JPH0820599A - New protein - Google Patents

Abstract

PURPOSE:To obtain a new protein separated from a thymic muscular cell, having a specific molecular weight, having proliferation-inducing activity of a thymic macrophage-based cell and pectoral microglia cell and useful for treating granulocytopenia, immunodeficiency, cerebral lesion, etc. CONSTITUTION:This new protein contains partial amino acid sequences expressed by formulas I, II, III (Xaa, Xab and Xac are each an unidentified amino acid selected from 20 amino acid including carboxy methyl cysteine), IV and V, etc., and has about 60000 dalton molecular weight measured by electrophoresis of SDS-polyacrylamide gel and has activity independently proliferating and inducing a thymic macrophage-based cell and a pectoral microglia cell and is useful for treating adverse effects due to chemotherapy and radiation therapy and granulocytopenia accompanied by hyperthyroidism, immunodeficiency, cerebral lesion, etc. The protein is obtained by separating and purifying the thymic muscular cell using anion exchange column, hydroxyapatite column and gel permeation column, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel protein having an action of promoting the differentiation and proliferation of thymic macrophage cells and brain microglial cells.

[0002]

BACKGROUND OF THE INVENTION In recent years, granulocyte colony stimulating factor (G-CSF) that induces differentiation and proliferation of neutrophils, and granulocyte / macrophage colony that promotes proliferation of granulocytes and macrophages. Stimulator (G
The existence of M-CSF) and macrophage colony stimulating factor (M-CSF) that promotes induction of macrophage differentiation has been discovered, and their roles are being clarified.

[0003] For example, human-derived G-CSF is a protein consisting of 174 amino acid residues and having a molecular weight of about 1.8 to 220,000. It is a side effect of anticancer drugs and radiation therapy, ie, granulocytopenia and immunodeficiency. Be useful as a remedy for diseases such as
Human-derived GM-CSF is a glycoprotein consisting of 125 amino acid residues and having a molecular weight of 1.4 to 35,000.
Like SF, it is expected to be useful as a therapeutic drug for side effects of cancer chemotherapy or radiation therapy and granulocytopenia associated with hyperthyroidism.

On the other hand, macrophage cells are a group of cells that differentiate and proliferate from bone marrow stem cells, perform phagocytosis, and protect the living body from invading microorganisms and the like. These cells are differentiated into organ-specific cells such as Kupffer cells in the liver and macrophages in the lung, and are involved in organ-specific functions. It is known that it exists as thymic macrophage cells in the thymus and is involved in "negative selection" in which T cells that recognize self-antigens are selectively excluded. However, the existence of factors that promote differentiation and proliferation of such organ-specific macrophage cells has not been known at all.

[0005]

[Means for Solving the Problems] The present inventors have searched for a novel differentiation and growth factor, and as a result, have proceeded to research, and as a result, muscle-like cells, which are thymic stromal cells, differentiated into thymic macrophage cells and brain microglial cells. It was discovered for the first time that a growth factor was produced, and the factor was isolated and purified to complete the present invention.

That is, the gist of the present invention resides in a novel protein characterized by having the following physicochemical properties. 1. The molecular weight by SDS-polyacrylamide gel electrophoresis is about 60,000 daltons. 2. It has the activity of independently inducing proliferation of thymic macrophage cells and brain microglial cells.

The present invention will be described in detail below. The novel protein of the present invention having an action of promoting the proliferation induction of thymus-derived macrophage cells and brain microglial cells (hereinafter sometimes referred to as “60K-CSF”) is
For example, as described in detail in Examples below, the culture supernatant of thymus-derived myoid cells is treated with DEAE-Sepharose CL-6B.
Fractionation by ion exchange chromatography (Pharmacia) and the like, followed by affinity chromatography on a hydroxyapatite column and a blue sepharose-conA column, TSK G4000S.
It can be obtained by further fractionating by molecular sieve chromatography such as W (manufactured by Tosoh Corporation) and then purifying by YMCPack C4 column chromatography (manufactured by YMC).

The purified protein of the present invention is SDS
-It has a molecular weight of about 60,000 daltons by polyacrylamide gel electrophoresis, and has the activity of independently inducing proliferation of thymic macrophage cells and brain microglial cells. G-CSF activity and GM-CSF activity were not found in the protein. Also M-CSF
Does not act on thymocytes even when it stimulates the proliferation of peritoneal exudate cells well, whereas 60K-CSF of the present invention acts on the differentiation and proliferation of macrophage cells of the thymus. From these, it was revealed that the factor is a novel factor having an unprecedented CSF-like activity.

Further, the 60K-CSF of the present invention contains the amino acid sequence as shown in SEQ ID NO: 1 in the sequence listing at the N-terminal and further the amino acid sequences as shown in SEQ ID NO: 2-5 in the sequence listing as an intermediate fragment. It is a waste. Take 60
K-CSF can be modified, such as by removing, substituting, modifying or adding a part of amino acids, as long as the function of inducing proliferation of thymic macrophage cells and brain microglial cells is not impaired.

When the 60K-CSF of the present invention is used as a medicine, it is preferably mixed with a suitable diluent or other additive in a suitable formulation form or dosage form and used. As a dosage form, a dosage form suitable for parenteral administration is generally used. Preferred examples include an ampoule for injection and a freeze-dried powder for injection (vial).

The above-mentioned various dosage forms can be prepared by a conventional method commonly used in this technical field.
As a pharmaceutical carrier used for formulation, diluents, additives and the like which are commonly used for preparing various dosage forms are used. For example, lyophilized powder for injection is purified 60K
-Dissolving an effective amount of CSF in a diluent such as distilled water, physiological saline, or an aqueous glucose solution, and if necessary, an excipient such as carboxymethyl cellulose or sodium alginate,
Add benzyl alcohol, benzalkonium chloride, preservatives such as phenol, soothing agents such as glucose, calcium gluconate, procaine hydrochloride, pH adjusting agents such as hydrochloric acid, acetic acid, citric acid, sodium hydroxide, etc. It is prepared by freeze-drying.

The ampoule for injection is 60K-CSF.
An effective amount of, for example, distilled water, physiological saline, dissolved in a diluent such as Ringer's solution, sodium salicylate, if necessary,
Solubilizing agents such as mannitol, buffering agents such as sodium citrate and glycerin, isotonic agents such as glucose and invert sugar,
It is prepared by adding additives such as a stabilizer such as polyethylene glycol, the above preservative, the soothing agent, the above pH adjuster, etc., and sterilizing it by ordinary heat sterilization, sterile filtration and the like.

[0013] Such a pharmaceutical composition may be administered parenterally to a patient in need thereof, generally by subcutaneous, intramuscular or intravenous injection, in a given amount in single or divided doses. , Or continuously. In the case of single or multiple administration, the dose as a drug is usually about 1 per day for an adult as an active ingredient (60K-CSF).
It is preferably administered in the range of 0 μg to 10 mg / kg, intravenously, subcutaneously, or intramuscularly in a patient to be administered, and may be increased or decreased depending on the pathological condition, nutritional status, age, weight, concomitant drug, etc. of the patient. Can be made. Further, in the case of continuous administration, an amount corresponding to the above-mentioned daily dose can be administered within the range of about 0.1 to 50 μg / kg per 1 hour for an adult.

As an administration method, in the case of an ampoule for injection, it can be directly administered subcutaneously, intramuscularly or intravenously. In the case of intravenous administration, a minipump for infusion is used if necessary. You can also do it. In the case of a freeze-dried powder for injection, the powder is dissolved in a diluent such as distilled water, physiological saline or Ringer's solution at the time of use, and then administered in the same manner as in the case of ampoule.

Further, the pharmaceutical composition of the present invention may be administered by pre-mixing a predetermined amount of glucose infusion with a glucose infusion upon treatment, or by administering an effective amount of the glucose infusion alone at the same time as a peripheral vein, etc. It is also possible to administer from.

[0016]

The novel protein 60K-CS of the present invention
Since F specifically induces proliferation of thymic macrophage cells, it is expected that F can be used to enhance the functions of various immunocompetent cells such as tumoricidal action and osteoclast proliferation activity of macrophages. Specifically, granulocytopenia after chemotherapy or radiation therapy, aplastic anemia, myelodysplastic syndrome, granulocytopenia associated with bone marrow transplantation, periodic neutropenia, granulocytes in AIDS-related syndromes Treatments include hyperactivity, opportunistic infections in the elderly, thrombocytopenia, combined use with anti-tumor agents, and hyperlipidemia.

When the protein is given to brain microglial cells, it exhibits a characteristic growth morphology, suggesting that the protein of the present invention also functions as a differentiation and growth factor for brain microglial cells. Therefore, the protein of the present invention can also be applied to the treatment of brain damage and the like. Furthermore, it is considered that conventional treatment with M-CSF is difficult for osteoporosis, which is bone atrophy associated with bone deformation or narrowing of the bone marrow cavity, and 60K-CSF of the present application can be applied to these diseases. Sexuality is also considered.

[0018]

EXAMPLES The present invention will be described in more detail below with reference to examples, but the invention is not limited to the examples as long as the gist thereof is not exceeded. Example 1 Purification of 60K-CSF Immunology, 79 , 103-106 (199)
3) The culture supernatant of rat thymus-derived myoid cells (871207B) cultured in RPMI 1640 medium according to the method described in 3) was equilibrated with 0.05M phosphate buffer (pH 7.3) to DEAE-Sepharose CL-6B. It was added to the column and eluted with a linear gradient elution method of 0 to 1.3 M NaCl in the same buffer, and the conductivity was 0.7 mmho to 1.
Fractions up to 6 mmho were collected, passed through hydroxyapatite (10 mM, PB, pH 7.0), passed through a blue sepharose-conA agarose gel, and 0.3 M α-methylmannoside elution fractions were collected. did. Next, after concentrating with a concentrator having a molecular weight of 30,000 (Centricon, Amicon), it was replaced with 0.05M phosphate buffer (pH 7.3), and then equilibrated with the same buffer TSK G4000SW (manufactured by Tosoh Corporation). It was added to the column and eluted with the same buffer at a flow rate of 1 ml / min, and the elution fraction from 45 minutes to 55 minutes was collected. Next, 0.1% of this fraction
YMC equilibrated with acetonitrile / isopropyl alcohol (3/7, v / v) mixture containing trifluoroacetic acid
C4 column (YMC-Pack C4-AP, 0.4
6 × 15 cm) and added with 0.1% trifluoroacetic acid in acetonitrile / isopropyl alcohol (3 /
7, v / v) mixed solution was eluted at a flow rate of 1 ml / min by the linear concentration gradient method for 20 minutes at 20 to 80% for 30 minutes. The elution pattern by ultraviolet absorption measurement at 215 nm is shown in FIG. The desired activity was observed in the fraction eluted at 15.5 minutes, and the fraction was dried under vacuum and then dissolved in 20 mM phosphate buffer (pH 7.4) containing 0.15 M NaCl. YMC C8 column (YMC-Pack equilibrated with 30% acetonitrile)
C8-AP, 0.46 × 15 cm), 0.1%
30 to 60 with acetonitrile containing trifluoroacetic acid
%, A linear concentration gradient method for 30 minutes was used to elute at a flow rate of 1 ml / min, and each peak was collected. The elution pattern by UV absorption measurement at 215 nm is shown in FIG. 1 of these
The desired activity was observed in the fraction eluted at 4.2 minutes,
The same fraction was dried under vacuum. Purified 60K-CSF
Was analyzed by SDS-polyacrylamide gel electrophoresis, and it was revealed that its molecular weight was about 60,000 daltons. The amino acid sequence was analyzed using this sample.

In each of the above-mentioned purification steps, 60K-CSF was fractionally purified by the method described in Examples below, using the activity of thymidine uptake into bone marrow cells as an index. Example 2 Amino acid sequence analysis of 60K-CSF The 60K-CSF purified in Example 1 was dissolved in 60 μL of 50% trifluoroacetic acid and added to a glass filter treated with polybrene, and Applied Biosystem was analyzed.
Edman decomposed with a 470A sequencer manufactured by ems,
The amino acid sequence in the N-terminal region was determined. Such a sequence is shown in SEQ ID NO: 1 in the sequence listing.

The purified sample was subjected to S-treatment with monoiodoacetic acid.
50 mM containing 4M urea after carboxymethylation
Dissolved in Tris-HCl buffer (pH 8.5) and treated with lysyl endopeptidase (manufactured by Wako Pure Chemical Industries), enzyme: substrate =
0.1% at 37 ° C for 6 hours at 1: 200
Bakerbond C8 equilibrated with trifluoroacetic acid
Column (Bakerbond ^™ WP Octyl,
0.46 × 25 cm), and the acetonitrile concentration is 0
Perform linear gradient elution from 60% to 60% for 60 minutes,
Each peak was collected. The elution pattern by absorbance at 215 nm is shown in FIG. Of these, 29.5 minutes, 40.
The peaks eluting at 7 minutes, 42.2 minutes, and 45.0 minutes were dried under vacuum, and similarly Edman-decomposed by a sequencer. As a result, 29.5 minutes, 40.7 minutes,
The internal partial sequences of the peaks eluted at 42.2 minutes and 45.0 minutes were as shown in SEQ ID NOs: 2, 3, 4 and 5 of the Sequence Listing, respectively.

Example 3 Evaluation of 60K-CSF activity The proliferation activity of Wistar rat brain microglial cells was measured by the tritium thymidine incorporation method using a microplate in the presence of purified 60K-CSF. OX42 as a marker for the identification of cell proliferation
(Macropage, granulocyte, d
endric cell), ED1 (monocy)
te), ED2 (macrophase), ED3 (m
An indirect fluorescent antibody method using a monoclonal antibody such as Acrophase) was used. The results are shown in the table below.

[0022]

[Table 1] ─────────────────────────── Marker Day 0 of culture Day 7 of culture ────────── ───────────────── OX42 ＋ ＋ ED1－ ＋ ED2－ ＋ ED3 － ＋＋ ────────────────────── ────── ＋: About 80% of the cells react −: Not react

From the above results, the 60K-CSF of the present application
Is found to have the ability to act on brain microglial cells to differentiate macrophage-like cells having the above markers.

Further, using the Wistar rat bone marrow cells, the method of Kamo et al. (Cell. Immunol.,
94 , 587 (1985)), a colony assay (cultured for 8 days) was attempted.
The formation of colonies was observed. This shows that 60K-CSF of the present application has the ability to act on bone marrow cells to differentiate macrophage-like cells.

[0025]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment Origin Biological name: Rat Tissue type: Thymus Sequence Ser Met Val Ser Asn Arg Pro Phe Ile Thr Val Trp Asn Ala Asp Thr 1 5 10 15 His Trp Asp Leu Lys 20

SEQ ID NO: 2 Sequence length: 7 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Origin Biological name: Rat Tissue type: Thymus

SEQ ID NO: 3 Sequence length: 18 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Origin Biological name: Rat Tissue type: Thymus Sequence Glu Gln Asn Phe Gln Gly Xaa Xab Met Thr Ile Phe Tyr Arg Glu Glu 1 5 10 15 Xac Gly Xaa, Xab, Xac are unidentified amino acids selected from 20 amino acids including carboxymethyl cysteine.

SEQ ID NO: 4 Sequence length: 11 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Origin Biological name: Rat Tissue type: Thymus

SEQ ID NO: 5 Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Origin Biological name: Rat Tissue type: Thymus

[Brief description of drawings]

FIG. 1 is a drawing showing an elution pattern when 60K-CSF of the present invention was purified by reverse phase HPLC using ultraviolet absorption at 215 nm as an index.

FIG. 2 is a drawing showing an elution pattern when the active fraction of FIG. 1 was further purified by reverse phase HPLC using ultraviolet absorption at 215 nm as an index.

FIG. 3: 60K-C treated with lysyl endopeptidase
SF is a reverse phase HPL using ultraviolet absorption at 215 nm as an index.
It is a figure showing the elution pattern at the time of purifying by C.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Jun Kondo 1000 Kamoshida-cho, Midori-ku, Yokohama, Kanagawa Sanryo Kasei Co., Ltd. (72) Inventor Kazushi Takahashi 1000 Kamoshida-cho, Midori-ku, Yokohama, Kanagawa (72) Inventor Hide Yamada, 1000 Kamoshida-cho, Midori-ku, Yokohama-shi, Kanagawa Sanryo Kasei Co., Ltd.

Claims (7)

[Claims] 1. A novel protein having the following physicochemical properties. 1. The molecular weight by SDS-polyacrylamide gel electrophoresis is about 60,000 daltons. 2. It has the activity of independently inducing proliferation of thymic macrophage cells and brain microglial cells. 2. The partial amino acid sequence is represented by the sequence set forth in SEQ ID NO: 1 in the sequence listing.
The described protein. 3. The partial amino acid sequence is represented by the sequence set forth in SEQ ID NO: 2 in the sequence listing.
The described protein. 4. The partial amino acid sequence is represented by the sequence set forth in SEQ ID NO: 3 in the sequence listing.
The described protein. 5. The partial amino acid sequence is represented by the sequence set forth in SEQ ID NO: 4 in the sequence listing.
The described protein. 6. The partial amino acid sequence is represented by the sequence set forth in SEQ ID NO: 5 in the sequence listing.
The described protein. 7. A pharmaceutical composition comprising the protein according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.