JP2002212099A - Tumor vaccine - Google Patents

Abstract

(57) [Problem] To provide a tumor vaccine having versatility applicable to prevention and treatment of recurrence irrespective of tumor type and capable of achieving high antitumor effect. SOLUTION: The following components: (a) tumor tissue, tumor cell,
And microparticles prepared from a solidified tumor material selected from the group consisting of these components or a lysate prepared from the tumor material, (b) at least one cytokine and / or cytokine inducer, (c) A tumor vaccine comprising at least one chemokine and / or chemokine inducer and (d) bone marrow cells and / or blood cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the prevention of tumor recurrence,
The present invention relates to tumor vaccines useful for metastasis inhibition and treatment.

[0002]

2. Description of the Related Art Tumor vaccine therapy involves killer lymphocytes, particularly cytotoxic T lymphocytes (hereinafter referred to as "C-lymphocytes"), which play a central role in the immune function in the body, especially the cellular immune response.
TL ”. ) To specifically kill tumor cells without damaging normal cells, prevent tumor recurrence,
It is a therapy that inhibits metastasis or is expected to cure existing tumors.

Many kinds of tumor vaccines have been developed (Pardoll, DM, Nature Med., 4 (5 Suppl), pp. 52).
5-531, 1998). Broadly classified, as tumor-specific, (1) a tumor antigen peptide whose properties are already known; (2) a tumor tissue extract containing an unidentified tumor antigen peptide is used (3) These are bound to antigen-presenting cells, particularly to dendritic cells having a strong antigen-presenting function (Nestle, FO, et al., Nature M.
ed., 4, pp.328-332, 1998); (4) dendritic cells loaded with tumor antigen protein and loaded; (5) dendritic cells fused with tumor cells; (6) tumor The antigen is bound to the liposome and incorporated together with the liposome (Nakanishi,
T., et al., Biochem. Biophys. Res. Comm., 240, pp.
793-797, 1997); (7) Inactivation treatment of tumor cells themselves with radiation or a fixative; (8) Cytokines that stimulate antigen-presenting cells or lymphocytes by gene therapy A gene that is introduced into a tumor cell and administered as a vaccine, or a tumor antigen gene is introduced into appropriate cells and a tumor cell that expresses the gene is administered as a vaccine; (9) tumor antigen gene Which is incorporated into a virus or bacterium to infect a patient; (10)
Live tumor cells, tumor antigen peptides or tumor cell extracts are administered, and large amounts of cytokines are separately administered (Rosenberg, SA, et al., Nature Med., 4, pp. 32
1-327, 1998), or a drug that is formulated as a sustained-release cytokine (Golumbek, PT, et al., Cance
r Res., 53, pp. 5841-5844, 1993).

[0004] However, each of the above tumor vaccines has advantages and disadvantages. For example, in method (1), a specific major histocompatibility antigen (hereinafter, abbreviated as “MHC”) to which the identified tumor antigen peptide applies, and in the case of Class I, “MHC-
In the case of "I" and Class II, it is described as "MHC-II". ) Is only applicable to tumors expressing The types of human MHC are enormous, and the cases to which the tumor antigen peptide is applicable are extremely limited. To overcome this difficulty, a method (2) using an extract of tumor tissue containing an unidentified tumor antigen peptide was developed, but the amount of tumor antigen peptide that can be extracted from tumor tissue is extremely small, When the amount of the raw material tumor is small, it cannot be concentrated in many cases. As a result, it cannot be administered in a large amount like the identified and synthesized tumor antigen peptide, and its effect is also limited.

[0005] If a tumor antigen peptide is previously bound to antigen-presenting cells as in method (3), the CTL activating effect is high. However, peripheral blood and bone marrow for the isolation and preparation of antigen-presenting cells, especially dendritic cells with strong antigen-presenting ability, are dangerous transplant-host rejection reactions (GVHD).
Abbreviated. ), It is necessary to be from the patient who has the tumor to which the tumor vaccine therapy is applied. Methods (4) and (5) also have the same problems as method (3), and method (5) requires a very complicated fusion operation. Although it is not necessary to consider the risk of GVHD in method (6), the efficiency of introducing tumor antigens into antigen-presenting cells is not always high, and a relatively large amount of tumor antigens is required for preparing a tumor vaccine.

The method (7) is also complicated and costly because the tumor cells are obtained in a large-scale culture, and has a problem that the amount of tumor antigen contained in the tumor cells themselves is very small. Also, this method is known to be successful if poly-L-lysine treatment is added to tumor cells with high antigenicity (Naito, M. and Seno, S., Cell Biol. International
Rep., 5, pp. 675-681, 1981), which is not successful with poorly antigenic tumor cells. In the gene therapy of the methods (8) and (9), not only the treatment operation, but also the procedure for obtaining approval for the treatment is extremely complicated. At this stage, method (10) is promising, but especially Rose
In the method of nberg et al., the side effects of a large amount of interleukin-2 administered simultaneously are severe, and the clinical results of tumors are not always high. Even when formulating sustained-release cytokines according to the method of Golumbek et al., The complexity of preparing irradiated tumor cells in living cells remains.

[0007] The tumor vaccine is preferably in a form that can be handled as simply as possible. At that point, the method of administering living tumor cells or antigen-presenting cells as a part of the vaccine requires an operation in a live state, and thus has a problem that it is technically very complicated. In the case of gene therapy, the operation is even more complicated. If the tumor antigen peptide is known, it can be synthesized and administered in large quantities, but there are a very large number of tumor antigen peptides,
Because of MHC restriction, it is often unclear which tumor antigen peptide can be applied to the target patient, and the application is limited. When a tumor antigen protein is used instead of a tumor antigen peptide, the protein is processed in antigen-presenting cells to select a tumor antigen peptide that matches the MHC. However, there is a problem that purification and large-scale preparation of the tumor antigen protein itself is difficult.

On the other hand, as a method of inducing CTL, a method of inducing CTL from peripheral blood mononuclear cell fraction on a fixed tumor tissue obtained by deparaffinizing a pathological section (Li)
u, SQet al., Nature Med., 2, pp.1283-1283, 199
6). Also, usually, even when the antigen protein in the dissolved state is given to the antigen-presenting cells, the antigen peptide derived from the antigen protein is bound to MHC-II, and the effect of stimulating humoral immunity leading to antibody production is high. Although the effect of stimulating the cellular immune response that activates killer cells by binding antigen peptides derived from antigen proteins is low, Falo et al. Bind egg white albumin, a heterologous protein and a strong antigen, to iron powder to form an adjuvant. Injection into mice without addition induced CTLs that responded to antigenic peptides derived from ovalbumin (Falo, Jr., L.
D., et al., Nat. Med., 1, pp. 649-653, 1995).

[0009] The present inventors fixed a soluble tumor antigen protein on fine polystyrene beads, and used an in vitro cell culture system to convert antigen-presenting cells in the human peripheral blood mononuclear cell fraction as microsolids. When phagocytosed, it was found that CTLs could be efficiently induced from peripheral blood lymphocytes of the same individual (K
im, C., et al., Cancer Immunol. Immunother., 47, p.
p.90-96, 1998). In addition, it is known that when dead cell-derived antigens are phagocytosed by immature dendritic cells in the state of dead cells, the efficiency of inducing an immune response is several thousand times higher than when they are not phagocytosed. (Inaba: December 2, 1998, Japanese Society of Immunology, title SI-3-3).

[0010] Furthermore, the present inventors can easily handle and prevent recurrence, inhibition of metastasis,
And research to provide a tumor vaccine with high versatility and high antitumor effect that can be applied to therapy, and using a material obtained by solidifying tumor tissue, tumor cells, or these components by a fixing operation, and using this material. Antigen-presenting cells are micronized to a size that can phagocytize, or are lysed by a lysis operation, and further used as a tumor vaccine in combination with at least one type of cytokine to prevent tumor recurrence with a high effective rate, inhibit metastasis, and They found that treatment could be achieved and filed a patent application for this invention (PCT / JP00 / 0692).

However, for tumors such as ovarian cancer, even if the above-mentioned tumor vaccine according to PCT / JP00 / 0692 is used, it may not always succeed in suppressing tumor formation and preventing metastasis. Was required.

[0012]

SUMMARY OF THE INVENTION The object of the present invention is to provide a versatility which can be easily handled, is applicable to prevention of recurrence, inhibition of metastasis, and treatment irrespective of the type of tumor. It is to provide a high tumor vaccine.

[0013]

Means for Solving the Problems The present inventors have made intensive efforts to solve the above-mentioned problems, and have found that tumor tissues, tumor cells,
Alternatively, using a material in which these components are solidified by a fixation operation, the material is micronized to a size capable of phagocytosis by antigen-presenting cells, or dissolved by a lysis operation, and at least one cytokine, and at least one more It has been found that, by combining with a chemokine, and adding bone marrow cells and / or blood cells thereto and using it as a tumor vaccine, tumor tissue formation inhibition can be achieved with a high efficiency. The present invention has been completed based on the above findings.

That is, the present invention provides the following components: (a) fine particles prepared from a solidified tumor material selected from the group consisting of tumor tissues, tumor cells, and these components; And (c) at least one type of chemokine and / or chemokine inducer, and (d) a tumor vaccine comprising bone marrow cells and / or blood cells.

[0015] In another embodiment, the following components: (a) a lysate prepared from a solidified tumor material selected from the group consisting of tumor tissue, tumor cells, and these components; The present invention provides a tumor vaccine comprising one cytokine and / or cytokine inducer, (c) at least one chemokine and / or chemokine inducer, and (d) bone marrow cells and / or blood cells.

According to a preferred embodiment of the present invention, the above-mentioned tumor vaccine further comprising an adjuvant which non-specifically elicits an immune response; the above-mentioned tumor vaccine for administration to the same local part in the body; The above tumor vaccine containing granulocyte / macrophage colony stimulating factor and / or interleukin-2 as a cytokine; the above tumor vaccine containing a sustained release chemokine preparation as a chemokine; secondary lymphoid-tissue chemokine as a chemokine The above tumor vaccine comprising: hematopoietic stem cells, peripheral blood mononuclear cells as bone marrow cells and / or blood cells,
And a tumor vaccine comprising cells selected from the group consisting of cell fractions derived therefrom.

In another aspect, there is provided a tumor vaccine for use in combination with at least one cytokine and at least one chemokine, comprising:
(e) tumor tissue, tumor cells, and microparticles prepared from a solidified tumor material selected from the group consisting of these components or a lysate prepared from the tumor material, and (f) bone marrow cells and / or The present invention provides a tumor vaccine containing blood cells as an active ingredient.

In still another aspect, a method for treating a tumor,
A method for preventing recurrence, and a method for inhibiting metastasis, wherein (a) a fine particle prepared from a solidified tumor material selected from the group consisting of tumor tissue, tumor cells, and these components, or a method prepared from the tumor material An effective amount of a lysate, (b) at least one cytokine and / or cytokine inducer, (c) at least one chemokine and / or chemokine inducer, and (d) an effective amount of bone marrow cells and / or blood cells. A method of administration; the above method of repeated administration to the same site; and the use of microparticles prepared from the solidified tumor material or a lysate prepared from the tumor material for the production of the tumor vaccine. Is done.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION The tumor vaccine of the present invention comprises the following components: (a) microparticles prepared from solidified tumor material selected from the group consisting of tumor tissue, tumor cells, and these components; A lysate prepared from tumor material, and (b) at least one cytokine and / or cytokine inducer, further comprising (c) at least one chemokine and / or chemokine inducer, and (d) bone marrow cells And / or blood cells. above
The tumor vaccine containing the components (a) and (b) is PCT / JP00 / 0692.
And the disclosure of which is incorporated herein by reference.

As the tumor cells or tumor tissues, for example, those derived from mammals, preferably humans, can be used. Seeds may be used. The type of tumor tissue is not particularly limited as long as it contains tumor cells. When a component of a tumor tissue or a tumor cell is used, its type is not limited as long as it contains a substance that can serve as a tumor antigen. Biological samples containing cancer cells separated or collected from a living body, such as solid cancer tissues, bone marrow, and leukocytes, can be used as tumor materials. As a component of a tumor tissue or tumor cell, for example, an antigen peptide or an antigen protein can be used.

[0021] The fixing method for preparing the solidified tumor material is not particularly limited, and any means available to those skilled in the art may be employed. For example, when a tissue fixative is used, neutral formalin, glutaraldehyde, methanol, alcohols such as ethanol and the like can be used, but in addition to these, living tissues or cells, or components thereof, are solid. Any method may be used as long as the method can be used. The tumor material may be solidified by a method such as paraffin embedding or freezing. Even when a tissue in a solid state, such as a bone tissue, is used as a solidified tumor material, it is desirable to perform an appropriate fixing method.

The method for preparing the fine particles is not particularly limited.
For example, besides a method of crushing solidified tumor tissue to prepare fine particles as fine fragments, a method of dissolving crushed fragments of tumor tissue or tumor cells and fixing them to solid fine particles, or a method such as an antigen peptide or an antigen protein And the like can be employed. As solid fine particles, for example, from 0.05 microns in diameter to 1000 microns
Micron-sized iron powder, charcoal powder, polystyrene beads, or the like can be used. Also, crushed tissue fragments, tumor cells,
Alternatively, a soluble tumor antigen is bound to a lipid particle such as a liposome so that antigen-presenting cells can recognize and phagocytose as fine particles, or a soluble tumor antigen itself is bound to each other by a binder or a cross-linking agent. Fine particles may be used.

The size of the fine particles is not particularly limited, but it is desirable that the fine particles have a size that allows phagocytic cells in the body to phagocytose. Originally, fixed tumor cells in a minute single cell state do not need to be particularly crushed, but when they are aggregated by the cell fixing operation, it is desirable to crush or disperse them. For the crushing or dispersion treatment, a homogenizer treatment, an ultrasonic treatment, a partial digestion method using a digestive enzyme, or the like can be used. Fine particles can also be prepared by passing through a mesh having a pore size of 1000 microns or less, preferably 380 microns or less. Methods for preparing these fine particles are well known to those skilled in the art, and those skilled in the art can prepare fine particles by using an appropriate method alone or by combining a plurality of methods.

As a method for preparing a lysate from a solidified tumor material, for example, a method using a proteolytic enzyme can be adopted. An example of the proteolytic enzyme is pronase K. Further, a method in which an enzyme other than the proteolytic enzyme, an acid, an alkali, or the like is appropriately combined may be used. Any method may be adopted as long as it can dissolve the solidified tumor material, and those skilled in the art can select an appropriate method. The lysate may be immobilized on the solid fine particles described above.

As used herein, the term “lysate” refers to a solidified tumor material that is water, saline,
It refers to a state in which a solid is dispersed in an aqueous medium such as a buffer to such an extent that a solid is not visually recognized.It is sufficient that the dispersion can be phagocytosed by antigen-presenting cells, but in any sense Should not be interpreted restrictively. The details of the method for preparing the immobilized tumor material, the method for preparing the microparticles, and the method for preparing the lysate are specifically shown in the examples of the present specification. It is possible to add desired modifications or alterations to these methods as needed, with reference to the specific description of Examples and Examples, to prepare desired fine particles or dissolved materials.

The type of cytokine contained in the tumor vaccine of the present invention is not particularly limited, and one or more cytokines can be used. For example, it is preferable to use granulocyte / macrophage colony stimulating factor (hereinafter abbreviated as “GM-CSF”) or interleukin-2 (hereinafter abbreviated as “IL-2”), and GM-CSF and IL- It is also preferable to use a combination of the two. In addition, other cytokines and cytokine inducers that stimulate local immunocompetent cells in the body and consequently achieve the same situation as when GM-CSF and / or IL-2 are administered can also be used. These two
Examples of cytokines other than the kind of cytokines or cytokine inducers include, but are not limited to, interleukin 12, interleukin 18, and interferons.

The types of chemokines contained in the tumor vaccine of the present invention are not particularly limited, and one or more chemokines can be used. For example, Secondary Lymphoid-Tissue Chemokine
hoid-tissue Chemokine, abbreviated as “SLC” below. Sometimes called CCL21: Zlotnik, A. and Yoshie, O., Im
munity 12: 121-127, 2000) or Epstein-Barr
Virus Induced Molecule-1 Ligand Chemokine (Epstein-Bar
r virus-induced molecule-1 ligand chemokine, abbreviated as “ELC” below. Sometimes called CCL19: Zlotnik,
A. and Yoshie, O., Immunity 12: 121-127, 2000), and preferably a combination of SLC and ELC. In addition, other chemokines capable of stimulating immunocompetent cells in the body and consequently achieving the same situation as when SLC and / or ELC is administered, chemokine inducers,
The chemokine receptor (CC for both SLC and ELC)
A substance that binds to R7) and has a chemokine-like stimulating action can also be used. Chemokines other than these two chemokines or chemokine inducers include, for example, MIP-3b
Examples include, but are not limited to, eta, 6-C-kine, LARK, and RANTES.

It is preferable that these cytokines and their inducers, and chemokines and their inducers are prepared as sustained-release preparations so that the local concentration can be kept as high as possible for as long as possible. Such a sustained release means has been reported, for example, by Golumbek et al. (Golumb
ek, PT, et al., Cancer Res., 53, pp.5841-5844,1
993), various sustained-release methods are known in the art, and any method may be employed.

The type of bone marrow cells and / or blood cells contained in the tumor vaccine of the present invention is not particularly limited, and bone marrow cells and / or blood cells containing cells having an antigen presenting function, or hematopoietic stem cells / hematopoietic progenitors Bone marrow cells and / or blood cells including cells or peripheral blood mononuclear cells can be used. Alternatively, bone marrow cells and / or blood cells containing a cell fraction derived from hematopoietic stem cells, hematopoietic progenitor cells, and peripheral blood mononuclear cells may be used. When administering the tumor vaccine of the present invention to animal individuals including humans,
It is particularly preferable to use bone marrow cells and / or blood cells derived from the individual, but it is not limited thereto.

[0030] The tumor vaccine of the present invention may contain an adjuvant which elicits a non-specific immune response. Adjuvants can be used alone or in combination of two or more. As an adjuvant, for example, Freund Complet
Bacterial preparations such as eAdjuvant, Freund Imcomplete Adjuvant, BCG, etc., bacterial component preparations such as tuberculin, keyhole limp
Natural polymer substances such as et hemocyanine and yeast mannan, Al
Examples thereof include synthetic adjuvant preparations such as um and TiterMax Gold. However, the present invention is not limited to these specific examples, and any substance having an adjuvant effect may be used. Whether or not to use an adjuvant can be determined by using, as an index, the strength of the inflammatory response at the administration site or the strength of the antitumor effect caused as a result of the administration. For example, it is also possible to alternately administer a tumor vaccine containing an adjuvant and a tumor vaccine not containing an adjuvant at the same location.

The formulation of the tumor vaccine of the present invention is not particularly limited. However, when provided as a composition containing all of the above components (a) to (d), the conditions under which the added cells can survive are provided. It is desirable that the preparation form is provided and that the preparation form is suitable for topical administration. The method of formulation is not particularly limited, and a formulation in a desired form can be prepared by using a method available in the art alone or in an appropriate combination. In formulating, one or more kinds of pharmaceutical additives available in the art can be used in addition to aqueous media such as distilled water for injection and physiological saline. For example, buffers, pH adjusters, solubilizers, stabilizers, soothing agents, preservatives, and the like can be used, the specific components of which are well known to those skilled in the art.

Further, cells are prepared in an appropriate form,
A composition containing other components may be separately prepared in the form of a solid preparation such as a lyophilized preparation, and a dissolving agent such as distilled water for injection may be added to both at the time of use to prepare an injection. The cells may be suspended in a cryopreservation medium to which a cryoprotectant has been added, and prepared as frozen cells. These specific formulation methods are well known to those skilled in the art. A method in which four types of preparations each containing the above-mentioned components (a) to (d) are prepared, and they are mixed at the time of use and topically administered, or the four types of preparations are topically administered to the same site; A preparation containing one or more components selected from the components (a) to (d) and one or more preparations containing other components are prepared and mixed at the time of use. Methods for local administration or local administration of the preparations to the same site are all included in the scope of the present invention.

When performing vaccine therapy using the tumor vaccine of the present invention, the tumor vaccine may be administered only once. However, in order to allow the tumor antigen and other tumor vaccine components to coexist for as long as possible, it is necessary to use a tumor vaccine. It is desirable to repeat the administration to the same local area. It is also possible to separately prepare a cell and a composition containing other components, and to administer each to the same site locally. For example, it is desirable that both components coexist for 3 hours or more so that an inflammatory reaction at the administration site is induced and immune cells are concentrated and remain there. When administering an adjuvant-free tumor vaccine, the adjuvant may be administered at the same location. In general, a tumor vaccine can be administered to a patient from which the tumor material is derived, but for pathological diagnosis, the tumor vaccine is administered to a tumor patient containing a tumor antigen of the same or related species as the tumor antigen contained in the tumor material. It is also possible.

The site to be administered is not particularly limited, but is preferably a site within a major organ such as intradermal, subcutaneous, intramuscular, intralymphatic, or spleen, where cytokines and the like are not easily diffused and eliminated. However, by selecting a dosage form that does not allow the active ingredient of the tumor vaccine to spread easily, local administration at any site may be possible, and systemic administration may be possible by applying a drug delivery system In some cases. The dose and the administration period of the tumor vaccine of the present invention are not particularly limited, but it is desirable to determine the dose and the administration period appropriately while confirming the effect of the vaccine therapy. Administration can be performed, for example, by injection or the like.

[0035]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples, but the scope of the present invention is not limited to the following Examples. Example 1: Effect of tumor vaccine of the present invention Syngeneic transplanted mouse ovarian cancer cell line OV2944-HM-1 (abbreviated as "HM-1";Hashi; widely known to be apt to metastasize to lymph nodes)
moto, M. et al., Jpn. J. Cancer Res., 80: 459-463,
1989; Shinji Ichii, Yasuyuki Imai: Manual for Research on Cancer Invasion and Metastasis-Techniques and Guidelines for Experiments, edited by Cancer Metastasis Research Group,
pp. 22-27, Kinyoshido, Kyoto, 1997), targeting fixed tumor cells, GM-CSF, IL-2,
We examined whether a tumor vaccine combining SLC, adjuvant and bone marrow cells could inhibit ovarian cancer formation and lymph node metastasis.

[Method] 1. Preparation of fixed tumor cells Ovarian cancer cells OV2944-HM-1 developed in B6C3F1 mice (female)
(Abbreviated HM-1, obtained from RIKEN Cell Development Bank), and cultured in Dulbecco's phosphate buffered saline (hereinafter, referred to as
Abbreviated as "PBS". ) And fixed with a 3% paraformaldehyde solution for 2 hours. The fixed cells were washed and sterilized once with 70% alcohol, then aseptically washed four times with PBS, further added with MEMα medium containing 10% fetal bovine serum, and incubated at 37 ° C. for 2 days in a carbon dioxide gas incubator. . After removing this medium, an aqueous solution of poly-L-lysine (50 μg / ml) was added to the cell layer, incubated at 37 ° C. for 2 hours, and washed four times with PBS. Thereafter, the cells were collected with a pipette and diluted to 1.25 × 10 ⁸ cells / ml with PBS. All fixed cells have a size of 100 microns or less, and are sized to be able to phagocytose phagocytic antigen-presenting cells.

2. Preparation of bone marrow cells B6C3F1 mice (females) were dislocated to the cervical vertebra, and femurs and tibias were removed. Both ends of each bone were cut, and a syringe with a 26G needle was inserted. The bone was washed with 10 ml of RPMI1640 medium per foot and bone marrow cells were collected. Centrifuge at 1000 rpm for 7 minutes, wash cells twice with RPMI medium, remove supernatant and remove PBS
Was added. Count the number of cells and immunize each mouse
The cells were adjusted to 1 × 10 ⁷ cells and centrifuged to form a pellet.

3. Preparation of Tumor Vaccine 50 μl of tuberculin (0.1 for human clinical use) was added to fixed HM-1 cells and incubated at 37 ° C. for 2 hours. This was mixed with 40 μl of a solution containing the following cytocans (abbreviations: rh, recombinant human; rm, recombinant m
ouse; rr, recombinant rat) group B rhIL-2, 1000 IU; rmGM-CSF, 1000 UC group rhIL-2, 1000 IU; rmGM-CSF, 1000 U; rmSLC, 5
00 ng D group rhIL-2, 1000 IU; rmGM-CSF, 1000 U; rmELC, 5
00ng E group rhIL-2, 1000 IU; rmGM-CSF, 1000 U; rrLARC,
500 ng A final volume of 90 μl per immunized mouse. This was added to a previously prepared bone marrow cell pellet and mixed. This was used as a tumor vaccine. In addition, for the group A (control group), only PBS was 90 μl.

4. Preparation of IL-2 / GM-CSF sustained-release preparation 1) Preparation of lecithin solution To 2 g of lecithin (Sigma Chemical Co., USA), 0.5 ml of ethanol was added, and the mixture was dissolved by heating to 45 ° C. The bacteria were removed by passing through a filter (pore size 0.22 μm, Millipore Co., USA). 2) Preparation of vitamin E solution 0.1 g of vitamin E (Sigma) dissolved in advance by heating to 45 ° C.
Chemical Co., USA) with a Millipore filter (pore size 0.22)
μm, Millipore Co., USA). 3) Preparation of cytokine solution rhIL-2 (manufactured by Shionogi & Co., Ltd.) was adjusted to 42 × 10 ⁴ IU / ml.
It was dissolved in a 5% human albumin solution (Baxter Co., USA). 100 μl of this rhIL-2 solution was added to rmGM-CSF (PEPROTech LT
D) Added and dissolved in 25 μg (41,666 U) to obtain a cytokine solution.

4) Preparation of Cholesterol Solution Cholesterol powder (Sigma Chemical Co., USA) 50 m
g was dissolved in ethanol to make 2 ml. 5) Preparation of IL-2 / GM-CSF sustained-release formulation Add 500 μl of lecithin solution, 100 μl of vitamin E solution and 24 μl of cholesterol solution, and aseptically knead with two syringes and a three-way stopcock (TERMO) Was mixed. Add 100 μl of cytokine solution (rhIL-2: 42,000 I
U, rmGM-CSF: 41,666 U) was added, and the mixture was aseptically kneaded with two syringes and a three-way stopcock (TERMO) and mixed well. It became a translucent and semi-solid grease-like composition which was hardly emulsified, and was used as an IL-2 / GM-CSF sustained-release preparation.

[0041] 5. Sensitization with tumor vaccine 90 μl of the tumor vaccine prepared in 3 above was inhaled with a 1 ml syringe equipped with a 26G injection needle,
The mice were injected intradermally on the right side of the tail (right side of the base of the tail). Thirty minutes later, 50 μl of the IL-2 / GM-CSF sustained-release preparation was injected intradermally into the same local area. One week later, the same treatment was performed on the left side of the tail of the same mouse.

6. Transplantation of HM-1 cell line subcutaneously into the footpad and measurement of tumor rejection: HM-1
-1 cells were transplanted. The method is as follows. 1) Collect the cultured HM-1 cells in suspension, wash with PBS
The cells were resuspended in S and adjusted to 5 × 10 ⁶ cells / ml. 2) After anesthetizing the B6C3F1 mouse with ether, disinfect the right forelimb footpad with alcoholic cotton, hold the mouse on your back, grasp the right forelimb of the mouse, fix it so that the palm is facing up, and inject 26G of the cell suspension. The cells were placed in a microsyringe equipped with a needle, and cells were slowly injected from near the wrist to the fingertip. The dose volume per mouse is 20 μl. From the tenth day, the vertical diameter of the foot pad was measured with a caliper twice a week.

[Results] As shown in FIG. 1, in the control group, primary tumors of footpads of all mice were formed (group A). In contrast, fixed HM-1 cells, mouse bone marrow cells, tuberculi
In group B containing n, IL-2, and GM-CSF, and additionally with a sustained-release formulation of IL-2 / GM-CSF, about half of the primary tumors were formed compared to group A. On the other hand, groups C (containing SLC), group D (containing ELC), and group E (containing LARC), each containing a chemokine in the same composition as group B, exhibited different effects. In particular, group C showed a stronger inhibitory effect on primary tumor formation than group B. Further, as shown in FIG. 2, the formation of regional lymph node metastases was suppressed in Group C to a level comparable to that of the regional lymph node of the opposite forefoot, which is a control within the same individual. Although an example of ovarian cancer was described in the examples of the present invention, it is apparent that the present invention can be easily applied to various tumors by changing the fine particles prepared from the solidified tumor material contained therein.

[0044]

Industrial Applicability The tumor vaccine of the present invention can be easily produced, has versatility applicable to prevention of recurrence, metastasis inhibition, and treatment regardless of the type of tumor, and is extremely excellent in antitumor effect. It has the characteristic that there is.

[Brief description of the drawings]

FIG. 1 is a graph showing the effects of various tumor vaccines on ovarian cancer primary tumor formation in forefoot footpads after HM-1 cell transplantation. The results in the figure show the results using three mice per group, and the vertical bar at each point indicates the standard deviation. Group A (-○-): control. Group B (-●-): tumor vaccine (no chemokine) + IL-2 /
GM-CSF sustained release formulation. Group C (-■-): Sustained-release formulation of tumor vaccine (containing SLC) + IL-2 / GM-CSF. Group D (-▽-): Sustained-release preparation of tumor vaccine (containing ELC) + IL-2 / GM-CSF. Group E (-▲-): Tumor vaccine (containing LARC) + IL-2 / GM-CSF
Sustained-release preparations.

FIG. 2 shows the effect of various tumor vaccines on the formation of metastatic lesions in regional lymph nodes after HM-1 transplantation. In the figure, the left column (gray) shows the result of the right upper arm lymph node, and the right column (white) shows the result of the left upper arm lymph node (control) of the same individual. The results in the figure show the results using three mice per group, and the vertical bar shows the standard deviation. Group A: control. Group B: tumor vaccine (without chemokine) + IL-2 / GM-CSF sustained-release preparation. Group C: tumor vaccine (containing SLC) + sustained-release formulation of IL-2 / GM-CSF. Group D: sustained-release formulation of tumor vaccine (containing ELC) + IL-2 / GM-CSF. Group E: sustained-release formulation of tumor vaccine (containing LARC) + IL-2 / GM-CSF.

Claims (9)

[Claims] 1. The following components: (a) microparticles prepared from a solidified tumor material selected from the group consisting of tumor tissue, tumor cells, and these components; (b) at least one cytokine and / or Or a cytokine inducer, (c) at least one chemokine and / or a chemokine inducer, and (d) a tumor vaccine comprising bone marrow cells and / or blood cells. 2. The following components: (a) a lysate prepared from a solidified tumor material selected from the group consisting of tumor tissue, tumor cells, and these components; (b) at least one cytokine; And / or a cytokine inducer, (c) at least one chemokine and / or a chemokine inducer, and (d) a tumor vaccine comprising bone marrow cells and / or blood cells. 3. The tumor vaccine according to claim 1, further comprising an adjuvant. 4. The tumor vaccine according to claim 1, which comprises a sustained-release cytokine preparation as the cytokine. 5. As a cytokine, granulocyte / macrophage colony stimulating factor and / or interleukin-2
The tumor vaccine according to any one of claims 1 to 4, comprising: 6. The tumor vaccine according to claim 1, which comprises a sustained release chemokine preparation as the chemokine. 7. The tumor vaccine according to claim 1, comprising a secondary lymphoid-tissue chemokine as a chemokine. 8. The bone marrow cells and / or blood cells include cells selected from the group consisting of hematopoietic stem cells, peripheral blood mononuclear cells, and a cell fraction derived therefrom.
The tumor vaccine according to any one of the above. 9. A tumor vaccine for use in combination with at least one cytokine and at least one chemokine, comprising: (e) tumor tissue, tumor cells, and a group consisting of these components A microparticle prepared from a solidified tumor material or a lysate prepared from the tumor material, and (f) a tumor vaccine comprising bone marrow cells and / or blood cells as active ingredients.