JPS62107796A - Production of novel human granulocyte-monocyte stem cell proliferation factor - Google Patents

Abstract

PURPOSE:To produce the titled factor in large quantities, by culturing a human lymphocyte cell imparted with spontaneous proliferability in a cell culture medium, heating the medium and recovering the active component having the activity to promote the proliferation of a human granulocyte-monocyte stem cell. CONSTITUTION:A human lymphocyte cell imparted with spontaneous proliferability is produced by the mixed culture of a normal human lymphocyte and a cell infected with adult T-lymphocyte leukemia virus. The obtained lymphocyte cell is cultured in a cell culture medium to produce a substance having the activity to promote the proliferation of a human granulocyte-monocyte stem cell. The medium is exchanged at least 5 times and the culture liquid is recovered and centrifuged at 500-2,500 G for 5min. The supernatant liquid is filtered with a filtration membrane having a pore size of 0.45-3mu. The filtrate is heat-treated at 6-8pH and 60+ or -2 deg.C for 10-15hr in the presence of 1-5%(v/w) albumin or 5-20%(v/v) serum and the heat-treated product is purified to obtain the objective factor.

Description

[Detailed Description of the Invention] [Industrial Applicability] The present invention relates to a novel method for producing human @granulocyte-monocyte cell growth factor. The present invention relates to a method for producing a novel hematopoietic factor useful for the treatment of hematopoietic disorders by culturing N11 human lymphocytes endowed with spontaneous proliferation.

[Technical background and prior art]

Human blood constituent cells are maintained by the direct stem KB foam present in the bone marrow, which proliferates and differentiates under the action of various specific cardiac blood factors, and is produced and released into the blood.

When stem cells receive secondary damage from their own defects or from drugs, radiation, etc., stem cell proliferation is targeted and cell production capacity in the bone marrow decreases, resulting in a so-called bone marrow hypoplasia state, which can lead to anemia, Disorders such as thrombocytopenia or granulocytopenia will appear. Granulocytopenia, which occurs after administration of drugs such as aplastic anemia, myeloid leukemia, or cancer drugs, is thought to be caused by an increase in snow granulocyte stem cells.

In recent years, colony stimulating factor (colony stimulating factor), a specific hematopoietic factor for granulocytic-monocytic stem cells, has been used for the treatment of hypocytosis.
one-stimulating factor)
, hereinafter referred to as C5F and W1] has been tried and its usefulness has been proven. The application of such specific cardiac and blood factors to the treatment of hematopoietic disorders and various diseases caused by them is expected to be an epoch-making cure, in combination with bone marrow transplantation, which is equivalent to stem cell transfusion.

C, a specific hematopoietic factor for granulocyte-carocyte stem W cells
5F is G-C5F, which produces granulocytes due to its action.
1 GM-C5F, ff that produces granulocytes and globules
M-C3F, which produces i-cell-macrophages; Mul, which also produces granulocytes, globules, erythroblasts, and megakaryocytes;
It is classified as ti-C5F. Until now, human C5F
Examples include human urine C3F, C5F produced by certain am cells, C3F produced by monocytes-macrophages,
Several types of C3F produced by antigen-stimulated lymphocytes are known.

Conventionally, methods for producing C5F in normal human T-lymphocytes include a method of stimulating normal human T-lymphocytes with various lectins, and a method of stimulating with antigens such as IR bacterial cells (Kaisho et al. 59-78122) is known.

However, stimulation with lectin or antigen is required for cell proliferation and CSP (mainly GM-csF) i production by normal human T-lymphocytes, and this method has drawbacks as a method for mass production of C5F.

In the search for new human C3F materials, the present inventors
When normal human lymphocytes are mixedly cultured with adult T-lymphocyte hematopoietic virus (hereinafter sometimes abbreviated as ATLV), their transformation occurs, resulting in the development of specific human granulocyte-A cell lineage stem cells. colony formation stimulating factor (C5)
F), and when the culture medium in which this C5F activity has been produced is heated in the presence of human serum albumin, bovine serum albumin, human serum, bovine blood, etc., the produced C5F activity can be maintained. The present invention has been achieved based on these findings.

[Object of the invention and summary of the invention]

An object of the present invention is to provide a method for producing large quantities of human granulocyte-globocyte stem cell growth factors.

Another object of the present invention is to provide a human granulocyte-111 stem cell growth factor that also has cM-C5F activity.

The present invention involves cultivating human lymphocytes endowed with spontaneous proliferation in a cell culture medium to produce a substance that promotes the proliferation of human granulocyte-111 stem cells, and heating this medium. This is a method for producing human granulocyte-monocytic stem cell growth factor, which comprises treating the human granulocyte-monocytic stem cell growth factor and recovering an active ingredient having an effect of promoting the proliferation of human granulocyte-monocytic stem cells.

Human lymphocytes endowed with spontaneous proliferation can be obtained by mixed culture of normal human lymphocytes with cells infected with adult T-lymphocyte leukemia virus, and heat treatment is performed using human serum albumin, bovine serum albumin, human This heat treatment can be carried out in the presence of serum, bovine blood or a mixture thereof, and the heat treatment is carried out at pH 6-8 and 6.
These heat treatments can be carried out for 10-15 hours at o±2°C, and these heat treatments can be carried out in the presence of 1-5% (f/V) albumin or under g pressure of 5-20% (v/V) serum. It can be done in

[Detailed description of the invention]

(1) Establishment of normal human lymphocytes endowed with spontaneous proliferation ability Normal lymphocytes are collected from peripheral blood of a normal person,
MT-2i of ATLV transformed cells pre-irradiated with +5000 R of Xi (Miyoshi-fu et al.: Neichi 7
(Nature): Volume 294, Nos. 770-77
15N (+98] year)] and these normal lymphocytes were added to RPMr-1640 medium containing fetal bovine serum at a rate of 5 Sow at the rate of holes, 5%
Cultured for 30 days at 37° C. under aeration of (v/v) CO. After culturing, cells with high proliferative properties were selected from among the cells that were transformed with MT-2 cells, acquired spontaneous proliferative properties, and proliferated, and 10% (v/v) fetal bovine serum was added to these cells. The cells were transferred to RPMI-1640 medium and further cultured in the same manner as above. After culturing, the C5F activity in the culture supernatant was measured using the method of Rhre et al.
, N., Ihre et al: The Jour
C3F activity was measured using mouse bone marrow cells, and cell lines with high C3F activity were further selected.

Next, this selected cell line was cloned by the limiting dilution method, and transformed T-cells with high C5F production ability were cloned.
Lymphoid cells QC1-1 and QCI-2 were isolated in pure form.

QC of transformed T-lymphocytes with high C3F production ability
1-1 and QCI-2 can be established by, in addition to the above methods,
The method of Chen et al. [Proceedings of the National Academy of Sciences (
Proceeding of the National
Aeademy of 5 Sciences) No. 80
Vol. 7006-7009 Shin (1983)], and in the same manner as described above, a pure cell line can be obtained.

(2) Manufacture of human m-granulocyte-Kura-cell stem cell growth factor (
1) Add 1.0 to 10% (v/d) of human serum, bovine serum, or beef tallow to the cells exposed in step 1 at a rate of 1 to 5 x 10 cells/d.
Cell culture medium (e.g. RPMr −+640 medium, SIF, M medium, α-medium or Ham's medium, etc.) containing a concentration of v): inoculated with this and incubated at 37°C under aeration of carbon dioxide gas. Culture for 2-7 days. These H cells are adsorbed to supports and proliferate by adsorbing them to glass, plastics, carrier beads, etc., but at the same time they proliferate, what is the effect of promoting the proliferation of two bottles of human granulocytes and monocytic stem cells? 1. After culturing, collect the medium supernatant, add new medium and culture again. This medium exchange is performed at least 5 times, and the culture solution is collected. Next, the collected culture solution was centrifuged at 500 to 2500 G for 5 minutes, the supernatant was suspended with a filter membrane with a pore size of 45 to 3 μm, and the ML-filtered supernatant was treated with human Yingshun albumin. , bovine serum albumin 1-5% (v/v)
or the molecule f! t10000
Concentrate this medium supernatant using an ultrafiltration membrane to exclude more than 5-20% (v/v) human blood serum, bovine serum or fetal bovine serum that was initially added to the medium. Adjust to.

Next, the pH was adjusted to 6-8 +, :, WII, and heat treated for 10 to 15 hours at around 60" (:).

The heat-treated culture supernatant has no risk of ATLV infection, and the culture supernatant can be subjected to known purification methods such as ion-exchange chromatography, gel filtration, or reversed-phase chromatography. Human granulocyte-monocytic stem cell growth factor can be highly purified.

(3) Biological activity of human granulocyte-monocyte stem cell growth factor (3-1) Colony formation stimulating effect on human bone marrow cells (C5F activity) Bone marrow cells were collected from normal volunteers, and in Example 1 GM-C5F of the obtained human normal granulocyte-Kurumyu stem cell growth factor
activity and Multi-C5F activity by R.C.
The method of Ash et al.
:n1ood) m Volume 58j'W 309-31
6 (1981)] containing 0.9% methylcellulose and 30% tallow serum.
s) tested using modified media.

The results are shown in the g+ table. In addition, in order to compare with known C5F, GM-C5F was prepared using 0CT-cell culture medium [hereinafter sometimes abbreviated as GCT-CM, Gibco Co., Ltd.
Co., Ltd. (commercial product)] and phytohemagglutinin (Phyt.

he+aagglutinin) Stimulated R leukocyte culture medium (hereinafter abbreviated as PHA-LCM) [Nie Waanschaf et al.: Experimental Hematology (A, Wa
hnschaffeet at: Experience
ntal nemato+ogy) Volume 12, No. 655
~659 paintings (1984)] were used.

(Margins below) According to these results, the growth factor of Example 1 shows GM-CSF activity against human bone marrow cells, but Multi
-'C3F activity was not shown.

(3-2) Growth-promoting effect on human granulocyte-monocytic stem cells Granulocyte-monocytic stem cells in human bone Vl cells (hereinafter referred to as C
The following test was conducted to compare the effect of the number (sometimes abbreviated as FU-CM) with the known GM-C3F.

The bone marrow cells of a normal human volunteer were used as non-adsorbed bone marrow cells by the method of R.C. Ash et al., and the growth factor and GCT-cell culture medium of Example 1 were added to Iooo m, respectively.
20% (v/v) beef tallow (l5cove's containing υ serum) and modified Dulbecco's (15% (v/v) with no factor added (control))
Dulbecco's) MEM medium (hereinafter referred to as rMDM)
(sometimes abbreviated as medium) was cultured at 37°C under aeration. At 0.24, 48 and 72 hours after the start of culture, each bone marrow cell was centrifugally washed to obtain a cell suspension. As a human granulocyte-monocyte stem cell (CFU-GM) stimulating factor, phytohemagglutinin-stimulated leukocyte culture medium (
PHA-LCM) 100041st (quantified using mouse bone marrow cells described below), 20% tallow serum and 0.3%
The above cell suspension was mixed at 10% (v/v) in IMDM medium containing (v/v) agar, and 7.5
The cells were cultured for 14 days at 37° C. under aeration of % CO.

After culturing, aggregates consisting of 50 or more cells were transformed into CFU-G.
As M, bone marrow cells! C included per 10 pieces
The number of FU-CMs was changed to the tt side.

The results were as shown in Table 2.

(Remaining days below) Second death According to these results for human granulocyte-monocyte stem cells, no increase in the number of CFU-CM was observed in GM-C5F C, CT-CM, and control. ,
It was found that the growth factor of Example 1 had the effect of increasing the number of CFU-CM.

(3-3) Colony formation stimulating effect (C3F activity) on mouse bone marrow cells (, M-C5P and 5lu
ltE C5F activity was tested by the method of Ehle et al. described above, except that C5713L/6N mouse bone marrow tissue was used.

Furthermore, in order to unify the biological potency of the various stimulating factors used in the tests of the present invention, the potency of each factor was expressed as a C5F titer against mouse bone marrow cells.

That is, C5713L/6N mouse bone marrow cells I×10
20% (v/v) fetal bovine serum containing /- and 0
．． 3% agar and matsukoi (McCode 5) 5A medium,
Each factor was mixed and cultured at 37°C under 7.5% CO aeration for 7 days, and 50 or more cell aggregates formed were counted as colonies.
It was expressed as a titer of 5F.

GM-C3F against mouse bone marrow cells (, CT
-CM, Multi-C5F is WEH
I-3 cells @ bud fluid supernatant [hereinafter sometimes abbreviated as WEHr-3-CM, J.N. Ihre et al.: The Journal of Immunology (J.N.Ihre.
etal: The Journal of f+
++unology) Volume 129 No. 2431-243
6 (1982)] was used.

The C5F activity of the growth factor of the present invention against mouse bone marrow cells was as shown in Table 3.

(3-4) Growth promoting effect on mouse interleukin 3-dependent cells Using FDC-P2 cells, which are mouse interleukin 3 (hereinafter sometimes abbreviated as ratio-3) dependent cells, the method of Ehle et al. [J, Ihre et al.: The Journal of Immunology
: The Journal of [mmunol
ogy) Volume 129, No. 2431-2436 (1982
radiolabeled thymidine (H-Thymid)
ine) Measuring the uptake of the human nuchal granulocytes of banquet example I -
The activity of mouse interleukin 3, a growth factor for B cells in the m cell lineage, was tested.

The results were as shown in Figure 1.

In FIG. 1, the vertical axis shows 11-thymidine incorporation 1, the horizontal axis shows the dilution factor in logarithm, (-·-) is the control, and (-Δ-) is GCT-CM.

(-[]-> indicates the growth factor of Example 1, and (-0-) indicates WEHI-3-CM.

The growth factor of Example 1 exhibited IL-3 activity similar to that of WEHT-3-CM, a mouse interleukin 3 preparation, and promoted proliferation of FDC-P2 cells.

On the other hand, GM-C3F Teal GCT-CM is IL-
3 activity was not shown at all.

(4) Physical and chemical properties and thermal stability of human granulocyte-mocyte stem cell growth factor (,i-1) Anion exchanger chromatogram Example 1
The heat-treated culture supernatant obtained by the method of
Equilibrated with M phosphate lik buffer (pH: 7.0) and subjected to column chromatography using DEAE-Sepharose, an anion exchanger, to measure C3F activity in mouse spinal cord cells and TL-3 activity in FDC-P2 cells. The measurements were performed using the methods described above.

The results were as shown in Figure 2.

In Figure 2, the left vertical axis shows the absorbance (represented by -〇-), the vertical axis shows C5F activity (represented by -mu-) and IL-3 activity (-・-), and the horizontal axis shows the fraction number. shows.

According to FIG. 2, the human granulocyte-mocyte stem cell growth factor of Example 1 was eluted as a fraction not adsorbed to DEAE-Sepharose, and the C5F activity and rL-3 activity were completely consistent.

(4-2) Gel filtration chromatogram The human granulocyte-mocyte stem cell growth factor of Example 1, which was eluted as a fraction not adsorbed to DEAE-Sepharose, was filtered using an ultrafiltration membrane A-ray device (manufactured by Amicon). , PH-10')
It was subjected to high performance liquid gel filtration chromatography using TELRALiC1TSK 30005W (manufactured by Toyo Soda Co., Ltd.), and its molecular weight was measured.

The results were as shown in Figure 3.

In Figure 3, the left vertical axis shows absorbance, and the right vertical axis shows C5F activity (represented by -0-) and Roux 3 activity (-×
-), and the axis shows the fraction number.

According to FIG. 3, the human granulocyte-monocyte stem cell growth factor of Example 1 had a molecular weight ranging from 25,000 to 40,000.

(4-3) Concanavalin A-Sepharose chromatogram Molecule obtained by the above gel filtration chromatography @
The fraction of 25,000 to 40,000 was concentrated using an ultrafiltration membrane (manufactured by Asahi Kasei Corporation, Mini Module NM-3), and o.
s M NaC11lls Kaho02 M phosphate buffer f(
pH near, 2) and equilibrated with concanavalin A-
It was subjected to Sepharose column chromatography.

The results were as shown in FIG.

In the $4 diagram, the left vertical axis is the absorbance (expressed as -・-)
The right vertical axis shows C3F activity (represented by -0-), and the horizontal axis shows the fraction number.

According to Figure 8g4, the M-enhancing factor of Example 1 was adsorbed on concanavalin A-Sepharose and eluted with a 0.15M α-methylmannoside solution.

(4-4> Thermal Stability Test The culture FIl solution obtained by the method of Example 1 was heated to 0.
After filtering through a 45μ membrane, the culture supernatant was filtered to 60±2
℃ for 10 hours and tested for thermal stability and the effect of stabilizers. Growth factor stability is determined by CSF activity and FDC activity against marrow bone marrow cells, as described above.
- Measured by IL-3 activity against P2 dish cells.

Table 4 shows the results of a test in which the heat stabilizing effect of the addition of human serum albumin and bovine serum albumin was expressed as the recovery rate of each sample when the non-heated control was set as 100.

(Margins below) According to Table 4, when the culture supernatant (fetal bovine serum 1%, pH near 1) is heat-treated as it is at 60°C for 10 hours, the biological activity of growth factors decreases by about 5096 flE. Therefore, when human serum albumin or bovine groove albumin is added at 1.0-5.0% (v/v), the pH of the medium changes to 6.4-7.0, but the heat stability increases. With this addition amount, a recovery rate of approximately 70% or more could be obtained.

Table 5 shows the results of the thermostabilizing effect of adding human serum or bovine serum in the same manner as above.

(Margins below) According to Table 5, if the culture supernatant (fetal bovine serum 1%, pH near I) is directly heat-treated at 60°C for 10 hours, the biological activity of growth factors decreases to about 40%. human serum or bovine serum at 5.0-20.0% (v
/v), a recovery of 70% or more could be obtained.

Example I ATLV transformed T-lymphocyte cells Q C1-1 at 1O
% (V/v) fetal bovine serum supplemented in RPMI-1640@4/), and the proliferated cells were grown in RPMI-1640 medium 1000 supplemented with 1% (v/v) fetal bovine serum, 5×1.
The seeds were sown at a ratio of 0 cells/-, transferred to a plastic culture vessel (manufactured by Nunc, 330 x 2000.2 stages), and 7.5
% (v/v) Co 2 for 4 days at 37°C under aeration. After culturing, the medium was collected, irradiated with ultraviolet light, and the resulting medium was centrifuged at 400 G for 20 minutes at 4°C. The obtained supernatant was filtered through a 0.45μ filter membrane, and human serum albumin (Sigma W) was added aseptically at a concentration of 2.5% (Y/V).
Heat treatment was performed at 0°C±2°C for 12 hours. After heat treatment, ultrafiltration bag fi (manufactured by Asahi Kasei Corporation, Lab Module SI)
P-1013) rratm, desalted, 0.02 M
IJ acid 1111ti'e was equilibrated. A DEAE-Sepha0-strength column equilibrated with the buffer (
2.5 x 40 cm) to remove proteins, and the non-adsorbed fraction containing human granulocyte-mocyte stem cell growth factor (approximately 400
− was recovered. Human IIa of the non-adsorbed fraction obtained here
The activity of the globular-monocyte stem cell growth factor was calculated from the C5F activity value of mouse bone marrow cells to 1.8×1081.

Example 2 Q C1-2 as ATLV-transformed T-lymphocyte cells
The proliferated cells were seeded in the same manner as in Example 1, except that 2.5% (v/v) tallow blood m was added to Eagle's MEM i at a rate of 5 x 10 @/mA. The cells were cultured and filtered in the same manner as in Example 1 to prepare a supernatant. The obtained supernatant liquid was condensed to 1 lll by 2@ using an ultrafiltration device (manufactured by Asahi Kasei Co., Ltd., Lab Module SIP-1013), and heat-treated at 60° C.±2° C. for 15 hours. Next, the same treatment as in Example 1 was carried out to collect the non-adsorbed fraction containing human granulocyte-mocyte stem cell growth factor, which had been deproteinized using a DEAE-Sepharose column. The total activity of human granulocyte-S cell stem cells in this non-adsorbed fraction was 2.4 x +07jli in terms of mouse C5F activity.

〔Effect of the invention〕

(1) Human granulocyte-monocyte stem cell growth factor can be produced in large quantities.

(2) The substance produced by the present invention is a human [;M
-Has the effect of promoting C5F activity and proliferation of granulocyte-monocytic stem cells themselves.

(3) The substance produced by the present invention has C,M-C3F activity against mouse bone marrow cells, Multi-C5
It has F activity and mouse interleukin 3 activity.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the proliferation-promoting effect of the human granulocyte-monocyte stem cell growth factor of Example 1 on mouse interleukin 3-dependent cells, and FIG. 2 is the heat-treated culture supernatant of Example 1. FIG. 3 is a chart showing the results of molecular weight measurement by high performance liquid gel filtration chromatography of the human granulocyte-mocyte stem cell growth factor of Example 1, and FIG. 4 is a chart showing the results of concanavalin A-Sepharose chromatography of the human granulocyte-mocyte stem cell growth factor of Example 1. Applicant: Morinaga Milk Industry Co., Ltd.

Claims (5)

[Claims] (1) Cultivating human lymphocytes endowed with spontaneous proliferation in a cell culture medium to produce a substance that promotes the proliferation of human granulocyte-monocytic stem cells, and heat-treating this medium. 1. A novel method for producing human granulocyte-monocytic stem cell growth factor, which comprises recovering an active ingredient having an action of promoting proliferation of human granulocyte-monocytic stem cells. (2) The scope of the patent claim, characterized in that the human lymphocytes endowed with spontaneous proliferation are obtained by culturing normal human lymphocytes in combination with adult T-lymphocyte leukemia virus-infected cells. 2. A method for producing the novel human granulocyte-monocytic stem cell growth factor according to item 1. (3) The heat treatment is performed at pH 6 to 8 and 60 in the presence of a substance selected from the group consisting of human serum albumin, bovine serum albumin, human serum, bovine serum and mixtures thereof.
3. The novel method for producing human granulocyte-monocytic stem cell growth factor according to claim 1 or 2, which is carried out at ±2° C. for 10 to 15 hours. (4) The novel human granule according to any one of claims 1 to 3, wherein the heat treatment is performed in the presence of 1 to 5% (w/v) albumin. A method for producing monocytic stem cell growth factor. (5) The novel human granule according to any one of claims 1 to 4, wherein the heat treatment is performed in the presence of 5 to 20% (v/v) serum. A method for producing monocytic stem cell growth factor.