Pharmaceutical composition for treating cancer, comprising vaccinia virus and granulopoiesis inhibitor as active ingredients

Abstract

To provide a pharmaceutical composition for treating cancer that enhances the antitumor effect of an oncolytic virus.SOLUTION: The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising a vaccinia virus and a granulopoiesis inhibitor as active ingredients. The pharmaceutical composition for treating cancer, comprising a vaccinia virus and a granulopoiesis inhibitor as active ingredients, of the present invention has an excellent anticancer effect and safety compared to a case of administering only the vaccinia virus. Therefore, the pharmaceutical composition comprising the vaccinia virus and the granulopoiesis inhibitor as active ingredients of the present invention may be effectively utilized in treating cancer.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition for treating cancer, comprising as active ingredients a vaccinia virus and a granulopoiesis inhibitor. [Background technology]

[0002] Oncolytic viruses have excellent tumor-specific targeting, proliferation and killing abilities in cancer cells. In recent years, various clinical trials based on oncolytic viruses have been carried out. In 2015, talimogene laherparepvec (T-Vec), an oncolytic virus based on herpes simplex virus, was successfully commercialized as a therapeutic agent for advanced melanoma, thus starting the era of oncolytic virus field in the United States and Europe.

[0003] In recent years, the usefulness of oncolytic viruses has exceeded their own efficacy, and the viruses activate tumor immunity, thereby showing their potential as therapeutic agents combined with other immunotherapeutic agents. Until 2000, which was the early stage of the development of oncolytic viruses, the direct killing effect of the virus brought about by its cancer cell-specific proliferation was relatively more important. However, subsequent clinical trials have found that the activation of tumor immunity is a more important mechanism than the direct killing effect of tumor cells. Based on this knowledge, therapeutic agents including oncolytic viruses and immunotherapeutic agents such as immune checkpoint inhibitors administered in combination have been developed in recent years. This is because oncolytic viruses are known to convert the immune-suppressed tumor microenvironment into a tumor microenvironment suitable for immunotherapy.

[0004] In some clinical trials of vaccinia virus-based oncolytic viruses, oncolytic virotherapy can result in acute tumor necrosis, durable response or complete response, but in some cases it can lead to difficult-to-predict outcomes (pharmacodynamic fluctuations), such as progressive disease or early death. For example, in the case of Pexa-vec based on vaccinia virus, in phase 1 clinical trials, some patients died early within one month of oncolytic virotherapy, which was associated with persistent systemic inflammatory response and major organ dysfunction. In addition, the transient cold symptoms (high fever) and low blood pressure observed after oncolytic virotherapy are the most frequent adverse events after oncolytic virotherapy.

[0005] On the other hand, in the treatment using oncolytic viruses, there have been no accurate reports on the effect of drug-induced increase in neutrophils on the treatment outcome. The first innate immune cells to respond to the administration of oncolytic viruses are neutrophils, which have a short half-life of less than 20 hours in the human body. Clinically, a large number of neutrophils have been observed in patients treated with drugs (e.g., clozapine) or in patients with acute inflammation and acute injury (Liao Y et al., PloS One, 8(7), 2013), but since this is not included in the Common Terminology Criteria of Adverse Events (CTCAE), an increase in absolute neutrophil count (ANC) has not been recognized as an abnormal response.

[0006] Therefore, there is a need for studies on the impact of changes in neutrophil counts on oncolytic virus therapy. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, as a result of carrying out research to enhance the anti-cancer effect of vaccinia virus used as oncolytic virus, the present inventor has found that when vaccinia virus is administered to an individual with cancer, if an inhibitor that reduces neutrophil levels is administered in combination, compared with the existing case of administering vaccinia virus alone, this simultaneous administration can significantly reduce systemic inflammatory response, ensuring safe use.Furthermore, the present inventor has found that when the inhibitor is administered in combination, the cancer cell specific selectivity and proliferation ability of vaccinia virus is improved, thereby completing the present invention.It is speculated that the inhibitor inhibits granulocyte production, thereby reducing neutrophil levels, thereby improving the anti-cancer effect of oncolytic virus. [Means for solving the problem]

[0008] To achieve the above object, in one aspect of the present invention, there is provided a pharmaceutical composition for treating cancer, comprising as active ingredients a vaccinia virus and a granulopoiesis inhibitor.

[0009] In another aspect of the invention, a method of treating cancer is provided comprising administering to an individual with cancer a vaccinia virus and a granulopoiesis inhibitor.

[0010] In yet another aspect of the present invention, there is provided a use of a composition comprising a vaccinia virus and a granulopoiesis inhibitor for the prevention or treatment of cancer.

[0011] In yet another aspect of the present invention, there is provided a use of a composition comprising a vaccinia virus and a granulopoiesis inhibitor for the manufacture of a medicament for preventing or treating cancer.

[0012] In yet another aspect of the present invention, there is provided an anti-cancer adjuvant comprising a granulopoiesis inhibitor as an active ingredient. Effect of the Invention

[0013] The pharmaceutical composition for cancer treatment of the present invention, which contains vaccinia virus and a granulopoiesis inhibitor as active ingredients, has superior anticancer effect and safety compared to the conventional case in which only vaccinia virus is administered. Therefore, the pharmaceutical composition of the present invention, which contains vaccinia virus and a granulopoiesis inhibitor as active ingredients, can be effectively used in the treatment of cancer. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 illustrates the results obtained by administering wild-type vaccinia virus (Western Reserve strain vaccinia virus, WR) and a granulopoiesis inhibitor (HU) to mice bearing mouse kidney cancer cells (Renca), followed by measuring the tumor volume on days 0, 3, 7, 10, and 14. [Diagram 2] FIG. 1 illustrates the results obtained by administering wild-type vaccinia virus (WR) and HU to mice (Renca) implanted with mouse kidney cancer cells, and measuring the body weight on days 0, 3, 7, 10, and 14 thereafter. [Diagram 3] FIG. 1 illustrates the results obtained by administering a recombinant vaccinia virus obtained by deleting the TK gene from WR (WR VVtk-) and HU (60 mg / kg) to mice transplanted with mouse kidney cancer cells (Renca), followed by measuring the tumor volume on days 0, 3, 7, 10, 14, 17, and 21. [Figure 4] FIG. 1 illustrates the results obtained by administering recombinant vaccinia virus (WR VVtk-) and HU (30 mg / kg) to mice implanted with mouse kidney cancer cells (Renca), followed by measuring the tumor volume on days 0, 3, 7, 10 and 14. [Diagram 5] FIG. 1 shows the results of measuring tumor volume 1 day before, and 4 and 7 days after administration of a recombinant vaccinia virus (VV_DD) obtained by simultaneously deleting the TK gene and vaccinia virus growth factor (VGF) gene from WR and HU to mice (B16F10) bearing mouse melanoma. [Figure 6]FIG. 1 illustrates the results obtained by administering recombinant vaccinia virus (WOTS-418) and HU to mice implanted with human lung cancer cells (NCI-H460), and measuring the tumor volume on days 0, 5, 10, 12, and 15 thereafter. [Figure 7] FIG. 1 illustrates the results obtained by administering recombinant vaccinia virus (WOTS-418) and HU to mice implanted with human lung cancer cells (NCI-H460) and then measuring the survival rate. [Figure 8] FIG. 1 illustrates the results obtained by administering recombinant vaccinia virus (VVtk-) and human granulocyte colony-stimulating factor (rhG-CSF) or HU to mice implanted with mouse kidney cancer cells (Renca), followed by measuring the tumor volume in the mice. [Figure 9] FIG. 1 illustrates the results obtained by isolating splenic lymphocytes from mice (Renca) implanted with mouse kidney cancer cells administered recombinant vaccinia virus (VVtk-) and human granulocyte colony-stimulating factor (rhG-CSF) or HU, administering the lymphocytes to new mice, and then measuring the tumor volume in the new mice. [Figure 10] FIG. 1 illustrates the results obtained by administering recombinant vaccinia virus (Wyeth VVtk-) and HU to mice implanted with mouse kidney cancer cells (Renca), followed by measuring tumor volume in the mice. [Figure 11] FIG. 1 illustrates the results obtained by isolating T lymphocytes from mice (Renca) implanted with mouse kidney cancer cells administered recombinant vaccinia virus (Wyeth VVtk-) and HU, administering the T lymphocytes to new mice, and then measuring the tumor volume in the new mice. [Figure 12] FIG. 1 illustrates the results obtained by isolating splenocytes from mice (Renca) implanted with mouse kidney cancer cells administered recombinant vaccinia virus (Wyeth VVtk-) and HU, administering the splenocytes to new mice, and then measuring the tumor volume in the new mice. [Figure 13]FIG. 1 illustrates the results obtained by administering recombinant vaccinia virus (Wyeth VVtk) and HU to mice implanted with mouse kidney cancer cells (Renca), followed by measuring the tumor volume 22 days later. [Figure 14] FIG. 1 illustrates the results obtained by administering recombinant vaccinia virus (Wyeth VVtk) and HU to mice (Renca) implanted with mouse kidney cancer cells, and then observing the proliferation of CD4+ T cells or CD8+ T cells in splenic tissue. [Figure 15] FIG. 1 illustrates the results obtained by administering recombinant vaccinia virus (OTS-412) and HU to mice (4T1) implanted with mouse breast cancer cells, and then observing the proliferation of CD4+ T cells or CD8+ T cells in blood and spleen tissues. [Figure 16] FIG. 1 illustrates the results obtained by administering recombinant vaccinia virus (WR VVtk-) and HU to the left tumor of mice (4T1) implanted with mouse breast cancer cells, followed by measuring the left tumor volume. [Figure 17] FIG. 1 illustrates the results obtained by administering recombinant vaccinia virus (WR VVtk-) and HU to the left tumor of mice (4T1) implanted with mouse breast cancer cells, followed by measuring the right tumor volume. [Figure 18] FIG. 1 illustrates the results obtained by administering recombinant vaccinia virus (WR) and HU to mice (Renca) implanted with mouse kidney cancer cells, and then performing staining on day 22 to identify the distribution of the recombinant vaccinia virus in the mouse tumor tissue. [Figure 19] FIG. 1 illustrates the results obtained by administering wild-type vaccinia virus (WR) or wild-type vaccinia virus (WR) and HU to normal mice and subsequently determining the distribution of wild-type vaccinia virus in liver and kidney tissue. [Figure 20]FIG. 1 illustrates the absolute neutrophil counts in mice in each group after administration of saline, HU, recombinant vaccinia virus (OTS-412), recombinant vaccinia virus and recombinant human granulocyte colony-stimulating factor (OTS-412+rhG-CSF), or recombinant vaccinia virus and HU (OTS-412+HU) to mice (Renca) implanted with mouse kidney cancer cells. [Figure 21] FIG. 1 shows an example of the number of blood neutrophils measured in each group of mice after administration of saline, recombinant vaccinia virus, or recombinant vaccinia virus (WR VVtk-) and HU to mice (Renca) implanted with mouse kidney cancer cells. [Figure 22] FIG. 1 shows an example of the number of neutrophils in the blood of mice measured in each group after administration of saline, recombinant vaccinia virus, or recombinant vaccinia virus (WOTS-418) and HU to mice (Renca) implanted with mouse kidney cancer cells. [Figure 23] FIG. 1 shows an example of the number of blood neutrophils measured in each group of mice (Renca) implanted with mouse kidney cancer cells after administration of saline, lenalidomide, or HU. [Figure 24] FIG. 1 shows an example of the number of neutrophils in the blood of mice measured in each group after administration of saline, recombinant vaccinia virus, recombinant vaccinia virus (WOTS-418) and lenalidomide, or recombinant vaccinia virus (WOTS-418) and HU to mice (Renca) implanted with mouse kidney cancer cells. [Diagram 25] FIG. 1 illustrates the tumor volume of mice (Renca) implanted with mouse kidney cancer cells, measured after administration of recombinant vaccinia virus (WR VVtk-) and lenalidomide. [Figure 26] FIG. 1 illustrates the tumor volume of mice (Renca) implanted with mouse kidney cancer cells, measured after administration of recombinant vaccinia virus (WR VVtk-) and palbociclib. [Figure 27]FIG. 1 shows an example of the body weight of mice (Renca) implanted with mouse kidney cancer cells, measured after administration of recombinant vaccinia virus (WR VVtk-) and palbociclib. [Figure 28] FIG. 1 shows an example of the tumor volume of mice (Renca) implanted with mouse kidney cancer cells, measured on days 0, 4, 10, 14, 17, and 21 after administration of an oncolytic virus (Wyeth VVtk-), a PD-1 inhibitor, and HU. [Figure 29] FIG. 1 shows an example of the tumor volume of mice (Renca) implanted with mouse kidney cancer cells, measured on days 0, 4, 10, 14, and 17 after administration of an oncolytic virus (Wyeth VVtk-), a CTLA-4 inhibitor, and HU. [Diagram 30] FIG. 1 shows an example of the tumor volume of mice (Renca) implanted with mouse kidney cancer cells, measured on days 0, 4, 10, 14, 17, and 21 after administration of an oncolytic virus (Wyeth VVtk-), a PD-L1 inhibitor, and HU. [Diagram 31] FIG. 1 shows an example of the tumor volume of mice (4T1) implanted with mouse breast cancer cells, measured on days 0, 3, 7, 10, and 14 after administration of a tumor-lytic virus (WR VVtk-), a CTLA-4 inhibitor, and HU. [Diagram 32] FIG. 1 shows an example of the tumor volume of mice (4T1) implanted with mouse breast cancer cells, measured on days 0, 3, 7, 10, 14, and 18 after administration of an oncolytic virus (WOTS-418), a PD-L1 inhibitor, and HU. [Diagram 33] FIG. 1 shows an example of the tumor volumes of mice (Renca) implanted with mouse kidney cancer cells, measured on days 0, 3, and 7 after administration of Western Reserve strain vaccinia virus (WR), a CTLA-4 inhibitor, and HU. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Hereinafter, the present invention will be described in detail.

[0016] In one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, comprising as active ingredients a vaccinia virus and a granulopoiesis inhibitor.

[0017] The vaccinia virus and the granulopoiesis inhibitor contained in the pharmaceutical composition can be administered simultaneously, sequentially, or in reverse order. Specifically, the vaccinia virus and the granulopoiesis inhibitor can be administered simultaneously. The granulopoiesis inhibitor can be administered first, and then the vaccinia virus. The vaccinia virus can be administered first, and then the granulopoiesis inhibitor. The granulopoiesis inhibitor can be administered first, and then the vaccinia virus, and the granulopoiesis inhibitor can be administered again.

[0018] The vaccinia virus may belong to, but is not limited to, Western Reserve (WR), New York vaccinia virus (NYVAC), Wyeth (The New York City Board of Health; NYCBOH), LC16m8, Lister, Copenhagen, Tian Tan, USSR, TashKent, Evans, International Health Division-J (IHD-J) or International Health Division-White (IHD-W) vaccinia virus strains. In one embodiment of the present invention, Western Reserve strain vaccinia virus and Wyeth strain vaccinia virus were used.

[0019] The vaccinia virus may be a wild-type vaccinia virus or a recombinant vaccinia virus. Specifically, the recombinant vaccinia virus can be obtained by deleting a gene from a wild-type vaccinia virus or by inserting a foreign gene into a wild-type vaccinia virus. Here, among the genes of the wild-type vaccinia virus, a gene related to viral pathogenicity, which encodes any one selected from the group consisting of thymidine kinase (TK), vaccinia growth factor (VGF), WR53.5, F13.5L, F14.5L, A56R, B18R, or a combination thereof, can be deleted.

[0020] In addition, the foreign gene to be inserted may be a gene that promotes immunity and encodes any one selected from the group consisting of herpes simplex virus thymidine kinase (HSV-TK), mutated HSV-TK, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), cytosine deaminase (CD), carboxylesterase type 1, carboxylesterase type 2, interferon beta (INF-β), somatostatin receptor 2, and combinations thereof.

[0021] Specifically, the recombinant vaccinia virus can be obtained by deleting the TK gene from a vaccinia virus belonging to the Western Reserve (WR), New York Vaccinia Virus (NYVAC), Wyeth (New York City Department of Health; NYCBOH), LC16m8, Lister, Copenhagen, Tiantan, USSR, Tashkent, Evans, International Health Department-J (IHD-J) or International Health Department-White (IHD-W) vaccinia virus strain. In one embodiment of the present invention, a recombinant vaccinia virus obtained by deleting the TK gene from a Western Reserve strain vaccinia virus was used, and this virus was named "WR VVtk-". In another embodiment of the present invention, a recombinant vaccinia virus obtained by deleting the TK gene from a Wyeth strain vaccinia virus was used, and this virus was named "Wyeth VVtk-".

[0022] In addition, the recombinant vaccinia virus can be obtained by deleting the TK gene and the VGF gene from a vaccinia virus belonging to Western Reserve, NYVAC, Wyeth, LC16m8, Lister, Copenhagen, Tiantan, USSR, Tashkent, Evans, IHD-J or IHD-W vaccinia virus strain. In one embodiment of the present invention, a recombinant vaccinia virus obtained by deleting the TK gene and the VGF gene from a Western Reserve strain vaccinia virus is used, and this virus is named "VV_DD".

[0023] Furthermore, recombinant vaccinia viruses can be obtained by deleting the TK gene from a vaccinia virus belonging to the Western Reserve, NYVAC, Wyeth, LC16m8, Lister, Copenhagen, Tiantan, USSR, Tashkent, Evans, IHD-J or IHD-W vaccinia virus strain and inserting the HSV-TK gene therein.

[0024] In addition, the recombinant vaccinia virus can be obtained by deleting the TK gene from a vaccinia virus belonging to the Western Reserve, NYVAC, Wyeth, LC16m8, Lister, Copenhagen, Tiantan, USSR, Tashkent, Evans, IHD-J or IHD-W vaccinia virus strain and inserting a mutated HSV-TK gene therein. In one embodiment of the present invention, a recombinant vaccinia virus obtained by deleting the TK gene from a Wyeth strain vaccinia virus and inserting a gene encoding the HSV-TK fragment (1-330aa) of SEQ ID NO: 1 at the deleted position is used, and this virus was named "OTS-412". In addition, in one embodiment of the present invention, a recombinant vaccinia virus obtained by deleting the TK gene from a Western Reserve strain vaccinia virus and inserting a gene encoding the HSV-TK variant of SEQ ID NO: 2 of the HSV-TK gene at the deleted position is used, and this virus was named "WOTS-418".

[0025] Furthermore, a recombinant vaccinia virus can be obtained by deleting the TK gene from a vaccinia virus belonging to the Western Reserve, NYVAC, Wyeth, LC16m8, Lister, Copenhagen, Tiantan, USSR, Tashkent, Evans, IHD-J or IHD-W vaccinia virus strain and inserting the GM-CSF gene therein.

[0026] Alternatively, a recombinant vaccinia virus can be obtained by deleting the TK gene from a vaccinia virus belonging to the Western Reserve, NYVAC, Wyeth, LC16m8, Lister, Copenhagen, Tiantan, USSR, Tashkent, Evans, IHD-J or IHD-W vaccinia virus strain and inserting the G-CSF gene therein.

[0027] Furthermore, recombinant vaccinia viruses can be obtained by deleting the TK gene from a vaccinia virus belonging to the Western Reserve, NYVAC, Wyeth, LC16m8, Lister, Copenhagen, Tiantan, USSR, Tashkent, Evans, IHD-J or IHD-W vaccinia virus strain and inserting a cytosine deaminase (CD) gene therein.

[0028] Alternatively, a recombinant vaccinia virus can be obtained by deleting the TK gene from a vaccinia virus belonging to the Western Reserve, NYVAC, Wyeth, LC16m8, Lister, Copenhagen, Tiantan, USSR, Tashkent, Evans, IHD-J or IHD-W vaccinia virus strain and inserting the somatostatin receptor 2 gene therein.

[0029] Furthermore, a recombinant vaccinia virus can be obtained by deleting the TK gene from a vaccinia virus belonging to the Western Reserve, NYVAC, Wyeth, LC16m8, Lister, Copenhagen, Tiantan, USSR, Tashkent, Evans, IHD-J or IHD-W vaccinia virus strain, and inserting therein any two or more genes selected from the group consisting of genes encoding herpes simplex virus thymidine kinase (HSV-TK), mutated HSV-TK, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), cytosine deaminase (CD), or somatostatin receptor 2.

[0030] In addition, the recombinant vaccinia virus can be obtained by deleting the TK gene and the VGF gene from a vaccinia virus belonging to the Western Reserve, NYVAC, Wyeth, LC16m8, Lister, Copenhagen, Tiantang, USSR, Tashkent, Evans, IHD-J or IHD-W vaccinia virus strain, and inserting therein any one gene selected from the group consisting of genes encoding herpes simplex virus thymidine kinase (HSV-TK), mutated HSV-TK, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), cytosine deaminase (CD), or somatostatin receptor 2, and any combination thereof.

[0031] As used herein, the term "gene deletion" means that a gene is not expressed due to partial or complete deletion of the gene or the insertion of a foreign gene therein. When a partial deletion occurs in a gene, some amino acids at the N-terminus or C-terminus of the polypeptide expressed by the gene may be deleted.

[0032] As used herein, the term "thymidine kinase (TK)" refers to an enzyme involved in nucleotide biosynthesis, called thymidine kinase. TK is an enzyme used in nucleotide biosynthesis in both cells and viruses. Here, for cells, TK is not present in normal cells because they no longer divide, and even in rapidly dividing cells such as hair follicle cells, TK is not present in sufficient amounts for viruses to utilize. From these perspectives, the deletion of the TK gene in the virus allows it to grow only in the presence of cancer cells in which TK is present, and therefore it can selectively kill cancer cells.

[0033] As used herein, the term "vaccinia growth factor (VGF)" refers to a polypeptide that has sequence homology with epidermal growth factor and stimulates cell proliferation around infected cells. Vaccinia virus replicates better in proliferating cells and can therefore be advantageously used for in vivo virus replication. In order to make oncolytic virus more specifically propagate only in cancer cells, the virus can further undergo deletion of VGF gene in addition to deletion of TK gene.

[0034] As used herein, the term "GM-CSF", referred to as granulocyte-macrophage colony-stimulating factor, refers to a protein secreted by macrophages, T cells, mast cells, natural killer cells, endothelial cells, and fibroblasts. GM-CSF stimulates stem cells to produce granulocytes (neutrophils, basophils, eosinophils) and monocytes. GM-CSF also rapidly increases the number of macrophages, thereby inducing an immune response. GM-CSF may be of human origin and may be a protein having the sequence of GenBank: AAA52578.1.

[0035] As used herein, the term "CD," referred to as cytosine deaminase, refers to an enzyme that catalyzes the hydrolytic deamination of cytosine to uracil and ammonia.

[0036] As used herein, the term "G-CSF", referred to as granulocyte colony-stimulating factor, refers to a cytokine produced by macrophages, fibroblasts, endothelial cells, etc., following stimulation by inflammation or endotoxin. G-CSF promotes the production of neutrophils. G-CSF may be of human origin (rhGCSF) and may be a protein having the sequence of GenBank: AAA03056.1.

[0037] As used herein, the term "somatostatin receptor 2" refers to the protein encoded by the human SSTR2 gene. Somatostatin receptor 2 is expressed primarily in tumors, and patients with neuroendocrine tumors that overexpress somatostatin receptor 2 exhibit improved prognosis. Somatostatin receptor 2 has the ability to stimulate apoptosis in many cells, including cancer cells.

[0038] The bone marrow cells may be granulocytes, specifically the bone marrow cells may be neutrophils, eosinophils or basophils.

[0039] A granulopoiesis inhibitor may be a substance that inhibits granulocytes (e.g., neutrophils, eosinophils, or basophils) that are primarily produced in the bone marrow. If it reduces or suppresses the number of neutrophils (i.e., one of the bone marrow cells) in the body, the granulopoiesis inhibitor may be referred to as or include a neutrophil inhibitor. Neutrophils, also called neutrophilic white blood cells, refer to neutrophil cells that circulate in the blood, which is a type of granulocyte that is primarily produced in the bone marrow. Neutrophils are the main component of granulocytes, and normal numbers are about 100 / mm of blood. 3 Neutrophils absorb foreign substances such as bacteria that have invaded the body through phagocytosis and break down the foreign substances using digestive enzymes (e.g., hydrogen peroxide, lysosomes, etc.).

[0040] As used herein, the term "absolute neutrophil count (ANC)" refers to the number obtained by multiplying the white blood cell count by the percentage of neutrophils.

[0041] The granulopoiesis inhibitor may be hydroxyurea, lenalidomide, thalidomide, tadalafil, palbociclib, an alkylating agent, anthracycline, an antimetabolite, camptothecin, epipodophyllotoxin, mitomycin C, a taxane, or vinblastine. The hydroxyurea may be a compound having the structure of Formula 1 below:

[0042] [ka]

[0043] Hydroxyurea is known as an anticancer drug that inhibits DNA synthesis, but its exact mechanism of action has not been elucidated. Hydroxyurea may also be included in the pharmaceutical composition in the form of a commercially available drug containing hydroxyurea. Examples of commercially available drugs containing hydroxyurea include, but are not limited to, Hydroxyurea®, Hydrea®, Droxia®, Mylocel®, Siklos® and Hydrine® capsules. Hydroxyurea can be taken orally, and can also be administered parenterally.

[0044] Lenalidomide may be a compound having the structure of Formula 2 below: [ka]

[0045] Lenalidomide is an anti-cancer drug used to treat multiple myeloma and other diseases. As an anti-cancer effect, lenalidomide activates tumor suppressor genes to stop the growth cycle of cancer cells and inhibit cancer proliferation. As an immunomodulatory effect, lenalidomide eliminates tumor cells by activating immune cells (e.g., T cells, natural killer cells (NK cells), B cells, etc.). Lenalidomide also has an anti-angiogenic effect that inhibits the formation of new blood vessels to supply nutrients to cancer cells.

[0046] Thalidomide may be a compound having the structure of Formula 3 below: [ka]

[0047] Thalidomide's exact mechanism of action is unknown, but it is used to treat severe skin lesions in patients with multiple myeloma and leprosy (hansen's disease).

[0048] Tadalafil may be a compound having the structure of Formula 4 below: [ka]

[0049] Palbociclib may be a compound having the structure of Formula 5 below: [ka]

[0050] Among the chemotherapy agents for malignant tumors, alkylating agents may be nitrogen mustards, ethylene derivatives, alkylsulfonic acid derivatives, nitrosourea or triazine compounds. These can be called alkylating agents because they replace hydrogen in many organic compounds, proteins or nucleic acids with alkyl groups. Alkylation by alkylating agents can inhibit DNA replication and mRNA transcription in tumor cells, and exhibit antitumor effects. In terms of general pharmacological action, alkylating agents act nonspecifically on each stage of the cell cycle and can inhibit cell division with high proliferation ability. Alkylating agents exhibit radiation-like effects, so they can cause severe hematopoietic abnormalities and immunosuppression.

[0051] Anthracyclines are drugs extracted from the bacterium Streptomyces that are used in cancer chemotherapy to treat many cancers, including leukemia, lymphoma, breast, stomach, ovarian, bladder, and lung cancer. The first anthracycline discovered was daunorubicin (brand name: Daunomycin), which is naturally produced by the Actinomycetes bacterium Streptomyces peucetius. The most clinically important anthracyclines include doxorubicin, daunorubicin, epirubicin, idarubicin, etc.

[0052] An antimetabolite may be a substance that inhibits cell development and proliferation by antagonizing a metabolite essential for the metabolism or growth of microorganisms or tumor cells. Historically, sulphamin was first developed, which was used as a chemotherapeutic agent and antagonized bacterial para-aminobenzoic acid (PABA). Antimetabolites may include sulfa drugs for bacteria, purine antimetabolites (8-azaguanine, 6-thioguanine, 6-mercaptopurine), pyrimidine antimetabolites (5-fluorouracil, cytarabine, azauridine), folate antimetabolites (4-aminopterin, methotrexate), or glutamine antimetabolites (azeline, DON) for malignant tumors.

[0053] Camptothecin may be a natural anti-cancer substance isolated from plants such as Camptotheca acuminate (Camptotheca, happy tree) and Chonemorpha fragrans.

[0054] Camptothecin may be a compound having the structure of Formula 6 below: [ka]

[0055] Epipodophyllotoxin may be a natural anticancer substance that is naturally produced in the roots of Podophyllum peltatum. Currently, derivatives of epipodophyllotoxin can be used in cancer treatment.

[0056] The epipodophyllotoxin may be a compound having the structure of Formula 7 below: [ka]

[0057] Mitomycin C may be an antibiotic isolated by Streptomyces griseus. Mitomycin C is heat stable, has the lowest toxicity, and has a strong anticancer effect. Mitomycin C inhibits the cell enzyme system and nucleic acid metabolism, thus inhibiting the division of cell nuclei, thereby blocking the proliferation of malignant tumor cells. Examples of side effects of mitomycin C include bleeding accompanied by leukopenia and thrombocytopenia, etc.

[0058] Taxanes, also called cytostatics or anti-microtubule inhibitors, are potential anti-cancer drugs that inhibit the growth of cancer cells by inhibiting cell division. Taxanes can kill cancer cells by destroying the microtubules along which chromosomes travel during cell mitosis. Taxanes are used to treat various cancers, such as breast cancer, ovarian cancer, and non-small cell lung cancer. Specifically, taxanes include paclitaxel, docetaxel, and the like.

[0059] Vinblastine is an anti-cancer drug that is a component of vinca alkaloids and is used to treat various cancers. Vinblastine was first extracted from the periwinkle plant, which belongs to the Oleander family, and is now used synthetically. Vinblastine is the most widely used anti-cancer drug and is also widely used in combination with other anti-cancer drugs. Vinblastine prevents cancer cells from dividing by interfering with the normal function of microtubules. Vinblastine can be widely used in testicular cancer, breast cancer, lymphoma, Kaposi's sarcoma, etc. The most important side effect of vinblastine is a decrease in white blood cells and platelets, and side effects such as gastrointestinal disorders, elevated blood pressure, excessive sweating, depression, muscle pain, nausea, and headache may occur.

[0060] The dosage of vaccinia virus varies depending on the condition and weight of the individual, the severity of the disease, the type of drug, the route and duration of administration, and can be appropriately selected by those skilled in the art. 5 ~1×10 18 of virus particles, infectious viral units (TCID 50 ) or plaque forming units (pfu) of vaccinia virus. Specifically, the dosage may be such that a patient receives 1×10 5 , 2×10 5 , 5×10 5 , 1×10 6 , 2×10 6 , 5×10 6 , 1×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , 5×10 8 , 1×10 9 , 2×10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 1×10 11 , 5×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×1016 , 1×10 17 or more viral particles, infectious viral units or plaque forming units of vaccinia virus, including various values ​​and ranges therebetween. Preferably, the vaccinia virus is administered at a concentration of 1×10 5 ~1×10 10 More preferably, the vaccinia virus is administered at a dose of 1×10 pfu. 5 Above and 1×10 9 In one embodiment of the invention, the vaccinia virus can be administered in a dose of less than 1×10 pfu. 5 Or 1×10 7 was administered at p.f.u.

[0061] Also, the granulopoiesis inhibitor can be administered at a dose of 1 mg / kg / day to 100 mg / kg / day, or 10 mg / kg / day to 90 mg / kg / day. Specifically, the granulopoiesis inhibitor can be administered at a dose of 10 mg / kg / day to 90 mg / kg / day, 15 mg / kg / day to 80 mg / kg / day, 20 mg / kg / day to 70 mg / kg / day, 25 mg / kg / day to 65 mg / kg / day, or 30 mg / kg / day to 60 mg / kg / day. In an embodiment of the present invention, hydroxyurea, lenalidomide or palbociclib is administered at a dose of 25 mg / kg / day, 30 mg / kg / day, 50 mg / kg / day, 60 mg / kg / day or 100 mg / kg / day as the granulopoiesis inhibitor. Depending on the dosage, the granulopoiesis inhibitor can be administered several times per day by divided doses. Specifically, the granulopoiesis inhibitor can be administered 1 to 4 times per day or 1 to 2 times per day.

[0062] The pharmaceutical composition may further comprise an immune checkpoint inhibitor (ICI).

[0063] An immune checkpoint inhibitor refers to a substance that inhibits the mechanism of cancer cells that interferes with the activation of T cells, and may be any one selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA4 antibody, anti-PD-L2 antibody, LTF2 modulating antibody, anti-LAG3 antibody, anti-A2aR antibody, anti-TIGIT antibody, anti-TIM-3 antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-VISTA antibody, anti-CD47 antibody, anti-BTLA antibody, anti-KIR antibody, anti-IDO antibody, and combinations thereof.

[0064] Cancer cells hijack the immune checkpoint system as a mechanism to avoid immune responses. Specifically, cancer cells use immune checkpoint receptors to avoid immune responses, representative receptors include PD-L1, PD-1, CTLA-4, etc. To block these immune evasions of cancer cells, immune checkpoint inhibitors, molecules that specifically bind to immune checkpoint receptors, are used in cancer treatment. The first immune checkpoint inhibitor was ipilimumab (Yervoy®), which is a monoclonal antibody that specifically binds to cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4). The next immune checkpoint therapy developed is a monoclonal antibody against programmed cell death-1 (PD-1) and its corresponding ligand, programmed death ligand-1 (PD-L1). Representative drugs include anti-PD-1 antibodies (e.g., nivolumab (Opdivo®), pembrolizumab (Keytruda®, etc.)) and anti-PD-L1 antibodies (e.g., avelumab (Bavencio®), atezolizumab (Tecentriq®), durvalumab (Imfinzi®, etc.).

[0065] In addition, research is ongoing on monoclonal antibodies that specifically bind to various immune checkpoint receptors (e.g., glucocorticoid-induced TNFR-related protein (GITR), killer cell immunoglobulin-like receptor (KIR), lymphocyte-activation gene-3 (LAG-3), T-cell immunoglobulin and mucin domain-containing-3 (TIM-3), tumor necrosis factor receptor superfamily member 4 (TNFRSF4)).

[0066] The dose of the immune checkpoint inhibitor can be administered according to the dosage regimen of each manufacturer. The dose of the immune checkpoint inhibitor can be 0.1 mg / kg to 10 mg / kg or 1 mg / kg to 5 mg / kg. For example, in the case of Opdivo injection containing nivolumab as an active ingredient, 3 mg / kg can be intravenously infused over 60 minutes at 2-week intervals, and in the case of the dosage regimen as a combination therapy, 1 mg / kg can be intravenously infused over 30 minutes. In addition, in the case of Keitruda injection containing pembrolizumab as an active ingredient, 200 mg can be intravenously infused over 30 minutes at 3-week intervals. Thus, even if the anti-PD-1 antibody is the same, different products have different dosage regimens, so it is preferable to administer the antibody according to the dosage regimen of the manufacturer.

[0067] When the pharmaceutical composition further comprises an immune checkpoint inhibitor, the oncolytic virus, the granulopoiesis inhibitor and the immune checkpoint inhibitor contained in the pharmaceutical composition can be administered simultaneously, sequentially or in the reverse order.

[0068] Specifically, the oncolytic virus, the granulopoiesis inhibitor and the immune checkpoint inhibitor can be administered simultaneously. Also, the granulopoiesis inhibitor can be administered first, followed by the immune checkpoint inhibitor, and then the oncolytic virus. The granulopoiesis inhibitor can be administered first, followed by the oncolytic virus, and then the immune checkpoint inhibitor. The granulopoiesis inhibitor can be administered first, and then the oncolytic virus and the immune checkpoint inhibitor can be administered simultaneously.

[0069] Additionally, the oncolytic virus can be administered first, followed by the granulopoiesis inhibitor, and then the immune checkpoint inhibitor. The oncolytic virus can be administered first, followed by the immune checkpoint inhibitor, and then the granulopoiesis inhibitor. The oncolytic virus can be administered first, followed by the granulopoiesis inhibitor and the immune checkpoint inhibitor simultaneously.

[0070] Also, the immune checkpoint inhibitor can be administered first, followed by the granulopoiesis inhibitor, and then the oncolytic virus. The immune checkpoint inhibitor can be administered first, followed by the oncolytic virus, and then the granulopoiesis inhibitor. The immune checkpoint inhibitor can be administered first, followed by the oncolytic virus and then the granulopoiesis inhibitor. The immune checkpoint inhibitor can be administered first, and then the oncolytic virus and the granulopoiesis inhibitor can be administered simultaneously.

[0071] Furthermore, the granulopoiesis inhibitor can be administered first, followed by the oncolytic virus, then the immune checkpoint inhibitor, and then the granulopoiesis inhibitor again. The granulopoiesis inhibitor can be administered first, followed by the immune checkpoint inhibitor, then the oncolytic virus, and then the granulopoiesis inhibitor again. The granulopoiesis inhibitor can be administered first, the oncolytic virus and the immune checkpoint inhibitor can be administered simultaneously, and then the granulopoiesis inhibitor can be administered again.

[0072] Alternatively, the granulopoiesis inhibitor can be administered first, followed by the oncolytic virus, then the granulopoiesis inhibitor again, and then the immune checkpoint inhibitor.The granulopoiesis inhibitor can be administered first, followed by the immune checkpoint inhibitor, then the granulopoiesis inhibitor again, and then the oncolytic virus.

[0073] Additionally, the granulopoiesis inhibitor can be administered first, followed by the oncolytic virus, then the granulopoiesis inhibitor again, then the immune checkpoint inhibitor, then the granulopoiesis inhibitor again.The granulopoiesis inhibitor can be administered first, followed by the immune checkpoint inhibitor, then the granulopoiesis inhibitor again, then the oncolytic virus, then the granulopoiesis inhibitor again.

[0074] Alternatively, the oncolytic virus can be administered first, followed by the granulopoiesis inhibitor, then the immune checkpoint inhibitor, and then the granulopoiesis inhibitor again.The immune checkpoint inhibitor can be administered first, followed by the granulopoiesis inhibitor, then the oncolytic virus, and then the granulopoiesis inhibitor again.

[0075] The cancer may be a solid cancer or a blood cancer. Specifically, the blood cancer may be any one selected from the group consisting of lymphoma, acute leukemia, and multiple myeloma. The solid cancer may be any one selected from the group consisting of lung cancer, colorectal cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymus cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, and combinations thereof.

[0076] In addition, the pharmaceutical composition of the present invention may further comprise a physiologically acceptable carrier.In addition, the pharmaceutical composition of the present invention may further comprise suitable excipients and diluents that are commonly used in the preparation of pharmaceutical compositions.In addition, the pharmaceutical composition may be formulated into the form of an injection by a conventional method.

[0077] When formulated as a preparation for parenteral administration, the pharmaceutical composition can be formulated into a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a freeze-dried preparation, a suppository, etc. In the case of a non-aqueous solution or suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, etc. can be used. As a base for a suppository, Witepsol (trademark), macrogol, Tween (trademark) 61, cacao butter, lauric fat, glycerogelatin, etc. can be used.

[0078] Regarding the administration route, dosage and frequency of administration, the pharmaceutical composition can be administered to the subject in various ways and amounts depending on the patient's condition and the presence or absence of side effects; and the optimal administration route, dosage and frequency of administration, and therefore can be selected by those skilled in the art within a suitable range. In addition, the pharmaceutical composition can be administered in combination with another drug or bioactive substance whose therapeutic effect is known for the disease to be treated, or can be formulated in the form of a combination preparation with other drugs.

[0079] The pharmaceutical composition can be administered parenterally, and such administration can be performed by any suitable method, such as intratumoral, intraperitoneal, subcutaneous, intradermal, intranodal, intravenous or intraarterial administration. Among these, intratumoral, intraperitoneal or intravenous administration may be preferred. On the other hand, the dosage of the pharmaceutical composition can be determined according to the administration schedule, the total dosage and the health condition of the patient.

[0080] The pharmaceutical composition for treating cancer may be characterized by increased cancer selectivity of the vaccinia virus.

[0081] Another aspect of the present invention provides a kit for preventing or treating cancer, comprising a first composition comprising a vaccinia virus as an active ingredient, a second composition comprising a granulopoiesis inhibitor as an active ingredient, and may further comprise a third composition comprising an immune checkpoint inhibitor as an active ingredient.

[0082] The vaccinia virus, the granulopoiesis inhibitor and the immune checkpoint inhibitor are the same as those described above in the pharmaceutical composition.

[0083] The second composition containing the granulopoiesis inhibitor as an active ingredient may be a commercially available drug. Examples of commercially available drugs containing hydroxyurea as an active ingredient as a granulopoiesis inhibitor include Hydroxyurea (registered trademark), Hydrea (registered trademark), Droxia (trademark), Mirocel (trademark), Cyclos (registered trademark) and Hydrin (registered trademark) capsules. The second composition can be taken orally, and its parenteral administration is also possible.

[0084] The dosage of the first composition varies depending on the condition and weight of the individual, the severity of the disease, the type of drug, the route and duration of administration, and can be appropriately selected by those skilled in the art. 5 ~1×10 18 of virus particles, infectious viral units (TCID 50 ) or plaque forming units (pfu) of vaccinia virus. Specifically, the dosage may be such that a patient receives 1×10 5 , 2×10 5 , 5×10 5 , 1×10 6 , 2×10 6 , 5×10 6 , 1×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , 5×10 8 , 1×10 9 , 2×10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 1×10 11 , 5×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×1017 or more viral particles, infectious viral units or plaque forming units of vaccinia virus, including various values ​​and ranges therebetween. Preferably, the first composition contains 1×10 5 ~1×10 10 More preferably, the first composition is administered in a dose of 1×10 pfu. 5 Above and 1×10 9 In one embodiment of the present invention, the first composition can be administered in a dose of less than 1×10 pfu. 5 Or 1×10 7 was administered at p.f.u.

[0085] Also, the second composition can be administered at a dose of 1 mg / kg / day to 100 mg / kg / day, or 10 mg / kg / day to 90 mg / kg / day. Specifically, the second composition can be administered at a dose of 10 mg / kg / day to 90 mg / kg / day, 15 mg / kg / day to 80 mg / kg / day, 20 mg / kg / day to 70 mg / kg / day, 25 mg / kg / day to 65 mg / kg / day, or 30 mg / kg / day to 60 mg / kg / day. In one embodiment of the present invention, the second composition is administered at 25 mg / kg / day, 30 mg / kg / day, 50 mg / kg / day, 60 mg / kg / day, or 100 mg / kg / day. Depending on the dosage, the second composition can be administered several times per day by divided doses. Specifically, the second composition can be administered 1 to 4 times per day or 1 to 2 times per day.

[0086] The dose of the third composition can be administered according to the respective manufacturer's dosing regimen of the immune checkpoint inhibitor contained in the third composition. The dose of the third composition can be 0.1 mg / kg to 10 mg / kg, or 1 mg / kg to 5 mg / kg.

[0087] The cancer may be a solid cancer or a blood cancer. Specifically, the blood cancer may be any one selected from the group consisting of lymphoma, acute leukemia, and multiple myeloma. The solid cancer may be any one selected from the group consisting of lung cancer, colorectal cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymus cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, and combinations thereof.

[0088] The first composition, the second composition and the third composition can further comprise a physiologically acceptable carrier.The composition included in the kit of the present invention can further comprise suitable excipients and diluents that are generally used in the preparation of pharmaceutical compositions.The composition can also be formulated into the form of an injection by conventional methods.

[0089] When formulated as a preparation for parenteral administration, the first composition, the second composition, and the third composition can be formulated as a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a freeze-dried preparation, a suppository, etc. In the case of a non-aqueous solution or suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, etc. can be used. As a base for a suppository, Witepsol (trademark), Macrogol, Tween (trademark) 61, cocoa butter, lauric fat, glycerogelatin, etc. can be used.

[0090] Regarding the administration route, dosage and frequency of administration, the first composition, the second composition and the third composition can be administered to the subject in various ways and amounts according to the patient's condition and the presence or absence of side effects; and the optimal administration route, dosage and frequency of administration, and therefore can be selected by those skilled in the art within a suitable range. In addition, the pharmaceutical composition can be administered in combination with another drug or bioactive substance whose therapeutic effect is known for the disease to be treated, or can be formulated in the form of a combination preparation with other drugs.

[0091] The second composition can be administered orally or parenterally. In particular, the second composition can be administered parenterally, and such administration can be performed by intraperitoneal, intraarterial or intravenous administration.

[0092] The first composition can be administered parenterally, and such administration can be carried out by any suitable method, such as intratumoral, intraperitoneal, subcutaneous, intradermal, intranodal, intraarterial or intravenous administration. Among these, intratumoral, intraperitoneal or intravenous administration may be preferred. Meanwhile, the dosage of the first composition and the second composition can be determined according to the administration schedule, the total dosage and the health condition of the patient.

[0093] The first composition can be administered 1 to 10 times or 2 to 5 times, and the administration to an individual can be performed at intervals of 7 to 30 days. Specifically, the first composition can be administered at intervals of 7, 14, 21, or 30 days.

[0094] The second composition can be administered before or after administration of the first composition. Specifically, the second composition can be administered continuously once a day starting 3-5 days before administration of the first composition, and can be administered continuously once a day starting within 24 hours or 24 hours after administration of the first composition for 9-28 days. In one embodiment of the present invention, the second composition can be administered continuously once a day starting 1-3 days before administration of the first composition, and can be administered once a day for 13 days, 17 days, 18 days, or 28 days after administration of the first composition.

[0095] The third composition can be administered continuously at least once a week for 1 to 10 weeks after administration of the first composition. Specifically, the third composition can be administered continuously at least twice a week for 1 to 8 weeks after administration of the first composition.

[0096] In yet another aspect of the invention, a method of treating cancer is provided comprising administering to an individual with cancer a vaccinia virus and a granulopoiesis inhibitor.

[0097] The method of treatment can further include administering an immune checkpoint inhibitor to the individual with cancer.

[0098] The oncolytic virus, the granulopoiesis inhibitor and the immune checkpoint inhibitor are the same as those described above in the pharmaceutical composition.

[0099] The vaccinia virus may belong to, but is not limited to, Western Reserve, NYVAC, Wyeth, LC16m8, Lister, Copenhagen, Tiantan, USSR, Tashkent, Evans, IHD-J or IHD-W vaccinia virus strains.

[0100] The vaccinia virus and the granulopoiesis inhibitor can be administered simultaneously, sequentially, or in the reverse order. Specifically, the vaccinia virus and the granulopoiesis inhibitor can be administered simultaneously. Alternatively, the granulopoiesis inhibitor can be administered first, followed by the vaccinia virus. Alternatively, the vaccinia virus can be administered first, followed by the granulopoiesis inhibitor. Alternatively, the granulopoiesis inhibitor can be administered first, followed by the vaccinia virus, followed by the granulopoiesis inhibitor again.

[0101] In addition, when the treatment method further includes a step of administering an immune checkpoint inhibitor, the oncolytic virus, the granulopoiesis inhibitor, and the immune checkpoint inhibitor can be administered simultaneously, sequentially, or in the reverse order. Specifically, the oncolytic virus, the granulopoiesis inhibitor, and the immune checkpoint inhibitor can be administered simultaneously. In addition, the granulopoiesis inhibitor can be administered first, followed by the immune checkpoint inhibitor, and then the oncolytic virus. The granulopoiesis inhibitor can be administered first, followed by the oncolytic virus, and then the immune checkpoint inhibitor. The granulopoiesis inhibitor can be administered first, and then the oncolytic virus and the immune checkpoint inhibitor can be administered simultaneously.

[0102] Additionally, the oncolytic virus can be administered first, followed by the granulopoiesis inhibitor, and then the immune checkpoint inhibitor. The oncolytic virus can be administered first, followed by the immune checkpoint inhibitor, and then the granulopoiesis inhibitor. The oncolytic virus can be administered first, followed by the granulopoiesis inhibitor and the immune checkpoint inhibitor, and then the granulopoiesis inhibitor is administered simultaneously.

[0103] Also, the immune checkpoint inhibitor can be administered first, followed by the granulopoiesis inhibitor, and then the oncolytic virus. The immune checkpoint inhibitor can be administered first, followed by the oncolytic virus, and then the granulopoiesis inhibitor. The immune checkpoint inhibitor can be administered first, followed by the oncolytic virus and then the granulopoiesis inhibitor. The immune checkpoint inhibitor can be administered first, and then the oncolytic virus and the granulopoiesis inhibitor can be administered simultaneously.

[0104] Furthermore, the granulopoiesis inhibitor can be administered first, followed by the oncolytic virus, then the immune checkpoint inhibitor, and then the granulopoiesis inhibitor again. The granulopoiesis inhibitor can be administered first, followed by the immune checkpoint inhibitor, then the oncolytic virus, and then the granulopoiesis inhibitor again. The granulopoiesis inhibitor can be administered first, then the oncolytic virus and the immune checkpoint inhibitor are administered simultaneously, and then the granulopoiesis inhibitor again.

[0105] Alternatively, the granulopoiesis inhibitor can be administered first, followed by the oncolytic virus, then the granulopoiesis inhibitor again, and then the immune checkpoint inhibitor.The granulopoiesis inhibitor can be administered first, followed by the immune checkpoint inhibitor, then the granulopoiesis inhibitor again, and then the oncolytic virus.

[0106] Additionally, the granulopoiesis inhibitor can be administered first, followed by the oncolytic virus, then the granulopoiesis inhibitor again, then the immune checkpoint inhibitor, then the granulopoiesis inhibitor again.The granulopoiesis inhibitor can be administered first, followed by the immune checkpoint inhibitor, then the granulopoiesis inhibitor again, then the oncolytic virus, then the granulopoiesis inhibitor again.

[0107] Alternatively, the oncolytic virus can be administered first, followed by the granulopoiesis inhibitor, then the immune checkpoint inhibitor, and then the granulopoiesis inhibitor again.The immune checkpoint inhibitor can be administered first, followed by the granulopoiesis inhibitor, then the oncolytic virus, and then the granulopoiesis inhibitor again.

[0108] The dosage of vaccinia virus varies depending on the condition and weight of the individual, the severity of the disease, the type of drug, the route and duration of administration, and can be appropriately selected by those skilled in the art. 5 ~1×10 18of virus particles, infectious viral units (TCID 50 ) or plaque forming units (pfu) of vaccinia virus. Specifically, the dosage may be such that a patient receives 1×10 5 , 2×10 5 , 5×10 5 , 1×10 6 , 2×10 6 , 5×10 6 , 1×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , 5×10 8 , 1×10 9 , 2×10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 1×10 11 , 5×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 or more viral particles, infectious viral units or plaque forming units of vaccinia virus, including various values ​​and ranges therebetween. Preferably, the vaccinia virus is administered at a concentration of 1×10 5 ~1×10 10 More preferably, the vaccinia virus is administered at a dose of 1×10 pfu. 5 Above and 1×10 9 In one embodiment of the invention, the vaccinia virus can be administered in a dose of less than 1×10 pfu. 5 Or 1×10 7 was administered at p.f.u.

[0109] Also, the granulopoiesis inhibitor can be administered at a dose of 1 mg / kg / day to 100 mg / kg / day, or 10 mg / kg / day to 90 mg / kg / day. Specifically, the granulopoiesis inhibitor can be administered at a dose of 10 mg / kg / day to 90 mg / kg / day, 15 mg / kg / day to 80 mg / kg / day, 20 mg / kg / day to 70 mg / kg / day, 25 mg / kg / day to 65 mg / kg / day, or 30 mg / kg / day to 60 mg / kg / day. In an embodiment of the present invention, hydroxyurea, lenalidomide, or palbociclib is administered as the granulopoiesis inhibitor at a dose of 25 mg / kg / day, 30 mg / kg / day, 50 mg / kg / day, 60 mg / kg / day, or 100 mg / kg / day. Depending on the dosage, the granulopoiesis inhibitor can be administered several times per day by divided doses. Specifically, the granulopoiesis inhibitor can be administered 1 to 4 times per day or 1 to 2 times per day.

[0110] In addition, the vaccinia virus can be administered 1 to 10 times or 2 to 5 times to an individual at intervals of 7 to 30 days. Specifically, the vaccinia virus can be administered at intervals of 7, 14, 21, or 30 days.

[0111] The granulopoiesis inhibitor can be administered before, during or after administration of the vaccinia virus. Specifically, the granulopoiesis inhibitor can be administered before or after administration of the vaccinia virus. The granulopoiesis inhibitor can be administered continuously once a day starting 3-5 days before administration of the vaccinia virus, and can be administered continuously once a day starting 24 hours after administration of the vaccinia virus for 9-28 days. In one embodiment of the present invention, the granulopoiesis inhibitor can be administered continuously once a day starting 1-3 days before administration of the vaccinia virus, and can be administered once a day for 13 days, 17 days, 18 days or 28 days after administration of the vaccinia virus.

[0112] The cancer may be a solid cancer or a blood cancer. Specifically, the blood cancer may be any one selected from the group consisting of lymphoma, acute leukemia, and multiple myeloma. The solid cancer may be any one selected from the group consisting of lung cancer, colorectal cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymus cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, and combinations thereof.

[0113] The granulopoiesis inhibitor can be administered orally or parenterally. Specifically, the granulopoiesis inhibitor can be administered parenterally, and such administration can be performed by intraperitoneal, intraarterial or intravenous administration.

[0114] The vaccinia virus and the granulopoiesis inhibitor can be administered parenterally, and such administration can be carried out by any suitable method, such as intratumoral, intraperitoneal, subcutaneous, intradermal, intranodal, intravenous or intraarterial administration. Among these, intratumoral, intraperitoneal or intravenous administration may be preferred. Meanwhile, the dosage of the vaccinia virus and the granulopoiesis inhibitor can be determined according to the administration schedule, the total dosage and the health condition of the patient.

[0115] As used herein, the term "individual" refers to a person having or suffering from a disease, a condition that can be alleviated, inhibited or treated by administering a pharmaceutical composition of the present invention.

[0116] As used herein, the term "administration" means introducing an effective amount of a substance into an individual by an appropriate method, and administration of vaccinia virus and granulopoiesis inhibitors can be carried out via any common route that allows the substance to reach the target tissue.

[0117] Additionally, the vaccinia virus and granulopoiesis inhibitors can be administered in combination with another drug or biologically active substance whose therapeutic effect is known for the disease being treated, or can be formulated in a combination formulation with other drugs.

[0118] In yet another aspect of the present invention, there is provided a use of a composition comprising a vaccinia virus and a granulopoiesis inhibitor for the prevention or treatment of cancer.

[0119] In yet another aspect of the present invention, there is provided a use of a composition comprising a vaccinia virus and a granulopoiesis inhibitor for the manufacture of a medicament for preventing or treating cancer.

[0120] In yet another aspect of the present invention, there is provided an anti-cancer adjuvant comprising a granulopoiesis inhibitor as an active ingredient, wherein the granulopoiesis inhibitor is as described above for the pharmaceutical composition. The anti-cancer adjuvant may also be characterized as being used as an anti-cancer adjuvant for an anti-cancer agent comprising a vaccinia virus as an active ingredient. The anti-cancer adjuvant may be characterized as improving, enhancing or increasing the anti-cancer activity of the vaccinia virus. The anti-cancer adjuvant may be characterized as increasing the cancer selectivity of the vaccinia virus.

[0121] The granulopoiesis inhibitor may be hydroxyurea, lenalidomide, thalidomide, tadalafil, palbociclib, an alkylating agent, anthracycline, an antimetabolite, camptothecin, epipodophyllotoxin, mitomycin C, a taxane, or vinblastine. EXAMPLES

[0122] Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereto.

[0123] Preparation Example 1. Recombinant vaccinia virus (Wyeth VV tk- , W.R.V.V. tk- ) Preparative Example 1.1. Construction of Shuttle Plasmid Vector To generate recombinant vaccinia viruses lacking the thymidine kinase (TK) gene, wild-type vaccinia viruses, i.e., Wyeth strain (NYC Department of Health) and Western Reserve strain, were purchased from the American Type Culture Collection (ATCC). For recombination, the TK region of wild-type vaccinia viruses was replaced with a shuttle plasmid vector containing a firefly luciferase reporter (p7.5 promoter) gene or a GFP gene.

[0124] Preparative Example 1.2. Generation of Recombinant Vaccinia Virus To obtain recombinant viruses, HeLa cells (ATCC) were cultured at 4 × 10 per well. 5 The cells were seeded in a 6-well plate, and then cultured in EMEM medium containing 10% fetal bovine serum. Then, treatment with wild-type vaccinia virus was performed at an MOI of 0.05. After 2 hours, the medium was replaced with EMEM medium containing 2% fetal bovine serum, and then the cells were transfected with 4 μg of the shuttle plasmid vector constructed and linearized in Preparation Example 1.1 using Xfect™ polymer (Clonetech631317, USA). Culture was performed for 4 hours. Then, the medium was replaced with EMEM medium containing 2% fetal bovine serum, and then cultured for another 72 hours. Finally, the infected cells were collected, and then freeze-thawed three times. Then, the cells were lysed by sonication, and the free recombinant vaccinia virus was obtained using the sucrose cushion method, and they were then transfected with Wyeth VV tk- or WR VV tk- and named it.

[0125] Preparative Example 2. Generation of recombinant vaccinia virus (OTS-412) To generate a recombinant vaccinia virus that lacks thymidine kinase (TK) gene and expresses mutated herpes simplex virus thymidine kinase (HSV-TK) gene, the TK region of the Wyeth strain wild-type vaccinia virus was replaced by using the pUC57amp+ plasmid (Genewiz, USA) as a shuttle vector, which is a synthetic mutated type 1 HSV-TK gene (pSE / L promoter) and a firefly luciferase reporter (p7.5 promoter) gene of SEQ ID NO: 1. Using the shuttle vector constructed above, a recombinant vaccinia virus was obtained in the same manner as in Preparation Example 1.2, and this virus was named OTS-412.

[0126] Preparative Example 3. Generation of recombinant vaccinia virus (WOTS-418) To generate a recombinant vaccinia virus in which the thymidine kinase (TK) gene was deleted and which expressed a mutated herpes simplex virus thymidine kinase (HSV-TK) gene, the TK region of the Western Reserve strain wild-type vaccinia virus was replaced with the synthetic mutated type 1 HSV-TK gene (pSE / L promoter) of SEQ ID NO:2 and the firefly luciferase reporter (p7.5 promoter) gene recombined with pUC57amp+ plasmid (Genewiz, USA) as a shuttle vector. Using the shuttle vector constructed above, a recombinant vaccinia virus was obtained in the same manner as in Preparation Example 1.2, and this virus was named WOTS-418.

[0127] Preparation Example 4. Generation of recombinant vaccinia virus (VV_DD) To generate a recombinant vaccinia virus lacking the thymidine kinase (TK) gene and the vaccinia growth factor (VGF) gene, the TK region of the Western Reserve wild-type vaccinia virus was replaced with a shuttle plasmid containing the enhanced green fluorescent protein (EGFP) gene, and the VGF gene region of the same virus was replaced with a shuttle plasmid containing the lacZ gene. Using the shuttle plasmid containing the EGFP gene and the shuttle plasmid containing the lacZ gene, a recombinant vaccinia virus was obtained as in Example 1.2, and this virus was named VV_DD.

[0128] I. Identification of synergistic anti-cancer effects by co-administration of vaccinia virus and hydroxyurea

[0129] Experimental Example 1. Identification of the cancer treatment effects of wild-type vaccinia virus (WR) and hydroxyurea in Renal cancer cell-implanted mice (Renca (I)) Experimental Example 1.1. Generation of mice implanted with mouse kidney cancer cells and drug administration Balb / c mice (female, 10 weeks old) purchased from ORIENT BIO (Busan, Korea) were acclimated for 2 days and then subcutaneously implanted with Renca cancer cell line (Korea Cell Line Bank) at 5 × 106 cells. 3 ~80mm 3 The tumor volume was monitored until it reached a volume of 100 μg / kg, after which administration of wild-type vaccinia virus was initiated. On the other hand, Western Reserve wild-type vaccinia virus (WR) has stronger proliferation ability than Wyeth wild-type vaccinia virus in the allograft model.

[0130] The resulting mouse kidney cancer cell-implanted mice were divided into three groups (n = 6). The group receiving intraperitoneal administration of saline was set as the negative control group, and the group receiving intraperitoneal administration of wild-type vaccinia virus (WR, 1 × 10 5 The group receiving 1 × 10 pfu of wild-type vaccinia virus (WR, 1 × 10 5The group receiving simultaneous administration of 1000 mg / kg pfu of 1000 sera and hydroxyurea (30 mg / kg) was set as the experimental group. Wild-type vaccinia virus was administered once intratumorally; hydroxyurea was administered intraperitoneally 5 times per week starting 1 day before administration of wild-type vaccinia virus and continuing through 14 days after administration, except on the day of administration of wild-type vaccinia virus.

[0131] Experimental Example 1.2. Examination of changes in tumor volume Tumor volumes were measured on days 0, 3, 7, 10 and 14 after drug administration to mice in each group of Experimental Example 1.1. As a result, it was determined that the tumor volumes of mice in the positive control group were suppressed compared to the negative control group, and that the tumor volumes of mice in the experimental group were significantly suppressed (Figure 1).

[0132] Experimental Example 1.3. Examination of body weight change Body weights were measured on days 3, 7, 10 and 14 after administration of each drug to the mice in the negative control group, positive control group and experimental group of Experimental Example 1.1. As a result, there was no significant weight loss in all three groups (Figure 2).

[0133] Experimental Example 2. Mouse renal cell transplantation mice: Recombinant vaccinia virus (WR VV) in Renca (II) tk- ) and the identification of the cancer treatment effects of hydroxyurea Experimental Example 2.1. Generation of mice implanted with mouse kidney cancer cells and drug administration Balb / c mice (female, 8 weeks old) purchased from ORIENT BIO (Busan, Korea) were acclimated for 1 week and then inoculated with Renca cancer cell line (Korea Cell Line Bank) at 5 × 10 6 The cells were transplanted into the 100 mm 3 ~150mm 3 The tumor volume was monitored until it reached 100 mg / kg, after which administration of recombinant vaccinia virus was started. On the other hand, Western Reserve strain-derived recombinant vaccinia virus (WR VV tk- ) has stronger proliferation ability than recombinant vaccinia virus derived from the Wyeth strain in an allograft model.

[0134] The mouse kidney cancer cell-implanted mice were divided into three groups (n=8). The group receiving intraperitoneal administration of saline was set as a negative control group, and the group receiving intraperitoneal administration of recombinant vaccinia virus (WR VV) was set as a negative control group. tk- , 1×10 7 The group receiving 100 mg / kg pfu of recombinant vaccinia virus was set as a positive control group, and the group receiving recombinant vaccinia virus and hydroxyurea (60 mg / kg) simultaneously was set as an experimental group. The recombinant vaccinia virus was administered twice intratumorally; hydroxyurea was administered intraperitoneally six times per week, starting one day before and ending 21 days after recombinant vaccinia virus administration, except on the day recombinant vaccinia virus was administered.

[0135] Experimental Example 2.2. Examination of changes in tumor volume The tumor volumes were measured on days 0, 3, 7, 10, 14, 17 and 21 after drug administration to the mice in each group of Experimental Example 2.1. As a result, it was determined that the tumor volumes of the mice in the experimental group were significantly suppressed compared to the tumor volumes of the mice in the positive control group ( FIG. 3 ).

[0136] Experimental Example 3. Mouse renal cell transplantation mice: Recombinant vaccinia virus (WR VV) in Renca (III) tk- ) and the identification of the cancer treatment effects of hydroxyurea Balb / c mice (female, 10 weeks old) purchased from ORIENT BIO (Busan, Korea) were acclimated for 2 days and then Renca cancer cell line (Korea Cell Line Bank) was subcutaneously injected into the left thigh at 5 × 10 6 The cells were transplanted. It was 50 mm 3 ~150mm 3 The tumor volume was monitored until it reached 100 mg / kg, after which administration of the recombinant vaccinia virus was initiated.

[0137] The mouse kidney cancer cell-implanted mice were divided into three groups (n=6). The group receiving intraperitoneal administration of saline was set as a negative control group, and the group receiving intraperitoneal administration of recombinant vaccinia virus (WR VV) was set as a negative control group. tk- , 1×10 5The group receiving 100 mg / kg pfu of recombinant vaccinia virus was set as a positive control group, and the group receiving recombinant vaccinia virus and hydroxyurea (30 mg / kg) simultaneously was set as an experimental group. The recombinant vaccinia virus was administered once intratumorally; hydroxyurea was administered intraperitoneally six times per week, starting one day before and ending 14 days after recombinant vaccinia virus administration, except on the day recombinant vaccinia virus was administered.

[0138] The tumor volumes were measured on days 0, 3, 7, 10, and 14 after drug administration to the mice in each group. As a result, it was determined that the tumor volume of the mice in the experimental group was suppressed by approximately 25% in growth compared to the tumor volume of the mice in the positive control group (Figure 4).

[0139] Experimental Example 4. Identification of the cancer treatment effect of recombinant vaccinia virus (VV_DD) and hydroxyurea in melanoma-bearing mice: B16F10 C57BL / 6 mice (female, 7 weeks old) purchased from KOATECH (Korea) were acclimated for 2 days and then inoculated with 5 × 10 5 The cells were then implanted subcutaneously. 3 ~100mm 3 The tumor volume was monitored until it reached 100 mg / kg, and then administration of a recombinant vaccinia virus (VV_DD) was started. The recombinant vaccinia virus (VV_DD) was obtained by performing a double deletion of the thymidine kinase (TK) and vaccinia growth factor (VGF) domains of the Western Reserve strain vaccinia virus, and has limited proliferation ability in an allograft model.

[0140] The generated mouse melanoma-implanted mice were divided into 4 groups (n=6). The group receiving intraperitoneal administration of saline was set as a negative control group, and the group receiving intraperitoneal administration of hydroxyurea (30 mg / kg) or recombinant vaccinia virus (VV_DD, 1×10 6A group receiving only 100 mg / kg pfu of recombinant vaccinia virus was set as a positive control group, and a group receiving recombinant vaccinia virus and hydroxyurea (30 mg / kg) simultaneously was set as an experimental group. The recombinant vaccinia virus was intraperitoneally administered on days 0 and 5; hydroxyurea was intraperitoneally administered 6 times per week starting 1 day before administration of recombinant vaccinia virus and continuing through day 15, except on the day of administration of recombinant vaccinia virus.

[0141] The tumor volume was measured 1 day before drug administration to the mice in each group and on the 4th and 7th days after administration. As a result, it was determined that the tumor volume of the mice in the experimental group was significantly suppressed compared to the tumor volume of the mice in the positive control group (Figure 5). From these results, it was determined that a synergistic effect was observed when recombinant vaccinia virus (VV_DD) and hydroxyurea were administered simultaneously.

[0142] Experimental Example 5. Identification of the cancer therapeutic effects of recombinant vaccinia virus (WOTS-418) and hydroxyurea in human lung cancer cell line-implanted mice: NCI-H460 Balb / c nu / nu mice (female, 7 weeks old) purchased from ORIENT BIO (Busan, Korea) were subjected to 2 days of acclimation and then subcutaneously inoculated with 5 × 10 6 The xenograft was performed with 100 mm 3 ~150mm 3 The tumor volume was monitored until it reached 100%, after which administration of recombinant vaccinia virus (WOTS-418) was started. On the other hand, Western Reserve strain-derived recombinant vaccinia virus (WOTS-418) has the ability to grow in human lung cancer cell line (NCI-H460) xenografted mice.

[0143] The resulting human lung cancer cell line-implanted mice were divided into two groups (n = 4). The group receiving intraperitoneal administration of saline was set as the control group, and the group receiving intraperitoneal administration of recombinant vaccinia virus (WOTS-418, 1 × 10 7The group receiving simultaneous administration of 1000 mg / kg of recombinant vaccinia virus (pfu) and hydroxyurea (30 mg / kg) was set as the experimental group. The recombinant vaccinia virus was administered intraperitoneally once; hydroxyurea was administered intraperitoneally six times per week starting from 1 day before administration of the recombinant vaccinia virus to 15 days after administration, excluding the day of administration of the recombinant vaccinia virus.

[0144] The tumor volume was measured on days 0, 5, 10, 12, and 15 after drug administration to the mice in each group. As a result, it was determined that the tumor volume of the mice in the experimental group was suppressed by about 40% compared to the control group (FIG. 6).

[0145] Experimental Example 6. Survival analysis of recombinant vaccinia virus (WOTS-418) and hydroxyurea in mouse colon cancer cell-implanted mice: CT-26 Balb / c mice (female, 7 weeks old) purchased from ORIENT BIO (Busan, Korea) were subjected to 2 days of acclimation and then subcutaneously injected with 5 × 10 6 The mice were transplanted with 1000 cells. After 7 days, administration of recombinant vaccinia virus (WOTS-418) and hydroxyurea was started. On the other hand, the Western Reserve strain-derived recombinant vaccinia virus (WOTS-418) had stronger proliferation ability than the Wyeth strain-derived recombinant vaccinia virus in the allograft model.

[0146] The mice implanted with the generated mouse colorectal cancer cell line were divided into two groups (n = 12): recombinant vaccinia virus (WOTS-418, 1 × 10 7 pfu) intraperitoneally, and a group receiving recombinant vaccinia virus and hydroxyurea (30 mg / kg) simultaneously. The recombinant vaccinia virus was administered intraperitoneally once; hydroxyurea was administered intraperitoneally five times consecutively starting on day 1 of recombinant vaccinia virus administration.

[0147] The survival curves of mice in each group were analyzed. As a result, in the group receiving recombinant vaccinia virus alone, all mice died 25 days after administration; however, more than 30% of mice receiving hydroxyurea and recombinant vaccinia virus simultaneously survived for more than 55 days (Figure 7). These results identified that the simultaneous administration of recombinant vaccinia virus and hydroxyurea provides enhanced safety compared to the administration of recombinant vaccinia virus alone.

[0148] Experimental Example 7. Mouse renal cell transplantation mice: Recombinant vaccinia virus (Wyeth VV) in Renca (IV) tk- ) and the identification of the cancer treatment effects of hydroxyurea Experimental Example 7.1. Generation of mice implanted with mouse kidney cancer cells and drug administration Balb / c mice (female, 7 weeks old) purchased from ORIENT BIO (Busan, Korea) were acclimated for 2 days and then inoculated with 5 × 10 Renca cancer cells (Korea Cell Line Bank). 6 The cells were transplanted into the 100 mm 3 ~150mm 3 The tumor volume was monitored until it reached 100 mg / kg, after which administration of the recombinant vaccinia virus was initiated.

[0149] The resulting mouse kidney cancer cell-implanted mice were divided into four groups (n=4). The group receiving intratumoral administration of saline was set as a negative control group, and the group receiving intratumoral administration of recombinant vaccinia virus (Wyeth VV tk- , 1×10 7 The group receiving pfu of recombinant vaccinia virus (Wyeth VV) was set as a positive control group. tk- , 1×10 7 pfu) and recombinant human granulocyte colony-stimulating factor (rhG-CSF, 75 μg / kg), and a group receiving recombinant virus (Wyeth VV tk- , 1×10 7The group receiving 100 mg / kg of recombinant vaccinia virus (pfu) and hydroxyurea (30 mg / kg) was set as the experimental group. The recombinant vaccinia virus was administered intratumorally, and rhG-CSF or hydroxyurea was administered intraperitoneally 5 times per week starting 3 days before administration of the recombinant vaccinia virus and until sacrifice.

[0150] Experimental Example 7.2. Examination of changes in tumor volume The mice in each group of experimental example 7.1 were sacrificed on the 16th day after drug administration, and tumor volume was measured. As a result, the mice in the positive control group and the mice in the experimental group administered recombinant vaccinia virus and rhG-CSF simultaneously showed an increase of approximately 10 times compared to the initial tumor volume. On the other hand, the mice in the experimental group administered recombinant vaccinia virus and hydroxyurea simultaneously showed an increase of approximately 8 times compared to the initial tumor volume, which was the most suppressed tumor volume observed (Figure 8).

[0151] Experimental Example 7.3. Characterization of antigen-specific cytotoxic T lymphocyte (CTL) activation To determine whether a tumor-specific anticancer effect can be obtained when recombinant vaccinia virus and hydroxyurea are administered simultaneously, the mice in each group of Experimental Example 7.1 were sacrificed on the 16th day, and then the lymphocytes in the spleen were isolated from each group. The isolated lymphocytes were then injected into new normal mice, respectively. Cancer transplantation was performed, and the tumor volume was observed. Specifically, after one week, the mice were inoculated with 5×10 Renca cancer cell line (Korea Cell Line Bank) at 10°C. 6 Cells were allografted and tumor volumes were measured on day 19.

[0152] As a result, tumor growth was significantly suppressed in mice injected with splenocytes collected from mice in the group that received recombinant vaccinia virus and hydroxyurea at the same time. On the other hand, tumor growth was not significantly suppressed in each of the mice injected with splenocytes collected from the remaining groups (Figure 9). These results indicate that in the group that received recombinant vaccinia virus and hydroxyurea at the same time, not only were immune cells such as cytotoxic T cells generated, but adaptive immunity was also activated.

[0153] Experimental Example 8. Mouse renal cell transplantation mice: Recombinant vaccinia virus (Wyeth VV) in Renca (V) tk- ) and the identification of the cancer treatment effects of hydroxyurea Experimental Example 8.1. Generation of mice implanted with mouse kidney cancer cells and drug administration Balb / c mice (female, 7 weeks old) purchased from ORIENT BIO (Busan, Korea) were acclimated for 1 week and then inoculated with Renca cancer cell line (Korea Cell Line Bank) at 5 × 10 6 The cells were transplanted into the 50 mm 3 ~100mm 3 The tumor volume was monitored until it reached 100 mg / kg / day, after which administration of the recombinant vaccinia virus was started. tk- ) barely proliferates in a mouse model implanted with mouse kidney cancer cells.

[0154] The resulting mouse kidney cancer cell-implanted mice were divided into 4 groups (n=4). The group receiving intratumoral administration of saline was set as a negative control group, and the groups receiving hydroxyurea (30 mg / kg) alone and recombinant vaccinia virus (Wyeth VV) were tk- , 1×10 7 A group receiving only pfu of recombinant vaccinia virus (Wyeth VV) was set as a positive control group. tk- , 1×10 7The group receiving simultaneous administration of 1000 mg / kg pfu of recombinant vaccinia virus and hydroxyurea (30 mg / kg) was designated as the experimental group. The recombinant vaccinia virus was administered intratumorally, and hydroxyurea was administered intraperitoneally 5 times per week starting 3 days before administration of recombinant vaccinia virus and until sacrifice.

[0155] Experimental Example 8.2. Examination of changes in tumor volume Tumor volumes were measured on days 0, 4, 10, 15, and 22 after drug administration to mice in each group of Experimental Example 8.1. As a result, the tumor volumes of mice in the positive control group increased by about 11 to 13 times compared to the initial tumor volumes. On the other hand, the tumor volumes of mice in the experimental group increased by about 4 times compared to the initial tumor volumes (Figure 10).

[0156] Experimental Example 8.3. Identification of Tumor-Specific Cytotoxic T Lymphocyte (CTL) Activation To determine whether a tumor-specific anticancer effect can be obtained when recombinant vaccinia virus and hydroxyurea are administered simultaneously, the mice in each group of Experimental Example 8.1 were sacrificed on the 16th day, and then splenocytes and cytotoxic T lymphocytes (CD8+ T cells) were isolated from each group. The isolated splenocytes or cytotoxic T lymphocytes were then injected into new normal mice, respectively. Cancer transplantation was performed, and the tumor volume was observed. Specifically, after one week, the mice were inoculated with 5×10 Renca cancer cell line (Korea Cell Line Bank) at 10°C. 6 Cells were allografted and tumor volumes were measured on days 7, 10, 14, 18 and 21.

[0157] As a result, tumor growth was significantly suppressed in mice injected with splenocytes or T lymphocytes collected from mice in the experimental group. On the other hand, tumor growth was not significantly suppressed in mice injected with splenocytes or T lymphocytes collected from mice in the remaining groups (Figure 11). These results identified that in the group receiving simultaneous administration of recombinant vaccinia virus and hydroxyurea, adaptive immunity with anti-cancer efficacy was activated not only by T lymphocytes but also by other immune cells formed in the spleen (Figures 11 and 12).

[0158] Experimental Example 9. Mouse renal cell transplantation mice: Recombinant vaccinia virus (Wyeth VV) in Renca (VI) tk- ) and the identification of the cancer treatment effects of hydroxyurea Experimental Example 9.1. Generation of mice implanted with mouse kidney cancer cells and drug administration Balb / c mice (female, 8 weeks old) purchased from ORIENT BIO (Busan, Korea) were acclimated for 1 week and then inoculated with Renca cancer cell line (Korea Cell Line Bank) at 5 × 10 6 The cells were transplanted into the 100 mm 3 ~150mm 3 The tumor volume was monitored until it reached 100 mg / kg / day, after which administration of the recombinant vaccinia virus was started. tk- ) barely proliferates in a mouse model implanted with mouse kidney cancer cells.

[0159] The resulting mouse kidney cancer cell-implanted mice were divided into three groups (n=6). The group receiving intratumoral administration of saline was set as a negative control group, and the group receiving intratumoral administration of recombinant vaccinia virus (Wyeth VV) was set as a control group. tk- , 1×10 7 The group receiving pfu of recombinant vaccinia virus (Wyeth VV) was set as a positive control group. tk- , 1×10 7 The group receiving 1000 mg / kg of recombinant vaccinia virus (pfu) and hydroxyurea (30 mg / kg) was set as the experimental group. The recombinant vaccinia virus was administered intratumorally, and hydroxyurea was administered intraperitoneally six times per week starting one day before administration of the recombinant vaccinia virus and until sacrifice.

[0160] Experimental Example 9.2. Examination of changes in tumor volume Mice in each group of experimental example 9.1 were sacrificed on the 22nd day after drug administration, and tumor volumes were measured. As a result, the tumor volumes of the mice in the positive control group were suppressed by about 25% compared to the tumor volumes of the mice in the negative control group. In particular, the tumor volumes of the mice in the experimental group were suppressed by about 37.5% compared to the tumor volumes of the mice in the negative control group, and by about 15% compared to the tumor volumes of the mice in the positive control group (Figure 13).

[0161] Experimental Example 9.3. Identification of the spleen tissue microenvironment The distribution of immune cells in the tumor microenvironment was analyzed when recombinant vaccinia virus and hydroxyurea were co-administered. For the analysis, immunohistochemical staining using diaminobenzidine (DAB) was performed. Specifically, spleens were collected from each group of mice. Spleen tissues were cut at 0.4 μm and dried. The tissues were then washed with PBS and then treated with bovine serum albumin (BSA). The tissues were subjected to treatment with primary antibodies (anti-CD3 antibody (Abcam), anti-CD4 antibody (BD Biosciences), anti-CD8 antibody (BD Biosciences)) diluted in a ratio of 1:50, and the reaction was allowed to proceed overnight at 4°C. The next day, the tissues were washed with PBS and then reacted with secondary antibodies (Dako) at room temperature for 30 minutes. The tissues were washed again with PBS and reacted using the ABC kit (Dako), then H. 2 O 2 Color was developed by the addition of 100 mM NaCl. The tissue was then dehydrated and then mounted.

[0162] As a result, it was determined that CD4+T cells and CD8+T cells were more abundantly distributed in the tumor tissues of the mice in the experimental group (Figure 14). These results indicate that when recombinant vaccinia virus and hydroxyurea are administered simultaneously, CD4+T cells and CD8+T cells in the spleen tissue are differentiated and activated more than when recombinant vaccinia virus alone is administered. In other words, it was determined that when recombinant vaccinia virus and hydroxyurea are administered simultaneously, adaptive immunity is more activated than when recombinant vaccinia virus alone is administered.

[0163] Experimental Example 10. Identification of antigen-specific cytotoxic T lymphocyte (CTL) activation induced by recombinant vaccinia virus (OTS-412) and hydroxyurea in mouse breast cancer cell-implanted mice: 4T1(I) Balb / c mice (female, 7 weeks old) purchased from ORIENT BIO (Busan, Korea) were acclimated for 1 week and then inoculated with 1 × 10 6 The cells were transplanted into the 100 mm 3 ~150mm 3 The tumor volume was observed until it reached 100%, after which administration of recombinant vaccinia virus was started. On the other hand, Wyeth strain-derived recombinant vaccinia virus (OTS-412) hardly grows in mouse models implanted with mouse breast cancer cells. In addition, breast cancer cell line implanted mice are animal models in which metastasis progresses throughout the body, including the lung tissue, and metastasis is generally evaluated by the number of nodules on the surface of the tumor.

[0164] The resulting mouse breast cancer cell-implanted mice were divided into four groups (n = 5). The group receiving intratumoral administration of saline was set as the negative control group, and the group receiving intratumoral administration of recombinant vaccinia virus (OTS-412, 1 × 10 7 The group receiving either 1000 mg / kg pfu or hydroxyurea (30 mg / kg) was set as the positive control group. The group receiving recombinant vaccinia virus and hydroxyurea was set as the experimental group. The recombinant vaccinia virus was administered intratumorally first, then second, on the 7th day after the first administration. Hydroxyurea was administered intraperitoneally once a day starting 3 days before administration of the recombinant vaccinia virus, except on the day of administration of the recombinant vaccinia virus, until 3 days before sacrifice.

[0165] On the 18th day after drug administration, the mice in each group were sacrificed, and blood and spleens were collected therefrom. The distribution of immune cells in blood and splenocytes was analyzed by flow cytometry. The results identified that the distribution of CD4+T cells and CD8+T cells in blood and spleen, which induce tumor immune responses, was highest in the mice in the experimental group. In addition, it was identified that the number of myeloid-derived suppressor cells (MDSCs), which have immune suppressive function, was significantly lower in the mice in the experimental group compared with the mice in the negative control group and positive control group (Figure 15).

[0166] Experimental Example 11. Recombinant vaccinia virus (WR VV) in mouse breast cancer cell-implanted mice: 4T1 (II) tk- ) and identification of the adaptive immunity enhancing effect of hydroxyurea Balb / c mice (female, 10 weeks old) purchased from ORIENT BIO (Busan, Korea) were acclimated for 2 days and then 4T1 cancer cell line (Korea Cell Line Bank) was subcutaneously injected into the left thigh at 1 × 10 6 Two days later, the same number of 4T1 cancer cell lines were subcutaneously transplanted into the right thigh of each mouse. The volume of the cells was 50 mm. 3 ~200mm 3 The tumor was subcutaneously implanted in the right thigh and observed until it reached 1 h, after which administration of the recombinant vaccinia virus was initiated.

[0167] The resulting mouse breast cancer cell-implanted mice were divided into three groups (n=6). The group receiving intratumoral administration of saline was set as a negative control group, and the group receiving intratumoral administration of recombinant vaccinia virus (WR VV) was set as a negative control group. tk- , 1×10 5 The group receiving 100 mg / kg pfu of recombinant vaccinia virus was set as a positive control group, and the group receiving recombinant vaccinia virus and hydroxyurea (90 mg / kg) simultaneously was set as an experimental group. The recombinant vaccinia virus was administered once to the left tumor, and hydroxyurea was administered intraperitoneally six times per week starting from one day before administration of the recombinant vaccinia virus to 14 days after administration, except on the day of administration of the recombinant vaccinia virus.

[0168] The volumes of the tumors implanted subcutaneously in both thighs were measured on days 0, 3, 7, 10, and 14 after drug administration to the mice in each group. As a result, it was determined that the growth of the left tumor volume of the mice in the experimental group was suppressed by about 35% compared to the left tumor volume of the mice in the positive control group (FIG. 16). It was also determined that the growth of the right tumor volume of the mice in the experimental group was suppressed by about 45% compared to the right tumor volume of the mice in the positive control group (FIG. 17). From these results, it was determined that the effect of simultaneous administration of recombinant vaccinia virus and hydroxyurea on the surrounding tumors.

[0169] That is, it was determined that when tumors are locally treated with co-administration of vaccinia virus and hydroxyurea, anti-cancer effects are observed even in tumors to which the virus was not administered.

[0170] Experimental Example 12. Identification of increased cancer selectivity following co-administration of wild-type vaccinia virus (WR) and hydroxyurea in mice bearing mouse kidney cancer cells (I). Balb / c mice (female, 8 weeks old) purchased from ORIENT BIO (Busan, Korea) were acclimated for 1 week and then inoculated with Renca cancer cell line (Korea Cell Line Bank) at 5 × 10 6 The cells were transplanted into the 100 mm 3 ~150mm 3 Tumor volumes were monitored until they reached a volume of 100 μg / mL, after which administration of wild-type Western Reserve strain vaccinia virus (WR) was initiated. On the other hand, wild-type Wyeth strain vaccinia virus has limited proliferation ability in syngeneic mice.

[0171] The resulting mouse kidney cancer cell-implanted mice were divided into three groups (n = 8). The group receiving intraperitoneal administration of saline was set as the negative control group, and the wild-type vaccinia virus (WR, 1 × 10 7A group receiving 100 mg / kg of 10000 pfu each was set as a positive control group, and a group receiving simultaneous administration of wild-type vaccinia virus and hydroxyurea (60 mg / kg) was set as an experimental group. Wild-type vaccinia virus was administered twice into the tumor, and hydroxyurea was administered intraperitoneally six times per week starting from 1 day before administration of wild-type vaccinia virus through 21 days after administration, except on the day of administration of wild-type vaccinia virus.

[0172] Mice from each group were sacrificed on day 22, and tumors were isolated therefrom. Viral proliferation was compared through immunohistochemical staining using diaminobenzidine (DAB). Specifically, tumor tissues were collected from mice from each group. The tumor tissues were cut at 0.4 μm and dried. The tissues were then washed with PBS and then treated with bovine serum albumin (BSA). The tissues were subjected to treatment with primary antibodies (catalog number ABIN1606294, Antibody-Online) diluted at a ratio of 1:50, and the reaction was allowed to proceed overnight at 4°C. The next day, the tissues were washed with PBS and then reacted with secondary antibodies (Alexa 594, catalog number A21205, Invitrogen) at room temperature for 30 minutes. The tissues were washed again with PBS and reacted using an ABC kit (Dako), then H 2 O 2 Color was developed by the addition of 100 mM NaCl. The tissue was then dehydrated and then mounted.

[0173] The results showed that wild-type vaccinia virus was more abundantly distributed in the tumor tissues of the experimental mice ( FIG. 18 ). These results showed that more effective tumor-specific proliferation of wild-type vaccinia virus was observed when hydroxyurea was administered simultaneously with the systemic administration of wild-type vaccinia virus.

[0174] Experimental Example 13. Identification of increased survival and cancer selectivity following co-administration of wild-type vaccinia virus (WR) and hydroxyurea in normal mice (II) Balb / c nu / nu mice (female, 7 weeks old) purchased from ORIENT BIO (Busan, Korea) were acclimated for 2 days and then administered wild-type Western Reserve strain vaccinia virus (WR), whereas wild-type Wyeth strain vaccinia virus has limited proliferation ability in syngeneic mice.

[0175] Mice were divided into two groups (n = 12). 7 The group receiving 1000 mg / kg of wild-type Western Reserve vaccinia virus (pfu) was set as the control group, and the group receiving wild-type Western Reserve vaccinia virus and hydroxyurea (50 mg / kg) simultaneously was set as the experimental group. Wild-type vaccinia virus was administered intranasally once; hydroxyurea was administered intraperitoneally five times per week, starting one day before administration of wild-type vaccinia virus and excluding the days of administration of wild-type vaccinia virus.

[0176] On the eighth day, the mice of the control and experimental groups were sacrificed, and kidney and liver tissues were isolated therefrom. Immunohistochemical staining was performed. Paraffin blocks were prepared, and each block was deparaffinized using xylene and ethyl alcohol. The resulting blocks were subjected to antigen retrieval using a decloaking chamber. Then, a primary antibody (catalog number ABIN1606294, Antibody-Online) was bound to the block, and a FITC-labeled secondary antibody (Alexa 594, catalog number A21205, Invitrogen) was bound to it. Then, observation was performed using a fluorescent microscope.

[0177] As a result, it was determined that the virus was distributed and proliferated in small numbers in the liver and kidney tissues of the experimental mice compared to the liver and kidney tissues of the control mice (Figure 19).

[0178] Experimental Example 14. Determination of absolute neutrophil count (ANC) in mice implanted with mouse kidney cancer cells after co-administration of recombinant vaccinia virus (OTS-412) and hydroxyurea Balb / c mice (female, 7 weeks old) purchased from ORIENT BIO (Busan, Korea) were acclimated for 7 days and then inoculated with 5 × 10 Renca cancer cell lines (Korea Cell Line Bank). 6 The cells were transplanted into the 100 mm 3 ~150mm 3 The tumor volume was monitored until it reached 100%, after which administration of recombinant vaccinia virus was started. On the other hand, recombinant vaccinia virus (OTS-412) hardly grows in mouse kidney cancer cell xenograft model.

[0179] The mice transplanted with mouse kidney cancer cells were divided into four groups (n = 4). The group receiving intratumoral administration of saline was set as the negative control group, and the group receiving intratumoral administration of hydroxyurea (30 mg / kg) was set as the positive control group. Recombinant vaccinia virus (OTS-412, 1 × 10 7 pfu), and a group receiving recombinant vaccinia virus (OTS-412, 1 × 10 7 pfu) and recombinant human granulocyte colony-stimulating factor (rhG-CSF, 75 μg / kg), and a group receiving recombinant vaccinia virus (OTS-412, 1 × 10 7 The group receiving 100 mg / kg of recombinant vaccinia virus (pfu) and hydroxyurea (30 mg / kg) was set as the experimental group. The recombinant vaccinia virus was administered intratumorally, and the second administration was performed 13 days after the first administration. rhG-CSF or hydroxyurea was administered intraperitoneally from 2 days before administration of the recombinant vaccinia virus until the sacrifice of the mice.

[0180] Complete blood counts (CBC) were performed by sacrificing three mice from each group on day 8 after administration of recombinant vaccinia virus, and it was confirmed that the absolute neutrophil count (ANC) in the positive control group was decreased compared to that in the negative control group. In the experimental groups, the absolute neutrophil count was measured to be similar to that of the positive control group only in the group co-administered with recombinant vaccinia virus and hydroxyurea (Figure 20).

[0181] Experimental Example 15. Recombinant Vaccinia Virus (WR VVtk- Measurement of neutrophil counts in mice implanted with mouse kidney cancer cells after simultaneous administration of hydroxyurea and rituximab Balb / c mice (female, 8 weeks old) purchased from Orient Bio (Busan, Korea) were subjected to 1 week of acclimation and then inoculated with Renca cancer cell line (Korea Cell Line Bank) at 5 × 10 6 The cells were transplanted into the 100 mm 3 ~150mm 3 The tumor volume was monitored until it reached 100%, after which administration of the recombinant vaccinia virus was started. tk- ) can grow in a mouse model implanted with mouse kidney cancer cells.

[0182] The mice transplanted with mouse kidney cancer cells were divided into three groups (n=8). The group receiving intratumoral administration of saline was set as the control group. tk- , 1×10 7 pfu, and the group receiving recombinant vaccinia virus (WR VV tk- , 1×10 7 The group receiving simultaneous administration of 1000 mg / kg of recombinant vaccinia virus (pfu) and hydroxyurea (60 mg / kg) was set as the experimental group. The recombinant vaccinia virus was administered intraperitoneally twice, and hydroxyurea was administered intraperitoneally six times per week from 1 day before administration of the recombinant vaccinia virus to the 21st day after administration, except for the day of administration of the recombinant vaccinia virus.

[0183] Eight days after administration of the recombinant vaccinia virus, three mice from each group were sacrificed and a complete blood count (CBC) was performed. The results showed that the blood neutrophil count of the mice administered only the recombinant vaccinia virus was increased compared to the control group, whereas the blood neutrophil count of the mice administered both the recombinant vaccinia virus and hydroxyurea was significantly decreased compared to the control group (Figure 21).

[0184] Experimental Example 16. Determination of absolute neutrophil count (ANC) in mice bearing mouse kidney cancer cells after co-administration of recombinant vaccinia virus (WOTS-418) and hydroxyurea Balb / c mice (female, 8 weeks old) purchased from Orient Bio (Busan, Korea) were subjected to 1 week of acclimation and then inoculated with Renca cancer cell line (Korea Cell Line Bank) at 5 × 10 6 The cells were transplanted into the 100 mm 3 ~150mm 3 The tumor volume was monitored until it reached 100 μg / mL, after which administration of Western Reserve strain vaccinia virus-derived oncolytic virus (WOTS-418) was initiated.

[0185] The mice transplanted with mouse kidney cancer cells were divided into three groups (n = 3). The group receiving intraperitoneal administration of saline was set as a negative control group, and the group receiving intraperitoneal administration of oncolytic virus (WOTS-418, 1 × 10 7 The group receiving 100 mg / kg pfu was set as the positive control group, and the group receiving oncolytic virus and hydroxyurea (30 mg / kg) simultaneously was set as the experimental group. Oncolytic virus was administered intraperitoneally once, and hydroxyurea was administered intraperitoneally daily from 1 day before administration of oncolytic virus to the 2nd day after administration.

[0186] Complete blood counts (CBC) were performed by sacrificing the mice from each group on day 3 after administration of the oncolytic virus, and it was confirmed that the neutrophil counts in the experimental group tended to decrease (Figure 22).

[0187] II. Identification of synergistic anti-cancer effects by co-administration of vaccinia virus and lenalidomide

[0188] Experimental Example 17. Determination of absolute neutrophil count (ANC) in mice bearing mouse kidney cancer cells after administration of lenalidomide Balb / c mice (female, 8 weeks old) purchased from Orient Bio (Busan, Korea) were subjected to 1 week of acclimation and then inoculated with Renca cancer cell line (Korea Cell Line Bank) at 5 × 10 6 After an additional 1-week acclimation period, mice were divided into 3 groups (n=3) and received daily intraperitoneal administration of saline, hydroxyurea (30 mg / kg), and lenalidomide (30 mg / kg), respectively, starting 1 day before and ending 4 days after oncolytic virus administration.

[0189] Complete blood counts (CBC) were performed by sacrificing the mice from each group on day 5 after administration of the oncolytic virus, and it was confirmed that the neutrophil counts of the mice in the lenalidomide-treated group were reduced compared to the saline-treated group, except for one outlier mouse, and that the neutrophil counts of the mice in the hydroxyurea-treated group were significantly lower than those in the saline-treated group (Figure 23).

[0190] Experimental Example 18. Determination of absolute neutrophil count (ANC) in mice implanted with mouse kidney cancer cells after co-administration of recombinant vaccinia virus (WOTS-418) and lenalidomide Balb / c mice (female, 8 weeks old) purchased from Orient Bio (Busan, Korea) were subjected to 1 week of acclimation and then inoculated with Renca cancer cell line (Korea Cell Line Bank) at 5 × 10 6 The cells were transplanted into the 100 mm 3 ~150mm 3 The tumor volume was monitored until it reached 100 mg / kg, after which administration of an anticancer drug derived from Western Reserve vaccinia virus (WOTS-418) was started.

[0191] The mice transplanted with mouse kidney cancer cells were divided into four groups (n = 5). The group receiving intraperitoneal administration of saline was set as a negative control group, and the group receiving intraperitoneal administration of oncolytic virus (WOTS-418, 1 × 10 7The group receiving 100 mg / kg pfu was set as the positive control group, and the group receiving the oncolytic virus and hydroxyurea (30 mg / kg) simultaneously, and the group receiving the oncolytic virus and lenalidomide (30 mg / kg) simultaneously were set as the experimental groups. The oncolytic virus was administered intraperitoneally once, and hydroxyurea or lenalidomide was administered intraperitoneally daily from 1 day before administration of the oncolytic virus to the second day after administration.

[0192] Complete blood counts (CBCs) were performed by sacrificing the mice from each group on day 5 after administration of the oncolytic virus, and it was confirmed that the neutrophil counts of the mice in the experimental group were significantly lower than that of the positive control group that was administered only the oncolytic virus (Figure 24).

[0193] Experimental Example 19. Recombinant vaccinia virus (WR VV) in mice transplanted with mouse kidney cancer cells tk- Identifying the efficacy of cancer treatment after co-administration of lenalidomide and rituximab Balb / c mice (female, 8 weeks old) purchased from Orient Bio (Busan, Korea) were subjected to 1 week of acclimation and then inoculated with Renca cancer cell line (Korea Cell Line Bank) at 5 × 10 6 The cells were transplanted into the 100 mm 3 ~150mm 3 The tumor volume was monitored until it reached 100 mg / kg / day, after which administration of the oncolytic virus was started. On the other hand, Western Reserve strain vaccinia virus-derived oncolytic virus (WR VV) was administered. tk- ) can grow in a mouse model implanted with mouse kidney cancer cells.

[0194] The prepared mouse kidney cancer cell-implanted mice were divided into 6 groups (n=8). The experiment was carried out by dividing the mice as follows: the group receiving intraperitoneal administration of saline was set as the control group, and the group receiving intraperitoneal administration of oncolytic virus (WR VV) was set as the control group. tk- , 1×10 7pfu), a group receiving oncolytic virus and hydroxyurea (60 mg / kg) simultaneously, and a group receiving oncolytic virus and lenalidomide (25 mg / kg) simultaneously. Oncolytic virus was administered intraperitoneally twice, and hydroxyurea or lenalidomide was administered intraperitoneally six times per week from 1 day before administration of oncolytic virus to the 21st day after administration, except on the day of administration of oncolytic virus.

[0195] On day 21, the mice in each group were sacrificed to measure the tumor volume. As a result, it was confirmed that the tumor growth was significantly inhibited in the group administered with oncolytic virus alone compared to the control group (p<0.001). The group administered with oncolytic virus and lenalidomide simultaneously showed a tendency to inhibit tumor growth compared to the group administered with lenalidomide alone. In particular, it was observed that the group administered with oncolytic virus and hydroxyurea simultaneously inhibited tumor growth significantly compared to the group administered with oncolytic virus alone (p<0.05), and it was confirmed that the group administered with lenalidomide and hydroxyurea simultaneously showed higher tumor growth inhibition than the group administered with oncolytic virus and hydroxyurea simultaneously (Figure 25).

[0196] III. Identification of synergistic anti-cancer effects by co-administration of vaccinia virus and palbociclib

[0197] Experimental Example 20. Recombinant vaccinia virus (WR VV) in mice transplanted with mouse kidney cancer cells tk- ) and palbociclib to identify the effects of cancer treatment Experimental Example 20.1. Preparation of mice implanted with mouse kidney cancer cells and drug administration Balb / c mice (female, 8 weeks old) purchased from Orient Bio (Busan, Korea) were subjected to 1 week of acclimation and then inoculated with Renca cancer cell line (Korea Cell Line Bank) at 5 × 10 6 The cells were transplanted into the 100 mm 3 ~150mm 3The tumor volume was monitored until it reached a volume of 100 μg / kg, after which administration of the oncolytic virus was initiated. On the other hand, the Western Reserve strain vaccinia virus-derived oncolytic virus (WOTS-418) can grow in mouse kidney cancer cell xenograft models.

[0198] The mice transplanted with mouse kidney cancer cells were divided into 5 groups (n=5). The experiment was carried out by dividing the mice as follows: a group receiving intraperitoneal administration of saline was set as the control group, a group receiving only oncolytic virus (WR VVtk-, 1×107 pfu), a group receiving simultaneous administration of oncolytic virus and hydroxyurea (60 mg / kg), and a group receiving simultaneous administration of oncolytic virus and palbociclib (50 mg / kg or 100 mg / kg). Oncolytic virus was administered intraperitoneally once, palbociclib was orally administered once a week from 5 days before administration of oncolytic virus, and hydroxyurea was administered intraperitoneally 6 times a week from 1 day before administration of oncolytic virus to the 19th day after administration, except for the day of administration of oncolytic virus.

[0199] Experimental Example 20.2. Identification of changes in tumor volume On the 19th day, the mice in each group of Experimental Example 20.1 were sacrificed to measure the tumor volume. As a result, it was confirmed that the tumor growth was statistically significantly inhibited in the group administered with oncolytic virus alone compared to the control group (p<0.05). It was observed that the group administered with oncolytic virus and hydroxyurea or palbociclib simultaneously inhibited tumor growth significantly compared to the group administered with oncolytic virus alone (p<0.0001) (Figure 26).

[0200] Experimental Example 20.3. Identification of weight change After administration of each drug to the control group and each group in Experimental Example 20.1, the weight of the mice was measured on days 3, 6, 9, 12, 16, and 19. As a result, there was no tendency for weight loss in all of the co-administration groups, and although there was a tendency for continuous weight loss in the group administered with the oncolytic virus alone, the weight on day 19 was maintained at nearly 90% of the weight at the start of administration, and therefore, it was confirmed that there was no concern about safety (Figure 27).

[0201] IV. Identification of synergistic anti-cancer effects by simultaneous administration of vaccinia virus, granulocyte production inhibitor (hydroxyurea) and immune checkpoint inhibitor

[0202] Experimental Example 21. Mouse renal cell transplantation mice: Oncolytic virus (Wyeth virus) in Renca (I) tk- ) Identifying the cancer treatment effects of simultaneous administration of PD-1 inhibitors and hydroxyurea To confirm the additional effect of administration of hydroxyurea when co-administered with an oncolytic virus and a PD-1 inhibitor (CD279, BioXCell) (i.e., an immune checkpoint inhibitor), experiments were performed using mice implanted with mouse kidney cancer cells.

[0203] First, Balb / c mice (female, 8 weeks old) purchased from Orient Bio (Busan, Korea) were acclimated for 7 days, and then Renca cancer cell line (Korea Cell Line Bank) was inoculated at 5 × 10 6 The cells were transplanted into the 200 mm 3 ~300mm 3 The tumor volume was monitored until it reached 100%, and then an oncolytic virus (Wyeth VV) was administered. tk- Oncolytic viruses have limited proliferation potential in allograft models.

[0204] The mice transplanted with mouse kidney cancer cells were divided into 5 groups (n=5). The group receiving intraperitoneal administration of saline was set as a negative control group, the group receiving a mouse PD-1 inhibitor (200 μg / mouse), the group receiving an oncolytic virus (Wyeth VV) tk- , 1×10 7 pfu, and an oncolytic virus (Wyeth virus VV tk- , 1×10 7 The group receiving pfu and PD-1 inhibitors simultaneously was set as a positive control group. tk- , 1×10 7 The group receiving simultaneous administration of pfu), PD-1 inhibitor and hydroxyurea (30 mg / kg) was set as the experimental group, in which the oncolytic virus was administered intratumorally once, the PD-1 inhibitor was administered intraperitoneally once every 2 days on days 14, 16, 18 and 20, and hydroxyurea was administered intraperitoneally 6 times per week.

[0205] The tumor volumes were measured on days 0, 4, 10, 14, 17, and 21 after drug administration to the mice in each group. As a result, it was confirmed that the tumor volumes of the mice in the experimental group were significantly suppressed compared to the tumor volumes of the mice in the positive control group (FIG. 28).

[0206] Experimental Example 22. Mouse renal cell transplantation mice: Oncolytic virus (Wyeth virus) in Renca (II) tk- ), Identifying the cancer treatment effects of simultaneous administration of CTLA4 inhibitors and hydroxyurea To confirm the additional effect of administration of hydroxyurea when co-administered with an oncolytic virus and a CTLA-4 inhibitor (B7-H1, BioXCell) (i.e., an immune checkpoint inhibitor), experiments were performed using mice implanted with mouse kidney cancer cells.

[0207] First, Balb / c mice (female, 8 weeks old) purchased from Orient Bio (Busan, Korea) were acclimated for 7 days, and then Renca cancer cell line (Korea Cell Line Bank) was inoculated at 5 × 10 6The cells were transplanted into the 50 mm 3 ~150mm 3 The tumor volume was monitored until it reached 100%, and then an oncolytic virus (Wyeth VV) was administered. tk- Oncolytic viruses have limited proliferation potential in allograft models.

[0208] The mice transplanted with mouse kidney cancer cells were divided into five groups (n=6). The group receiving intraperitoneal administration of saline was set as a negative control group, the group receiving CTLA-4 inhibitor (150 g / mouse), the group receiving tumor lytic virus (Wyeth VV) tk- , 1×10 7 pfu, and an oncolytic virus (Wyeth virus VV tk- , 1×10 7 The group receiving pfu and CTLA-4 inhibitors simultaneously was set as a positive control group. tk- , 1×10 7 The group receiving simultaneous administration of oncolytic virus (pfu), CTLA-4 inhibitor and hydroxyurea (30 mg / kg) was set as the experimental group, in which the oncolytic virus was administered intratumorally once, the CTLA-4 inhibitor was administered intraperitoneally once every 2 days on days 3, 5, 7 and 9, and hydroxyurea was administered intraperitoneally 6 times per week.

[0209] The tumor volume was measured on days 0, 4, 7, 10, 14, and 17 after drug administration to the mice in each group. As a result, it was confirmed that the tumor volume of the mice in the experimental group was significantly suppressed compared to the tumor volume of the mice in the positive control group (Figure 29). These results confirmed that when the tumor-lytic virus and the immune checkpoint inhibitor (CTLA-4 inhibitor) were administered simultaneously, the additional administration of hydroxyurea exerted an excellent effect of suppressing mouse kidney cancer.

[0210] Experimental Example 23. Mouse renal cell transplantation mice: Oncolytic virus (Wyeth virus) in Renca (III) tk-) Identifying the cancer treatment effects of simultaneous administration of PD-L1 inhibitors and hydroxyurea To confirm the additional effect of administration of hydroxyurea when co-administered with an oncolytic virus and a PD-L1 inhibitor (CD152, BioXCell) (i.e., an immune checkpoint inhibitor), experiments were performed using mice implanted with mouse kidney cancer cells.

[0211] First, Balb / c mice (female, 8 weeks old) purchased from Orient Bio (Busan, Korea) were acclimated for 7 days, and then Renca cancer cell line (Korea Cell Line Bank) was inoculated at 5 × 10 6 The cells were transplanted into the 50 mm 3 ~100mm 3 The tumor volume was monitored until it reached 100%, and then an oncolytic virus (Wyeth VV) was administered. tk- Oncolytic viruses have limited proliferation potential in allograft models.

[0212] The mice transplanted with mouse kidney cancer cells were divided into five groups (n=6). The group receiving intraperitoneal administration of saline was set as the negative control group, the group receiving PD-L1 inhibitor (300 μg / mouse), the group receiving tumor lytic virus (Wyeth VV) tk- , 1×10 7 pfu, and an oncolytic virus (Wyeth virus VV tk- , 1×10 7 The group receiving pfu and PD-L1 inhibitors simultaneously was set as a positive control group. tk- , 1×10 7 The group receiving simultaneous administration of oncolytic virus (pfu), PD-L1 inhibitor and hydroxyurea (30 mg / kg) was set as the experimental group, in which the oncolytic virus was administered intratumorally once, the PD-L1 inhibitor was administered intraperitoneally on days 0, 3, 7, 10, 14, 17 and 21, and hydroxyurea was administered intraperitoneally six times per week.

[0213] Tumor volumes were measured on days 0, 3, 7, 10, 14, 17, and 21 after drug administration to mice in each group. As a result, it was confirmed that the tumor volumes of mice in the experimental group were significantly suppressed compared to the tumor volumes of mice in the positive control group (Figure 30). In particular, when comparing the tumor volumes before the mice were sacrificed, it was confirmed that the tumor volumes of the experimental group were approximately 46% smaller than those of the group that received simultaneous administration of an oncolytic virus and a PD-L1 inhibitor.

[0214] These results confirmed that when an oncolytic virus and an immune checkpoint inhibitor (PD-L1 inhibitor) were administered simultaneously, the additional administration of hydroxyurea exerted a superior effect in suppressing mouse kidney cancer.

[0215] Experimental Example 24. Oncolytic virus (WR VV) in mouse breast cancer cell-implanted mice: 4T1(I) tk- ), Identifying the efficacy of CTLA-4 inhibitors and hydroxyurea in treating cancer Experimental Example 24.1. Preparation of mice implanted with mouse breast cancer cells and drug administration To confirm the additional effect of administration of hydroxyurea when co-administered with an oncolytic virus and a CTLA-4 inhibitor (B7-H1, BioXCell), experiments were performed using mice implanted with mouse breast cancer cells.

[0216] First, Balb / c mice (female, 8 weeks old) purchased from Orient Bio (Busan, Korea) were acclimated for 7 days, and then 1 × 10 4T1 cancer cells (Korea Cell Line Bank) were inoculated into the mice. 6 The cells were transplanted into the 50 mm 3 ~150mm 3 The tumor volume was monitored until it reached 100%, and then an oncolytic virus (WR VV) was administered. tk- Western Reserve strain vaccinia virus-derived oncolytic virus (WR VV) was administered. tk- ) has stronger proliferation ability than oncolytic viruses derived from Wyeth strain vaccinia virus in an allograft model.

[0217] The prepared mouse breast cancer cell-implanted mice were divided into 5 groups (n=5). The group receiving intraperitoneal administration of saline was set as a negative control group, the group receiving CTLA-4 inhibitor (300 μg / mouse), the group receiving tumor lytic virus (WR VV) tk- , 1×10 7 pfu, and an oncolytic virus (Wyeth virus VV tk- , 1×10 7 The group receiving both pfu and CTLA-4 inhibitors was set as a positive control group. tk- , 1×10 7 The group receiving simultaneous administration of oncolytic virus (pfu), CTLA-4 inhibitor and hydroxyurea (30 mg / kg) was set as the experimental group, where the oncolytic virus was administered intraperitoneally twice, the CTLA-4 inhibitor was administered intraperitoneally on days 3, 5, 7 and 9, and hydroxyurea was administered intraperitoneally six times per week.

[0218] Experimental Example 24.2. Identification of changes in tumor volume The tumor volumes of the mice in each group were measured on days 0, 3, 7, 10, and 14 after drug administration. As a result, it was confirmed that the tumor volumes of the mice in the experimental group were significantly suppressed compared to the tumor volumes of the mice in the positive control group (FIG. 31).

[0219] Experimental Example 24.3. Survival Analysis Furthermore, it was confirmed that during the survival period, the survival time and survival rate of the mice in the experimental group were shown to be the highest.The results confirmed that when the oncolytic virus and the CTLA-4 inhibitor were simultaneously administered to mice implanted with mouse breast cancer cells, the additional administration of hydroxyurea showed a significant effect.

[0220] Experimental Example 25. Identification of the cancer therapeutic effects of oncolytic virus (WOTS-418), PD-L1 inhibitor and hydroxyurea in mouse breast cancer cell-implanted mice: 4T1 (II) Experimental Example 25.1. Preparation of mice implanted with mouse breast cancer cells and drug administration To confirm the additional effect of administration of hydroxyurea when co-administered with an oncolytic virus and a PD-L1 inhibitor (CD152, BioXCell), experiments were performed using mice implanted with mouse breast cancer cells.

[0221] First, Balb / c mice (female, 8 weeks old) purchased from Orient Bio (Busan, Korea) were acclimated for 7 days, and then 1 × 10 4T1 cancer cells (Korea Cell Line Bank) were inoculated into the mice. 6 The cells were transplanted into the 50 mm 3 ~100mm 3 The tumor volume was monitored until it reached 100%, and then administration of oncolytic virus (WOTS-418) was initiated. The Western Reserve strain has stronger proliferation ability than the Wyeth strain in the allograft model.

[0222] The prepared mouse breast cancer cell-implanted mice were divided into five groups (n = 6). The group receiving intraperitoneal administration of saline was set as the negative control group, the group receiving PD-L1 inhibitor (300 μg / mouse), the group receiving tumor lytic virus (WOTS-418, 1 × 10 7 pfu) and an oncolytic virus (WOTS-418, 1 × 10 7 The group receiving both 1×10 pfu and PD-L1 inhibitor was set as a positive control group. 7 The group receiving simultaneous administration of oncolytic virus (pfu), PD-L1 inhibitor and hydroxyurea (30 mg / kg) was set as the experimental group, in which the oncolytic virus was intraperitoneally administered twice, the PD-L1 inhibitor was intraperitoneally administered on days 3, 5, 7 and 9, and hydroxyurea was intraperitoneally administered six times per week.

[0223] Experimental Example 25.2. Identification of changes in tumor volume Tumor volumes were measured on days 0, 3, 7, 10, and 14 after drug administration to the mice in each group of Experimental Example 5.1. As a result, it was confirmed that the tumor volumes of the mice in the experimental group were significantly suppressed compared to the tumor volumes of the mice in the positive control group (Figure 32). In particular, when comparing the tumor volumes before the mice were sacrificed, it was confirmed that the tumor volumes of the experimental group were approximately 30% smaller than those of the group that received the oncolytic virus and the PD-L1 inhibitor simultaneously.

[0224] These results confirmed that when an oncolytic virus and an immune checkpoint inhibitor (PD-L1 inhibitor) were administered simultaneously, the additional administration of hydroxyurea produced a synergistic effect in suppressing mouse breast cancer.

[0225] Experimental Example 25.3. Survival Analysis The survival rate of mice in each group of Experimental Example 5.1 for 30 days was analyzed, and the results showed that the survival rate of mice in the experimental group was higher than that of mice in the negative and positive control groups.

[0226] Experimental Example 26. Analysis of survival rate of mice implanted with mouse colorectal cancer cells: CT-26I treated with oncolytic viruses (WR, WOTS-418), PD-L1 inhibitors and hydroxyurea To confirm the safety of co-administration of Western Reserve vaccinia virus (WR), PD-L1 inhibitors and hydroxyurea, we analyzed survival time using mice implanted with mouse colorectal cancer cells.

[0227] First, Balb / c mice (female, 8 weeks old) purchased from Orient Bio (Busan, Korea) were acclimated for 7 days and then inoculated with 1 × 10 6The mice were subcutaneously implanted with 100% vaccinia virus (WVV) and 100% PD-L1 inhibitor. After 7 days, the mice were intraperitoneally administered with oncolytic virus (WR) and PD-L1 inhibitor, and hydroxyurea was administered daily for 5 days. On the other hand, the Western Reserve strain vaccinia virus has stronger proliferation ability than the Wyeth strain vaccinia virus in the allograft model.

[0228] The mice were divided into 5 groups (n=13). The group receiving intraperitoneal administration of saline was set as a negative control group, and the group receiving PD-L1 inhibitor (300 μg / mouse) alone and the group receiving oncolytic virus (WOTS-418) and hydroxyurea (30 mg / kg) simultaneously were set as positive controls. In addition, the group receiving oncolytic virus (WR, 1×10 6 pfu or WOTS-418, 1 × 10 7 The group receiving simultaneous administration of pfu), PD-L1 inhibitor and hydroxyurea was set as the experimental group, in which the oncolytic virus was intraperitoneally administered once, the PD-L1 inhibitor was intraperitoneally administered on days 1, 4, 8 and 11, and hydroxyurea was intraperitoneally administered five times per week.

[0229] Analysis of the survival curves of the mice in each group showed that the survival time of the mice in the experimental group was the longest compared to the mice in the negative control group and the positive control group. These results confirmed that the simultaneous administration of oncolytic virus, immune checkpoint inhibitors and hydroxyurea improved safety.

[0230] Experimental Example 27. Analysis of survival rate in Renca (IV) mice implanted with mouse kidney cancer cells using Western Reserve strain vaccinia virus (WR), CTLA-4 inhibitors, and hydroxyurea To confirm the additional effect of administration of hydroxyurea when Western Reserve vaccinia virus (WR) and a CTLA-4 inhibitor (B7-H1, BioXCell) (i.e., one of the immune checkpoint inhibitors) were co-administered, experiments were performed using mice implanted with mouse kidney cancer cells.

[0231] First, Balb / c mice (female, 8 weeks old) purchased from Orient Bio (Busan, Korea) were acclimated for 7 days, and then Renca cancer cell line (Korea Cell Line Bank) was inoculated at 5 × 10 6 The cells were transplanted into the 30 mm 3 ~50mm 3 The tumor volume was monitored until it reached 100 μg / kg, and then Western Reserve vaccinia virus (WR) was administered. WR has stronger proliferation ability than Wyeth vaccinia virus in the allograft model.

[0232] The mice transplanted with mouse kidney cancer cells were divided into four groups (n = 4). The group receiving intraperitoneal administration of saline was set as a negative control group, and the group receiving intraperitoneal administration of Western Reserve strain vaccinia virus (WR, 1 × 10 5 The group receiving simultaneous administration of 1000 mg / kg of 10000 pfu (1000 mg / kg) and hydroxyurea (30 mg / kg), and the group receiving simultaneous administration of Western Reserve strain vaccinia virus and CTLA-4 inhibitor (150 μg per mouse) were set as positive control groups. The group receiving simultaneous administration of Western Reserve strain vaccinia virus, CTLA-4 inhibitor, and hydroxyurea were set as experimental groups. In this case, Western Reserve strain vaccinia virus was intraperitoneally administered once, CTLA-4 inhibitor was intraperitoneally administered on days 2, 4, 6, and 8, and hydroxyurea was intraperitoneally administered four times per week.

[0233] The tumor volumes of the mice in each group were measured on days 0, 3, and 7 after drug administration. As a result, it was confirmed that the tumor volumes of the mice in the experimental group were significantly suppressed compared to the tumor volumes of the mice in the positive control group (FIG. 33).

Claims

[Claim 1] A pharmaceutical composition for treating cancer, comprising as active ingredients a vaccinia virus and a granulopoiesis inhibitor.

Images