JPS6388199A - Human differentiation inducing factor k-buf - Google Patents

Abstract

NEW MATERIAL:A polypeptide having activity of differentiating and maturing mouse leukemic cells to normal cells as well as the following physico-chemical properties. Molecular weight; 16+ or -1kd [measured by sodium dodeyl sulfate (SDS)-electrophoresis in the presence of 1.0% mercaptoethanol] and 25+ or -1kd (measured by the SDS-electrophoresis in the absence of mercaptoethanol). Amino acid sequence at the amino terminal; Expressed by the formula (X is amino acid which could not be identified). USE:A remedy for leukemia and anemia. PREPARATION:For example, human chronic myelomatous leukemic cells are cultivated in the coexistence of a differentiation inducer, e.g. actinomycin D, etc., in a culture medium and the resultant culture fluid is then concentrated by an ultrafiltration method, etc., to salt out proteins. Contaminating proteins are removed by dialysis and then ion exchange chromatography using an anion exchanger, etc. Purification is further carried out by reversed phase high-speed liquid chromatography to afford the aimed K-BUF.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a polypeptide having physiological activity, and more particularly to a polypeptide having an effect of causing differentiation and maturation of tumor cells or promoting erythroblast production. . That is, in the polypeptide of the present invention, -BUF is a factor that has an effect of differentiating and maturing human white-face disease cells into normal cells, that is, a shedding effect, and also has an effect of promoting erythroblast production, 1, different from those derived from animal cells or microorganisms,
Since it is produced by human cells, it has the potential to be used as a therapeutic agent for human leukemia and anemia.

(Prior Art) For the purpose of cancer treatment, research on decancer agents, that is, differentiation inducers, which convert cancer cells into normal cells, is being actively pursued internationally. Mousefriend leukemia cells and human monocytic white deer disease cells are treated with drugs and biological factors, respectively.
It is known that the cells differentiate and mature into macrophages, granulocytoid cells, or erythrocyte-like cells and lose their cancerous properties, ie, their transplantability and proliferative properties.

The above drugs include polar compounds such as DMSO, lipopolysaccharides, immunostimulants, antibiotics such as bleomycin and actinomycin D, and vitamins TPA (12-0-
tetradecanoylphorbol-13-i
Phorbol esters such as cetate), proteins such as arginase, and histones are known. On the other hand, biological factors include the molecule JIT 40,000-50,00 (1) glycoprotein produced by mouse fetal cells (MI cells), factor D (Ship and Chemotherapy, 9,105-114
゜1982) is well known. On the other hand, when human mononuclear leukocytes are stimulated with mitodane such as concanavalin A, the molecular weight of 125,000 ft and 40,000 ft of C8F.
(leukocyte-inducing factor) and factor D are produced, and it has been revealed that factor D differentiates human acute promyelocytic leukemia cells (HL-60) into macrophage-like cells. Similarly, a factor has been announced that induces the differentiation of human myeloid leukemia, monocytic leukemia cells, etc. when human mononuclear leukocytes are stimulated with mitogens such as lipopolysaccharide (Japanese Cancer Society General Meeting Article, 43rd General Meeting tiger 639,
P 190 (1984) and JP-A-60-28934
Publication No.). However, there is no known human-derived differentiation inducer that induces the differentiation of Friend's disease cells, which is generally well known.

On the other hand, the drugs used to treat anemia vary depending on the cause of the anemia, but iron supplements are generally used for iron deficiency anemia.

For pernicious anemia, vitamin B, 29 folic acid, and aplastic.

Corticosteroids such as corticoids are used for hemolytic anemia. Of these, steroid hormones have been confirmed to have a hematopoiesis-promoting effect and are effective therapeutic agents, but the main problem is their strong side effects, and long-term administration is said to be problematic.

Ru. Erythropoietin is a glycoprotein with a molecular length of 45,000 that exists in the n1α-globulin fraction produced in the kidney, and is a humoral regulatory factor that acts on hematopoietic stem cells to promote differentiation into erythroblast cells and erythroblast production. It is expected to be a new anemia treatment.

However, since the raw material is human urine and the amount of raw material is very small, it is difficult to supply it in large quantities. On the other hand, production by genetic recombination is also being studied, but because it is a glycoprotein, it has not been put to practical use.

(Problems to be Solved by the Invention) Therefore, the aim of the present invention is to use various human cells to discover new tumor cell differentiation factors, as well as effective factors useful in the treatment of anemia, and to clarify their chemical entities. There is something to do.

(Means for Solving the Problems) In order to solve the above problems, the present inventors investigated the productivity of differentiation factors in various human cells. discovered that -BUF is produced as a human differentiation-inducing factor that causes murine leukemia cells to differentiate and mature into normal cells by culturing or inducing them in the culture medium. We were able to clarify the characteristics of

Furthermore, the present inventors discovered that the -BUF produced in this manner has an effect of improving anemia, leading to the completion of the present invention. The present invention will be explained below.

The human leukemia cells referred to in the present invention include established strains derived from human leukemia or human bone marrow cells that have been artificially made malignant, and more specifically include the following.

Human histiocytic lymphoma cells (U-937ATCCCRL
1593. Int, J, Cancer 17:565
(1976) ), human chronic myeloid leukemia cells (K5
62゜Blood 45:321 (1975)), human monocytic leukemia cells (J-111.Blood 10:1
010 (1955)), human acute monocytic leukemia cells (
THP-1. Int, J Cancer 26:171-
176 (1980)). Specific differentiation-inducing substances are substances that have the effect of producing K-BUF when brought into contact with human leukemia cells, and specifically include actinomycin D, mitomycin C, concanavalin A, and phorbol ester (TPA). is a specific differentiation-inducing substance.

The method of producing K-BUF of the present invention is carried out by culturing human leukemia cells in the coexistence of at least one or more of the above-mentioned specific differentiation-inducing substances, and
F is produced in the culture medium (extracellularly).

As the medium for culturing the human leukemia cells of the present invention, a usual medium for culturing animal cells can be used. For example, Rosewell Noyak Memorial Institute)
1640 medium (Roswell park Memory
al In5tituFe 1640, hereinafter referred to as RPMI-
It is abbreviated as 1640. ) is preferred. Others Dulbetsko's modified Eagle's basal medium (Eagle's Fviinlm)
um FJs++ entia1M@dium), click medium (C11ck Medium), etc. are also used. Usually, these media contain fetal bovine serum (hereinafter referred to as FB).
Abbreviated as S. ), neonatal bovine decomposition, or horse decomposition may be added, but in the case of the present invention, good results can be obtained even without the addition. Human leukemia cells are usually cultured at a cell density of 1 to 5 x 106 cells/d at 35 to 38°C in a stream of 4 to 6% carbon dioxide gas while standing still or with gentle stirring. A specific differentiation-inducing substance may be added to the medium at the beginning of normal culture, or may be added during the culture. The amount added varies depending on the type of differentiation-inducing substance, but in the case of actinomycin D, mitomycin C, etc., it is 0.1 to 10 μg/me, and in the case of TPA, it is 1
~500 ng/rnl. In this way 1-5
BUF accumulates in the culture solution over days of culture.

'The activity of K-BUF was detected in mouse Fr1end virus-induced Shiroka disease cells F5-5 (Bib1.Haemat,
, 43.37 (1976)) using the known method (Pr.
oc, Natle, Acad, Sci, , 71.9
8 (1975)). In addition, to display the activity, the value of the reciprocal of the dilution rate of the sample stock solution where F5-5 cell differentiation is clearly confirmed is the stock solution 1.0! III "5" activity.

When -BUF was produced by the method of this invention, the culture solution was
It shows an activity of 1000 units/rnl.

Purification of K-BUF is carried out according to the usual protein purification method. For example, a crude protein sample can be obtained by concentrating a culture solution by ultrafiltration, salting out proteins from this concentrated solution, and performing ion exchange chromatography using a dialysis-extracted ion exchanger. Most of the contaminant proteins are removed from this crude sample by hydrophobic chromatography or the chromatography method. Also, if you combine these two, even more KPM
Magnification can be improved. The prepared specimens were analyzed using reverse phase high performance liquid chromatography or FPLC (Fast Prot manufactured by Pharmacia) equipped with a Superrose or Mono Q HR5//'') column.
ein Pept-1ds Polynucl+ot
lde Liquld Chromatography
fh D'J can be obtained by performing high performance rA/filtration methods or ion exchange chromatography using a ) system.

Ni-BUF prepared in mM in this manner has the following physicochemical properties.

(1) Molecule 5: 16±lkd (SDB polyacrylamide gel turtle electrophoresis in the presence of 1.0% mercaptoethanol) 25±lkd (lower right SDS in the absence of mercaptoethanol)
Electrophoresis method) (2) etc.: pras ± 0.5 (chromatographic focusing method) pH 7,3 ± 0.5 (isoelectric focusing method) (3) pH stability: pF (2,0 Stable in the range of ~10.0 (4) Thermal stability: Stable when heated at 65°C for 960 minutes (
5) Organic solvent stability: Stable against lower alcohols and acetonitrile (6) Protease resistance: Completely inactivated by pronase treatment (7) Specific activity: 2X10U/π-riprotein (8) Amino terminal amino acid sequence Gly-Lsu- Glu-X-Asp-Gly-Lys
-Val-Asn-11eX is an unidentified amino acid (action) In the present invention -BUF has the effect of differentiating and maturing Mouse Friend leukemia cells into normal cells, and also has the effect of promoting erythroblast production. .

That is, when K-BUF is administered to anemic mice, the anemia of the mice is improved. When Mouse Friend virus leukemia cells are transplanted into mice, the mice's hematocrit value (a number of 11 that represents the red blood cell volume percentage and more accurately indicates the degree of anemia than the number of red blood cells) gradually decreases, and the mouse's hematocrit value (a number of 11, which represents the red blood cell volume ratio and more accurately represents the degree of anemia than the number of red blood cells) decreases, and the mouse's hematocrit value decreases from 1 to 1.
The patient becomes anemic within 2 weeks. Compared to these controls, when -BUF was administered intravenously after transplantation, the hemadcrit value of the mice hardly decreased, and a clearly significant difference was observed in the 21-day-old mice. Furthermore, when -BUF is administered to mice exhibiting poor facial appearance one week after transplantation, the decrease in hematocrit value is suppressed, and an increase in the hematocrit value is observed 2 or 3 days later, indicating a clear therapeutic effect on hematocrit.

The therapeutic agent of the present invention is effective in preventing and treating anemia caused by decreased red blood cell production, is not toxic to cultured mouse and human cells, and is effective in preventing and treating anemia in humans.

The anemia therapeutic agent of the present invention is mainly administered parenterally (intramuscularly, subcutaneously, intravenously). The dosage of the active ingredient -BUF varies depending on the symptoms, but is usually 0.051n9 per adult.
Dose ranges of ~25 m9 are generally divided into several doses, so the typical daily dose is 0.1 to 50 m9. The dosage will vary depending on the degree of anemia, the patient's weight, and other factors recognized by those skilled in the art.

BUF for use in the present invention is formulated by a conventional method, and is mainly used as an injection, but can also be formulated into capsules, tablets, and other dosage forms. When preparing injections, it is necessary to add a pH adjusting agent to the herbal medicine.
Add a buffer, stabilizer, preservative, etc. and make it into a subcutaneous, intramuscular, or intravenous injection by a conventional method. In addition, when preparing oral preparations, excipients and, if necessary, binders, disintegrants, coloring agents, etc. are added to Ni-BUF, and tablets are prepared by a conventional method.
Use as capsules, etc.

Example 1 I x 106 -562 to human chronic myeloid Yamako disease cells
RPM containing 5x FBS at a cell concentration of cells/ml.
I-1640 medium or Dulbecco's modified Eagle's basal medium containing 5% FBS (suspended and placed in a Falcon flat-bottomed 24-well microtiter plate for 3 hours).
Cultured at 7°C. At this time, from the start of culture, TPA 'i
Add sea urchin to a concentration of 10 ng/ml or 100 n, 97 mt. After 91 days, 2 days, 3 days, 4 days, and 5 days after the start of culture, collect the culture fluid and centrifuge it to obtain each culture supernatant. The differentiation-inducing activity of dll against F5-5 cells was determined. The results are shown in Table 1.

Table 1: Human Chronic Myeloid Leukemia Cells - BUF Productivity TPA Concentration Number of Culture Days Activity As shown in the table, strong activity was observed at all TPA i levels between 3 and 5 days in culture. .

Next, the effects of various differentiation-inducing substances were studied using K-562 cells according to the above method. The offspring are shown in Table 2.

Table 2 Effect of differentiation inducer Actinomycin D 5μg/ml
3 4 Might Mine-C1ol
3 2ConA
251 3 4LPS*5
0 t 3 0TPA
100n9/ml 3
64* LPS derived from canine coliform bacteria (E, coll) Example 2 RPMI-1640 sterile medium with 5% tallow serum 5
, OL was put into a 2O4g spinner flask, and Ni-562 cells obtained by culturing in this medium according to the method of Example 1 were suspended to a density of 4 x 105 cells/ml. This was cultured at 37°C for 4 days, and the resulting culture solution was centrifuged to collect 1562 cells aseptically.

5. The cells were placed in a separate spinner flask in the above RPMI-1640 medium without serum. Gun to OL and TPA'? : Add 10 o9/ml and stir the solution gently (10-Or, p, m) at 37°.
Culture (induction) was performed for 2 hours at C. The culture solution thus obtained was centrifuged to separate and remove the cells.
A culture medium with an activity of 1/G was obtained. Ammonium sulfate was added to 100"t of the culture solution thus obtained (70% saturation), and the resulting precipitate was centrifuged at 19 t (10,00 rpm,
10 minutes) and a small amount of 0.05 M IJ
After dissolving in HCl buffer 'H(p) (7,8), it was thoroughly dialyzed against the buffer. DEAE-Toyopur #650M (2,5
x30 cm). Add this column to the same buffer solution 5.
．． After washing with Ot, gradient elution was performed with the same buffer containing 0-0.4 M NaCl. Differentiation inducing activity is 0.
It was eluted with 1M salt. The elution pattern of ion exchange chromatography using D to E-Toyo Pearl 650M is shown in FIG. Ammonium sulfate was deposited by adding solid ammonium sulfate to 70% saturation.

The precipitate was collected by centrifugation and dissolved in 2Qmt water. Add 20ml of 80% saturated ammonium sulfate solution to this solution,
0.05 k containing 0% saturated ammonium sulfate! ) Squirrel-HC11
It was loaded onto a Butyl Toyono medium 650M column (2×160) which had been pre-equilibrated with Jjl buffer (pH 7,8). After lowering the ammonium sulfate concentration stepwise, elution is performed with 20% ethanol. Differentiation induction method 18: Ensure that the substance is eluted. The elution pattern of hydrophobic chromatography using this Butyl Toyo Pearl is shown in FIG. The active fraction was collected and lyh gled under reduced pressure to remove the ethanol.
The lt tr3 solution was diluted with 0.05M tris-hydrochloride mild % solution (p
Pharmacia FPLC (Farmacia FPLC) using a Mono Q HR575 column (Pharmacia anion exchanger) dialyzed against H8,0) and equilibrated with the same buffer.
st Protein, Peptide, Polynu
el-eotide, Liquid Chromato
It was purified using a graphy system. Elution is 0.
A gradient elution of salt from 0.05 M to 0.1'M was performed.K-BUF activity was eluted at around 0.1 M of salt.The seminal p magnification in this step was approximately 5. This sample was prepared using Hynor RP304 (manufactured by Bio-Rad, C-4).
Reverse phase high performance liquid chromatography was performed using a reverse phase Kawa column). Elution was carried out using 0.1 difluoroacetic acid as a developing solution and changing the concentration of n-zolo/4'nol linearly from O to 80L4. The elution pattern is shown in FIG.

The protein peak shown in FIG. 3 and the activity completely matched.

This active peak was collected to obtain a purified sample of approximately 70 μy.

This sample was subjected to SDS-polyacrylamide gel electrophoresis (dal concentration: 15.0 in the presence of 1.0 thymercaptoethanol). As a result, a single band (silver staining method) was observed at 16 kd, and no other protein bands were detected. Moreover, it was 25 kd in the absence of mercaptoethanol. The specific activity of the sample thus purified was approximately 2xlOU apricot protein.

10 μg of this electrophoretically purified sample was collected using a gas phase amino acid sequencing analyzer (Model 470A) manufactured by Applied Biocystenos.
Edman degradation was performed sequentially on the N-terminus. The liberated phenylthiohydantoin-amino acids were collected using high-performance liquid chromatography (HPLC apparatus 5P8100° manufactured by Svectrophysics Co., Ltd. The column was Solpax OD manufactured by DuPont Co., Ltd.
The analysis was conducted at S. As a result, in the present invention -BU
The amino acid sequence from the amino terminus of F was as follows.

Gly-Leu-Glu-X-Asp-Gly-Lys
-Val-Asn-11e Note that X in the amino acid sequence represents one amino acid residue that cannot be analyzed by a gas phase amino acid analyzer.

Next, other physical and chemical properties X such as isoelectric point and pH stability were examined for this sample. One fruit of this is shown below.

(1) Isoelectric point: I) I6.5 ± 0.5 (Chromatography 7 oven method) 7.3 ± 085 (Isoelectric focusing method) (2) pH stability: pH 2.0 to 10.00f (3) Thermal stability = Stable when heated at 65°C for 160 minutes (pH
7,4) (4) Solvent stability Niacetonitrile, stable against lower alcohols (25°C) (5) Protease: Stable, completely lost by glonase treatment Active.

(Dissolve a certain amount of -BUF'k in 1% NaHCO3 solution, add 1/10 amount of -BUF of pronase to this, and completely inactivate by enzymatic treatment at 37゛0 overnight.) The isoelectric point was measured by chromatography as follows. T! s sample for 25m
Polypanophr exchanger P dialyzed against M bistris hydrochloride buffer (pH 7.0) and equilibrated with the same buffer.
BE 94 (manufactured by Pharmacia) column (1.0/40
eluted with pH 5.0 polybuffer (manufactured by Pharmacia) diluted to 1/10. The isoelectric point is expressed in terms of the pH at the elution site of activity.

On the other hand, measurement of isoelectric point by isoelectric focusing method is LKB
Isoelectric focusing device (LKB-
Multiphore) and reagents (Ampholin
The measurement was carried out using LKB-Ampholine PAG plate according to the standard method. The sample is plated (LKB)
iso4-xol, pH 3, s ~ 9.5) After K addition 1
Electrophoresis was performed at 500 V for 2.5 hours, and the isoelectric point was measured using a Pharmacia Isofix Point Measurement Kit (pH 3 to 10) as a marker. As a result, etc., the score is 7.3
The isoelectric point when measured in the coexistence of β-mercaptoethanol was 7.3, with no noticeable difference.

Ni-562 used in the present invention is Blood 45:
321 (1975).

In addition, K-562 is ATCC (CCL243) and JCR
B (JCRB0019) and can be obtained.

Example 3 Using ddY mice (male, 5 weeks old, Tokyo Experimental Animals ■),
Five animals per group were used as test animals. 2×10 mouse friend leukemia cells F5-5, which had been subcultured in the ascites of the same mouse, were intraperitoneally transplanted into the mouse.

Ni-BUF' obtained in Experimental Example 2 was dissolved in sterilized physiological saline to prepare a 5000 U/mi injection drug.

In the K-BUF administration group, F5-5 cells were transplanted, and from the next day, 0.2 ml (100 OU) of the above injection drug was administered intraperitoneally and intravenously for 3 days.

On the 14th and 21st days after F5-5 cell transplantation, blood was collected from the tail vein into a hematocrit tube and incubated at 12,0 OOr, p, m.
The mixture was centrifuged for a minute and the hematocrit value was measured by a conventional method. Physiological saline was administered to the control group. The results are shown in Table 3.

Table 3 Before transplantation 14th day 21st day Control group 45 30 28 Intravenous administration group 46 41 42 In parallel, another group of mice was administered 2 ml of Q intravenously on the 14th day after PS-SA cyst transplantation. administration, and the hematocrit value was measured on the 21st day. As a result, the hematocrit value was 3
5, a clear increase in hematocrit value was confirmed.

On the other hand, 50μ of -BUF was given to normal mice! 9 (2,5
When the mice were administered intravenously and kept for two months to examine toxicity, no abnormalities were observed as the mice were normal.

(Actions and Effects of the Invention) The anemia therapeutic agent of the present invention has the effect of preventing and curing anemia caused by Friend's leukemia.

Therefore, the anemia therapeutic agent of the present invention can be used for anemia caused by a decrease in red blood cells and hemoglobin caused by malignant tumors such as leukemia, multiple myeloma, and lymphoma.

[Brief explanation of the drawing]

FIG. 1 shows the DEA of BUF in the human differentiation-inducing factor of the present invention.
This is the elution/setan of ion exchange chromatography using E-Toyo Pearl 650M. FIG. 2 shows the elution pattern of -BUF, the human differentiation-inducing factor of the present invention, in hydrophobic chromatography using Butyl Toyopearl. FIG. 3 shows four turns of elution of -BUF, a human differentiation-inducing factor of the present invention, in reverse phase high performance liquid chromatography.

Claims (1)

[Claims] (1) A polypeptide having the activity of causing mouse leukemia cells to differentiate and mature into normal cells and having the following physicochemical properties. (a) Molecular weight: 16 ± 1 kd (in the presence of 1.0% mercaptoethanol, SDS electrophoresis method) 25 ± 1 kd (in the absence of mercaptoethanol, SDS
(electrophoresis method) (b) Amino acid sequence of the amino terminal: [There is an amino acid sequence] X is an amino acid that could not be identified