JP2005281266A - Pharmaceutical composition containing thermocell vaccine and cytokine and method for preparing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a thermocell vaccine and a kit of cytokine excellent in immune effects and containing various kinds of antigens and having high immune effects on antigens each having low immunogenecity. <P>SOLUTION: The method for preparing the thermocell vaccine includes to harvest cells from an individual living body, treat the cells with heat to exhibit a heat shock protein and dissolve the treated cells. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a pharmaceutical composition comprising a thermal cell vaccine for cancer cells that can be used for vaccine therapy, particularly cancer treatment, and cytokines.

  Immunotherapy has been actively studied as the fourth cancer treatment method following surgery, chemotherapy, and radiation therapy. Immunotherapy includes treatment using non-specific immunostimulators, cytokine therapy, adoptive immunotherapy, monoclonal antibody therapy, gene therapy, etc. One of them is induction of cancer-specific immunity by administration of cancer-specific antigen. Intended vaccine therapy is also included. Many epitopes (cancer antigen peptides) of proteins that can become human cancer antigens have been identified so far, and vaccine therapy using these cancer antigen peptides has been performed. However, since the type of cancer antigen varies from patient to patient, there is almost no antitumor effect even when only one type of identified cancer antigen peptide is administered, and a mixture containing many types of antigen peptides is desired. In addition, since cancer cells of a patient contain a cancer antigen peptide unique to the patient, there is a method of using the cancer cells themselves as a vaccine. In this case, the cancer cells are subjected to a treatment such as freezing and thawing and used as a vaccine in the state of a cell lysate. Although this method may contain many types of patient-specific cancer antigens, the amount of cancer-specific antigen protein in the cell lysate is low, while the amount of protein that can be administered is limited. Therefore, there is a problem in that high specific immunity against the anti-tumor effect cannot be obtained. In order to solve these problems of vaccine therapy, development of a vaccine containing a large amount of various cancer antigen peptides is desired.

Heat shock protein (Heat shook protein, HSP) is a protein family that is expressed by stress such as heat. Among HSPs, HSP70, HSP90, gp96 and the like are known to bind to antigenic peptides in cancer cells, and to be taken up by antigen-presenting cells when released from cancer cells to promote presentation of antigenic peptides. . And, until now, the expression of HSP70 is increased in cancer cells that have been subjected to thermothermal treatment, and when this is transplanted into rats, the engraftment is suppressed (Non-patent Document 1), It has been reported that when cancer cells transplanted into rats are heat-treated, anti-tumor immunity of the host is strongly induced, and engraftment of the transplanted cancer cells is strongly suppressed thereafter (Non-patent Document 2). However, it was unclear whether cancer cells expressing a large amount of HSP70 could be directly inoculated to obtain a strong antitumor effect. Therefore, the inventors of the present invention, in the present invention, in the present invention, a heat-treated cancer cell lysate such as vaccination for infectious diseases (hereinafter, heat treatment including cancer cells to be inoculated into a living individual like vaccination for infectious diseases) It was clarified that the antitumor effect on transplanted cancer cells can be induced by inoculating rats and mice with the lysate of the obtained cells called a hyperthermic cell vaccine. However, it has been clarified that even though the antitumor effect of this thermal cell vaccine is effective for certain cancer cells, it is not necessarily applicable to all cancer cells. They found that the cause is the low immunogenicity of the antigen. Therefore, a substance that can further enhance the immune effect of the thermal cell vaccine is searched, and the interleukin 12, which is one of the cytokines, is not effective in a vaccine for cells that are not subjected to thermal treatment, but a thermal cell vaccine for cells that are subjected to thermal treatment. Was found to have an immune enhancing effect.
Ito A, Shinkai M, Honda H, Wakabayashi T, Yoshida J, Kobayashi T. Augmentation of MHC class I antigen presentation via heat shock protein expression by hyperthermia. Cancer Immunol Immunother. 2001 Dec; 50 (10): 515-22. Ito A, Shinkai M, Honda H, Yoshikawa K, Saga S, Wakabayashi T, Yoshida J, Kobayashi T. Heat shock protein 70 expression induces antitumor immunity during intracellular hyperthermia Using magnetite nanoparticles. Cancer Immunol Immunother. 2003 Feb; 52 (2) : 80-8.

  An object of the present invention is to provide a thermal cell vaccine and cytokine kit having an excellent immune effect, which includes various types of antigens and has a high immune effect against a low immunogenic antigen.

The inventors of the present application have found that the lysate of cells in which HSP protein is induced has a high immune effect, and even when the immunogenicity of the antigen is low, by using a cytokine together, We found an immunopotentiating effect that was not seen, and succeeded in developing a kit containing a thermal cell vaccine and a cytokine containing an excellent antitumor effect, including a variety of antigens and a high immune effect. That is,
(1) The present invention provides a method for preparing a thermal cell vaccine, comprising collecting cells from an individual organism, thermothermally treating the cells, expressing heat shock protein, and lysing the treated cells About.
(2) The present invention relates to the method for preparing a thermal cell vaccine according to (1), wherein the thermothermal treatment of the cells is a temperature treatment of 42 to 60 ° C. and 15 to 60 minutes.
(3) In the present invention, (1) or (2) is that the heat-shock protein is expressed by culturing the cells after the thermothermal treatment described above at 37 to 45 ° C. for 0.5 to 72 hours. A method for preparing a hyperthermic cell vaccine according to claim 1.
(4) The present invention relates to a method for preparing a thermal cell vaccine according to any one of (1) to (3), wherein the heat shock protein is HSP70, HSP90, or gp96.
(5) The present invention relates to a thermal cell vaccine prepared by the method for preparing a thermal cell vaccine according to any one of (1) to (4).
(6) The present invention relates to an immunopotentiator for a warm cell vaccine for an organism individual after administration of the warm cell vaccine according to (5), which contains a cytokine as an active ingredient.
(7) The present invention relates to an immune enhancer for a hyperthermic cell vaccine, wherein the cytokine according to (6) is IL-12.
(8) The present invention relates to a kit comprising the thermal cell vaccine according to (5) and the immunopotentiator of the thermal cell vaccine according to (6).
(9) The present invention relates to a kit comprising the thermal cell vaccine according to (5) and the immune enhancer of the thermal cell vaccine according to (7).
(10) The present invention relates to the thermal cell vaccine according to (5) for the prevention or treatment of cancer.
(11) The present invention relates to the kit according to (8) or (9) for preventing or treating cancer.
(12) The present invention relates to a pharmaceutical composition comprising the thermal cell vaccine according to (5) or (10) or the kit according to any one of (8), (9) or (11).
(13) The present invention provides an adoptive immunotherapy comprising contacting the thermal cell vaccine according to (5) or (10) or the kit according to any one of (8), (9) or (11) The present invention relates to a method for preparing immune cells for an agent.
(14) The present invention relates to immune cells prepared by the method according to (13).

  By using cells as a vaccine antigen in the form of a lysate, it is possible to include many types of patient-specific antigens, and by heat-treating the cells, induction of heat shock protein expression that promotes antigen presentation ability is induced. In addition, the combined use of cytokines further enhances the immune effect not seen in untreated cell vaccines, resulting in superior immune effects against small amounts of antigens and antigens with low immunogenicity. This makes it possible to produce a kit for a thermal cell vaccine and a cytokine.

-The individual organism used in the present invention is preferably a human suffering from infection or cancer.

-Cells collected from an individual organism used in the present invention may be collected intact cells, cultured cells, or established cells, but those of humans suffering from infection or cancer A cell derived from a diseased part is preferable, and a cancer cell is particularly preferable.

-The preferable conditions for the thermothermal treatment of the cells used in the present invention are 42-60 ° C, 15-60 minutes, more preferably 42-45 ° C, 30-60 minutes, and particularly preferably 43 ° C, 30 minutes. Temperature treatment. In addition to heat treatment of cells, if induction of heat shock protein can be induced, irradiation treatment; kelanylgeranylacetone treatment; isoprenoid treatment such as retinoic acid; lower aliphatic alcohol treatment such as technol; zinc and cadmium Treatment with heavy metals such as arsenic; treatment with selenium; treatment with cytokines such as tumor necrosis factor α and interferon β; treatment with anticancer agents such as cisplatin, 5-FU (fluorouracil), adriamycin, geldanamycin, and herbimycinad; Local anesthetic treatment or the like may be used, and administration may be directly performed on the cells of HSP itself such as HSP70, HSP90, gp96.

-The expression of the heat shock protein used in the present invention is performed by culturing the cells under certain conditions after giving the cells stimulation such as heat treatment. Therefore, the conditions for the expression of heat shock protein by culturing after thermothermal treatment used in the present invention are 37 to 45 ° C. and 0.5 to 72 hours in a 5% carbon dioxide addition type incubator. Preferable is culture at 37 to 43 ° C. for 6 to 48 hours, and particularly preferable is culture at 37 ° C. for 12 hours.

-The heat shock protein expressed in the present invention includes HSP70, HSP90, gp96, HSP60, HSP110, HSP27, etc., preferably HSP70, HSP90, gp96.

-Lysis of cells used in the present invention is preferably boiled or freeze-thawed, but freeze-thaw is more preferred.

-The freeze-thaw treatment used in the present invention is repeated 1 to 10 times, more preferably 3 to 7 times, and particularly preferably 5 times alternately in a water bath maintained at 37 ° C and liquid nitrogen. Freezing and thawing by soaking and destroying cells.

-IL-1β, IL-2, IL-4, IL-6, IL-12 and other interleukins; interferons such as IFNβ and IFNγ; TNFα Tumor necrosis factors such as; colony stimulating factors such as granulocyte macrophage-colony stimulating factor (GM-CSF); and HSPs such as HSP70, preferably IL-2, IL-12, GM-CSF More preferably, it is IL-12. The origin is not limited to the kind of organism, but is preferably derived from humans, and may be naturally derived or genetically modified, but genetically modified is preferred from the viewpoint of purity and price.

-Conditions for administration of IL-12 in the mouse used in the present invention are preferably 0.1 to 10 µg / dose, and more preferably 4 µg / dose immediately after administration of the cell lysate.

-The number of cells used for one vaccine in the mouse used in the present invention is preferably 1 x 10 ⁴ to 1 x 10 ¹⁰ , more preferably 5 x 10 ⁵ to 1 x 10 ⁶ .
The vaccine used in the present invention can be used for the prevention or treatment of infectious diseases such as viruses, bacteria, fungi, etc. and cancer, but the more preferred use is cancer that has metastasized or is about to metastasize after surgical resection. Use for prevention and treatment.

・ Adoptive immunotherapy used in the present invention refers to culturing human lymphocytes in the presence of IL-2, IL-4, etc. to induce killer cells that exhibit non-specific cytotoxic activity against tumor cells. It means one of the cancer treatment methods to return these cells to patients and apply them to cancer treatment.

-Adoptive immunotherapy immune cells used in the present invention include lymphocytes (T cells, B cells), macrophages (including dendritic cells), NK cells, killer cells (including LAK cells, TIL cells), It includes phagocytic cells, Langerhans cells and the like.

1. Culture of cancer cells As the cancer cells, mouse B16 melanoma cells, which are malignant melanomas, and rat T-9 glioma cells, which are gliomas, were used. Subculture was performed at 37 ° C. in a 5% carbon dioxide incubator using a 100 mm tissue culture dish (Asahi Techno Glass) containing 10 ml of medium. B16 cells are Dulbecco's modified Eagle medium (Gibco BRL) containing 10% fetal bovine serum and penicillin G potassium (100 U / ml) and streptomycin sulfate (90 mg / ml) as antibiotics, T-9 cells Used a medium containing 10% fetal bovine serum, 1% non-essential amino acids, penicillin G potassium (100 U / ml) and streptomycin sulfate (90 mg / ml) as antibiotics in Eagle's minimal medium (Gibco BRL).

2. Heat treatment method The cells in sub-confluent state are kept in a constant temperature (41 ° C, 43 ° C, 45 ° C) in a water bath (PROTERMO BATH, Tokyo Rika Kikai) in advance. After soaking for a period of time (30 minutes, 60 minutes, 120 minutes), it was allowed to stand for a certain period of time (12 hours, 18 hours, 24 hours) in a 5% carbon dioxide addition incubator at 37 ° C.

3. How to collect cancer cells Cells were detached from the dish by trypsin treatment, and 4-5 ml of medium was added to stop the enzyme reaction. The cell suspension was collected in a centrifuge tube (IWAKI), and the cells were sedimented by centrifugation (1000 rpm, 5 minutes), and the medium was removed. Thereafter, the cells were suspended in 10 ml of PBS and washed by removing PBS by centrifugation (1000 rpm, 5 minutes). This was repeated twice and the resulting cell pellet was suspended in PBS. Thereafter, the cells were counted, and the cells were transferred to a centrifuge tube.

4). Preparation of vaccine After heat-treating B16 cells at 43 ° C. for 30 minutes or T-9 cells at 43 ° C. for 60 minutes, 5 × 10 ⁵ B16 cells or 1 × 10 ⁶ T-9 cells were maintained at 37 ° C. The cells were disrupted by freezing and thawing by alternately immersing them in a water bath and liquid nitrogen (repeated 5 times). This lysate was used as a thermal cell vaccine.

5). Method of administration to mice The mice were anesthetized with Nembutal using C57BL / 6 (female 4-6 weeks old) and the rats F344 (female 6 weeks old). The thermal cell vaccine was administered twice a week by a syringe (29G × 1/2 ″, Becton Dickinson) subcutaneously in the right back, which had been previously shaved. One week after the second administration, B16 cells (5 × 10 ⁵ cells) or T-9 cells (1 × 10 ⁶ cells) were transplanted subcutaneously into the shaved left back of the back using a syringe, and then the tumor was measured for engraftment. The time when the diameter became 2 mm or more was regarded as engraftment.

6). Preparation of cytokine IL-2 (human recombinant, Wako) and IL-12 (mouse recombinant, Wako) were used as cytokines. Each was dissolved in PBS to a concentration of 40 μg / ml.

7). The first dose of cytokine was administered immediately after the vaccine was administered. 100 μl of the solution was taken so that the cytokine was 4 μg, and administered to the abdominal cavity of the mouse with a syringe.

8). Quantitative method for HSP70 The thermal conditions under which B16 cells highly express HSP70 were examined by ELISA. HSP70 was quantified using an HSP70 ELISA kit (Stressgen). The conditions examined were the temperature (41 ° C, 43 ° C, 45 ° C) and time (30 minutes, 60 minutes, 120 minutes) and the time to incubate after heating (12 hours, 18 hours, 24 hours). is there.

Results Examination of thermal conditions Thermal conditions under which H16 of B16 cells are highly expressed were examined by ELISA. FIG. 1 shows the results of comparison of the expression level of HSP70 with respect to the temperature and time for heating, and the incubation time after heating. First, the amount of HSP70 was compared at 41 ° C, 43 ° C, and 45 ° C. When heated at 43 ° C, the expression level of HSP70 in the cells was markedly increased compared to those not heated (37 ° C), and when heated at 45 ° C, the cells were almost dead. Not detected. Heating time was 30 minutes, 60 minutes, and 120 minutes, and HSP70 expression level was compared. As a result, HSP70 expression level was significantly increased by heating for 30 minutes. L, cells were killed by heating for 60 minutes and 90 minutes. As a result, the expression level of HSP70 was decreased. When the expression level of HSP70 in the incubation time of 12 hours, 18 hours, and 24 hours was compared L, the expression level of HSP70 became the maximum 12 hours after the ripening, and the amount of HSP70 decreased with the passage of time. Therefore, the condition for high expression of HSP70 in B16 cells was to heat at 43 ° C. for 30 minutes and then to incubate for 12 hours. After freezing and thawing, the expression of HSP70 was confirmed and used as a thermal cell vaccine.
Although not shown in the figure, the high expression conditions of HSP70 of T-9 cells were also incubated for 12 hours after heating at 43 ° C. for 60 minutes, as in the case of B16 cells. The expression of HSP70 was confirmed and used as a thermal cell vaccine.

Effect of Heat Cell Vaccine The effect of the vaccine was examined by comparing the time to engraftment of the transplanted cancer cells between the rat and mouse administered with the heat cell vaccine and the rat and mouse administered with PBS as a control. Here, rat T-9 glioma cells, which are known to be highly immunogenic cancer cells, were also used. FIG. 2 shows the ratio of rats (T-9) or mice (B16) in which tumors did not engraft with respect to the number of days elapsed after cancer cell transplantation (n = 5). In T9 glioma cells, all rats formed tumors in the control group, whereas 60% did not form tumors in the vaccine group. From this, the effect of the vaccine was recognized in T9 glioma cells.
On the other hand, in the case of B16 cells, which are very high grade malignant (low immunogenicity) mouse melanoma cells, there was no mouse in which no tumor formation was observed in the vaccine administration group, and the delay in tumor survival compared to the control group. Since it was only about 2 or 3 days, it was judged that the effect of the vaccine on B16 cells was hardly seen.

Examination of vaccine effect by cytokine combination B16 melanoma is a cancer cell with very low immunogenicity, and is considered to be a model close to the state of human terminal cancer. Therefore, a substance that enhances the effect of the vaccine against B16 melanoma was searched, and a vaccine that was effective against such highly malignant cancer was developed. Cytokines were used in combination with mice administered with the thermal cell vaccine to examine the vaccine effect (FIG. 3). As the cytokine, IL-2 having an action of generally activating the immune system, or IL-12 particularly activating cellular immunity or the like was used. In combination with IL-2, compared to untreated mice, delayed survival was observed in both mice administered with the thermal cell vaccine and mice administered with the cell vaccine without heat, both of which had some vaccine effect. Admitted. However, there was not much difference in the effect between the thermal cell vaccine with high expression of HSP and the non-thermal cell vaccine without HSP expression. On the other hand, surprisingly, a result was obtained that the thermal cell vaccine was specifically enhanced by the combined use with IL-12. That is, in the combined use with IL-12, the effect of the cell vaccine administration group without heat was hardly seen, whereas in the treatment of the heat cell vaccine administration group, compared with the non-treatment group or the cell vaccine administration group without heat, A special effect effective only in combination with the thermal cell vaccine was obtained, in which tumor formation was significantly suppressed in the group administered with the thermal cell vaccine, and no tumor was formed in one of the 5 mice.

  It can be used as a vaccine against the fourth cancer treatment following surgery, chemotherapy, and radiation therapy. In particular, it is effective for use in treating cancer that has metastasized or is about to metastasize after surgical resection.

Examination of thermal conditions. The thermal conditions under which HSP70 of B16 cells are highly expressed were examined by ELISA. The result of having compared the expression level of HSP70 about the temperature to heat, time, and the incubation time after a heat is shown. In the figure of warm temperature, warm time and incubation time after warm are 30 minutes and 12 hours, respectively. In the diagram of the heat time, the heat temperature and the incubation time after the heat are 43 ° C. and 12 hours, respectively. In the incubation time chart, the heating time and the heating temperature are 30 minutes and 43 ° C., respectively. The effect of a thermal cell vaccine. The vaccine effect which compared the period after transplanting a cancer cell to a mouse | mouth to engraftment between the prepared thermal cell vaccine administration mouse | mouth and the PBS administration mouse | mouth as a control is shown. Vaccine effect by cytokine combination. The vaccine effect when a cytokine is combined with the mouse | mouth which administered the hyperthermia cell vaccine is shown.

Claims (14)

  A method for preparing a hyperthermic cell vaccine, comprising collecting cells from a living organism, thermothermally treating the cells, expressing heat shock protein, and lysing the treated cells.   The method for preparing a thermal cell vaccine according to claim 1, wherein the thermothermal treatment of the cells is a temperature treatment of 42 to 60 ° C and 15 to 60 minutes.   The method for preparing a thermal cell vaccine according to claim 1 or 2, wherein expressing the heat shock protein is culturing the cells after the thermal treatment described above at 37 to 45 ° C for 0.5 to 72 hours. .   The method for preparing a thermal cell vaccine according to any one of claims 1 to 3, wherein the heat shock protein is HSP70, HSP90, or gp96.   A warm cell vaccine prepared by the method for preparing a warm cell vaccine according to any one of claims 1 to 4.   The immunity enhancement agent of the warm cell vaccine for the living individual after administration of the warm cell vaccine of Claim 5 which contains cytokine as an active ingredient.   An immune enhancer for a thermal cell vaccine, wherein the cytokine according to claim 6 is IL-12.   A kit comprising the warm cell vaccine according to claim 5 and the immune enhancer of the warm cell vaccine according to claim 6.   A kit comprising the warm cell vaccine according to claim 5 and the immune enhancer of the warm cell vaccine according to claim 7.   The hyperthermic cell vaccine according to claim 5 for preventing or treating cancer.   The kit according to claim 8 or 9 for prevention or treatment of cancer.   A pharmaceutical composition comprising the thermal cell vaccine according to claim 5 or 10 or the kit according to any one of claims 8, 9 or 11.   A method for preparing immune cells for an adoptive immunotherapeutic agent, comprising contacting the thermal cell vaccine according to claim 5 or 10 or the kit according to any one of claims 8, 9 or 11.   An immune cell prepared by the method of claim 13.

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