Pharmaceutical composition for preventing or treating cancer

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer, which comprises a reovirus or a biological sample treated with a reovirus as an active ingredient, and a method for preventing or treating cancer using the same. The present invention also relates to a combined use of the pharmaceutical composition and an immune checkpoint inhibitor for cancer treatment.

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition for preventing or treating cancer, and a method for preventing or treating cancer using the same. The present invention also relates to a combined use of the pharmaceutical composition and an immune checkpoint inhibitor for treating cancer.

[0002] This application claims priority to U.S. Provisional Application No. 63 / 027,574, filed May 20, 2020, the entire contents of which are incorporated herein by reference in their entirety in the specification and drawings. [Background technology]

[0003] Cancer is generally defined as a disease, malignant tumor, or neoplasm in which cells divide too rapidly and function abnormally. Standard treatments include surgery to remove the affected cancerous tissue, chemotherapy, and radiation therapy. Certain cancers do not respond or become resistant to such chemotherapy, radiation therapy, and other treatments. Although some human tumors are sensitive to conventional chemo / radiotherapy, various solid tumors (e.g., colon cancer, brain tumors, breast tumors, ovarian tumors, and other tumors) and hematological tumors are known to be refractory to conventional treatment regimens. Recently, immunotherapy, which is different from conventional cancer treatments and aims to treat cancer using the immune system, has attracted attention.

[0004] Unlike conventional anticancer drugs that directly attack the cancer itself, immune anticancer therapy is a method of injecting artificial immune proteins into the body to stimulate the immune system and induce immune cells to selectively attack only cancer cells. It can be broadly divided into passive immune therapy and active immune therapy. Passive immune therapy includes immune checkpoint inhibitors, immune cell therapy, and therapeutic antibodies, among which immune checkpoint inhibitors are drugs that block the activation of immune checkpoint proteins involved in suppressing T cells, activate T cells, and attack cancer cells, and include CTLA-4, PD-1, and PD-L1 inhibitors. In 2016, a PD-L1 antibody drug (Atezolizumab) was approved by the FDA for anticancer treatment, but there is a limitation in that immune checkpoint inhibitors alone have limited therapeutic effects. Active immunotherapy includes cancer treatment vaccines and immune-modulating agents, among which cancer treatment vaccines are made from cancer cells or substances derived from cancer cells and injected into the human body to activate the body's natural defense system. However, the production process of cancer treatment vaccines is complicated, making it difficult to apply to various types of cancer, and since it is a treatment tailored to each individual patient, it places a financial burden on the patient.

[0005] Therefore, it is important to develop more effective approaches, especially combination approaches, to improve cancer prevention and treatment.

[0006] Colon cancer is a malignant tumor formed in the large intestine, and symptoms include changes in bowel habits, diarrhea, constipation, bloody stools, abdominal pain, abdominal distension, fatigue, loss of appetite, or indigestion. To treat colon cancer, the treatment method is determined based on the degree of tissue penetration of cancer cells, and most primary cancers are treated with resection and anticancer chemotherapy or radiation therapy. However, side effects of surgery include pulmonary complications due to general anesthesia after surgery, anastomotic leakage, bleeding, or ileus. Side effects of anticancer chemotherapy include leukopenia and thrombocytopenia, hair loss, nausea, vomiting, and fatigue, and side effects of radiation therapy include pelvic pain, changes in bowel habits, urinary problems, anal pain, diarrhea, or hair loss.

[0007] Even after radical resection, colon cancer will recur in about 20-50% of cases, and this can occur in three forms: local recurrence, distant metastasis, and recurrence involving both local and distant metastasis. Generally, there are many cases of widespread recurrence involving local recurrence and distant metastasis, but when metastasis occurs, it is stage 4 colon cancer, which is classified as the most advanced stage of the disease and is known to have a poor prognosis. Therefore, research is needed into new treatment methods to effectively treat colon cancer while complementing current treatment methods that have many side effects.

[0008] On the other hand, viruses are one of the biological therapeutic agents, and have the concept of targeted therapeutic agents that attack genetic mutations in tumor cells. Anticancer viruses selectively grow in cancer cells and induce tumor necrosis and death.

[0009] Oncolytic viruses are viruses used in cancer treatment, and the anticancer method using them is called oncolytic viral therapy. Research on cancer treatment using wild-type oncolytic viruses is different from conventional gene therapy, which inserts a therapeutic gene into a virus and aims to obtain tumor killing effects mainly through the expression of the therapeutic gene. Research on wild-type oncolytic viruses began after it was discovered that some wild-type viruses have strong intrinsic tumor-destroying ability.

[0010] There have been reports for a long time that natural infection with various kinds of viruses can treat cancer naturally, and since the start of full-scale research into the tumor-specific killing mechanism of wild-type viruses, research into cancer treatment using wild-type Reovirus has progressed to Phase 3 clinical application. Other viruses that have been developed include adenovirus, poliovirus, herpes simplex virus, and vesicular stomatitis virus, and methods to improve the efficacy and stability of viruses are under investigation. Summary of the Invention [Problem to be solved by the invention]

[0011] The inventors have not only confirmed that treatment is possible through oral administration of Reovirus, but also that combination with immune checkpoint inhibitors can exert a remarkable synergistic effect in cancer treatment and enhance the anti-cancer effect, thereby completing the present invention.

[0012] Therefore, an object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises a Reovirus or a biological sample treated with a Reovirus as an active ingredient.

[0013] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating recurrent cancer, which comprises a Reovirus or a biological sample treated with a Reovirus as an active ingredient.

[0014] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art to which the present invention pertains from the following description. [Means for solving the problem]

[0015] To achieve the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising a Reovirus or a biological sample treated with a Reovirus as an active ingredient.

[0016] The present invention also provides a pharmaceutical composition for preventing or treating recurrent cancer, comprising a Reovirus or a biological sample treated with a Reovirus as an active ingredient.

[0017] The present invention also provides a method for preventing or treating cancer or recurrent cancer, comprising administering to an individual a composition comprising, as an active ingredient, Reovirus or a biological sample treated with Reovirus.

[0018] Furthermore, the present invention provides use of a composition comprising, as an active ingredient, a Reovirus or a biological sample treated with a Reovirus for the prevention or treatment of cancer or recurrent cancer.

[0019] Furthermore, the present invention provides use of a composition comprising a Reovirus or a biological sample treated with a Reovirus as an active ingredient for the manufacture of a medicament for preventing or treating cancer or recurrent cancer.

[0020] In one embodiment of the present invention, the cancer may be one or more selected from the group consisting of cervical cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, liver cancer, colon cancer, colorectal cancer, bone cancer, skin cancer, head cancer, neck cancer, skin melanoma, intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, brain tumor, blood cancer, gastric cancer, perianal cancer, breast cancer, fallopian tube cancer, endometrial tumor, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic cancer, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma, and pituitary adenoma, but is not limited thereto.

[0021] In another embodiment of the present invention, the pharmaceutical composition may further comprise, but is not limited to, an immune checkpoint inhibitor.

[0022] In yet another embodiment of the present invention, the immune checkpoint inhibitor may be one or more selected from the group consisting of a PD-L1 inhibitor, a PD-1 inhibitor, and a CTLA-4 inhibitor, but is not limited thereto.

[0023] In yet another embodiment of the present invention, the reovirus, or a biological sample treated with the reovirus; and The immune checkpoint inhibitors may be administered simultaneously, separately, or sequentially, but are not limited thereto.

[0024] In yet another embodiment of the present invention, the reovirus or a biological sample treated with the reovirus may be administered orally, but is not limited thereto.

[0025] In yet another embodiment of the present invention, the pharmaceutical composition can increase the infiltration of CD8+ T cells into tumors, but is not limited thereto.

[0026] In yet another embodiment of the present invention, the immune checkpoint inhibitor may be administered parenterally, but is not limited thereto.

[0027] In yet another embodiment of the present invention, the pharmaceutical composition may be administered simultaneously, separately or sequentially with the immune checkpoint inhibitor, but is not limited thereto.

[0028] In yet another embodiment of the present invention, the biological sample may be prepared by applying an effective amount of reovirus to an ex vivo biological sample to kill cancer cells, but is not limited thereto.

[0029] In yet another embodiment of the present invention, the biological sample may be, but is not limited to, a bone marrow sample, an adipose-derived stem cell sample, or a blood sample. Effect of the Invention

[0030] The present inventors have not only confirmed the cancer therapeutic effect of reovirus alone, but also confirmed that when reovirus is orally administered in combination with an immune checkpoint inhibitor, the cancer therapeutic effects such as reduction in tumor volume, inhibition of tumor growth rate, and increase in survival rate are significantly increased in colon cancer, skin cancer, kidney cancer, etc., and in particular, when reovirus is combined with PD-1 antibody and CTLA-4 antibody, not only can cancer be put into complete remission, but also cancer recurrence can be suppressed. Therefore, it is expected that the reovirus of the present invention can be usefully used as a therapeutic agent and therapeutic adjuvant for various cancers. [Brief description of the drawings]

[0031] [Figure 1] This figure shows the process of creating an azoxymethane (AOM) / dextran sulfate sodium (DSS)-induced colorectal cancer animal model and the administration schedule of reovirus (RC402) and anti-PD-L1 antibody. [Diagram 2] FIG. 1 shows the Disease Activity Index (DAI) criteria for measuring the severity of colon cancer-related disease in colon cancer animal models. [Diagram 3] This figure shows the results of measuring the Disease Activity Index (DAI) in the normal control group, the AOM / DSS-induced colon cancer group (Vehicle), the reovirus oral administration group (RC402), the immune checkpoint inhibitor intraperitoneal administration group (αPD-L1), and the reovirus / immune checkpoint inhibitor combined administration group (RC402+αPD-L1). [Figure 4] This figure shows the results of measuring the survival rate of the normal control group, the AOM / DSS-induced colon cancer group (Vehicle), the reovirus oral administration group (RC402), the immune checkpoint inhibitor intraperitoneal administration group (αPD-L1), and the reovirus / immune checkpoint inhibitor combined administration group (RC402+αPD-L1). [Diagram 5] Figure 1 shows the administration schedule of reovirus (RC402) and anti-PD-L1 antibody in an animal model of colon cancer using CT26 colon cancer cell transplants. [Figure 6] FIG. 6 shows the results of measuring survival rates according to the administration schedule of FIG. 5. [Figure 7] This shows the administration schedule of reovirus (RC402) and anti-PD-L1 antibody in an animal model of skin cancer using B16F10 melanoma cell transplants. [Figure 8] FIG. 8 shows the results of measuring tumor volume according to the administration schedule of FIG. 7. [Figure 9] FIG. 8 shows the results of measuring survival rates according to the administration schedule of FIG. 7. [Figure 10]FIG. 1 shows the schedule for administering reovirus alone in an animal model of kidney cancer using RENCA kidney cancer cell transplants. [Figure 11] FIG. 11 shows the results of measuring tumor volume and growth rate according to the administration schedule of FIG. 10. [Figure 12] FIG. 1 shows the schedule for administration of reovirus alone in an animal model of colon cancer using transplanted MC38 colon adenocarcinoma cells. [Figure 13] FIG. 13 shows the results of measuring tumor volume and growth rate according to the administration schedule of FIG. 12. [Figure 14] FIG. 1 shows the percentage of CD8+ T cells that infiltrated into tumors following administration of reovirus (RC402) in an MC38-xenograft colon cancer animal model. [Figure 15] FIG. 1 shows the results of observing the degree of CD8+ T cell infiltration in tumors and lymph nodes obtained from an MC38-implanted colon cancer animal model by administration of reovirus (RC402) using a fluorescent microscope after immunohistochemical staining and DAPI staining. [Figure 16] FIG. 1 shows the results of immunohistochemical staining and DAPI staining followed by fluorescence microscopy to examine the infiltration of CD8+ T cells into tumors obtained from an MC38-implanted colon cancer animal model by administration of reovirus (RC402). [Figure 17] FIG. 1 shows the administration schedule of reovirus (RC402) and anti-PD-1 antibody and / or anti-CTLA-4 antibody in an animal model of colon cancer using transplanted CT26 colon cancer cells. [Figure 18] FIG. 18 shows the results of measuring tumor volume and growth rate according to the administration schedule of FIG. 17. [Figure 19] FIG. 1 shows the results of immunohistochemical staining and flow cytometry analysis of the degree of CD8+ T cell infiltration in tumors obtained from a CT26-implanted colorectal cancer animal model following administration of reovirus (RC402) and anti-PD-1 antibody and / or anti-CTLA-4 antibody. [Figure 20]FIG. 1 shows the results of re-transplanting CT26 after complete remission of the tumor in order to confirm the preventive effect on CT26 colon cancer recurrence. [Figure 21] This figure shows the percentage of memory T cells (CD44+ CD62+ CD8+) in the spleens harvested from CT26-reimplanted mice after complete remission, as determined by flow cytometry analysis. [Figure 22] FIG. 1 shows the percentage of CD8+ T cells expressing the T cell receptor as determined by flow cytometry analysis using tetrameric synthetic MHC complexes loaded with MC38-specific antigenic peptides. [Figure 23] FIG. 1 shows PD-1 expression in CD8+ cells in the mesenteric lymph nodes and blood of an MC38 colon cancer model following administration of reovirus (RC402). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The present invention provides a pharmaceutical composition for preventing or treating cancer or recurrent cancer, comprising a Reovirus or a biological sample treated with a Reovirus as an active ingredient.

[0033] The present invention will be described in detail below.

[0034] As used herein, the term "reovirus" refers to any virus that has a double-stranded segmented RNA genome and is classified in the Reoviridae family. The reovirus virion is 60-80 nm in diameter and possesses two concentric capsid shells. The genome is composed of 10-12 discontinuous segments of double-stranded RNA with a total genome size of 16-27 kbp, with each RNA segment having a different size.

[0035] In the present invention, the reovirus includes naturally occurring reoviruses as well as modified or recombinant reoviruses. The reovirus is said to be "naturally occurring" if it can be isolated from nature and has not been artificially altered by humans. For example, the reovirus can be derived from a "field source," i.e., a human infected with the reovirus.

[0036] The reovirus can be modified but can lytically infect mammalian cells that have an active ras pathway. The reovirus can also be pretreated chemically or biochemically (e.g., by treatment with a protease such as chymotrypsin or trypsin) before administration to growing cells. Pretreatment with a protease removes the viral outer membrane or capsid and can increase the infectivity of the virus. The reovirus can be coated in liposomes or micelles; for example, virions can be treated with chymotrypsin in the presence of micelle-forming concentrations of alkyl sulfate detergent to generate new infectious subviral particles.

[0037] In the present invention, the reovirus may be, but is not limited to, a wild-type reovirus or an attenuated reovirus.

[0038] The attenuated reovirus comprises infectious, replication-competent reovirus virions lacking detectable reovirus sigma 1 capsid protein due to a reovirus genome lacking the S1 gene of wild-type reovirus. Thus, the attenuated reovirus results from the surprising observation that a mutated reovirus lacking detectable reovirus sigma 1 capsid protein unexpectedly retains the ability to productively infect target tumor cells while advantageously avoiding cytopathic effects on non-malignant cells. As previously discussed, prior to the present disclosure, reovirus particles lacking sigma 1 were understood to be non-infectious.

[0039] In certain embodiments, the attenuated reovirus can include a mutated reovirus S4 gene. The reovirus wild-type S4 gene encodes a reovirus capsid sigma 3 polypeptide involved in virion processing during replicative reovirus infection of a host cell.

[0040] In certain embodiments, the attenuated reovirus can comprise a mutated reovirus S4 gene that includes one or more mutations in the genomic sequence encoding a reovirus sigma3 polypeptide compared to a wild-type S4 gene sequence.

[0041] The attenuated reoviruses are unexpectedly infectious, even though they lack detectable sigma 1 capsid protein. As previously described, we show that sigma 1 is involved in the binding and attachment of reoviruses to cells through cell surface sialic acid residues during the early stages of viral replication and infection. Despite lacking detectable sigma 1, the attenuated reoviruses described herein are capable of host cell entry and cytolytic viral replication. The attenuated reoviruses also exhibit the surprising property of inducing reduced (i.e., statistically significant) levels of one or more cytopathic effects on non-malignant cells compared to the levels of cytopathic effects exhibited by naturally occurring non-attenuated reoviruses on non-malignant cells.

[0042] The attenuated reovirus can be derived from any reovirus, meaning a member of the reovirus family, including reoviruses with different tropisms, which can be obtained from a variety of sources.

[0043] In certain embodiments, the reovirus may be a mammalian reovirus, and in other embodiments, a human reovirus. In other embodiments (e.g., for use in animal models relevant to human disease or for veterinary applications), the attenuated reovirus can be derived from one or more reoviruses that exhibit tropism for cells of other mammalian species, including non-human primates (e.g., chimpanzees, gorillas, macaques, monkeys, etc.), rodents (e.g., mice, rats, Kerbil mice, hamsters, rabbits, guinea pigs, etc.), dogs, cats, common domestic animals (e.g., cows, horses, pigs, goats), etc., or, alternatively, a reovirus with a distinct tropism (e.g., avian reovirus) can be used.

[0044] Attenuated reoviruses can be derived by other methodologies, including the generation and identification of sigma 1-deficient and / or sigma 1-defective mutants by molecular biological approaches (and, in certain embodiments, additionally or alternatively, the generation and identification of sigma 3-deficient and / or sigma 3-defective mutants), isolation of naturally occurring sigma 1-deficient and / or sigma 1-defective mutants and / or sigma 3 mutants, and / or artificial induction of such sigma 1 (and / or sigma 3) mutants by chemical, physical and / or genetic techniques (e.g., selective recombination of reovirus genes in productively infected host cells).

[0045] The attenuated reovirus comprises infectious, replication-competent reovirus virions (i.e., virus particles containing the viral genome, core protein and protein coat) that are deficient in the wild-type reovirus S1 gene and, consequently, lack detectable reovirus sigma-1 capsid protein.

[0046] In certain embodiments, the attenuated reovirus is deficient in the wild-type reovirus S4 gene and expresses a mutated reovirus sigma 3 capsid protein. As is known in the relevant art, an infectious, replication competent reovirus, upon introduction into a suitable host cell under appropriate conditions and for a sufficient period of time, is capable of binding to and internalizing the host cell and then, upon release from the host cell, directing the replication of the reovirus genome and the biosynthesis of reovirus structural proteins in a manner that permits the assembly of complete progeny reoviruses that are capable of productively infecting other host cells to perpetuate the viral replication cycle.

[0047] Attenuated reoviruses for cancer treatment are described, for example, in US Application Publication Nos. US2009 / 0214479 and US2009 / 0104162, each of which is incorporated by reference in its entirety.

[0048] The human reovirus consists of three serotypes: type 1 (Lang strain or T1L), type 2 (Jones strain, T2J), and type 3 (Dearing strain, T3D or Abney strain, T3A). The three serotypes can be easily identified based on neutralization and hemagglutinin inhibition assays.

[0049] In the present invention, the reovirus can be obtained using baby hamster kidney (BHK) cells (e.g., BHK-21 cells), but is not limited thereto. In the term "BHK-21 cells" used herein, BHK is an abbreviation of Baby Hamster Kidney. BHK cells were originally isolated by polyoma transformation of hamster cells and can be used as a substrate for virus transmission and virus-mediated expression for vaccines. BHK cells are also useful as a host cell line for stable expression of various recombinant proteins. The BHK-21 (BHK Strain 21) cell line was derived from the neonatal hamster kidneys of five unsexed, one-day-old hamsters in March 1961 by IA Macpherson and MGP Stoker. The hamsters used to generate the BHK-21 cells are commonly known as golden hamsters (Mesocricetus auratus).

[0050] As used herein, the term "reovirus-treated biological sample" refers to any biological sample obtained from an individual, cell line, tissue culture, or other source of cells, such as adult stem cells (adipose-derived stem cells, bone marrow stem cells) or umbilical cord blood stem cells. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. For example, adipose-derived stem cells can be pretreated with an oncolytic virus and administered to a cancer patient.

[0051] In the present invention, the biological sample may be prepared by applying an effective amount of reovirus to the biological sample ex vivo to kill multiple cancer cells, but is not limited thereto.

[0052] In the present invention, the biological sample may be, but is not limited to, a bone marrow sample, an adipose-derived stem cell sample, or a blood sample.

[0053] The pharmaceutical composition of the present invention may be administered orally, but is not limited thereto. Oral administration can minimize side effects caused by repeated administration. Specifically, when the same amount of virus is administered intravenously 2 to 3 times continuously, side effects such as cytokine storm may become fatal. In contrast, when the same or more amount of virus is administered orally repeatedly, such side effects may not occur.

[0054] Furthermore, the pharmaceutical composition for oral administration of the present invention can be taken in its naked form without the need for enteric coating for intestinal administration.

[0055] Moreover, the pharmaceutical composition for oral administration of the present invention has excellent virus delivery efficiency into cancer, and can maximize the anti-cancer effect. The efficiency of virus delivery into cancer is considered to be the most important factor that determines the anti-cancer effect. Attempts have been made to treat cancer through intravenous administration of anti-cancer viruses, but it has been reported that such an administration method results in the virus being largely removed by the anti-viral immune system in the blood, which significantly reduces the delivery efficiency into the cancer. In contrast, in the case of oral administration, the anti-viral effect of the immune system is relatively low, and therefore it is expected that the virus delivery efficiency will be good.

[0056] In the present invention, the pharmaceutical composition for oral administration may be administered in combination with an immune checkpoint inhibitor. 80-90% of metastatic colorectal cancers are microsatellite stable (MSS) type that do not respond to immune checkpoint inhibitors, and there is an urgent need for a combination therapeutic agent that enhances and maximizes the reactivity. Since reovirus has an excellent priming effect that converts such carcinomas into responsive carcinomas, when the pharmaceutical composition of the present invention is used in combination with an immune checkpoint inhibitor, it may be a revolutionary turning point in cancer treatment through a synergistic effect that maximizes the viral transmissibility through oral administration and dramatically enhances the reactivity of the immune checkpoint inhibitor.

[0057] The term "immune checkpoint inhibitors" as used herein refers to substances that totally or partially suppress, interfere with or modulate one or more immune checkpoint proteins, also referred to as immune anticancer drugs. Immune checkpoint proteins regulate the activation or function of T cells. Numerous immune checkpoint proteins are known, such as PD-1, PD-L1 and CTLA-4 (Nature Reviews Cancer 12:252-264,2012). These proteins are involved in co-stimulatory or inhibitory interactions in T cell responses. Immune checkpoint inhibitors include and can be derived from antibodies.

[0058] In the present invention, the immune checkpoint inhibitor may be, but is not limited to, a drug that specifically binds to PD-1 (Programmed cell death protein 1). In a specific example, the drug that specifically binds to PD-1 may be an anti-PD-1 antibody, and examples of the anti-PD-1 antibody may be selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, INCMGA00012, AMP-224, and AMP-514, but are not limited thereto.

[0059] In the present invention, the immune checkpoint inhibitor may be, but is not limited to, a drug that specifically binds to PD-L1 (Programmed Death-Ligand 1). In a specific example, the drug that specifically binds to PD-L1 may be an anti-PD-L1 antibody, and examples of the anti-PD-L1 antibody may be selected from the group consisting of atezolizumab, avelumab, durvalumab, envafolimab, cosibelimab, AUNP12, CA-170, and BMS-986189, but are not limited thereto.

[0060] In the present invention, the immune checkpoint inhibitor may be, but is not limited to, a drug that specifically binds to CTLA-4 (cytotoxic T-lymphocyte-associated protein 4). In a specific example, the drug that specifically binds to CTLA-4 may be an anti-CTLA-4 antibody, and examples of the anti-CTLA-4 antibody may be, but are not limited to, ipilimumab, tremelimumab, etc.

[0061] The term "antibody" as used herein refers to a substance that specifically binds to immune checkpoint proteins such as PD-1, PD-L1, and CTLA4, and exhibits inhibitory activity against immune checkpoints. The scope of the antibody includes not only complete antibodies, but also antigen-binding sites of antibody molecules. The antibody also includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and the like.

[0062] The term "cancer" as used in the present invention refers to a disease related to the regulation of cell death, which occurs when the balance of normal cell apoptosis is broken and cells proliferate excessively. Such abnormally proliferating cells may invade surrounding tissues and organs to form a mass, which may cause the destruction or deformation of normal structures in the body, and such a condition is collectively called a tumor.

[0063] Generally, tumors can be divided into benign and malignant tumors. Malignant tumors grow much faster than benign tumors, invade surrounding tissues, and cause metastasis, posing a threat to life. Such malignant tumors are usually called "cancer."

[0064] In the present invention, the cancer may be one or more selected from the group consisting of cervical cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, liver cancer, colon cancer, colorectal cancer, bone cancer, skin cancer, head cancer, neck cancer, skin melanoma, intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, brain tumor, blood cancer, gastric cancer, perianal cancer, breast cancer, fallopian tube cancer, endometrial tumor, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic cancer, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma, and pituitary adenoma, but is not limited thereto.

[0065] In the present invention, the term "synergy" means that the effect produced when the components are administered in combination is greater than the sum of the effects produced when each component is administered alone as a single component, as described in the literature (Chou and Talalay, Adv. Enzyme. Regul., 22:27-55, 1984).

[0066] As used herein, the term "administered in combination" refers to different components administered to a subject together, meaning that each component can be administered at the same time or in any order or sequentially at different times to achieve the desired therapeutic effect.

[0067] In the present invention, the pharmaceutical composition may include, but is not limited to, a reovirus and a PD-L1 inhibitor as active ingredients, and the pharmaceutical composition including the reovirus may be administered in combination with a PD-L1 inhibitor, but is not limited to this.

[0068] In the present invention, the pharmaceutical composition may contain a reovirus and a PD-1 inhibitor as active ingredients, but is not limited thereto. Also, in the present invention, the pharmaceutical composition containing a reovirus may be administered in combination with a PD-1 inhibitor, but is not limited thereto.

[0069] In the present invention, the pharmaceutical composition may contain, but is not limited to, a reovirus, a PD-1 inhibitor, and a CTLA-4 inhibitor as active ingredients, and in the present invention, the pharmaceutical composition containing a reovirus may be administered in combination with a PD-L1 inhibitor and a CTLA-4 inhibitor, but is not limited to this.

[0070] In the present invention, the pharmaceutical composition for oral administration may be administered simultaneously, separately, or sequentially with the immune checkpoint inhibitor.

[0071] The immune checkpoint inhibitor can be administered to an individual by various routes. All modes of administration are predictable, for example, oral administration, intranasal administration, intratumoral administration, transbronchial administration, intraarterial injection, intravenous injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. The daily dose can be administered once or several times a day.

[0072] In the present invention, the reovirus may include, but is not limited to, a heterologous gene for cancer therapy. The term "heterologous gene" is used to mean accepting any gene not found in the viral genome. The heterologous gene may be an allelic variant of a wild-type gene or a mutant gene. The heterologous gene for cancer therapy may be inserted into an essential region or a non-essential region in the reovirus gene to increase anti-cancer activity.

[0073] The foreign gene may be operably linked to a control sequence that allows the foreign gene to be expressed in cells in vivo. Thus, the virus of the present invention may be used to deliver a foreign gene / genes to cells in vivo where the foreign gene can be expressed. The gene usually encodes a protein that can increase the tumor-destroying properties of the virus. The gene may be a cytotoxin itself or may encode a protein that can promote / enhance an anti-tumor immune response.

[0074] Exogenous genes include any gene that can be inserted into replication-competent oncolytic viruses or traditional replication-incompetent viral vectors to treat cancer.

[0075] The term "therapeutic gene" as used herein is considered to describe any broad range of genes whose expression influences a favorable outcome, e.g., an anti-cancer effect. The reoviruses of the invention may contain one or more sequences of interest that encode a therapeutic gene. Therapeutic genes can have pharmacological or prophylactic activity when properly administered to a patient, particularly a patient suffering from a disease or disease state or a patient to be protected from such a disease or disease state.

[0076] Such pharmacological or preventive activity means that it is expected to be associated with a beneficial effect on the course or symptoms of said disease or said condition. The sequence of interest may be homologous or heterologous to the target cell into which it is introduced and encodes all or part of a polypeptide, in particular a therapeutic or preventive polypeptide that confers therapeutic or preventive properties. Polypeptide is understood to be any translational product of a polynucleotide, regardless of size, and whether or not it is glycosylated, and includes peptides and proteins. Therapeutic polypeptides include polypeptides that can compensate for defective or deficient proteins in an animal or human organism or those that act through a toxic effect to limit or eliminate harmful cells from the body. They may also be immunity-conferring polypeptides that act as endogenous antigens to elicit humoral or cellular responses, or both, including, but not limited to, drug-sensitizing genes, proapoptotic genes, cytostatic genes, cytotoxic genes, tumor suppressor genes, antigenic genes, anti-angiogenic genes, cytokine genes, and the like.

[0077] The active ingredients of the present invention are administered to an individual in a pharma- ceutically effective amount.

[0078] As used herein, the term "pharmacologically effective amount" means an amount sufficient to treat a disease at a reasonable benefit-risk ratio applicable to any medical treatment, which can be determined by factors including the type and severity of the disease, activity of the drug, sensitivity to the drug in an individual, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical art.

[0079] An effective amount of a virus is the dosage required for a sufficient time to alleviate, improve, mitigate, ameliorate, stabilize, inhibit the spread of, inhibit or slow the progression of, or cure a disease. For example, an effective amount can be an amount sufficient to achieve the effect of reducing or destroying the number of cancer cells, or reducing or destroying the number of cells chronically infected with the virus, or inhibiting the growth and / or proliferation of such cells.

[0080] The effective amount can vary depending on many factors such as the pharmacokinetic properties of the virus, the method of administration, the age, health condition and weight of the patient, the nature and extent of the disease state, the number of treatments and the current treatment form, and can also vary depending on the virulence and potency of the virus. Those skilled in the art can adjust the appropriate amount based on the above factors. The virus can be administered in an appropriate amount as needed, depending on the initial clinical response of the patient. The effective amount of the virus can be determined empirically and can be determined by the maximum amount of the virus that can be safely administered and the minimum amount of the virus that induces a favorable result.

[0081] The concentration of virus administered can vary depending on the virulence of the virus strain to be administered and the characteristics of the target cell.

[0082] In the present invention, the reovirus is 2 ~10 15 The reovirus may contain 10 PFU. 2 ~10 15 TCID 50 , 10 3 ~10 14 TCID 50 , 10 3 ~10 13 TCID 50 , 10 3 ~10 12 TCID 50 , 10 4 ~10 12 TCID 50 , 105 ~10 11 TCID 50 , 10 5 ~10 10 TCID 50 , 10 6 ~10 10 TCID 50 , or 10 7 ~10 10 TCID 50 It may be included in.

[0083] An effective amount of the virus may be administered repeatedly depending on the effect of the initial therapeutic regimen. Generally, the administration is periodically performed while monitoring any reaction. Those skilled in the art can easily understand that lower or higher doses than those indicated above may be administered depending on the administration schedule and the selected route.

[0084] In one embodiment of the present invention, it was demonstrated that administration of reovirus alone or in combination with an anti-PD-L1 antibody significantly alleviated the severity of colon cancer-related disease in an animal model of colon cancer (Figures 2 and 3).

[0085] In another example of the present invention, it was confirmed that administration of reovirus alone or in combination with an anti-PD-L1 antibody in an animal model of colon cancer significantly increased the survival rate of the animal model (Figure 4).

[0086] In another example of the present invention, it was confirmed that administration of reovirus alone or in combination with an anti-PD-L1 antibody in an animal model of colon cancer significantly increased the survival rate of the animal model (Figure 6).

[0087] In yet another example of the present invention, it was confirmed that the combined administration of reovirus and anti-PD-L1 antibody significantly reduced the volume of skin cancer in an animal model of skin cancer (Figure 8).

[0088] In yet another example of the present invention, it was confirmed that the combined administration of reovirus and anti-PD-L1 antibody in an animal model of skin cancer significantly increased the survival rate of the animal model (Figure 9).

[0089] In yet another example of the present invention, it was confirmed that the combined administration of reovirus and anti-PD-L1 antibody in an animal model of renal cancer significantly reduced the volume and growth rate of renal cancer (Figure 11).

[0090] In yet another example of the present invention, when reovirus was orally administered alone to an animal model of colon cancer, it was confirmed that the effects of reducing the volume and growth rate of colon cancer were significantly increased (Figure 13).

[0091] In still another example of the present invention, it was confirmed that reovirus alone was orally administered to an animal model of colon cancer, resulting in a significant increase in the percentage of CD8+ T cells that had infiltrated into the tumor (FIGS. 14 to 16).

[0092] In yet another example of the present invention, it was confirmed that the combined administration of reovirus and anti-PD-1 antibody and / or anti-CTLA-4 antibody to an animal model of colon cancer significantly increased the effects of reducing the volume and growth rate of colon cancer (Figure 18).

[0093] In yet another example of the present invention, it was found that the combination of reovirus and anti-PD-1 antibody and / or anti-CTLA-4 antibody in an animal model of colon cancer significantly increased the percentage of CD8+ T cells that infiltrated into the tumor (Figure 19).

[0094] In yet another embodiment of the present invention, retransplantation of CT26 after complete remission of colon cancer using reovirus, anti-PD-1 antibody, and anti-CTLA-4 antibody confirmed that CT26 colon cancer did not recur (Figure 20).

[0095] In yet another example of the present invention, retransplantation of CT26 mice after complete remission of colon cancer using reovirus, anti-PD-1 antibody, and anti-CTLA-4 antibody resulted in a significant increase in the percentage of memory T cells (CD44+ CD62+ CD8+) and CD8+ T cells in the spleen ( FIG. 21 ).

[0096] In yet another example of the present invention, it was confirmed that administration of reovirus increased PD-1 expression in CD8+ cells in the mesenteric lymph nodes of a colon cancer model (Figure 23).

[0097] Based on the above results, it is expected that the pharmaceutical composition of the present invention can be used for the prevention and treatment of cancer or recurrent cancer.

[0098] The pharmaceutical composition according to the present invention may be formulated in the form of a single composition, or in the form of separate compositions, including the reovirus, the reovirus-treated biological sample, and the immune checkpoint inhibitor. Preferably, the compositions may be formulated in the form of separate compositions. Methods for formulating these may be any technique commonly known in the art.

[0099] The reovirus of the present invention; the reovirus-treated biological sample or the total effective amount of the immune checkpoint inhibitor may be administered in a single dose or in a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may vary in the content of the components (the PD-L1 inhibitor, PD-1 inhibitor, and / or CTLA-4 inhibitor of the present invention) depending on the severity and / or purpose of the disease, and may be administered in an effective dose of 0.01 μg to 10,000 mg, preferably 0.1 μg to 1,000 mg, several times a day. However, the effective dose of the pharmaceutical composition is determined in consideration of various factors such as the formulation method, administration route, and number of treatments, as well as the patient's age, weight, health condition, sex, severity of the disease, diet, and excretion rate. Therefore, a person having ordinary skill in the art can determine an appropriate effective dose of the composition of the present invention in consideration of these points. The pharmaceutical composition according to the present invention is not particularly limited in its dosage form, administration route and administration method, so long as it exhibits the effects of the present invention.

[0100] The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions, such as one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials and controlled release additives.

[0101] The pharmaceutical composition according to the present invention can be formulated by a conventional method into the form of a powder, granule, sustained-release granule, enteric-coated granule, liquid, eye drop, elixir, emulsion, suspension, spirit, troche, aromatic perfume, lemonade, tablet, sustained-release tablet, enteric-coated tablet, sublingual tablet, hard capsule, soft capsule, sustained-release capsule, enteric-coated capsule, pill, tincture, soft extract, dry extract, fluid extract, injection, capsule, perfusion solution, plaster, lotion, paste, spray, inhalant, patch, sterile injection solution, or external preparation such as aerosol, and the external preparation may have the form of a cream, gel, patch, spray, ointment, plaster, lotion, liniment, paste, or cataplasm.

[0102] Carriers, excipients and diluents that may be included in pharmaceutical compositions according to the invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0103] When the formulation is made, it is prepared by using a diluent or excipient such as a commonly used filler, extender, binder, wetting agent, disintegrant, surfactant, etc.

[0104] Additives for the tablets, powders, granules, capsules, pills and lozenges according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, iodine, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethylcellulose (HPMC), HPMC1928, HPMC2208, HPMC2906, HPMC2910, propylene glycol, casein, calcium lactate, Primogel, etc.; gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, ... Binders such as sodium cellulose acetate, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch paste, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, and polyvinylpyrrolidone can be used; disintegrants such as hydroxypropylmethylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, L-hydroxypropylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelling starch, gum arabic, amylovectin, pectin, sodium polyphosphate, ethylcellulose, white sugar, magnesium aluminum silicate, D-sorbitol solution, and hard anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium, kaolin, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, DL-leucine, and hard silicic anhydride can be used.

[0105] Examples of additives that can be used in the liquid preparation according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, and sodium carboxymethylcellulose.

[0106] The syrup according to the present invention may contain a solution of sucrose, other sugars or sweeteners, and may contain, if necessary, flavorings, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, thickening agents, etc.

[0107] In the emulsion of the present invention, purified water can be used, and emulsifiers, preservatives, stabilizers, fragrances, etc. can be used as necessary.

[0108] The suspension according to the present invention may contain suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC1828, HPMC2906, and HPMC2910, and may contain surfactants, preservatives, stabilizers, colorants, and fragrances as necessary.

[0109] Injections according to the present invention may contain solvents such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, benzene benzoate; solubilizing agents such as sodium benzoate, sodium salicylate, sodium acetate, iodine, urethane, monoethylacetamide, butazolidine, propylene glycol, teulenes, nicotinamide, hexamine, diethylacetamide; buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptones, gums; isotonicity agents such as sodium chloride; sodium bisulfite (NaHSO 3 ) Carbon dioxide gas, sodium metabisulfite (Na 2 S 2 O 5 ), Sodium sulfite (Na 2 SO 3 ), nitrogen gas (N 2 ), stabilizers such as ethylenediaminetetraacetic acid; sulfating agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, sodium ethylenediaminetetraacetate, and acetone sodium bisulfite; soothing agents such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium CMC, sodium alginate, Tween 80, and aluminum monostearate.

[0110] Suppositories according to the invention may contain any of the following ingredients: cocoa butter, lanolin, witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, mixture of stearic and oleic acids, Subanal, cottonseed oil, peanut oil, coconut oil, cocoa butter + cholesterol, lecithin, lanet wax, glycerol monostearate, Tween or Span, Imhausen, Monolen (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, 16), Hexalide Base 95, Cotomar, Hydrokote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrokote 25, Hydrokote 711, Idropostal, Massa Estralium Bases such as estrarium, A, AS, B, C, D, E, I, T), Massa-MF, Maspol, Maspol-15, Neospostal-En, Paramound-B, Sposil (OSI, OSIX, A, B, C, D, H, L), suppository base type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Spostal (N, Es), Wecoby (W, R, S, M, Fs), and tegester triglyceride base (TG-95, MA, 57) can be used.

[0111] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations are prepared by mixing the extract with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0112] Liquid preparations for oral administration include suspensions, liquid preparations for internal use, emulsions, syrups, etc., and may contain various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc. in addition to water and liquid paraffin, which are frequently used simple diluents. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of non-aqueous solvents and suspensions that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0113] In the present invention, the pharmaceutical composition of the present invention may contain albumin and a buffer in addition to the active ingredient.

[0114] The pharmaceutical composition according to the present invention is administered in a pharmacologic effective amount. In the present invention, the term "pharmacologic effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to any medical treatment, and the effective dose level can be determined by factors including the type and severity of the patient's disease, the activity of the drug, sensitivity to the drug, administration time, administration route and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0115] The composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered as a single or multiple doses. It is important to administer an amount that can obtain maximum effect at a minimum amount without side effects, taking into consideration all of the above factors, and this can be easily determined by a person skilled in the art to which the present invention pertains.

[0116] The pharmaceutical compositions of the present invention may be administered to an individual by a variety of routes, including, for example, oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intradural) injection, sublingual administration, buccal administration, rectal insertion, vaginal insertion, intraocular administration, otic administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, and the like, although all modes of administration are foreseeable.

[0117] The pharmaceutical composition of the present invention will be determined by the type of drug as the active ingredient, as well as various related factors such as the disease to be treated, the administration route, the patient's age, sex, weight and the severity of the disease.

[0118] In the present invention, the term "individual" means a subject requiring treatment for a disease, and more specifically may be a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited thereto.

[0119] In the present invention, "administration" means providing a given composition of the present invention to an individual by any suitable method.

[0120] In the present invention, "prevention" means any action to suppress or delay the onset of the target disease, "treatment" means any action to improve or beneficially change the target disease and the associated metabolic abnormality symptoms by administering the pharmaceutical composition according to the present invention, and "amelioration" means any action to reduce parameters related to the target disease, for example, the severity of symptoms, by administering the composition according to the present invention.

[0121] All documents mentioned herein are hereby incorporated by reference as if fully set forth herein. When introducing elements of the present invention or preferred embodiments thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements. Although the present invention has been described with respect to specific embodiments or aspects, it should not be construed as limiting the details of these embodiments.

[0122] In the following, preferred examples are presented to aid in understanding the present invention. However, the following examples are merely provided to facilitate understanding of the present invention, and the contents of the present invention are not limited to the following examples. EXAMPLES

[0123] Example 1. Creation of an animal model of colon cancer induced by Azoxymethane (AOM) / Dextran Sulfate Sodium (DSS) The animals used in the experiment were 7-8 week-old female BALB / C mice purchased from Nara Biotech., Seoul, South Korea. The mice were subjected to a 7-day adaptation period in the animal laboratory of Biocure Research Institute before the experiment, during which water and food were not restricted. The experimental animals were provided with a standardized environment, with day and night maintained at 12-h intervals, and the room temperature (23 ± 2°C) was maintained at an appropriate level. As shown in Figure 1, mice were given a single intraperitoneal injection of the carcinogen azoxymethane (AOM) (Merk, Cat# 25843-45-2) at a concentration of 12 mg / kg. One week later, the mice were given 2.5% (W / V) dextran sulfate sodium (DSS) instead of regular drinking water for one week, and then given a two-week rest period. Colitis associated colon rectal cancer was induced by repeating the one-week DSS treatment and two-week rest period excluding AOM treatment four times.

[0124] Example 2. Oral administration of a composition containing a reovirus and systemic administration of an immune checkpoint inhibitor (anti-PD-L1 antibody) The mice used in the experiment were divided into a normal control group, an AOM / DSS-induced colon cancer group, a reovirus oral administration group, an immune checkpoint inhibitor (anti-PD-L1 antibody) intraperitoneal administration group, and a reovirus / immune checkpoint inhibitor combination administration group. The reovirus was administered to the colon cancer animal model by oral administration, and the antibody was administered by intraperitoneal injection. Reovirus (type 3, Dearing) is 1×10 8 TCID 50The mice were orally administered Reovirus / 100μL PBS (Reovirus / 0.1%(w / v)Human serum albumin(HAS)in 1xPBS) for 5 consecutive days as shown in Figure 1, and anti-PD-L1 antibody (BioXcell, Cat# BE0101) was intraperitoneally administered at 5mg / kg every other day for 3 doses. This administration was repeated a total of 4 times as shown in Figure 1.

[0125] Example 3. Analysis of the therapeutic effect of an orally administered composition containing a reovirus using an AOM / DSS-induced colon cancer animal model The experiment was continued from the 39th day after the initial AOM treatment, during which the presence or absence of colon cancer and the severity of symptoms were observed through weight measurement, fecal and anal conditions, survival rate, and cancer formation and growth. Statistical analysis was performed using GraphPad Prism 6. Differences among the normal control group, AOM / DSS-induced colon cancer group, reovirus oral administration group, immune checkpoint inhibitor intraperitoneal administration group, and reovirus / immune checkpoint inhibitor combination administration group were compared using Dunnett's multiple comparison test in a one-way ANOVA test. Differences with p values ​​less than 0.05 were considered statistically significant. Data are presented as mean and SEM.

[0126] 3-1. Macroscopic evaluation by Disease Activity Index (DAI) measurement To assess the severity of colon cancer-related disease in the colon cancer animal model mice that had been orally administered the reovirus-containing composition of Example 2 and intraperitoneally treated with the anti-PD-L1 antibody, the disease activity index (DAI) was measured by checking changes in body weight, the presence or absence of stool masses, and bloody stools observed macroscopically in the stool or anus according to the standard disease activity index (DAI) grades shown in Figure 2. As shown in Figure 3, in mice administered AOM / DSS, loose stools and macroscopic bloody stools began to appear from day 4 after administration, and diarrhea and bloody stools were observed in all mice by day 7. Meanwhile, it was confirmed that the severity of diarrhea and bloody stools was significantly improved in the group that received oral administration of reovirus and intraperitoneal administration of anti-PD-L1 antibody. These results indicate that combined administration of a composition containing a reovirus (orally administered) and an immune checkpoint inhibitor (e.g., an anti-PD-L1 antibody) shows excellent anti-cancer effects against colorectal cancer.

[0127] 3-2. Evaluation by measuring survival rate Animal models of colon cancer lead to death through persistent inflammation in the colon and subsequent cancer formation. As shown in Figure 4, in this colon cancer animal model, as the disease progressed, deaths occurred in the AOM / DSS-induced colon cancer group and the single-administration group (virus or antibody administration group), but no deaths occurred in the combined administration group. These results indicate that combined administration of a composition containing a reovirus (orally administered) and an immune checkpoint inhibitor (e.g., an anti-PD-L1 antibody) can significantly improve survival rates in colorectal cancer.

[0128] 3-3. Evaluation by measuring cancer formation and growth In this animal model of colon cancer, it is expected that as the disease progresses, the single administration group (virus or antibody administration group) will have a partial inhibition of cancer formation or a slight effect, while the combined administration group will have a significant inhibition of such inflammation-induced cancer formation. Therefore, combined administration of a composition containing reovirus (orally administered) and an immune checkpoint inhibitor (e.g., an anti-PD-L1 antibody) is expected to significantly suppress the formation and growth of colorectal cancer.

[0129] Example 4. Creation of a colon cancer animal model through intramural surgical implantation of a colon cancer cell line (CT26) The animals used in the experiment were 7-8 week-old female BALB / C mice purchased from Nara Biotech., Seoul, South Korea. The mice were subjected to a 7-day adaptation period in the animal laboratory of Biocure Research Institute before the experiment, during which water and food were not restricted. The experimental animals were provided with a standardized environment, with day and night maintained at a 12-h interval and the room temperature (23±2°C) maintained at an appropriate level. The mice were inoculated with a colon cancer cell line (CT26 cells, 1×10 6 We then injected 100 μL of PBS into the colon wall and transplanted the cells. Two weeks after transplantation, tumor formation was confirmed, and a colon cancer animal model was established.

[0130] Example 5. Oral administration of a composition containing a reovirus and systemic administration of an immune checkpoint inhibitor (anti-PD-L1 antibody) The mice used in the experiment were divided into a normal control group, a CT26 cell-injected colon cancer group, a reovirus oral administration group, and a reovirus / immune checkpoint inhibitor combination administration group. The reovirus was administered to the colon cancer animal model by oral administration, and the antibody was administered by intraperitoneal injection. Four days after CT26 cell transplantation, reovirus (type 3, Dearing) was administered at 1 × 10 9 The mice were orally administered Reovirus / 100μL PBS (Reovirus / 0.1%(w / v)Human serum albumin(HAS)in 1x PBS) for three consecutive weeks, and anti-PD-L1 antibody (BioXcell, Cat# BE0101) was administered intraperitoneally at 2.5mg / kg three times every three days (Figure 5). In this animal model of colon cancer, as the disease progressed, we observed that the survival rate improved in the treatment groups (virus alone or in combination with anti-PD-L1 antibody) compared to the CT26 cell-injected colon cancer group, as shown in Figure 6. These results indicate that administration of a composition containing reovirus (orally administered) alone or in combination with an immune checkpoint inhibitor (e.g., an anti-PD-L1 antibody) can significantly improve survival rates in colorectal cancer.

[0131] Example 6. Creation of a skin cancer animal model through transplantation of a melanoma cell line (B16F10) The animals used in the experiment were 5-8 week-old female C57BL / 6 mice purchased from Nara Biotech., Seoul, South Korea. The mice were subjected to a 7-day adaptation period in the animal laboratory of Biocure Research Institute before the experiment, during which water and food were not restricted. The experimental animals were provided with a standardized environment, with day and night maintained at a 12-h interval, and the room temperature (23 ± 2°C) was maintained at an appropriate level. The mice were inoculated with melanoma cell lines (B16F10 cells, 1 × 10 5 cells / 50 μL PBS) were injected subcutaneously into the flank.

[0132] Example 7. Oral administration of a composition containing a reovirus and systemic administration of an immune checkpoint inhibitor (anti-PD-L1 antibody) For the anticancer effect test, the subjects were divided into a control group administered with vehicle or 0.1% (w / v) Human serum albumin (HAS) in 1x PBS, a reovirus oral administration group, an immune checkpoint inhibitor (anti-PD-L1 antibody) intraperitoneal administration group, and a reovirus / immune checkpoint inhibitor combination administration group. The reovirus was administered to the colorectal cancer animal model by oral administration, and the antibody was administered by intraperitoneal injection. As shown in FIG. 7, one week after B16F10 cell transplantation, reovirus (type 3, Dearing) was administered at 1×10 9 TCID 50The mice were orally administered Reovirus / 100μL PBS (Reovirus / 0.1%(w / v)Human serum albumin(HAS)in 1x PBS) six times on days 1, 2, 3, 6, 7, and 8, and anti-PD-L1 antibody (BioXcell, Cat# BE0101) was administered intraperitoneally at 5mg / kg three times every other day.

[0133] Example 8. Analysis of the therapeutic effect of an orally administered composition containing a reovirus in an animal model of skin cancer 8-1. Evaluation by measuring cancer growth As shown in Figure 8, in this skin cancer animal model, it was confirmed that tumors continued to grow after they were formed in the untreated control group. As the disease progressed, a tumor growth retardation effect was observed in the single-treatment group (virus or antibody treatment group) compared to the control group. Meanwhile, in the combined treatment group, tumor growth was significantly suppressed. Therefore, combined administration of a composition containing a reovirus (orally administered) and an immune checkpoint inhibitor (e.g., an anti-PD-L1 antibody) is expected to be useful in preventing or treating skin cancer.

[0134] 8-2. Evaluation by viability measurement As shown in FIG. 9, in this skin cancer animal model, as the disease progressed, the survival rate was significantly improved in the combination administration group compared to the control group and the single administration groups (virus or antibody administration groups). Therefore, combined administration of a composition containing reovirus (orally administered) and an immune checkpoint inhibitor (e.g., an anti-PD-L1 antibody) is expected to significantly improve survival rates in skin cancer.

[0135] Example 9. Anticancer effect of single administration in a mouse model of kidney cancer The animals used in the experiment were male BALB / C mice aged 5 to 8 weeks, purchased from Nara Biotech., Seoul, South Korea. Mice were inoculated with renal cancer cell line (RENCA cells, 2 × 10 5 cells / 50 μL PBS) were injected subcutaneously into the flank. One week after RENCA cell transplantation, reovirus (type 3, Dearing) was administered at 1 × 10 9 TCID 50 The mice were orally administered Reovirus / 100 μL PBS (Reovirus / 0.1% (w / v) Human serum albumin (HAS) in 1x PBS) once a day for 10 days (Figure 10). As shown in Figure 11, in this kidney cancer animal model, it was confirmed that the untreated control group showed continuous growth after tumors were formed in the mice. In addition, as the disease progressed, the reovirus-administered group showed a delayed effect on tumor growth compared to the control group.

[0136] Example 10. Oral administration of reovirus induces increased immune cell penetration into tumors in an animal model of colon cancer transplanted with colon cancer cells In this example, it was confirmed in vivo whether oral administration of reovirus induces increased infiltration of anti-cancer immune cells, CD8+ T cells, into tumors.

[0137] 10-1. Construction of tumor mouse model MC38 cell line, derived from C57BL6 colon adenocarcinoma cells, was diluted at 1.0 × 10 in 50 μl PBS. 5 The cells were resuspended at a concentration of 100 cells and injected subcutaneously into the flank of 6-week-old male C57BL6 mice. One week after MC38 cell transplantation, reovirus (type 3, Dearing) was injected at a concentration of 1 × 10 9 TCID 50 The mice were orally administered Reovirus / 100 μL PBS (Reovirus / 0.1% (w / v) Human serum albumin (HAS) in 1x PBS) once a day for 10 days ( FIG. 12 ). As shown in Figure 13, in this colon cancer animal model, it was confirmed that the untreated control group showed continuous growth after tumors were formed in the mice. In addition, as the disease progressed, the reovirus-administered group showed a delayed effect on tumor growth compared to the control group.

[0138] 10-2. Evaluation of immune cell penetration into tumors Tumors were taken from mice 3 and 10 days after administration, washed with PBS, cut into 2-4 mm pieces, and then 1 ml of PBS containing 0.1-0.5% type I collagenase (Gibco, Cat No. P2031) was added and treated at 37°C for 30 minutes to 1 hour. 10 ml of FACS buffer (0.1% BSA + 0.01% sodium azide in PBS) was added and mixed, then centrifuged at 300-400g for 5 minutes, the supernatant was discarded, and the precipitate was collected in 7 ml of FACS buffer and filtered through a 70 μm cell strainer. The filtered single cells were mixed with 10 ml of FACS buffer, centrifuged at 300-400g for 5 minutes, and the precipitate was collected. After discarding the supernatant, 1-2 ml of ACK lysis buffer (Lonza cat no. 10-548E) was added and left at room temperature for 1 minute, after which 10 ml of FACS buffer was added and mixed, and then centrifuged at 300-400 g for 5 minutes to recover. After resuspending in an appropriate amount of FACS buffer, the cells were stained with CD8 monoclonal antibody (BD Biosciences, Cat no. 563234) to confirm the degree of increase in CD8+ T cells, which was analyzed by flow cytometry using FACSDiVa software (BD Biosciences). As shown in FIG. 14, it was confirmed that the amount of CD8+ T cells that had infiltrated into the tumor 10 days after administration was increased by more than 5-fold in the reovirus oral administration group. In other words, through an increase in CD8+ T cells that have infiltrated into the tumor, which is a representative biomarker for the tumor-suppressing effect of immune anticancer drugs, it is expected that an orally administered composition containing reovirus will act in the treatment of cancer by activating anti-cancer immunity.

[0139] 10-3. Evaluation of immune cell infiltration in tumors and lymph nodes Tumors and lymph nodes were harvested from the mice 3 and 10 days after administration, fixed with acetone, placed in OCT solution (Sakura Finetek, Cat No. 4583), and stored in a -80°C ultra-low temperature freezer to prepare frozen tissue blocks. The frozen tissue blocks were cut to a thickness of 5-10 μm using a Cryostat (Leica Biosystems, CM3050S) to prepare slides. The tissue section slides were washed with PBS using a Coplin Jar, covered with 200 μl Superblock (ThermoFisher Scientific, Cat no. 37515), and left at room temperature for 10 minutes for processing. Then, the sections were stained with fluorescently labeled CD8 monoclonal antibody (Biolegend, Cat no. 100723) and CD31 monoclonal antibody (Biolegend, Cat no. 102416) diluted in PBS for 1-2 hours at room temperature in the dark, washed three times with PBS, coated with ProLong Diamond Antifade Mountant (Thermofisher Scientific, Cat no. P36965), and covered with a coverslip. The degree of staining was then observed under a fluorescent microscope. Nuclear staining was carried out with DAPI solution (BD Biosciences, Cat no. 564907) for 1 minute. As shown in Figure 15, after administration, the amount of CD8+ T cells that had infiltrated into the mesenteric lymph nodes tended to increase in the reovirus oral administration group, and the degree of increase was particularly significant on the 10th day. In addition, as shown in FIG. 16, the amount of CD8+ T cells that had infiltrated into the tumor 10 days after administration was significantly increased in the reovirus oral administration group, consistent with the flow cytometry analysis results. Since an increase in CD8+ T cells that have infiltrated into a tumor is a representative biomarker for the tumor-suppressing effect of an immunosuppressant anticancer drug, it is expected that an orally administered composition containing reovirus will act in the treatment of cancer through the activation of anticancer immunity.

[0140] Example 11. Therapeutic effect in colon cancer animal model The animals used in the experiment were male BALB / C mice aged 5 to 8 weeks, purchased from Nara Biotech., Seoul, South Korea. The mice were subjected to a 7-day adaptation period in the animal laboratory of Biocure Research Institute before the experiment, during which water and food were not restricted. The experimental animals were provided with a standardized environment, with day and night maintained at a 12-h interval and the room temperature (23 ± 2°C) maintained at an appropriate level. The mice were inoculated with a colon cancer cell line (CT26 cells, 2 × 10 5 cells / 50 μL PBS) were injected subcutaneously into the flank.

[0141] 11-1. Analysis of the therapeutic effect of an orally administered composition containing reovirus in an animal model of colon cancer For the anti-cancer effect test, the subjects were divided into a control group administered with vehicle or 0.1% (w / v) Human serum albumin (HAS) in 1x PBS, a reovirus oral administration group, an immune checkpoint inhibitor (anti-PD-1 antibody) intraperitoneal administration group, and a reovirus / immune checkpoint inhibitor combination administration group (anti-PD-1 antibody alone or anti-CTLA-4 antibody combination). The reovirus was administered to the colorectal cancer animal model by oral administration, and the antibody was administered by intraperitoneal injection. One week after CT26 cell transplantation, reovirus (type 3, Dearing) was administered at 1 × 10 9 TCID 50 The mice were orally administered Reovirus / 100μL PBS (Reovirus / 0.1%(w / v)Human serum albumin(HAS)in 1x PBS) once a day for 12 days. Anti-PD-1 antibody (BioXcell, Cat# BE0033-2) was administered at 8mg / kg, and anti-CTLA4 antibody (BioXcell, Cat# BE0164) was administered at 4mg / kg intraperitoneally four times at 3-day intervals (Figure 17).

[0142] 11-2. Evaluation by measuring cancer growth As shown in Figure 18, in this colon cancer animal model, it was confirmed that the tumor volume continued to grow after tumors were formed in the mice in the untreated control group. However, as the disease progressed, the single-treatment group (virus or antibody administration group) was observed to have a delayed effect on cancer growth compared to the control group. On the other hand, in the combination treatment group, especially in the group that received two types of immune checkpoint inhibitors simultaneously (RC402+aPD-1+aCTLA-4), cancer growth was suppressed by 84.6%, and some individuals achieved complete remission (CR).

[0143] 11-3 Evaluation of immune cell penetration into tumors Thirteen days after administration, tumors were harvested from the mice and the degree of CD8+ T cell infiltration was assessed using flow cytometry or histoimmunochemical staining. As shown in FIG. 19, the degree of tumor penetration of CD8+ T cells in the virus alone or combination treatment groups was confirmed to increase in correlation with the degree of cancer growth inhibition compared to the control group or antibody alone treatment group. In particular, it was confirmed that the degree of tumor penetration of CD8+ T cells was significantly increased in the group treated with two types of immune checkpoint inhibitors simultaneously.

[0144] Example 12. Induction of long-term anti-cancer immunity to prevent cancer recurrence after treatment 12-1. Evaluation by measuring cancer growth CT26 was reimplanted into mice that achieved complete tumor remission through combined administration of a composition containing reovirus (oral administration) and an immune checkpoint inhibitor (anti-PD-1 antibody). Fresh BALB / C mice served as the control group. As shown in Figure 20, in the control group, the CT26 colon cancer cell line grew normally, whereas when the CT26 colon cancer was re-implanted after complete remission, the tumor did not grow, indicating that the anti-cancer immune effect was sustained. Therefore, it is expected that combined administration of a composition containing reovirus (orally administered) and an immune checkpoint inhibitor (e.g., anti-PD-1 antibody, anti-CTLA4 antibody) can activate clinically long-term anti-cancer immunity.

[0145] 12-2. Splenic immune cell analysis Spleens were collected from mice that had achieved complete remission, homogenized with 10 ml of lysate, and filtered through a 70 μm cell strainer. The spleens were collected by centrifugation at 300-400 g for 5 min. The supernatant was discarded, and 1-2 ml of ACK lysis buffer (Lonza cat no. 10-548E) was added. The cells were left at room temperature for 1 min, and then 10 ml of FACS buffer was added, mixed, and centrifuged at 300-400 g for 5 min. The cells were resuspended in an appropriate amount of FACS buffer, stained with CD8 monoclonal antibody (BD Biosciences, Cat no. 563234), CD44 antibody (Biosciences, Cat no. 560569), and CD62 antibody (BD Biosciences, Cat no. 553150), and analyzed by flow cytometry using FACSDiVa software (BD Biosciences). As shown in Figure 21, the percentage of memory T cells (CD44+Cd62+CD8+) was confirmed to increase in the spleens of mice transplanted with CT26 colon cancer cell lines, suggesting that memory T cells are involved in long-term anti-cancer immunity. In other words, it is expected that by increasing CD8+ T cells, the tumor-suppressing effect of immune anticancer drugs can activate long-term anti-cancer immunity, including preventing recurrence.

[0146] Example 13. Immune response to oral administration of reovirus MC38 cell line, 1.0 × 10 in 50 μl PBS 5 The cells were resuspended at a concentration of 1 × 10 cells and injected subcutaneously into the flank of 6-week-old male C57BL6 mice. One week after transplantation of the MC38 cell line, reovirus (type 3, Dearing) was injected at a concentration of 1 × 10 9 TCID 50The mice were orally administered Reovirus / 100μL PBS (Reovirus / 0.1%(w / v)Human serum albumin(HAS)in 1x PBS) once a day for 10 days.

[0147] 13-1. Increase in tumor antigen-specific CD8+ T cells Ten days after administration, mesenteric lymph nodes were harvested from the mice, and changes in CD8+ T cells expressing T-cell receptors (TCRs) that specifically bind to the MC38-specific antigen peptide KSPWFTTL-loaded tetramer synthetic MHC complex (MBL, Cat No. TS-M507) were analyzed by flow cytometry. Mesenteric lymph nodes collected from mice were washed with PBS, lightly crushed, and filtered through a 70 μm cell strainer. The filtered single cells were added to 10 ml of FACS buffer, mixed, and then centrifuged at 300-400 g for 5 min to collect the cells. 2 × 10 7 The cells were resuspended at a concentration of 10 ... As shown in FIG. 22, it was confirmed that tumor antigen-specific CD8+ T cells (KSP-tetramer+ CD8+) were significantly increased in the group administered the composition for oral administration containing reovirus.

[0148] Increased expression of PD-1 on CD8+ T cells in lymph nodes Mesenteric lymph nodes were harvested, washed with PBS, and lightly disrupted, then filtered through a 70 μm cell strainer. The filtered single cells were added to 10 ml of FACS buffer, mixed, and collected by centrifugation at 300-400 g for 5 min. Blood was placed in a tube coated with EDTA (or heparin), mixed with PBS at a 1:1 ratio, transferred to a 15mL tube containing Ficoll-Paque PLUS, centrifuged, and mouse peripheral blood mononuclear cells (PBMCs) were collected. PBMCs were centrifuged at 400g to remove the supernatant, discarded, and then 1-2mL of ACK lysis buffer (Lonza cat no. 10-548E) was added and left at room temperature for 1 minute. Then, 10mL of FACS buffer was added and mixed, and centrifuged at 300-400g for 5 minutes to collect. Immune cells isolated and collected from lymph nodes and blood were resuspended in an appropriate amount of FACS buffer and stained with CD8 monoclonal antibody (BD Biosciences, Cat no. 563234) and PD-1 monoclonal antibody (Biosciences, Cat no. 25-9985-80), and the degree of increase in CD8+ T cells expressing PD-1 was confirmed using flow cytometry. As shown in Figure 23, no change in CD8+ T cells expressing PD-1 was observed in PBMCs isolated from blood, but a clear change was observed in mesenteric lymph nodes, with a 2.5-fold or greater increase in CD8+ T cells.

[0149] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical concept or essential features of the present invention. Therefore, the above-described embodiments should be understood to be illustrative in all respects and not restrictive. [Industrial Applicability]

[0150] The present inventors have not only confirmed the cancer therapeutic effect of reovirus alone, but also confirmed that when reovirus is orally administered in combination with an immune checkpoint inhibitor, the cancer therapeutic effects such as reduction in tumor volume, inhibition of tumor growth rate, and increase in survival rate are significantly increased in colon cancer, skin cancer, kidney cancer, etc., and in particular, when reovirus is combined with PD-1 antibody and CTLA-4 antibody, not only can cancer be put into complete remission, but also cancer recurrence can be suppressed. Thus, the reovirus of the present invention can be usefully used as a therapeutic agent and therapeutic adjuvant for various cancers, and has industrial applicability.

Claims

1. A pharmaceutical composition for preventing or treating colon cancer or kidney cancer, comprising a Reovirus as an active ingredient, The reovirus is administered orally. composition.

2. The composition described in claim 1, further comprising one or more immune checkpoint inhibitors selected from the group consisting of anti-PD-L1 antibodies, anti-PD-1 antibodies, and anti-CTLA-4 antibodies.

3. The composition of claim 2, wherein the reovirus and the immune checkpoint inhibitor are administered simultaneously, separately or sequentially.

4. The composition of claim 1, wherein the pharmaceutical composition increases intratumoral penetration of CD8+ T cells.

5. A pharmaceutical composition for preventing recurrent cancer, comprising reovirus and an anti-PD-1 antibody as active ingredients, the reovirus being administered orally.

6. The composition of claim 5, wherein the reovirus and the anti-PD-1 antibody are administered simultaneously, separately, or sequentially.

7. 1. Use of a composition comprising a reovirus as an active ingredient for producing a medicament for preventing or treating colorectal cancer or kidney cancer, comprising: The reovirus is administered orally. use.

8. Use of a composition comprising a reovirus and an anti-PD-1 antibody as active ingredients for the manufacture of a medicament for preventing recurrent cancer, comprising: The reovirus is administered orally. use.

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