JP2005022997A - Therapy of tumor by cointroduciton of chemotherapeutic or the like and cytokine gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new immune gene therapeutic method which is effective not only for treated tumors but also for the therapy of metastatic focuses and the inhibition of tumor metastasis, and to provide an anti-tumor medicine and a kit used for performing the method. <P>SOLUTION: This method for treating tumor or metastatic tumor or inhibiting the metastatis of the tumor is characterized by introducing both (a) a cytotoxic agent and (b) a cytokine gene for inducing and/or activating Th1 cell into a tumor cell to induce an anti-tumor immune reaction. The cytotoxic agent and the cytokine gene are used for producing anti-tumor medicines, kits and medicines for treating tumors. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention induces an anti-tumor immune response by introducing both a cytotoxic agent and a cytokine gene that induces and activates Th1 cells into tumor cells, treats tumors or metastatic tumors, or treats tumors. The present invention relates to a method for suppressing metastasis. Furthermore, it is related with the antitumor agent and the kit used for the said method.
[0002]
[Prior art]
There is chemotherapy as an existing treatment for malignant tumors. However, systemic administration of a chemotherapeutic agent has strong side effects such as bone marrow suppression, and the dose is limited, so that sufficient effects are often not obtained.
[0003]
In order to reduce systemic side effects and increase the killing effect of tumors, treatments in which an anticancer agent is injected into the tumor site and then electroporation is performed to incorporate the drug into cells have already been clinically applied.
[0004]
Electroporation is a drug that does not sufficiently penetrate into cells due to its high molecular weight or electrostatic or physicochemical properties due to high voltage electrical impact on the cells to increase the permeability of the plasma membrane. Can be introduced into cells. Therefore, it becomes possible to administer the chemotherapeutic agent locally and effectively into the tumor cells, and it is possible to avoid severe side effects from high doses of the chemotherapeutic agent.
[0005]
Already, trials of electroporation using bleomycin or cisplatin for superficial tumors have been conducted, and therapeutic efficacy has been reported in many of these studies (see Non-Patent Documents 1 and 2). ). However, chemotherapeutic agents introduced by electroporation show tumoricidal activity only in the introduced tumor cells, so that local effects can be expected, but therapeutic effects on metastases cannot be expected.
[0006]
On the other hand, for the purpose of inducing or enhancing the anti-tumor immune response, genes such as cytokines can be treated by retrovirus vectors, adenovirus vectors, cationic liposomes, electroporation, etc. to treat malignant tumors It is. The greatest merit of this method is that antitumor immunity works systemically, so that it can be expected to be effective against metastatic lesions. In order to reduce the side effects of cytokines, the gene must be accurately delivered and / or expressed within the tumor. In such situations, electroporation is very suitable for introducing cytokine genes into tumor cells. Already, experiments have been carried out to introduce genes encoding IL-12, IL-2 and IL-18 into tumor cells using electroporation, and their effectiveness and safety have been demonstrated. (See Non-Patent Documents 3 and 4)
[0007]
Moreover, as a combination therapy, a combination of systemic administration of a chemotherapeutic agent and cytokine gene therapy is already known, but the problem of side effects due to systemic administration of the chemotherapeutic agent remains.
[0008]
In addition, after introducing a cytotoxic agent into a tumor cell, an experiment is conducted in which not an IL-2 gene itself but an IL-2 producing cell (IL-2 gene introduced cell) is injected around the tumor cell. (Refer nonpatent literature 5).
[0009]
However, in this method, there is a risk of infection from the cells themselves or the culture medium, growth factor, serum, etc. used for the culture, and strict safety inspection is required. Moreover, in order to obtain complete remission, it was necessary to repeat a series of treatments a plurality of times because the amount of cytokine production in the tissue is low, the duration of production is short, or both.
[0010]
In addition, experiments have been conducted in which both chemotherapeutic agents and cytokine genes are introduced into tumor cells by electroporation (see Non-Patent Document 6).
[0011]
In this experiment, mice were transplanted with B16 melanoma subcutaneously, and bleomycin and GM-CSF or IL-2 expression vector were injected back and forth into the tumor, and then an electric pulse was given. Suppression of tumor growth and survival of mice were confirmed. Cytokine genes can be handled much more easily and cheaply in terms of production, storage, and transport than cytokine-producing cells. Moreover, since the cytokine gene and the antitumor agent can be injected into the tumor on the same day, the burden on the patient is considered to be small.
[0012]
However, in this experiment, as compared with bleomycin alone or cytokine gene alone, a synergistic effect was not obtained even when both were used in combination, and only a slight additive effect was observed. Also, experiments were conducted only with subcutaneously transplanted tumors, and no therapeutic effect on metastatic tumors or tumor metastases has been reported.
[0013]
[Non-Patent Document 1]
Heller, R.A. , Et al. (1996). Phase I / II trial for the treatment of cutaneous and subtumorous tumors using electrochemotherapy. Cancer 77: 964-971.
[Non-Patent Document 2]
Domenge, C.I. , Et al. (1996). Anti-electrochemotherapy: new advance in the clinical protocol. Cancer 77: 956-963.
[Non-Patent Document 3]
Kishida, T .; , Et al. (2001). In vivo electroporation-mediated transfer of interleukin-12 and interleukin-18 gene genesisignificant effects effects alanomain. Gene Ther 8: 1234-1240.
[Non-Patent Document 4]
Lucas, M.M. L. , Heller, L .; , Coppola, D.C. , And Heller, R .; (2002). IL-12 plasmid delivery by in vivo electrophoretic for the successful full treatment of established subcutaneous B16. F10 melanoma. Mol Ther 5: 668-675.
[Non-Patent Document 5]
J. et al. Immunotherapy: 30-38, 1995Mir et al, 17 (1).
[Non-Patent Document 6]
Melanoma Res 10: 577-583, Heller, L .; Pottinger, C.I. , Jaroszeski, M .; J. et al. Gilbert, R .; , And Heller, R .; (2000).
[0014]
[Problems to be solved by the invention]
Therefore, a new immune gene therapy system that is effective for metastases and postoperative residual tumors has been desired.
[0015]
[Means for Solving the Problems]
As a result of repeated studies to solve the above problems, the present inventors have introduced both a cytokine gene that induces and activates Th1 cells and a cytotoxic agent such as an antitumor chemotherapeutic agent into tumor cells. Thus, the present inventors have found that the antitumor immune response is synergistically enhanced and a very strong tumor suppressive effect can be obtained as compared with a single treatment.
[0016]
That is, the present invention is characterized in that both (a) a cytotoxic agent and (b) a cytokine gene that induces and / or activates Th1 cells are introduced into tumor cells to induce an antitumor immune response. The invention relates to a method of treating a tumor or metastatic tumor or suppressing tumor metastasis.
[0017]
The principle of the present invention is considered as follows. Tumor cells are damaged by cytotoxic agents such as antitumor chemotherapeutic agents introduced into the cells, and tumor antigens are released outside the cells. This tumor antigen is captured by the immune system, particularly antigen presenting cells. Alternatively, the tumor cells are efficiently phagocytosed or processed by phagocytic cells. In this way, tumor antigens are processed and presented more efficiently than when no cytotoxic agent has been introduced.
[0018]
In recent years, it has been shown that the death of tumor cells by apoptosis is particularly important for inducing the presentation of tumor-specific antigens. Apoptotic cells are efficiently phagocytosed and antigen-treated by dendritic cells (DCs). Antigens are presented by class I and class II major histocompatibility complex (MHC), which elicits a tumor-specific T cell response.
[0019]
On the other hand, when a cytokine gene that induces and / or activates Th1 is introduced into tumor cells, the cytokine induces and activates Th1 cells. That is, the following is expected by co-introduction of a cytotoxic agent such as an antitumor chemotherapeutic agent and a cytokine gene that induces and activates Th1 cells. (1) By introducing a cytotoxic agent, presentation of a tumor-specific antigen is promoted, and as a result, T cells are stimulated. (2) Next, T cells differentiate into Th1 under the influence of cytokines that induce and activate Th1 cells. These two processes are thought to act synergistically to elicit strong cellular immunity induction and anti-tumor immune responses.
[0020]
That is, the present invention is characterized by the introduction of both a cytotoxic agent and a cytokine gene that induces and activates Th1 cells into a tumor cell, thereby preventing an unexpected complex effect (suppression of metastatic tumor, cytotoxic T A significant increase in cell activity). This could not be obtained with GM-CSF or IL-2 genes. Since gene therapy for malignant tumors focuses on treating metastatic tumors, this combined effect is very significant. Therefore, the present invention does not merely obtain the expected effect by combining the two existing methods, but a high effect far exceeding the expectation can be induced by a synergistic effect that has not been known so far. Is.
[0021]
In addition to cytokine genes other than those that induce and / or activate Th1 cells, by using genes such as cytokines that enhance one place or multiple places of any step that induces an anti-tumor immune response, It is considered that the anti-tumor immune response is synergistically enhanced and a strong tumor suppressing effect can be obtained as compared with the single treatment. That is, the following is foreseen. Releases tumor antigens to the outside when tumor cells are proliferated by cytotoxic agents such as antitumor chemotherapeutic agents, or the cell cycle is stopped or suppressed, or metabolism such as protein synthesis is suppressed or dies To do. This tumor antigen is captured by the immune system, particularly antigen presenting cells. Alternatively, the tumor cells are efficiently phagocytosed or processed by phagocytic cells. In this way, tumor antigens are processed and presented more efficiently than when no cytotoxic agent is received. Cytokines, etc. enhance tumor antigen presentation by enhancing one or more of these steps. That is, cytokines induce antigen-presenting cells or phagocytic cells locally in the tumor, or remain in the tumor, or proliferate, or increase the activity of phagocytosis / processing, or produce cytokines, chemokines, peroxide radicals, etc. To increase the effect. Cytokines also act on effector cells such as T cells and NK cells to induce or proliferate locally in the tumor, or increase activities such as antigen recognition / target damage, or cytokines, chemokines, their receptors, etc. It exerts effects such as enhancing expression. Which immunocompetent cell and how it works depends on the cytokine used, and therefore, an appropriate gene such as cytokine may be selected according to the purpose. This enhancement of antitumor immunity is thought to result in further tumor cell damage or death resulting in the formation of positive feedback. Such cytokines include IFN-gamma, IL-12, IL-15, IL-18, IL-21, IL-27, transforming growth factor-beta, tumor necrosis factor-alpha, and interferon ( IFN) -alpha, IFN-beta, interleukin (IL) -1, IL-7, IL-8, IL-9, IL-11, IL-14, IL-16, IL-17, IL-19, IL -20, IL-22, 1L-23, IL-24, IL-25, IL-26, IL-28, IL-29 and the like are possible. These are considered not only to induce and activate Th1 cells, but also to exert a synergistic effect by enhancing any step that induces an anti-tumor immune response. The step of enhancing may be one place or a plurality of places.
[0022]
In the above treatment / suppression method, the method for introducing a cytotoxic agent and a cytokine gene into tumor cells is preferably an in vivo electroporation method. Introducing the cytotoxic agent into the tumor cell using electroporation, etc., rather than giving it from outside the cell, the molecular weight is large, or due to electrostatic and physicochemical properties, Even drugs that can't penetrate can work. In addition, the effect can be expected for tumors that have acquired resistance to drugs by normal administration.
[0023]
Examples of cytokine genes that induce and / or activate Th1 cells include IFN-gamma, IL-12, IL-15, IL-18, IL-21, IL-27, transforming growth factor-beta, and tumor necrosis. Factor-alpha can be mentioned, and these may be introduced alone into tumor cells, or multiple kinds may be introduced. A particularly preferred cytokine gene is the IL-12 gene. IL-12 is a cytokine that plays an important role in the induction of cellular immune responses and enhances the proliferation and injury activity of CTLs as well as NK cells. IL-12 strongly induces and activates Th1 cells, enhances the Th1 response, and induces production of interferon γ. Therefore, the combined use of the IL-12 gene and the cytotoxic agent can significantly enhance the immune response against the tumor.
[0024]
The cytotoxic agent is preferably an antitumor chemotherapeutic agent, more preferably an antitumor antibiotic or a platinum preparation. Moreover, it may be used only by 1 type and multiple types may be used. More preferred are bleomycin and cisplatin. Bleomycin and cisplatin can induce apoptosis in target cells.
[0025]
In performing the above method, the cytokine gene to be introduced into the tumor cells is preferably IL-12 gene, and the cytotoxic agent is more preferably bleomycin, and the method of introducing into the tumor cells is the in vivo electroporation method. More preferably.
[0026]
The present invention also relates to an antitumor drug containing as active ingredients both (a) a cytotoxic agent and (b) a cytokine gene that induces and / or activates Th1 cells. The antitumor agent is preferably for administration by in vivo electroporation.
[0027]
In addition, the present invention relates to a kit of a preparation that contains (a) a cytotoxic agent and (b) a cytokine gene that induces and / or activates Th1 cells, which are used for carrying out the above treatment / suppression method.
[0028]
Furthermore, the present invention treats tumors or metastatic tumors by introducing into a tumor cell both (a) a cytotoxic agent and (b) a cytokine gene that induces and / or activates Th1 cells. The present invention relates to the use of the above cytotoxic agent and cytokine gene for producing a medicament for suppressing metastasis of cancer.
[0029]
DETAILED DESCRIPTION OF THE INVENTION
Cytotoxic agents are chemical substances that damage cells in general, such as cholera toxin, and preferably antitumor chemotherapeutic agents such as antitumor antibiotics, platinum preparations, alkylating agents, antifolates, pyrimidines Analogues, purine analogs, antitumor plant component preparations, and the like, more preferably antitumor antibiotics and platinum preparations. Examples of antitumor antibiotics include mitomycins, anthracyclines, actinomycins, aurelaric acids, and the like, and bleomycin is particularly preferable. Examples of the platinum preparation include carboplatin, and cisplatin is particularly preferable. These pharmaceutically acceptable salts are also included. For example, the bleomycin of the present invention includes bleomycin hydrochloride, bleomycin sulfate and the like.
[0030]
Cytokines that induce and / or activate Th1 cells include, for example, IFN-gamma, IL-12, IL-15, IL-18, IL-21, IL-27, transforming growth factor-beta, tumor necrosis factor -Alpha and the like. More preferably, they are IFN-gamma, IL-12, IL-15, IL-18, More preferably, it is IL-12.
[0031]
Tumors to which the present invention can be applied include not only superficial tumors such as malignant melanoma, skin cancer, head and neck cancer, but also tumors that can be reached endoscopically (esophageal cancer, stomach cancer, lung cancer, colon cancer). Etc.), tumors that can be reached under ultrasound guidance (prostate cancer, renal cancer, liver cancer, lung cancer, etc.), tumors that can be operated during surgery (liver cancer, pancreatic cancer, intracranial tumor, etc.), and the like. It can also be used for the treatment of metastatic tumors that have spread from these tumors to various organs, peritoneal dissemination, pleural dissemination, and the like. It can also be used to treat various metastatic tumors or residual tumors after surgery. Moreover, when metastasis is not clear, it can be used for prevention of metastasis.
[0032]
An anti-tumor immune response is a cell (T cell, B cell, NK cell, NKT cell, macrophage, neutrophil, etc.) or substance (antibody, complement, cytokine, active oxygen, etc.) involved in immunity and body defense However, it refers to the overall effect of causing cell death or growth suppression specifically or non-specifically on tumor cells. In this process, dendritic cells may present tumor antigens.
[0033]
The method of introducing a cytotoxic agent and a cytokine gene into tumor cells is preferably a method that can be locally introduced into tumor cells in order to avoid systemic side effects. For example, as a method for locally introducing a cytokine gene, in-vivo electroporation, gene gun, ultrasonic introduction, and viral vectors, cationic liposomes, cationic polymers, or as naked DNA Or a method of locally introducing a cytotoxic agent, such as an in-vivo electroporation method or a cationic liposome into a tumor or tumor blood vessel. There are injection methods. An in-vivo electroporation method that can introduce both a cytotoxic agent and a cytokine gene is particularly preferred, but the method of the present invention may be performed by combining two or more introduction methods.
[0034]
When introducing a cytotoxic agent by electroporation, it is common to energize with a high voltage (about 1350 V / cm) and a short pulse (about 100 μsec). May mutate. Therefore, it is preferable to use a low voltage (about 125 V / cm) and a long pulse (about 50 msec) when simultaneously introducing both the cytotoxic agent and the cytokine gene by electroporation. When the cytotoxic agent and the cytokine gene are separately introduced by electroporation, a high voltage / short pulse may be used for introduction of the cytotoxic agent, and a low voltage / long pulse may be used for introduction of the cytokine gene.
[0035]
The cytokine gene of the present invention may be any as long as it contains a base sequence encoding a cytokine. For example, it may be any of genomic DNA, genomic DNA library, or mammalian, preferably human cell / tissue-derived cDNA, isolated from a cDNA library by a known method, PCR product, or synthetic DNA. Moreover, the gene which changed the base sequence artificially so that an amino acid sequence may not be changed may be sufficient. Moreover, even if an amino acid sequence changes, the gene which changed the base sequence artificially may be sufficient so that the function as a cytokine may not be changed. These genes may be DNA or RNA. Specifically, cDNA described in GenBank or the like can be used. The IL-12 gene of the present invention is already known, and can be obtained from, for example, a human lymphocyte-derived cDNA library. These genes can be prepared by ordinary genetic engineering techniques, for example, by inserting them into a plasmid vector and transforming them into E. coli. The gene thus prepared can also be purified, for example, by ion exchange chromatography.
[0036]
When a cytokine gene is introduced into a tumor cell using in vivo electroporation, gene gun, or ultrasonic method, it is preferable to use a plasmid vector that expresses the cytokine gene. Examples of plasmid vectors include pCMV, pRSV, pSV, and pCAGGS. Examples of the promoter include cytomegalovirus IE promoter, b-actin promoter, and EF-1 promoter, and an appropriate promoter can be selected according to the type of tumor cell to be targeted. A more preferred plasmid vector is the EBV-based plasmid vector, such as pGEG shown in FIG. In addition, when a cytokine gene is injected into a tumor, or into a tumor blood vessel and introduced into a tumor cell, an adenovirus vector, a retrovirus vector, an adeno-associated virus vector, a herpes virus vector, a lentivirus vector, HVJ Viral vectors such as vectors, or artificial virus particles can be used. Cytokine genes can also be introduced in the form of DNA fragments or RNA.
[0037]
When performing the method of the present invention using in vivo electroporation, for example, after injecting a cytotoxic agent and a cytokine gene into a tumor, both can be introduced into tumor cells by electroporation. At this time, both the cytotoxic agent and the cytokine gene may be injected into the tumor at the same time, or each may be injected into the tumor alone, continuously or at an appropriate interval. In terms of reducing the burden on the patient, it is preferable to inject both at the same time. When injected alone, the cytotoxic agent may be injected first or the cytokine gene may be injected first, and when both are injected at intervals, the interval is preferably 1 second to 6 weeks. More preferably, it is 1 minute to 2 weeks, and more preferably 1 minute to 3 days.
[0038]
Furthermore, the cytotoxic agent and / or cytokine gene may be applied and pasted to the tumor as a coating agent or a patch, and then electroporation may be performed. For example, it is possible to apply and affix both a cytotoxic agent and a cytokine gene to a tumor and then perform electroporation. In addition, the cytokine gene is applied to or applied to the tumor as an application agent or patch, and before, during, or after that, a cytotoxic agent is injected into the tumor or injected into the tumor blood vessel. Electroporation may be performed by attaching electrodes so that an electric field is applied to the tumor, or conversely, a cytotoxic agent may be applied and applied to the tumor, and before, during or after the cytokine. The gene may be injected into a tumor, injected into a blood vessel of the tumor, or administered by intravenous injection or the like, and an electrode may be attached so that an electric field is applied to the tumor.
[0039]
It is also possible to introduce cytokine genes into tumor cells using methods other than electroporation. For example, a cytotoxic agent is injected into a tumor, or applied or pasted to a tumor as an application agent or a patch, and electroporation is performed, and before, during or after that, a cytokine gene is electroporated It can also be introduced into tumor cells by other methods. For example, a cytokine gene may be introduced into a tumor with a gene gun, and the cytokine gene is injected into the tumor together with microbubbles, injected into the blood vessel of the tumor, or administered by intravenous injection, etc. to target the tumor May be introduced by applying ultrasound, or the cytokine gene may be adenovirus vector, retrovirus vector, adeno-associated virus vector, herpes virus vector, lentivirus vector, HVJ vector or other virus vector, or artificial virus They may be incorporated into particles and injected into tumors or injected into tumor blood vessels.
[0040]
As described above, both can be introduced separately into tumor cells and introduced in different ways. Moreover, when introducing both by the same method, for example, when introducing each by electroporation, you may adjust buffer and pulse conditions suitably. It is preferable to introduce both at the same time from the viewpoint of simplicity and reduction of burden on the patient.
[0041]
Each of the cytokine gene and cytotoxic agent of the present invention may be one kind or two or more different kinds. For example, two or more types of cytokine genes and / or two or more types of cytotoxic agents may be introduced at once, or when introduction is performed a plurality of times, different cytokine genes and / or cytotoxic agents may be introduced each time. May be introduced. Moreover, cytokine genes other than the cytokine gene of the present invention can also be introduced. The types and combinations of cytokine genes and / or cytotoxic agents to be used can be appropriately selected depending on the type of tumor, the degree of symptoms, and the like. In addition, when introduction is performed a plurality of times, it is possible to determine an appropriate type and combination in consideration of therapeutic effects, side effects, and the like by the introduction up to the previous time.
[0042]
When two or more cytokine genes are introduced, for example, from IFN-gamma, IL-12, IL-15, IL-18, IL-21, IL-27, transforming growth factor-beta, tumor necrosis factor-alpha In addition to at least one cytokine gene selected from the group consisting of other cytokines (for example, interferon (IFN) -alpha, IFN-beta, interleukin (IL) -1, IL-7, IL-8, IL) -9, IL-11, IL-14, IL-16, IL-17, IL-19, IL-20, IL-22, IL-23, IL-24, IL-25, IL-26, IL-28 , IL-29, etc., or cytokines that induce and / or activate Th2, such as IL4, IL-10, IL-13, Inhibitory cytokines, inflammatory cytokines such as IL-1 and IL-6, hematopoietic cytokines such as GM-CSF and G-CSF, and growth factors such as IL-2) in tumor cells It may be introduced.
[0043]
The amount of cytotoxic agent / cytokine gene used, the number of introductions, and the interval between introductions can be appropriately determined depending on the sex, age, weight, type of tumor, severity of symptoms, therapeutic effect, etc. Further, the amount used, the number of introductions, and the introduction interval can be appropriately selected depending on the active ingredient to be administered, the dosage form, and the administration method.
[0044]
As an example of the amount used, in the case of a cytokine gene, the amount used per day is usually in the range of 1 pmol to 100 nmol, preferably 10 pmol to 10 nmol, more preferably 100 pmol to 3 nmol. In addition, the amount of cytotoxic agent used per day can be determined in an appropriate amount in consideration of the dosage and administration described in the package insert of each drug. About 0.002% to 100%, preferably 0.02% to 100%, more preferably about 0.2% to 40%, even more preferably about 2% to 10% of the daily dose given Dosage amount. For example, when the cytotoxic agent is bleomycin, bleomycin can be administered in a range of 0.3 μg to 30 mg per day, preferably 3 μg to 30 mg, more preferably 30 μg to 10 mg, more preferably 300 μg to It can be administered in the range of 3 mg. When the cytokine gene and cytotoxic agent are injected into a tumor, they are usually dissolved in an appropriate diluent and used preferably at a high concentration.
[0045]
The introduction frequency is usually 1 to 5 times, preferably 3 to 4 times, and the introduction interval is usually 1 to 28 days, preferably 2 to 7 days, but is not limited thereto. is not. Further, the number of administrations of the cytotoxic agent and the cytokine gene need not be the same, and can be appropriately adjusted in consideration of the degree of therapeutic effect, the presence or absence of side effects, the patient's condition, and the like.
[0046]
Furthermore, the methods of the invention can be performed in combination with other anti-tumor therapies. For example, it can be performed at the same time as, or before or after, chemotherapy, surgery, radiation therapy, hormone therapy, and the like. For example, after surgery, treatment for residual tumor and / or suppression of tumor metastasis, or in combination with oral administration of an anticancer drug, etc., to treat the tumor more efficiently and / or anticancer drug It is also possible to carry out for the purpose of reducing the amount of use.
[0047]
Furthermore, the method of the present invention can be performed in parallel with supportive therapy such as administration of a drug for reducing side effects and administration of morphine for pain relief.
[0048]
The antitumor agent of the present invention can be produced by directly mixing a cytokine gene and a cytotoxic agent with a pharmaceutically acceptable carrier according to known means generally used in the production of pharmaceutical preparations. it can. For example, each active ingredient is mixed with water or other pharmaceutically acceptable liquids to form a sterile solution or suspension, or each active ingredient is freeze-dried to form a solid mixture for dissolution at the time of use. It can be used parenterally as an injection. Examples of the form of the antitumor agent of the present invention include an injection agent, a coating agent, a patch agent and the like.
[0049]
The kit of the present invention is prepared by individually formulating each of the cytotoxic agent and the cytokine gene, for example, each active ingredient in a solid or aqueous solution, either of which is an aqueous solution and another active ingredient is Examples thereof include a solid injection kit. In addition, each active ingredient formulated individually is used to mix and administer using a diluent, etc. at the time of use, and each active ingredient formulated individually, separately or separately at a time difference A kit product for administration to the same subject is also included in the kit of the present invention. The kit of the present invention is not limited to injection, and includes, for example, a kit for mixing each active ingredient at the time of use and using it as a coating agent or a patch. The preparation contained in the kit of the present invention can be produced according to known means generally used in the production of pharmaceutical preparations.
[0050]
Examples of the pharmaceutically acceptable carrier that may be used for the preparation of the antitumor agent of the present invention or the preparation contained in the kit include an excipient in a solid preparation, and a solvent in a liquid preparation. Examples include solubilizers, isotonic agents, buffers, stabilizers, preservatives, suspending agents, soothing agents and the like. Examples of the solvent include water for injection, sesame oil, soybean oil, and corn oil. Examples of the solubilizer include potassium iodide, sodium benzoate, and sodium nicotinate. Examples of isotonic agents include sodium chloride. Glucose, etc., buffering agents include sodium salts such as citric acid, acetic acid, and phosphoric acid, stabilizers include human serum albumin, polyethylene glycol, etc., and preservatives include, for example, paraoxybenzoic acid There are acid esters, benzyl alcohol, chlorobutanol, phenol and the like. Examples of the suspending agent include carmellose sodium and povidone. Examples of the soothing agent include benzalkonium chloride and carbocaine hydrochloride. Examples of coating agents include ointment bases such as oleaginous bases, emulsion bases and water-soluble bases, stabilizers such as antioxidants and preservatives, and moisturizers. In the patch, adhesives, skin permeation promoting substances, stabilizers and the like can be mentioned.
[0051]
When the administration method is the above-mentioned in vivo electroporation method, the preferred form of the antitumor agent / kit according to the present invention is an injectable agent, a coating agent, and a patch agent, and particularly preferably for injection. It is an agent.
[0052]
The antitumor agent / kit of the present invention may contain two or more different cytotoxic agents and / or cytokine genes of the present invention. In that case, in a kit, you may formulate for every 1 type, or may be mixed and it may be set as the one agent cytotoxic agent containing formulation and the one cytokine gene containing formulation.
[0053]
The cytokine gene in the antitumor drug and kit of the present invention may be included in the form of a DNA fragment or RNA, but is incorporated into a gene expression vector suitable for the administration method of the antitumor drug / kit. It is preferable that For example, in the case of a preparation introduced into tumor cells by in vivo electroporation, gene gun, or ultrasonic method, it is preferably incorporated into a plasmid vector. In addition, in the case of a preparation to be introduced into tumor cells by injection into a tumor or tumor blood vessel, it is preferably incorporated into a viral vector or artificial virus particle, or a plasmid vector, cationic lipid, cationic More preferably, it is included as a complex of a carrier such as a nick polymer. Furthermore, in the case of preparations that are introduced into tumor cells by injection into tumors or tumor blood vessels, each preparation contains a plasmid vector and a carrier such as cationic lipid or cationic polymer separately. May be. Further, it may be bound to particles such as gold particles for introduction by a gene gun, or may be bound to microbubbles for introduction of ultrasonic waves. Alternatively, each preparation may contain a plasmid vector for introduction by a gene gun and gold particles separately. Alternatively, each preparation may contain a plasmid vector and a microbubble for ultrasonic introduction separately.
[0054]
The antitumor agent and kit of the present invention may contain other drugs in addition to the cytotoxic agent and cytokine gene of the present invention. For example, it may contain a drug for preventing / reducing side effects, a cytokine gene other than the cytokine gene of the present invention, and the like. In addition, other medicaments may be formulated by mixing with the cytotoxic agent and cytokine gene of the present invention, or may be formulated alone and combined with the antitumor drug or kit of the present invention.
[0055]
The antitumor drug and kit of the present invention are preferably for local administration, more preferably for local administration of tumor tissue. Particularly preferred is for local administration of tumor tissue by in vivo electroporation.
[0056]
Overview of pharmacological experiments
1. A solid tumor is formed under the skin of a mouse, a cytokine expression vector and / or cytotoxic agent is introduced into the tumor cell by in vivo electroporation, and then the serum cytokine concentration is measured to examine gene expression efficiency (Fig. 4).
2. In the same manner as in 1, a solid tumor is formed under the skin of a mouse, a cytokine expression vector and / or a cytotoxic agent is introduced into the tumor cell by in vivo electroporation, and then the tumor is collected, homogenized, and then tumor tissue The internal cytokine concentration is measured to examine gene expression efficiency in mouse tumors (see FIG. 5).
3. For the purpose of examining the antitumor effect, a cytokine expression vector and / or cytotoxic agent is introduced into a solid tumor formed subcutaneously in a mouse by in vivo electroporation, and the volume of the subcutaneous tumor is measured (see FIG. 6). .
4.3 Check the survival of mice in each group every day and examine the survival rate. On the other hand, by introducing both a cytotoxic agent and a cytokine expression vector, the same tumor is transplanted again into a surviving mouse, and the presence or absence of acquisition of specific immunity against the same tumor is examined (see FIG. 7).
5. In order to investigate the therapeutic effect on tumor metastasis or metastatic tumor, lung metastasis mice were prepared, cytokine expression vectors and / or cytotoxic agents were introduced into tumor cells by in vivo electroporation, The lungs are removed and the number of metastases is counted (see FIG. 8).
6). The survival of lung metastasis mice is confirmed daily and the survival rate is examined (see FIG. 9).
7). Cytotoxic tumors and / or cytokine genes are introduced into subcutaneous tumor mice by in vivo electroporation, and CTL assay and NK assay are performed to analyze induction of anti-tumor immune response (see FIGS. 10 and 11).
[0057]
【Example】
EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated concretely, this invention is not limited to these.
[0058]
"Vector used for experiment"
As a plasmid vector, pGEG. mIL-12 was used (FIG. 1 left). This is described in a non-patent document (Kishida et al., Gene Ther. 8: 1234-1240, 2001). In brief, it is a plasmid vector composed of the following components. That is, CAG promoter, mouse IL-12 p40 gene cDNA, internal ribosomal entry site (IRES) sequence, mouse IL-12 p35 gene cDNA, SV40 polyA signal, Epstein-Barr virus (EBV) oriP sequence, EBV EBNA1 gene A CAG promoter, an ampicillin resistance gene, and an ori sequence. As a control, pGEG. GL3 was also used (FIG. 1 right). This is described in a non-patent document (Cui et al., Gene Ther. 8 (19): 1508-1513, 2001). In brief, it is a plasmid vector composed of the following components. That is, it has a CAG promoter, luciferase GL3 gene, SV40 polyA signal, EBV oriP sequence, EBV EBNA1 gene, CAG promoter, ampicillin resistance gene, and ori sequence. All of these plasmids were amplified by transforming into E. coli HB101, and then purified according to the manual using an end-free plasmid purification kit manufactured by Qiagen.
[0059]
"Examination of introduction conditions by luciferase gene introduction"
The following experiment was conducted for the purpose of examining the conditions for in vivo gene introduction into mouse tumors. 5 × 10 5 B16 melanoma cells were subcutaneously subcutaneously in the flank of C57BL6 mice.⁵Individuals were transplanted by injection. 7 days later, solid tumors (average 75 mm)³) Was formed (this day is day 0). The mice were randomly divided into 4 groups of 3 mice, and each tumor was treated as follows. (1) K-PBS (30 mM NaCl, 120 mM KCl, 3 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 5 mM MgCl₂50 ml) was injected. (2) 5.5 pmol of pGEG. GL3 (dissolved in 50 ml K-PBS) was injected. Thereafter, tweezers-type electrodes were attached to the tumors of these mice (Neppagene, stainless steel, diameter 1.0 cm, distance between electrodes, 0.2 cm). An electric pulse was applied using an electroporation apparatus (manufactured by CUY21 Nepagene). Voltage (1) was 25 V, and (2) mice were further divided into 4 groups, and 0 V, 12.5 V, 25 V, 50 V, or 75 V was given to each group (3 mice in each group). The frequency was once per second, the pulse width was 50 milliseconds, a square wave was given three times, the polarity was reversed, and the pulse was further given three times. Three days later, the mice were euthanized and the tumors were removed. 200 μL of lysis buffer (Promega) was added to the tumor and homogenized using a sonicator. After freezing and thawing twice, the extract was centrifuged at 14000 × g for 5 minutes, and the supernatant was collected. The luciferase activity was performed according to the protocol using a Promega luciferase assay kit (catalog number, E1483). Photoluminescence for 10 seconds was measured using a luminometer. In addition, the total amount of protein in the same tumor extract was measured according to the method of Bradford et al., And the amount of luciferase was corrected by the amount of total protein. The result is shown in FIG. Tumors applied with a voltage of 25 V after injection of PBS, and pGEG. In the tumors to which no voltage was applied after injection of GL3, the expression of luciferase was below the measurement limit in both cases, but the activity of luciferase was observed in the tumors to which 25 to 75 V was applied. When compared between different voltages, the 25V group showed the highest expression. From this, when electroporation was performed using the above electrodes under the conditions of a frequency of once per second, a pulse width of 50 milliseconds, and a total of 6 square waves, a low voltage long pulse of 25 V is the most plasmid DNA. It was found that the conditions were suitable for introducing.
[0060]
"Introduction of bleomycin by low voltage long pulse"
It was investigated whether bleomycin could be efficiently introduced under the optimum conditions for introduction of the above plasmid. Tumor-bearing mice were prepared in the same manner as described above, and (1) K-PBS (50 ml) or (2) 20 μg of bleomycin (dissolved in 50 ml of K-PBS) was injected. In the same manner as above, (1) was divided into two groups of 25V, and (2) was further divided into two groups, each of which was applied with a voltage of 0V or 25V (four mice in each group). The same treatment was further repeated on the 5th and 10th days. The volume of the subcutaneous tumor of the mouse was measured by the following method. Using a digital caliper, the major axis (a) and the minor axis (b) of each tumor are measured every two days, and V = a × b²Tumor volume (V) was calculated as / 2. The result is shown in FIG. The arrow in the figure indicates the day on which electroporation was performed. In addition, the graphs represented by rhombuses, squares, and triangles are respectively the group rhombus in which PBS was injected and a pulse of 25 V was added, the group (square) in which bleomycin was injected and no pulse was given, and the pulse of 25 V injected with bleomycin. The mean and SD of the tumor volumes of the 3 groups of the added group (triangle) are shown. The group given only one of the electric pulse and bleomycin showed a marked increase in tumor, but the group given both significantly suppressed the tumor growth. Therefore, when electroporation was not performed, this tumor local chemotherapy had no antitumor effect, and this antitumor chemotherapeutic agent suppressed tumor growth by performing electroporation. I understand that. Therefore, when electroporation was performed using the above electrodes under the conditions of a voltage of 25 V, a frequency of once per second, a pulse width of 50 milliseconds, and a total of 6 square waves, bleomycin was strongly taken into tumor cells. It was suggested.
[0061]
"Example 1" IL-12 expression vector introduction experiment into tumor by electroporation (1)
(A) The following experiment was conducted for the purpose of examining the efficiency of in vivo gene introduction into mouse tumors. 5 × 10 5 B16 melanoma cells were subcutaneously subcutaneously in the flank of C57BL6 mice.⁵Individuals were transplanted by injection. 7 days later, solid tumors (average 75 mm)³) Was formed (this day is day 0). The mice were randomly divided into 4 groups of 3 mice, and each tumor was treated as follows. (1) K-PBS (30 mM NaCl, 120 mM KCl, 3 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 5 mM MgCl₂50 ml) was injected. (2) 20 μg of bleomycin (dissolved in 50 ml of K-PBS) was injected. (3) 5.5 pmol of pGEG. GL3 (dissolved in 50 ml K-PBS) was injected. (4) 20 μg of bleomycin and 5.5 pmol of pGEG. mIL-12 (dissolved in 50 ml K-PBS) was injected. Thereafter, tweezers-type electrodes were attached to the tumors of these mice (Neppagene, stainless steel, diameter 1.0 cm, distance between electrodes, 0.2 cm). An electric pulse was applied using an electroporation apparatus (manufactured by CUY21 Nepagene). The voltage was 25 V, the frequency was once per second, the pulse width was 50 milliseconds, a square wave was applied three times, the polarity was reversed, and the pulse was further performed three times.
(B) Mice were euthanized on day 2, 4, or 6, serum was collected, and IL-12 concentration was measured by ELISA. A mouse IL-12 ELISA kit manufactured by R & D Systems was used for the ELISA. The result is shown in FIG. In groups (1) and (2), no significant increase in serum IL-12 concentration was observed, but in groups (3) and (4), a high concentration of 400 pg / mL or more was observed on days 2 and 4 after introduction. IL-12 was detected. On the sixth day after introduction, the IL-12 concentration decreased, but was significantly higher than that in the non-introduction group. From this experiment, it was found that introduction of an IL-12 expression vector into a tumor by electroporation led to very high IL-12 production, and this IL-12 production was not suppressed by co-introduction of bleomycin.
[0062]
"Example 2" IL-12 expression vector introduction experiment into tumor by electroporation (2)
After performing the same experiment as in Example 1 (A), the mice were euthanized 4 days later, and tumors were collected. After homogenizing the tumor using a sonicator, the concentration of IL-12 in the extract was measured by ELISA. A mouse IL-12 ELISA kit manufactured by R & D Systems was used for the ELISA. In order to correct the IL-12 concentration, the protein concentration in the same extract was measured based on the method of Bradford et al. The result is shown in FIG. As a result consistent with the above 3, no significant increase in serum IL-12 concentration was observed in the groups (1) and (2), but in the groups (3) and (4), the total protein was more than 1500 pg / mg. A high concentration of IL-12 was detected. This experiment further confirmed that introduction of an IL-12 expression vector into a tumor by electroporation led to very high IL-12 production, and that this IL-12 production was not suppressed by co-introduction of bleomycin.
[0063]
"Example 3" Examination of antitumor effect
(A) The following experiment was conducted for the purpose of examining the antitumor effect by introduction of bleomycin and / or IL-12 gene. Subcutaneous flank of 5-6 week old C57BL6 mice received 5 × 10 B16 melanoma cells.⁵An individual was transplanted. 7 days later, solid tumors (average 75 mm) at the transplant site³) Was formed (this day is day 0). On day 0, mice were divided into 4 groups and each tumor was treated as follows. (1) pGEG. GL3 was dissolved in 5.5 pmol, 50 ml of K-PBS, and this solution was injected (7 animals). (2) pGEG. mIL-12 was dissolved in 5.5 pmol, 50 ml of K-PBS, and this solution was injected (7 animals). (3) 20 μg of bleomycin was dissolved in 50 ml of K-PBS, and this solution was injected (8 animals). (4) 20 μg of bleomycin and 5.5 pmol of pGEG. mIL-12 was dissolved in 50 ml K-PBS and this solution was injected (8 animals). Thereafter, tweezers-type electrodes were attached to the tumors of these mice (Neppagene, stainless steel, diameter 1.0 cm, distance between electrodes, 0.2 cm). An electric pulse was applied using an electroporation apparatus (manufactured by CUY21 Nepagene). The voltage was 25 V, the frequency was once per second, the pulse width was 50 milliseconds, a square wave was applied three times, and then the polarity was changed and further pulsed three times. Similar treatments were performed on the fifth and tenth days.
(B) The volume of the subcutaneous tumor of the mouse in (A) was measured by the following method. Using a digital caliper, the major axis (a) and the minor axis (b) of each tumor are measured every 2-3 days, and V = a X b²Tumor volume (V) was calculated as / 2. The result is shown in FIG. The arrow in the figure indicates the day on which electroporation was performed. In addition, the graphs represented by diamonds, squares, triangles, and circles are the four groups of (1)-(4) ((1) ◆, (2) ■, (3) ▲, (4) ●) in (A). Mean tumor volume and SD. Although only bleomycin introduction or IL-12 gene introduction alone suppresses tumor growth, the inhibitory effect is not perfect. On the other hand, it can be seen that tumor growth was suppressed very efficiently in tumors into which both bleomycin and an IL-12 expression vector were introduced.
[0064]
"Example 4" Examination of survival rate and specific immunity
The survival rate of each group of mice in Example 3 (A) was confirmed daily and Kaplan-Meier analysis was performed. The result is shown in FIG. The arrow in the figure indicates the day on which electroporation was performed. In addition, the graphs represented by diamonds, squares, triangles, and circles are marked with (1)-(4) ((1) ◆, (2) ■, (3) ▲, (4) ●) of Example 3 (A). 4 represents the mean and SD of the volumes of the 4 groups of tumors. The IL-12 gene transfer alone did not significantly improve the survival rate of tumor-bearing mice. In the bleomycin-introduced group only, the survival rate improved significantly, but all mice died within 49 days and no complete relief was obtained. On the other hand, in the tumors into which both the chemotherapeutic agent and the IL-12 expression vector were introduced, all mice survived for 50 days or more, and 3 out of 8 mice (37.5%) rejected the tumor. Therefore, on the 150th day, 5 × 10 5 of B16 was again applied to the flank of these surviving mice (on the opposite side to the site where the tumor was implanted).⁴Individual transplants (Fig. 7, large arrows). All mice rejected the tumor and survived for over 50 days after reimplantation. Therefore, bleomycin and pGEG. It was found that by introducing both of mIL-12 by electroporation, 37.5% of mice were completely relieved and acquired specific immunity against the same tumor.
[0065]
[Example 5] Examination of therapeutic effect on tumor metastasis or metastatic tumor
(A) The following experiment was conducted for the purpose of investigating the therapeutic effect on tumor metastasis or metastatic tumor. Subcutaneous flank of 5-6 week old C57BL6 mice received 5 × 10 B16 melanoma cells.⁵An individual was transplanted. 7 days later, solid tumors (average 75 mm) at the transplant site³) Was formed (Day 0). On day 0, 5 x 10⁴B16 cells were injected from the tail vein to create lung metastasis mice. On the same day, the mice were divided into 4 groups, and each tumor was treated as follows: (1) 50 ml of PBS was injected (control group) (4 mice). (2) pGEG. mIL-12 was dissolved in 5.5 pmol, 50 ml of K-PBS, and this solution was injected (5 animals). (3) 20 μg of bleomycin was dissolved in 50 ml of K-PBS, and this solution was injected (4 animals). (4) 20 μg of bleomycin and 5.5 pmol of pGEG. mIL-12 was dissolved in 50 ml of K-PBS and this solution was injected (5 animals). Thereafter, tweezers-type electrodes were attached to the tumors of these mice (Neppagene, stainless steel, diameter 1.0 cm, distance between electrodes, 0.2 cm). An electric pulse was applied using an electroporation apparatus (manufactured by CUY21 Nepagene). The voltage was 25 V, the frequency was once per second, the pulse width was 50 milliseconds, a square wave was applied three times, and then the polarity was changed and further pulsed three times. Similar treatments were performed on the fifth and tenth days.
(B) The mice in (A) were euthanized on day 14, the lungs on both sides were removed, and the number of metastatic foci of melanoma was counted. The result is shown in FIG. The number of metastatic lesions in each experimental group is expressed as mean and SD. In this system, no significant inhibition of metastasis was observed in the bleomycin monotherapy as compared with the control, and similar suppression was observed in the IL-12 gene alone introduction group and the bleomycin + IL-12 gene co-introduction group. This indicates that IL-12 expressed locally in the tumor induces anti-tumor immunity, and the immunity acts to suppress metastasis to distant organs and / or the growth of metastatic tumors.
[0066]
"Example 6" Examination of survival rate in lung metastasis model
(A) The same experiment as in Example 5 (A) was performed. However, as a difference from Example 5 (A), 5 mice were used in all groups (1) to (4).
(B) The survival rate of each group of mice in (A) was confirmed daily and Kaplan-Meier analysis was performed. The result is shown in FIG. The arrow in the figure indicates the day on which electroporation was performed. In addition, the graphs represented by diamonds, squares, triangles, and circles are the four groups of (1)-(4) ((1) ◆, (2) ■, (3) ▲, (4) ●) in (A). Mean tumor volume and SD. No significant improvement was observed in the survival rate of the tumor-bearing mice in the group with only IL-12 gene transfer and only with bleomycin transfer. On the other hand, the survival rate was significantly improved in tumors into which both the chemotherapeutic agent and the IL-12 expression vector were introduced. Therefore, it was found that bleomycin + IL-12 gene co-introduction has the most therapeutic effect.
[0067]
"Example 7" Examination of antitumor immune response induction effect
(A) A CTL assay and an NK assay were performed for the purpose of analyzing an anti-tumor immune response induced by bleomycin and / or IL-12 gene transfer. For this purpose, the same treatment experiment as “Example 3” (A) was performed. However, as a difference from “Example 3” (A), three mice were used in all groups (1) to (4).
(B) On the 15th day, the mouse was euthanized, the spleen was removed, the spleen cell suspension was adjusted, and erythrocytes were lysed. After washing, the number of viable cells was counted by trypan blue dye exclusion method to obtain effector cells. On the other hand, as target cells, B16 cells were suspended in RPMI 1640 medium containing 10% fetal calf serum, and 370 kBq of Na.₂ ⁵¹CrO₄Add 5% CO₂Incubated for 60 minutes in an atmosphere containing / 95% saturated water vapor. The B16 cells were washed 3 times with RPMI 1640 medium containing 10% fetal calf serum, and then added to each well 10 wells in a 96-well culture plate (U-shaped bottom).⁴It was sowed so that it might become a piece / 100 ml. In these wells, the above effector cells are added 10⁶5 x 10⁵2.5 x 10⁵Or 1.25 x 10⁵  It adjusted and added so that it might become / 100 ml. As a control, wells were prepared by adding 100 ml of RPMI 1640 medium containing 10% fetal calf serum without effector cells to the effector cells. As another control, a well was prepared by adding 100 ml of 0.1% Triton-X-100 to the effector cells. (All these wells were made in triplicate). After 4 hours, 100 ml of the supernatant of each well was collected, and the radioactivity of gamma rays (corresponding to chrome release) was measured with a gamma counter. The% cell injury was calculated based on the following formula: % Cytotoxicity = 100 × (Chrome release of supernatant of wells with each effector added) − (Chrome release of supernatant of wells added with RPMI 1640 medium containing 10% fetal calf serum instead of effector) / (effector (Chrome release of supernatant of wells added with 0.1% Triton-X-100 instead)-(Crime release of supernatant of wells added with RPMI 1640 medium containing 10% fetal calf serum instead of effector). FIG. 10 shows the result. % Cell injury is significantly increased only in the bleomycin + IL-12 gene co-introduced group, indicating that this treatment enhanced the CTL activity specific to B16. On the other hand, no significant CTL enhancement was observed in the bleomycin alone introduction group or the IL-12 gene alone introduction group.
(C) The same experiment as (B) was conducted. However, instead of B16 cells, YAC-1 cells, which are NK-sensitive lymphoma cell lines, were used as target cells. FIG. 11 shows the result. NK activity was greatly enhanced in the bleomycin + IL-12 gene co-introduced group. In the IL-12 gene alone introduction group, statistically significant enhancement was observed, but the degree of enhancement was lower than that in the bleomycin + IL-12 gene introduction group. In contrast, no significant NK enhancement was observed in the bleomycin-introduced group.
[0068]
"result"
According to this example, co-introduction of cytokine genes and cytotoxic agents using in vivo electroporation induces specific and non-specific anti-tumor immune responses, resulting in against subcutaneous and metastatic tumors in mice. It has been demonstrated that significant therapeutic effects can be obtained.
[0069]
【The invention's effect】
By introducing both a cytotoxic agent and a cytokine gene that induces and activates Th1 cells into tumor cells, not only the therapeutic effect of the introduced tumor, but also the suppression of metastatic tumors and the remarkable cytotoxic T cell activity As a result, the survival rate of mice is significantly improved. Moreover, fully ameliorated mice show specific immunity against the same tumor.
[Brief description of the drawings]
FIG. 1 shows a map of a plasmid.
FIG. 2 is a bar graph showing the expression of a luciferase gene within a tumor.
FIG. 3 is a line graph showing the growth inhibitory effect of subcutaneous tumors.
FIG. 4 is a bar graph showing serum IL-12 concentration.
FIG. 5 is a bar graph showing IL-12 concentration in tumor tissue.
FIG. 6 is a line graph showing the growth inhibitory effect of subcutaneous tumors.
FIG. 7 is a graph showing the survival rate of mice transplanted with subcutaneous tumors.
FIG. 8 is a bar graph showing the effect of reducing the number of lung metastases.
FIG. 9 is a graph showing the survival rate of lung metastasis mice.
FIG. 10 is a line graph showing CTL activity.
FIG. 11 is a line graph showing NK activity.

Claims (32)

A tumor or metastatic tumor characterized by introducing both (a) a cytotoxic agent and (b) a cytokine gene that induces and / or activates Th1 cells into a tumor cell to induce an antitumor immune response Or treating tumor metastasis. The method according to claim 1, wherein the method of introducing a cytotoxic agent and a cytokine gene into tumor cells is in vivo electroporation. The cytokine gene is at least selected from the group consisting of IFN-gamma, IL-12, IL-15, IL-18, IL-21, IL-27, transforming growth factor-beta, tumor necrosis factor-alpha. The method according to claim 1, which is a kind of cytokine gene. The cytokine gene is at least selected from the group consisting of IFN-gamma, IL-12, IL-15, IL-18, IL-21, IL-27, transforming growth factor-beta, tumor necrosis factor-alpha. The method according to claim 2, which is a gene of a kind of cytokine. The method according to claim 1, wherein the cytokine gene is an IL-12 gene. The method according to claim 2, wherein the cytokine gene is an IL-12 gene. The method according to claim 1, wherein the cytotoxic agent is at least one antitumor chemotherapeutic agent. The method according to claim 2, wherein the cytotoxic agent is at least one antitumor chemotherapeutic agent. The method according to claim 1, wherein the cytotoxic agent is at least one antitumor antibiotic and / or at least one platinum preparation. The method according to claim 2, wherein the cytotoxic agent is at least one antitumor antibiotic and / or at least one platinum preparation. The method according to claim 1, wherein the cytotoxic agent is bleomycin and / or cisplatin. The method according to claim 2, wherein the cytotoxic agent is bleomycin and / or cisplatin. The method according to claim 1, wherein the cytokine gene is an IL-12 gene and the cytotoxic agent is bleomycin. The method according to claim 2, wherein the cytokine gene is an IL-12 gene and the cytotoxic agent is bleomycin. An antitumor agent containing both (a) a cytotoxic agent and (b) a cytokine gene that induces and / or activates Th1 cells as active ingredients. The antitumor agent according to claim 15, which is for administration by in vivo electroporation. The cytokine gene is at least selected from the group consisting of IFN-gamma, IL-12, IL-15, IL-18, IL-21, IL-27, transforming growth factor-beta, tumor necrosis factor-alpha. The antitumor agent according to claim 15, which is a kind of cytokine gene. The cytokine gene is at least selected from the group consisting of IFN-gamma, IL-12, IL-15, IL-18, IL-21, IL-27, transforming growth factor-beta, tumor necrosis factor-alpha. The antitumor agent according to claim 16, which is a kind of cytokine gene. The antitumor agent according to claim 15, wherein the cytokine gene is an IL-12 gene. The antitumor agent according to claim 16, wherein the cytokine gene is an IL-12 gene. The antitumor agent according to claim 15, wherein the cytotoxic agent is at least one antitumor chemotherapeutic agent. The antitumor agent according to claim 16, wherein the cytotoxic agent is at least one antitumor chemotherapeutic agent. The antitumor agent according to claim 15, wherein the cytotoxic agent is at least one antitumor antibiotic and / or at least one platinum preparation. The antitumor agent according to claim 16, wherein the cytotoxic agent is at least one antitumor antibiotic and / or at least one platinum preparation. The antitumor agent according to claim 15, wherein the cytotoxic agent is bleomycin and / or cisplatin. The antitumor agent according to claim 16, wherein the cytotoxic agent is bleomycin and / or cisplatin. The antitumor agent according to claim 15, wherein the cytokine gene is an IL-12 gene, and the cytotoxic agent is bleomycin. The antitumor agent according to claim 16, wherein the cytokine gene is an IL-12 gene, and the cytotoxic agent is bleomycin. A kit of a preparation for separating and comprising (a) a cytotoxic agent and (b) a cytokine gene that induces and / or activates Th1 cells for carrying out the method according to claim 1. A kit of a preparation for separating and comprising (a) a cytotoxic agent and (b) a cytokine gene that induces and / or activates Th1 cells for carrying out the method according to claim 2. By introducing both (a) a cytotoxic agent and (b) a cytokine gene that induces and / or activates Th1 cells into a tumor cell, the tumor or metastatic tumor is treated or tumor metastasis is suppressed. Use of the cytotoxic agent and cytokine gene for the manufacture of a medicament. 32. The use according to claim 31, wherein the method of introducing (a) a cytotoxic agent and (b) a cytokine gene into tumor cells is in vivo electroporation.

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