KR101605176B1 - Novel cancer favored oncolytic virus and use thereof - Google Patents

Abstract

The present invention relates to a novel vaccinia virus, which is deficient in thymidine kinase from recombinant vaccinia virus SLJ496, and to a composition for preventing or treating cancer, comprising the same. In addition, the present invention relates to a method for manufacturing a novel vaccinia virus with excellent effects in inhibiting cancer metastasis and oncotropic properties on the basis of the recombinant vaccinia virus SLJ496. According to the present invention, the novel vaccinia virus has high oncotropic properties, and exhibits the activities of inhibiting the cancer metastasis. Particularly, the novel vaccinia virus exhibits effective cytotoxicity in a cancer cell strain which exhibits anticancer drug resistance. Therefore, the novel vaccinia virus can be used as a virus drug, which exhibits significantly higher effects than conventional vaccinia virus.

Description

[0001] The present invention relates to novel anti-cancer viruses having high tumor affinity,

The present invention relates to a composition for preventing or treating a novel vaccinia virus and cancer comprising the same, wherein the thymidine kinase is deleted in the recombinant vaccinia virus SLJ496.

The present invention also relates to a method for producing a novel vaccinia virus having excellent tumor affinity and cancer metastasis inhibitory effect based on recombinant vaccinia virus SLJ496.

The first example of a medical use of a virus is the development of a vaccine against naturally occurring poxviruses for the prevention of smallpox. Until the vaccine against vaccinia was developed, smallpox was the most deadly disease in human history, causing 10% mortality and permanent damage to skin for thousands of years. In the 1700s Edward Zenner first showed that a smallpox virus could become a vaccine that suppresses the onset of smallpox when it grows in cattle (vaccinia virus), producing cross-immunity with smallpox viruses, without lethal effects on humans . Since then, it has been named the vaccinia virus, and in 1979 the World Health Organization (WHO) became the occasion to declare that the smallpox disease was conquered.

As a result of recent molecular biologic developments, the genetic comparison between smallpox virus and vaccinia virus has shown that the two viruses are genetically very similar and that the vaccinia virus is the first virulent virus produced by a selective evolutionary method It was found to be a virus antivirus. Based on this principle, techniques for selectively evolving attenuated viruses in vitro have been used to develop preventive vaccines such as polio, measles, and mumps. The basic principle of the vaccine using the attenuated virus is that when the pathogenic virus is isolated from human cells and continuously cultured in xenogeneic cells such as monkeys, the virus selectively accumulating in the xenogeneic cells is selectively accumulated, Although the proliferation is good in xenogeneic cells, attenuated viruses are produced in human cells, and these attenuated viruses do not cause disease in the human body, but cause wild-type pathogenic viruses and cross-immune functions, .

Along with the development of molecular biology, various kinds of viruses that have been recently used as vaccines are being genetically modified using genetic engineering techniques to develop them as anticancer drugs. David Kirn et al. Have developed ONYX-015, an anticancer drug that uses tumor-selective replication of adenovirus variants, and have conducted clinical phase II studies. However, the anticancer effect of the primary tumor-killer adenovirus was not sufficient to be used clinically, and it was reported that the repeated administration of the tumor-associated adenovirus was rapidly eliminated by the host immune system and the anti-cancer effect was decreased. Despite these limitations, it can be seen that the therapeutic efficacy of the tumor-killer adenovirus principle has been commercialized in China, reflecting the expectation of the development of tumor-killer viruses with improved clinical efficacy. In recent years, the selective development of ONYX-015 has led to the successful development of a more potent anticancer virus, and clinical trials are currently underway in the United States.

On the other hand, the tumor-shrinking viruses derived from Vaccinia virus are superior to adenoviruses in terms of anticancer function. Currently, the multinational clinical phase 2 is completed in the USA, Canada and Korea for liver cancer, colon cancer and skin cancer patients. It is in preparation. However, when repeated administration of blood vessels occurs, the anticancer efficacy is deteriorated due to the formation of neutralizing antibodies, so that there is a limit in continuous administration, and development of more advanced tumor-specific viruses is urgently required.

Application No. 10-2010-0064718

Accordingly, the present inventors have conducted a study to develop a novel vaccinia virus that solves the above problems and has improved anticancer effect. As a result, it has been found that the virus is able to induce the deficiency of the thymidine kinase in the recombinant vaccinia virus SLJ496 And found that Mars is greatly increased, and the present invention has been completed.

Accordingly, one aspect of the present invention is to provide a novel vaccinia virus.

Another aspect of the present invention is to provide a composition for preventing or treating cancer comprising Vaccinia virus.

Another aspect of the present invention is to provide a method for producing a new vaccinia virus based on recombinant vaccinia virus SLJ496.

One aspect of the present invention provides a novel vaccinia virus wherein the thymidine kinase is defective in the recombinant vaccinia virus SLJ496.

Hereinafter, the present invention will be described in detail.

Vaccinia virus is a large, complex, enveloped virus that has about 190 Kbp of the linear double helix DNA gene and codes for about 250 genes. The role of vaccinia virus as a vaccine against smallpox is well known.

After the eradication of smallpox, scientists have been studying the use of vaccinia virus as a tool to deliver genes into biological tissues (gene therapy and genetic engineering). Vaccinia virus is unique among DNA viruses because it replicates only in the cytoplasm of host cells. Thus, large genomes are required to code viral enzymes and proteins necessary for viral DNA replication. During the replication process, Bacillus cereus has several forms of infection that differ from their outer membranes: intracellular mature virion (IMV), intracellular epithelial virion (IEV), cell associated epithelial virion (CEV) (EEV). IMV is the most abundant form of infection and is thought to be involved in host-to-host distribution. On the other hand, CEV seems to play a role in the distribution of cells and cells, and EEV is thought to be important for long range propagation in host organism.

Bacsinia encodes several proteins that produce interferon-resistant viruses. Among them, K3L is a protein having homology with eIF-2α. The K3L protein inhibits the action of PKR, an activator of interferon. E3L is another vaccinia protein that inhibits PKR activity, which can also bind to double-stranded RNA.

In the present invention, vaccinia virus is a novel virus that deletion of thymidine kinase in the known virus, SLJ496.

Vaccinia virus SLJ496 (KCTC 11704BP) is an anticancer virus isolated from blood when viruses are detected in the blood after tumor infiltration of Vaccinia virus into a host animal having tumor tissue under immunosuppressive conditions and the tumor size of the animal decreases.

The 'Vaccinia virus' strain may be Western Reserve, Copenhagen, Wyeth, but is particularly preferred. The vaccinia virus may be naturally occurring or genetically modified.

The term "host animal" is any host animal (mammal or bird) of Vaccinia virus, but it may be any kind of animal, but is preferably a horse, a cow, a goat, a sheep, a dog, a chicken, a turkey, a rabbit, a mouse, Rabbits are particularly preferred.

The 'tumor tissue' may be, but not limited to, gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, skin cancer, colon cancer or cervical cancer.

In one embodiment, the 'host animal with tumor tissue' may be a rabbit with VX2 cancer cells. This rabbit is a host animal in which preclinical toxicity and preclinical pharmacological testing of JX-594 has been performed, and the proliferation of vaccinia virus is known to be similar to that of humans.

The 'immunosuppression condition' may be variously set depending on the kind of the host animal and the immunosuppressant. Immunosuppression conditions can be induced by administering one or more immunosuppressive agents to the host animal. For example, cyclosporine and prednisone may be combined to induce immunosuppressive conditions. In this case, cyclosporin at 3-5 mg / kg / 2d and prednisone at 0.5-1.5 mg / kg / , But is not limited thereto.

On the other hand, in order to prepare SLJ496, it is preferable to inject low-concentration vaccinia virus into a host animal having a tumor tissue, and it is particularly preferable to inject vaccinia virus at a concentration of 10 ^{7 to 9} . SLJ496 can be isolated from all tissues in which the virus is detected, including the tumor tissue of the host animal, but is preferably isolated from the blood.

In one embodiment of the invention, vector is used to disrupt thymidine kinase in SLJ496. The homologous portion between the DNA sequence of the vector and the sequence of the vaccinia virus is exchanged by introducing a homologous sequence and a vector in which a foreign gene is inserted between both (left and right) of the gene of thymidine kinase, By introducing the foreign gene into the region of the kinase, a new vaccinia virus in which the thymidine kinase is deleted can be obtained.

The vector may be a shuttle vector, and preferably consists of a cleaved map of FIG.

The vaccinia virus is characterized in that the gene encoding the thymidine kinase of SEQ ID NO: 1 is missing. The entire sequence of thymidine kinase may be represented by SEQ ID NO: 5.

The novel vaccinia virus according to the present invention has high tumor affinity and exhibits an activity of inhibiting cancer metastasis. In particular, it shows effective cytotoxicity even in cancer cell lines showing resistance to anticancer drugs, and thus can be used as a viral therapeutic agent which shows a significantly higher effect than conventional vaccinia viruses.

Accordingly, one aspect of the present invention provides a composition for preventing, ameliorating or treating cancer comprising Vaccinia virus.

The composition may be a pharmaceutical composition or a food composition.

Namely, the novel vaccinia virus according to the present invention can be used for the treatment and prevention of hyperproliferative diseases such as cancer. Examples of the diseases to be considered for treatment include gallbladder cancer, colon cancer, pancreatic cancer, leukemia, ovarian cancer, stomach cancer, lung cancer, breast cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, skin cancer, colon cancer, cervical cancer, head and neck cancer, But are not limited to, bone cancer, testicular cancer, gastrointestinal cancer, lymphoma, precancerous lesions in the lung, melanoma, bladder cancer and any other cancer or tumors that can be treated.

An effective amount of a pharmaceutical composition in the present invention is defined as being sufficient to induce tumor killing or destruction or dissolution of cancer cells as well as to slowly inhibit or reduce tumor growth or size thereof, .

The route of administration of the compositions of the present invention will vary according to the location and nature of the lesion and can be varied depending on the location and nature of the lesion and can be varied depending on the location and nature of the lesion, , Intramuscular, intraperitoneal, intra-arterial, intravesical, intratumor, inhalation, perfusion, oral, and oral administration and formulations, which are in close proximity to the tumor, particularly through blood vessels or adjacent blood vessels of the tumor.

Intratumoral injection or direct injection into tumor blood vessels is considered especially for isolated, accessible solid tumors. Topical, regional or systemic administration may also be appropriate. For tumors larger than 4 cm, the volume administered will be about 4 to 10 ml (preferably 10 ml), and for tumors smaller than 4 cm, a volume of about 1 to 3 ml (preferably 3 ml) Will be used. Multiple infusions delivered in a single dose comprise from about 0.1 to about 0.5 ml volumes. Viral particles can be advantageously contacted through multiple infusion administrations into tumors spaced about 1 cm apart. In the case of surgical intervention, the present invention enables the resection of tumor subjects that are used prior to surgery and are not operable. For example, continuous administration by inserting a catheter into a tumor or tumor vasculature may also be applied where appropriate. Such continuous perfusion may be from about 1 to 2 hours, from about 2 to 6 hours, from about 6 to 12 hours, from about 12 to 24 hours, from about 1 to 2 days, from about 1 to 2 weeks, or Can be done for a longer time. In general, the dosage of the composition through continuous perfusion will be equivalent to that given through single or multiple infusion, which is controlled over time during perfusion. It is also contemplated that limb perfusion may be used to administer compositions of the invention, particularly in the treatment of melanoma and sarcoma.

Therapeutic regimens can also vary, and often depend on tumor type, tumor location, disease progression, and patient health and age.

Therapeutic regimens may include various "unit dose ". Unit dosages are defined as containing a predetermined amount of the composition. The amount administered and the particular route and formulations are conventional to those of ordinary skill in the clinical arts. Unit dosages need not be administered as a single infusion and may include continuous infusion over a set period of time. A unit dose of the present invention can be described typically as plaque forming units (pfu) for viral constructs. The unit dose is in the range of 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ pfu or more. In addition, depending on the type of virus and the attainable titer, it may be 1 to 100, 10 to 50, 100 to 1000 or about 1 x 10 ⁴ , 1 x 10 ⁵ , 1 x 10 ⁶ , 1 x 10 ⁷ , 1 x 10 ⁸ , ^{x 10 9, 1 x 10 10} , 1 x 10 11, 1 x 10 12, 1 x 10 13, 1 x 10 14, or 1 x 10 ¹⁵ or less, or at least about ^{1 x 10 4, 1 x 10} 5, 1 x ^{10 6, 1 x 10 7,} 1 x 10 8, 1 x 10 9, 1 x 10 10, 1 x 10 11, 1 x 10 12, 1 x 10 13, 1 x10 14, or 1 x 10 ¹⁵ or more The infectious viral particles (vp) may be delivered to the tumor or tumor site.

A preferred method of delivering an expression construct or virus encoding all or a portion of the novel vaccinia virus genome to a cancer or tumor cell in the present invention is through intratumoral injection and can be carried out by parenteral, intravenous, intradermal, intramuscular, Transdermally or intraperitoneally.

Injection of the nucleic acid construct may be delivered via a syringe or any other method used to inject the solution, as long as the expression construct can pass the specific needle gauge required for injection.

Pharmaceutical forms suitable for injectable use include sterile aqueous liquids or dispersions and sterile powders for the extemporaneous preparation of injectable sterile solutions or dispersions. In all cases, the form must be sterile and must be fluid enough to be readily injectable. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), a suitable mixture thereof, and / or a solvent containing vegetable oil or dispersed daily. Proper fluidity can be maintained, for example, through the use of coatings such as lectins, the maintenance of the required particle size in the case of dispersions, and the use of surfactants. Various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc., can prevent the action of microorganisms. In many cases, it will be desirable to include isotonic agents, for example, sugars or sodium chloride. The absorption of injectable compositions can be extended by the use of absorption delaying agents, for example, aluminum monostearate and gelatin in the composition.

For parenteral administration of aqueous solutions, for example, the solution should be suitably buffered if necessary and the liquid diluent should be made isotonic with sufficient saline or glucose. This particular aqueous solution is particularly suitable for intravenous, intramuscular, subcutaneous, intratumoral and intraperitoneal administration. In addition, sterile aqueous media that may be used will be known to those skilled in the art in view of the present disclosure. For example, a daily dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of hypodermoclysis fluid or injected at the proposed injection site (see, for example, Remington's Pharmaceutical Sciences "15th Edition, pages 1035-1038 and 1570-1580). The dosage will vary depending on the condition of the patient being treated. In any case, the person responsible for the administration will determine the appropriate dosage for the individual patient. Further, in human administration, the formulation must meet the standards of purity required by aseptic, pyrogenic, general safety, and biologics standards of the FDA agency.

The active ingredient in the required amount is incorporated into the appropriate solvent along with the various other ingredients enumerated above, and if necessary after that, filter sterilization is performed to produce an injectable sterile solution. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and the other required ingredients listed above. In the case of sterile powders for the preparation of injectable sterile solutions, preferred methods of manufacture include vacuum drying and lyophilization methods which obtain powders from these pre-sterilized filtered solutions of the active ingredient plus any additional desirable ingredients.

The compositions of the invention may be used in conjunction with other therapies, such as chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy and / or alternative therapies.

Cervical surgery involves resection that partially removes, excises, and / or destroys all cancer tissues or some cancer tissues. Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor resection, surgical treatment includes laser surgery, cryosurgery, electrosurgery, and microscopic controlled surgery (Mohs surgery). It can also be used with the removal of surface tumors, precancerous or accidental amounts of normal tissue.

The composition of the present invention can be used in combination with other agents, and other agents are intended to improve therapeutic efficacy, including, for example, immunomodulatory agents, cell surface receptors and agents that affect upregulation of GAP junctions, cell proliferation inhibitors, Cell adhesion inhibitors, agents that increase the sensitization of hyperproliferative cells to apoptosis inducing agents, or other biological agents. Immunomodulatory agents include tumor necrosis factor; Interferon-alpha, -beta and-gamma; IL-2 and other cytokines; F24K and other cytokine analogs; Or MIP-I, MIP-I-beta, MCP-I, RANTES, and other chemoline. Upregulation of cell surface receptors or their ligands, such as Fas / Fas ligand, DR4 or DR5 / TRAIL (Apo-2 ligand), may also be achieved through the establishment of autocrine or periplasmic secretion on hyperproliferative cells, It is also thought that it will enhance the induction ability. An increase in intracellular signaling through an increase in the number of GAP junctions will increase the anti-hyperplasia effect on neighboring hyperproliferative cell numbers. In another embodiment, a cell proliferation inhibitor or fragmentation agent can be used in conjunction with the present invention to improve anti-hyperprogesteral efficacy. Examples of cell adhesion inhibitors include topically applied kinase (FAK) inhibitors and lovastatin. It is also contemplated that other agents, such as antibody c225, which increase the sensitivity to apoptosis of hyperproliferative cells may be used in conjunction with the present invention to increase therapeutic efficacy. The Apo-2 ligand (also referred to as Apo2L, TRAIL) is a member of the tumor necrosis factor (TNF) cytokine family. TRAIL activates rapid apoptosis in many cancer cell types, but is not toxic to normal cells. TRAIL mRNA is produced in various tissues. Most normal cells appear to be resistant to the cytotoxic effects of TRAIL, suggesting a mechanism to protect against induction of apoptosis by TRAIL. The first receptor described for TRAIL, called death receptor 4 (DR4), contains the cytoplasmic "death domain " and DR4 propagates the apoptotic signal carried by TRAIL. Another receptor was found to bind to TRAIL. One receptor, termed DR5, contains a cytoplasmic killing domain and a signal apoptotic domain very similar to DR4. DR4 and DR5 mRNA are expressed in normal tissues and tumor cell lines. Recently, decoy receptors such as DcR1 and DcR2 have been found to prevent TRAIL from inducing apoptosis via DR4 and DR5. Thus, these attractor receptors represent a novel mechanism that directly regulates the sensitization of cytolines on the cell surface. The desirable expression of these inhibitory receptors in normal tissues suggests that TRAIL may be useful as an anti-cancer agent that induces apoptosis in cancer cells while leaving normal cells (Marsters et al., 1999).

Cytotoxic chemotherapy There has been much progress in cancer treatment after the introduction of drugs. However, one of the consequences of chemotherapy is the development / acquisition of the drug resistant phenotype and the development of multiple drug resistance. The development of drug resistance remains a major obstacle to the treatment of these tumors and therefore a separate approach such as gene therapy is clearly needed.

Another form of therapy for use with chemotherapy, radiation therapy or biological therapy includes hyperthermia, which is a method of exposing the parent tissue to high temperatures (below 106 [deg.] F). External or internal heating devices may be involved in the application of hyperthermia to a local, regional or whole body. Local hyperthermia involves the application of heat to small areas such as the tumor. Heat can be generated externally with high frequency wavelengths that target the tumor from the extracorporeal device. The internal heat may be accompanied by a sterilized probe, including a thin heated wire or hollow tube filled with warm water, an inserted microwave antenna or a high frequency electrode. The patient's organ or limb is heated by regional therapy, which is performed using a device that produces high energy, such as a magnet. Alternatively, after removing some of the patient ' s blood and heating, it may be perfused internally into the area to be heated. Whole body heating can also be performed when the cancer is distributed throughout the body. Heating blanket, hot wax, inductive coil and thermal chamber can be used for this purpose.

Hormone therapy may also be used with or in conjunction with any of the other cancer therapies described above. The use of hormones can be used to treat cancers such as breast cancer, prostate cancer, ovarian cancer, or cervical cancer to lower the concentration of any hormone, such as testosterone or estrogen, or to block its effect. This treatment is used as an alternative therapy or in combination with one or more other cancer therapies to reduce the risk of metastasis.

The novel vaccinia virus according to the present invention has high tumor affinity and exhibits an activity of inhibiting cancer metastasis. In particular, it shows effective cytotoxicity even in cancer cell lines showing resistance to anticancer drugs, and thus can be used as a viral therapeutic agent which shows a significantly higher effect than conventional vaccinia viruses.

1 is a schematic diagram for explaining a process for producing a virus of the present invention.
2 is a clear map of vectors used to produce the virus of the present invention.
FIG. 3 is a graph showing GFP expression in a BSC cell line infected with the virus constructed in the present invention and a normal BSC cell line (not infected) infected with the virus constructed in the present invention.
FIG. 4 is a graph showing that the virus (496GFP) of the present invention has anticancer activity in a colon cancer cell line exhibiting anticancer drug resistance. The virus of the present invention selectively infects colon cancer cell lines and kills the cells in comparison with VR1536, a wild-type vaccinia virus.
(A and B) It is confirmed that the virus (496GFP) of the present invention shows high cytotoxicity in various colorectal cancer cell lines.
(C) It is confirmed that the virus (496GFP) of the present invention exhibits high infectivity and reproducibility in a colon cancer cell line than a primary cultured MEF (mouse embryonic fibroblast), which is a normal cell line.
(D) After injecting the virus (496GFP) of the present invention into the abdominal cavity of a nude mouse, the active virus was traced and it was confirmed that the virus appeared only in the cancer tissue.
FIG. 5 shows that the cytotoxicity of the virus of the present invention (496GFP) in CPT11-resistant cells can be effective on a group of tumor cells similar to stem cells.
(A) high anticancer effect of the virus (496GFP) of the present invention in colorectal cancer cells.
(B) the expression of a stem cell-like cancer marker in colorectal cancer cells and the relationship of the drug resistance gene ABCG2.
FIG. 6 shows that the stem cell-like cancer cells form a sphere but are more sensitive to the virus (496GFP) of the present invention than CPT11.
(A) The expression of the stem cell markers Nanog, Sox2, Oct4, c-Myc.
(B) shows the sphere forming ability of stem cell-like cancer cells (CD133 + CD44 +).
(C) high cytotoxicity of the virus (496GFP) of the present invention against stem cell-like colon cancer spear cells.
Fig. 7 is a graph showing that sphere forming ability is related to stem cell and tumorigenicity of cancer cells. Fig.
(A) shows that stem cell continuity is maintained in accordance with sequential spear formation.
(B) the expression of stem cell markers in sphere using immunofluorescence staining.
(C & D) sphere has high tumor-forming ability.
8 is a graph showing cytotoxicity of virus (496GFP) of the present invention to colon cancer cells (HT-29) and cytotoxicity against mouse embryonic stem cells (mES).
9 is a graph showing the anticancer effect of the virus (496GFP) of the present invention in the HT29 xenograft model. The mice were injected with the virus (496GFP) and after 21 days complete inhibition of the tumor was confirmed. The arrow on the graph represents the 496 GPF scan (it) time point.
10 shows the expression of CD44 in various types of hepatocellular carcinoma (HCC) cell lines.
(A) The expression of CD44 was relatively high in SNU 449 and Sk-Hep1, and this is consistent with the expression of c-Met, a gene showing metastasis.
(B) FACS data showed that expression of CD44 was high in SNU449 and Sk-Hep-1.
Figure 11 is a graph showing that expression of CD44 is associated with hepatic tumorigenesis.
(A) SNU449 and SK-Hep-1.
(B) SNU449 and SK-Hep-1, direct cell injection (2x10 ⁶ cells) induces tumors.
(C) SK-Hep-1 alone showed tumorigenicity 4 weeks after cell scintillation (sc).
Figure 12 shows high cytotoxicity of the vaccinia virus (496GFP) of the present invention in various kinds of HCCs. While most of the HCC expressing CD44 showed high resistance to the anticancer agents sorafenib and cisplatin, the four HCC cell lines showed high sensitivity to 496 GFP.
Figure 13 is a graph showing that CD44 is associated with metastasis in the liver.
(A) High expression of CD44 in HCC was associated with metastasis. SNU449 and SK-HEp1 cells were injected into the large intestine. Five weeks later, the cells showed metastatic appearance.
(B) A decrease in body weight was observed according to the cancer risk.
Figure 14 is a graph showing that the vaccinia virus (496GFP) of the present invention completely inhibits liver tumor formation and colon transposition.
(A) 496GPF and Sorafenib. Fig.
(B) Unlike sorafenib, treatment with 496GFP completely inhibited metastasis.
FIG. 15 shows the results of comparing anticancer efficacy of wild-type vaccinia viruses, JX594 and SLJ496, and vaccinia virus 496GFP of the present invention.

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

Example  One: New Vaccinia  Production and identification of viruses

Thymidine kinase (TK) was removed from SLJ-496 in order to produce a novel vaccinia virus that has enhanced tumor affinity and also has an effect on tumor stem cells or advanced metastatic cancers having anticancer drug resistance.

Specifically, a new vaccinia virus was prepared by the following method.

In order to delete the thymidine kinase in the virus, homologous sequences between the DNA sequence of the vector and the sequence of the vaccinia virus are introduced by introducing a vector having a homologous sequence and a foreign gene therebetween on both sides (left and right) of the gene of thymidine kinase And a foreign gene was inserted at the site of thymidine kinase to obtain vaccinia virus lacking thymidine kinase. This vector contains the sequence Tk-L (Tk-L 4516-4754 of SEQ ID NO: 2) which is identical to the left portion sequence of the thymidine kinase for homologous recombination (homologous recombination) and the sequence homologous to the right sequence has a GFP gene between Tk-L and vTk-R to insert the foreign gene GFP into the locus of thymidine kinase, with vTk-R (vTk-R 901-1120 of SEQ ID NO: 3) And a vaccinia virus promoter P3 for expression of GFP. In order to select and isolate only vaccinia virus deficient in thymidine kinase by the insertion of the vector, gpt which allows growth only in the selective medium was inserted, and the expression of gpt could be carried out by the vaccinia virus promoter P4.

For homologous recombination, BSC-1 cells were used as host cells. Upon reaching 80% confluence, 0.01 MOI of SLJ496 virus was infected into the cells for 1 hour, then the medium was removed and Lipofectamine 2000 (Life Technologies) was used And transformed with the above vector. The transformed mixture was prepared by mixing Lipofectamine 2000 with 1-5 μg of the insertion vector in a serum-free medium to a final volume of 150 μL, and transformed into the virus-infected cells, Lt; RTI ID = 0.0 > recombination < / RTI > After 4 hours, the cells are cultured in a growth medium containing 10% serum for 3 days to induce the generation of new virus. In the selective medium, only new viruses that produce gpt together with GFP can grow, so only new viruses generate plaques. Generation of new vaccinia virus (hereinafter referred to as 496GFP) lacking thymidine kinase was confirmed through the expression of GFP in the plaques generated in the selective medium, and only a single new virus plaque was selected by purifying plaques of three or more steps Can be obtained.

1 is a schematic diagram illustrating a process for producing vaccinia virus of the present invention.

2 is a cleavage map of a vector used for producing vaccinia virus of the present invention. SEQ ID NO: 4 shows nucleotide sequence information of the vector.

In addition, the expression of vaccinia virus of the present invention was confirmed by expression of GFP (green fluorescent protein) contained in the vector. The results are shown in Fig.

As shown in FIG. 3, the expression of GFP in the BSC cell line infected with the novel vaccinia virus constructed in the present invention and in the BSC cell line (uninfected) infected were confirmed.

Example  2: According to the invention Vaccinia  High anticancer efficacy compared to conventional anticancer drugs

In order to confirm whether the vaccinia virus of the present invention can exhibit the anticancer effect in the colon cancer cell line exhibiting the anticancer drug resistance, the anticancer drug resistant cell lines HT-29, DLD-1, SNUC4 and SNUC5 cell lines were obtained in Example 1 The vaccinia virus 496GFP of the present invention was treated. As a control, wild type vaccinia virus VR1536 was treated. The results are shown in Fig.

4 (A) and (B), it can be confirmed that the vaccinia virus of the present invention shows high cytotoxicity to various colon cancer cell lines.

FIG. 4C shows that 496GFP shows high infectivity and reproducibility in colon cancer cell lines compared with MEF (mouse embryonic fibroblast) cultured as a normal cell line.

4 (D), the vaccinia virus of the present invention was injected into the abdominal cavity of the nude mouse, and the active virus was traced. As a result, it can be confirmed that the virus only appears in the cancer tissue.

As shown in FIG. 4, it was confirmed that the vaccinia virus of the present invention has anticancer activity in most of the colon cancer cell lines exhibiting anticancer drug resistance. The vaccinia virus of the present invention selectively infects colon cancer cell lines and kills the cells in comparison with wild type vaccinia virus VR1536.

Example  3: 496 for stem cell-like tumor cell population GFP Anticancer effect

In order to confirm whether the vaccinia virus 496GFP of the present invention exhibits anticancer activity in the colon cancer cells HT-29, LOVO, DND-1 and HCT-116 showing resistance to CPT11, the vaccinia virus 496GFP Was treated with each of the above colorectal cancer cells to confirm cell viability. As a control, each cell was treated with CPT11. The results are shown in Fig.

FIG. 5 (A) is a graph showing a high anti-cancer effect of the vaccinia virus of the present invention in colon cancer cells.

FIG. 5 (B) is a graph showing the relationship between the expression of a stem cell-like cancer marker and the drug resistance gene ABCG2 in colorectal cancer cells. FIG. As shown in FIG. 5 (B), it was confirmed that the ratio of CD133 + CD44 + cells was related to the drug resistance gene ABCG2. In addition, it was confirmed that the vaccinia virus of the present invention exhibited greater cytotoxicity in cells having a high expression ratio of CD133 + CD44 + and ABCG2.

In addition, the relationship between the anti-cancer effect of stem cell-like cancer cells and the vaccinia virus 496GFP of the present invention was confirmed in relation to the sphere formation ability of stem cell-like cancer cells.

The expression of stem cell markers was measured in each of the untreated cell, CD133-CD44-cell group or CD133 + CD44 + cell group, and spear forming ability was confirmed. In addition, anti-cancer effect of 496GFP on spear cells formed by stem cell-like cancer cells was measured.

The results are shown in Fig.

6 (A) shows the expression of the stem cell markers Nanog, Sox2, Oct4, and c-Myc. As shown in FIG. 6 (A), the expression of Nanog, Sox2, Oct4, and c-Myc was found to be higher in the CD133 + CD44 + cell group than in the other cell groups.

FIG. 6 (B) is a diagram showing the sphere forming ability of a stem cell-like cancer cell (CD133 + CD44 +). It was confirmed that the size of the spear was significantly larger in CD133 + CD44 + cells than in CD133-CD44-.

Figure 6 (C) shows the high cytotoxicity of 496GFP to stem cell-like colon cancer spear cells. Spear cells were treated with 496GFP, and cell viability was measured. Control groups were treated with 5-fu and CPT-11. As can be seen in FIG. 6 (C), it was confirmed that 496GFP showed a higher anticancer effect on stem cell-like colon cancer spear cells.

In addition, we confirmed the ability of sphere formation to correlate with stem cell and tumorigenicity of cancer cells. The results are shown in Fig.

FIG. 7 (A) is a diagram showing that stem cell continuity is continuously maintained in accordance with sequential sphere formation. In other words, the expression of the stem cell markers Nanog, Sox2, Oct4, c-Myc was also increased as the spear formation continued.

FIG. 7 (B) shows the results of confirming the expression of stem cell markers in sphere using immunofluorescence staining.

FIGS. 7C and 7D are graphs showing that the cells isolated from the sphere exhibit high tumor-forming ability.

In addition, cytotoxicity of new viruses to cancer stem cells requires confirmation of cytotoxicity against normal stem cells. Thus, the cytotoxicity of the vaccinia virus of the present invention against cancer stem cells and normal stem cells was compared with each other, and the results are shown in FIG.

FIG. 8 is a chart showing cytotoxicity of vaccinia virus of the present invention on colon cancer cells (HT-29) and mouse embryonic stem cells (mES). As shown in FIG. 8, the cytotoxic response to mouse embryonic stem cells, which are normal cells, is insignificant compared with cytotoxicity against HT-29 of Vaccinia virus of the present invention. That is, it was confirmed that the toxicity of vaccinia virus of the present invention to cancer stem cells is due to cytotoxicity due to tumor affinity.

In addition, it was confirmed whether the vaccinia virus of the present invention exhibited anticancer effect in HT29 heterotransplantation model. 9 is a graph showing the anticancer effect of the vaccinia virus of the present invention in the HT29 xenograft model. Mice were vaccinated with vaccinia virus and complete inhibition of the tumor was confirmed after 21 days.

Based on the above results, the resistance of the anticancer agent seems to be related to the tumor stem cells constituting the cancer. However, the vaccine virus 496GFP of the present invention exhibits a high anticancer effect regardless of the resistance of the colon cancer to the existing anticancer agent Thus, it has been confirmed that the tumor-affinity enhanced 496GFP shows an effective anticancer activity in cancer stem cells showing anticancer drug resistance.

Example  4: 496 in advanced metastatic cancer GPF Anticancer effect

Expression of CD44 in various types of hepatocellular carcinoma (HCC) cell lines was confirmed. Expression patterns of CD133, CD44 and c-Met in HepG2, SNU354, SNU449 and sk-Hep1 as hepatocellular carcinoma were confirmed, and the results are shown in Fig.

10 (A) shows that the expression of CD44 is relatively high in SNU 449 and Sk-Hep1, which is consistent with the expression of c-Met, a gene showing metastasis. 9 (B) shows the results of confirming the high expression of CD44 in SNU449 and Sk-Hep-1 on the FACS data.

In addition, the relationship between the expression of CD44 and hepatic tumorigenesis was confirmed. The results are shown in Fig.

FIG. 11 (A) shows the results of confirming that high motility was observed in SNU449 and SK-Hep-1.

Figure 11 (B) shows the results of direct cell injection (2 x 10 ⁶ cells) inducing tumors only in SNU449 and SK-Hep-1.

FIG. 11 (C) shows that only SK-Hep-1 shows tumorigenicity 4 weeks after cell scintillation (sc).

In order to evaluate the cytotoxicity against HCC of Vaccinia virus 496GFP according to the present invention, 496GFP was treated with HepG2, SNU354, SNU449 and sk-Hep1, respectively. As a control, each cell was treated with VR1536, a vaccinia virus wild type, and treated with anticancer agents sorafenib and cisplatin to show anticancer resistance. The results are shown in Fig.

Figure 12 shows the high cytotoxicity of 496GFP in various types of HCC. While most of the HCC expressing CD44 showed high resistance to the anticancer agents sorafenib and cisplatin, all four HCC cell lines showed high sensitivity to 496GFP.

In order to confirm the relationship between expression of CD44 and metastasis, HepG2, SNU354, SNU449, and sk-Hep1 were injected into the large intestine of the animal model, and after 5 weeks, the animal model was sacrificed and its metastasis was evaluated Respectively. Also, the body weight of the animal model for 5 weeks was observed. The results are shown in Fig.

As shown in Fig. 13 (A), SNU449 and SK-HEp1 cells were injected into the large intestine and after 5 weeks, the metastatic appearance was confirmed. This is the result of confirming that high expression of CD44 in HCC is associated with metastasis.

FIG. 13 (B) shows the results of confirming the decrease in body weight in the animal model injected with SNU449 and SK-HEp1 in which the metastatic disease has been induced.

Finally, in order to confirm whether 496GFP inhibits tumor formation in the liver and metastasis to the large intestine, SK-HEp1 cells were directly administered to the liver and 496GFP was administered ip. Inject the Sorafenib o.p. Respectively.

Figure 14 (A) is a schematic diagram of an animal experiment for confirmation of effects on 496 GPF and Sorafenib.

The results are shown in Fig. 14 (B). As shown in Fig. 14, unlike sorafenib, treatment with 496GFP completely inhibited the metastasis.

As described above, it was found that CD44 expression and migration ability and tumor formation ability were correlated with each other on the basis of confirming the correlation between CD44 expression and metastasis-related expression of cMet in liver cancer cell lines there was.

CD44 expressing cell lines expressing metastasis were also relatively resistant to anticancer drugs. However, 496GFP enhanced tumor affinity regardless of cell resistance to chemotherapeutic agents showed excellent anticancer functions.

In addition, in an animal study, sorafenib does not seem to prevent liver-to-colon metastasis, but administration of 496GFP appears to completely inhibit metastasis as well as tumor reduction. In combination therapy, we could not find metastatic cancer as well as in the liver.

Therefore, tumor-affinity enhanced 496GFP showed excellent anticancer function to overcome advanced metastatic cancer.

Example  5: wild type Vaccinia  virus, JX594  And SLJ496 And 496 of the present invention GFP Of anticancer efficacy

The present invention relates to wild-type vaccinia virus VR1536 and TK deficiency viruses JX594 and SLJ496 against Hepatocellular carcinoma cell lines, Chlangiocarcinoma, Cervical Cancer cell line and Osteocarcinoma cell line, that is, HepG2, SNU478, HeLa and U2OS cell lines, The anti-cancer effects of the vaccinia virus 496GFP were compared with each other, and the results are shown in Fig.

As shown in FIG. 15, it was confirmed that the cytotoxic effect on hepatocellular carcinoma cell lines (HCC), HepG2, was significantly higher in 496 GFP than in VR1536 and JX594 at the same concentration. The cytotoxicity of 496GFP enhanced tumor affinity compared to SLJ496 was also confirmed by applying SNU478, HeLa and U2OS to the cytotoxicity of SLJ496 compared to SLJ496.

<110> Pusan National University Industry-University Cooperation Foundation <120> Novel cancer favored oncolytic virus and use thereof <130> 208 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 531 <212> DNA <213> Artificial Sequence <220> <223> Coding region of thymidine kinase <400> 1 atgaacggcg gacatattca gttgataatc ggccccatgt tttcaggtaa aagtacagaa 60 ttaattagac gagttagacg ttatcaaata gctcaatata aatgcctgac tataaaatat 120 tctaacgata atagatacgg aacgggacta tggacgcatg ataagaataa ttttgaagca 180 ttggaagcaa ctaaactatg tgatgtcttg taatcaatta cagatttctc cgtgataggt 240 atcgatgaag gacagttctt tccagacatt gttgaattct gtgagcgtat ggcaaacgaa 300 ggaaaaatag ttatagtagc cgcactcgat gggacatttc aacgtaaacc gtttaataat 360 attttgaatc ttattccatt atctgaaatg gtggtaaaac taactgctgt gtgtatgaaa 420 tgctttaagg aggcttcctt ttctaaacga ttgggtgagg aaaccgagat agaaataata 480 ggaggtaatg atatgtatca atcggtgtgt agaaagtgtt acatcgactc a 531 <210> 2 <211> 239 <212> DNA <213> Artificial Sequence <220> <223> Tk-L 4516 - 4754 <400> 2 atgaacggcg gacatattca gttgataatc ggccccatgt tttcaggtaa aagtacagaa 60 ttaattagac gagttagacg ttatcaaata gctcaatata aatgcgtgac tataaaatat 120 tctaacgata atagatacgg aacgggacta tggacgcatg ataagaataa ttttgaagca 180 ttggaagcaa ctaaactatg tgatgtcttg gaatcaatta cagatttctc cgtgatagg 239 <210> 3 <211> 220 <212> DNA <213> Artificial Sequence <220> <223> vTk-R 901 - 1120 <400> 3 agtagccgca ctcgatggga catttcaacg taaaccgttt aataatattt tgaatcttat 60 tccattatct gaaatggtgg taaaactaac tgctgtgtgt atgaaatgct ttaaggaggc 120 ttccttttct aaacgattgg gtgaggaaac cgagatagaa ataataggag gtaatgatat 180 gtatcaatcg gtgtgtagaa agtgttacat cgactcataa 220 <210> 4 <211> 5696 <212> DNA <213> Artificial Sequence <220> <223> pYOO-GFP Orig_Yoo101114 <400> 4 gtcgacttcg agcttattta tattccaaaa aaaaaaaata aaatttcaat ttttaagctt 60 tcactaattc caaacccacc cgctttttat agtaagtttt tcacccataa ataataaata 120 caataattaa tttctcgtaa aagtagaaaa tatattctaa tttattgcac ggtaaggaag 180 tagatcataa ctcgaggaat tggggatctc tataatctcg cgcaacctat tttcccctcg 240 aacacttttt aagccgtaga taaacaggct gggacacttc acatgagcga aaaatacatc 300 gtcacctggg acatgttgca gatccatgca cgtaaactcg caagccgact gatgccttct 360 gaacaatgga aaggcattat tgccgtaagc cgtggcggtc tggtaccggg tgcgttactg 420 gcgcgtgaac tgggtattcg tcatgtcgat accgtttgta tttccagcta cgatcacgac 480 aaccagcgcg agcttaaagt gctgaaacgc gcagaaggcg atggcgaagg cttcatcgtt 540 attgatgacc tggtggatac cggtggtact gcggttgcga ttcgtgaaat gtatccaaaa 600 gcgcactttg tcaccatctt cgcaaaaccg gctggtcgtc cgctggttga tgactatgtt 660 gttgatatcc cgcaagatac ctggattgaa cagccgtggg atatgggcgt cgtattcgtc 720 ccgccaatct ccggtcgcta atcttttcaa cgcctggcac tgccgggcgt tgttcttttt 780 aacttcaggc gggttacaat agtttccagt aagtattctg gaggctgcat ccatgacaca 840 ggcaaacctg cggatcccag cttttgttcc ctttagtgag ggttaattgc gcgcagttat 900 agtagccgca ctcgatggga catttcaacg taaaccgttt aataatattt tgaatcttat 960 tccattatct gaaatggtgg taaaactaac tgctgtgtgt atgaaatgct ttaaggaggc 1020 ttccttttct aaacgattgg gtgaggaaac cgagatagaa ataataggag gtaatgatat 1080 gtatcaatcg gtgtgtagaa agtgttacat cgactcataa tattatattt tttatctaaa 1140 aaactaaaaa taaacattga ttaaatttta atataatact taaaaatgga tgttgtgtcg 1200 ttagataaac cgtttatgta ttttgaggaa attgataatg agttagatta cgaaccagaa 1260 agtgcaaatg aggtcgcaaa aaaactgccg tatcaaggac agttaaaact attactagga 1320 gaattatttt ttcttagtaa gttacagcga cacggtatat tagatggtgc caccgtagtg 1380 tatataggat ctgctcccgg tacacatata cgttatttga gagatcattt ctataattta 1440 ggagtgatca tcaaatggat gctaattgac ggccgccatc atgatcctat tttaaatgga 1500 ttgcgtgatg tgactctagt gactcggttc gttgatgagg aatatctacg atccatcaaa 1560 aaacaactgc atccttctaa gattatttta atttctgatg tgagatccaa acgaggagga 1620 aatgaaccta gtacggcgga tttactaagt aattacgctc tacaaaatgt catgattagt 1680 attttaaacc ccgtggcgtc tagtcttaaa tggagatgcc cgtttccaga tcaatggatc 1740 aaggactttt atatcccaca cggtaataaa atgttacaac cttttgctcc ttcatattca 1800 gctgaaatga gattattaag tatttatacc ggtgagaaca tgagactgac tcgggccgcg 1860 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 1920 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 1980 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 2040 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 2100 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 2160 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 2220 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 2280 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 2340 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 2400 ggtagcggtg gtttttttgt ttgcaagcag cagattacac gcagaaaaaa aggatctcaa 2460 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 2520 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 2580 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 2640 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 2700 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 2760 atgataccgc gagacccacg ctcaccgggc tccagattta tcagcaataa accagccagc 2820 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 2880 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 2940 cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 3000 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 3060 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 3120 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 3180 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 3240 ggcgtcaaca cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 3300 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 3360 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 3420 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 3480 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 3540 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 3600 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta 3660 taaaaatagg cgtatcacga ggccctttcg tcttcgaata aatacctgtg acggaagatc 3720 acttcgcaga ataaataaat cctggtgtcc ctgttgatac cgggaagccc tgggccaact 3780 tttggcgaaa atgagacgtt gatcggcacg taagaggttc caactttcac cataatgaaa 3840 taagatcact accgggcgta ttttttgagt tatcgagatt ttcaggagct aaggaagcta 3900 aaatggagaa aaaaatcact ggatatacca ccgttgatat atcccaatgg catcgtaaag 3960 aacattttga ggcatttcag tcagttgctc aatgtaccta taaccagacc gttcagagct 4020 tttgcgatca ataaatggat cacaaccagt atctcttaac gatgttcttc gcagatgatg 4080 attcattttt taagtatttg gctagtcaag atgatgaatc ttcattatct gatatattgc 4140 aaatcactca atatctagct agactttctg ttattattat tgatccaatc aaaaaataaa 4200 ttagaagccg tgggtcattg ttatgaatct ctttcagagg aatacagaca attgacaaaa 4260 ttcacagact ttcaagattt taaaaaactg tttaacaagg tccctattgt tacagatgga 4320 agggtcaaac ttaataaagg atatttgttc gactttgtga ttagtttgat gcgattcaaa 4380 aaagaatcct ctctagctac caccgcaata gatcctgtta gatacataga tcctcgtcgc 4440 aatatcgcat tttctaacgt gatggatata ttaaagtcga ataaagtgaa caataattaa 4500 ttctttattg tcatcatgaa cggcggacat attcagttga taatcggccc catgttttca 4560 ggtaaaagta cagaattaat tagacgagtt agacgttatc aaatagctca atataaatgc 4620 gtgactataa aatattctaa cgataataga tacggaacgg gactatggac gcatgataag 4680 aataattttg aagcattgga agcaactaaa ctatgtgatg tcttggaatc aattacagat 4740 ttctccgtga taggcgcgcg taatacgact cactataggg cgaattggag ctctttttat 4800 ctgcgcggtt aaccgccttt ttatccatca ggtgatctgt ttttattgtg gagtctagaa 4860 ctagtaggcc tattattttt gacaccagac aagttggtaa tggtagcgac cggcgctcag 4920 ttggaattct agagtcgcgg ccgctttact tgtacagctc gtccatgccg agagtgatcc 4980 cggcggcggt cacgaactcc agcaggacca tgtgatcgcg cttctcgttg gggtctttgc 5040 tcagggcgga ctgggtgctc aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga 5100 tgggggtgtt ctgctggtag tggtcggcga gctgcacgct gccgtcctcg atgttgtggc 5160 ggatcttgaa gttcaccttg atgccgttct tctgcttgtc ggccatgata tagacgttgt 5220 ggctgttgta gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga 5280 tgcccttcag ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg 5340 tcttgtagtt gccgtcgtcc ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca 5400 tggcggactt gaagaagtcg tgctgcttca tgtggtcggg gtagcggctg aagcactgca 5460 cgccgtaggt cagggtggtc acgagggtgg gccagggcac gggcagcttg ccggtggtgc 5520 agatgaactt cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc 5580 tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga 5640 acagctcctc gcccttgctc accatggtgg cgaccggtac ccggggatcc tctaga 5696 <210> 5 <211> 794 <212> DNA <213> Artificial Sequence <220> <223> Thymidine kinase <400> 5 ttaaccaggt ccctattgtt acagatggaa gggtcaaact taataaagga tatttgttcg 60 actttgtgat tagtttgatg cgattcaaaa aagaatcctc tctagctacc accgcaatag 120 atcctgttag atacatagat cctcgtcgca atatcgcatt ttctaacgtg atggatatat 180 taaagtcgaa taaagtgaac aataattaat tctttattgt catcatgaac ggcggacata 240 ttcagttgat aatcggcccc atgttttcag gtaaaagtac agaattaatt agacgagtta 300 gacgttatca aatagctcaa tataaatgcc tgactataaa atattctaac gataatagat 360 acggaacggg actatggacg catgataaga ataattttga agcattggaa gcaactaaac 420 tatgtgatgt cttgtaatca attacagatt tctccgtgat aggtatcgat gaaggacagt 480 tctttccaga cattgttgaa ttctgtgagc gtatggcaaa cgaaggaaaa atagttatag 540 tagccgcact cgatgggaca tttcaacgta aaccgtttaa taatattttg aatcttattc 600 cattatctga aatggtggta aaactaactg ctgtgtgtat gaaatgcttt aaggaggctt 660 ccttttctaa acgattgggt gaggaaaccg agatagaaat aataggaggt aatgatatgt 720 atcaatcggt gtgtagaaag tgttacatcg actcataata ttgtattttt tatctagaac 780 taaataacat tgat 794

Claims (12)

A novel vaccinia virus characterized in that the gene encoding the thymidine kinase consisting of the nucleotide sequence of SEQ ID NO: 1 is deleted in the recombinant vaccinia virus SLJ496.
The vaccinia virus according to claim 1, wherein the vaccinia virus exhibits tumor-affinity and cancer metastasis-suppressing effect.
delete 2. The vaccinia virus according to claim 1, wherein said vaccinia virus is transformed with a vector comprising Tk-L 4516-4754 of SEQ ID NO: 2 and vTk-R 901-1120 of SEQ ID NO: 3.
5. The vaccinia virus according to claim 4, wherein said vector is a shuttle vector.
5. The vaccinia virus according to claim 4, wherein said vector comprises a cleavage map of Fig.
7. The vaccinia virus according to claim 6, wherein said vector comprises the nucleotide sequence of SEQ ID NO: 4.
A pharmaceutical composition for preventing or treating cancer, which comprises the vaccinia virus according to any one of claims 1, 2 and 4 to 7.
The method of claim 8, wherein the cancer is at least one selected from the group consisting of gallbladder cancer, colon cancer, pancreatic cancer, leukemia, ovarian cancer, stomach cancer, lung cancer, breast cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, skin cancer, colon cancer and cervical cancer &Lt; / RTI &gt; or a pharmaceutically acceptable salt thereof.
A food composition for preventing or improving cancer, which comprises the vaccinia virus according to any one of claims 1, 2 and 4 to 7.
A recombinant vaccinia virus, SLJ496, comprising a step of deleting thymidine kinase, wherein the recombinant vaccinia virus SLJ496 exhibits tumor-affinity and cancer metastasis inhibitory effect.
12. The method according to claim 11, wherein said step is by transforming recombinant vaccinia virus SLJ496 with a vector consisting of cleaved map of Fig.

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