Oncolytic virus with improved safety and anti-cancer effect

Abstract

The present invention relates to an oncolytic virus with improved safety and anti-cancer efficacy, and its use. The oncolytic virus with improved safety and anti-cancer efficacy of the present invention can express an HSV-TK fragment containing an effector domain consisting of a minimal amino acid sequence that lacks thymidine kinase (TK) activity but can phosphorylate GCV or ACV, or a variant thereof that phosphorylates GCV or ACV, and can kill cancer cells infected with the oncolytic virus as well as adjacent cancer cells. Furthermore, GCV or ACV is involved in the suppression of viral proliferation, thereby controlling virus-induced side effects even when high doses of the virus are administered. Furthermore, even if the number of viral particles is reduced due to the suppression of viral proliferation by GCV, the anti-cancer efficacy is enhanced. Therefore, the oncolytic virus with improved safety and anti-cancer efficacy of the present invention can be advantageously used in cancer treatment.

Description

Technical Field

[0001] The present invention relates to an oncolytic virus with improved safety and anti-cancer effect, and its use.

Background Art

[0002] By the full-scale use of gene recombination technology, clinical studies using oncolytic viruses with increased tumor selectivity and anti-cancer efficacy have been initiated. The first recombinant oncolytic virus reported in the literature was herpes simplex virus. Since then, studies on tumor lysis using other viruses have been actively conducted.

[0003] In recent years, the usefulness of oncolytic viruses has attracted attention since T-Vec (talimogene laherparepvec), which is based on herpes virus, has been commercialized for the treatment of advanced melanoma in Europe and the United States. On the other hand, although the thymidine kinase (TK) gene-deficient vaccinia virus has high clinical usefulness, its therapeutic range is narrow, so there is a limit to maximizing the clinical effect. In the case of the TK-deficient vaccinia virus, the narrow therapeutic range means that a high dose of the virus has great clinical effectiveness but is accompanied by clinical risks due to the toxicity of the virus.

[0004] Actually, in the Phase II clinical trial of Pexa-Vec (JX-594; SillaJen, Inc.) for 30 patients with primary liver cancer, the high-dose group (10 9 pfu) was shown to have a higher survival rate compared to the low-dose group (10 8 pfu). However, dose-limiting toxicity (DLT) was observed at 3×10 9 pfu in the Phase I clinical trial by intratumoral administration, and this determined the maximum tolerated dose (MTD) to be 1×10 9It was restricted to pfu. There are reports that there is no relevance to the drug. However, early deaths after treatment with oncolytic viruses have been frequently reported, indicating that unwanted viral growth may lead to unpredictable results. These dose-dependent increases in efficacy and dose-limiting toxicity imply the need to develop a safer and more effective vaccinia virus.

[0005] On the other hand, ganciclovir (GCV) is an antiviral drug effective against herpes simplex virus, cytomegalovirus, and varicella-zoster virus. When GCV binds to the TK of herpes simplex virus, its 5'-end is phosphorylated and converted to ganciclovir-triphosphate (GCV-TP). GCV-TP inhibits the activity of DNA polymerase and binds to the 3'-end of viral DNA to terminate DNA elongation. GCV-TP, which is a highly toxic substance, also inhibits DNA synthesis in cells and thereby exhibits cytotoxicity.

[0006] Recently, anti-cancer research has been conducted in which HSV1-TK is inserted into an oncolytic virus, and the resulting oncolytic virus is co-administered with GCV to induce tumor cell death. According to the research, first, the oncolytic virus infects tumor cells and induces a direct anti-cancer effect. GCV phosphorylated by HSV1-TK (suicide gene) exhibits an additional anti-cancer effect by suppressing tumor cell growth (Oliver W et al., Human Gene Therapy, Vol.10, No.16, 1999). The HSV1-TK / GCV system has mainly been used in oncolytic virus therapy using adenovirus as a vector. However, there is still room for discussion regarding the additional cytotoxic effects expected by the co-administration of GCV.

[0007] Specifically, when a oncolytic virus obtained by inserting the HSV-TK gene into an adenovirus having replication ability was co-administered with GCV, a significant enhancement of cytotoxicity was observed in glioma cells. On the other hand, in many other studies in which HSV-TK was inserted into an adenovirus having replication ability, the additional anti-cancer effect caused by the administration of GCV has not been consistently shown (Lambright ES et al., Gene Ther, 8:946 - 53). This is reported to be because the HSV-TK / GCV system is involved not only in the suppression of tumor cell growth but also in the suppression of virus growth, and these effects are opposite and cancel each other out.

[0008] Therefore, when applying the HSV-TK / GCV system to an oncolytic virus, it is necessary to examine a specific method that can enhance the anti-cancer effect while ensuring safety.

[0009] [Disclosure of the Invention] [Technical Problem] Therefore, as a result of examining the development of an oncolytic virus with improved safety and anti-cancer effect, the present inventors developed an oncolytic virus that does not have thymidine kinase (TK) activity and can phosphorylate GCV or ACV, and confirmed that when this oncolytic virus was co-administered with GCV, it exhibited excellent safety and anti-cancer effect, and completed the present invention.

[0010] [Means for Solving the Problems] In one aspect of the present invention, to achieve the above object, there is provided an oncolytic virus comprising a nucleotide sequence encoding a polypeptide containing an effector domain derived from herpes simplex virus thymidine kinase (HSV-TK), wherein the effector domain is represented by SEQ ID NO: 1 and is derived from herpes simplex virus thymidine kinase (HSV-TK).

[0011] In another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, comprising an oncolytic virus as an active ingredient.

[0012] In yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, which comprises, as an active ingredient, an oncolytic virus and ganciclovir (GCV) or acyclovir (ACV).

[0013] In yet another aspect of the present invention, there is provided a method for producing an oncolytic vaccinia virus containing a gene encoding a mutant HSV-TK, the method comprising: i) removing the TK gene from the vaccinia virus and recombining the vaccinia virus with the wild-type HSV-TK gene; and ii) continuously passaging the recombinant vaccinia virus in the presence of bromodeoxyuridine (BrdU) in a host cell.

[0014] [Advantages of the Invention] The oncolytic virus with improved safety and anticancer effect according to the present invention does not have thymidine kinase (TK) activity but contains an effector domain consisting of a minimum amino acid sequence capable of phosphorylating GCV or ACV, or can express a variant thereof for phosphorylating GCV or ACV, whereby it can kill cancer cells infected with the oncolytic virus and adjacent cancer cells. Further, GCV or ACV is also involved in inhibiting virus growth, and thus can control virus-induced side effects even when a high dose of the virus is administered. Furthermore, the oncolytic virus exhibits an increased anticancer effect even when the number of virus particles decreases due to the inhibition of virus growth caused by GCV. Therefore, the oncolytic virus with improved safety and anticancer effect according to the present invention can be effectively used for the treatment of cancer. [Brief Description of the Drawings]

[0015]

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Mode for Carrying Out the Invention

[0016] Hereinafter, the present invention will be described in detail.

[0017] In an embodiment of the present invention, there is provided an oncolytic virus comprising a nucleotide sequence encoding a polypeptide containing an effector domain, wherein the effector domain is represented by SEQ ID NO: 1 and is derived from herpes simplex virus thymidine kinase (HSV-TK).

[0018] As used herein, the term "herpes simplex virus thymidine kinase (HSV-TK)" refers to an enzyme involved in the initial phosphorylation reaction during DNA synthesis in herpes simplex virus. HSV-TK is also involved in the phosphorylation of antiviral drugs such as ganciclovir (GCV) and acyclovir (ACV). In particular, HSV1-TK reacts with GCV or ACV with approximately 10-fold higher sensitivity than TK present in other viruses. HSV is herpes simplex virus type 1 (HSV1) or herpes simplex virus type 2 (HSV2). Specifically, HSV can be HSV1. Furthermore, HSV-TK can be herpes simplex virus type 1 thymidine kinase (HSV1-TK).

[0019] As used herein, the term "effector domain" refers to a protein domain consisting of 145 consecutive amino acids at positions 1 to 145 of wild-type HSV-1 TK, represented by SEQ ID NO: 15 and consisting of 376 amino acids. The effector domain has low or no TK activity compared to wild-type HSV-1 TK. Therefore, an oncolytic virus containing a nucleotide sequence encoding a polypeptide containing the effector domain cannot proliferate on its own. However, the effector domain contains an ATP binding site and can phosphorylate GCV or ACV. When cells infected with an oncolytic virus containing a nucleotide sequence encoding a polypeptide containing the effector domain are treated with GCV or ACV, GCV or ACV can be phosphorylated.

[0020] A polypeptide containing the effector domain may further have 0 to 231 amino acids linked to the C-terminus of the effector domain. Here, the polypeptide has low or no TK activity compared to wild-type HSV-1 TK. Therefore, an oncolytic virus containing a nucleotide sequence encoding the polypeptide cannot proliferate on its own. Furthermore, the polypeptide can phosphorylate GCV or ACV. When cells infected with an oncolytic virus containing a nucleotide sequence encoding the polypeptide are treated with GCV or ACV, GCV or ACV can be phosphorylated.

[0021] Specifically, a polypeptide containing the effector domain may further have 0 to 231, 10 to 200, 20 to 150, or 40 to 100 amino acids linked to the C-terminus of the effector domain. Preferably, a polypeptide containing the effector domain may further have 36, 82, or 231 amino acids linked to the C-terminus of the effector domain.

[0022] The polypeptide containing the effector domain may consist of the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13. The nucleotide sequence encoding the polypeptide may be the nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13. In particular, the nucleotide sequence encoding the polypeptide may be the nucleotide sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14.

[0023] In order to develop an oncolytic virus with improved safety and anti-cancer effect, the inventors prepared a recombinant vaccinia virus containing the wild-type HSV1-TK gene and then adaptively evolved the virus in the absence of TK. The adaptively evolved vaccinia virus was subjected to experiments on luciferase activity, genomic analysis, and sensitivity to GCV. Through the experiments, mutant vaccinia viruses (C1, C3, C40, C45, C52, and C57) expressing the HSV1-TK fragment or its variant were selected. In addition, mutant vaccinia viruses (WOTS-418, OTS-418) containing a gene encoding an HSV-TK variant obtained by mutating a part of the amino acid sequence shown in SEQ ID NO: 15 were prepared. The nucleotide sequences of the mutant vaccinia viruses (C1, C3, C40, C45, C52, C57, WOTS-418) were analyzed. As a result, although the amino acid sequence up to position 145 (reference point) of the wild-type HSV-1TK shown in SEQ ID NO: 15 does not have TK activity, it was confirmed that it phosphorylates GCV or ACV.

[0024] As used herein, the term "oncolytic virus" refers to a recombinant virus whose gene is engineered to specifically replicate in cancer cells so that the virus can destroy cancer cells. Oncolytic viruses can be derived from adenovirus, herpes simplex virus, measles virus, lentivirus, retrovirus, cytomegalovirus, baculovirus, reovirus, adeno-associated virus, myxoma virus, vesicular stomatitis virus, poliovirus, Newcastle disease, parvovirus, coxsackievirus, Seneca virus, vaccinia virus, or poxvirus. Preferably, the oncolytic virus can be derived from vaccinia virus.

[0025] Vaccinia virus can be a vaccinia virus strain, namely, 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).

[0026] In another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, an oncolytic virus comprising a nucleotide sequence encoding a polypeptide containing an effector domain, wherein the effector domain is represented by SEQ ID NO: 1 and is derived from herpes simplex virus thymidine kinase (HSV-TK).

[0027] The oncolytic virus contained as an active ingredient in the pharmaceutical composition is as described above.

[0028] The dosage of the oncolytic virus varies depending on the individual's condition and body weight, the severity of the disease, the type of drug, the administration route, and the administration period, and can be appropriately selected by those skilled in the art. The dosage may be such that the patient receives 1×10 3 ~1×10 18 virus particles, infectious virus units (TCID 50 ), or plaque-forming units (pfu). Specifically, for the patient, dosages such as 1×10 3 , 2×10 3 , 5×10 3 , 1×10 4 , 2×10 4 , 5×10 4 , 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 , 1×10 18 or more virus particles, infectious virus units, or plaque-forming units may be administered, and various numerical values and ranges may be included between these numerical values. Preferably, the oncolytic virus can be administered at a dosage of 1×10 3 ~1×10 10 pfu.

[0029] The cancer may be any one selected from the group consisting of lung cancer, colorectal cancer, prostate cancer, thyroid cancer, breast cancer, brain tumor, 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, bile duct cancer, pancreatic cancer, non-small cell lung cancer, bone cancer, intraocular melanoma, perianal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, small intestine cancer, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, kidney cancer, ureteral cancer, renal cell cancer, renal pelvis cancer, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, and combinations thereof.

[0030] The pharmaceutical composition of the present invention can further contain a physiologically acceptable carrier. Furthermore, the pharmaceutical composition of the present invention can further contain suitable excipients and diluents commonly used in the preparation of pharmaceutical compositions. Also, the pharmaceutical composition can be formulated in the form of an injection according to a conventional method.

[0031] When formulated as a preparation for parenteral administration, the pharmaceutical composition can be formulated into a sterile aqueous solution, non-aqueous solution, suspension, emulsion, lyophilized preparation, suppository, etc. As the non-aqueous solution or suspension, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. can be used. As the base of the suppository, Witepsol (registered trademark), macrogol, Tween (registered trademark) 61, cocoa butter, lauric fat, glycerogelatin, etc. can be used.

[0032] 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 condition of the patient and the presence or absence of side effects. Also, the optimal administration route, dosage, and frequency of administration can be selected by those skilled in the art within an appropriate range. Further, the pharmaceutical composition may be administered in combination with other drugs or physiologically active substances whose therapeutic effects are known, or may be formulated in combination with other drugs.

[0033] The pharmaceutical composition can be administered parenterally, and such administration can be carried out by any suitable method, for example, intratumoral, intraperitoneal, subcutaneous, intradermal, intranodal, and intravenous administration. Among these, intratumoral, intraperitoneal, or intravenous administration is preferred. On the other hand, the dosage of the pharmaceutical composition can be determined according to the administration schedule, total dosage, and the health status of the patient.

[0034] In yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, comprising a oncolytic virus and ganciclovir (GCV) or acyclovir (ACV) as active ingredients, wherein the oncolytic virus comprises a nucleotide sequence encoding a polypeptide containing an effector domain, and the effector domain is represented by SEQ ID NO: 1 and is derived from herpes simplex virus thymidine kinase (HSV-TK).

[0035] The oncolytic virus and GCV or ACV contained in the pharmaceutical composition can be administered simultaneously, or co-administered sequentially or in the reverse order. Specifically, the oncolytic virus contained in the pharmaceutical composition and GCV or ACV can be administered simultaneously. Further, the administration of the pharmaceutical composition can be such that the oncolytic virus is administered first, then GCV or ACV is administered, and these components are contained in the pharmaceutical composition. Further, the administration of the pharmaceutical composition can be such that the oncolytic virus is administered first, then GCV or ACV is administered, and then the oncolytic virus is administered again, where these components are contained in the pharmaceutical composition.

[0036] The oncolytic virus contained as an active ingredient in the pharmaceutical composition is as described above.

[0037] The dosage of the oncolytic virus varies depending on the individual's condition and body weight, the severity of the disease, the type of drug, the administration route, and the administration period, and can be appropriately selected by those skilled in the art. The dosage is such that the patient is 1×10 3 ~1×10 18virus particles, infectious virus units (TCID 50 ), or plaque-forming units (pfu) may be such that it can receive. Specifically, for a patient, 1×10 3 , 2×10 3 , 5×10 3 , 1×10 4 , 2×10 4 , 5×10 4 , 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 , 1×10 18 or more virus particles, infectious virus units (TCID 50 ), or plaque-forming units (pfu) may be a dosage to be administered, and various numerical values and ranges may be included between these numerical values. Preferably, the oncolytic virus can be administered at a dosage of 1×10 3 ~1×10 10 pfu.

[0038] As used herein, the term "GCV" refers to ganciclovir, an antiviral agent effective against herpes simplex virus, cytomegalovirus, and varicella-zoster virus. GCV is phosphorylated at the 5' end by viral TK and converted to ganciclovir triphosphate (GCV-TP). GCV-TP inhibits the activity of viral DNA polymerase, binds to the 3' end of viral DNA, and terminates DNA elongation. Furthermore, phosphorylated GCV can stop cellular DNA replication, thus inhibiting cell proliferation. GCV is represented by Formula 1.

[0039] [Formula 1] [Chemical formula]

[0040] As used herein, the term "ACV" refers to acyclovir, an antiviral agent effective against herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus. ACV is phosphorylated by viral TK and converted to acyclovir triphosphate (ACV-TP). ACV-TP inhibits the activity of viral DNA polymerase, binds to the 3' end of viral DNA, and terminates DNA elongation. ACV is represented by Formula 2.

[0041] [Formula 2] [Chemical formula]

[0042] Furthermore, GCV or ACV can be administered at a dose of 0.1 μg / kg to 50 mg / kg. Specifically, GCV or ACV can be administered at a dose of 0.1 μg / kg to 50 mg / kg, 1 μg / kg to 40 mg / kg, 5 μg / kg to 30 mg / kg, or 10 μg / kg to 20 mg / kg.

[0043] The cancer can be any selected from the group consisting of lung cancer, colorectal cancer, prostate cancer, thyroid cancer, breast cancer, brain tumor, 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, bile duct cancer, pancreatic cancer, non-small cell lung cancer, bone cancer, intraocular melanoma, perianal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, small intestine cancer, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, kidney cancer, ureteral cancer, renal cell cancer, renal pelvis cancer, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, and combinations thereof.

[0044] The pharmaceutical composition of the present invention can further contain a physiologically acceptable carrier. Further, the pharmaceutical composition of the present invention may further contain appropriate excipients and diluents commonly used in the preparation of pharmaceutical compositions. Also, the pharmaceutical composition can be formulated in the form of an injection according to a conventional method.

[0045] When formulated as a preparation for parenteral administration, the pharmaceutical composition can be formulated into a sterile aqueous solution, non-aqueous solution, suspension, emulsion, lyophilized preparation, suppository, etc. As the non-aqueous solution or suspension, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. can be used. As the base of the suppository, Witepsol (registered trademark), macrogol, Tween (registered trademark) 61, cocoa butter, laurin fat, glycerogelatin, etc. can be used.

[0046] 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 condition of the patient and the presence or absence of side effects. Also, the optimal administration route, dosage, and frequency of administration can be selected by those skilled in the art within an appropriate range. Further, the pharmaceutical composition may be administered in combination with other drugs or bioactive substances whose therapeutic effects are known, or may be formulated in combination with other drugs.

[0047] The pharmaceutical composition can be administered parenterally, and such administration can be carried out by any suitable method, for example, intratumoral, intraperitoneal, subcutaneous, intradermal, intranodal, and intravenous administration. Among these, intratumoral, intraperitoneal, or intravenous administration is preferred. On the other hand, the dosage of the pharmaceutical composition can be determined according to the administration schedule, total dosage, and the health status of the patient.

[0048] In yet another aspect of the present invention, there is provided a method for treating cancer, which comprises the step of administering a oncolytic virus and ganciclovir (GCV) or acyclovir (ACV), wherein the oncolytic virus comprises a nucleotide sequence encoding a polypeptide comprising an effector domain, wherein the effector domain is represented by SEQ ID NO: 1 and is derived from herpes simplex virus thymidine kinase (HSV-TK).

[0049] The oncolytic virus, GCV, and ACV are as described above.

[0050] As used herein, the term "individual" refers to a person having or suffering from a disease in a condition that can be alleviated, inhibited, or treated by administering the oncolytic virus and GCV or ACV of the present invention.

[0051] In yet another aspect of the present invention, there is provided the use of an oncolytic virus for the treatment of cancer, wherein the oncolytic virus comprises a nucleotide sequence encoding a polypeptide comprising an effector domain, wherein the effector domain is represented by SEQ ID NO: 1 and is derived from herpes simplex virus thymidine kinase (HSV-TK).

[0052] In yet another aspect of the present invention, there is provided a method for producing an oncolytic vaccinia virus containing a gene encoding a mutant HSV-TK, the method comprising: i) removing the TK gene from the vaccinia virus and recombining the vaccinia virus with a wild-type HSV-TK gene; and ii) serially culturing the recombinant vaccinia virus in the presence of bromodeoxyuridine (BrdU) in a host cell.

[0053] The wild-type HSV-TK gene may be a nucleotide sequence represented by SEQ ID NO: 16.

[0054] The oncolytic vaccinia virus has low or no TK activity and can phosphorylate GCV or ACV.

[0055] The serial culturing can be carried out continuously at least 3 times, preferably at least 10 times continuously.

[0056] In yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, comprising an oncolytic virus and hydroxyurea as active ingredients, wherein the oncolytic virus contains a nucleotide sequence encoding a polypeptide containing an effector domain, and the effector domain is represented by SEQ ID NO: 1 and is derived from herpes simplex virus thymidine kinase (HSV-TK). For the oncolytic virus, refer to the above description.

[0057] As used herein, the term "hydroxyurea" refers to a compound having the following formula.

[0058] [Formula 3] [Chemical formula]

[0059] Hydroxyurea is known as an anticancer agent that inhibits DNA synthesis. However, its exact mechanism has not been elucidated. Also, hydroxyurea may be contained in a pharmaceutical composition as a commercial drug containing hydroxyurea. Examples of commercial drugs containing hydroxyurea may include, but are not limited to, Hydroxyurea®, Hydrea®, Droxia™, Mylocel™, Siklos®, and Hydrine® caps. Hydroxyurea can be administered orally and can also be administered parenterally.

[0060] The oncolytic virus and hydroxyurea contained in the pharmaceutical composition can be administered simultaneously, or co-administered sequentially or in the reverse order. Specifically, the oncolytic virus and hydroxyurea can be administered simultaneously. Further, hydroxyurea can be administered first, followed by the oncolytic virus. Further, the oncolytic virus can be administered first, followed by hydroxyurea. Further, hydroxyurea can be administered first, followed by the oncolytic virus, and then hydroxyurea can be administered again.

[0061] Furthermore, hydroxyurea 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, hydroxyurea 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, 30 mg / kg / day to 60 mg / kg / day. In an embodiment of the present invention, hydroxyurea was administered at 30 mg / kg / day or 60 mg / kg / day. Depending on the dose, the pharmaceutical composition can be administered in several divided doses per day. Specifically, the pharmaceutical composition can be administered in divided doses such as 1 to 4 times a day or 1 to 2 times a day.

[0062] Cancer may be any selected from the group consisting of lung cancer, colorectal cancer, prostate cancer, thyroid cancer, breast cancer, brain tumor, head and neck cancer, esophageal cancer, skin cancer, thymic cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, non-small cell lung cancer, bone cancer, intraocular melanoma, perianal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, small intestine cancer, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, kidney cancer, ureteral cancer, renal cell cancer, renal pelvis cancer, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, and combinations thereof.

[0063] The pharmaceutical composition of the present invention can further contain a physiologically acceptable carrier. Further, the pharmaceutical composition of the present invention may further contain appropriate excipients and diluents generally used in the preparation of pharmaceutical compositions. Also, the pharmaceutical composition can be formulated in the form of an injection according to a conventional method.

[0064] When formulated as a preparation for parenteral administration, the pharmaceutical composition can be formulated into a sterile aqueous solution, non-aqueous solution, suspension, emulsion, freeze-dried preparation, suppository, etc. As the non-aqueous solution or suspension, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. can be used. As the base of the suppository, Witepsol (registered trademark), macrogol, Tween (registered trademark) 61, cocoa butter, lauric fat, glycerogelatin, etc. can be used.

[0065] 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 condition of the patient and the presence or absence of side effects. Also, the optimal administration route, dosage, and frequency of administration can be selected by those skilled in the art within an appropriate range. Further, the pharmaceutical composition may be administered in combination with other drugs or physiologically active substances whose therapeutic effects are known, or may be formulated in combination with other drugs.

[0066] The pharmaceutical composition can be administered parenterally, and such administration can be carried out by any suitable method, for example, intratumoral, intraperitoneal, subcutaneous, intradermal, intranodal, and intravenous administration. Among these, intratumoral, intraperitoneal, or intravenous administration is preferred. On the other hand, the dosage of the pharmaceutical composition can be determined according to the administration schedule, total dosage, and the health status of the patient.

[0067] In yet another aspect of the present invention, there is provided a method for treating cancer, comprising the step of administering an oncolytic virus.

[0068] In yet another aspect of the present invention, there is provided the use of an oncolytic virus for the treatment of cancer.

[0069] In yet another aspect of the present invention, there is provided the use of an oncolytic virus for the manufacture of a medicament for treating cancer.

[0070] In yet another aspect of the present invention, there is provided a method for treating cancer, comprising the step of administering a pharmaceutical composition for preventing or treating cancer.

[0071] In yet another aspect of the present invention, there is provided the use of a pharmaceutical composition for preventing or treating cancer for the treatment of cancer.

[0072] In yet another aspect of the present invention, there is provided the use of a pharmaceutical composition for preventing or treating cancer for the manufacture of a medicament for treating cancer.

[0073] [Examples] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereto.

[0074] Example 1. Preparation of a mutant vaccinia virus Example 1.1. Construction of a shuttle plasmid vector The shuttle plasmid vector used was a shuttle plasmid vector prepared by synthesizing the type 1 HSVTK gene (pSE / L promoter) and the firefly luciferase reporter (p7.5 promoter) gene and incorporating these genes into the pUC57amp+ plasmid (Genewiz, USA).

[0075] Example 1.2. Construction of Mutant Vaccinia Viruses (C1, C3, C40, C45, C52, and C57) The method for producing the mutant vaccinia virus is shown in FIG. 1. Specifically, first, to obtain a recombinant virus, HeLa cells (ATCC) were seeded in a 6-well plate at 4×10 5 cells / well, and EMEM medium containing 10% fetal bovine serum was added. Treatment was carried out at 0.05 MOI with the Wyeth strain wild-type vaccinia virus (NYC Department of Health strain, WR-1536, ATCC). After 2 hours, the medium was replaced with EMEM medium containing 2% fetal bovine serum, and 4 μg of the shuttle plasmid vector constructed in Example 1.1 was delivered to the cells using Xfect™ polymer (Clonetech 631317, USA). After culturing for 4 hours, the medium was replaced with EMEM medium containing 2% fetal bovine serum, and the HeLa cells were cultured for an additional 72 hours. The recombinant vaccinia virus containing the HSV1-TK gene was obtained by checking the luciferase activity in the HeLa cells.

[0076] Next, mutations that cause HSV1-TK lacking TK activity were induced in the TK-osteosarcoma (143B TK-) cell line (ATCC) by performing 10 consecutive subcultures under a biochemical environment (TK-selection pressure) that enables selection of cells lacking TK function in the presence of BrdU (thymidine analog, 15 μg / ml). Lysates of mutant vaccinia virus clones having luciferase activity were dispensed onto plates, and then 100 single plaques having luciferase activity were isolated. Next, the single plaques were amplified, and then clones (S3C4#1_C1 to S3C4#1_C100) expressing HSV-TK fragments of different protein sizes were identified by Western blotting.

[0077] Specifically, 100 clones were each treated with 15 μl and infected into the HeLa cell line. Then, after 24 hours, the cells were collected, lysed, and proteins were extracted. The extracted proteins were denatured, and 40 μg of each sample was loaded onto an SDS-PAGE gel for electrophoresis. After electrophoresis, the samples were transferred to a PVDF membrane and reacted with the anti-HSV1-TK antibody (Bethyl A50-101P), which is the primary antibody. Then, after washing with PBST, it was reacted with the HRP-labeled anti-goat antibody (SantaCruz, sc-28037), which is the secondary antibody. The samples were washed again with PBST, treated with a chemiluminescence reagent, and checked using a chemiluminescence imaging system (Davinch K). As a result, it was confirmed that the HSV1-TK fragment protein was expressed in the virus (Figs. 2 to 13).

[0078] 100 different clones basically do not have TK activity because they were cultured in a biochemical environment where cells lacking the TK function could be selected. On the other hand, in order to screen for clones sensitive to GCV, while culturing in the presence of GCV in a 96-well plate for 4 days, 10 clones in which apoptosis occurred by real-time imaging were first selected using the Incucyte® (Essen Biosciences), a real-time cell imaging and analysis system. Generally, virus-induced cytotoxicity gradually increases after plaque formation. However, in the case of GCV-induced cytotoxicity, overall cytotoxicity occurs all at once within 15 hours after plaque formation. Therefore, GCV-induced cytotoxicity was qualitatively checked.

[0079] As a result of image analysis, for the colonies first selected, suppression of virus growth and cytotoxicity by GCV administration was confirmed. Then, the mutant vaccinia viruses in colonies S3C4#1_C1, S3C4#1_C3, S3C4#1_C40, S3C4#1_45, S3C4#1_C52, and S3C4#1_C57 were finally selected. The mutant vaccinia viruses in colonies S3C4#1_C1, S3C4#1_C3, S3C4#1_C40, S3C4#1_45, S3C4#1_C52, and S3C4#1_C57 were named "C1", "C3", "C40", "C45", "C52", and "C57", respectively.

[0080] The amino acid sequencing of C1, C3, C40, C45, C52, and C57 was requested from Macrogen (Seoul, Korea). As a result, it was confirmed that the amino acid sequences of C40 and C45 were the same. Also, it was confirmed that C1, C3, C40 / C45, C52, and C57 had the amino acid sequences of SEQ ID NOs: 15, 17, 19, 21, 23, and 25. In particular, for the HSV1-TK of the selected mutant vaccinia virus, it was confirmed that the mutation starts from the 145th amino acid at the N-terminus (Figure 14).

[0081] Production of Mutant Vaccinia Virus (WOTS-418 / OTS-418) The most frequently reported mutations in HSV-TK are frameshift mutations caused by insertions or deletions of bases occurring at the nucleotide sequence sites at positions 430 - 436 (7G) and 548 - 553 position (6C). After inserting wild-type HSV-TK into vaccinia virus, more than 98% of the mutations occurred at these sites. Therefore, in order to introduce silent mutations at the nucleotide sequence sites, the nucleotide sequence GGGGGGG at positions 430 - 436 was changed to GGTGGTG, and the nucleotide sequence CCCCCCC at positions 548 - 553 position was changed to CCCCTC. The shuttle plasmid vector was synthesized by combining a gene encoding an HSV-TK variant in which alanine at position 167 of HSV-TK of SEQ ID NO: 15 was replaced with tyrosine and a firefly luciferase reporter (p7.5 promoter) gene, and these genes were recombined into a pUC57amp+ plasmid (Genewiz, USA). Subsequently, a mutant vaccinia virus was prepared in the same manner as in Example 1.2 using the shuttle plasmid vector and the Western Reserve strain (ATCC) or the Wyeth strain vaccinia virus. The mutant vaccinia virus prepared using the Western Reserve strain vaccinia virus was designated "WOTS-418", and the mutant vaccinia virus prepared using the Wyeth strain vaccinia virus was designated "OTS-418".

[0082] As a result, luciferase activity was confirmed in both the supernatant and pellet separated from the culture solution of the HeLa cancer cell line treated with the shuttle plasmid vector and the Western Reserve strain vaccinia virus (Figs. 15 and 16). In addition, luciferase activity was confirmed in the supernatant separated from the culture solution of the HeLa cancer cell line treated with the shuttle plasmid vector and the Wyeth strain vaccinia virus (Fig. 17). From these results, it was confirmed that the gene encoding the HSV-TK variant was introduced into the Western Reserve strain or the Wyeth strain vaccinia virus.

[0083] Example 2. Confirmation of growth inhibition of mutant vaccinia viruses (C1, C3, C45, C52) after GCV administration (in vitro) In Example 1.2, the inhibitory effect of GCV on the growth ability of C1, C3, C45 and C52 was confirmed. Here, the mutant vaccinia virus in colony S3C4#1_C19 was used as a negative control and named "C19". To confirm the difference in the number of virus particles after C1, C3, C19, C45 or C52 was treated with GCV, quantitative PCR analysis (qPCR) was performed using the E9L gene that is specifically expressed only in vaccinia virus.

[0084] Specifically, probes (SEQ ID NOs: 19 and 20) that recognize the E9L gene while binding to only one of the two complementary strands of DNA were prepared. The prepared probes measured one luminescence each time virus growth occurred. The NCI-H460 cancer cell line was seeded at 1.5×10 4 cells / well, and then infected with virus C1, C3, C19, C45 or C52 at an MOI of 0.01 - 1. After 2 hours, co-treatment with GCV at a concentration of 60 μM was performed. After culturing for 48 hours, extraction was performed using a virus extraction kit (QIAamp MinElute Virus Spin, QIAGEN, 57704). The extracted DNA was diluted to a concentration of 1 ng / 5 μl and subjected to qPCR.

[0085] As a result, for C19, virus growth was not inhibited despite GCV administration. On the other hand, for C1, C3, C45 and C52, virus growth was inhibited following GCV administration (Figure 18). From these results, it was confirmed that the growth ability of C1, C3, C45 and C52 decreased due to GCV administration.

[0086] Example 3. Confirmation of decreased growth ability of mutant vaccinia viruses (C40, C57) after GCV administration (in vitro) The growth capabilities of C40 and C57 produced in Example 1.2 were confirmed by administering GCV. To examine the changes in the growth levels of C40 or C57 when treated with GCV, quantitative PCR analysis (qPCR) was performed using the E9L gene.

[0087] Specifically, a probe that recognizes the E9L gene was prepared while binding to only one of the two complementary strands of DNA. The prepared probe was one that measured one luminescence each time viral growth occurred. The HeLa cancer cell line was seeded at 1×10 4 cells / well, or the NCI-H460 cancer cell line was seeded at 1.5×10 4 cells / well. Subsequently, the cells were infected with virus C40 or C57 at an MOI of 0.1 (0.1 pfu / cell). Two hours later, co-treatment with GCV at a concentration of 50 μM was performed. After culturing for 48 hours, DNA was extracted using a virus extraction kit. The extracted DNA was diluted to a concentration of 1 ng / 5 μl and subjected to qPCR.

[0088] As a result, for C40 and C57, it was confirmed that viral growth was suppressed by GCV administration (Figs. 19 and 20). From these results, it was confirmed that the growth capabilities of C40 and C57 decreased due to GCV administration.

[0089] Example 4. Confirmation of the cytotoxicity of mutant vaccinia viruses (C1, C3, C45, C52) after GCV administration (in vitro) C1, C3, C45, and C52 showed growth inhibition by GCV in Example 2. To confirm whether C1, C3, C45, and C52 maintain cytotoxicity upon administration of GCV, co-treatment of C1, C3, C19, C45, or C52 with GCV was performed for comparison of cytotoxicity. Specifically, the NCI-H460 cancer cell line was seeded at 1.5×10 4Cells were seeded in wells and then infected with C1, C3, C19, C45 or C52 at an MOI of 0.01 - 1. After 2 hours, co-treatment was performed with GCV at a concentration of 60 μM. After culturing for 72 hours, cytotoxicity was analyzed using a CCK8 kit (Cell Counting Kit8, Tongrentang, Kumamoto, Japan).

[0090] As a result, when co-treatment with C1, C3, C45 or C52 and GCV was performed, the NCI-H460 cancer cell line was killed even more, by about 25% or more, despite the inhibition of virus growth by GCV in C1, C3, C45 and C52. On the other hand, when co-treatment with C19 and GCV was performed, the NCI-H460 cancer cell line was killed to the same extent as when treated with C19 alone (Figure 21). From these results, it was confirmed that the cytotoxicity of C1, C3, C45 or C52 increased when co-administered with GCV.

[0091] Example 5. Confirmation of cytotoxicity of mutant vaccinia virus (C40, C57) after administration of GCV (in vitro) Although C40 and C57 showed inhibition of virus growth by GCV in Example 3, co-treatment with C40 or C57 and GCV was performed to confirm whether C40 and C57 maintain cytotoxicity upon administration of GCV, and their cytotoxicity was analyzed.

[0092] Specifically, the HeLa cancer cell line was seeded at 1×10 4 cells / well, or the NCI-H460 cancer cell line was seeded at 1.5×10 4 cells / well. Then, co-treatment with C40 or C57 at an MOI of 0.1 (0.1 pfu / cell) and GCV at a concentration of 50 μM was performed for 2 hours to infect the cells. After culturing for 48 hours, cytotoxicity was analyzed using a CCK8 kit (Cell Counting Kit8).

[0093] As a result, when co-treatment with C40 or C57 and GCV was performed, although virus growth inhibition by GCV was observed in C40 and C57, the HeLa cancer cell line and the NCI-H460 cancer cell line were killed to the same extent as when treated with C40 or C57 alone (Figs. 22 and 23). From these results, it was confirmed that when C40 or C57 and GCV were co-administered, the cytotoxicity of C40 or C57 increased.

[0094] Example 6. Confirmation of cytotoxicity of mutant vaccinia virus (WOTS-418) (in vitro) To confirm the cytotoxicity of WOTS-418 prepared in Example 1.3 against cancer cells, 10 cancer cell lines, namely human lung cancer cell lines (A549, NCI-H460), human kidney cancer cell lines (A498, Caki-1), human colorectal cancer cell lines (HT-29, HCT116), human breast cancer cell lines (MDA-MB-231, MCF), mouse breast cancer cell line (4T1), and mouse kidney cancer cell line (Renca), were seeded in a 96-well plate at 3×10 3 cells / well, and the cells were treated with WOTS-418 at 1 MOI (1 pfu / cell). After culturing for 72 hours, cytotoxicity was analyzed using a CCK8 kit (Cell Counting Kit8). Here, the human lung cancer cell line (A549) and the mouse breast cancer cell line (4T1) were obtained from the American Type Culture Collection (ATCC). Also, from the Korea Cell Line Bank (KCLB), human kidney cancer cell line (A498), human colorectal cancer cell lines (HT-29, HCT-116), human lung cancer cell line (NCI-H460), human breast cancer cell lines (MDA-MB-231, MCF), and mouse kidney cancer cell line (Renca) were obtained.

[0095] As a result, cytotoxicity of 60% or more was observed in human lung cancer cell line (A549), human kidney cancer cell lines (A498, Caki-1), human colorectal cancer cell lines (HT-29, HCT-116), and human breast cancer cell lines (MDA-MB-231, MCF) (Fig. 24).

[0096] Example 7. Confirmation of reduced growth ability of mutant vaccinia virus (WOTS-418) after administration of ACV, GCV, or BrdU (in vitro) The growth ability of WOTS-418 prepared in Example 1.3 was confirmed upon administration of GCV. To examine the difference in the number of virus particles after treating WOTS-418 with GCV, quantitative PCR analysis (qPCR) was performed using the E9L gene.

[0097] Specifically, a probe that recognizes the E9L gene was prepared while binding to only one of the two complementary strands of DNA. The prepared probe measured one luminescence each time virus growth occurred. The A549 cancer cell line was seeded at 1×10 4 cells / well and then infected with WOTS-418 at 0.1 MOI (0.1 pfu / cell). After 2 hours, co-treatment was performed with ACV, GCV, or BrdU at concentrations of 200 μM or 300 μM. After culturing for 48 hours, DNA was extracted using a virus extraction kit. The extracted DNA was diluted to a concentration of 1 ng / 5 μl and subjected to qPCR.

[0098] As a result, it was confirmed that in WOTS-418, virus growth was significantly inhibited after co-administration of ACV, GCV, or BrdU (Figure 25).

[0099] Furthermore, the NCI-H460 cancer cell line was seeded at 1.5×10 4 cells / well and then infected with WOTS-418 at 0.1 MOI (0.1 pfu / cell). After 2 hours, co-treatment was performed with GCV at a concentration of 100 μM. After culturing for 48 hours, DNA was extracted using a virus extraction kit. The extracted DNA was diluted to a concentration of 1 ng / 5 μl and subjected to qPCR.

[0100] As a result, it was confirmed that for WOTS-418, virus growth was significantly suppressed after co-administration of WOTS-418 and GCV (Figure 26). From these results, it was confirmed that WOTS-418 is sensitive to ACV, GCV, and BrdU and shows a decrease in growth ability upon administration of ACV, GCV, or BrdU.

[0101] Example 8. Confirmation of reduced growth ability of mutant vaccinia virus (WOTS-418) after GCV administration (in vitro) The growth ability of WOTS-418 was confirmed by qPCR analysis in a mouse (Balb / c nu / nu) model transplanted with human colorectal cancer cell line (HCT-116, 5×10 6 cells / ml).

[0102] Specifically, mice (balb / c nu / nu) purchased from KOATECH (Korea) were subjected to a 1-week acclimation period, and then the HCT-116 cancer cell line, a human colorectal cancer cell line (Korea Cell Line Bank), was xenografted with 5×10 6 cells. The tumor volume was observed until it reached 100 mm 3 ~150 mm 3 and then WOTS-418 and GCV were co-administered intratumorally. Here, WOTS-418 (1×10 6 pfu) was administered intraperitoneally. From 4 days later, GCV (50 mg / kg) was administered once a day for 3 days (D5, D6, D7). From the 5th day, three mice in each group were sacrificed and the tumors were isolated. The isolated tumors were homogenized using a sample homogenization system (OMNI Bead Ruptor24), and then the number of virus particles was quantified by qPCR.

[0103] As a result, it was confirmed that virus growth inhibition by GCV treatment occurred from the 3rd day (Figure 27).

[0104] Example 9. Confirmation of cytotoxicity of mutant vaccinia virus (WOTS-418) (in vitro) Although WOTS-418 showed growth inhibition by GCV in Examples 7 and 8, to confirm whether this virus maintains cytotoxicity upon administration of GCV, co-treatment with WOTS-418 and GCV was performed and cytotoxicity was compared. Specifically, the NCI-H460 cancer cell line was seeded at 1.5×10 4Cells were seeded in wells. Subsequently, the cells were infected with WOTS-418 at 0.02 MOI (0.02 pfu / cell). After 2 hours, co-treatment was performed with GCV at a concentration of 60 μM or 100 μM. After culturing for 72 hours, cytotoxicity was analyzed using a CCK8 kit (Cell Counting Kit8).

[0105] As a result, when co-treated with WOTS-418 and GCV, the NCI-H460 cancer cell line was killed more by about 30% or more despite the inhibition of virus growth by WOTS-418 by GCV (Figure 28). From these results, it was confirmed that when WOTS-418 and GCV were co-administered, the cytotoxicity of WOTS-418 increased.

[0106] Example 10. Confirmation of cytotoxicity of mutant vaccinia virus (OTS-418) (in vitro) To confirm the cytotoxicity of OTS-418 upon administration of GCV, co-treatment with OTS-418 and GCV was performed and cytotoxicity was compared. Specifically, the NCI-H460 cancer cell line was seeded at 3×10 3 cells / well. Subsequently, the cells were infected with OTS-418 at 0.02 MOI (0.02 pfu / cell). After 2 hours, co-treatment with GCV at a concentration of 6 μM or 60 μM was performed. After culturing for 72 hours, cytotoxicity was analyzed using a CCK8 kit (Cell Counting Kit8).

[0107] As a result, when co-treated with OTS-418 and GCV, it was confirmed that the cytotoxicity of OTS-418 was increased by GCV. In particular, when co-treated with OTS-418 and GCV at a concentration of 60 μM, the NCI-H460 cancer cell line was killed more by about 20% or more (Figure 29). From these results, it was confirmed that cytotoxicity increased when OTS-418 and GCV were co-administered.

[0108] Example 11. Confirmation of anti-cancer effects of recombinant vaccinia virus (WOTS-418) and hydroxyurea in human breast cancer cell line xenograft mice: MDA-MB-231 Balb / c nu / nu mice (female, 8 weeks old) purchased from ORIENT BIO (Busan, South Korea) were subjected to a one-week acclimation period and then xenografted with 5 × 10 6 cells of the human breast cancer cell line (MDA-MB-231). Observation was carried out until the tumor volume reached 50 mm 3 , and drug administration was started. After 31 days, the tumor volume was measured.

[0109] The mice transplanted with the prepared human breast cancer cell line were divided into 4 groups (n = 5). The group administered with physiological saline intraperitoneally was used as the control group, and the groups administered with hydroxyurea (HU, 30 mg / kg), WOTS-418 (1 × 10 7 pfu), and the group administered with both WOTS-418 and HU were used as the experimental groups. WOTS-418 was administered intraperitoneally a total of 2 times, on day 0 and intratumorally on day 10 (D0, D10). Hydroxyurea was administered intraperitoneally twice a day every day (6 times / week) (Figure 30).

[0110] The tumor volume was measured for 31 days. As a result, in the control group, the tumor volume increased slowly, increased dramatically after the 17th day, and the tumor volume on the 31st day was more than 6 times larger than the initial tumor volume. Also, in the mice of the HU administration group, the tumor volume increased gradually, and the tumor volume on the 31st day increased to more than 5 times the initial tumor volume.

[0111] On the other hand, regarding the tumor volume of the mice in the experimental group administered with WOTS-418, the growth of the tumor was suppressed from the 14th day, and the tumor did not change until the 31st day. Also, regarding the tumor volume in the mice of the experimental group administered with WOTS-418 and hydroxyurea, the growth of the tumor was suppressed from the 17th day, and a tendency for the tumor volume to decrease was observed (Figure 31).

[0112] From these results, it was found that WOTS-418 has an effect of suppressing tumor growth. When treated with hydroxyurea alone, tumor growth was not suppressed, but when WOTS-418 and hydroxyurea were co-administered, it was confirmed that the anti-cancer effect was improved compared to the case of treatment with WOTS-418 or hydroxyurea alone.

[0113] Example 12. Confirmation of the Anticancer Effects of Recombinant Vaccinia Virus (WOTS-418) and Hydroxyurea in Mouse Renal Cancer Cell Line-Transplanted Mice: Renca Balb / c nu / nu mice (female, 8 weeks old) purchased from ORIENT BIO (Busan, Korea) were subjected to a 1-week acclimation period and then orthotopically transplanted with a mouse renal cancer cell line (Renca, Korea Cell Line Bank). The tumor volume was observed until it reached 100 mm 3 and drug administration was initiated. After 28 days, the tumor volume was measured.

[0114] The prepared mouse renal cancer cell line-transplanted mice were divided into 3 groups (n = 7). The group administered with physiological saline intraperitoneally was used as the control group, and the groups administered with WOTS-418 and hydroxyurea (1×10 6 pfu, 30 mg / kg) once and the groups administered with WOTS-418 and hydroxyurea (1×10 6 / 1×10 7 pfu, 30 mg / kg) repeatedly were used as the experimental groups. WOTS-418 was administered intraperitoneally on day 0 and day 14, and hydroxyurea was administered intraperitoneally twice a day (6 times / week).

[0115] As a result, it was confirmed that in the mice of the experimental groups administered with WOTS-418 and hydroxyurea, the tumor volume was significantly suppressed compared with the control group, and the tumor volume was further suppressed in the repeatedly administered group compared with the single-dose group. Also, in the repeatedly administered group, it was confirmed that the higher the concentration at the second administration of WOTS-418, the stronger the anticancer effect (Figure 32).

[0116] Example 13. Confirmation of the Anticancer Effects of Recombinant Vaccinia Virus (WOTS-418) and Hydroxyurea in Mouse Colorectal Cancer Cell Line-Transplanted Mice: CT-26 Example 13.1. Preparation of Mouse Colorectal Cancer Cell-Transplanted Mice and Drug Administration Balb / c mice (female, 8 weeks old) purchased from ORIENT BIO (Busan, Korea) were subjected to a 1-week acclimation period and then xenografted with a mouse colorectal cancer cell line (CT-26, Korea Cell Line Bank). The tumor volume was observed until it reached 100 mm 3 and drug administration was started.

[0117] The prepared mice transplanted with mouse colorectal cancer cells were divided into 4 groups (G1, G2, G3: n = 15, and G4: n = 14). The group administered with physiological saline intraperitoneally was used as the control group, the group administered with hydroxyurea (30 mg / kg) only intraperitoneally, the group administered with WOTS-418 only twice intraperitoneally (1×10 8 pfu, intraperitoneal (i.p.), D0, D3) and the group administered with it twice intratumorally (1×10 7 pfu, intratumoral (i.t.), D14, D21), and the group co-administered with WOTS-418 and hydroxyurea at the same dose and regimen was used as the experimental group. After administering WOTS-418 intraperitoneally on days 0 and 3, it was administered intratumorally on days 14 and 21, and hydroxyurea was administered intraperitoneally once a day (6 times a week) from immediately before the virus administration until day 26 (Figure 33).

[0118] On day 25, the tumor volume was measured and the mice were sacrificed. Then, immune cells were isolated from them and subjected to an interferon-γ assay.

[0119] Example 13.2 Confirmation of changes in tumor volume In Example 13.1, drugs were administered to the mice in each group, and the tumor volume was measured on day 25. As a result, it was confirmed that the tumor volume of the experimental groups administered with WOTS-418 only, the experimental group administered with hydroxyurea only, and the experimental group co-administered with WOTS-418 and hydroxyurea was suppressed compared with the control group. In particular, it was confirmed that the tumor volume of the mice in the experimental group co-administered with WOTS-418 and hydroxyurea was significantly suppressed (Figure 34).

[0120] Example 13.3 Interferon-γ assay Mice in each group of Example 13.1 were administered with drugs. Then, immune cells were isolated from them and subjected to an interferon-γ assay.

[0121] Specifically, CD8+ T cells in the spleen of mice in each group were isolated using the (Ly-2) MicroBeads kit and magnetic-activated cell sorting (MACS), and then co-cultured with the CT-26 cell line (1×10 4 cells). CD8+ T cells secreting IFN-γ were subjected to an ELISPOT (MABTECH, Sweden) experiment, the spots were scanned, and counted by LK Bioscience (Seoul, Korea).

[0122] As a result, compared with CD8+ T cells isolated from the spleen of control mice, CD8+ T cells secreting IFN-γ were confirmed to be abundant in CD8+ T cells isolated from the spleen of mice in the experimental group administered only with WOTS-418, the experimental group administered only with hydroxyurea, and the experimental group co-administered with WOTS-418 and hydroxyurea. In particular, it was confirmed that the tumor volume was significantly suppressed in the mice in the experimental group co-administered with WOTS-418 and hydroxyurea (Figs. 35 and 36).

Claims

1. A oncolytic virus comprising a nucleotide sequence encoding a polypeptide containing an effector domain, wherein the polypeptide consists of an amino acid sequence represented by SEQ ID NO: 3, 5, 7, 9, 11, or 13, and the nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 13 contains a nucleotide substitution from CCC to CTC at positions 551 to 553, the effector domain is represented by SEQ ID NO: 1 and is derived from herpes simplex virus thymidine kinase (HSV-TK), the oncolytic virus is derived from vaccinia virus.

2. The oncolytic virus according to claim 1, wherein the HSV is herpes simplex virus type 1 (HSV1).

3. The oncolytic virus according to claim 1 or 2, wherein the polypeptide further has a sequence of 36, 82, or 231 amino acids linked to the C-terminus of the effector domain.

4. The oncolytic virus according to any one of claims 1 to 3, wherein the nucleotide sequence encoding the polypeptide is a nucleotide sequence represented by SEQ ID NO: 4, 6, 8, 10, 12, or 14.

5. A pharmaceutical composition for preventing or treating cancer, comprising the oncolytic virus according to any one of claims 1 to 4 as an active ingredient.

6. The pharmaceutical composition according to claim 5, wherein the cancer is any one selected from the group consisting of lung cancer, colorectal cancer, prostate cancer, thyroid cancer, breast cancer, brain tumor, 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, bile duct cancer, pancreatic cancer, non-small cell lung cancer, bone cancer, intraocular melanoma, perianal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, small intestine cancer, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, kidney cancer, ureteral cancer, renal cell cancer, renal pelvis cancer, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, and combinations thereof.

7. An oncolytic virus according to any one of claims 1 to 4; and ganciclovir (GCV) or acyclovir (ACV), as an active ingredient, A pharmaceutical composition for preventing or treating cancer.

8. The pharmaceutical composition according to claim 7, wherein the oncolytic virus and GCV or ACV contained in the pharmaceutical composition are administered simultaneously or sequentially.

9. The pharmaceutical composition according to claim 7 or 8, wherein GCV or ACV is administered at a dose of 0.1 μg / kg / day to 50 mg / kg / day.

10. The cancer is any one selected from the group consisting of lung cancer, colorectal cancer, prostate cancer, thyroid cancer, breast cancer, brain tumor, head and neck cancer, esophageal cancer, skin cancer, thymic cancer, gastric cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, non-small cell lung cancer, bone cancer, intraocular melanoma, perianal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, small intestine cancer, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft tissue cancer, urethral cancer, penile cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, kidney cancer, ureteral cancer, renal cell cancer, renal pelvis cancer, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, and combinations thereof. The pharmaceutical composition according to any one of claims 7 to 9.

11. A method for producing an oncolytic virus, comprising: i) removing the TK gene from the virus and recombining the virus with a wild-type HSV-TK gene; and ii) performing continuous passage culture of the recombinant virus in a host cell in the presence of bromodeoxyuridine (BrdU), wherein the virus comprises a nucleotide sequence encoding a polypeptide comprising an effector domain, the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3, 5, 7, 9, or 11, and the effector domain is represented by SEQ ID NO: 1 and is derived from herpes simplex virus thymidine kinase (HSV-TK).

12. The method according to claim 11, wherein the virus is vaccinia virus.

13. The method according to claim 11 or 12, wherein the wild-type HSV-TK gene is the nucleotide sequence represented by SEQ ID NO:

16.

14. The method according to any one of claims 11 to 13, wherein the oncolytic virus has no TK activity and phosphorylates GCV or ACV.

15. Use of the oncolytic virus according to any one of claims 1 to 4 for the manufacture of a medicament for treating cancer.

16. Use of a pharmaceutical composition according to any one of claims 7 to 10 for the manufacture of a medicament for treating cancer.

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