JPH06305972A - Antitumor agent containing cell injuring macrophage - Google Patents

Abstract

PURPOSE:To provide a cell injuring macrophage remarkably useful for adoptive immunity therapy to a tumor bearing patient. CONSTITUTION:The antitumor agent containing a cell injuring macrophage obtained by culturing bone marrow cells of a mammalian animal in the presence of interleukin 2 and other cytokine participating in differentiation or activation of monocyte/macrophage by using a nutritive medium.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a cytotoxic macrophage.
More specifically, the present invention relates to an antitumor agent containing diene, containing bone marrow cells (Bone Marrow cell (s)) of a mammal.
Cytotoxic macrophages obtained by culturing in a nutrient medium in the presence of leukin-2 (Interleukin-2: IL-2) and other cytokines involved in monocyte / macrophage differentiation or activation The present invention relates to an antitumor agent containing

[0002]

2. Description of the Related Art Cells involved in the immune system of a living body are proliferated and activated by a biological response modifier (BRM) such as a cytokine, and the activated cells are used to necrose the tumor or metastasize the tumor. It is conventionally known to use peripheral blood lymphocytes or tumor tissue infiltrating lymphocytes as an immunotherapy for suppressing the so-called adoptive immunotherapy, and an excellent antitumor effect is recognized especially in mouse experimental systems. Has been reported (J.Immunolo
gy, 132, 2123-2128, 1984 and J. Exp. Med., 156, 385-397, 19
82 etc.). For activated lymphocytes, the study by Rosenberg et al. Of the National Cancer Institute (NCI) is well known, and they reported that IL-2 was added to unstimulated peripheral blood lymphocytes. It was found that cells having high antitumor activity were induced, and the cells having the antitumor activity were induced by lymphokine activated killer (Lymphokine Activated K
iller: LAK) cells (J.Exp.Med., 155,1823-).
1841,1982 and Science, 223,1412-1415,1984). After that, because recombinant IL-2 became easy to obtain by gene recombination technology (Nature, 302, 305-310, 1983),
Clinical application of adoptive immunotherapy for tumors using the LAK cells has started, and its usefulness has been recognized (N. Engl. J. Med., 313, 1485-1492, 1985 and N. .En
gl.J.Med., 316,898-905,1987 etc.). For activated macrophages, monocytes isolated from human peripheral blood /
The macrophage fraction was transferred to Interleukin-1 (Interl
eukin-1; IL-1), IL-2, tumor necrosis factor (Tumo
ur Necrosis Factor (TNF) or interferon-
In nutrient medium in the presence of γ (Interferon-γ; TNF-γ)
It has been reported that macrophages having cytotoxic activity are induced by culturing for 18 to 48 hours (Na
ture, 325,262-265,1987, Nature, 323,86-89,1986, JI
mmunology, 140, 1345-1349, 1988 and Jpn. J. Cancer Res.,
80,59-64,1989). In addition, monocytes isolated from human peripheral blood were treated with IL-2, or IL-2 and colony-stimulating factor-1.
(Colony Stimulating Factor-1; CSF-1) or by culturing in a nutrient medium in the presence of IL-2 and IL-1 for 6 days or more, a higher cytotoxic activity than that of the activated macrophages is obtained. It has been reported that the macrophages possessed therein are induced (Japanese Patent Application Laid-Open No. 4-4870). In addition, Lohmann-Matthes et al. Used a mixed cell group of various cells including bone marrow cells collected from a mouse thigh for IL-2.
And weakly adherent LAK or NK morphologically similar to large granule lymphocytes (Large Glanule Lymphoid Cells; LGL) that have their origin in lymphoid stem cells by culturing in a nutrient medium in the presence of CSF-1 (Natural Kille
r) It has been reported that cells having cell-like cytotoxic activity are induced (Scand. J. Immunol., 33, 511-520,
1991 and J. Exp. Med., 142, 118-125, 1989).

[0003]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention The present inventors focused their attention on bone marrow cells that are the origin cells of macrophages, which are one of the immunocompetent cells of mammals, and as a result of earnest research,
Induction of mammalian bone marrow cells from an undifferentiated state by long-term culture in nutrient medium in the presence of other cytokines involved in the differentiation or activation of interleukin-2 and monocytes / macrophages The macrofuge
The inventors have found that they have high cytotoxic activity against a wide range of tumor cells in vitro and in vivo, and have completed the present invention.

[0004]

[Means for Solving the Problems] That is, according to the present invention, mammalian bone marrow cells are transformed from an undifferentiated state to an interleukin-
2 and an antitumor agent containing cytotoxic macrophages obtained by long-term culture in a nutrient medium in the presence of other cytokines involved in monocyte / macrophage differentiation or activation It is provided.

As used herein, "mammalian bone marrow cells", "other cytokines involved in monocyte / macrophage differentiation or activation", "interleukin-2 (IL-2)". , "Macrophage colony
Stimulator (M-CSF) "," cytotoxic macropha
The terms "di" and "tumor" are as follows.

A) Mammalian bone marrow cells "Mammalian bone marrow cells" used in the present invention mean bone marrow cells of healthy animals or cancer-bearing animals which are not limited by species difference, sex difference and age. However, examples thereof include bone marrow cells of human, monkey, cow, horse, rabbit, guinea pig, rat, mouse and the like, and particularly preferable are human bone marrow cells.

B) Other cytokines involved in monocyte / macrophage differentiation or activation "The other cytokines involved in monocyte / macrophage differentiation or activation" used in the present invention are: MC
It means SF, IL-1, TNF, IFN and the like, and particularly preferably M-CSF. c) Inter-leukin-2 (IL-2) "Inter-leukin-2 (IL-)" used in the present invention.
The term “2)” includes a natural type derived from a human or an animal such as a mouse or a rat, or a recombinant type obtained by a gene recombination technique.

D) Macrophage-colonie-stimulating factor (M-CSF) The “macrophage-colonie-stimulating factor (M-CSF)” used in the present invention is of human origin or animal origin such as mouse or rat. A natural type or a recombinant type obtained by a gene recombination technique is included.

E) Cytotoxic macrophages The cytotoxic macrophages in the present invention have the ability to damage not only the cell membrane but also the DNA of a wide range of tumor cells which are not limited to NK cell sensitivity or insensitivity. Means macrophage.

F) Tumor The "tumor" to which the present invention is applicable includes, for example, cervical cancer, erythroid leukemia, myeloblastoma, histiocytoma, mastocytoma, melanoma and the like.

The antitumor agent containing the cytotoxic macrophage of the present invention is administered as an injection, similar to the administration method in adoptive immunotherapy such as LAK therapy. As an injection, according to a conventional method, it can be produced by suspending the cytotoxic macrophage of the present invention as an active ingredient and suspending it in a physiological saline solution for injection or the like usually used for dissolving or suspending the active ingredient. , Or a stabilizer depending on the purpose,
Preservatives, excipients, etc. can also be added. As a route of administration by injection, it can be injected intradermally, subcutaneously, intramuscularly, intravenously, intraarterially, intravertebral cavity or the like depending on the site of injection, and local administration is preferred. The dose varies depending on the type of tumor, condition, age, weight, etc. of the tumor-bearing living body. For example, when administered locally to the tumor, the number of cells per administration is in the range of 10 ^{6 to} 10 ^7. be able to. Further, since the antitumor agent containing the cytotoxic macrophage of the present invention is a cell derived from a living body, it can be used as a medicine without concern about toxicity of the cell.

[0012]

EXAMPLES Hereinafter, the embodiments of the present invention will be described more specifically by way of examples, but the present invention is not limited to the specific embodiments described herein.

A. Induction and Collection of Cytotoxic Macrophages Bone marrow cells were collected from the bone marrow collected from the femur of female C57BL / 6 mice 6-8 weeks old. The bone marrow cells
000 U / ml recombinant IL-2 (recombinant IL
-2: rIL-2, Shionogi Pharmaceutical Co., Ltd.) and 10 U / ml
RPMI16 containing M-CSF (manufactured by Genzym)
96-well multititer plate (manufactured by Falcon) by adding to 40 medium and adjusting to 1 × 10 ⁵ cells / ml
Alternatively, using a φ5 cm culture dish (manufactured by Falcon), each 1 ×
Cytotoxic macrophages were obtained by culturing at a concentration of 10 ⁴ cells / well and 5 × 10 ⁵ cells / culture dish at 37 ° C. in 5% CO ₂ for 6 days. After washing the induced cytotoxic macrophages with PBS, 2 mM ED
TA-PBS (pH 7.2) was added, and under 5% CO ₂ .
Incubated at 37 ° C for 15 minutes. After culturing, the suspended cytotoxic macrophages were collected. If necessary, a rubber policeman was used to collect the cytotoxic macrophages remaining on the plate.

B. Differences in Culture Produced Cells Depending on Presence or Absence of IL-2 and / or M-CSF In the induction of cytotoxic macrophages, differences in the number of produced cells after culture and the properties of the cells depending on the presence or absence of IL-2 and / or M-CSF were measured investigated. The mouse bone marrow cells obtained in A were treated with IL-2 at a concentration of 1 × 10 ⁵ cells / ml.
(1000 U / ml) and M-CSF (10 U / m
l), IL-2 (1000 U / ml) only, M-CSF
RPMI1640 each containing only (10 U / ml)
The cells were cultured in a medium at 37 ° C. under 5% CO ₂ for 6 days, and the number of viable cells recovered and the properties of the cells were examined. As a control experiment, RPMI1 containing neither IL-2 nor M-CSF was used.
Culturing was carried out in 640 medium at 37 ° C. under 5% CO ₂ for 6 days. The results are shown in Table 1.

[0015]

[Table 1]

In the cultured cell group in the presence of M-CSF (10 U / ml), remarkable growth of adherent cells was observed. Furthermore, IL-2 (1000 U / ml) and M-CSF
In the cultured cell group in the presence of (10 U / ml), the proliferation of non-adhesive cells was observed along with the proliferation of adherent cells. In the cell group cultured in the absence of M-CSF, no significant proliferation was observed in both adherent cells and non-adherent cells.

C. Cytological Properties of Cytotoxic Macrophages Cells such as T lymphocytes, B lymphocytes, and macrophages express a unique surface antigen (Surface Marker) on their cell surface at the stage of cell differentiation and maturation. It is possible to understand what cell lineage the cell belongs to and what kind of differentiation and maturation process it is in.
For example, macrophages, NK cells, T cells and B cells are Mac-1, NK1.1, Thy-1, B22, respectively.
It expresses a surface antigen called 0.

In order to examine the cytological properties of the cytotoxic macrophages of the present invention, a fluorescent dye-labeled antibody method using a monoclonal antibody against each surface antigen of various cells was used to obtain the above in A using a flow cytometer. The expression state of the cell membrane surface antigen of the cytotoxic macrophages was analyzed.

The monoclonal antibody used in this experiment was a Picoerythrine (PE) -labeled anti-mouse Mac-1 monoclonal antibody (Boehringer Mamheim G).
mbH), PE-labeled anti-mouse NK1.1 monoclonal antibody (Phar Mingen), Fluorscein (FI)
TC) labeled anti-mouse Thy-1 monoclonal antibody (Phar
Mingen) and FITC-labeled anti-mouse B220 monoclonal antibody (Phar Mingen).

As a control, M-CSF in the above B was used.
The macrophages obtained by culturing the cells alone were used. The results are shown in FIGS.

The cytotoxic macrophages obtained in the present invention are strongly positive for Mac-1 (FIGS. 1 and 5), like the control macrophages, and are NK1.1, Thy-.
1 and B220 were all negative (FIGS. 2 and 6, respectively).
3 and 7, FIG. 4 and FIG. 8). From these results, the cytotoxic cells obtained in the present invention are NK cells,
It was confirmed to be purely macrophages, which were neither T cells nor B cells.

D. Cytotoxic activity (antitumor activity) The cytotoxic activity of the cytotoxic macrophages of the present invention is described below in the ³ H-thymidine release test (Tritiated Thymid).
ine Release Test). ( 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. 2 for this target cell
³ H-thymidine-labeled tumor cells obtained by adding 5 μCi / bottle of ³ H-thymidine and culturing for 4 to 5 hours were treated with ³ H-.
³ H-thymidine-labeled tumor cells used in this test were obtained by culturing in a fresh medium containing no thymidine for 1 hour. The ³ H-thymidine-labeled tumor cells were suspended in RPMI-1640 medium containing 10% fetal bovine serum, added to the cytotoxic macrophages induced by A, and 100 μl of supernatant was collected from each well. The radioactivity of < ³ > H-thymidine released in E. coli was measured by liquid scintillation counter. The cytotoxic activity can be calculated by the following formula.

[0023]

[Equation 1]

A) Strength of cytotoxic activity In order to examine the strength of cytotoxicity (antitumor activity) of the cytotoxic macrophage of the present invention, B16 derived from mouse is used.
For melanoma (melanoma) 1 × 10 ⁴ cells, the above A
2.5 x 1 for each cytotoxic macrophage obtained in
The antitumor effect when applied at cell concentrations of 0 ⁴ , 5 × 10 ⁴ , 1 × 10 ⁵ and 2 × 10 ⁵ was measured. The results are shown in Table 2.

[0025]

[Table 2]

As a control, M-CS in the above B was used.
Macrophages obtained by culturing with F (10 U / ml) only were used. Macrophages induced by culturing M-CSF alone showed no cytotoxic activity, whereas macrophages cultured and induced in the presence of IL-2 and M-CSF showed high cytotoxic activity.

B) Cytotoxic activity against various tumor cells Cytotoxic macrophages (1 × 10 5) obtained in the above A.
⁵ cells) were used to examine the cytotoxic activity (antitumor activity) against various tumor cells (1 × 10 ⁴ cells). In addition,
M-CSF (10 U / ml) in B as a control
The macrophages obtained by culturing and inducing the cells alone were used. The target tumor cells used in this test are as follows. Mouse origin: B16 melanoma (melanoma), LP3, P815 and YAC-1, human origin H
ela (cervical cancer), and K562 (erythroid leukemia)
Is. Of the above target tumor cells, K562 and YAC-1 are sensitive to NK cells, and Hela and B1
6 Melanoma, LP3 and P815 are NK cell insensitive. The results are shown in Table 3.

[0028]

[Table 3]

The control macrophages cultured and induced with M-CSF alone showed almost no cytotoxic activity against all tumor cells, but the cytotoxic macrophages of the present invention were limited to NK cell sensitive and insensitive cells. However, it showed high cytotoxic activity against all tumor cells. This indicates that the cytotoxic macrophage of the present invention has high cytotoxic activity against a wide range of tumor cells without being limited to NK cell sensitive or insensitive.

C) Cytotoxic activity (antitumor effect) in animal model The cytotoxic activity (antitumor activity) of the cytotoxic macrophages of the present invention was examined in an animal model (in vivo). The cytotoxic macrophages obtained in A and the macrophages obtained by culturing in B only with M-CSF (10 U / ml) were each washed twice with physiological saline for injection (Otsuka Pharmaceutical Co., Ltd.), Each suspension was suspended in the same physiological saline to prepare an injection. B16 melanoma cells suspended in physiological saline were intravenously injected into C57BL / 6 mice (5 mice per group) at a concentration of 2 × 10 ⁵ cells / mouse, and 7 days later, each injection prepared above Using the agent
IL-2 and M-CS at a concentration of 2 × 10 ⁶ cells / mouse
Macrophages induced by F, and M-CSF (1
Macrophages induced only with 0 U / ml) were injected intravenously into each group. Seven days after macrophage administration, each mouse was thoracotomized and lung metastases colonies were counted. As a control, a group of mice to which no macrophage was administered after intravenous injection of B16 melanoma tail was used. The results are shown in Table 4.

[0031]

[Table 4]

In the group administered with the cytotoxic macrophages obtained in the above A, tumor metastasis was significantly suppressed as compared with the group administered with M-CSF-only macrophages and the control group.

[0033]

The cytotoxic macrophages of the present invention have been shown to be extremely useful in the application of adoptive immunotherapy to cancer-bearing patients.

[Brief description of drawings]

1 to 8 show the expression state of the cell surface antigen of the cytotoxic macrophages of the present invention measured by a fluorescent dye-labeled antibody method using a flow cytometer. 1 to 8, the horizontal axis represents fluorescence intensity and the vertical axis represents cell number.

FIG. 1 shows the expression state of Mac-1 by an anti-Mac-1 monoclonal antibody in cytotoxic macrophages cultured in the presence of IL-2 and M-CSF.

FIG. 2 shows M of anti-Mac-1 monoclonal antibody in cytotoxic macrophages cultured in the presence of M-CSF.
It shows the state of expression of ac-1.

FIG. 3 shows the expression state of NK1.1 by an anti-NK1.1 monoclonal antibody in cytotoxic macrophages cultured in the presence of IL-2 and M-CSF.

[Fig. 4] N by anti-NK1.1 monoclonal antibody of cytotoxic macrophages cultured in the presence of M-CSF.
It shows the state of expression of K1.1.

FIG. 5 shows the expression state of Thy-1.2 by anti-Thy-1.2 monoclonal antibody in cytotoxic macrophages cultured in the presence of IL-2 and M-CSF.

FIG. 6 shows the expression state of Thy-1.2 by anti-Thy-1.2 monoclonal antibody in cytotoxic macrophages cultured in the presence of M-CSF.

FIG. 7 shows the expression of B220 by anti-B220 monoclonal antibody in cytotoxic macrophages cultured in the presence of IL-2 and M-CSF.

FIG. 8: B2 of anti-B220 monoclonal antibody on cytotoxic macrophages cultured in the presence of M-CSF.
20 shows the expression state of 20.

Claims (5)

[Claims] 1. Cells obtained by culturing mammalian bone marrow cells in a nutrient medium in the presence of interleukin-2 and other cytokines involved in the differentiation or activation of monocytes / macrophages. An antitumor agent comprising a damaging macrophage. 2. The antitumor agent according to claim 1, which has a culture period of 6 days or more. 3. The antitumor agent according to claim 2, wherein the culture period is 6 to 11 days. 4. The antitumor agent according to any one of claims 1 to 3, wherein the other cytokine involved in monocyte / macrophage differentiation or activation is M-CSF. 5. The antitumor agent according to claim 1, wherein the tumor is cervical cancer, erythroblastic leukemia, myeloblastoma, histiocytoma, mastocytoma or melanoma.