JPH0789907B2 - Cytokine-activated macrophage and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel method for producing cytokine-activated macrophages, and more specifically, to monocytes isolated from human peripheral blood, interleukin-2 (Interleukin-2). −2: IL−
Culture using a human serum-containing nutrient medium in the presence of 2),
The present invention relates to a novel method for producing cytokine-activated macrophages, which comprises separating and collecting the cytokine-activated macrophages produced in the culture medium.

[Prior Art] Biological Response Modifiers (B) such as cytokines are used as one of the immunotherapy for tumors.
It has been previously known to use peripheral blood lymphocytes or tumor tissue-infiltrating lymphocytes activated or proliferated by RM), and it has been reported that an excellent antitumor effect is observed particularly in mouse experimental systems. (J.Immunology, 132 , 2123
-2128, 1984 and J. Exp. Med., 156 , 385-397, 1982).

Rosenberg et al. Of the National Cancer Institute (NCI) reported that addition of IL-2 to unstimulated peripheral blood lymphocytes induced cells with high antitumor activity (Science , 223 , 1412-1415, 1984). The cells do not require antigenic stimulation, have a broad antitumor activity that is not defined by the major histocompatibility complex (MHC), and the presence of IL-2 when inducing the cells Given the necessary and sufficient conditions, they named the induced cells Lymphokine Activated Killer (LAK) cells. (J. Exp. Med., 155 , 1823-1841, 1982).

Since then, it became easier to obtain recombinant IL-2 by gene recombination technology (Nature, 302 , 305-310, 198).
3), clinical application of so-called adoptive immunotherapy, which is one of immunotherapy for tumors using LAK cells, has started,
Its usefulness has come to be recognized (N.Engl.JM
ed., 313 , 1485-1492, 1985 and N. Engl. J. Med., 316 , 898-
905, 1987).

LAK cells used in this adoptive immunotherapy are usually induced by culturing lymphocytes isolated from human peripheral blood in a nutrient medium containing or without serum in the presence of IL-2. It is considered that cell groups other than lymphocytes used as progenitor cells, particularly monocytes, act suppressively on the induction of LAK cells, and it is desirable to remove them from progenitor cells (JP-A-63- 28388, JP-A-63-202
378, JP-A-63-295510, etc.).

Recently, monocyte / macrophage fractions isolated from human peripheral blood were used to convert cytokines such as IL-1, IL-2, Tumo
r Necrosis Factor (TNF) or Interferon-γ (IFN-
It has been reported that macrophages with cytotoxic activity are induced by culturing in serum-containing nutrient medium for 18 to 48 hours in the presence of BRM such as γ) (Nature, 325 , 2
62-265, 1987, Nature, 323 , 86-89, 1986, J. Immunolog
y, 140 , 1345-1349, 1988 and Jpn. J. Cancer Res., 80 , 59-
64, 1989).

However, the cytotoxic activity of activated macrophages induced by these methods is not always high and is transient, and it has an effect only on specific tumor cells.

[Problems to be Solved by the Invention] The present inventors have focused their attention on monocytes, which have been conventionally thought to act suppressively during LAK cell induction, and as a result of intensive research, Isolated monocytes from undifferentiated state
It was found that macrophages induced by long-term culture in a human serum-containing nutrient medium in the presence of IL-2 have high cytotoxic activity against a wide range of tumor cells, and completed the present invention. It was

[Means for Solving Problems] That is, according to the present invention, monocytes isolated from human peripheral blood are used for a long period of time using a human serum-containing nutrient medium in the presence of interleukin-2 from an undifferentiated state. It is intended to provide a novel method for producing a cytokine-activated macrophage, which comprises culturing and separating and collecting the cytokine-activated macrophage produced in the culture medium.

In addition, as used herein, peripheral blood, monocytes, IL-
2. Human serum and cytokine-activated macrophages are as follows.

a) Peripheral blood The "peripheral blood" used in the present invention is peripheral blood collected from healthy persons, cancer-bearing patients and the like, which are not limited by age, species difference, sex difference and the like.

b) Monocytes "Monocytes" as used in the present invention are isolated from the peripheral blood as defined in a).

That is, "peripheral blood" is centrifuged to obtain a buffy coat (light yellow leukocyte layer), and this buffy coat is layered on a specific gravity separation liquid (Percoll (Pharmacia) or the like), and if necessary, a phosphate buffer (Phosphate). Buffered Saline: PB
After overlaying S), perform density gradient centrifugation. A group of cells with a low specific gravity (monocytes, which gather at the interface between the specific gravity separation liquid and PBS).
Macrophage, Natural Killer: N
(K) cells and platelets) are collected, and the obtained cell group is washed several times by centrifugation. The density gradient centrifugation can be repeated if necessary. 5-10% after washing
Suspended in a nutrient medium containing human serum (RPMI-1640, etc.) and added to a 96-well microtiter plate, 5% CO ₂
Incubate at 37 ° C for 60-90 minutes. After culturing, the non-adhesive cells in the supernatant are removed, and the monocytes with high purity can be prepared by washing several times with the nutrient medium kept at 37 ° C.

c) IL-2 The term “IL-2” used in the present invention includes natural ones derived from humans or animals such as mice and rats, or recombinant ones obtained by gene recombination technology. To be done.

d) Human serum "Human serum" used in the present invention means adult human serum which is not limited to blood group.

e) Cytokine-activated macrophage "Cytokine-activated macrophage" in the present invention
Is induced by culturing monocytes isolated from human peripheral blood for a long period of time using a human serum-containing nutrient medium in the presence of interleukin-2, and is not limited to NK cell sensitive or insensitive. It means a cytokine-activated macrophage having the ability to damage tumor cells.

[Examples] Hereinafter, the embodiments of the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the specific embodiments described herein.

A. Isolation of monocytes 200 ml of peripheral blood was collected from human and the red blood cells were removed by centrifugation to obtain a buffy coat. 1 / buffy coat
Mix with 4 ml of 2 volumes of Percoll solution (specific gravity 1.128), and
Overlay 3 ml of Percoll solution (specific gravity 1.064) with 1 ml of PBS, 400
Density gradient centrifugation was performed for 20 minutes at xg. After centrifugation, the cell fraction collected at the interface between Percoll solution (specific gravity 1.064) and PBS was collected. This fraction was washed by centrifugation at 150 xg for 10 minutes. After washing, the cells were resuspended in PBS, overlaid with Percoll solution (specific gravity 1.064), and subjected to density gradient centrifugation at 400 × g for 20 minutes to collect cell screens collected at the interface between Percoll solution and PBS. This cell fraction was centrifuged (150 × g, 5
10 minutes human serum after washing 3 times)
Suspended in RPMI-1640 medium (Gibco), 1 × 10 ⁵ cells
It was added to a 96-well flat-bottomed microtiter plate at a concentration of / 100 μl / well, and cultured for 60 minutes at 37 ° C. in 5% CO ₂ or less. After culturing, the non-adhesive cell population in the supernatant was removed, and the mixture was incubated at 37 ° C.
Washed 3 times with RPMI-1640 medium to obtain extremely high purity (99%
Above) Monocytes were obtained.

B. Induction of Cytokine-Activated Macrophages The monocytes obtained in A above were treated with 10-1,000 IU / ml of recombinant IL.
-2 (recombinant IL-2: rIL-2, Shionogi & Co., Ltd.)
And manufactured in RPMI-1640 medium containing 10% human serum,
Cytokine-activated macrophages were obtained by culturing at 37 ° C. under 5% CO ₂ for 3 to 17 days.

C. Separation and collection of cytokine-activated macrophages
After washing with, 2 mM EDTA-PBS (pH 7.2) was added at 0.5 ml / well, and the mixture was incubated at 37 ° C. for 15 minutes under 5% CO ₂ . After culturing, the floating cytokine-activated macrophages were collected. If necessary, a rubber policeman was used to collect the cytokine-activated macrophages remaining on the plate.

D. Characteristics of Cytokine-Activated Macrophages The characteristics of cytokine-activated macrophages obtained by the present invention were analyzed by intracellular non-specific esterase (Non Specif
ic Esterase (NSE) staining and fluorescent dye-labeled antibody method (Fluor
luminescence-labelled Antibody Method).

a) NSE staining method: Using α-naphthylbutyrate (manufactured by Hanai Chemical Co., Ltd.) as a substrate, the method of CYLi et al. (J. Histochem. Cytoche)
m., 21 , 1-22, 1973).

Monocytes obtained in A above 0.5 × 10 ^{6 to} 1.0 × 10 ⁶ / well
Lab Tek Camber (Sanko Junyaku Co., Ltd., # 4804), and RP containing 10% human serum and 1000 IU / ml rIL-2
Cytokine-activated macrophages were obtained by culturing in MI-1640 medium under 5% CO ₂ at 37 ° C for 8 days. The cells were fixed with a fixative (cold acetone: formalin: water = 9: 5: 6) for about 1 minute, then washed with water and air dried. Then, the cells were stained with a pararosaniline solution for about 60 minutes, washed with water and air-dried to prepare a sample of cytokine-activated macrophages.

The controls include monocytes, human peripheral blood mononuclear cells and rIL-
Samples of macrophages cultured in RPMI-1640 medium containing 10% human serum containing no 2 were prepared.

These specimens were examined using a phase contrast microscope (magnification: × 12
5) Monocytes and macrophages were identified.

Results: Shown in FIGS. 1-a to 1-d (photographs).

According to this staining method, normally, monocytes and macrophages have NSE in the cells and thus are stained in purple red, but the NSE in the cells is stained.
Cell groups such as lymphocytes that do not have no stain remain uncolored.

Therefore, monocytes and cytokine-activated macrophages in this study were stained very well in purple-red,
% Or more was confirmed to be NSE positive, and it was confirmed to be monocytes and macrophages.

b) Fluorescent dye-labeled antibody method This method is one of the immunohistochemical methods, and it is used as a marker for antibodies against cell surface antigens.
It is a method of confirming the location of cells having a surface antigen by observing the fluorescence excited under a fluorescence microscope using a fluorescent dye such as othiocyanate). Monocytes and macrophages in human peripheral blood have CD14 as a surface antigen, and NK cells and neutrophils have CD16. In addition, CD14 and CD16
As a monoclonal antibody that recognizes, for example,
MO2-FITC (Coulter Corp.) and anti Leulla-FITC
(Becton Dickinson) is used.

Method: Cytokine-activated macrophages obtained according to B) a) were loaded with MO2-FITC at 0.1% sodium azide and 0.
1% maggot serum albumin (Fraction V, Seikagaku Corporation)
(Manufactured by Mitsui Chemical Co., Ltd.) was dissolved in PBS and added, and the mixture was reacted for 30 minutes under ice cooling. After completion of the reaction, the specimen was prepared by washing with PBS.

As a control, anti Leulla-FITC was used to prepare a sample in the same manner as above.

These specimens were observed using a fluorescence microscope (magnification: × 400) to identify cytokine-activated macrophages.

Results: Shown in Figures 2-a and 2-b (photo).

The cytokine-activated macrophages of the present invention are MO2-F
ITC was found to emit yellow-green fluorescence very well and to be MO2-positive, that is, to be mostly macrophages.

Further, since the sample using anti Leulla-FITC does not emit fluorescence at all, the cytokine-activated macrophages of the present invention are completely contaminated with cells having anti-tumor activity such as anti Leulla-positive NK cells. It was found that there was no.

As described above, from the results obtained by the methods a) and b),
Cells having cytotoxic activity induced by the present invention,
It was confirmed to be activated macrophages induced by cytokines from extremely high purity monocytes.

E. Cytotoxic activity The cytotoxic activity of the cytokine-activated macrophages of the present invention is measured by ³ H-described below unless otherwise specified.
Tritirated Thymidine Release Tes
t).

The target tumor cells used in this test were human-derived Hela cells unless otherwise specified, and the numbers of cytokine-activated macrophages and target tumor cells were 1 × 10 ⁵ cells and 1 × 10 ⁵ , respectively. ⁴ cells were used.

( ³ H-thymidine release test method) Tumor cells were cultured in a Falcon culture bottle, and the tumor cells obtained 2-3 days after passage were used as target cells. To this target cell, 25 μCi / bottle of ³ H-thymidine was added and cultured for 4 to 5 hours, and the resulting ³ H-thymidine-labeled tumor cells were cultured in a new medium containing no ³ H-thymidine for 1 hour.
The cells were cultured for a period of time to obtain ³ H-thymidine-labeled tumor cells used in this test.

The ³ H-thymidine-labeled tumor cells were suspended in RPMI-1640 medium containing 10% fetal bovine serum, added to the cytokine-activated macrophages induced in a 96-well flat bottom microtiter plate, and cultured at 37 ° C for 48 hours. 100 μl of the supernatant was collected from each well, and the radioactivity of ³ H-thymidine released in the supernatant was measured by a liquid scintillation counter.

The cytotoxic activity can be calculated by the following formula.

a) Cytotoxic activity of macrophages activated by IL-2. IL-2 concentration Method: Monocytes isolated from human peripheral blood were treated with 10% human serum or
10% fetal bovine serum and 10, 100 and 1,000 IU / ml, respectively
The cytotoxic activity of cytokine-activated macrophages obtained by culturing for 8 days in RPMI-1640 medium containing rIL-2 was measured.

As a control, 10% human serum containing no rIL-2 or 10% human serum was used.
A culture in RPMI-1640 medium containing% fetal bovine serum was used.

Results: Shown in Table 1.

When rIL-2 was added, at each concentration, the cytokine-activated macrophages obtained by culturing in the human serum-containing medium had higher cytotoxic activity than those obtained by culturing in the FCS-containing medium. Further, regarding the rIL-2 concentration, a remarkable effect was observed at 10 IU / ml or more, particularly at 10 IU / ml or more.

・ Time of IL-2 addition Method: 10% human serum containing monocytes isolated from human peripheral blood
After preculturing in RPMI-1640 medium for 0, 1, 3, 5 and 7 days, respectively, in RPMI-1640 medium containing 10% human serum supplemented with 500 IU / ml of rIL-2, 8, 7, 5, respectively. The cytotoxic activity of cytokine-activated macrophages obtained by culturing for 3 and 1 day was measured.

In addition, as a control, R containing 10% human serum without rIL-2 was used.
Macrophages obtained by culturing in PMI-1640 medium for 8 days were used.

Results: Shown in Table 2.

The cytotoxic activity was determined by rIL- immediately after isolation of monocytes (day 0).
Cytokine-activated macrophages induced by culturing in a medium containing 2 for 8 days showed the maximum value, and the cytotoxic activity decreased as the preculture period became longer.

From this, when inducing macrophages having cytotoxic activity with rIL-2 using monocytes / macrophages, it has the highest cytotoxic activity by culturing with rIL-2 from an undifferentiated state in the early stage of induction. It was found that cytokine-activated macrophages can be induced.

-Comparison with LAK cells For the purpose of comparing the difference in the mechanism of cytotoxic action between cytokine-activated macrophages obtained according to the present invention and LAK cells, the following ⁵¹ Cr release test and ³ H-thymidine release test were used. The experiment was done.

Method: Cytokine-activated macrophages obtained by culturing monocytes isolated from human peripheral blood for 3 or 10 days in RPMI-1640 medium containing 10% human serum and 1,000 IU / ml rIL-2 And LAK cells obtained by operating the lymphocytes isolated from human peripheral blood in the same manner as described above to determine the cytotoxic activity of both for 4 hours ⁵¹ Cr release test and 48 hours ³ H- It was measured by the thymidine release test.

Results: Shown in Tables 3 and 4. ^{In the 51} Cr release test, when target tumor cells were damaged by the effector having cytotoxic activity such as LAK cells or cytokine-activated macrophages obtained in the present invention on the cell membrane, ⁵¹ Cr incorporated into the cytoplasm of tumor cells was detected. It is released and the cytotoxic activity is calculated by measuring its radioactivity. On the other hand, in the ³ H-thymidine release test, DNA in the nucleus of the target tumor cell was detected by the effector as described above.
When is destroyed, ³ H-thymidine incorporated into DNA is released, and the cytotoxic activity is calculated by measuring the radioactivity.

The cytokine-activated macrophages of the present invention showed low cytotoxic activity in any culture period in the short-time ⁵¹ Cr release test, but were obtained by culturing for 10 days in the 48-hour ³ H-thymidine release test. The obtained product showed extremely high cytotoxic activity.

On the other hand, LAK cells showed high cytotoxic activity in the ⁵¹ Cr release test, but almost no cytotoxic activity in any culture period in the ³ H-thymidine release test.

From the above results, the cytokine-activated macrophages obtained in the present invention have a mechanism completely different from the tumor cytotoxicity of known LAK cells, that is, by destroying the DNA in the nucleus of the target tumor cell. It was suggested to have a mechanism of necrosis of tumor cells.

-Culture period in IL-2 containing medium Method: Monocytes isolated from human peripheral blood were treated in RPMI-1640 medium containing 10% human serum and 500 IU / ml rIL-2 for 3, 6, 10 and The cytotoxic activity of cytokine-activated macrophages obtained by culturing for 17 days was measured.

Results: Shown in Table 5.

When IL-2 is used to induce macrophages having cytotoxic activity, it shows almost no cytotoxic activity in culture within 3 days, but the cytotoxic activity rapidly increases after 6 days, and after 10 days, The highest value was shown, and extremely high cytotoxic activity was maintained on day 17 thereafter.

From this, it was confirmed that cytokine-activated macrophages having extremely high cytotoxic activity were induced by culturing for 6 days or more, as compared with the conventional short-term (1 to 3 days) culturing.

・ Cytotoxic activity against various tumor cells Method: Monocytes isolated from human peripheral blood were treated with 500 IU / ml of rIL
-8 and 8 in RPMI-1640 medium containing 10% human serum
Using cytocan-activated macrophages obtained by culturing for a day, the cytotoxic activity against various tumor cells was examined.

As a control, macrophages obtained in the same manner in RPMI-1640 medium containing 10% human serum without rIL-2 were used.

The target tumor cells used are as follows.

Hela, K562, A375, HL60, U937, P815, MethA, LP3 Among the target tumor cells, K562 is sensitive to NH cells and Hela, A375, HL60 and U937 are insensitive to NK cells.

Results: Shown in Table 6.

It showed cytotoxic activity against all tumor cells regardless of NK cell sensitivity or insensitivity. In particular, Hela of human origin
It showed extremely high cytotoxic activity against cells and K562 cells.

From this, it was confirmed that the cytocan-activated macrophages of the present invention have cytotoxic activity against a wide range of tumor cells without being limited to NK cell sensitivity or insensitivity.

F. Antitumor activity in animal model The antitumor activity of the cytocan-activated macrophages of the present invention was measured using an animal model. The cytocan-activated macrophages, tumor cells and animals used in this experiment are as follows.

(Cytocan-activated macrophage) The monocytes obtained in Example A were treated with RPMI-16 containing 500 IU / ml of rIL-2 (Shionogi Pharmaceutical Co., Ltd.) and 10% human serum.
Culture in 40 medium, 5% CO ₂ at 37 ° C. for 6 to 10 days,
Cytocan-activated macrophages obtained in the same manner as in Example C were washed twice with a washing solution (HANKS; manufactured by Nissui Co., Ltd.),
It was suspended in the same washing solution.

(Tumor cells) A375 human melanoma cell line cultured in RPMI-1640 medium containing 10% human serum was used.

(Animal) Balb / c nu / nu nude mice aged 4 to 6 weeks were used.

Method 1: One Balb / c nu / nu nude mouse (5 per group)
To the cytokine-activated macrophages (2 x
10 ⁶ cells) and A375 human melanoma cells (1 × 10 ⁶ cells)
The mixture suspended in HANKS (200 μl) was subcutaneously injected on the back.

On the 7th, 12th, 14th and 20th days after administration, the tumor area of the sample mouse was measured based on the following tumor area calculation formula.

As a control, A375 human maranoma cells (1 × 10 ⁶ cell /
Balb / c nu / nu nude mice to which only 200 μl / mouse were administered were used.

The results are shown in Fig. 3.

Method 2: the above Balb / c nu / nu nude mice (6 mice per group)
A375 human melanoma cells (1 × 10 ⁶ cells / 200 μl /
2), and cytokine-activated macrophages (2 × 10 ⁶ cells / 200 μl / animal) were also subcutaneously administered to the back.

On day 4, 7, 10 and 14 after administration, the tumor diameter of the sample mouse was measured in the same manner as in Method 1.

As a control, A375 human melanoma cells (1 × 10 ⁶ cell /
Balb / c nu / nu nude mice to which only 200 μl / mouse were administered were used.

The results are shown in Fig. 4.

In the control group in which only the A375 human melanoma cells were administered and the cytokine-activated macrophages of the present invention were not administered, a marked increase in tumor was observed, whereas the A375 human melanoma cells were mixed with the cytokine-activated macrophages of the present invention. A remarkable suppression of tumor growth was observed in the group of animals (method 1) that were administered afterwards. In addition, in a group of animals (method 2) in which the cytokine-activated macrophage of the present invention was administered to the tumor site after pre-administration of A375 human melanoma cells assuming a human cancer patient, suppression of tumor growth was suppressed compared to the control group. Admitted.

This experiment demonstrated that the cytokine-activated macrophages of the present invention are effective in treating cancer in tumor-bearing animals.

[Effect of the Invention] The cytokine-activated macrophage of the present invention has a high cytotoxic activity against a wide range of tumor cells, and is useful as a cell type used in adoptive immunotherapy in the field of cancer treatment.

[Brief description of drawings]

1-a to 1-d are microscopic photographs showing the biological morphology of human peripheral blood mononuclear cells, macrophages and the like in the NSE staining test. That is, FIG. 1-a is a microscopic photograph of a human peripheral blood mononuclear cell specimen, FIG. 1-b is a microscopic photograph of a macrophage specimen, and FIG. 1-c is a microscopic photograph of a monocyte specimen. d is rIL
2 shows a micrograph of a macrophage specimen activated by -2. 2-a and 2-b are microscopic photographs showing the biological morphology of cytokine-activated macrophages in the fluorescent dye-labeled antibody test. That is, FIG. 2-a shows a microscopic photograph of a specimen by MO2-FITC, and FIG. 2-b shows a spectroscopic photograph of a specimen by anti Leulla-FITC. FIGS. 3 and 4 show the antitumor activity of the cytokine-activated macrophages of the present invention in animal models by increasing or decreasing the tumor area. That is, FIG. 3 shows increase / decrease in tumor area after administration of a mixture of the cytokine-activated macrophage of the present invention and A375 human melanoma cells to Balb / c nu / nu nude mice. The vertical axis represents the tumor area (mm ² ) obtained based on the tumor area calculation formula, and the horizontal axis represents the number of days elapsed after administration of A375 human melanoma cells. Figure 4 shows Balb / c nu / nu
Pre-administered A375 human melanoma cells to nude mice,
2 shows increase / decrease in tumor area after administration of the cytokine-activated macrophages of the present invention after 2 days. The vertical axis is the tumor area (mm ² ) obtained based on the tumor area calculation formula, and the horizontal axis is A375.
It represents the number of days elapsed after administration of human melanoma cells.

Claims (2)

[Claims] 1. A method for producing a cytokine-activated macrophage, comprising monocytes isolated from human peripheral blood in a human serum-containing nutrient medium in the presence of interleukin-2 at a concentration of at least 100 IU / ml. It is characterized in that it is cultured for 17 days, and the obtained cytokine-activated macrophage is ³ H by mixed culture for 48 hours at a cell number ratio of the cytokine-activated macrophage and ³ H-thymidine-labeled tumor cells of 10: 1. -A method for producing a cytokine-activating macrophage that exhibits cytotoxic activity against the following tumor cells in the thymidine release test: Hela, K562, A375, HL60, U937, P815, MethA and LP3. 2. A method for culturing monocytes isolated from human peripheral blood in a human serum-containing nutrient medium for 8 to 17 days in the presence of interleukin-2 at a concentration of at least 100 IU / ml. a cytokine-activated macrophages, the cytokine-activated macrophages, ³ H- thymidine cell number ratio of the cytokine-activated macrophages and ³ H- thymidine-labeled tumor cells by mixed culture of 48 hours at 10 to 1 In a release test, a cytokine-activated macrophage characterized by exhibiting cytotoxic activity against the following tumor cells: Hela, K562, A375, HL60, U937, P815, MethA and LP3.