JP2009091348A - How to treat cancer - Google Patents

Abstract

The present invention provides an immune / gene combined treatment method that has an effect of suppressing the growth of refractory solid cancer and is useful for the treatment, and a therapeutic agent for use in the treatment method.
A method for treating solid cancer, comprising performing interferon gene therapy on a tumor or in the vicinity of the tumor after performing treatment with lymphopenia and / or autologous hematopoietic stem cell transplantation, and the method A therapeutic agent used in the treatment method.
[Selection figure] None

Description

  The present invention relates to a combined immuno-gene therapy method that has an effect of suppressing the growth of refractory solid cancer and is useful for the treatment, and a therapeutic agent for use in the therapy method.

  Cancer treatment includes surgical treatment, radiation therapy, chemotherapy, immunotherapy, and the like. Among them, chemotherapy using an anticancer drug is common for advanced cancer. Many of the anticancer drugs have many side effects because they damage not only cancer cells but also normal cells. For example, a side effect called hematologic toxicity may occur due to bone marrow suppression caused by an anticancer agent, which reduces neutrophils, platelets, lymphocytes, and the like in peripheral blood to below normal values. In such cases, it is effective and often practiced to administer as much anticancer agent as possible while repeating administration while minimizing the accumulation of side effects after a certain recovery period. Usually, in such treatment, a plurality of anticancer agents are used in combination.

  Among cancers, hematopoietic stem cell transplantation is often performed in combination with the above-described chemotherapy and radiotherapy as a treatment for hematopoietic malignant tumors. At this time, the patient falls into a lymphocytopenia, but the remaining or transplanted T cells proliferate rapidly in order to maintain the T cell count (this is called Homeostatic Proliferation; HP). Under normal conditions, low-affinity self-antigens cannot induce T cell proliferation, but during HP, the signal threshold required for lymphocyte proliferation is reduced, so that self-antigens can also induce T cell proliferation. . Originally, cancer antigens are self-antigens with low affinity. It is believed that when lymphocytes are exposed to a cancer antigen during HP, the proliferation of lymphocytes that are toxic to the cancer antigen is promoted and an immune response against the cancer is induced (non-) Patent Documents 1 and 2). And even in autologous hematopoietic stem cell transplantation, when the immunosuppressive environment is destroyed and the fresh immune system is reconstructed, the recognition of tumor-specific antigens can be promoted to obtain an antitumor effect. It is clear.

  Interferon (IFN) has been developed more than 10 years ago as a protein preparation because it has various physiological functions such as antiviral activity, cell growth inhibitory effect, and tumor-specific immunostimulation. It is widely used as a therapeutic agent for various diseases such as malignant tumors such as myeloma, chronic myelogenous leukemia, renal cancer, melanoma, and multiple sclerosis. Among the IFNs, IFN-α and IFN-β classified as type I-IFN exert their effects via a common receptor. In addition, IFN-α is known to have ten or more subtypes in terms of specificity. Currently, IFN-α and IFN-β pharmaceuticals are on the market. These protein preparations are administered systemically by subcutaneous or intramuscular administration, but systemic side effects due to various physiological activities (flu-like symptoms such as fever, dullness, fatigue, headache, muscle pain, convulsions, and fatal ones) Interstitial pneumonia) is often a problem.

  It has been reported in animal models that an adenoviral vector (Ad-IFNα) expressing IFN-α is effective against pancreatic cancer by a single local administration of tumor and has very little systemic toxicity. (Non-patent Documents 3 and 4, Patent Document 1).

US Patent Application Publication No. 2005/0260167 Dummer et al. J Clin Invest 110: 185-192, 2002 Hu et al. Cancer Research 62: 3914-3919, 2002 Ohashi M et al. British J Cancer 93: 441-449, 2005 Hara H et al. Cancer Science 98: 455-463, 2007

  Chemotherapy and radiation therapy, and their subsequent hematopoietic stem cell transplantation, are accompanied by a marked decrease in lymphocytes, but the recovery of tumor-specific antigens by immune cells is promoted during the recovery process, resulting in an antitumor effect. Is obtained. However, the clinical problem is that these anti-tumor effects are not sustained. Anticancer agents kill many cancer cells and many normal cells are also damaged, and cells in the blood such as lymphocytes are killed and their number decreases. In some cases, the administration of anticancer agents must be stopped to recover lymphocytes and the like. Recurrence is a major unresolved problem even in treatment with anticancer drugs.

  Since IFN has various physiological actions such as antiviral activity, cell growth inhibitory effect, and tumor specific immune activation, systemic side effects due to various physiological activities often become a problem. However, by administering an IFN gene-containing vector in or near a tumor, IFN is strongly expressed locally, and by increasing its concentration, a more effective therapeutic effect can be obtained while avoiding adverse events. Furthermore, it is possible to exert an immunostimulatory effect systemically while suppressing side effects caused by systemic administration by local administration.

  The present invention relates to a tumor-specific immunostimulatory effect by local administration of this IFN gene expression vector in or near a tumor and a method for treating cancer with lymphopenia and / or subsequent autologous hematopoietic stem cell transplantation. The objective is to synergistically promote specific anti-tumor immunity effects against refractory solid cancer by combining them rationally.

  Briefly describing the present invention, the first invention of the present invention includes the steps of performing treatment with lymphopenia on a patient and administering an IFN gene to a local area in or near the tumor of the patient. The present invention relates to a method for treating cancer characterized by comprising:

  In addition, the second invention of the present invention is a step of performing treatment with lymphopenia in a patient, a step of transplanting autologous hematopoietic stem cells to a patient, and administering an IFN gene to a local area in or near the tumor of the patient. The present invention relates to a method for treating cancer, comprising a step.

  In the first or second aspect of the present invention, the treatment accompanied by a decrease in the number of lymphocytes includes administration of an anticancer agent, an immunosuppressive agent and / or irradiation. The anticancer agent administration is selected from the group consisting of fluorouracil, methotrexate, gemcitabine, fludarabine, bleomycin, adriamycin, mitomycin, paclitaxel, docetaxel, vincristine, irinotecan, etoposide, cisplatin, carboplatin, nedaplatin, and cyclophosphamide. The immunosuppressive agent and the like is at least selected from the group consisting of azathioprine, mizoribine, mycophanol mofetil, methotrexate, cyclosporine, tacrolimus, gusperimus hydrochloride, and muromonab-CD3. Administration of one immunosuppressive agent is mentioned. In the first or second aspect of the present invention, IFN gene administration includes IFN gene administration using an IFN gene-containing vector, and the vector is used for high expression of the IFN gene used locally. Any suitable vector may be used, including viral vectors such as adenovirus vectors, retrovirus vectors, and Sendai virus vectors, and the use of liposome-encapsulated plasmid vectors. Further, in the first or second aspect of the present invention, examples of the IFN include IFN-α and IFN-β. In the first or second aspect of the present invention, the cancer includes solid cancer. For example, the present invention provides a method for treating colorectal cancer, renal cancer, etc., which are intractable solid cancer.

  A third invention of the present invention is a therapeutic agent for administering an IFN gene to a local area in or near a tumor of a patient who has been treated with lymphopenia, comprising an IFN gene-containing vector According to a fourth aspect of the present invention, there is provided a therapeutic agent for cancer, wherein the fourth aspect of the invention relates to a local area in or near a tumor of a patient who has undergone autologous hematopoietic stem cell transplantation following a treatment involving lymphopenia. The present invention relates to a therapeutic agent for administering an IFN gene, comprising an IFN gene-containing vector.

  In the third and fourth aspects of the present invention, examples of the IFN include IFN-α and IFN-β. The vector may be in the form of a vector suitable for high expression of the IFN gene to be used locally, and examples include viral vectors such as adenovirus vectors, retrovirus vectors, and Sendai virus vectors. Moreover, the use of a plasmid vector encapsulated in liposomes can be mentioned.

  The fifth invention of the present invention relates to the use of the IFN gene in the manufacture of a therapeutic agent for cancer administered to the local area in or near the tumor of a patient who has been treated with lymphopenia. The sixth invention of the present invention relates to the use of the IFN gene in the manufacture of a therapeutic agent for cancer administered to a local area in or near a tumor of a patient who has undergone autologous hematopoietic stem cell transplantation following treatment with lymphopenia. Regarding use.

  In the fifth and sixth aspects of the present invention, the present invention can be used for the production of a therapeutic agent capable of suppressing the growth of solid cancer. In the above embodiment, the IFN gene may be an IFN gene-containing vector. Examples of IFN include IFN-α and IFN-β. The vector may be in the form of a vector suitable for high expression of the IFN gene to be used locally, and examples thereof include the use of viral vectors such as adenovirus vectors, retrovirus vectors, and Sendai virus vectors. And the use of a plasmid vector encapsulating liposomes.

The present invention relates to a method of treating solid cancer, characterized by performing IFN gene therapy on a tumor or in the vicinity of the tumor after carrying out treatment with lymphopenia and / or autologous hematopoietic stem cell transplantation, and treatment therefor A therapeutic agent or the like for use in the method is provided.
For colon cancer and renal cancer which are intractable solid cancers, the present invention provides a synergistic and powerful anti-tumor by introducing an IFN gene after treatment with lymphopenia and / or autologous hematopoietic stem cell transplantation. An effect can be obtained. This antitumor effect is also observed in a subcutaneous tumor at a distant site where the IFN gene has not been introduced, and has a particular effect in that it can induce a systemic antitumor immune response.

  The present invention will be specifically described below.

  In the present invention, the treatment accompanied by lymphopenia includes administration of an anticancer agent or an immunosuppressive agent, irradiation, pretreatment for hematopoietic stem cell transplantation combining them. The anticancer agent is selected from the group consisting of fluorouracil, methotrexate, gemcitabine, fludarabine, bleomycin, adriamycin, mitomycin, paclitaxel, docetaxel, vincristine, irinotecan, etoposide, cisplatin, carboplatin, nedaplatin, and cyclophosphamide One anticancer agent is exemplified, and the immunosuppressive agent or the like is at least one immunosuppressive selected from the group consisting of azathioprine, mizoribine, mycophanol mofetil, methotrexate, cyclosporine, tacrolimus, gusperimus hydrochloride, and muromonab-CD3. Agents. Examples of pretreatment for hematopoietic stem cell transplantation include pretreatment in combination with irradiation, thiotepa, cyclophosphamide, rabbit antithymocyte globulin and the like.

  In the present invention, the decrease in the number of lymphocytes by performing a treatment involving lymphopenia on a patient means that the number of lymphocytes in the blood is decreased as compared with that before the treatment. For example, it means a state in which the number of lymphocytes in the blood is reduced to 1000 / μL or less for adults and 3000 / μL or less for children.

Autologous hematopoietic stem cell transplantation (HSCT: Hematopoietic Stem Cell Transplantation) performed in the present invention is often performed as a treatment method for hematopoietic malignant tumors such as leukemia. As used herein, the term “self” refers to the same individual. In addition, autologous hematopoietic stem cell transplantation can be reproduced by syngeneic hematopoietic stem cell transplantation by syngeneic animals belonging to the same species and having the same genetic background, and the same technique is often used in evaluation by animal experiments. Therefore, in the present specification, autologous hematopoietic stem cell transplantation is not limited to transplantation of hematopoietic stem cells derived from the same individual, but includes transplantation of hematopoietic stem cells derived from a syngeneic individual. Hematopoietic stem cells used in autologous hematopoietic stem cell transplantation may be collected from bone marrow or collected from peripheral blood after induction of remission by administration of an anticancer agent. In the latter method, a drug having a stem cell mobilization effect (cytarabine, cyclophosphamide, etoposide, etc.) and granulocyte growth factor (G-CSF) are combined and mobilized into peripheral blood, and a component blood collection device is used. To collect. For autologous hematopoietic stem cell transplantation, a necessary and sufficient amount of hematopoietic stem cells may be transplanted. Usually, 2.0 × 10 ⁶ or more of CD34 positive cells per kg body weight of the patient may be transplanted.
Prior to transplantation, a combination of anticancer drugs, immunosuppressants, called pre-transplant therapy, and radiotherapy is performed 7 to 4 days before transplantation, aiming at complete removal of tumor cells. . For this pretreatment, a combination of CBV (cyclophosphamide, BCNU, etoposide), BEAM (BCNU, etoposide, cytarabine, melphalan), melphalan alone, etc. are used. Along with the destruction, blood cells including lymphocytes in peripheral blood are greatly reduced.

The present invention also provides an anticancer agent or a refractory solid cancer treatment kit for use in the method of the present invention comprising an IFN gene-containing vector as an active ingredient.
In this specification, IFN is IFN-α or IFN-β. IFN-α is known to have 13 types of subtypes (α1, 2, 4, 5, 6 and others) from the viewpoint of specificity, and is not limited to any one. As for IFN-β, only β1 is known. These genes encoding IFN are all known genes and may be prepared according to a conventional method. Moreover, the vector used in the present invention may be in the form of a vector suitable for high expression of the IFN gene locally, and both viral vectors and non-viral vectors can be used. Examples of viral vectors include adenovirus vectors, retrovirus vectors, and Sendai virus vectors. Non-viral vectors include liposomes (cationic liposomes, membrane fusion liposomes, etc.), ligand-bound polylysine, and cationic polymers (polyethyleneimine). Etc.) and the like are exemplified. Furthermore, the plasmid vector may be administered locally as it is (naked-DNA method). As these vectors, known vectors may be used, and an IFN gene may be incorporated according to a conventional method. In the case of a viral vector, the non-proliferating type is preferable. Various serotypes are known as adenovirus vectors and are not particularly limited, but those derived from type 2, type 5 and type 35 are preferred. As the non-proliferating type, those lacking the E1 region are preferable. The Sendai virus vector is preferably SeV / dF lacking the fusion gene (F).
The IFN gene is incorporated into a vector under the control of an appropriate promoter so that IFN can be expressed in cells at the administration site. The promoter is not particularly limited, but CAG promoter [Gene, Vol. 108, 193-200 (1991)], cytomegalovirus promoter, SV40 promoter, 3-phosphoglycerate kinase (PGK) promoter, etc. Is mentioned. Furthermore, known regulators such as terminators and enhancers may be added to the vector.
The solid cancer is not particularly limited as long as the treatment method of the present invention is effective. For example, colorectal cancer, renal cancer, pancreatic cancer, esophageal cancer, bladder cancer, prostate cancer, which are refractory solid cancers. Cancer, melanoma, head cancer, neck cancer, gastric cancer, lung cancer, and hepatocellular carcinoma.

The local dose of the IFN gene in or around the tumor may be a dose that exhibits effectiveness, and can be appropriately determined depending on the IFN gene-containing vector to be used. For example, in the case of an IFN-α gene-containing adenoviral vector (Ad-IFNα) prepared according to the method described in Gene Therapy (2003) 10, 765-773, the dose is 1 × 10 ⁴ to 1 × 10 ¹¹ per tumor weight. pfu / g, more preferably 1 × 10 ⁶ to 1 × 10 ¹⁰ pfu / g.
In the case of plasmid vectors containing IFN-α and IFN-β genes (IFN-α expression plasmid and IFN-β expression plasmid), the dosage is 0.01 to 20,000 μg / g per tumor weight, More preferably, it is 0.1-2,000 microgram / g.
Local administration of the IFN gene may be performed once or multiple times at appropriate intervals. The number of administrations can be appropriately set in consideration of the type and size of the tumor and other conditions.

  The method of administering the IFN gene-containing vector to the subject is local administration within the tumor or in the vicinity of the tumor, and the administration preparation is preferably in the form of an injectable preparation suitable for the IFN gene-containing vector to be used. For example, in the case of a viral vector, the above preparation can be produced as a solution preparation containing a pharmaceutically acceptable carrier. As one embodiment, a preparation in which an adenovirus vector is suspended in distilled water for injection, physiological saline or phosphate buffered saline is exemplified. Further, the preparation may contain components for stabilizing the active ingredient (glycerol, saccharides, etc.), a component for adjusting osmotic pressure, an antibacterial agent and the like.

  According to the present invention, the tumor is killed by the action of IFN expressed by introducing the IFN gene into the tumor or in the vicinity of the tumor, the cancer antigen is exposed, the proliferation of lymphocytes against the cancer antigen is promoted, and the cancer An immune response can be induced. In addition, lymphocytes are activated and proliferated by the action of the expressed IFN itself. Furthermore, a tumor-specific immune response is induced by IFN-α gene introduction, and the antitumor effect of HP is synergistically enhanced and sustained. In addition, transplantation of hematopoietic stem cells destroys the immunosuppressive environment and reconstructs a new immune system. Tumor-specific immunity is remarkably enhanced by the synergistic action of IFN expressed in the tumor and autologous hematopoietic stem cell transplantation by this IFN gene introduction. By the action described above, the growth of cancer (tumor) is suppressed and the cancer is degenerated.

For example, Ad-IFNα gene therapy is characterized by the direct antitumor effect of inducing significant cell death in tumor cells by the method of locally injecting Ad-IFNα into tumors, as well as the effects of NK cells and cytotoxic T cells. Various anti-tumor mechanisms such as induction of systemic specific tumor immune effect by activation and activation / maturation promotion of antigen-presenting cells and suppression of tumor angiogenesis can be exhibited. That is, Ad-IFNα gene therapy can take measures against strong local tumor control and systemic metastasis. In addition, since IFN-α expressed in the tumor hardly leaks into the blood, high safety is one of the usefulness of Ad-IFNα gene therapy.
In actual clinical practice, solid cancer is often resistant to immunotherapy. Due to the induction of regulatory T cells and the influence of cytokines, the tumor has acquired an immunosuppressive environment, but this problem of conventional immunotherapy has been solved by using autologous hematopoietic stem cell transplantation in combination. . By completely reconstructing the immune system after completely destroying the immunosuppressive environment by hematopoietic stem cell transplantation, the effect of immunotherapy can be enhanced. Furthermore, autologous hematopoietic stem cell transplantation exerts an antitumor effect, and by combining immunogene therapy such as IFN-α gene introduction into the tumor, (1) induces tumor cell death and promotes exposure of tumor antigens (2) It is possible to induce a strong and long-lasting antitumor effect by a mechanism such as enhancing tumor antigen presentation in antigen-presenting cells and (3) activating specific lymphocytes. Autologous hematopoietic stem cell transplantation is excellent in safety because it does not cause graft-versus-host disease (GVHD), and no adverse events have been observed in animal experiments.
As described above, the present inventors have not only simply performed Ad-IFNα gene therapy for solid cancer but also developed a combined therapy with autologous hematopoietic stem cell transplantation, and have a high antitumor effect on refractory solid cancer. Provide new immune gene / cell combination therapy that exerts

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples at all.

Example 1
When a mouse is irradiated with a lethal dose of radiation and then bone marrow and T cells are injected intravenously to promote the proliferation of T cells in transplanted mice, the growth of subcutaneous tumors such as colorectal cancer and renal cancer is clearly suppressed. It has been shown that autologous hematopoietic stem cell transplantation exerts an antitumor effect. However, although autologous hematopoietic stem cell transplantation alone or Ad-IFNα gene introduction alone has a tumor suppressive effect, it cannot ultimately extend the survival rate of mice. Therefore, when the Ad-IFNα gene is administered at a time when the immune system is reconstructed early after hematopoietic stem cell transplantation, the growth of the subcutaneous tumor at the administered site is markedly suppressed. It was clarified that the growth inhibitory effect was also observed over a long period of time in a subcutaneous tumor at a distant site, and that the survival rate of mice could be significantly prolonged. In addition, transplantation of syngeneic hematopoietic stem cells using genetically identical syngeneic mice can reproduce autologous hematopoietic stem cell transplantation.
Details are described below.
(1) Female ^{BALB / c (H-2 d} , Ly-1.2) Animals and transplantation 7-9 week old mice (Cha - Rusuriba Inc.) were bred in a sterile environment. Animal experiments were conducted with the approval of the Animal Experimentation Ethics Committee in accordance with the National Cancer Center Animal Experiment Guidelines. BALB / c mice received a lethal dose (9 Gy) of whole body irradiation on the day of transplantation. To irradiated mice, 5 × 10 ⁶ T cell-depleted bone marrow cells derived from donor BALB / c and 2 × 10 ⁶ spleen T cells were added to a total volume of 0.2 ml of Dulbecco's PBS solution (Nissui Pharmaceutical). (HSCT). Bone marrow cells are separated by washing the femur and tibia of donor mice with RPMI 1640 (manufactured by Nissui Pharmaceutical) supplemented with 5% fetal bovine serum (ICN Biomedicals Inc.), and spleen cells are loosened with tweezers. Prepared. After hemolysis of erythrocytes, bone marrow cells and spleen cells were each incubated with anti-Thy-1.2 magnetic beads (Miltenyi Biotec) at 4 ° C. for 15 minutes, and then T cells were removed from bone marrow cells. Cell sorting was performed using AutoMACS (Miltenyi Biotec). More than 90% of T cells were removed from bone marrow cells.

(2) Tumor cells and recombinant adenoviral vectors CT26 and Renca are low immunogenic BALB / c-derived colon cancer cell lines and kidney cancer cell lines, respectively, obtained from ATCC (American Type Culture Collection). did. It was confirmed by flow cytometry that both cell lines expressed MHC class I molecules (H-2K ^d and H-2D ^d ) well. Cells were maintained in RPMI containing 10% fetal bovine serum, 2 mmol / L L-glutamine, and 0.15% sodium bicarbonate.
Recombinant adenovirus vector (Ad-mIFN) expressing mouse IFN-α, recombinant adenovirus vector (Ad-AP) incorporating alkaline phosphatase cDNA, adenovirus vector (Ad-ΔE1) not incorporating gene are Aoki K Et al., Mol. Med. Vol. 5, p. 224-231 (1999) and Suzuki K et al., Gene Ther. Vol. 10, p. Each was prepared according to 765-773 (2003). These recombinant adenoviruses lack the 3E region of the Ad5 virus and contain the CAG promoter, which is a hybrid of the cytomegalovirus immediate early enhancer sequence and the chicken β-actin / rabbit β-globulin promoter. Have.
The virus was purified by the cesium chloride method, and the purified virus was desalted with a sterilized Bio-GelP-6 DG chromatography column (Econopac DG10, manufactured by BioRad) and then stored in a 13% glycerol / PBS solution for storage. Diluted. All virus preparations were confirmed by PCR assay to be free of E1 ⁺ adenovirus contamination.

(3) Administration of IFN-α gene to living body 5 × 10 ⁶ Renca cells or 1 × 10 ⁶ CT26 cells were prepared in a total volume of 50 μL PBS and injected subcutaneously into the mouse leg. The engraftment / proliferation of the subcutaneous tumor was confirmed (diameter 0.6 cm or less), and 50 μl of Ad-mIFNα or a control vector (5 × 10 ⁶ PFU) was administered once into the tumor. The shortest diameter (r) and longest diameter (l) of the tumor were measured, and the tumor volume was measured as r ² l / 2.

(4) Suppression of colon cancer growth in lymphocyte-reduced mice First, it was examined whether HP of T cells can induce anti-tumor immunity in mice with decreased lymphocytes. After BALB / c mice were irradiated with a lethal dose (9 Gy) of radiation, bone marrow cells and T cells of syngeneic BALB / c mice were injected intravenously, and 3 days later, 1 × 10 ⁶ CT26 colon cancer cells were added to the legs. Transplanted subcutaneously. Tumor growth was significantly suppressed in syngeneic hematopoietic stem cell transplanted mice (FIG. 1). When lymphocyte infusion (LI) was given to non-irradiated mice with no lymphocyte reduction, a slight antitumor effect was observed on the 14th day after tumor transplantation, but the tumor growth was fast and the control on the 24th day. The mice caught up with tumor growth. This suggests that T cell proliferation plays an important role in anti-tumor immunity of hematopoietic stem cell transplanted mice.

(5) Antitumor effect of mouse IFN-α gene administration to subcutaneous tumor In order to examine the in vivo antitumor effect of IFN-α gene transfer treatment, various amounts of Ad-mIFN were administered to Renca and CT26 subcutaneous tumors. Administered. A single administration of Ad-mIFN showed a significant growth inhibitory effect on both tumors in a dose-dependent manner (FIG. 2).

(6) Induction of synergistic anti-tumor effect by administering IFN-α gene at an early stage after transplantation of syngeneic hematopoietic stem cells Next, IFN-α at the time when early immune reconstitution after syngeneic hematopoietic stem cell transplantation occurs The antitumor effect by gene administration was examined. In practice, when a hematopoietic stem cell transplant is performed, the recipient has relapsed or residual tumor cells. To match its clinical situation and to avoid the effects of irradiation on tumor cells, recipient mice were transplanted with CT26 cells subcutaneously immediately after irradiation, followed by intravenous transplantation of bone marrow and T cells. I did it. Subsequently, 5 × 10 ⁶ PFU of Ad-mIFNα was administered to the subcutaneous CT26 tumor. When a high dose of Ad-mIFNα is administered into a tumor, Ad-mIFNα alone exerts a strong antitumor effect, and the synergistic effect with IFN-α gene transfer in the transplanted mouse becomes unclear. Therefore, in this experiment, a low dose of Ad-mIFNα was used to suppress the antitumor effect of Ad-mIFNα (FIGS. 3a and 3b). The tumor volume on day 5 was 60-100 mm ³ . Tumor growth was suppressed in transplanted mice compared to naive mice, and tumor suppression by IFN-α gene transfer was significantly enhanced in transplanted mice (FIG. 3a). These results indicate that a synergistic anti-tumor effect was induced by intratumoral IFN-α gene transfer during immune reconstitution.
In order to evaluate whether tumor local IFN-α gene administration can exert an antitumor effect on a tumor at a distant site, Renca cells were transplanted subcutaneously in both feet at the time of syngeneic hematopoietic stem cell transplantation, and 5 days later, the right leg Ad-mIFN vector was administered only to these tumors. The administration of the IFN-α gene significantly suppressed the growth of the tumor in the left leg to which the vector was not administered, similarly to the tumor in the right leg to which the vector was administered (FIG. 3b). This result indicates that intratumoral IFN-α gene administration can induce systemic anti-tumor immunity in autologous hematopoietic stem cell transplant recipients.

(7) Prolonging survival period of autologous HSCT recipient by IFN-α gene administration Next, the survival extension effect of intratumoral IFN-α gene administration in autologous hematopoietic stem cell transplant recipients was examined. By the 30th day after tumor inoculation, syngeneic hematopoietic stem cell transplant recipients showed significant tumor growth inhibition even when administered control adenovirus, but since the tumor grew rapidly thereafter, No extension was observed (Figure 4). On the other hand, the antitumor effect of syngeneic hematopoietic stem cell transplantation was significantly enhanced in the intratumoral IFN-α gene transfer complex group. Subcutaneous tumors disappeared at 43%, clearly extending the survival time of recipient mice (FIG. 4). The mean survival time was 76.2 days in the hematopoietic stem cell transplantation and intratumoral IFN-α gene transfer complex group, and other control groups and treatment groups (No treatment group: 53.7 days, Ad-ALP group: 54. 8 days, Ad-IFN group: 56.5 days, HSCT group: 53.1 days, HSCT + Ad-ALP group: 55.9 days). There were no adverse events in the treated mice.

(8) Induction of synergistic anti-tumor effect by administration of IFN-α expression plasmid or IFN-β expression plasmid into the tumor Next, instead of the adenovirus vector, IFN-α expression plasmid or IFN-β expression plasmid was used. We investigated whether the anti-tumor effect of autologous hematopoietic stem cell transplantation could be enhanced by mixing with liposomes and injecting into the tumor. First, IFN-α expression plasmid and IFN-β expression plasmid were prepared according to Molecular Cloning (Cold Spring Harbor Laboratory Press). The IFN-α expression plasmid is a plasmid vector used when preparing the adenoviral vector Ad-mIFN described in Example 1- (2) above, and pAT153 (GenBank Accession: L08853) is used as a backbone, and the adenoviral genome 0 ˜1 mu, CAG promoter, IFN-α gene and adenovirus genome 9-16 mu. In the IFN-β expression plasmid, the IFN-α gene of the IFN-α expression plasmid is replaced with an IFN-β gene (GenBank Accession: X14455).
After BALB / c mice were irradiated with a lethal dose of 9 Gy, CT26 cells were implanted subcutaneously in the legs, and bone marrow cells and T cells were injected intravenously. After 7 days, 9 days and 11 days, IFN-α or IFN-β expression plasmid was mixed with liposomes and directly injected into the tumor. A mixture of plasmid and liposome was prepared by mixing 25 μL of a solution containing 10 μg of IFN-α or IFN-β expression plasmid dissolved in PBS buffer with 25 μL of 0.15 mM liposome (DMRIE-DOPE, Vital Incorporated). The entire amount of this solution was administered. The tumor volume at the start of administration was 60-100 mm ³ . In naive mice that had not been irradiated and transplanted with bone marrow cells and T cells, the antitumor effect of 10 μg of IFN-α or IFN-β plasmid was not clear (FIG. 5). However, in mice transplanted with bone marrow cells and T cells, CT26 subcutaneous tumor growth is markedly suppressed by injecting an IFN-α or IFN-β expression plasmid into the tumor, and exhibits a synergistic antitumor effect. (FIG. 5).

  Sustained anti-tumor effect by administering the IFN-α gene in or near solid tumors during recovery of lymphocytes after autologous hematopoietic stem cell transplantation, a treatment involving lymphopenia I found that it can be strongly promoted. The present invention is extremely useful in the treatment of refractory solid cancer.

FIG. 3 shows that bone marrow and T cell syngeneic hematopoietic stem cell transplantation clearly inhibits the growth of subcutaneous tumors in mice exposed to lethal doses of radiation. After irradiation with 9 Gy (lethal dose), the mice were transplanted with bone marrow cells and T cells. As controls, non-irradiated mice were left untreated or T lymphocyte infusion (LI). (N = 4-6) It is a figure which shows the direct antitumor effect of mouse | mouth IFN- (alpha) gene administration to Renca and CT26 subcutaneous tumor. Subcutaneous tumors were made in the legs and various amounts of Ad-mIFN or control virus (Ad-ΔE1 or Ad-AP) were administered once into the tumor. (N = 5-6) It is a figure which shows that an IFN- (alpha) gene administration improves an antitumor effect in a mouse | mouth with a syngeneic hematopoietic stem cell transplant. (A) CT26 cells were inoculated into the right leg immediately prior to syngeneic hematopoietic stem cell transplantation, and 5 days later, Ad-mIFN was administered to CT26 tumors. (N = 7-8) (b) Renca cells were inoculated into both legs just prior to syngeneic hematopoietic stem cell transplantation, and 5 days later, Ad-mIFN was administered only to the right leg tumor. (N = 7-8) It is a figure which shows that IFN- (alpha) gene administration at the time of immune reconstruction prolongs the survival of a transplanted mouse. IFN-α gene transfer clearly prolongs the survival of syngeneic hematopoietic stem cell transplanted mice. Following intratumoral administration of the Ad-IFNα vector, the survival time of the mice was observed. (N = 7-8) It is a figure which shows that administration of an IFN expression plasmid raises an anti-tumor effect in a mouse | mouth with a syngeneic hematopoietic stem cell transplant.

Claims (16)

  A method for treating cancer, comprising a step of performing treatment with lymphopenia on a patient, and a step of administering an interferon gene to a local area in or near the tumor of the patient.   The method includes a step of performing treatment with lymphopenia in a patient, a step of performing autologous hematopoietic stem cell transplantation on the patient, and a step of administering an interferon gene to a local area in or near the tumor of the patient. How to treat cancer.   The treatment method according to claim 1 or 2, wherein the treatment accompanied by a decrease in the number of lymphocytes is administration of an anticancer agent, administration of an immunosuppressive agent and / or irradiation.   The method for treating cancer according to any one of claims 1 to 3, wherein the interferon gene is administered using an interferon gene-containing vector.   The method for treating cancer according to any one of claims 1 to 4, wherein the interferon is interferon-α and / or interferon β.   The method for treating cancer according to claim 4, wherein the vector is an adenovirus vector and / or a plasmid vector.   The cancer treatment method according to any one of claims 1 to 6, wherein the cancer is solid cancer.   A therapeutic agent for administering an interferon gene to a local area in or near a tumor of a patient who has been treated with lymphopenia, comprising an interferon gene-containing vector. Agent.   A therapeutic agent for administering an interferon gene to a local area in or near a tumor of a patient who has undergone autologous hematopoietic stem cell transplantation following treatment with lymphopenia, comprising an interferon gene-containing vector A cancer therapeutic agent characterized by.   The therapeutic agent for cancer according to claim 8 or 9, wherein the interferon is interferon-α and / or interferon β.   The cancer therapeutic agent according to any one of claims 8 to 10, wherein the vector is an adenovirus vector and / or a plasmid vector.   Use of an interferon gene in the manufacture of a therapeutic agent for cancer administered to a local area in or near a tumor of a patient who has been treated with lymphopenia.   Use of an interferon gene in the manufacture of a therapeutic agent for cancer administered in or near a tumor of a patient who has undergone autologous hematopoietic stem cell transplantation following treatment with lymphopenia.   The use according to claim 12 or 13, wherein the interferon gene is used as an interferon gene-containing vector.   The use according to any one of claims 12 to 14, wherein the interferon is interferon-α and / or interferon β.   Use according to claim 14, wherein the vector is an adenoviral vector and / or a plasmid vector.

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