JPH07188292A - Glycoprotein derived from human, physiologically active factor composed of the same glycoprotein and pharmaceutical preparation containing the same active ingredient - Google Patents

Abstract

PURPOSE:To obtain a new glycoprotein, available from a culture supernatant of a fibroblast derived from a human and useful as an antltumor agent, a cell immunopotentiator, etc. CONSTITUTION:This glycoprotein is obtained from a culture supernatant of a fibroblast derived from a human and has the following properties: molecular weight: 78000+ or -2000 or 74000(+)2000 in a nonreduced state and a common band (A) of 52000(+)2000 and two bands of a band (B) of 30000+ or -2000 and a band (C) of 26000+ or -2000 in a reduced state (measured by the SDS-polyacrylamide electrophoresis); isoelectric point: 7.4-8.6; thermal stability:stable even by heating at 60 deg.C for 10min; pH stability: stable within the range of pH6-9; saccharide chain: manifesting the adsorptivity for concanavalin A-Sepharosce(R); physiological activity: capable of suppressing the proliferation of a KB cell, an HeLa cell and an L-929 cell without suppressing the proliferation of an IMR-90 cell; reactivity to antibody: without neutralizing the disorder activity by an anti-TNF antibody, an antilymphotoxin antibody and an antiinterferon beta antibody and amino acid sequence at the N-terminal; formula I or II (X is unidentified). Furthermore, the protein is obtained by a genetic engineering technique.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a glycoprotein obtained from a culture supernatant of human-derived fibroblasts, a physiologically active factor comprising the glycoprotein, and a preparation containing the same as an active ingredient.
The glycoprotein of the present invention has a damaging effect on tumor cells,
In addition, it is a novel tumor cell damaging factor, a leukemia cell line differentiation inducing factor, a cell immunopotentiating factor, a vascular endothelial cell growth factor, and a hepatocyte growth factor that show no damage to normal cells. This substance is an antitumor agent, an antileukemia agent, a cell immunity enhancer,
It is useful as a wound healing agent, a liver regeneration promoter, etc., or as a biochemical or pharmacological agent.

[0002]

2. Description of the Related Art As a physiologically active substance produced by human-derived fibroblasts, for example, β-interferon is a typical substance as a tumor cytotoxic factor. This is a glycoprotein secreted extracellularly after culturing fibroblasts, stimulating the cells and stimulating with poly I-poly C or Sendai virus, and exerts various physiological activities in addition to antiviral and antitumor effects. It has become clear to show. JP-A-58-146293 discloses a fibroblast-derived tumor cytotoxic glycoprotein called CBF. Japanese Patent Application Laid-Open No. 61-33120 discloses a tumor growth inhibitory factor (INF) having a molecular weight of 35,000 to 45,000 extracted from a human tissue-derived fibroblast culture medium. Further, JP-A-61-56131 discloses a tumor necrosis factor-like substance extracted from fibroblasts.
Japanese Patent Laid-Open Publication No. Sho.
62-103021 discloses a physiologically active substance produced from animal fibroblasts having a molecular weight of 40,000 to 60,000 and an isoelectric point of 5.0 ± 0.5 and having a cytotoxic action. Further, JP-A-64-10998 discloses a complete amino acid sequence of a tumor cytotoxic factor having a molecular weight of 36,000 ± 1,000 and an isoelectric point of 10.5 or more obtained from the culture supernatant of human-derived fibroblasts and the same. The cDNA sequence is disclosed.

[0003]

DISCLOSURE OF THE INVENTION The present inventors have conducted a search for physiologically active substances contained in the culture supernatant of human-derived fibroblasts, and as a result, the previously reported substances have molecular weight and isoelectricity. The present invention has been accomplished by discovering glycoproteins having various physiological activities that differ in points and the like. Therefore,
It is an object of the present invention to provide a novel glycoprotein obtained from a culture supernatant of human-derived fibroblasts, a physiologically active factor comprising the glycoprotein, and a preparation containing the physiologically active factor as an active ingredient. To do.

[0004]

A novel glycoprotein derived from human fibroblasts (hereinafter referred to as TCF-II) according to the present invention is a glycoprotein specified by the physicochemical properties shown below. a. Molecular weight; measured by SDS polyacrylamide gel electrophoresis, 78,000 ± 2,000 or 74,0 for non-reducing
It has a molecular weight of 00 ± 2000 and, when reduced, 52,000 ± 2,000
0 common band A and 30,000 ± 2,000 band B and 2
Two bands of 6,000 ± 2,000 band C are shown. b. Isoelectric point; 7.4 to 8.6 c. Thermal stability Stable by heating at 60 ° C for 10 minutes d. pH stability; stable in the range of pH 6 to 9 e. Sugar chain: Adsorbs to concanavalin A (ConA) sepharose. f. Physiological activity; suppresses proliferation of KB cells, HeLa cells, L-929 cells, but does not inhibit proliferation of IMR-90 cells g. Reactivity with antibody: The damaging activity is not neutralized by anti-TNF antibody, anti-lymphotoxin antibody, and anti-interferon β antibody. Furthermore, the TCF-II of the present invention preferably has the following N-terminal amino acid sequence and amino acid composition. h. N-terminal amino acid sequence: Bands B and C described above are subchains of band A, and band A has the N-terminal amino acid blocked. Subchains B and C both have the following N-terminal amino acid sequences: Val-Val-Asn-Gly-Ile-Pro-Thr- or Val-Val-Asn-Gly-Ile-Pro-Thr-X-Thr-Asn. -Ile-Gly-X-Me
t-Val-Ser-Leu- However, X means unidentified. i. Amino acid composition: Shows the following amino acid composition when hydrolyzed with hydrochloric acid. AA nmol mol% Asp 10.375 12.97 Glu 7.750 9.69 Ser 5.000 6.25 Gly 7.250 9.06 His 3.000 3.75 Arg 5.375 6.72 Thr 5.125 6.41 Ala 2.625 3.28 Pro 5.625 7.03 Tyr 3.875 4.84 Val 4.125 5.16 Met 1.875 2.34 Cys ND-Ile 5.000 6.25 Leu 4.875 6.09 Phe 2.250 2.81 Trp ND-Lys 5.875 7.34 Total 80.000 100 (99.99)

[0005] The amino acid sequence of TCF-II of the present invention was obtained from human fetal lung-derived fibroblasts (IM
R-90), after purifying the mRNA encoding the TCF-II,
The gene was cloned to determine the nucleotide sequence, and deduced from the nucleotide sequence. (1) Extraction of poly (A) ⁺ RNA from IMR-90 cells Dulbecc supplemented with 5% New born calf serum (NBCS)
IMR-90 cultured in o's modified eagle (DME) medium
Total RNA was prepared from 2 × 10 ⁸ cells by the guanidine thiocyanate-cesium chloride method (Biochemistry 18 5294-5299 (1979)). IMR-90 cells were homogenized by adding 6 M guanidine thiocyanate, 5 mM sodium citrate, 0.5% sarcosyl and 28 ml of 0.1 M β-mercaptoethanol solution. 4 ml of 5.7 M cesium chloride and 0.1 M EDTA solution was placed in a polyallomer centrifuge tube, 7 ml of the homogenizing solution was placed thereon, and ultracentrifugation was carried out at 35,000 rpm, 20 ° C. for 16 hours using a Beckman ultracentrifuge 40 Tl rotor. After centrifugation, the precipitate was washed twice with 95% ethanol,
Dissolve by heating with 200 μl of 10 mM Tris-HCl buffer (pH 7.5), 1 mM EDTA solution at 65 ° C for 5 minutes to give a total solution.
RNA solution was used. Poly (A) ⁺ RNA was purified from total RNA by oligo (dT) cellulose column chromatography. Apply oligo (dT) cellulose column to 10mM Tris-HCl buffer (pH7.4), 1mM EDTA, 0.5M sodium chloride, 0.05% SDS.
Equilibrate with, pass through total RNA, and adsorb fraction to 10 mM
Elution was performed with Tris-HCl buffer (pH 7.4), 1 mM EDTA, 0.05% SDS to obtain a poly (A) ⁺ RNA solution.

(2) Synthesis of cDNA Using the poly (A) ⁺ RNA obtained in (1) as a template, a double-stranded cDNA was prepared with a cDNA synthesis kit (Pharmacia) and an EcoRl adapter was added. The method of preparation was in accordance with the company's protocol, but an addition of reverse transcriptase (AMV RTase) derived from avian myeloblastosis virus was added during the synthesis of single-stranded cDNA (40 units / reaction system, Life Science Company).

(3) Preparation of cDNA library The cDNA obtained in (2) was transformed with EcoRI of the phage vector λ gt10.
Incorporated into arm (Promega). 3.3 μg poly (A) ⁺
The cDNA synthesized from RNA was dissolved in 150 μl of 66 mM Tris-HCl buffer (pH 7.6), 1 mM spermidine, 10 mM magnesium chloride, 15 mM dithiothreitol, 0.2 mg / ml bovine serum albumin solution (column buffer). Out of 5.2
μl was mixed with 1 μg of λ gt10 EcoRI arm and then precipitated with ethanol. This precipitate was redissolved in 9 μl of column impaction solution, 1 μl of 10 mM adenosine triphosphate, 1 μl of TM
DNA ligase (350 units / μl) was added, and the mixture was reacted overnight at 16 ° C to prepare recombinant phage DNA of λgt10 and cDNA.

(4) Screening of cDNA library (i) Preparation of oligonucleotide probe 17mer complementary strand oligonucleotide mixture (3) corresponding to the first to sixth amino acid sequences at the N-terminus of TCF-II β chain
84 kinds of mix) were synthesized and 5 ′ was prepared using T4 polynucleotide kinase (Takara Shuzo) and [γ- ³² p] ATP (Amersham).
The end was labeled and used as a probe. This probe is shown below. Complementary strand used as probe: 3'-C
ACCACTTACCGTAGGG -5 ' (ii) Screening of recombinant phages The recombinant phage DNA solution prepared in (3) was added to Gigapack Gol.
d (Stratagene) in vitro packaging,
E. coli C600hfl was infected and about 500,000 phage plaques were obtained. Plaque with Hybond-N filter (Amersh
(Company am), the filter was alkali-denatured and neutralized, and then baked at 80 ° C. for 2 hours. Hybridization was performed according to the method of Bell et al. (Nature 31 775-777 (1984)).
Primary screening was performed with the probe prepared in (i).
TC in the plaques positive in the primary screen
One clone was suspected of containing the F-II cDNA fragment.

(5) TC containing the entire region translated into amino acids
Cloning of F-II cDNA β-chain N-terminal amino acid sequence of TCF-II and partial internal amino acid sequences (α) (one letter) NYMGNLSQTR obtained by lysyl endopeptidase treatment of α-chain and β-chain, respectively
Since SGL and (β) TSXSVYGWGYTGLINYDGLL (X indicates terminal identification) are in good agreement with the amino acid sequence of human hepatocyte growth factor (hHGF), TCF-II was considered to be one of the gene families of hHGF. For hHGF, see Miyazawa et al. (Bioc
hemicaland Biophysical Research Communication 163
967-973 (1989)), Nakamura (Nature 342 440-443 (1989)) reported the nucleotide sequence of the cDNA, but the amino acid sequences differed at 14 positions in both, suggesting the existence of the hHGF gene family. It was Therefore, there is a match between the two, 5′- and 3′- around the polynucleotide chain coding region.
An oligonucleotide that serves as a primer was synthesized based on the DNA sequence of the untranslated region, and Polymerase Chain Reactio
The TCF-II cDNA was searched by the n (PCR) method. First, DNA
Using a synthesizer (Applied), a Sal-77 primer having a recognition sequence for the restriction enzyme SalI and a Sph2203 primer having a recognition sequence for the restriction enzyme Sph1 were synthesized. These primers are shown below. Sal-77 primer: 5'-G GTCGAC TAGGCACTGACTCCGAACAGGATTC-3 'Sall Sph2203 Primer: 5'-G GCATGC ACAGTTGTATTGGTGGGTGCTTCAG-3' SphI

Cloning by the PCR method was performed according to the following procedure. (i) cDNA synthesized in PCR (2) (dissolved in 150 μl column buffer) 1 μl 20 μM Sal-77 primer 2.5 μl 20 μM Sph2203 primer 2.5 μl 10x PCR reaction solution (500 mM potassium chloride, 100 mM Tris-HCl buffer (pH 8 .3), 15 mM magnesium chloride, 0.1% (w / v) gelatin) 10 μl 1.25 mM dGTP, dATP, dTTP, dCTP mixture 16 μl Ampli Taq (5 units / μl Takara Shuzo) 0.5 μl distilled water 67.5 μl

After mixing the above solution in a microcentrifuge tube for 0.5 ml, the surface of the liquid was covered with about 100 μl of mineral oil (Sigma), and then PCR was performed using Quick Thermo System (Nippon Genetics). . The reaction conditions are shown below.
After pretreatment at 94 ℃ for 7 minutes, 55 ℃ for 3 minutes (annealing reaction),
After the three-step reaction of 72 ° C for 4 minutes (polymerase reaction) and 94 ° C for 2 minutes (denaturation) was repeated 35 times, the post-treatment was 55 ° C 3
Min, 72 ° C for 11 minutes, and returned to room temperature ((Note) Each time includes the time when the temperature changes). When a portion of the reaction mixture was subjected to agarose gel electrophoresis, about 2.
A 3 kilobase (Kb) DNA fragment is obtained, which is the desired TC
It was considered to be F-II cDNA. So I got it from 4 reaction liquids
After the DNA was precipitated with ethanol, the restriction enzymes SalI and SphI
Digested with agarose gel and subjected to agarose gel electrophoresis to recover a DNA fragment of about 2.3 Kb using DE81 paper (Whatman).

(Ii) Subcloning About 2.3 Kb digested with the restriction enzymes SalI and SphI obtained in (i)
The cDNA fragment of Escherichia coli was inserted into a vector fragment obtained by digesting the plasmid vector pUC18 (Nippon Gene Co., Ltd.) with restriction enzymes SalI and Sphl using a ligation kit (Takara Shuzo).
DH5α (BRL) was transformed (following the protocol attached to BRL). As a result, 20 or more subclones could be obtained.

(Iii) Nucleotide sequence determination The obtained subclone was subjected to the dideoxy method (Sequena
The base sequence was determined by seVer.2.0 Toyobo). Ampi T
The nucleotide incorporation error of aq (Takara Shuzo) was corrected by analyzing the nucleotide sequences of multiple subclones. Figures 15 and 16 show the nucleotide sequence of the TCF-II cDNA obtained as described above and the amino acid sequence predicted from the sequence.
It was shown to. From translation start signal ATG to stop signal TAG 2172
It has a base pair (bp), and when translated into an amino acid, it has a 723 amino acid sequence, and it was predicted that the signal sequence extends from the 1st methionine residue to the 29th alanine residue. TCF-II
It was found that, although two polypeptide chains, α chain and β chain, were disulphide-bonded, as shown in FIGS. 15 and 16, the polypeptide was initially synthesized as one polypeptide chain. TCF-
It is unclear because the N-terminus of the α chain of II is blocked, but the N-terminus of the β chain and the partial internal amino acid sequences of the α chain and β chain have been determined as described above. It was shown to. The base sequence of the obtained TCF-II cDNA is shown in Miyazawa et al. [Bioc
hemical and Biophysical Research Communication 163
967-973 (1989)], the amino acid sequence of hHGF of Miyazawa et al. Is very similar to that of hHGF found, but the five residues from phenylalanine at the 162nd position to serine at the 166th position (FL
-PSS) was deleted in the TCF-II cDNA of this time, and TCF-II cDNA was found to be one of the genes of the new HGF gene family. Amino acid sequence of TCF-II proposed from the above nucleotide sequence and hHGF from Miyazawa
Comparison of the amino acid sequences of is shown in FIG.

The method for obtaining the novel glycoprotein TCF-II specified by the above physicochemical properties will be described below. As the cells used for producing the substance according to the present invention, any human-derived fibroblasts can be used. Suitable cells include human fetal lung-derived cell lines, human fetal kidney-derived cell lines, human fetal foreskin-derived cell lines, and the like. Among these cells, IMR-90 (ATCC CCL 186), WI-38 (ATCC CCL 75) and the like are particularly suitable for practicing the present invention. These cells are grown in a serum medium or a serum-free medium used for ordinary culture. An example of a typical medium is Dulbecco's modified Eagle medium (DMEM) supplemented with 5% calf serum.
In addition to these, hormones such as amino acids, transferrin, fatty acids, and insulin may be added if necessary.

The cells are cultivated in this medium. For the culturing, static culturing using a T-flask or the like, suspension culturing using a microcarrier, or continuous culturing using a hollow fiber or a ceramic carrier is used. Can be adopted. The culturing conditions are a CO ₂ 5% air atmosphere, a temperature of 20 to 37 ° C.,
It is preferable to change the medium every 2-3 days. After reaching the desired cell density in this way, the medium is replaced every 7 to 10 days and the culture solution is collected. The target glycoprotein is extracted and purified from the recovered culture solution. The collected culture solution is concentrated about 10 times by UF membrane treatment that cuts off the molecular weight of 6.000 or less, then adsorbed on a cation exchanger, and then eluted with a buffer solution having a salt concentration of 0.3M to 0.6M. Examples of the ion exchanger include CM Sephadex (manufactured by Pharmacia). Of the active fractions thus eluted, the fractions showing the strongest tumor cell growth inhibitory activity using the tumor cell growth inhibitory activity as an index are collected and further subjected to sugar affinity chromatography. ConA-Sepharose is particularly suitable for sugar affinity chromatography. Sugar affinity chromatography column is 0.5MN
After equilibration with 0.05M Tris-HCl buffer (pH 7.0) containing aCl, the collected fraction is loaded and the column is washed. After that, elution is carried out with an eluent corresponding to the sugar chain having sugar affinity. When ConA Sepharose described above is used, it is eluted with a buffer containing methylmannopyranoside. The eluted active fraction is dialyzed against water and freeze-dried. After that, it was dissolved in 0.05M Tris-hydrochloric acid buffer of pH 6.0 to 7.0, and HPLC was performed using strong cation exchange resin as the packing material.
To further separate and purify. MonoS (Pharmacia) is particularly suitable as a column packed with a strong cation exchange resin. For elution from the MonoS column, a gradient elution of OM → 1.0M sodium chloride is performed, and the active fractions are collected.

The substance of the present invention is eluted in the salt strength portion of 0.6M to 0.9M. The active fraction thus obtained is further purified by affinity chromatography using heparin-sepharose (Pharmacia).
Elution from heparin-sepharose column is 0.3M → 2.0M
The target substance is eluted in the salt strength part of 1.0 to 1.5M. Next, the measurement of the tumor cell-damaging activity and hepatocyte proliferation activity of TCF-II of the present invention will be described below.

Measurement of Tumor Cytotoxicity Mouse L929 (ATCC CCL1) was selected for the clone most sensitive to TCF-II of the present invention. Thus, a tumor cell damaging factor hypersensitive clone L 929-18 was obtained. L 929-18
Incubate in DMEM containing 10% FCS until confluent, and then trypsinize to detach and collect cells.
DMEM containing% FCS and 1 μg / ml actinomycin D
Resuspend at a cell density of 6 x 10 ⁵ cells / ml. 50 μl of DMEM prepared in the same manner as the cell suspension was placed in each well of a 96-well microplate (Falcon),
A sample containing the substance TCF-II of the present invention is also dissolved or diluted with the same DMEM, 50 μl is added to the first hole of the dilution column, and after mixing,
Add 50 μl to the second well and mix. Repeat this operation to create a dilution series. For each well in the sample dilution series,
50 μl of the cell suspension is added, and the cells are cultured at 37 ° C. for 2 days in a CO ₂ incubator. After culturing, the supernatant was gently discarded, washed twice with physiological saline, and the viable cells adhered to each well were dissolved in a solution prepared by methanol: water = 1: 4 0.5
Add 50 μl of the% crystal violet solution to each well, and fix by staining. Each well was washed with distilled water, the staining plate was air-dried, and the dye was stained with Serenson's buffer (6.1 ml,
Elute with 0.1 M disodium citrate (3.9 ml, 0.1 N hydrochloric acid, 10 ml ethanol) and measure the absorbance at 570 nm with a microtiter spectrophotometer. The dilution ratio showing 50% cell death rate is defined as the unit number of TCF-II (u / ml).

Measurement of Hepatocyte Proliferative Activity Segren's Method (Method in cell biology, vo1.13, p2
9, Academic Press, New York, 1976), hepatic parenchymal cells were isolated from Wistar male rats. The hepatic parenchymal cells were seeded on a 24-well plastic plate (Falcon) at a concentration of 8.8 × 10 ⁴ cells / 0.5 ml / well, and 5
Culturing was carried out at 37 degrees in the presence of% CO ₂ . The medium is 10% bovine neonatal serum (Hycron), 10 μM dexamethasone,
Williams E medium (Flow Laboratories) containing 100 U / ml penicillin and 100 ug / ml streptomycin was used (hereinafter abbreviated as basal medium). After culturing for 24 hours, the medium is replaced with a basal medium containing the test sample, and after culturing for another 24 hours, ³ H
-Thymidine (Amersham) was replaced with a basal medium containing 4 µCi / ml (86 Ci / m mol) and cultured for 2 hours, after which DNA synthesis was measured. In addition, in the above ³ H-thymidine label, each test group was incorporated in the presence or absence of 10 mM hydroxyurea,
The difference in the counts was taken as the uptake amount. After labeling with the above culture, the cells were washed twice with cold PBS, 2% perchloric acid and 95% ethanol, and then air-dried, and 2 mM EDT was added.
A, solubilized with 0.8 ml of 2% SDS containing 20 mM NaHCO _3, and measured with a liquid scintillation counter. The results are shown in Table 1.

[0019]

[Table 1] (n = 4) hEG as a positive control for hepatocyte proliferation
F (Yunaga Pharmaceutical) was used. From the table, it is found that the hepatocyte proliferation activity of TCF-II is stronger than that of hEGF.

Next, the preparation containing the physiologically active factor of ICF-II of the present invention as an active ingredient will be explained. The bioactive factor in the present invention exhibits the following drug activity. Antitumor activity Suppresses the growth of human-derived tumor cells KB, HeLa, MCF-7 and BG-1, and is mouse-derived L-929 cells and tumor cells Sa
It has cytotoxic activity on rcoma 180, Meth A Sarcoma, P388 but does not suppress the growth of human normal cells IMR-90. Leukemia cell differentiation-inducing activity It induces differentiation of human leukemia cells HL-60 into granulocytes. Cellular immunity enhancing activity Enhancement of human cytotoxic T cells Promoting proliferation of human vascular endothelial cells Promotes proliferation of human umbilical cord-derived vascular endothelial cells. Proliferation activity of liver parenchymal cells Promotes proliferation of rat liver-derived liver parenchymal cells. These activities are expressed in trace amounts in the range 1-1000 ng / ml.

The substance is expected as an antitumor agent, an antileukemia agent, a cell immunity enhancer, a wound therapeutic agent, a liver disease therapeutic agent including a liver regenerating agent, and the like. However, this bioactive factor (hereinafter referred to as TCF-II), which is a high molecular weight glycoprotein, is a substance that is significantly adsorbed to glass containers such as vials and polypropylene resin containers such as syringes and is unstable. . Its activity remarkably decreases due to temperature and humidity, and it is easily deactivated.
Therefore, it is expected to be formulated in a stable form. That is, the formulation according to the present invention comprises one or more selected from the group consisting of proteins and nonionic surfactants as an adsorption inhibitor, and one selected from the group consisting of proteins, sugars and amino acids, or Containing two or more stabilizers, one or more selected as an adsorption inhibitor and one or two selected as a stabilizer
It is a TCF-II preparation characterized by containing a combination of three or more thereof.

The formulation according to the present invention may be such that the above-mentioned adsorption inhibitor and stabilizer are mixed with TCF-II, and the formulation may be freeze-dried, aqueous solution or powder. TCF-II, which is the active ingredient in the present invention, may be produced and purified by any method, such as TCF-II extracted and isolated and purified from the culture solution of TCF-II producing cells, Escherichia coli, yeast, Chinese by genetic engineering techniques. What is produced by using mammalian cells such as hamster ovary cells as a host, extracted by various methods, separated and purified is used. Among the TCF-II adsorption inhibitors used in the present invention, proteins such as albumin and gelatin can be used, and nonionic surfactants such as Tween 80 and Tween 20 can be used, and TCF-II used in the present invention can be used. Among the stabilizers of II, albumin, gelatin and the like can be used as proteins, sorbitol, mannitol, xylitol and the like as sugars, and glycine, alanine and the like as amino acids.

Of these adsorption inhibitors, the amount of proteins added is 0.1% or more, preferably 0.1 to 20%, and the amount of nonionic surfactants added is 0.001% or more, preferably
It is 0.001 to 1.0%. Of the stabilizers, the amount of proteins added is 0.1% or more, preferably 0.1 to 20.0%.
Addition amount of saccharides is 5-40%, addition amount of amino acids is 1%
It is preferable to mix the above. Even when used in combination with one or more of the anti-adsorption agents and one or more of the stabilizers, or when used with two or more stabilizers, respectively The amount of the additive added may be within the above range. These anti-adsorption agents and stabilizers should be used in suitable concentrations and pH.
To prepare an aqueous solution of. The osmotic pressure ratio of this solution is 0.
It is 1 to 3.0, more preferably 1.0. Since the pH stability range of TCF-II is pH 6-9, the pH of the aqueous solution during formulation is 6.0.
It is preferable to adjust to ~ 9.0.

Next, experimental examples will be used to explain the adsorption prevention property and stability of the preparation of the present invention in more detail.

[Experimental Example 1] Adsorption prevention test IMR-90 cells (ATCC.CCL) which are human fetal lung-derived fibroblasts
186) was cultured in a medium containing 5% calf serum for 7 days, and 200 μg of TCF-II separated and purified by extracting from the culture supernatant was added.
Dissolve it in 100 ml of 0.01 M phosphate buffer pH 7 (PBS) containing 15 M sodium chloride, and dispense 0.5 ml each into a glass test tube and a polypropylene resin tube. Separately, Table 2- (1) a, b,
(2) Prepare a solution with twice the concentration of each additive substance shown in c with PBS. 0.5 ml of the TCF-II solution dispensed above
Add 0.5 ml of a solution of each concentration of the added substance to and mix well. The final concentration of TCF-II is 1 μg / ml, and the final concentration of each additive is adjusted to the concentration shown in Table 2- (1) a, b, (2) c. As a control, 0.5 ml of PBS was added to 0.5 ml of the TCF-II solution. Prepare two for each concentration of each additive,
After incubating at 37 ° C. for 1 hour, TCF-II activity was measured, and the result was calculated as the average value.

The activity of TCF-II was measured by its tumorigenic activity as follows. TCF-II hypersensitive clone L929 obtained by subcloning mouse L929 (ATCC, CCL1)
-18 was used as the target cell. L929-18 to 10% FC
Culture in Dulbecco's modified Eagle's medium (S) containing S until confluent, and then trypsinize the cells to detach and collect them, then add 6 x 10 ⁵ cells / ml to DMEM containing 10% FCS and 1 μg / ml actinomycin D. Suspend to the cell density of. A sample sample is diluted using DMEM used for cell suspension preparation to prepare a series of 20-fold or more serial dilutions. 50 μl of each sample in a series of 20-fold or more dilution is added to each well of a 96-well microplate (Falcon). 50 μl of the cell suspension is added to each well of the dilution series of the sample, and the cells are cultured at 37 ° C. for 2 days in a CO ₂ incubator. After culturing, the supernatant was gently discarded, and the cells were gently washed twice with PBS and the viable cells adhered to each well were dissolved in a mixture of methanol: water = 1: 4 0.5%
Add 50 μl of crystal violet solution to each well, and fix by staining. After washing each well with distilled water and air-drying, dye was mixed with Selenium buffer (6.1 ml, 0.1 M disodium citrate, 3.9 ml, 0.1 N hydrochloric acid, 10 ml ethanol mixed).
Elute with and measure the absorbance at 570 nm with a microtiter spectrophotometer. The dilution factor showing 50% cell death rate was TCF-
The number of units of II (u / ml) is defined, and the activity (u / ml) immediately after sample preparation is defined.
ml) as 100% and the residual activity after the test treatment is the relative activity
(%) Was calculated.

As shown in Table 2- (1) a, b, (2) c, TCF-II is easily adsorbed on the surface of glass or polypropylene resin. It has been found that has an effect of preventing adsorption of the preparation.

[0027]

[Table 2] (1) Glass test tube a. Effect of polymer activator Additive Concentration Human serum Low molecular weight ^* Gelatin Polyethylene dext (%) Albumi Gelatin glycol run (HSA) 4000 40 ……………… Residual relative activity (%) ……………………………… 0 8.3 8.3 8.3 8.3 8.3 0.01 16.7 8.3 25.0 8.3-0.05 25.0 8.3 37.5 8.3 8.3 0.10 50.0 16.7 50.0 16.7-0.25 100.0 16.7 100.0 16.7 8.3 0.50 100.0 33.3 100.0 16.7 8.3 1.00 100.0 50.0 100.0 16.7 12.5 2.00 100.0 100.0 100.0 33.3 12.5 10.00 100.0 100.0 100.0-75.0 20.00 100.0 100.0 100.0--*: Average molecular weight 6,000 (Nippi strain)

B. Effect of nonionic surfactant Additive concentration Tween 80 Tween 20 (%) …… Residual relative activity (%) ……… 0 8.3 8.3 0.0001 16.7 16.7 0.0005 25.0 25.0 0.001 50.0 50.0 0.005 100.0 100.0 0.01 100.0 100.0 0.05 100.0 100.0 0.10 100.0 100.0 1.00 100.0 100.0

(2) Polypropylene resin tube c. Effect of human serum albumin and Tween 80 Additive Additive Concentration Human serum concentration Tween (%) Albumin (HSA) (%) 80 Residual relative activity (%) Residual relative activity (%) 0 25.0 0 25.0 0.01 50.0 0.0001 33.3 0.10 75.0 0.0005 50.0 0.25 100.0 0.001 75.0 0.50 100.0 0.005 100.0 1.00 100.0 0.01 100.0 2.00 100.0 0.05 100.0 10.00 100.0 0.1 100.0 20.00 100.0 1.0 100.0

[0030]

[Experimental Example 2] Stability test The effect of various additives on the stability of TCF-II was tested under the condition that the adsorption of TCF-II on the glass wall could be completely prevented. That is, 120 μg of TCF-II derived from IMR-90 was dissolved in 30 ml of PBS containing 0.02% Tween 80, sterilized by filtration with a 0.22 μ filter, and then 0.5 ml was dispensed into sterile glass test tubes. Table 2- (1)
For each additive substance shown in a, b, (2) c, a solution with double concentration was prepared by dissolving in PBS, sterilized by filtration with 0.22μ filter, and 0.5 ml each of TCF-II solution 0.5 ml.
After mixing well and mixing well, the glass test tube was sealed to prevent contamination of various bacteria. As a control, 0.5 ml of TCF-II solution added with 0.5 ml of PBS containing no Tween 80 was used. TCF-
The final concentration of II is 2 μg / ml and the final concentration of Tween 80 is 0.
At 01%, the final concentration of each additive is as shown in Table 3a, b, c. Prepare 2 tubes for each test area and store at 40 ℃ for 1 week
Measure the TCF-II activity and calculate the TCF-II activity (μ / ml) before storage.
The activity after storage was set as 100% and shown as relative activity (%). The result was calculated as an average value. The results are shown in Table 3. Table 3a,
From the results of b and c, it is understood that the stabilizer used in the present invention has an effect of stably maintaining the TCF-II activity which is the active ingredient of the preparation in an aqueous solution state.

[0031]

[Table 3] a. Effect of polymer activators on TCF-II stability Storage period (days) 40 ℃ Addition concentration 0 3 7 (% W / V) …… Residual relative activity (%) …… 0.0 100.0 25.0 16.7 Human serum 0.1 100.0 50.0 33.3 Albumin 0.25 100.0 100.0 100.0 0.5 100.0 100.0 100.0 0.0 100.0 25.0 16.7 Gelatin 0.1 100.0 25.0 25.0 0.25 100.0 100.0 100.0 0.5 100.0 100.0 100.0 0.0 100.0 25.0 16.7 Low molecular weight gelatin 0.5 100.0 25.0 16.7 (Average molecular weight 6,000) 2.5 100.0 33.3 25.0 0.0 100.0 25.0 16.7 Polyethylene 0.5 100.0 16.7 12.5 Glycol 4000 2.5 100.0 16.7 12.5 Note: Tween 80 has a final concentration of 0.01% in all test plots.
include.

B. Effect of sugar addition Storage period (days) 40 ° C Sugar addition concentration 0 3 7 (% W / V) …… Residual relative activity (%) …… 0 100.0 25.0 16.7 Dextran 40 2 100.0 25.0 8.3 10 100.0 12.5 4.2 0 100.0 25.0 16.7 2 100.0 33.3 25.0 Sorbitol 10 100.0 66.7 66.7 20 100.0 100.0 100.0 40 100.0 100.0 100.0 0 100.0 25.0 16.7 2 100.0 33.3 16.7 Mannitol 10 100.0 66.7 50.0 20 100.0 100.0 95.0 40 100.0 100.0 100.0 0 100.0 25.0 16.7 Glucose 2 100.0 16.7 8.3 10 100.0 12.5 4.2 0 100.0 25.0 16.7 Fructose 2 100.0 12.5 6.3 10 100.0 <2.0 <2.0 0 100.0 25.0 16.7 Mannose 2 100.0 16.7 4.2 10 100.0 <2.0 <2.0 0 100.0 25.0 16.7 Xylitol 2 100.0 33.3 16.7 10 100.0 100.0 66.7 20 100.0 100.0 96.5

C. Effect of amino acid addition Storage period (days) 40 ° C Addition concentration 0 3 7 Amino acid (%) …… Residual relative activity (%) …… 0 100.0 25.0 16.7 Arginine 1 100.0 25.0 16.7 5 100.0 50.0 33.3 0 100.0 25.0 16.7 Glycine 1 100.0 33.3 25.0 5 100.0 100.0 66.7 10 100.0 100.0 100.0 0 100.0 25.0 16.7 Lysine 1 100.0 25.0 16.7 5 100.0 66.7 16.7 0 100.0 25.0 16.7 Alanine 1 100.0 25.0 16.7 5 100.0 100.0 50.0 10 100.0 100.0 90.0 Note: All tests The final concentration of Tween 80 in the ward is 0.01%
include.

Next, the results of testing the pharmaceutical activity of the present invention will be shown. Human novel cytokine TCF-II against Sarcoma 180 i
n vivo antitumor test material and test method : Experimental animal ICR mice were purchased from Charles River Japan, and 7-week-old females were used. Tumor cells Sarcoma 180 were used after being subcultured and maintained by mice once a week at our institute, which was distributed by the National Cancer Center. Test sample TCF-II was dissolved in 0.01M phosphate buffer containing 0.01% Tween, 0.25% human serum albumin and 0.8% sodium chloride, pH 7.0 to prepare a formulation. 0.2 μg TCF-II / 0.2 ml and 1.0
Two kinds of TCF-II samples of μg TCF-II / 0.2 ml were prepared. In order to examine the effect of pyrogen, a standard pyrogen (manufactured by Difco) sample (940 pg / 0.2 ml) in an amount equivalent to pyrogen contained in 1.0 μg TCF-II was prepared in the same manner. Preliminary toxicity test Preliminary toxicity test was conducted with 2 animals per group. 10 μg and 20 μg / mouse of TCF-II was administered once into the tail vein of an IRO mouse, and toxicity was determined using the death of the animal as an index. Antitumor test The antitumor test was carried out with 7 animals per group. Sarcoma 180 (10 ⁶ / mouse
E) was subcutaneously transplanted into ICR mice, and when the engraftment was confirmed, the mice were divided into groups and the sample was administered once a day into the tail vein for 7 consecutive days. The growth inhibitory effect was determined by determining the inhibition rate (1-T / C%) from the average tumor weight (MTW) of the administration group with respect to the control group.

Test results : Preliminary toxicity test No toxicity was observed with both 10 μg and 20 μg / mouse administration. Antitumor test Table 4 shows the test results 3 weeks after the start of administration.

[0036]

[Table 4] Sample dose MTW (mg) 1-T / C (%) Reference 0.0 3024.71-Pyrogen 940 pg / mouse 3036.00 -0.37 TCF-II 0.2 μg / mouse 1787.71 49.92 TCF-II 1.0 μg / mouse 1984.21 34.40

In the above test, the optimal dose of TCF-II was unknown, so there was some problem in setting the dose, but when looking at this result, it seems that the lower dose is more effective. Seem.

Further, the antitumor effect of TCF-II by intratumoral administration was examined by the following method. Test method : ICR mouse subcutaneously 1 x 10 ⁶ / mouse Sarcom
a 180 cells were transplanted, and after 1 week, mice in which solid tumor had engrafted were selected. 0.2 μg of TCF-II was administered to each mouse once a day for 7 consecutive days. When observed for 2 weeks after the completion of administration, the tumorous part showed blackening necrosis and showed a remarkable antitumor effect. In addition, mice with disappeared tumors were also observed.

The present invention will be described more concretely with reference to the following examples.

Example 1 Preparation of glycoprotein TCF-II (1) Culture of human fibroblast cell line IMR-90 Human fibroblast IMR-90 (ATCC CCL 186) cells containing 5% calf serum (CS) in DMEM 100 ml 3 × 10 ⁶ cells were transplanted into an 11-volume roller bottle containing the mixture, and the culture was continued for 7 days while rotating at a rotation speed of 0.5 to 2 rotations / minute. When the total number of cells reached 1 × 10 ⁷ , the cells were detached with trypsin, the cells were collected on the bottom of the bottle, and 100 g of 5-9 mesh ceramic (Toshiba Ceramic Co., Ltd.) was sterilized and put in for 24 hours. It was left to stand and cultured. Then, 500 ml of DMEM medium containing 5% of the above CS was added and the culture was continued. The entire amount of the medium was collected every 7 to 10 days, and the medium was replenished with fresh medium. In this way, the production was continued for 2 months, and 4 l of the culture solution was recovered per roller bottle. The specific activity per culture medium thus obtained was 32 u / ml.

(2) Purification of glycoprotein TCF-II 75 l of the culture solution obtained in (1) above was UF-concentrated by treatment with an Amicon membrane filter (MW 6,000 cut) to reduce the volume to 1 /
I made it 10. Then, CM Sephadex C-50 (Pharmacia) was equilibrated with 0.05M Tris-HCl buffer having a pH of 7.0, and a resin having a wet weight of 1.5 kg was added to 10 L of the UF concentrate to give a pH of 6.5. 4 ~ 7.0 under gentle stirring
It was allowed to adsorb at 24 ° C. for 24 hours. After adsorption, resin
The resin collected by filtration at 0.2 was washed with 0.05 Tris-hydrochloric acid buffer at pH 7. Approximately 1500 g of washed resin is 7 cm in diameter
Packed in a 40 cm column, 0.01% Tween 20 and 0.3 M
Elution was performed with 0.05 M Tris-hydrochloric acid buffer pH 7.0 containing salt.
The absorption at 280 nm was monitored, and when the protein was almost completely eluted, the salt strength was further changed to 0.6 M and elution was performed.
The tumor cytotoxic activity of each fraction was measured, and the tissue plasminogen activator (t-PA) activity produced by IMR-90 was measured. The elution pattern thus obtained is shown in FIG. The fraction eluted at a salt strength of 0.6 M showed strong tumor cytotoxic activity. This fraction is TCF-
It was designated as the II fraction.

Next, ConA-Sepharose CL-6B (manufactured by Pharmacia) was equilibrated with a pH 7.0, 0.05 M Tris-hydrochloric acid buffer containing 0.5 M sodium chloride and packed in a column having a diameter of 2.5 cm × 8 cm. Wash the column better with the same buffer,
TCF-II fraction eluted at CM-Sephadex column (pH
7.0) loaded. After that, 0.5M of 10 times the column volume again
After washing the column with pH 7.0, 0.05 M Tris-hydrochloric acid buffer containing sodium chloride, and 0.5 M sodium chloride and 0.3 M α-methyl-D-mannopyranoside-containing pH 7.0, 0.05 M Tris-hydrochloric acid buffer solution. Elution was performed at a flow rate of 70 ml per hour. Tumor cytotoxic activity of each elution fraction was measured, and protein absorption at 280 nm was monitored. The elution pattern shown in FIG. 2 was shown.

The first eluted fraction was recovered, dialyzed against distilled water at 4 ° C. for 48 hours, and then freeze-dried to obtain a white powder. This powder was dissolved in a minimum amount of 0.01% Tween 20 at pH 7.0 and 0.05M Tris-HCl buffer, and equilibrated with 0.01M Tween 20 at pH 7.0 and 0.01M phosphate buffer. (Made by Pharmacia)
Loaded on. After loading 0.01% Tween 20 included pH 7.0, 0.01
After washing with M phosphate buffer for 20 minutes at a flow rate of 0.5 ml / min, elution was performed for 60 minutes at a flow rate of 0.5 ml / min with a concentration gradient such that the final salt concentration was 1 M salt. The elution pattern is shown in FIG. The active fraction was eluted with 0.76M sodium chloride at the top. The active fraction was collected and loaded on the MonoS column again,
Elution was performed again with the same buffer with a concentration gradient up to a salt concentration of 1.0 M.

The active fraction was collected and then 1.0 cm in diameter x 7
Heparin-Sepharose (manufactured by Pharmacia) packed in a 5 cm column was equilibrated with 10 mM Tris-HCl buffer, pH 7.5 containing 0.3 M sodium chloride, and the active fraction in this column had a sodium chloride concentration of 0.3 M. As 0.01M Tris-HCl buffer
It was diluted with (pH 7.0) and loaded on the above heparin sepharose column. Thereafter, the gel was washed with 0.01 M Tris-hydrochloric acid buffer containing pH 7.5 and 0.3 M sodium chloride at 10 times the packed gel amount. Further, it was eluted with a buffer having the same pH by a salt concentration gradient from 0.3 M to 2.0 M at a flow rate of 20 ml per hour. The elution pattern is shown in FIG.

Thus, a glycoprotein was obtained. As shown in Table 5, 0.12 mg of active protein could be obtained using 75 l of culture medium as a starting material. At the same time, this glycoprotein
It was a tumor cytotoxic factor with a specific activity of 5,248,000u / mg.

[0045]

[Table 5]

The results of measuring the physicochemical properties of TCF-II obtained as described above are shown below. Molecular weight measurement by SDS polyacrylamide gel electrophoresis The molecular weight was measured by electrophoresis using polyacrylamide gel containing 0.1% SDS. 78,000 and 7 for glycoproteins
It showed 4,000 closely spaced bands. Further, when subjected to reduction treatment with 2-mercaptoethanol and similarly subjected to electrophoresis, three bands having a molecular weight of 52,000 and 28,000 and 32,000 were obtained (see FIG. 5). From this, TCF-II
It is expected to be a complex in which a common subunit having a molecular weight of 52,000 and a subunit having a molecular weight of 32,000 or a subunit having a molecular weight of 28,000 are bound. Isoelectric point The isoelectric point was measured by Phast Gel IEF3-9 using an LKB isoelectric focusing device, and it showed an isoelectric point of 7.4 to 8.55.

Thermostable 0.1 M Tris-containing 0.01% Tween 20 adjusted to pH 7.5
TCF-II dissolved in an activity of 51,200 u / ml was added to a hydrochloric acid buffer solution to prepare a solution of 512 u / ml. A liquid with this activity
It was treated for 10 minutes at each temperature of 25, 35, 50, 60, 70, 80, 90 and 95 ° C, and the relative activity to the activity at 25 ° C was measured. As shown in FIG. 6, it was heat stable up to 60 ° C. pH stability Each buffer solution with the composition shown in Table 6 (both 0.01% Tween 20
) Was added to the buffer solution of each pH, and the
TCF-II corresponding to 00 u / ml was dissolved and the activity was measured after standing at 37 ° C. for 1 hour, and the relative activity was measured as compared with the case of standing at pH 8 and room temperature for 1 hour. As shown in FIG. 7, pH 6-9
It was stable in the range.

[0048]

[Table 6] Preparation buffer pH 1-3 1 / 10M glycine-hydrochloric acid pH 4-6 1 / 10M acetate buffer pH 7-8 1 / 10M Tris-hydrochloric acid pH 9-12 1 / 10M glycine-sodium hydroxide

50 μg of N-terminal amino acid sequence of TCF-II was reduced and separated by electroplot into 3 proteins of A having a molecular weight of 52,000, B having a molecular weight of 32,000, C having a molecular weight of 28,000, and 477A type protein sequencer manufactured by Applied Co. for each protein. The N-terminal amino acid sequence was analyzed by. Since A was blocked at the N-terminal, analysis of the N-terminal amino acid sequence could not be measured, but B and C both showed the common N-terminal amino acid sequence shown below. Val-Val-Asn-Gly-Ile-Pro-Thr- X -Thr-Asn-Ile-Gly- X -Met-Val-Ser-Leu 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 X indicates unidentified. Since the N-terminal amino acid sequences of B and C are completely the same, TCF-II is A with a molecular weight of 52,000.
And B having a molecular weight of 32,000 or C having a molecular weight of 28,000 are considered to have a double-chain structure in which they are bound by an SS bond.

Amino Acid Composition An amount corresponding to 10 μg of the amount of protein measured by a protein assay kit manufactured by Bio-Rad was decomposed by hydrochloric acid hydrolysis, and the amino acid composition was measured by Hitachi L-8500 type amino acid analyzer. The following amino acid composition was obtained. Amino acid composition: AA nmol mol% Asp 10.375 12.97 Glu 7.750 9.69 Ser 5.000 6.25 Gly 7.250 9.06 His 3.000 3.75 Arg 5.375 6.72 Thr 5.125 6.41 Ala 2.625 3.28 Pro 5.625 7.03 Tyr 3.875 4.84 Val 4.125 5.16 Met 1.875 2.34 Cys ND-Ileu 5.000 6.25 Leu 4.875 6.09 Phe 2.250 2.81 Trp ND-Lys 5.875 7.34 Total 80.000 100 (99.99)

[0051]

Example 2 This example shows the tumor cytotoxic activity of the glycoprotein TCF-II obtained in Example 1. Tumor cell growth suppression Tumor cell lines HeLa, KB, human diploid cells IMR-
90 was adjusted to a cell density of 10 ⁵ / ml in DMEM containing 10% FCS to prepare a cell suspension. Micro well plate
(Falcon) was seeded with 50 μl of each cell. To each well, 5,120 u / ml TCF-II from the above medium 10, 20,
Add 50 μl of 40,80,160-fold diluted mixture and mix.
The cells were cultured at 37 ° C for 3 days in a CO ₂ incubator. The cells that survived in each well were dissolved in a solution adjusted to methanol: water = 1: 4, and a 0.5% crystal violet solution was added to each well in an amount of 50 μl to fix the cells. After washing each well with distilled water, the plate was air-dried, the dye was eluted with Selenium buffer, and the absorbance at 570 nm was measured with a microtiter spectrophotometer. The cell growth inhibition rate (%) was calculated for each cell using the TCF-II non-added group as a control, and the relationship with the TCF-II concentration was determined. As shown in FIG. 8, normal cells, IMR-90, did not show growth inhibition, but KB and HeLa cells showed strong inhibition.

Reaction with antibody against existing substance TCF-II was dissolved and prepared in DMEM containing 10% FCS so as to have a concentration of 320 u / ml. On the other hand, anti-LT antibody
Add the titer value to neutralize 1000u / ml and add 37
The reaction was allowed to stand at 1 ° C for 1 hour. In the same way, anti-TNF antibody
1 × 10 ⁶ u / ml and anti-INF β antibody were added at 1000 u / ml and reacted. In addition, each antibody used the commercially available thing. After the reaction, the neutralizing effect of each antibody was confirmed by measuring TCF-II activity, but no neutralizing effect of the activity was observed.

[0053]

Example 3 This example shows the cytotoxic activity of the glycoprotein TCF-II obtained in Example 1 against various mouse-derived tumor cells. Sarcoma180, MethA Sarcom as mouse-derived tumor cell lines
3 strains of a and P-388 were used. 10% for Sarcoma 180 cells
MethA and P388 are cells 1 in DMEM containing fetal bovine serum
2x each in RPMI 1640 medium containing 0% fetal bovine serum
The cells were suspended at 10 ⁴ cells / ml to prepare each cell suspension. 50 μl of each cell suspension was seeded in a 96-well flat-bottomed microwell plate (Falcon). TCF-II contains 10% fetal calf serum for Sarcoma 180
TCF-II solution was prepared by dissolving and diluting in DMEM or RPMI1640 medium containing 10% fetal bovine serum for MethA and P-388. TC in each well seeded with each cell suspension
Add 50 μl of F-II solution to the final concentration of TCF-II.
0, 2, 4, 8, 16, 31, 62, 125, 250, 500, 1000ng / ml
Was prepared. After mixing, the cells were cultured in a CO ₂ incubator at 37 ° C for 3 days. The cells in each well were stained with trypan blue, only live cells were counted using a hemocytometer, and the average of two experimental values was obtained. For each cell
Using the TCF-II non-added group as a control, the cytotoxic activity (%) was calculated by the following formula to determine the relationship with the TCF-II concentration.

[0054]

[Equation 1]

As a result, the cytotoxic activity of TCF-II thus obtained against Sarcoma 180 is shown in FIG. 9 and that against Meth A Sarcoma and P-388 is shown in FIG. All cells were highly sensitive to TCF-II, and TCF-II-induced Sarcoma18
0, cytotoxic activity IC against Meth A Sarcoma and P-388
₅₀ were 6, 40 and 460 ng / ml respectively.

[0056]

Example 4 In this example, the glycoprotein TCF-obtained in Example 1 was used.
2 shows the growth inhibitory effect of the human tumor cell line of II on ovarian cancer cell line BG-1 and breast cancer cell line MCF-7. BG-1 is 10% FC
Each cell suspension was prepared in McCoy's medium containing S such that MCF-7 was 2 × 10 ⁴ / ml in Eagle MEM containing 10% FCS, non-essential amino acid mixture, pyruvic acid and Eagle salt. On the other hand, TCF-II was dissolved in McCoy's medium containing 10% FCS for BG-1 to prepare a TCF-II solution at a concentration of 4 μg / ml, which was sequentially diluted 2-fold in the same medium to prepare TCF-II. A serial dilution series of was prepared. Similarly, for MCF-7, a serial dilution series of TCF-II was prepared in the above MCF-7 growth medium. 50 μl of each cell suspension was seeded on a 96-well microwell plate (Falcon). Next, 50 μl of each serially diluted solution of TCF-II prepared for BG-1 was added to each well in which BG-1 was seeded and mixed. The same was done for MCF-7. The cells were cultured in a CO ₂ incubator at 37 ° C for 5 days. After culturing, remove the culture solution, wash the microwell plate twice with PBS, and dissolve the cells attached to each well in a mixture of methanol: water = 1: 4 with 0.5% crystal violet solution in each well. 50 μl of each was added and fixed by staining. After washing each well with distilled water, the plate was air-dried, the dye was eluted with Selenium buffer, and the absorbance at 570 nm was measured with a microtiter spectrophotometer. The cell growth inhibition rate of each cell was calculated based on the following formula using the TCF-II non-added group as a control, and the relationship with the TCF-II concentration was calculated. The results are shown in Fig. 11. As a result, BG-1,
It was confirmed that the growth of both MCF-7 tumor lines was suppressed by TCF-II.

[0057]

[Equation 2]

[0058]

Example 5 This example is the glycoprotein TCF-obtained in Example 1.
2 shows the differentiation-inducing activity of HL-60, a promyelocytic leukemia strain by II. HL-60 cells in RPMI1640 medium containing 10% fetal bovine serum
The cells were suspended at 3.5 × 10 ⁵ / ml to prepare a cell suspension. 96 hole flat bottom microtiter plate (Falcon)
100 μl of the cell suspension was dispensed into each well. Then add 100 μl of the TCF-II solution dissolved and diluted in the same medium to the final concentration.
15.6, 62.5, 125, 250, 500 and 1000 ng / ml were added. After culturing at 37 ℃ for 3 and 7 days, the HL-60 differentiation-inducing activity of TCF-II was measured by nitroblue tetrazolium (N
BT) Measured by reducing ability. The morphological changes were also examined.

1) NBT reducing ability The NBT reducing ability is shown in Table 7.

[Table 7]

The numerical values in the table indicate the proportion of cells in which at least 200 cells were counted, NBT was reduced, and blue-black formazan was contained. (The average value of a total of 2 experiments is shown). (When HL-60 differentiates into normal cells, it acquires NBT reducing ability and accumulates blue-black formazan in the cells.) As a result, TCF-II induces differentiation of promyelocytic leukemia line, HL-60, and 250ng / It was found that ml had the highest differentiation-inducing activity.

2) Morphological Changes It is known that HL-60 exhibits two types of differentiation, a macrophage system and a monosite system, depending on the differentiation inducer.
As a result of examining the morphology or nuclear change of cells differentiated with TCF-II after culturing at 37 ° C for 7 days by Wright-Giemsa staining, TCF-II was
It was confirmed that HL-60 was induced to differentiate into monosite.

[0062]

Example 6 shows the cell immunological activity by the glycoprotein TCF-II obtained in Example 1. That is, a mixed lymphocyte culture test was performed under the condition that TCF-II was added to examine the activity of TCF-II on the lymphocyte blast transformation reaction. Ficall from human peripheral blood
-The lymphocytes were separated by the Conray method and RPMI 1640-10% FC
Suspended in S medium. 2 individual lymphocytes, 1: 1 after addition to the round bottom microplate for a total of 1 × 10 ⁵ / 100μl / well in a ratio, by adding TCF-II at various concentrations, CO
₂ Cultured in RPMI-10% FCS medium under incubator. 16 H before the end of culture, ³ H thymidine was added at 0.25 μCi / well. After completion of the culture, the cells were collected by a cell harvester, washed with PBS, and the radioactivity of ³ H thymidine incorporated into the cells was measured by a scintillation counter.

The results are shown in FIGS. 12 and 13. As shown in FIG. 12, no effect of TCF-II was observed on the 5th day of culture, but as shown in FIG. 13 on the 8th day of culture, the control was performed in the TCF-II-added group with a final concentration of 100 ng / ml. Significantly compared to the group
An increase in ³ H uptake was observed, and it was confirmed that TCF-II has an effect of enhancing cytotoxic T cell proliferation, that is, cell-mediated immunity.

[0064]

Example 7 shows the vascular endothelial cell proliferation activity by the glycoprotein TCF-II obtained in Example 1. Human umbilical cord-derived vascular endothelial cells, HUVEC, were used as test cells. Human vascular endothelial cells
2.5x10 ⁴ HUVEC in E-GM medium containing 2% fetal calf serum
It was suspended so that the amount became ml. 50 μl of the above cell suspension was dispensed into each well of a 96-well flat bottom microwell plate (Falcon). TCF-II was dissolved in E-GM medium containing 2% fetal bovine serum, 50 μl of each was added to each well containing cell suspension, and the final concentration of TCF-II was 0, 4, 8, 1.
It was adjusted to 6, 31, 62, 125, 250, 500 and 1000 ng / ml. The cells were cultured at 37 ° C in a CO ₂ incubator for 6 days. For the number of cells in each well, remove the medium in each well,
After washing with PBS, the cells were removed by trypsin treatment, and the number of viable cells was counted with a hemocytometer.

As a result, the action of TCF-II thus obtained on normal human vascular endothelial cells HUVEC is shown in FIG. It was confirmed that TCF-II has no cytotoxic activity on vascular endothelial cells, which are normal cells, and conversely has a growth promoting activity.
In particular, the growth promoting activity was maximized when the TCF-II concentration was 125 ng / ml.

The following example illustrates the formulation of the formulation according to the present invention.

Example 8 TCF-II 20 μg Human serum albumin 100 mg The above composition was dissolved in 0.01 M phosphate buffer (PBS) at pH 7.0,
After the total amount was adjusted to 20 ml and sterilized, 2 ml each was dispensed into vials, lyophilized and sealed.

[0067]

Example 9 TCF-II 40 μg Tween 80 1 mg Human serum albumin 50 mg The above composition was dissolved in physiological saline for injection, the total amount was adjusted to 20 ml, sterilized by filtration, dispensed into vials in 2 ml portions, lyophilized and sealed. did.

[0068]

Example 10 TCF-II 20 μg Tween 80 2 mg sorbitol 4 g The above composition was dissolved in PBS, the total amount was adjusted to 20 ml, sterilized, and then dispensed in 2 ml aliquots, freeze-dried and sealed.

[0069]

Example 11 TCF-II 40 μg Tween 80 2 mg Glycine 2 g The above composition was dissolved in physiological saline for injection, the total amount was adjusted to 20 ml, sterilized by filtration, dispensed in 2 ml aliquots, freeze-dried and sealed. .

[0070]

Example 12 TCF-II 40 μg Tween 80 1 mg Sorbitol 2 g Glycine 1 g The above composition was dissolved in physiological saline for injection, the total amount was adjusted to 20 ml, and after sterilization by filtration, 2 ml was dispensed into vials and freeze-dried. And sealed.

[0071]

Example 13 TCF-II 20 μg Sorbitol 4 g Human serum albumin 50 mg The above composition was dissolved in PBS, adjusted to 20 ml in total, sterilized by filtration, dispensed in 2 ml aliquots, freeze-dried and sealed.

[0072]

Example 14 TCF-II 40 μg Glycine 2 g Human serum albumin 50 mg The above composition was dissolved in physiological saline for injection, the total amount was adjusted to 20 ml, sterilized by filtration, dispensed into vials, lyophilized and sealed.

[0073]

INDUSTRIAL APPLICABILITY The present invention provides a novel glycoprotein, wherein the glycoprotein of the present invention is a tumor cytotoxic factor,
It becomes a leukemia differentiation-inducing factor, a cell immunopotentiating activator, a vascular endothelial cell growth factor and the like, and a novel drug can be provided by preparing a drug product according to a conventional drug preparation technique. Further, the glycoprotein of the present invention is also used as a reagent for biochemical or pharmacological action.

[0074]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 723 Sequence type: Amino acid Sequence type: Protein sequence Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu 1 5 10 15 Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln 20 25 30 Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr 35 40 45 Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val 50 55 60 Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu 65 70 75 80 Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys 85 90 95 Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe 100 105 110 Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys 115 120 125 Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys 130 135 140 Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His 145 150 155 160 Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg 165 170 175 Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser Asn Pro Glu Va l Arg 180 185 190 Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu Val Glu Cys Met Thr 195 200 205 Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp His Thr Glu Ser Gly 210 215 220 Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro His Arg His Lys Phe 225 230 235 240 Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp Asp Asn Tyr Cys Arg 245 250 255 Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr Thr Leu Asp Pro His 260 265 265 270 Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys Ala Asp Asn Thr Met 275 280 285 Asn Asp Thr Asp Val Pro Leu Glu Thr Thr Glu Cys Ile Gln Gly Gln 290 295 300 Gly Glu Gly Tyr Arg Gly Thr Val Asn Thr Ile Trp Asn Gly Ile Pro 305 310 315 320 Cys Gln Arg Trp Asp Ser Gln Tyr Pro His Glu His Asp Met Thr Pro 325 330 335 Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn Tyr Cys Arg Asn Pro 340 345 350 Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr Asp Pro Asn Ile Arg 355 360 365 Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp Met Ser His Gly Gln 370 375 380 Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met Gly Asn Leu Ser G ln 385 390 395 400 Thr Arg Ser Gly Leu Thr Cys Ser Met Trp Asp Lys Asn Met Glu Asp 405 410 415 Leu His Arg His Ile Phe Trp Glu Pro Asp Ala Ser Lys Leu Asn Glu 420 425 430 Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala His Gly Pro Trp Cys Tyr 435 440 445 Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys Pro Ile Ser Arg Cys 450 455 460 Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu Asp His Pro Val Ile 465 470 475 480 Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val Asn Gly Ile Pro Thr 485 490 495 Arg Thr Asn Ile Gly Trp Met Val Ser Leu Arg Tyr Arg Asn Lys His 500 505 510 Ile Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp Val Leu Thr Ala Arg 515 520 525 Gln Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr Glu Ala Trp Leu Gly 530 535 540 Ile His Asp Val His Gly Arg Gly Asp Glu Lys Cys Lys Gln Val Leu 545 550 555 560 Asn Val Ser Gln Leu Val Tyr Gly Pro Glu Gly Ser Asp Leu Val Leu 565 570 575 Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp Phe Val Ser Thr Ile 580 585 590 Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu Lys Thr Ser Cys S er 595 600 605 Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn Tyr Asp Gly Leu Leu 610 615 620 Arg Val Ala His Leu Tyr Ile Met Gly Asn Glu Lys Cys Ser Gln His 625 630 635 640 His Arg Gly Lys Val Thr Leu Asn Glu Ser Glu Ile Cys Ala Gly Ala 645 650 655 Glu Lys Ile Gly Ser Gly Pro Cys Glu Gly Asp Tyr Gly Gly Pro Leu 660 665 670 Val Cys Glu Gln His Lys Met Arg Met Val Leu Gly Val Ile Val Pro 675 680 685 Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly Ile Phe Val Arg Val 690 695 700 Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile Leu Thr Tyr Lys Val 705 710 715 720 Pro Gln Ser

[Brief description of drawings]

FIG. 1 shows the elution profiles of protein, plasminogen activator and TCF-II obtained from CM-Sephadex C-50 chromatography of IMR-90 culture containing 5% calf serum. (1) is the fraction eluted with 0.05M Tris-HCl buffer (pH 7.0) containing 0.3M NaCl, and (2) is 0.6M.
The fractions eluted with 0.05 M Tris-HCl buffer containing NaCl (pH 7.0) are shown.

FIG. 2 shows the results of ConA affinity chromatography of the elution fraction of 0.05 M Tris-hydrochloric acid buffer solution (pH 7.0) containing 0.6 M NaCl obtained from CM-Sephadex C-50 chromatography of IMR-90 culture solution. (1) is 0.5M NaCl containing 0.
The wash fraction with 05M Tris-HCl buffer (pH 7.0) was added to (2)
Indicates an elution fraction with 0.05 M Tris-HCl buffer (pH 7.0) containing 0.5 M NaCl and 0.3 M α-methyl-D-mannopyranoside.

FIG. 3 shows the elution pattern by MonoS-HPLC of the TCF-II fraction obtained from ConA Sepharose affinity chromatography.

FIG. 4 shows the elution pattern of heparin-sepharose affinity chromatography of the TCF-II fraction obtained from MonoS-HPLC. (1) for washing, (2) for salt concentration gradient
Elution by (0.3M → 2.0M) is shown.

FIG. 5 shows SDS electrophoresis of TCF-II (unreduced and reduced).

FIG. 6 shows the thermal stability of TCF-II.

FIG. 7 shows pH stability of TCF-II.

FIG. 8 shows the effect of TCF-II on the damaging activity of human tumor cells in vitro.

FIG. 9 shows the cytotoxic activity of TCF-II against Sarcoma 180.

FIG. 10 shows the cytotoxic activity of TCF-II against Meth A and P388.

FIG. 11 shows the growth inhibition rate of TCF-II on human tumor cells.

FIG. 12 shows the radioactivity concentration of ³ H thymidine incorporated into lymphocytes on day 5 of mixed lymphocyte culture. Six samples were measured for each sample, and shown as the average value ± SD.

FIG. 13 shows the radioactivity concentration of ³ H thymidine incorporated into lymphocytes on day 8 of culture. Six samples were measured for each sample and are shown as the average value ± SD.

FIG. 14 shows proliferative activity of TCF-II on vascular endothelial cell HUVEC.

FIG. 15 shows the nucleotide sequence of TCF-II cDNA and the amino acid sequence of TCF-II deduced from this sequence.

FIG. 16 shows the nucleotide sequence of TCF-II cDNA and the amino acid sequence of TCF-II deduced from this sequence. This is continued from FIG. 15, and in FIGS. 15 and 16, the nucleotide sequence of a series of TCF-II cDNAs and the amino acid sequence deduced therefrom are shown. Amino acids are shown in single letter code.

FIG. 17: TCF-II deduced from the nucleotide sequences of FIGS. 15 and 16
3 shows a comparison between the amino acid sequence of hHGF and that of Miyazawa et al. Amino acids are shown in single letter code.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location A61K 38/00 ADT ADU ADV C12N 5/10 15/09 C12P 21/02 ZNA H 9282-4B // (C12P 21/02 C12R 1:91) A61K 37/02 ADT ADU ADV 7729-4B C12N 5/00 B 9281-4B 15/00 A (72) Inventor Masaya Nagao 578-15 Ishibashi, Ishibashi-cho, Shimotsuga-gun, Tochigi Prefecture − 3-4

Claims (22)

[Claims] 1. A glycoprotein obtained from a culture supernatant of human-derived fibroblasts and having the following properties a. To h .: a. Molecular weight; molecular weight measured by SDS polyacrylamide gel electrophoresis, 78,000 ± 2,000 or 74,000 without reduction
With a molecular weight of ± 2,000, 52,000 ± 2,000 when reduced
0 common band A and 30,000 ± 2,000 band B and 2
Two bands of 6,000 ± 2,000 band C are shown. b. Isoelectric point; 7.4-8.6 c. Thermal stability; stable even by heating at 60 ° C. for 10 minutes d. pH stability; stable in the range of pH 6 to 9 e. Sugar chain: Adsorbs to concanavalin A (ConA) sepharose. f. Physiological activity: Suppresses the proliferation of KB cells, HeLa cells and L-929 cells, but does not inhibit the proliferation of IMR-90 cells. g. Reactivity with antibodies; anti-TNF antibody, anti-lymphotoxin antibody, anti-interferon β antibody do not neutralize the damaging activity. h. N-terminal amino acid sequence; B and C above are subchains of band A, and band A has the N-terminal amino acid blocked. Subchains B and C both have the following N-terminal amino acid sequences: Val-Val-Asn-Gly-Ile-Pro-Thr- or Val-Val-Asn-Gly-Ile-Pro-Thr-X-Thr-Asn. -Ile-Gly-X-Me
t-Val-Ser-Leu- However, X means unidentified. 2. The glycoprotein according to claim 1 as an active ingredient, and one or more kinds selected from the group consisting of proteins and nonionic surfactants as an adsorption inhibitor. Or one or two selected from the group consisting of proteins, sugars and amino acids
Human fibroblast-derived bioactive factor preparations containing the above-mentioned species as stabilizers and exhibiting the following physiological activities; Antitumor activity Proliferation of human-derived tumor cells KB, HeLa, MCF-7 and BG-1 , Which is a mouse-derived L-929 cell and a tumor cell Sa
It has cytotoxic activity on rcoma 180, Meth A Sarcoma, P388 but does not suppress the growth of human normal cells IMR-90. Leukemia cell differentiation-inducing activity It induces differentiation of human leukemia cells HL-60 into granulocytes. Cellular immunity enhancing activity Enhancement of human cytotoxic T cells Growth promoting activity of human vascular endothelial cells Promotes proliferation of human umbilical cord-derived vascular endothelial cells. Proliferation activity of liver parenchymal cells Promotes proliferation of rat liver-derived liver parenchymal cells. 3. The physiologically active agent preparation according to claim 2, wherein the protein selected as the adsorption inhibitor of the preparation is either albumin or gelatin. 4. The bioactive agent preparation according to claim 2, wherein the nonionic surfactant selected as an adsorption inhibitor of the preparation is either Tween 80 or Tween 20. 5. The protein selected as a stabilizer of the preparation is either albumin or gelatin.
The physiologically active factor preparation according to. 6. The bioactive agent preparation according to claim 2, wherein the saccharide selected as a stabilizer of the preparation is any one of sorbitol, mannitol and xylyl. 7. The bioactive factor preparation according to claim 2, wherein the amino acid selected as a stabilizer of the preparation is either glycine or alanine. 8. One or more selected from proteins and nonionic surfactants as an adsorption inhibitor and one or more selected from proteins, saccharides and amino acids as a stabilizer. The bioactive factor preparation according to claim 2, which contains various combinations thereof with. 9. A DNA containing a base combination encoding TCF-II.
. 10. A DNA containing a nucleotide sequence encoding the amino acid sequence shown in FIGS. 11. A cDNA containing a nucleotide sequence encoding the amino acid sequence shown in FIGS. 12. Includes the nucleotide sequences shown in FIGS. 15 and 16.
cDNA sequence. 13. An antitumor agent comprising the protein according to claim 1 as an active ingredient. 14. A leukemia cell differentiation inducer comprising the protein according to claim 1 as an active ingredient. 15. A cellular immunity enhancer comprising the protein according to claim 1 as an active ingredient. 16. A wound healing agent containing the protein according to claim 1 as an active ingredient. 17. A hepatocyte proliferating agent comprising the protein according to claim 1 as an active ingredient. 18. A microorganism having the DNA according to claim 10. 19. The protein according to claim 1, which is produced by expressing the DNA according to claim 10. 20. The protein according to claim 1, wherein the entire amino acid sequence of subchain B or C has the following amino acid sequence. Val Val Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly Trp Met Val Ser 1 5 10 15 Leu Arg Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu Ile Lys Glu 20 25 30 Ser Trp Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg Asp Leu Lys 35 40 45 Asp Tyr Glu Ala Trp Leu Gly Ile His Asp Val His Gly Arg Gly Asp 50 55 60 Glu Lys Cys Lys Gln Val Leu Asn Val Ser Gln Leu Val Tyr Gly Pro 65 70 75 80 Glu Gly Ser Asp Leu Val Leu Met Lys Leu Ala Arg Pro Ala Val Leu 85 90 95 Asp Asp Phe Val Ser Thr Ile Asp Leu Pro Asn Tyr Gly Cys Thr Ile 100 105 110 Pro Glu Lys Thr Ser Cys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu 115 120 125 Ile Asn Tyr Asp Gly Leu Leu Arg Val Ala His Leu Tyr Ile Met Gly 130 135 140 Asn Glu Lys Cys Ser Gln His His Arg Gly Lys Val Thr Leu Asn Glu 145 150 155 160 Ser Glu Ile Cys Ala Gly Ala Glu Lys Ile Gly Ser Gly Pro Cys Glu 165 170 175 Gly Asp Tyr Gly Gly Pro Leu Val Cys Glu Gln His Lys Met Arg Met 180 185 190 Val Leu Gly Val Ile Val Pro Gly Arg Gly Cys Ala Ile Pro Asn Arg 195 200 205 Pro Gly Ile Phe Val Ar g Val Ala Tyr Tyr Ala Lys Trp Ile His Lys 210 215 220 Ile Ile Leu Thr Tyr Lys Val Pro Gln Ser 225 230 21. The protein according to claim 1, wherein the purified protein has a binding property to a weak cation exchanger. 22. It has a growth promoting activity on rat liver parenchymal cells, and this growth promoting activity exhibits a maximum growth promoting effect at a concentration of about 10 ng / ml, and the promoting effect does not decrease beyond the above concentration. Claim 1 characterized by the above-mentioned.
The described protein.