having improved stability or modified physiological activity. Furthermore, the present invention also provides a therapeutic agent for thrombocytopenia containing the modified interleukin-6 as an active ingredient.

[0006]

The present invention provides a modified interleukin-6 containing at least three Asn-X-Thr or Asn-X-Ser sequences (where X represents an amino acid other than Pro). And a therapeutic agent for thrombocytopenia containing the same as an active ingredient. That is, the present invention relates to Asn (45) -Lys (46) -Ser (47) which already exists in the primary structure of interleukin-6.
In addition to the sequence of Asn (144) -Ala (145) -Ser (146), one or more sequences of Asn-X-Thr or Asn-X-Ser are newly generated artificially and three sequences are generated. The above relates to a modified interleukin-6 characterized by adding a sugar chain to the asparagine residue.

Interleukin-6 in the present invention means the conventional BSF-2, IFN-β2, 26K protein,
It refers to proteins that have been called by the names such as hybridoma / plasma cytoma growth factor and hepatocyte growth factor.
The specific amino acid sequence is described in the report by Hirano et al. (Nature 324, 73-76 (1986)). However, those containing mutations that do not impair the original activity of interleukin-6, such as mutants lacking the N-terminal amino acid (Brakenhoff et al.
(1989) J. Immunol. 143, 1175-1182) or a variant in which a cysteine residue is replaced with another amino acid (Jambou et al. (1988) Pro
c.Natl.Acad.Sci.USA, 85, 9426-9430) and the like are also included in the interleukin-6 of the present invention.

New Asn-X-Th on Interleukin-6
The position on the primary structure that produces r or Asn-X-Ser is
Although it may be any site, it is preferably a site where the activity of interleukin-6 is not impaired by the production of its amino acid sequence. In other words, it is preferable to introduce a mutation into a site exposed on the surface of the molecule that is considered not to inhibit structure formation of interleukin-6 protein or a site not involved in secondary structure formation, but interleukin- Under the present circumstances where the three-dimensional structure of 6 has not been elucidated, it is difficult to accurately predict such a site. More preferably, it is preferable to introduce a sugar chain addition site within 30 residues from the N-terminal of the interleukin-6 protein or in the vicinity of the 106th glutamic acid.

As a method for obtaining the DNA encoding interleukin-6, there is a method of preparing mRNA from animal cells and synthesizing cDNA, or a method of chemically synthesizing it, and any method may be used. Modified interleukin from this DNA
In order to obtain the gene encoding 6, a new chemically total synthesis method, a method of chemically synthesizing and replacing the sequence encoding the mutant portion, and a synthetic DNA encoding the mutant portion
Although a method of introducing a site-specific mutation using a primer is known (Molecular Cloning (1989) 15.2-1
5.113), any method may be used.

The modified interleukin-6 obtained here
The gene encoding is integrated into an expression vector for eukaryotic cells, and by introducing it into eukaryotic cells,
Interleukin-6 modified with a sugar chain can be obtained. Eukaryotic cells include yeast, insect, amphibian and mammalian cells, but any cell may be used. In the present invention, cells derived from mammals such as Chinese hamster ovary cells (CHO), mouse C127
It is preferable to use cells, mouse L cells, monkey COS cells, human PC8 cells, human PC12 cells and the like.
The expression vector used is also not particularly limited, and any one suitable for the host used for expression may be used. From the culture supernatant of the cells thus obtained, the modified interleukin-6 can be obtained by various chromatography.

The therapeutic agent for thrombocytopenia of the present invention contains the above-mentioned modified interleukin-6 as a main component. As the other components, general pharmaceutical additives are selected. Of course, the object of the present invention can be achieved without additives. Additives are generally added primarily for stabilization. Such a pharmaceutical additive is selected from the proteins and / or saccharides described in the local law that can be used as a pharmaceutical additive. Human serum albumin (HS
A), gelatin, mannitol, sorbitol, lactose and the like are appropriately selected or combined, but are not limited thereto.

In order to specifically achieve the platelet-increasing action, which is one of the objects of the present invention, the composition containing the above-mentioned modified interleukin-6 as the main component is administered to the living body. The administration method is not particularly limited, but an appropriate one is selected from general injections such as intravenous injection, subcutaneous injection, intramuscular injection, and intravenous drip infusion. In some cases, transmucosal administration methods such as oral, nasal, pulmonary, and enteral administrations are also suitably performed.

An effective dose is 1K body weight per day
It is selected in the range of 0.0001 to 300 μg, preferably 0.001 to 10 μg per g. The above-mentioned dose varies depending on the symptoms and is not limited to these values. The number of doses is usually 1-2 per day
It may be selected once, or once every 2 to several days, but is not limited thereto.

[0014]

EXAMPLES The present invention will be described in more detail below with reference to examples. For general experimental methods, refer to the experimental manual (Sambrook,
J. Fritsch, EF Maniatis, T. (1989) "Molecular Clon
ing: a laboratory manual "Cold Spring Harbor Labora
toryPress).

Example 1 (1). Cloning of interleukin-6 gene: Human thyroid-derived cell line NIM-1 which produces interleukin-6, G-CSF and GM-CSF.
No. 103), total RNA was prepared by a known method using guanidinium thiocyanate, and further mRNA was purified by a known method using oligo dT cellulose column chromatography. Using 1 μg of this mRNA as a material,
Double-stranded cDNA was synthesized using a cDNA synthesis kit (Boehringer) utilizing a known method. Next, interleukin-6 containing an EcoRI cleavage site as shown below.
Synthetic oligomers (A, B) encoding the portions corresponding to the N-terminal and C-terminal of were prepared.

A, 5′-CCGAATTCGAGCCCAGCTATGAACTCC-3 ′ B, 5′-CCGAATTCGCCCATGCTACATTTGCC-3 ′ Synthetic oligomer was synthesized using an automatic DNA synthesizer (Applied Biosystems) and then purified by reverse phase HPLC. Using. Using these as primers, 1/3 of the synthesized cDNA was used to amplify the interleukin-6 cDNA having EcoRI cleavage sites at both ends by PCR. The composition of the PCR reaction solution was previously reported (Saiki, RK et al. Science.
239,487-491 (1988)). The reaction was performed using DNA Thermal Cycler DJ1000 (Perkin Elmer Cetus), heat denaturation 94 ° C for 1 minute, annealing 50 ° C for 2 minutes,
Extension reaction 40 cycles of reaction were repeated under the conditions of 72 ° C. for 3 minutes. The resulting reaction mixture was extracted twice with phenol / chloroform and once with chloroform, and then EcoRI.
Digested. This DNA was separated using a low melting point agarose gel electrophoresis and the desired DNA fragment of about 570 bp was collected.

(2). Introduction into animal cell expression vector: Vector pcDL-SR α296 for expressing animal cells (Takebe, Y. et al. Mo.
l.Cell.Biol.8,466-472 (1988)) after digestion with EcoRI, BAP
The 5'end was dephosphorylated by treatment with (Bacterial alkaline phosphatase), and the interleukin-6 cDNA obtained in (1) above was mixed and ligated with T4 DNA ligase, and then E. coli HB101 was transformed. The obtained transformant showing ampicillin resistance was cultivated, and plasmid DNA was prepared by the alkali extraction method. Interleukin-6cD
In order to select a clone in which NA was inserted in the vector in the correct orientation, the prepared DNA was digested with PstI and the DNA was separated by agarose gel electrophoresis. Interleukin-6cD
NA has a PstI site on the 3'side of the vector and on the 5'side of the cDNA insertion position in the vector, and if the interleukin-6 cDNA is in the correct orientation, a band of about 500 bp will be detected. By such a method, the expression plasmid pSRα in which interleukin-6 cDNA was inserted in the correct direction.
I chose IL6.

(3). Preparation of M13-IL6: plasmid pSR αIL
6 digested with EcoRI and agarose gel electrophoresis
A 0 bp DNA fragment was collected. Separately, M13mp19 (commercially available from Takara Shuzo) was digested with EcoRI, the vector DNA fragment and the EcoRI fragment obtained above were mixed, ligated with T4DNAligase, and E.coli JM109 (available from Takara Shuzo) was transfec.
tion was done. Transfection methods are described in the literature (Messing et al.
Methods Enzymol. 101, 20-78). At this time, 2% each of X-gal and IPTG was added to the upper agar. A double-stranded DNA was prepared from the obtained white plaque, and a plasmid in which the insertion direction of the DNA fragment was opposite to the transcription direction of lacZ was selected by PstI digestion to obtain M13-IL6. Furthermore, single-stranded DNA was prepared from plaques carrying this plasmid and used as a template for mutant production.

(4). Encrypt the interleukin-6 mutant
(1): Site-directed mutagenesis was carried out in order to prepare a gene encoding a mutant in which proline at the 18th residue from the N-terminus of interleukin-6 was converted to asparagine. First, according to a known method, the DNA oligomer
5'-CACAGACAGAATCTCACCTCT was synthesized and its 5'end was phosphorylated using T4 polynucleotide kinase. this
Eckstein using M13-IL6 as a template using a DNA oligomer
The method developed by et al. (Nucleic AcidsRes. 16, 791-802)
The mutation was introduced using a kit (commercially available from Amersham Japan) utilizing the above. Single-stranded DN from the obtained plaque
A was prepared and the nucleotide sequence of the mutation site was confirmed using a DNA sequencer (commercially available from Aloka). By this mutation operation, the 18th proline of interleukin-6 was converted into asparagine, and therefore Asn-Leu-Thr was found at this site.
Will be generated. Double-stranded DNA was prepared from the plaque in which the mutation was confirmed, digested with EcoRI, and then a DNA fragment of about 650 bp was fractionated by agarose gel electrophoresis. This was inserted into pcDL-SR α296 by the same procedure as in 2 to obtain a plasmid pSR αIL6-P18N expressing the mutant.

(5). Encrypt the interleukin-6 mutant
Of the gene that controls (2): 106 of interleukin-6
The gene encoding the mutant in which the glutamic acid was converted to asparagine at the th position was obtained by the same method as in 4. As a DNA oligomer for mutation, 5'-GAACAGATTTAACAGTAG
TGAG was used. This mutation operation converts glutamic acid at position 106 into asparagine, and thus Asn-Ser-Ser is generated in this portion. The gene encoding this mutant was incorporated into an expression vector in the same manner as in (4) above to obtain pSR αIL6-E106N.

(6). Expression in COS-1: In order to analyze interleukin-6 and interleukin-6 variants, the plasmid prepared by the procedure up to (5) above was introduced into COS-1 cells and expressed. Tried. Dulbecco's modified Eagle medium containing 10% fetal calf serum (FCS) (D.
COS-1 cells (CRL-165) suspended in MEM)
0) was seeded on a 6-well plate (Corning Co., Ltd., 25810-6) by 3.5 × 10 ⁵ pieces / 5 ml at 37 ° C.
Culture was performed overnight in the presence of 5% CO ₂ . This COS-1
The cell culture medium is removed, 10% New Salem (Collaborative), 2 μg plasmid DNA, 100 μ
M chloroquine, DEAE-dextran 400 μg / ml
Solution (1 ml) was added, and transfection was carried out under the conditions of 37 ° C. and 5% CO ₂ for 4 hours. The cells were treated with D. After washing twice with MEM, 10% FCS
Including D. After adding MEM and culturing for 2 days and exchanging the medium, the supernatant obtained by culturing for 3 days was used as interleukin-6 or interleukin-6 mutant,
The activity and molecular weight were analyzed.

(7). Bioassay interleuk
Quantification of Kin-6: RPMI1640 medium containing 10% FCS and 5 × 10 ⁻⁵ M 2-mercaptoethanol was added with several serially diluted sample solutions to make 50 μl, and placed in a 96-well plate (for cell culture). 2 × 10 ³ cells / 50 μl of 7TD1 cells (interleukin-6-dependent cell line: Van Damme et al. (1987) J. Exp. Med. 165, 914)
-919) was added, and the mixture was cultured at 37 ° C in the presence of 5% CO ₂ for 3 days. After adding 10 μl of 5 mg / ml MTT to each well and culturing for 5 hours, 150 μl of 0.04N hydrochloric acid-isopropanol was added to lyse the cells, and then the absorption at 570 nm was measured. As the standard interleukin-6, natural interleukin-6 (IL-6 manufactured by Toray Fuji Bionics Co., Ltd.)
The standard IL-6) attached to the EIA kit was used.

As a result, wild-type interleukin-6
The concentration of interleukin-6 in the supernatant of COS-1 cells into which the expression vector pSRαIL6 was introduced was 710 ng / ml, and the vector expressing the mutant pSRαIL6-P18N or pSRαIL6-
In COS-1 with E106N introduced, both were 300 ng /
It was ml.

(8). Measurement of molecular weight: 7.5 μl of the supernatant obtained in (6) above was subjected to SDS-polyacrylamide gel electrophoresis (gel concentration 12%) to separate proteins, and then a blotting apparatus manufactured by Atto was used. Then, the protein on the gel was blotted on a Diapore filter (Millipore). After transfer, filter is 5% skim milk / PBS
Anti-interleukin-6 monoclonal antibody IC-67, IG-61 (Ida et al. (1989) Bio
chem. Biophys. Res. Comm. 165, 728-734) 2 μg / ml
Then, detection was performed using a Gold Immun Blot Kit manufactured by Bio-Rad.

As a result, while the main bands at 22 kDa and 27 kDa were obtained from the culture supernatant of COS-1 transformed with the wild-type interleukin-6 expression vector pSRαIL6, pSRαIL6-P18N or pSRαIL was obtained.
2 from the culture supernatant of COS-1 transformed with 6-E106N
The main band was detected at 7 kDa and the band at 22 kDa disappeared, suggesting glycosylation.

(9). Purification of modified interleukin-6:
In order to investigate the drug efficacy of the sugar chain-added interleukin-6 mutant, the interleukin-6 mutant and wild-type interleukin-6 expressed by COS-1 cells were purified. Wild type interleukin-6 (pSRα
IL6 expression product (hereinafter abbreviated as IL6W) and interleukin-6 mutant protein (pSR α) to which two types of sugar chains are added
IL6-P18N expression product (hereinafter abbreviated as IL6P18N) and pSR αIL6-
E106N expression product (hereinafter abbreviated as IL6E106N)) was expressed by each transformed COS-1 cell and cultured in D.MEM medium containing 2% fetal bovine serum at 37 ° C for 3 days. 10 L and 10 L were obtained (the total amount of interleukin-6 was about 3 mg in each case).

The supernatant was centrifuged at 10,000 rpm for 10 minutes to remove the precipitate, and then an ultrafiltration device (Amicon) equipped with a UK-10 filter (Advantech) that allows only proteins having a molecular weight of 10,000 or less to pass through. Was concentrated to about 20 times. Then, the whole amount of the natural interleukin-6
Antibody column (10 mg protein bound to Affigel 10 (Bio-Rad))
/ ml gel) applied to 3 ml gel.

After washing the column with 10 mM phosphate buffer (pH 7.4) containing 0.15 M sodium chloride, followed by 15 ml of water each time
Interleukin-6 was eluted with 6 ml of 15 mM HCl (pH 2.0). The amount of total interleukin-6 in the eluted fraction (about 6 ml) was 2.5 mg for IL6W, 2.3 mg for IL6P18N, and IL6E106N.
Was 2.3 mg, and the yields were 70%, 75%, and 65%, respectively. The Interleukin-6 purity of this fraction was determined by reversed phase-high performance liquid chromatography (HPLC) using a C18 column.
All were over 70%. To further increase the purity, purification was then carried out by reverse phase-HPLC. Each sample 6
ml was applied to a Zorbox C18 column (4.6x250mm, Mitsui Toatsu) in 2 portions and eluted with acetonitrile containing 0.1% trifluoroacetic acid (10-60% / 25 minutes, 60% / 25-50 minutes). It was
The flow rate of the mobile phase was 1 ml / min, and under these conditions, the elution time of interleukin-6 was about 28 minutes. The elution fraction was 1.5 ml and the yield in this step was 70% for IL6W and IL
6P18N is 70%, IL6E106N is 78%, and the purity is about 95% by HPLC.
That was all. In the collected interleukin-6 solution,
Immediately after adding 1/10 volume of 1 M ammonium carbonate solution (pH 8.0) to neutralize, Sephadex G-25 (2.5x100 cm, Pharmacia) was used to add 10 mM phosphate buffer (pH 7) containing 0.15 M NaCl. .4) was desalted to obtain fractions of about 3 ml each. The concentration of interleukin-6 obtained was 440 μg / ml for IL6W and IL6P1.
8N is 500 μg / ml, IL6E106N is 440 μg / ml, and finally 3
About 1.0 mg of each type of interleukin-6 was obtained.

(10). Confirmation of platelet-increasing action: C57BL was obtained by using the interleukin-6 obtained in the previous section as a test sample.
/ 6 mice were administered to examine the platelet increasing activity. C57
Male BL / 6 mice, 5 weeks old, were purchased from SLC, and 6 mice per group were used for a week for 6 weeks.

Interleukin-6 modified form I obtained in the preceding paragraph
L6P18N and IL6E106N solutions each 1 day
It was administered subcutaneously in the back at a volume of 10 ml / Kg. 1 group 6
Each animal was started from Day-0 and administered continuously for 7 days. The start time of administration was set at 9 am each time. After the lapse of 3 hours or more after the predetermined number of administrations, the platelet count was measured. The mice were anesthetized with ether gas, fixed in the supine position, the axilla was wiped with alcohol cotton, and then the left axillary arteries and veins were cut. The blood flowing out after cutting was collected in a silicon-coated glass blood collection tube (Beneject vacuum blood collection tube, manufactured by Terumo Corp.) in which 10 μl of 15% EDTA-2Ka solution was dispensed in advance. Immediately after that, the number of platelets in the collected blood was measured using an automatic hematology analyzer Celltac (Nihon Kohden). When 2.5 μg / kg of each modified interleukin-6 was administered per day, the modified IL6P
For 18N and IL6E106N, respectively, a 30% and 31% increase in platelets was observed with respect to the control (saline administration) group.

[0031]

INDUSTRIAL APPLICABILITY The modified interleukin-6 obtained according to the present invention has an amino acid sequence which induces the binding of the asparagine-linked sugar chain introduced in the primary structure thereof, and thus has a larger amount of sugar than the natural type. It is possible to have chains. As a result, changes in physicochemical properties such as increase in molecular weight, change in isoelectric point, change in stability, etc. can be expected to improve blood stability, increase drug efficacy, and promote uptake into target organs. .. In addition, the modified interleukin-6 of the present invention has a platelet increasing activity and is useful as a therapeutic agent for thrombocytopenia.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location G01N 33/53 P 8310-2J (C12P 21/02 C12R 1:91) (72) Inventor Ritsuko Sawada 1111 Tehiro, Kamakura City, Kanagawa Prefecture

Claims (3)

[Claims] 1. Asn-X-Thr or Asn-X-
An interleukin-6 modified product comprising at least three Ser sequences (where X represents an amino acid other than Pro). 2. A gene encoding the modified interleukin-6 according to claim 1. 3. A therapeutic agent for thrombocytopenia containing the modified interleukin-6 according to claim 1 as an active ingredient.