JP2000248000A - New nervous cell differentiation promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject agent capable of more strongly differentiating a nervous precursor cell into nervous cell such as astrocyte, compared with case when a mixture of a specific interleukin with the receptor in which these both components are not fused is used by including a fusion protein of the specific interleukin with the receptor as an active ingredient. SOLUTION: This agent contains a fusion protein of interleukin-6 (IL-6) with interleukin-6 receptor (IL-6R) as an active ingredient. It is preferable that the protein is a fusion protein in which IL-6 is directly bonded to IL-6R. The protein is obtained by integrating a gene encoding the protein into an expression vector, introducing the integrated protein into a host such as yeast, etc., of Pichia pastoris, culturing the transformed host and purifying the protein from the culturing supernatant, etc. The daily dose of the agent is generally 1-500 μg/kg.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a fusion protein of interleukin-6 receptor (hereinafter referred to as IL-6R) and interleukin-6 (hereinafter referred to as IL-6). (Abbreviated as IL-6 fusion protein)
The present invention relates to an agent for promoting differentiation of neural progenitor cells into nervous system cells, comprising as a main component. The present invention also relates to IL-6
The present invention relates to the promotion of neural cell differentiation by administering an agent for promoting differentiation of neural progenitor cells to neural cells containing an R • IL-6 fusion protein as an active ingredient.

[0002]

2. Description of the Related Art The gp130 protein is a glycoprotein with a molecular weight of 130,000 expressed on the cell membrane and is known to be a molecule that transmits various signals to target cells. Utilizing this fact, for example, a method has been reported in which neurites are extended from neurons by stimulating the gp130 protein (see Japanese Patent Application Laid-Open No. 9-87198).

Recently, neural progenitor cells prepared from mouse embryos have been prepared in the presence of IL-6R extracellular region and IL-6, or CNTF (ciliary neurotrope).
It has been reported that when cultured under stimulation with gp130 protein in the presence of chick factor, it differentiates into astrocytes (neural cells) (Bonni et al.,
Science, 278, 477, 1997).
This report indicates that neural precursor cells are differentiated into neural cells by stimulating the gp130 protein.

[0004]

As described above, IL-
It has been clarified that stimulating the gp130 protein in the presence of 6R and IL-6 causes neural progenitor cells to differentiate into nervous system cells, but its differentiation promoting effect is not sufficient, and the gp130 protein is not effective. In order to provide an agent for promoting differentiation of nervous system cells by stimulating, it is necessary to achieve a further differentiation promoting effect.

Accordingly, an object of the present invention is to provide a differentiation promoting agent for differentiating neural progenitor cells into neural cells such as astrocytes by stimulating the gp130 protein.
An object of the present invention is to provide a differentiation promoting agent having a stronger differentiation promoting effect as compared with the case where L-6R and IL-6 coexist. Another object of the present invention is to provide a method for promoting differentiation of neural progenitor cells into neural cells using such a powerful differentiation promoting agent.

[0006]

Means for Solving the Problems To achieve the above object, the invention of claim 1 of the present invention is directed to IL-6R and IL-R.
An agent for promoting the differentiation of neural progenitor cells into neural cells, comprising the fusion protein of No. 6 as an active ingredient. In order to achieve the other object, the invention of claim 2 of the present application provides an IL-
This is a method for promoting differentiation of neural progenitor cells into neural cells, which comprises administering a neural cell differentiation promoting agent containing a fusion protein of 6R and IL-6 as an active ingredient. Hereinafter, the present invention will be described in detail.

[0007] Examples of the IL-6R / IL-6 fusion protein used in the present invention include those in which IL-6R and IL-6 are linked by a disulfide bond. Further, for example, a fusion protein produced by a genetic engineering technique described below, in which IL-6R is located on the N-terminal side and IL-6 is located on the C-terminal side, and an arbitrary amino acid is located between both proteins. Examples thereof include those linked via a sequence linker or an amino acid sequence linker having an α-helical structure. Does not have a linker sequence,
Since a fusion protein in which both proteins are directly bound does not have antigenicity due to the linker sequence, it is preferable when the differentiation promoter of the present application is administered to a human body or the like.

[0008] IL-6R is a membrane protein composed of a total of 468 amino acid residues and comprises a signal region, an extracellular region, a transmembrane region and an intracellular region (Yamasaki).
Et al., Science, 241, 825, 1988), binding to IL-6 further increases gp130 on the cell membrane.
It binds to proteins and transmits signals into cells. IL-6
Only the extracellular region is required for binding to gp130 protein, and even soluble IL-6R lacking a transmembrane region or an intracellular region, after binding to IL-6,
Further, it can transmit a signal by binding to the gp130 protein (see Patent No. 2865300).
In the case of human IL-6R, the signal region extends from around the N-terminal first methionine residue to around the 19th alanine residue, and the extracellular region extends from around the 20th leucine residue to 35th.
Up to the vicinity of the eighth aspartic acid residue, the transmembrane region is 3
It is thought that the region from around the 59th serine residue to around the 386th leucine residue and the intracellular region from around the 387th arginine residue to around the 468th arginine residue. The extracellular region is divided into an immunoglobulin-like region and a cytokine receptor region. To the vicinity of the alanine residue at position 323. In addition, IL-6
It is known that an essential component for binding to is a cytokine receptor region, and an immunoglobulin-like region is unnecessary. The cytokine receptor region consists of seven β
Short barrel-like structure composed of sheets 2
A connected structure (Yawata et al., EMBO
J. , 12, 1705, 1993).

[0009] From the above viewpoint, IL-6 used in the present invention.
As the IL-6R constituting the R.IL-6 fusion protein, not only the full-length IL-6R but also the whole extracellular region or the cytokine receptor region, that is, a partial IL-6R can be used. . This is because it is the cytokine receptor region that binds to IL-6 to form its signal transduction system, and the extracellular region is a region containing this region. According to the findings of the present inventors, for example,
Specifically, the N-terminus includes a leucine residue at the 20th N-terminus, a valine residue at the 112th N-terminus, and an N-terminus 11
The sixth glutamic acid residue can be exemplified. According to the findings of the present inventors, for example, as the C-terminus of IL-6R, 361 to 361 from the alanine residue at the N-terminal 323rd position
Any one of the 39 amino acid residues up to the th serine residue, particularly preferably the 323 th alanine residue, the 333 th alanine residue, the 334 th leucine residue, the 335 th threonine residue For example, it is possible to use any one of the six amino acid residues of the 338th lysine residue or the 343rd isoleucine residue. That is, from the amino acid residue exemplified as the N-terminal to the C
The amino acid sequence up to the amino acid residue exemplified as the terminus was changed to IL-6R / IL-6 fusion protein IL-6.
It is used as the R part, and the N-terminus of IL-6 is bound to the C-terminal side. The N-terminus of IL-6R can be appropriately deleted in consideration of the effect of the fusion protein on signal transduction.

[0010] IL-6, which constitutes the IL-6R / IL-6 fusion protein, is a secretory protein composed of four α helices and having a total length of 212 amino acid residues (Hiran).
O et al., Nature, 324, 731 (1986)).
It is known that all helices are required. Therefore, the IL-6 used in the present invention is not particularly limited as long as it has all four α helices. That is, not only full-length IL-6 but also partial IL-6 in which, for example, some amino acid residues at the N-terminal and C-terminal are deleted.
It may be. For example, IL-6 from the N-terminal 28th alanine residue or the 29th proline residue to the 212th methionine residue which is specifically known as a secretory IL-6 structure can be exemplified. . In addition, other examples of expression of IL-6 (for example, Yaskawa
Et al., Biotech. Lett. 12, 419, 1
990) and IL-6R / IL-6 fusion protein expression examples (Fisher et al., Nature Biotech.,
15, 142, 1997).
It can be determined what sequence to use as 6.

The above-mentioned single-chain IL-6R · IL bound via a linker or without a linker.
The -6 fusion protein can be easily prepared by performing a gene recombination operation using a gene encoding the same. The gene encoding IL-6R or IL-6 has already been isolated, and its nucleotide sequence is also well known. Therefore, when preparing the fusion protein of the present invention, a necessary gene sequence is prepared from the amino acid sequences of IL-6R and IL-6 constituting the fusion protein, and these are ligated using a restriction enzyme. good. Here, in addition to the use of a natural gene sequence, any codon may be replaced with one which encodes the same amino acid residue but has a different base sequence in consideration of codon condensation. This is because when a protein is expressed in a host by genetic recombination, use of a specific codon may improve the expression rate and the translation rate.

There are no particular restrictions on the host used when the fusion protein of the present invention is produced by genetic recombination, and Escherichia coli and CHO cells used in ordinary genetic recombination operations can be used with reference to the conventional reports. Animal cells represented by, for example, (Yasukawa et al., J. Bio
tech. , 108, 673, 1990). Among them, the yeast of the species Pichia pastoris shown in the present example is a yeast that can grow using methanol as a sole carbon source, and can be cultured at a lower cost than animal cells such as CHO cells. It can be exemplified as a preferred host.

The above-described gene for producing the fusion protein of the present invention by genetic recombination is used by incorporating it into an expression vector when introducing (transforming) it into a host. The expression vector incorporates, in addition to the gene, an expression control gene, a gene serving as an indicator for selecting a transformed host, and the like. Such a gene may be appropriately selected and used in relation to the host to be used. . For example, if yeast of the species Pichia pastoris is used as a host, upstream and downstream sequences of an alcohol oxidase gene for introducing a gene encoding an IL-6R / IL-6 fusion protein into its chromosomal DNA, The histidine synthesis gene and the promoter sequence of the alcohol oxidase gene for expression control are used as an indicator of ampicillin resistance gene and Lac promoter / operator sequence for expression control, which are selection indicators if Escherichia coli is used as a host. Etc. can be exemplified. The gene of the present invention can be introduced into a commercially available expression vector (for example, pPIC9, an expression vector for Pichia pastoris yeast, manufactured by Invitrogen) and used.

In the present invention, preferably, when a yeast of the species Pichia pastoris is transformed with an expression vector containing a gene encoding an IL-6R / IL-6 fusion protein to produce a fusion protein, the expression vector may contain It is preferable to incorporate an intrinsic signal peptide of IL-6R or a signal peptide of α-factor as a signal peptide of the fusion protein. In particular, it is preferable to use a signal peptide of α-factor because high expression can be realized.

The above-mentioned transformed host is cultured under appropriate conditions, and the fusion protein can be produced by inducing the expression of the fusion protein as required in relation to the expression control gene in the expression vector. . In the present invention, when a yeast of Pichia pastoris is preferably used as a host, a method using a jar fermenter can be exemplified. More specifically, IL-6 prepared beforehand was placed in a 500 mL shake flask charged with 100 mL of medium.
A 20% glycerol frozen strain of Pichia pastoris yeast expressing the R.IL-6 fusion protein was inoculated with 28
Incubate with shaking at ３０30 ° C. for 20 hours. Next, 100 mL of the above culture solution is inoculated into a 16 L capacity jar fermenter charged with 6 to 9 L of medium, and aeration and stirring culture is started at 28 to 30 ° C. In order to monitor the state of the yeast during the culture, it is preferable to monitor and control the OD600, pH, dissolved oxygen concentration, stirring speed, and temperature. The type of the medium is not particularly limited as long as it contains a carbon source derived from a natural product, but the specific composition may be referred to the examples. As a jar fermenter for performing the culture, a commercially available device can be used. After the start of the culture, methanol may be added at the stage when the glycerol in the culture solution is depleted. Glycerol depletion can be determined by monitoring dissolved oxygen. It is desirable to add methanol within 5 hours after depletion. If the amount of methanol added is too large, it is toxic to yeast, but if the amount is too small, considering that the promoter sequence of the alcohol oxidase gene does not function sufficiently,
0.5% or more and 5% or less (mass / volume) are preferable.

The fusion protein produced by culturing can be obtained from a medium or the like by an appropriate method. When Escherichia coli is used as a host, the expressed fusion protein accumulates as an insoluble mass in Escherichia coli. Therefore, after the cells are disrupted, refolding and purification operations may be performed under appropriate conditions. When a yeast of the Pichia pastoris species is preferably used as a host, the fusion protein can be purified and obtained from the culture supernatant. The purification raw material is not particularly limited as long as it is a solution containing the fusion protein, and examples thereof include a culture solution of a yeast of Pichia pastoris containing the fusion protein. The solution may be used as it is,
Or dilute them with buffer or pure water,
It may be used after being concentrated with an ultrafiltration membrane or ammonium sulfate. As a purification operation, a liquid chromatography operation can be exemplified. Preferably, a combination of three types of chromatography, such as ion exchange chromatography, hydrophobic chromatography, and gel filtration chromatography, can be exemplified.

Since the culture supernatant of the yeast of the species Pichia pastoris is large in volume, it is preferred that the yeast is first subjected to ion exchange chromatography. Ion exchange chromatography is divided into cation exchange chromatography and anion exchange chromatography, and can be appropriately selected in consideration of protein removal efficiency. The former can be exemplified by SP as a cation exchange group, and the latter can be exemplified by DEAE as an anion exchange group. Here, the flow rate can be increased to 100 ml or more per minute and 1 μm
Considering that it is possible to add a sample that has not passed through a filter having a size of not more than m, as shown in the examples below, the adsorption fluidized bed S
A preferred example is a streamline SP C-50 column (manufactured by Amersham Pharmacia). The fraction containing the fusion protein obtained by the above method is then subjected to hydrophobic chromatography, whereby a fraction having a higher purity of the fusion protein can be obtained. This fraction
As shown in the examples below, gel filtration chromatography can be performed efficiently by concentrating by, for example, cation exchange chromatography.

The IL-6R / IL-6 fusion protein can be used as a nervous system cell differentiation promoting agent by formulating it. Formulation is achieved by mixing with normal excipients such as saline, glucose solution, mansitol, methylcellulose, gelatin, human serum albumin, and the like. The amount of the IL-6R / IL-6 fusion protein in the preparation can be, for example, about 0.01% or more by weight, but the upper limit is not particularly limited. The formulation contains IL-6R
-Components other than the IL-6 fusion protein may be present. Further, the IL-6R / IL-6 fusion protein may be a lyophilized product. In this case, it may be redissolved with an isotonic solution such as physiological saline, glucose solution or Ringer's solution immediately before use.

The neural cell differentiation promoting agent of the present invention is preferably administered parenterally to mammals such as humans, for example, by intravenous administration, intramuscular administration, transdermal administration, and the like. The dose is appropriately determined depending on the type of disease showing a deficient symptom of neural progenitor cells, the state of the patient to be administered, and the like.
The dose is in the range of ５００500 μg / kg / day, and continuous administration may be performed while observing the state of increase in neural cells.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail with reference to embodiments of the present invention, but the present invention is not limited thereto.

EXAMPLE 1 Neuroepithelial cells of ICR mouse embryos (purchased from CLEA Japan) on day 14.5 were removed and bF of 10 ng / ml was obtained.
GF (basic fibroblast growth)
h factor) was added to 6 ml of an N2-DMEM / F12 medium (manufactured by Gibco) and cultured for 4 days to prepare neural progenitor cells.

Next, the above cells were prepared at 0.8 × 10 ⁵ cells / well, and IL-6 consisting of the N-terminal 20th leucine residue to the N-terminal 333th alanine residue was prepared.
IL-6R-IL-6 fusion protein 50 ng / ml in which IL-6 consisting of the N-terminal 28th alanine residue to the N-terminal 212th methionine residue is bound to the C-terminal side of R.
In the presence (A), the IL-6R / IL-6 fusion protein 20
In the presence of ng / ml (B), IL-6 (20 ng / ml) containing the N-terminal 28th to 212th amino acid sequence and IL containing the N-terminal 20th to 344th amino acid sequence
IL-6 in the presence of -6R (125 ng / ml) (C)
(50 ng / ml) and the same IL-6R (125 ng / m2).
1) in the presence (D) and the fusion protein or IL-
6 and further cultured for 3 days in the absence of IL-6R (E).

This is prepared by adding PB containing 4% formaldehyde
G is immobilized with S and is an astrocyte marker
P (grill fibrillary acidic)
The protein was stained with an antibody (DAKO) and a rhodamine-conjugated anti-rabbit antibody (CHEMICON) and observed with a fluorescence microscope. FIG. 1 shows the results of (A), FIG. 2 shows the results of (B), and FIG. 3 shows the results of (C).
FIG. 4 shows the result of (D), and FIG. 5 shows the result of (E).
Are shown below. In FIG. 1 to FIG. 5, the neural progenitor cells appear as white granules, and the cells having a string-like projection are cells differentiated into astrocytes.

As is apparent from FIGS. 1 to 5, IL-6R
-It was observed that the IL-6 fusion protein promoted astrocyte differentiation very strongly as compared to the case where the unfused IL-6 and IL-6R were mixed and administered.
That is, as an agent for promoting nervous system cell differentiation by stimulating gp130 protein, unfused IL-6
IL-6R and IL-6R are mixed and administered.
The R • IL-6 fusion protein has a stronger differentiation promoting effect.

[0025]

Conventionally, it has been very difficult to differentiate neural progenitor cells into neural cells, and the degree of differentiation has been weak. Required a long time and a great deal of effort.

IL-6R and IL-6 provided by the present invention
The fusion protein comprises unfused IL-6R and IL-6.
Has a differentiation promoting effect of more strongly differentiating neural progenitor cells into nervous system cells such as astrocytes as compared with the case where they are used in combination. Therefore, IL-6R IL-
The differentiation promoting agent of the present invention comprising 6 fusion protein as an active ingredient is not only useful as a differentiation promoting agent for neural progenitor cells per se, but also studies on the cerebral nervous system in mammals including humans, It is extremely important for the development of new therapeutic agents or diagnostic agents based on the results of the above.

In the activity of promoting differentiation into astrocytes, CNTF is not fused with IL-6 and IL-6.
It has been reported that it has an activity equivalent to a mixture of -6R (Bonni et al., Science, 278, 477).
P. 1997). Therefore, the IL-6R / IL-6 fusion protein is considered to be superior to CNTF as an agent for promoting neural cell differentiation by stimulating the gp130 protein.

[Brief description of the drawings]

FIG. 1 shows that neuroepithelial cells were prepared by the method described in Example 1 by using 50 ng / IL-6R / IL-6 fusion protein.
It is a figure which shows the result when culture | cultivating in the presence of ml.

FIG. 2 shows that neuroepithelial cells were transformed into (B) IL-6R / IL-6 fusion protein 20 by the method described in Example 1.
It is a figure which shows the result when culture | cultivating in presence of ng / ml.

FIG. 3 shows that neuroepithelial cells were treated with (C) IL-6 (20 ng / ml) and IL by the method described in Example 1.
It is a figure which shows the result at the time of culture in the presence of -6R (125 ng / ml).

FIG. 4 shows that neuroepithelial cells were treated with (D) IL-6 (50 ng / ml) and IL by the method described in Example 1.
It is a figure which shows the result at the time of culture in the presence of -6R (125 ng / ml).

FIG. 5 is a diagram showing the results obtained by culturing neuroepithelial cells in the absence of (E) cytokine and its receptor by the method described in Example 1.

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Claims (4)

[Claims] An agent for promoting the differentiation of neural progenitor cells into neural cells, comprising a fusion protein of interleukin-6 receptor and interleukin-6 as an active ingredient. 2. A method for promoting differentiation of neural progenitor cells into neural cells, which comprises administering a neural cell differentiation promoting agent containing a fusion protein of interleukin-6 receptor and interleukin-6 as an active ingredient. 3. The differentiation promoting agent according to claim 1, wherein the fusion protein of interleukin-6 receptor and interleukin-6 is a fusion protein in which both are directly bound. 4. The method according to claim 2, wherein the fusion protein of interleukin-6 receptor and interleukin-6 is a fusion protein in which both are directly bound.