JP2747979B2 - Pharmaceutical containing a bioactive factor consisting of human-derived glycoprotein as an active ingredient - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a pharmaceutical comprising as an active ingredient the glycoprotein obtained from the culture supernatant of fibroblasts derived from human. Since the glycoprotein of the present invention has a damaging effect on tumor cells and does not show damage on normal cells, it has antitumor agents, antileukemia agents, cell immunity enhancers, wound healing agents, liver regeneration promoters, etc. Useful as a medicine or as a biochemical or pharmacological agent.

[0002]

2. Description of the Related Art As a bioactive substance produced by human-derived fibroblasts, for example, β-interferon is a typical example of a tumor cytotoxic factor. This is a glycoprotein secreted extracellularly when the cells are harvested and stimulated with poly I-poly C or Sendai virus after culturing fibroblasts, and have various physiological activities besides antiviral and antitumor effects. It is clear to show. JP-A-58-1462
No. 93 discloses a fibroblast-derived tumor cytotoxic glycoprotein called CBF. JP-A-61-
No. 33120 discloses a tumor growth inhibitor (INF) having a molecular weight of 35,000 to 45,000 extracted from a human tissue-derived fibroblast culture solution. JP-A-61-56131 discloses a tumor necrosis factor-like substance extracted from fibroblasts, and JP-A-61-1872 discloses a fibroblast-derived necrosis factor FNF. -1
No. 03021 discloses a molecular weight of 40,000 to 60,000 produced from animal fibroblasts and an isoelectric point of 5.0 ±.
A physiologically active substance having a cytotoxic effect of 0.5 is disclosed. Furthermore, Japanese Patent Application Laid-Open No. 64-10998 discloses all amino acids of a tumor cell-damaging factor having a molecular weight of 36,000 ± 1,000 and an isoelectric point of 10.5 or more obtained from a culture supernatant of human-derived fibroblasts. The sequence and the cDNA sequence encoding it are disclosed.

[0003]

DISCLOSURE OF THE INVENTION The present inventors have conducted a search for bioactive substances contained in the culture supernatant of human-derived fibroblasts and found that glycoproteins having various bioactivities
Found quality . When its pharmacological action was examined, it did not show any damage to normal cells, but had tumor cell damaging action, leukemia differentiation inducing action, cell immunity activating action, vascular endothelial cell proliferating action, hepatic parenchymal cell proliferating action. And found to be used as a medicament based on these pharmacological actions. That is, an object of the present invention is to provide a novel and useful medicament such as an antitumor agent, an antileukemia agent, a cell immunity enhancer, a wound treatment agent, and a liver regeneration promoter.

[0004]

The glycoprotein derived from human fibroblasts discovered by the present inventors is a glycoprotein (hereinafter referred to as TCF-II) specified by the following physicochemical properties. a. Molecular weight; measured by SDS polyacrylamide gel electrophoresis, 78,000 ± 2.0 for non-reduced
A common band A of 52,000 ± 2,000 with a molecular weight of 00 or 74,000 ± 2,000 and reduced
And band B of 30,000 ± 2,000 and 26.0
Two bands of band C of 00 ± 2,000 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 pH range of 6-9 e. Sugar chain: Shows adsorptivity to Concanavalin A (ConA) Sepharose. f. Physiological activity; suppresses the growth of KB cells, HeLa cells, L-929 cells and does not suppress the growth of IMR-90 cells g. Reactivity with antibodies; anti-TNF antibody, anti-lymphotoxin antibody, anti-interferon β antibody do not neutralize the damaging activity. Further, 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 are a subchain 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 sequence: Val-Val-Asn-Gly-Ile-Pro-T
hr- or Val-Val-Asn-Gly-Ile
-Pro-Thr-X-Thr-Asn-Ile-Gl
y-X-Met-Val-Ser-Leu-where X means unidentified. i. Amino acid composition: Hydrolysis with hydrochloric acid gives the following amino acid composition.

[0005] The amino acid sequence of TCF-II can be determined by the following procedure according to the following procedure.
MR-90), the mRN encoding the TCF-II
After purifying A, the gene was cloned to determine the nucleotide sequence, and deduced from the nucleotide sequence. (1) Poly (A) ⁺ RNA from IMR-90 cells
Extraction of 5% New bone calf serum (NB
CS) added Dulbecco's modifier
IM cultured using ed eagle (DME) medium
From 2 × 10 ⁸ R-90 cells, guanidine thiocyanate-cesium chloride method (Biochemistry)
Total RNA was prepared by 18 5294-5299 (1979)). IMR-90 cells were treated with 6M guanidine thiocyanate, 5mM sodium citrate, 0.
28 ml of 5% Sarkoseal and 0.1 M β-mercaptoethanol solution were added and homogenized. 4ml
5.7M cesium chloride, 0.1M EDTA solution was placed in a polyallomer centrifuge tube, 7 ml of the homogenizing solution was placed thereon, and ultracentrifugation was performed at 35,000 rpm, 20 ° C., 16 ° C. for 16 hours with a Beckman ultracentrifuge 40 Tl rotor. . After centrifugation, the precipitate was washed twice with 95% ethanol, and 200 µl of 10 mM Tris-HCl buffer (pH 7.0).
5) The resultant was dissolved by heating at 65 ° C. for 5 minutes with a 1 mM EDTA solution to obtain a total RNA solution. Poly (A) ⁺ RNA was purified from total RNA by oligo (dT) cellulose column chromatography. The oligo (dT) cellulose column was equilibrated with 10 mM Tris-HCl buffer (pH 7.4), 1 mM EDTA, 0.5 M sodium chloride, 0.05% SDS, and total RN was added.
A, pass the adsorbed fraction to 10 mM Tris-HCl buffer (pH
7.4), eluted with 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 using a DNA synthesis kit (Pharmacia), and an EcoRl adapter was added. The method of preparation was in accordance with the company's protocol, but was modified to add reverse transcriptase (AMV RTase) derived from avian myeloblastosis virus during the synthesis of single-stranded cDNA (40 units / reaction system, Life Science).
e).

(3) Preparation of cDNA library The cDNA obtained in (2) was inserted into EcoRI arm (Promega) of phage vector λgt10. CDNA synthesized from 3.3 μg of poly (A) ⁺ RNA was mixed with 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), and 5.2 μl of this was dissolved in 1 μg of λgt10 Ec
After mixing with oRI arm, it was precipitated with ethanol.
The precipitate was redissolved in 9 μl of a column impact solution, 1 μl of 10 mM adenosine triphosphate, 1 μl of TM DNA ligase (350 units / μl) were added, and the mixture was reacted at 16 ° C. overnight, and the recombinant phage DN of λgt10 and cDNA was added.
A was created.

(4) Screening of cDNA Library (i) Preparation of Oligonucleotide Probe 17-mer complementary oligonucleotide mixture (384 kinds of mix) corresponding to the N-terminal 1st to 6th amino acid sequences of TCF-II β chain were synthesized, T4 polynucleotide kinase (Takara Shuzo), [.gamma. ³² p] ATP (a
5'-end was labeled using Mersham Co., Ltd. and used as a probe. This probe is shown below. Complementary strand used as probe: 3'-CACCACTTAC
CGTAGGG-5 '(384 kinds of mixes) GGGCAAAATTTT (ii) Screening of recombinant phage The recombinant phage DNA solution prepared in (3) was subjected to Gig.
The cells were packed in vitro using an appack Gold (Stratagene), and infected with Escherichia coli C600hfl to obtain about 500,000 phage plaques. After adsorbing the plaque to a Hybond-N filter (Amersham), the filter was denatured with alkali, neutralized, and then baking at 80 ° C. for 2 hours.
did. Hybridization was performed using Bell et al. (Nat
ure 31 775-777 (1984)), and primary screening was performed with the probe prepared in (i). One clone was found to contain the TCF-II cDNA fragment among the plaques that were positive in the primary screening.

(5) Cloning of TCF-II cDNA Containing the Whole Region Translated into Amino Acids N-Terminal Amino Acid Sequence of β Chain of TCF-II and Partially Internal Amino Acids Obtained by Lysyl Endopeptidase Treatment of α and β Chains Array (α) (1 character display) NYMGNLSQT
RSGL and (β) TSXSVYGWGYTGLI
TCF-II was considered to be a member of the hHGF gene family because NYDGLL (X indicates identification) closely matches the amino acid sequence of human hepatocyte growth factor (hHGF). About hHGF, Miyazawa et al. (Bioc
chemicaland Biophysical Re
search Communication 1639
67-973 (1989)), Nakamura (Nature 34 )
2 440-443 (1989)).
Although the nucleotide sequence of A has been reported, the amino acid sequence differs between them at 14 positions, suggesting the existence of the hHGF gene family. Therefore, an oligonucleotide serving as a primer was synthesized based on the nucleotide sequence of the 5′- and 3′-untranslated regions around the polynucleotide chain coding region, which were identical to each other, and the polymerase was synthesized.
TCF-I by in Reaction (PCR) method
A search for IcDNA was performed. First, a DNA synthesizer (A
(Pplied) to synthesize a Sal-77 primer having a recognition sequence for a restriction enzyme SalI and a Sph2203 primer having a recognition sequence for a restriction enzyme Sph1. These primers are shown below.

[0010] Cloning by the PCR method was performed in the following procedure. (I) PCR cDNA synthesized in (2) (dissolved in 150 μl of column buffer) 1 μl 20 μM Sal-77 primer 2.5 μl 20 μM Sph2203 primer 2.5 μl 10 × 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 mixture was mixed with about 1 part of mineral oil (Sigma).
After covering the liquid surface with 00 μl, Quick Therm
PCR by oSystem (Nippon Genetics)
Was done. The reaction conditions are shown below. After pre-treatment at 94 ° C. for 7 minutes, a three-step reaction of 55 ° C. for 3 minutes (annealing reaction), 72 ° C. for 4 minutes (polymerase reaction), and 94 ° C. for 2 minutes (denaturation) was repeated 35 times, and then 55 times as post-treatment. ℃ 3 minutes, 72
The mixture was treated at 11 ° C. for 11 minutes and returned to room temperature ((Note) Each time includes a time during which the temperature changes). When a part of the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 2.3 kilobase (Kb) was obtained.
It was considered CF-II cDNA. Therefore, DNAs obtained from four reaction solutions were precipitated with ethanol, digested with restriction enzymes SalI and SphI, and subjected to agarose gel electrophoresis, and a DNA fragment of about 2.3 Kb was recovered using DE81 paper (Whatman).

(Ii) Subcloning The cDNA fragment of about 2.3 Kb digested with the restriction enzymes SalI and SphI obtained in (i) was converted into a plasmid vector pUC18 (Nippon Gene) with the restriction enzymes SalI and S
ph5 digested with a ligation kit (Takara Shuzo) and inserted into Escherichia coli DH5α (BRL
Was performed (according to the protocol attached to BRL). As a result, more than 20 subclones could be obtained.

(Iii) Determination of base sequence The obtained subclone was subjected to the dideoxy method (Seq
uenaseVer. 2.0 Toyobo). Mistakes in nucleotide incorporation of Ampi Taq (Takara Shuzo) were corrected by analyzing the nucleotide sequences of a plurality of subclones. The nucleotide sequence of the TCF-II cDNA obtained as described above and the amino acid sequence deduced from the sequence are shown in SEQ ID NOS: 1 and 2 in the Sequence Listing, FIG.
5 and 16. There are 2172 base pairs (bp) from the translation start signal ATG to the stop signal TAG, and when translated into amino acids, it consists of 723 amino acid sequences and the signal sequence from the first methionine residue to the 29th alanine residue was predicted. . TCF-II was found to be synthesized as a single polypeptide chain initially, as shown in FIGS. 15 and 16, although two polypeptide chains, an α chain and a β chain, are disulfide bonded. Although the N-terminus of the α-chain of TCF-II is unknown because it is blocked,
The N-terminus of the chain and the amino acid sequences of some of the α and β chains have been determined as described above, and are shown in FIGS. The nucleotide sequence of the obtained TCF-II cDNA was determined by Miyazawa et al. [Biochemical and Biophys].
icalResearch Communicatio
n 163 967-973 (1989)], but the amino acid sequence of Miyazawa et al.'s hHGF is 5 residues (F-L-P) from phenylalanine at position 162 to serine at position 166. -S-
S) differs from the present TCF-II cDNA only in that it is deleted, indicating that the TCF-II cDNA is one of the genes of the new HGF gene family. A comparison between the amino acid sequence of TCF-II proposed from the above nucleotide sequence and the amino acid sequence of hHGF from Miyazawa is as shown in FIG.

[0014] describes a method of obtaining a sugar蛋<br/> white matter TCF-II that will be identified by the physical and chemical properties below. TCF
As the cells used for producing -II, 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. In the practice of the present invention, I
MR-90 (ATCC CCL186), WI-38
(ATCC CCL 75) and the like are particularly suitable. These cells are grown in serum medium or serum-free medium used for normal culture. An example of a typical medium is a medium obtained by adding 5% of calf serum to Dulbecco's modified Eagle medium (DMEM). In addition, if necessary, hormones such as amino acids, transferrin, fatty acids, and insulin may be added.

The cells are cultured in this medium. The culture is performed by static culture using a T-flask or the like, suspension culture using microcarriers, or continuous culture using hollow fibers or ceramic carriers. Can be adopted. The culturing conditions are 20 to 37 ° C. in a CO ₂ 5% air atmosphere.
The temperature and medium are preferably changed every two to three days. After reaching the desired cell density in this way, 7
The medium is changed every 10 days, and the culture solution is collected. The target substance, glycoprotein, is extracted and purified from the collected culture solution. The recovered culture solution was concentrated about 10-fold by UF membrane treatment for cutting the molecular weight of 6,000 or less, and then adsorbed to a cation exchanger.
Elute with buffer. Examples of the ion exchanger include CM Sephadex (manufactured by Pharmacia). Among the eluted active fractions, fractions showing the strongest tumor cell growth inhibitory activity are collected using the tumor cell growth inhibitory activity as an index, and further subjected to sugar affinity chromatography. ConA-Sepharose is particularly suitable as sugar affinity chromatography. Sugar affinity chromatography columns are pH 0.5M NaCl
After equilibration with 7.0 0.05M Tris-HCl buffer,
The collected fraction is loaded, and the column is further washed. Thereafter, elution is carried out with an eluate corresponding to the sugar chain bound to the sugar affinity. When using ConA Sepharose described above,
It is eluted with a buffer containing methyl mannopyranoside.
The eluted active fraction is dialyzed against water and freeze-dried. Thereafter, the resultant is dissolved in a 0.05 M Tris-HCl buffer having a pH of 6.0 to 7.0, and further separated and purified by HPLC using a strong cation exchange resin as a filler. MonoS (manufactured by Pharmacia) is particularly suitable as a column packed with a strong cation exchange resin. Elution from MonoS column
Gradient elution from 0M to 1.0M salt is performed, and the active fraction is collected.

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

Measurement of Tumor Cytotoxic Activity Mouse L929 (ATCC CCL1) was ligated with TCF-I
The clone most sensitive to I was selected. Thus, the tumor cytotoxic factor hypersensitive clone L929-18
I got L 929-18 in DM containing 10% FCS
After culturing until confluent in EM, the cells are detached and collected by trypsin treatment, and suspended in DMEM containing 10% FCS and 1 μg / ml actinomycin D to a cell density of 6 × 10 ⁵ cells / ml. Let it. 96-well microplate (Falcon)
DMEM prepared in the same manner as the cell suspension
A sample containing TCF-II in 0 μl
After dissolving or diluting with M, 50 μl is added to the first well of the dilution row, and after mixing, 50 μl is added to the second well and mixed. Create a dilution series by repeating this operation. Add 50 μl of cell suspension per well to the dilution series of sample and add CO
Incubate at 37 ° C for 2 days in a ₂ incubator. After the culture, the supernatant was gently discarded, washed twice with physiological saline, and a 0.5% crystal violet solution obtained by dissolving viable cells adhered to each well in a solution prepared with methanol: water = 1: 4 was added to each well. And fix by staining. Each well was washed with distilled water, the stained plate was air-dried, and the dye was eluted with Selenson's buffer (6.1 ml, 3.9 ml of 0.1 M disodium citrate, 0.1 N hydrochloric acid, 10 ml of ethanol), The absorbance at 570 nm is measured with a microtiter spectrophotometer. The dilution factor showing 50% cell death rate is defined as the number of units of TCF-II (u / ml).

Measurement of Hepatocyte Proliferation Activity Segren's method (Method in cell bi)
logic, vol. 13, p29, Academic
Press, New York, 1976)
Liver parenchymal cells were isolated from male Wistar rats. The hepatocytes were seeded at a concentration of 8.8 × 10 ⁴ cells / 0.5 ml / well in a 24-well plastic plate (Falcon) and cultured at 37 ° C. in the presence of 5% CO ₂ .
Medium was 10% newborn bovine serum (Hycron), 10 μM
Dexamethasone, 100 U / ml penicillin, 100 u
Williams E medium (Flow Laboratories) containing g / ml streptomycin was used (hereinafter abbreviated as basal medium). After culturing for 24 hours, the medium was replaced with a basal medium containing the test sample, and after culturing for further 24 hours, ³ H-thymidine (Amersham) was replaced with a basal medium containing 4 μCi / ml (86 Ci / mmol), followed by culturing for 2 hours. DNA synthesis was measured. In addition, at the time of the above-mentioned ³ H-thymidine label, each test group was taken in with or without the presence of 10 mM hydroxyurea, and the difference between the counts was taken as the uptake amount. After labeling with the above culture, cells were cooled with cold PBS, 2% perchloric acid and 95%
% Ethanol each, then air-dry and wash twice.
2% S containing mM EDTA, 20 mM NaHCO ₃
It was solubilized with 0.8 ml of DS and measured with a liquid scintillation counter. The results are as shown in Table 1.

[0019]

[Table 1] HEGF as a positive control for hepatocyte proliferation
(Yukinaga Pharmaceutical) was used. The table shows that the hepatocyte proliferation activity of TCF-II is stronger than that of hEGF.

Next, a preparation containing ICF-II as an active ingredient will be described. The bioactive factor in the present invention exhibits the following drug activity. Anti-tumor activity Suppresses the growth of human-derived tumor cells KB, HeLa, MCF-7 and BG-1, and inhibits mouse-derived L-929 cells and tumor cells Sarcoma 180, Meth
A Sarcoma, P388 has cytotoxic activity, but does not suppress the growth of normal human cells, IMR-90. Leukemia cell differentiation-inducing activity Induces differentiation of human leukemia cells HL-60 into granulocytes. Cell-mediated 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. Hepatocyte proliferation activity Promotes the proliferation of rat liver-derived hepatocytes. These activities are expressed in trace amounts in the range of 1-1000 ng / ml.

TCF-II is used as an antitumor agent, an anti-leukemia agent, an agent for enhancing cell immunity, an agent for treating wounds, an agent for treating liver diseases including a liver regeneration agent, and the like. However, TCF-II, which is a high molecular weight glycoprotein, is a substance which is remarkably adsorbed on glass containers such as vials and polypropylene resin containers such as syringes, and is unstable. Its activity is remarkably reduced by temperature, humidity, etc., and easily deactivated. Therefore, it is expected to be formulated in a stable form. That is, in the preparation of the present invention, one or two or more selected from the group consisting of proteins and nonionic surfactants are used as adsorption inhibitors, and one or more selected from the group consisting of proteins, saccharides and amino acids.
One or two or more selected as stabilizers, and a combination of one or two or more selected as adsorption inhibitors and one or two or more selected as stabilizers Good.

The preparation of the present invention may contain the above-mentioned adsorption inhibitor and stabilizer in combination with TCF-II, and the dosage form may be lyophilized, aqueous solution or powder. TCF-II as active ingredient in the present invention
May be produced and purified by any method.
It was extracted from the culture of CF-II producing cells and separated and purified, and mammalian cells such as Escherichia coli, yeast and Chinese hamster ovary cells were produced as hosts by genetic engineering techniques, and extracted and separated and purified by various methods. Things are used. Among the TCF-II adsorption inhibitors used in the present invention, albumins and gelatin are used as proteins, and Tween 8 is used as a nonionic surfactant.
0, Tween 20, etc., among the TCF-II stabilizers used in the present invention, albumin, gelatin and the like as proteins, sorbitol, mannitol, xylitol and the like as sugars, and glycine and alanine as amino acids. Etc. can be used.

[0023] Of these adsorption inhibitors, the amount of protein added is 0.1% or more, preferably 0.1 to 20%.
The amount of the nonionic surfactant added is 0.001% or more, preferably 0.001 to 1.0%. In addition, among the stabilizers, the amount of protein is 0.1% or more, preferably 0.1 to 20.0%, the amount of saccharide is 5 to 40%, and the amount of amino acid is 1% or more. It is preferable to mix them. Even when one or two or more of the adsorption inhibitors are used in combination with one or more of the stabilizers, or when two or more of the stabilizers are used in combination, The amount of the additive may be within the above range. These anti-adsorption agents and stabilizers are used in the form of aqueous solutions having an appropriate concentration and pH in appropriate amounts. The osmotic pressure ratio of this solution is 0.1-3.0, more preferably 1.0. The pH stability range of TCF-II is pH
Since it is 6 to 9, the pH of the aqueous solution at the time of formulation is 6.0 to 6.0.
It is preferable to adjust to 9.0.

Next, the anti-adsorption property and the stability of the preparation of the present invention will be described in more detail with reference to experimental examples.

[Experimental Example 1] Adsorption prevention test IMR-90 cells (A) which are human embryonic lung-derived fibroblasts
TCC. CCL 186) was cultured in a medium containing 5% calf serum for 7 days, and 200 μg of TCF-II separated and purified from the culture supernatant was separated and purified with 0.01 M phosphate buffer (pH 7) containing 0.15 M sodium chloride. (PBS) 100ml
And dispensed 0.5 ml each into a glass test tube and a polypropylene resin tube. Table 2-
(1) A solution having twice the concentration of each of the additive substances shown in (a), (b) and (2) c is adjusted with PBS. To 0.5 ml of the above-dispensed TCF-II solution, 0.5 ml of each concentration of the additive substance is added and mixed well. TCF-
The final concentration of II was adjusted to 1 μg / ml, and the final concentration of each additive was adjusted to the concentrations described in Tables 2- (1) a, b, and (2) c. As a control, 0.5 ml of TCF-II solution was added with P
0.5 ml of BS was added. Two tubes were prepared for each concentration of each additive substance, and after incubating at 37 ° C for 1 hour, TCF-II
The activity was measured, and the result was calculated as the average value.

The activity of TCF-II was measured by its tumor damage activity as follows. Mouse L929 (ATCC,
TCL-I obtained by subcloning CCL1)
Clone L929-18 highly sensitive to I was used as target cells. L929-18 was cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FCS until it became confluent, and the cells were detached by trypsin treatment, and 6 × 10 5 cells were added to DMEM containing 10% FCS and 1 μg / ml actinomycin D. ⁵ cells
Suspend to a cell density of s / ml. The sample sample is diluted using the DMEM used for preparing the cell suspension to make a series of dilution series of 20 times or more. 50 μl of each sample in a series of dilution series of 20 times or more is added to each well of a 96-well microplate (Falcon). Add 5 cell suspensions per well to the dilution column of the sample.
Add 0 μl, and put in a CO ₂ incubator at 37 ° C.
Incubate for 2 days. After the culture, discard the supernatant gently, and add
After washing gently twice, a 0.5% crystal violet solution obtained by dissolving viable cells adhered to each well in a mixture of methanol: water = 1: 4 was added to each well in an amount of 50 pl.
Fix by staining. After washing each well with distilled water and air-drying, the dye was eluted with Selenson's buffer (6.1 ml, 0.1 M disodium citrate, 3.9 ml, 0.1N hydrochloric acid, 10 ml ethanol mixed) and microtiter The absorbance at 570 nm is measured with a spectrophotometer. The dilution ratio indicating the cell killing rate of 50% was calculated as the number of units of TCF-II (u / ml).
The activity (u / ml) immediately after sample preparation is 100%
The residual activity after the test treatment was determined as a relative activity (%).

The results show that, as shown in Tables 2- (1) a, b and (2) c, TCF-II is easily adsorbed on the surface of glass or polypropylene resin. Has an effect of preventing the preparation from being adsorbed.

[0027]

[Table 2]

[0028]

[0029]

[0030]

EXPERIMENTAL EXAMPLE 2 Stability Test The effect of various additives on the stability of TCF-II was tested under conditions that completely prevented TCF-II from adsorbing on the glass wall. That is, TCF-II 12 derived from IMR-90
0 μg of PBS 30m containing 0.02% Tween 80
The solution was sterilized by filtration through a 0.22 μ filter, and then dispensed into sterile glass test tubes in 0.5 ml portions. Table 2-
(1) Various additives shown in a, b and (2) c
After dissolving and preparing a double-concentration solution in PBS, 0.22 μl
Sterilized by filtration with a filter of 0.5m for each
One by one was added to 0.5 ml of the TCF-II solution and mixed well, and then the glass test tube was sealed to prevent bacterial contamination. As a control, 0.5 ml of TCF-II solution and 0.5 ml of PBS without Tween 80 were used. T
The final concentration of CF-II was 2 μg / ml, Tween 80
Is 0.01%, and the final concentration of each additive is shown in Table 3.
The density is as shown in a, b, and c. Two were prepared for each test plot. After storage at 40 ° C. for 1 week, TCF-II activity was measured.
TCF-II activity (μ / ml) before storage was defined as 100%, and activity after storage was shown as relative activity (%). The results were obtained as average values. The results are shown in Table 3. From the results in Tables 3a, 3b and 3c, it can be seen that the stabilizer used in the present invention has an effect of stably maintaining the activity of TCF-II, which is the active ingredient of the preparation, in an aqueous solution.

[0031]

[Table 3]

[0032]

[0033]

Next, the results of testing the pharmacological action of the present invention will be shown. Sarcoma, a new human cytokine TCF-II
In vivo antitumor test material against
And test methods : Experimental animals ICR mice were purchased from Charles River Japan, and female 7-week-old females were used. The tumor cells Sarcoma 180 used were those distributed from the National Cancer Center and subcultured and maintained once a week at our laboratory. Test sample TCF-II was dissolved in 0.01M phosphate buffer, pH 7.0 containing 0.01% Tween, 0.25% human serum albumin and 0.8% saline, and formulated. 0.2
μg TCF-II / 0.2 ml and 1.0 μg T
Two types of TCF-II samples of CF-II / 0.2 ml were prepared. To investigate the effect of pyrogen,
g Sample of a standard pyrogen (manufactured by Difco) equivalent to pyrogen contained in TCF-II (940 pg /
0.2 ml) was prepared similarly. Preliminary toxicity test The preliminary toxicity test was performed on two animals per group. 10 μg of TCF-II in the tail vein of IRO mouse once
And 20 μg / mouse was administered, and toxicity was determined using the death of the animal as an index. Anti-tumor test The anti-tumor test was performed on 7 animals per group. Sarcoma 180 (10 ⁶ / mouse) IC
R mice were implanted subcutaneously, and when engraftment was confirmed, they were divided into groups and 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 relative to the control group.

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

[0036]

[Table 4]

Since the optimal dose of TCF-II was unknown in the above test, there was some problem in setting the dose. In view of the results, it was found that the smaller dose was more effective. Seem.

Further, the antitumor effect of TCF-II by intratumoral administration was examined by the following method. Test Method : 1 × 10 ⁶ ICR mice subcutaneously / mouse Sa
rcoma 180 cells were transplanted, and one week later, mice in which a solid tumor had survived were selected. 1 per day per mouse
0.2 μg of TCF-II was administered for seven consecutive days. Observation for 2 weeks after the completion of the administration revealed that the tumor part had blackened necrosis and showed a remarkable antitumor effect. In addition, mice in which tumors disappeared were also observed.

Hereinafter, the present invention will be described in more detail with reference to Reference Examples and Examples.

Reference Example 1 Preparation of glycoprotein TCF-II (1) Culture of human fibroblast cell line IMR-90 Human fibroblast cell IMR-90 (ATCC CCL 18
6) DMEM100 containing 5% calf serum (CS)
3 × 10 ⁶ cells were transplanted into an 11-volume roller bottle containing ml and cultured for 7 days while rotating at a rotation speed of 0.5 to 2 rotations / min. When the total cell number reached 1 × 10 ⁷ cells, the cells were peeled off with trypsin, the cells were collected on the bottom of the bottle, and a 5 to 9 mesh ceramic 1
00g (made by Toshiba Ceramics Co., Ltd.)
The culture was allowed to stand for a period of time. Then, D containing 5% of the above CS
500 ml of MEM medium was added, and the culture was continued. 7-1
Every day, the entire medium was collected and supplemented with fresh medium. In this way, the production for two months was continued, and 41 culture solutions were collected per roller bottle. The specific activity per culture obtained in this way was 32 u / ml.

(2) Purification of Glycoprotein TCF-II The culture solution 751 obtained in the above (1) was UF-concentrated by a membrane filter (MW 6,000 cut) manufactured by Amicon to reduce the volume to 1/10. Subsequently, CM Sephadex C-50 (manufactured by Pharmacia) was added to 0.0 of pH 7.0.
After equilibration with 5M Tris-HCl buffer, the UF
1.5 kg of resin per wet weight of the concentrated liquid 101 was added, and the solution was stirred for 4 hours with gentle stirring at pH 6.5 to 7.0.
Adsorbed at 24 ° C. for 24 hours. After adsorption, the resin is converted to Whatman N
o. The resin collected by filtration in step 2 was washed with 0.05 Tris-HCl buffer (pH 7). About 1500 g of the washed resin was packed in a column having a diameter of 7 cm × 40 cm,
Eluted with 0.05 M Tris-HCl buffer pH 7.0 containing 20% Tween 20 and 0.3 M NaCl. The absorption at 280 nm was monitored, and when the protein was almost completely eluted, the salt was further eluted with a salt strength of 0.6 M salt. Each fraction measures the tumor cytotoxic activity and the IM
The tissue plasminogen activator (t-PA) activity produced by R-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 was designated as TCF-II fraction.

Then, ConA-Sepharose CL-6
B (manufactured by Pharmacia) at pH 7.
Equilibrate with 0, 0.05M Tris-HCl buffer, size 2.
A 5 cm × 8 cm column was packed. The column was further washed with the same buffer, and loaded with a TCF-II fraction (pH 7.0) eluted with a CM-Sephadex column. Thereafter, the column was washed again with 10 M column volume of 0.5 M NaCl containing pH 7.0, 0.05 M Tris-HCl buffer, and then 0.5 M NaCl and 0.3 M α-methyl-D-mannopyram. Nocide-containing pH 7.0, 0.05
Elution was carried out with M Tris-HCl buffer at a flow rate of 70 ml per hour. Each eluted fraction was measured for tumor cytotoxic activity and monitored for protein absorption at 280 nm. The elution pattern shown in FIG. 2 was shown.

The fraction eluted first was collected, dialyzed against distilled water at 4 ° C. for 48 hours, and then freeze-dried to obtain a white powder. Use this powder in a minimum amount of 0.01%
Dissolve with 0.05M Tris-HCl buffer containing Tween 20 at pH 7.0, and add 0.01% Tween 20
HPLC equilibrated with 7.0 0.01M phosphate buffer
MonoS column (manufactured by Pharmacia). After loading, 0.01% Tween 20-containing pH 7.0,
After washing with a 0.01 M phosphate buffer for 20 minutes at a flow rate of 0.5 ml / min, for 60 minutes at a flow rate of 0.5 ml / min,
Elution was performed with a concentration gradient such that the final salt concentration was 1 M of sodium chloride. The elution pattern is shown in FIG. The active fraction is 0.7
It was eluted with 6M salt as the top. Collecting the active fraction,
The sample was again loaded on a MonoS column, and eluted again with the same buffer in a concentration gradient up to 1.0 M salt concentration.

The active fraction was collected, and then,
Heparin-Sepharose (manufactured by Pharmacia) packed in 5 ml of a × 7 cm column was filled in with pM containing 0.3 M salt.
H7.5, equilibrated with 10 mM Tris-HCl buffer, the active fraction was diluted on this column with 0.01 M Tris-HCl buffer (pH 7.0) to a salt concentration of 0.3 M, and the above heparin Loaded on Sepharose column. Thereafter, the resultant was washed with a 0.01 M Tris-HCl buffer containing 0.3 M sodium chloride at pH 7.5, which was 10 times the amount of the packed gel. Further, elution was performed with a buffer having the same pH at a flow rate of 20 ml per hour by a salt concentration gradient from 0.3 M to 2.0 M. The elution pattern is shown in FIG.

Thus, TCF-II was obtained. Table 5
As shown in FIG.
mg of active protein could be obtained. At the same time, this glycoprotein is a tumor cytotoxic factor and has a specific activity of 5.2
48,000 u / 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 a polyacrylamide gel containing 0.1% SDS. The glycoprotein is 78,0
00 and 74,000 adjacent bands were shown. Further, a reduction treatment was performed with 2-mercaptoethanol, and electrophoresis was performed in the same manner.
Three bands of 8,000 and 32,000 were obtained (see FIG. 5). From this, TCF-II has a molecular weight of 5
2,000 common subunits with a molecular weight of 32,000
Is predicted to be a complex in which subunits having a molecular weight of 28,000 or subunits having a molecular weight of 28,000 are bound. Isoelectric point Using an LKB isoelectric point electrophoresis apparatus, Phase Gel
When the isoelectric point was measured by IEF3-9, 7.4 was obtained.
It showed an isoelectric point of ~ 8.55.

Thermostability [0047] TCF-II dissolved at 51,200 u / ml activity was added to 0.1 M Tris-HCl buffer containing 0.01% Tween 20 adjusted to pH 7.5, and a 512 u / ml solution was added. Was prepared. 25, 35, 5
The cells were treated at 0, 60, 70, 80, 90 and 95 ° C. for 10 minutes, and the relative activity to the activity at 25 ° C. was measured.
As shown in FIG. 6, it was thermally stable up to 60 ° C. pH stability Each buffer having the composition shown in Table 6 (each containing 0.01% Tween 20) was purified, and TCF-II corresponding to 51,200 u / ml at the time of pH 8 adjustment was added to each pH buffer. Dissolve and measure the activity after standing at 37 ° C for 1 hour.
8. The relative activity was compared with that when left at room temperature for 1 hour. As shown in FIG. 7, it was stable in the pH range of 6 to 9.

[0048]

[Table 6]

N-terminal amino acid sequence 50 μg of TCF-II was reduced, separated into 3 proteins by electroplotting: A having a molecular weight of 52,000, B having a molecular weight of 22,000, and C having a molecular weight of 28,000. The N-terminal amino acid sequence was analyzed using a 477A type protein sequencer. In A, the N-terminal amino acid sequence could not be measured because the N-terminal was blocked, but B and C both showed the following common N-terminal amino acid sequence. X indicates unidentified. Since the N-terminal amino acid sequences of B and C are completely identical, TCF-II has a molecular weight of 5
It is considered that 2,000 A and B having a molecular weight of 32,000 or C having a molecular weight of 28,000 have a double-stranded structure in which they are bonded by an SS bond. Furthermore, the present invention has revealed that the amino acid sequence at this point is as follows. The cDNA was as shown in SEQ ID NO: 2 in the Sequence Listing, and the amino acids deduced therefrom were as shown in SEQ ID NO: 1 in the Sequence Listing (see FIGS. 15 and 16).

Amino acid composition An amount corresponding to a protein amount of 10 μg measured using a protein assay kit manufactured by Bio-Rad was decomposed by hydrolysis with hydrochloric acid, and the amino acid composition was measured using an L-8500 amino acid analyzer manufactured by Hitachi. The following amino acid composition was obtained.

[0051]

[Test Example 1] This example shows that the glycoprotein TCF-
2 shows tumor cytotoxic activity of II. HeLa is a tumor cytostatic tumor cell lines, KB, to produce a cell suspension adjusted to a cell density of ¹⁰ 5 / ml in IMR-90 containing 10% FCS DMEM respectively are human diploid cells. 50 in microwell plate (Falcon)
Each cell was seeded in μl. 5,120u in each well
/ Ml of TCF-II from the above medium by 10,20,4
50 μl each of a dilution of 0,80,160 was added, mixed, and cultured in a CO ₂ incubator at 37 ° C. for 3 days. The cells surviving in each well were replaced with methanol: water =
50 μl of a 0.5% crystal violet solution dissolved in a 1: 4 solution was added to each well and stained and fixed. After washing each well with distilled water, the plate was air-dried, the dye was eluted with Seleson 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 group without TCF-II 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 suppression, but KB and HeLa cells showed strong suppression.

Reaction of Existing Substance with Antibody TCF-II was added to DMEM containing 10% FCS at 320 u /
Dissolution was adjusted to a concentration of ml. On the other hand, an anti-LT antibody was added to the same culture solution so as to have a titer of neutralizing 1000 u / ml of LT, and left at 37 ° C. for 1 hour to react. Similarly, anti-TNF antibody was added to 1 × 10 ⁶ u / m
1) An anti-INF β antibody was added and reacted at 1000 u / ml. In addition, each antibody used the commercially available thing. After the reaction, the neutralizing effect of each antibody was determined using TCF-II.
The activity was confirmed by measurement, but no neutralizing effect of the activity was observed.

[0053]

Test Example 2 This example shows the glycoprotein TCF-I obtained in Reference Example 1.
1 shows the cytotoxic activity of I on various tumor cells derived from mice. Sarcoma18 is a mouse-derived tumor cell line.
0, 3 of MethA Sarcoma and P-388
The strain was used. Sarcoma 180 cells in DMEM containing 10% fetal calf serum and MethA and P388 in RPMI 1640 medium containing 10% fetal calf serum.
Each was suspended at 2 × 10 ⁴ cells / ml to prepare respective cell suspensions. Each cell suspension of a 96-well flat bottom microwell plate (manufactured by Falcon) was seeded in an amount of 50 μl. TCF-II is Sarcoma
The TCF-180 was dissolved and diluted in DMEM containing 10% fetal calf serum for 180 and RPMI1640 medium containing 10% fetal calf serum for MethA and P-388.
A II solution was prepared. 50 μl of the TCF-II solution was added to each well inoculated with each cell suspension,
When the final concentration of CF-II is 0, 2, 4, 8, 16, 31,
It was adjusted to be 62, 125, 250, 500, 1000 ng / ml. After mixing, the cells were cultured in a CO ₂ incubator at 37 ° C. for 3 days. Cells in each well were stained with trypan blue, only viable cells were counted using a hemocytometer, and the average of two experimental values was determined. The cytotoxic activity (%) of each cell was calculated by the following formula using the group without TCF-II as a control, and the relationship with the TCF-II concentration was determined.

[0054]

(Equation 1)

As a result, Sar of the obtained TCF-II
FIG. 9 shows the cytotoxic activity against coma180.
That for eth A Sarcoma and P-388 is shown in FIG. All cells were TCF-II
Are highly sensitive to Sarcom by TCF-II.
a180, Meth A Sarcoma and P-3
The cytotoxic activity IC ₅₀ against 88 was 6, ₄₀ , respectively.
And 460 ng / ml.

[0056]

[Test Example 3] This example relates to the glycoprotein TC obtained in Reference Example 1.
FIG. 9 shows the growth inhibitory effect of F-II human tumor cell line on ovarian cancer cell line BG-1 and breast cancer cell line MCF-7. BG-1 was added to McCoy's medium containing 10% FCS,
F-7 is 2 × 10 in Eagle MEM containing 10% FCS, non-essential amino acid mixture, pyruvic acid and Eagle salt.
Each cell suspension was prepared to be ⁴ / ml. On the other hand, TCF-II is 10% FC for BG-1.
Dissolved in McCoy's medium containing S and 4 μg / ml TC
An F-II solution was prepared and sequentially diluted 2-fold with the same medium to prepare a serial dilution series of TCF-II. Similarly, M
For CF-7, use the TCF-growth medium described above for MCF-7.
A serial dilution series of II was created. 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. It carried out similarly about MCF-7. 37 in a CO ₂ incubator
C. for 5 days. After the culture, the culture solution was removed, the microwell plate was washed twice with PBS, and a 0.5% crystal violet solution obtained by dissolving the cells attached to each well in a mixture of methanol: water = 1: 4 was added to each well. 50 μl was added to each well to fix the stain. After washing each well with distilled water, the plate was air-dried and the dye was eluted with Selenson's buffer and 570 in a microtiter spectrophotometer.
The absorbance in nm was measured. TCF-II for each cell
Using the non-added group as a control, the cell growth inhibition rate was calculated based on the following formula, and the relationship with the TCF-II concentration was determined. The results were as shown in FIG. As a result, BG-1, MCF
-7 It was confirmed that the growth was suppressed by TCF-II in both tumor lines.

[0057]

(Equation 2)

[0058]

[Test Example 4] This example relates to the glycoprotein TC obtained in Reference Example 1.
The activity of F-II for inducing differentiation of a promyelocytic leukemia strain, HL-60, is shown. HL-60 cells were suspended in RPMI1640 medium containing 10% fetal calf serum at a concentration of 3.5 × 10 ⁵ / ml to prepare a cell suspension. 100 μl of the cell suspension was dispensed into each well of a 96-well flat bottom microtiter plate (Falcon). Then, 100 μl of the TCF-II solution dissolved and diluted in the same medium was used to give a final concentration of 15.
6,62.5,125,250,500 and 1000
ng / ml. After culturing at 37 ° C. for 3 and 7 days, the activity of inducing HL-60 differentiation by TCF-II was measured by nitroblue tetrazolium (NBT) reducing ability. The morphological changes were also examined.

1) NBT reducing ability Table 7 shows the NBT reducing ability.

[Table 7]

The numerical values in the table indicate the percentage of cells in which at least 200 or more cells were counted, in which NBT was reduced and blue-black formazan was contained. (Indicates the average of two experiments). (HL-60 acquires NBT reducing ability when differentiated into normal cells and accumulates blue-black formazan in the cells.) As a result, TCF-II induces differentiation of a promyelocytic leukemia strain, HL-60, at 250 ng / hr. ml showed the highest differentiation-inducing activity.

2) Morphological change It is known that HL-60 exhibits two types of differentiation of a macrophage system and a monocytic system depending on the difference of the differentiation inducer. As a result of examining the morphology or nuclear change of the cells differentiated by TCF-II after culturing at 37 ° C. for 7 days by light Giemsa staining, it was found that TCF-II induced differentiation of HL-60 into monocytes.

[0062]

[Test Example 5] Glycoprotein TCF-II obtained in Reference Example 1
1 shows the cell immunity activity. That is, a lymphocyte mixed culture test was performed under the conditions of TCF-II addition, and the activity of TCF-II on lymphocyte blastogenesis was examined. Lymphocytes were separated from human peripheral blood by the Ficall-Conray method and suspended in RPMI 1640-10% FCS medium. Lymphocytes from two individuals were combined in a 1: 1 ratio for a total of 1 ×
10 ^5/100 [mu] l / well and was added to a round bottom microplate so, the addition of TCF-II in various concentrations, RPMI-10% FC under CO ₂ incubator
The cells were cultured in the S medium. The ³ H-thymidine for 16 hours prior to the completion of the culture was added 0.25MyuCi / well. After completion of the culture, the cells were collected with 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 FIG. 12 and FIG. FIG.
As shown in FIG. 2, 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 group was added to the group added with a final concentration of 100 ng / ml TCF-II. Comparative significantly ³ increase in ^H uptake was observed in, TCF-II site toxic T cell proliferation and,
That is, it was confirmed that it had an effect of enhancing cellular immunity.

[0064]

Test Example 6 Glycoprotein TCF-II obtained in Reference Example 1
1 shows the vascular endothelial cell proliferation activity of the present invention. Human umbilical cord-derived vascular endothelial cells, HUVEC, were used as test cells. E-G containing human vascular endothelial cells HUVEC containing 2% fetal bovine serum
It was suspended in M medium at a concentration of 2.5 × 10 ⁴ / ml.
The above cell suspension was dispensed at 50 μl into each well of a 96-well flat bottom microwell plate (manufactured by Falcon). TCF-II was dissolved in E-GM medium containing 2% fetal bovine serum, and 50 μl of the solution was added to each well containing the cell suspension, so that the final concentration of TCF-II was 0,4,8,
16, 31, 62, 125, 250, 500 and 10
It was prepared to be 00 ng / ml. 37 ° C, CO ₂
The cells were cultured in an incubator for 6 days. The number of cells in each well was determined by removing the medium in each well, washing with PBS, removing the cells by trypsin treatment, and counting the number of viable cells using a hemocytometer.

FIG. 14 shows the effect of the obtained TCF-II on normal human vascular endothelial cells HUVEC. It was confirmed that TCF-II did not show cytotoxic activity on vascular endothelial cells, which are normal cells, but had growth promoting activity. In particular, a TCF-II concentration of 125 n
The growth promoting activity was maximum at g / ml.

This shows the formulation of the preparation according to the present invention.

Example 1 TCF-II 20 μg human serum albumin 100 mg The above composition was converted to a 0.01 M phosphate buffer (PB) having a pH of 7.0.
After dissolving in S), the total amount was adjusted to 20 ml, and sterilized, 2 ml was dispensed into vials, lyophilized and sealed. Note that T
CF-II was obtained in Reference Example 1 (T
The same applies to CF-II) .

[0067]

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

[0068]

Example 3 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, dispensed in vials 2 ml at a time, freeze-dried and sealed.

[0069]

Example 4 TCF-II 40 μg Tween 80 2 mg Glycine 2 g The above composition was dissolved in physiological saline for injection, the whole amount was adjusted to 20 ml, sterilized by filtration, dispensed into vials 2 ml at a time, freeze-dried and sealed.

[0070]

Example 5 TCF-II 40 μg Tween 80 1 mg Sorbitol 2 g Glycine 1 g The above composition was dissolved in saline for injection, the total amount was adjusted to 20 ml, sterilized by filtration, dispensed into vials 2 ml at a time, freeze-dried and sealed. did.

[0071]

Example 6 TCF-II 20 μg Sorbitol 4 g Human serum albumin 50 mg The above composition was dissolved in PBS, the total amount was adjusted to 20 ml, sterilized by filtration, dispensed into vials 2 ml at a time, freeze-dried and sealed.

[0072]

Example 7 TCF-II 40 μg Glycine 2 g Human serum albumin 50 mg The above composition was dissolved in physiological saline for injection, the whole volume was adjusted to 20 ml, sterilized by filtration, dispensed into vials, freeze-dried and sealed. Ltd. agent obtained in Examples 1 to 7, anti-tumor agents by intravenous or intramuscular injection, antileukemic agents,
It can be used as a medicine such as a cell immunity enhancer and a liver regeneration promoter.

[0073]

Industrial Applicability The present invention is based on the use of TCF-II for tumor cell damage, leukemia differentiation induction, cell immunity activation, vascular endothelial cell proliferation, etc. , Anti-tumor agent, anti-leukemia agent,
New drugs such as a vesicle immunity enhancer and a liver regeneration promoter can be provided. TCF-II is also used as a biochemical or pharmacological reagent.

[0074]

[Sequence list]

[0075]

[Brief description of the drawings]

FIG. 1: Protein, plasminogen activator and T obtained from CM-Sephadex C-50 chromatography of an IMR-90 culture containing 5% calf serum
3 shows the elution profile of CF-II. (1) is 0.
0.05M Tris-HCl buffer containing 3M NaCl (p
H7.0), (2) 0.6M NaC
The fractions eluted with 1M 0.05M Tris-HCl buffer (pH 7.0) are shown.

FIG. 2: CM-Sephadex C of IMR-90 culture solution
0.6M Na obtained from -50 chromatography
The result of ConA affinity chromatography of a fraction containing 0.05 M Tris-HCl buffer (pH 7.0) containing Cl was eluted. (1) is a 0.5M NaCl content 0.0
The washed fraction with a 5 M Tris-HCl buffer (pH 7.0) was used, and (2) was a 0.05 M Tris-HCl buffer (pH 7.0) containing 0.5 M NaCl and 0.3 M α-methyl-D-mannopyranoside. ) Shows the eluted fraction.

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

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

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

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

FIG. 7 shows the pH stability of TCF-II.

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

FIG. 9 shows the cytotoxic activity of TCF-II on Sarcoma180.

FIG. 10 shows the cytotoxic activity of TCF-II on 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. Each sample was measured for 6 samples, and is shown as an average value ± SD.

FIG. 13 shows the radioactivity concentration of ³ H-thymidine incorporated into lymphocytes on day 8 of culture. Each sample was measured for 6 samples, and is shown as an average value ± SD.

FIG. 14 shows the proliferative activity of TCF-II on vascular endothelial cells 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 continues from FIG. 15 and a series of T
1 shows the base sequence of CF-II cDNA and the amino acid sequence deduced therefrom. Amino acids are indicated by single letter code.

FIG. 17: T deduced from the nucleotide sequences of FIGS. 15 and 16
2 shows a comparison of the amino acid sequence of CF-II with the amino acid sequence of hHGF of Miyazawa et al. Amino acids are indicated by single letter code.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C07K 14/47 A61K 37/00 ADV C12N 15/09 ZNA C12N 15/00 ZNAA (56) References Biochemical and Biophysical Research Communications, Vol. 163, no. 2 (Septmber 15, 1989) 967-973

Claims (5)

(57) [Claims] 1. A glycoprotein having the following properties (hereinafter referred to as TC
F-II) as an active ingredient. Obtained from the culture supernatant of human-derived fibroblasts , the following a. ~ H. A glycoprotein having the properties of: a. Molecular weight; measured by SDS polyacrylamide gel electrophoresis, 78,000 ± 2.0 for non-reduced
A common band A of 52,000 ± 2,000 with a molecular weight of 00 or 74,000 ± 2,000 and reduced
And band B of 30,000 ± 2,000 and 26.0
Two bands of band C of 00 ± 2,000 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 pH range of 6-9 e. Sugar chain: Shows adsorptivity to Concanavalin A (ConA) Sepharose. f. Physiological activity; suppresses the growth of KB cells, HeLa cells, and L-929 cells, and does not suppress the growth 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 constitute a subchain of band A, and band A has an N-terminal amino acid blocked. Subchains B and C both have the following N-terminal amino acid sequence: Val-Val-Asn-Gly-Ile-Pro-T
hr- or Val-Val-Asn-Gly-Ile
-Pro-Thr-X-Thr-Asn-Ile-Gl
y-X-Met-Val-Ser-Leu-where X means unidentified. 2. An anti-leukemia containing TCF-II as an active ingredient.
Agent . 3. A fine 胞免疫増strong agent comprising as an active ingredient the TCF-II. 4. A wound treatment agent comprising TCF-II as an active ingredient. 5. A regeneration promoter comprising TCF-II as an active ingredient.