KR20200092866A - Pharmaceutical Composition for preventing or treating cancer comprising Squirrel fibroma virus and Reovirus - Google Patents

Abstract

The present invention relates to a use of squirrel fibroma virus and reovirus to prevent or treat cancer. A pharmaceutical composition containing the squirrel fibroma virus and the reovirus of the present invention, or a biological sample treated with the two viruses specifically infects cancer cells without affecting normal human cells and exhibits oncolytic activity, thereby suppressing the growth of cancer cells.

Description

Pharmaceutical composition for preventing or treating cancer comprising Squirrel fibroma virus and Reovirus

The present invention relates to a cancer prevention or treatment use of Squirrel fibroma virus and Reovirus.

Due to the development of industry, changes in the global ecosystem and diet, cancer incidence is continuously increasing compared to the past, and research on anticancer drugs continues to grow.

Currently used medicines can be divided into chemotherapeutic agents and biological agents, and biological anti-cancer agents using genes, enzymes, vaccines, and viruses are still insufficient in the commercialization stage, and chemical agents have various side effects due to pharmacological and toxic effects. The indicated problems are pointed out.

Accordingly, various treatment methods such as surgical therapy, radiation therapy, and chemotherapy have been studied to treat cancer, but they have not yet been able to fully cure cancer. Of these, surgical therapy and radiation therapy are useful methods in the early stages of cancer, so the dependence on chemotherapy is gradually increasing for cancers that are difficult to detect early, and chemotherapy has a history of more than 50 years. There have been hundreds of anti-cancer drugs developed to date and have been used clinically, but there are still few cases where clinically satisfactory effects are obtained.

Virus is one of the biotherapeutic agents, and has the concept of targeted therapeutics that attack using genetic variation in tumor cells. Anti-cancer virus selectively proliferates in cancer cells and induces tumor necrosis and death.

Anti-cancer virus (oncolytic virus) is a virus used to treat cancer, and the anti-cancer method using the same is called OV therapy (oncolytic viral therapy). Cancer treatment research using wild type oncolytic virus is distinguished from gene therapy, which mainly aims to see tumor killing effect by expression of therapeutic gene by inserting therapeutic gene into virus. Research into wild-type tumor-destructive viruses began when it became known that some wild-type viruses had inherently strong tumor-destructive abilities.

There has been a long history of reports that cancer is naturally cured due to natural infection by various types of viruses, and since studies on tumor-specific killing mechanisms by wild-type viruses have begun in earnest, cancer using wild-type reovirus Therapeutic studies have reached clinical phase III applications. In addition, adenovirus, poliovirus, herpes simplex virus, vesicular stomatitis virus, etc. have been developed, and methods for improving the efficacy and stability of the virus are under study.

Republic of Korea Publication No. 10-2009-0125103

Accordingly, the present inventors continued to study to find an effective tumor-destructive virus, and when using a combination of Squirrel fibroma virus and Reovirus, cancer cells do not infect human normal cells due to host specificity The present invention was completed by confirming that it exhibits a high stability and tumor destructive power that is infected only with, and that when squirrel fibroadenoma virus is used in combination with a lyovirus, it can exhibit a much better synergistic effect than virus monotherapy.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention (a) Squirrel fibroma virus (Squirrel fibroma virus) and Rio virus (Reovirus); Or (b) provides a pharmaceutical composition for preventing or treating cancer comprising the biological sample treated with (a) as an active ingredient.

In addition, the present invention (a) squirrel fibroma virus and lyovirus; Or (b) administering a biological sample treated with (a) to a subject.

In addition, the present invention (a) squirrel fibroma virus and lyovirus; Or (b) the use of a biological sample treated with (a) to prevent, ameliorate or treat cancer.

In one embodiment of the present invention, the cancer is selected from the group consisting of gastric cancer, glioma, lung cancer, liver cancer, melanoma, prostate cancer, blood cancer, breast cancer, colon cancer, pancreatic cancer, brain cancer, ovarian cancer and combinations thereof. It may be, but is not limited thereto.

In another embodiment of the present invention, the squirrel fibroma virus or lyovirus may include a foreign gene for cancer treatment, but is not limited thereto.

In another embodiment of the present invention, the foreign gene for cancer treatment is inserted into a squirrel fibroma virus gene or a lyovirus gene in an essential region for replication or a non-essential region for anti-cancer activity. Can be increased, but is not limited thereto.

In another embodiment of the present invention, the squirrel fibroma virus or lyovirus is mounted on virus carrier cells and may be administered in vivo, but is not limited thereto.

In another embodiment of the present invention, the squirrel fibroma virus may be included in 10 ² to 10 ¹³ PFU, but is not limited thereto.

In another embodiment of the present invention, the lyovirus may be included in 10 ² to 10 ¹⁵ PFU, but is not limited thereto.

In another embodiment of the present invention, the titer ratio of the squirrel fibroma virus and the riovirus may be 1:1 to 1000:1 (riovirus: squirrel fibroma virus), but is not limited thereto.

In another embodiment of the present invention, the titer ratio of the squirrel fibroma virus and the riovirus may be 1:1 to 1:1000 (riovirus: squirrel fibroma virus), but is not limited thereto.

In another embodiment of the present invention, the biological sample may be prepared to kill a plurality of cancer cells by applying effective amounts of squirrel fibroadenoma virus and riovirus to an ex vivo biological sample, but is not limited thereto. It does not work.

In another embodiment of the present invention, the biological sample may be a bone marrow sample, an adipose-derived stem cell sample, or a blood sample, but is not limited thereto.

In another embodiment of the present invention, the squirrel fibroma virus may be administered simultaneously with, but not limited to, a riovirus (simultaneous), separately (separate), or sequential.

In another embodiment of the present invention, the squirrel fibroma virus or lyovirus may be obtained using BHK (baby hamster kidney) cells, but is not limited thereto.

In another embodiment of the present invention, the pharmaceutical composition may further include a chemotherapeutic agent or an immunotherapeutic agent, but is not limited thereto.

The pharmaceutical composition comprising the squirrel fibroadenoma virus and lyovirus of the present invention or a biological sample treated with the two viruses is specifically infected with cancer cells without affecting normal cells of humans and has oncolytic activity (oncolytic activity) ) To suppress the growth of cancer cells.

In addition, when the combination of the squirrel fibroma virus and the lyovirus shows a very good synergistic effect than the virus monotherapy, the present invention can be useful for the prevention and treatment of cancer.

Figure 1 shows a synergistic oncolytic effect of the combination of squirrel fibroma virus and lyovirus in gastric cancer cells, gastric cancer cells (MKN28, AGS, SNU668) no treatment (Mock), lyovirus alone (REO), squirrel fibroma virus alone (SFV) and the combination of lyovirus and squirrel fibroma virus (REO+SFV) confirms the death of cancer cells when infected by CPE assay, and is a graph shown through WST assay.
Figure 2 shows a synergistic oncolytic effect of squirrel fibroma virus and lyovirus combination in glioma cells, glioma cells (U87MG, SNU489) untreated (Mock), lyovirus alone (REO), squirrel fibroma virus alone (SFV) and the combination of lyovirus and squirrel fibroma virus (REO+SFV) confirms the death of cancer cells when infected by CPE assay, and is a graph shown through WST assay.
Figure 3 shows the synergistic oncolytic effect of the combination of squirrel fibroma virus and lyovirus in lung cancer cells, lung cancer cells (A549) no treatment (Mock), lyovirus alone (REO), squirrel fibroma virus alone (SFV) and This is a graph showing the death of cancer cells when infected with a combination of riovirus and squirrel fibroma virus (REO+SFV) by CPE assay and graphed through WST assay.
Figure 4 shows the synergistic oncolytic effect of the combination of squirrel fibroma virus and lyovirus in liver cancer cells, liver cancer cells (Hep3B) untreated (Mock), lyovirus alone (REO), squirrel fibroma virus alone (SFV) and This is a graph showing the death of cancer cells when infected with a combination of riovirus and squirrel fibroma virus (REO+SFV) by CPE assay and graphed through WST assay.
Figure 5 shows a synergistic oncolytic effect of the combination of squirrel fibroma virus and lyovirus in melanoma, in vivo, lyovirus alone (REO), squirrel fibroma virus in C57BL/6 female mice induced by B16F10 melanoma allograft It is a graph showing the change in tumor size after infection with single (SFV) and the combination of riovirus and squirrel fibroma virus (REO+SFV).

The present invention (a) Squirrel fibroma virus (Squirrel fibroma virus) and Rio virus (Reovirus); Or (b) provides a pharmaceutical composition for preventing or treating cancer comprising the biological sample treated with (a) as an active ingredient.

The present inventors continued research to find an effective tumor destruction (tumor lytic activity) virus, and when a combination of squirrel fibroadenoma virus and lyovirus is used, the host specificity does not infect human normal cells but infects cancer cells only. In addition to confirming stability and tumor destruction, it was confirmed that when the squirrel fibroma virus was used in combination with the lyovirus, a synergistic effect superior to that of the virus monotherapy was shown (see Examples 1 to 5).

As used herein, the term “squirrel fibroma virus” is isolated from the American gray squirrel as a host, is included in the Chordopox virinae of the Poxviridae, and is classified as a genus of Parapoxvirus. . The squirrel fibroma virus of the present invention can be isolated through the host mentioned above, and can also be isolated through cell culture.

The squirrel fibroma virus of the present invention uses a known technique such as in vivo passage using a squirrel/woodchuck, primary squirrel kidney cells, or choroid plexus cells. Can be obtained.

In the present invention, the squirrel fibroma virus may be a wild-type squirrel fibroma virus or an attenuated squirrel fibroma virus, but is not limited thereto.

As used herein, the term “riovirus” is a virus having a double-stranded segmented RNA genome, and refers to any virus classified into the Reoviridae family. The virion of the lyovirus is 60-80 nm in diameter and has two concentric capsid shells. This genome consists of 10 to 12 discontinuous double-stranded RNAs, has a total genomic size of 16 to 27 kbp, and each RNA segment has a different size.

In the present invention, the lyovirus includes not only a naturally occurring lyovirus, but also a modified or recombinant lyovirus. Said lyovirus can be isolated from nature and is said to be "naturally occurring" when no artificial changes have been made by humans. For example, the lyovirus can be derived from a “field source”, ie, a person infected with this lyovirus.

The lyovirus can be modified, but can lytically infect mammalian cells with an active ras pathway. In addition, the lyovirus can be pretreated chemically or biochemically (e.g., by treatment with a protease such as chymotrypsin or trypsin) prior to administration to proliferating cells. The outer membrane or capsid of the virus can be removed by pre-treatment with a protease to increase the infectivity of the virus. The lyovirus may be coated with liposomes or micelles, and for example, virions may be treated with chymotrypsin in the presence of an alkyl sulfate detergent having a micelle-forming concentration to generate new infectious subviral particles. have.

In the present invention, the lyovirus may be a wild-type lyovirus or an attenuated lyovirus, but is not limited thereto.

The attenuated lyovirus comprises an infectious replicable lyovirus virion deficient in the lyovirus sigma 1 capsid protein detectable by the genome of the lyovirus deficient in the wild type lyovirus S1 gene. As such, the attenuated lyovirus has a cytopathic effect on non-malignant cells while unexpectedly maintaining the ability of the mutated lyovirus deficient in detectable lyovirus sigma 1 capsid protein to infect the target tumor cells productively. effect). As described above, prior to the present disclosure, it was understood that particles of lyovirus deficient in Sigma 1 are non-infectious.

In certain embodiments, the attenuated lyovirus may include the S4 gene of the mutated lyovirus. The lyovirus wild type S4 gene encodes a lyovirus capsid sigma 3 polypeptide that is involved in virion processing during lyovirus replication infection of the host cell.

In certain embodiments, the attenuated lyovirus can comprise a mutated lyovirus S4 gene comprising one or more mutations in the genomic sequence encoding the sigma 3 polypeptide of the lyovirus relative to the wild-type S4 gene sequence. have.

Attenuated lyovirus lacks detectable Sigma 1 capsid protein, but is unexpectedly infectious. As described above, it is shown that sigma 1 is involved in the binding and attachment of lyovirus to cells through cell surface sialic acid residues in the early stages of viral replication infection. Despite the lack of detectable Sigma 1, the attenuated lyovirus described herein is capable of host cell entry and replication of cytolytic viruses. In addition, the attenuated lyovirus is a reduced (ie, statistical) one or more cytopathic effects on non-malignant cells compared to the level of cytopathic effect seen on non-malignant cells by naturally occurring non-attenuated lyovirus. It exhibits surprising properties that induce levels of reduced significance).

The attenuated lyovirus can be derived from any lyovirus, which means a member of the lyoviridae family, and includes lyoviruses with various affinity, which can be obtained from various sources.

In certain embodiments, it can be a mammalian lyovirus, and in other embodiments it can be a human lyovirus. In other embodiments (eg, for use in animal models associated with human disease or for veterinary applications), the attenuated lyovirus is a non-human primate (eg, chimpanzee, gorilla, macaque, monkey, etc.) Cells of other mammalian species, including rodents (e.g., mice, rats, gerbils, hamsters, rabbits, guinea pigs, etc.), dogs, cats, and general livestock (e.g., cattle, horses, pigs, goats), etc. It can be derived from one or more lyoviruses that exhibit affinity for, or alternatively, lyoviruses (eg, avian reovirus) with distinctly distinct affinity can be used.

Attenuated lyoviruses include the generation and identification of sigma 1-deficient and/or sigma 1-deficient mutants by molecular biological approaches (and in certain embodiments, additionally or alternatively, sigma 3-deficiency and/or Or generation and identification of sigma 3-defective mutants), also separation of naturally occurring sigma 1-deficient and/or sigma 1-defective mutants and/or sigma 3 mutants, and/or chemical, physical And/or artificial induction of such Sigma 1 (and/or Sigma 3) mutants by genetic techniques (eg, selective recombination of lyovirus genes in productively infected host cells). have.

The attenuated lyovirus lacks the wild-type lyovirus S1 gene and consequently lacks detectable lyovirus sigma 1 capsid protein, including infectious replicable lyovirus virions (i.e., the viral genome, core protein and protein envelope). Virus particles).

In certain embodiments, the attenuated lyovirus lacks the wild-type lyovirus S4 gene and expresses a mutated lyovirus sigma 3 capsid protein. As is known in the art, infectious replicable lyovirus is one that can be internalized by binding to a host cell upon introduction into a suitable host cell for a sufficient period of time under appropriate conditions, followed by the cycle of replication of the virus upon release from the host cell. It directs the replication of the genome of the lyovirus and the biosynthesis of the lyovirus structural protein in a manner that allows the assembly of a complete progeny lyovirus capable of productively infecting other host cells to persist.

Attenuated lyoviruses for the treatment of cancer are described, for example, in US applications published US2009/0214479 and US2009/0104162, each of which is incorporated herein by reference in its entirety.

The human lyovirus can be composed of three serotypes as follows: type 1 (Lang strain or T1L), type 2 (Jones strain, T2J) and type 3 (Dearing strain, T3D or Abney strain, T3A). The three serotypes can be easily identified based on neutralization reactions and hemagglutination inhibition assays.

In the present invention, the squirrel fibroma virus or lyovirus can be obtained using BHK (baby hamster kidney) cells (eg, BHK-21 cells), but is not limited thereto.

The term BHK in the term “BHK-21 cells” as used herein, stands for Baby Hamster Kidney. BHK cells were originally isolated by polyoma transformation of the hamster cells, It can be used as a substrate for viral propagation and viral mediated expression. In addition, BHK cells are useful as host cell lines for stable expression of various recombinant proteins. The BHK-21 (BHK Strain 21) cell line was derived from the baby hamster kidney of 5 unsexed, 1-day-old hamsters in March 1961 by IA Macpherson and MGP Stoker. The hamster was used to generate BHK-21 cells and is generally known as the Syrian golden hamster (Mesocricetus auratus).

As used herein, “biological sample” in “biological sample treated with (a)” means any biological sample obtained from an individual, cell line, tissue culture, or other source of cells, such as adult stem cells (fat Stem cells, bone marrow stem cells) or cord blood stem cells. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. For example, adipose derived stem cells may be pretreated with an oncolytic virus and administered to cancer patients.

In the present invention, the biological sample may be prepared to kill a plurality of cancer cells by applying effective amounts of a squirrel fibroma virus and a lyovirus to an ex vivo biological sample, but is not limited thereto.

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

In the present invention, the squirrel fibroma virus or lyovirus may include, but is not limited to, a heterologous gene for cancer treatment.

The term "heterologous gene" is used to mean accepting a gene not found in the viral genome. The foreign gene may be an allelic variant of the wild type gene or a mutant gene.

The foreign gene for cancer treatment may be inserted into a squirrel fibroma virus gene or a lyovirus gene in an essential region for replication or a non-essential region for replication, thereby increasing anticancer activity.

The foreign gene can be operably linked to a control sequence that allows the expression of an electric foreign gene in cells in vivo. Thus, the virus of the present invention can be used to deliver foreign genes/genes to cells of in vivo conditions where foreign genes can be expressed. The gene typically encodes a protein that can enhance the virus's tumor-destructive properties. The gene itself may be a cytotoxin or may encode a protein that can promote/enhance an anti-tumor immune response.

The foreign gene includes all genes that treat cancer by inserting into a replicable tumor-destructive virus or an existing non-replicable virus vector.

As used herein, the term “therapeutic gene” is considered to describe any wide range of genes whose expression results in desirable results, eg, anti-cancer effects. The squirrel fibroma virus or lyovirus of the present invention may include one or more desired sequences encoding a therapeutic gene. The therapeutic gene can have pharmacological or prophylactic activity when properly administered to a patient, particularly a patient suffering from a disease or disease state, or a patient to be protected from such disease or condition.

Such pharmacological or prophylactic activity is meant to be expected to be associated with a beneficial effect on the disease or the course of the condition or symptom. The desired sequence can be homologous or heterologous to the target cell into which it is introduced, and encodes all or part of a polypeptide, particularly a therapeutic or prophylactic polypeptide that gives therapeutic or prophylactic properties. Polypeptides are understood to be any translational product of a polynucleotide regardless of size and whether it is glycosylated and include peptides and proteins. Therapeutic polypeptides include those that act through polypeptides or toxic effects that can compensate for defective or deficient proteins in an animal or human organism to limit or eliminate harmful cells from the body. These can also be immuno-imparting polypeptides that act as endogenous antigens to induce a humoral or cellular response, or both, for example, drug-sensitizing genes, proapoptotic genes, cells Proliferation inhibitor genes, cytotoxic genes, tumor suppressor genes, antigenic genes, anti-angiogenic genes, cytokine genes, and the like. It does not work.

The drug-sensitizing gene is a gene for an enzyme that converts a non-toxic precursor into a toxic substance and is also called a suicide gene because the cell into which the gene has been transferred dies. In other words, when a non-toxic precursor is administered systemically to a normal cell, the precursor is converted into a toxic metabolite only to the cancer cell, thereby destroying the cancer cell by changing the sensitivity to the drug. Typically, the HSVtk (Herpes simplex virus-thymidine kinase) gene, ganciclovir, and E. coli cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) It is possible.

The apoptotic gene refers to a nucleotide sequence that is expressed and induces programmed apoptosis. Apoptosis gene known to those of skill in the art, encoding p53, adenovirus E3-11.6K (from Ad2 and Ad5) or adenovirus E3-10.5K (from Ad), adenovirus E4 gene, p53 pathway gene and caspase Gene.

The cell proliferation inhibitory gene refers to a nucleotide sequence that is expressed in a cell and stops the cell cycle during the cell cycle. Representatively, p21, retinoblastoma gene, E2F-Rb fusion protein gene, gene encoding a cyclin-dependent kinase inhibitor (e.g., p16, p15, p18 and p19), growth arrest specific homeobox , GAX) gene.

The cytotoxic gene refers to a nucleotide sequence expressed in a cell and exhibiting a toxic effect. For example, there are nucleotide sequences encoding pseudomonas exotoxin, lysine toxin, diphtheria toxin, and the like.

The tumor suppressor gene refers to a nucleotide sequence that can be expressed in a target cell to suppress a tumor phenotype or induce apoptosis. Representative tumor necrosis factor (α, TNF-α), p53 gene, APC gene, DPC-4/Smad4 gene, BRCA-1 gene, BRCA-2 gene, WT-1 gene, and retinoblastoma gene (Lee) et al., Nature, 329,642, 1987), MMAC-1 gene, adenomatous polyposis coil protein, missing colon tumor (DCC) gene, MMSC-2 gene, NF-1 gene, chromosome 3p21 Non-throat tumor suppressor genes located in .3, MTS1 gene, CDK4 gene, NF-1 gene, NF-2 gene and VHL gene are included.

The antigenic gene refers to a nucleotide sequence that is expressed in a target cell and produces a cell surface antigenic protein recognizable by the immune system. Examples of antigenic genes known to those of ordinary skill in the art include cancer-bearing antigens (carcinoembryonic antigen, CEA) and p53 (Levine, A., International Patent Application Publication WO 94/02167).

Examples of polypeptides encoded by more specific therapeutic genes include cytokines (alpha, beta or gamma interferon, interleukin, especially IL-2, IL-6, IL-10 or IL-12, tumor necrosis factor (TNF), chemokines , Colony stimulating factors GM-CSF, C-CSF, M-CSF), immunostimulatory polypeptides (B7.1, B7.2, etc.), coagulation factors (FVIII, FIX), growth factors (transformation growth factor TGF, fiber Subcellular growth factor FGF, etc.), enzymes (urease, renin, thrombin, metalloproteinase, nitric oxide synthase (NOS, SOD, catalase), enzyme inhibitors (alpha1-antitrypsin, antithrombin III, Viral protease inhibitor, plasminogen activator inhibitor PAI-1), CFTR (cystic fibrosis transmembrane conductivity regulator) protein, insulin, dystrophin, MHC class I or II antigen, polypeptide capable of modulating/regulating expression of cellular genes , Polypeptides capable of inhibiting bacterial, parasitic or viral infections or their development (antigenic polypeptides, antigenic epitopes, transdominant variants that inhibit the action of natural proteins in competition), apoptosis inducers or inhibitors (Bax, Bcl2, BclX...), cell stoppers (p21, p 16, Rb), apolipoprotein (ApoliAI, ApoAIV, ApoE...), inhibitors of angiogenesis (angiostatin, endostatin), blood vessels Forming polypeptides (vascular endothelial growth factors VEGF family, FGF family, CTGF family, CCN family including Cyr61 and Nov), oxygen radical scavengers, polypeptides with anti-tumor effect, antibodies, toxins, immunotoxins and markers (beta) Genes encoding galactosidase, luciferase) or any other gene of interest recognized in the art to be useful in the treatment or prevention of a clinical condition Suitable anti-tumor genes include tumor suppressor genes (eg, Rb , p53, DCC, NF-1, Will These include, but are not limited to, those encoding the polypeptides that inhibit the Wilms' tumor, NM23, BRUSH-1, p16, p21, p56, p73 and their mutants), suicide gene products, antibodies, cell division or transduction signals. , But is not limited to this. The suicide gene includes a gene encoding a protein having cytosine deaminase activity, thymidine kinase activity, uracil phosphoribosyl transferase activity, purine nucleoside phosphorylase activity and/or thymidylate kinase activity , But is not limited to this. The therapeutic gene may be inserted into a squirrel fibroma virus or a lyovirus gene into a replication-requiring essential region or a non-essential region to increase its anticancer activity.

In the present invention, the squirrel fibroma virus or lyovirus is mounted on virus carrier cells (for example, Molecular Therapy. 2009 Vol 17 :1667-1676) and can be administered in vivo, through which The therapeutic effect of can be further increased.

Meanwhile, the present invention relates to a pharmaceutical composition for preventing or treating cancer and a method for treating cancer.

As used herein, the term “prevention” refers to all actions that inhibit cancer or delay progression by administration of the composition of the present invention.

As used herein, the term “treatment” refers to any action in which cancer is improved or beneficially altered by administration of a composition of the present invention, and means an attempt to obtain useful or desirable results, including clinical results. Useful or desirable clinical results, although detectable or not, can alleviate or ameliorate one or more symptoms or conditions, reduce the extent of the disease, stabilize the disease state, suppress disease occurrence, inhibit disease spread, delay or slow disease progression , Delaying or slowing the onset of disease, improving or alleviating disease conditions, and decay (part or all), but is not limited thereto. In addition, “treatment” may mean prolonging patient survival beyond what is predicted in the absence of treatment. In addition, “treatment” may mean suppression of disease progression, temporary slowing of disease progression, and may be associated with forever stopping disease progression.

Cancer diseases that can be treated in the present invention include blood cancer, colorectal cancer, brain cancer, glioma, neuroeducation species, anaplastic astrocytoma, medulloblastoma, stomach cancer, lung cancer, small cell lung carcinoma, cervical carcinoma, colon cancer, Rectal cancer, chordoma, throat cancer, Kaposi's sarcoma, lymphatic sarcoma, lymphatic endothelial sarcoma, colorectal cancer, endometrial cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, hepatocarcinoma, bile duct carcinoma, chorionic carcinoma, normal hematoma, Testicular tumor, Wilm's tumor, Ewing's tumor, bladder carcinoma, angiosarcoma, endothelial sarcoma, adenocarcinoma, glandular carcinoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, cystic sarcoma, bronchial carcinoma, medulla carcinoma, mastocytoma, mesothelioma, synovioma, black Species, leiomyoma, rhabdomyoma, neuroblastoma, retinoblastoma, oligodendroma, auditory glioma, hemangioblastoma, meningioma, pinealoma, epithelioma, parietal head, epithelial carcinoma, embryonic carcinoma, squamous cell carcinoma, basal cell carcinoma, fibrosarcoma, myxoma , Mucus sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma or leukemia.

In the present invention, the cancer may be selected from the group consisting of gastric cancer, glioma, lung cancer, liver cancer, melanoma, prostate cancer, blood cancer, breast cancer, colon cancer, pancreatic cancer, brain cancer, ovarian cancer, and combinations thereof. It is not limited thereto.

Viruses of the invention can be administered in combination or in combination with other treatments, including chemotherapy, radiation therapy, or other anti-viral therapies. For example, the virus can be administered before or after removal of the primary tumor through surgery, or before, concurrently, or after treatment with radiotherapy or conventional chemotherapy drugs. The virus can be administered in combination with other oncolytic viruses that are specific for various tumor cell types, or in a sequential manner.

Therapies commonly used to treat, prevent or treat cancer include, but are not limited to, surgery, chemotherapy, radiation therapy, hormone therapy, biological therapy and immunotherapy.

In the present invention, the pharmaceutical composition may additionally include a chemotherapeutic agent or an immunotherapeutic agent, but is not limited thereto.

As used herein, the term “administration” means providing a subject composition of the invention to an individual in any suitable way.

As used herein, the term “individual” refers to all animals, such as humans, monkeys, dogs, goats, pigs, or rats, with diseases that can improve cancer by administering the composition of the present invention.

The active ingredient of the present invention is administered to a subject in a pharmaceutically effective amount.

As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to treat a disease at a reasonable benefit or risk ratio applicable to medical treatment, which is the type, severity, and activity of the drug, The sensitivity to the drug, the time of administration, the route of administration and discharge rate, the duration of treatment, factors including the drugs used simultaneously, and other factors well known in the medical field may be determined.

The effective amount of the virus is to alleviate, improve, mitigate, stabilize, spread the disease, slow or delay the progression of the disease, and require sufficient time to cure the disease. Is the capacity. For example, an effective amount will be an amount sufficient to achieve the effect of reducing the number of cancer cells or reducing or destroying the number of cells chronically infected with a virus, or inhibiting the growth and/or proliferation of such cells. Can.

The effective amount can be varied by a number of factors, such as the pharmacokinetic properties of the virus, the method of administration, age, the health and weight of the patient, the nature and extent of the disease state, the number of treatments, and recent treatment modalities, such as the viral toxicity and It may also vary depending on the titer. Those skilled in the art can adjust the appropriate amount based on the above factors. The virus may initially be administered in appropriate amounts depending on the patient's clinical response. The effective amount of virus can be determined empirically and can be determined according to the maximum amount of virus that can be safely administered and the minimum amount of virus that produces the desired result.

When administering the virus systemically, administration of a significantly higher dose of virus may be required to induce a clinical effect similar to that realized by injecting the virus at the site of the disease. However, a suitable dose level should be the minimum amount that can achieve the desired result.

The concentration of the virus to be administered may vary depending on the pathotoxicity of the squirrel fibroma virus or lyovirus strain to be administered and the characteristics of the target cell.

In the present invention, the squirrel fibroma virus may be included in 10 ² to 10 ¹³ PFU (plaque forming unit). For example, the squirrel fibroma virus is 10 ² to 10 ¹² , 10 ² to 10 ¹¹ , 10 ² to 10 ¹⁰ , 10 ² to 10 ⁹ , 10 ² to 10 ⁸ or 10 ² to 10 ⁷ PFU; 10 ³ to 10 ¹³ , 10 ³ to 10 ¹² , 10 ³ to 10 ¹¹ , 10 ³ to 10 ¹⁰ , 10 ³ to 10 ⁹ , 10 ³ to 10 ⁸ or 10 ³ to 10 ⁷ PFU; 10 ⁴ to 10 ¹³ , 10 ⁴ to 10 ¹² , 10 ⁴ to 10 ¹¹ , 10 ⁴ to 10 ¹⁰ , 10 ⁴ to 10 ⁹ , 10 ⁴ to 10 ⁸ or 10 ⁴ to 10 ⁷ PFU; Or 10 ⁵ to 10 ¹³ , 10 ⁵ to 10 ¹² , 10 ⁵ to 10 ¹¹ , 10 ⁵ to 10 ¹⁰ , 10 ⁵ to 10 ⁹ , 10 ⁵ to 10 ⁸ or 10 ⁵ to 10 ⁷ PFU may be included.

In addition, in the present invention, the squirrel fibroma virus may be included as 0.01 to 10 multiplicity of infection (MOI). For example, the squirrel fibroma virus may contain 0.01 to 9, 0.01 to 8, 0.01 to 7, 0.01 to 6 or 0.01 to 5 MOI; 0.05 to 10, 0.05 to 9, 0.05 to 8, 0.05 to 7, 0.05 to 6 or 0.05 to 5 MOI; Or 0.1 to 10, 0.1 to 9, 0.1 to 8, 0.1 to 7, 0.1 to 6, or 0.1 to 5 MOI.

In the present invention, the lyovirus may be included in 10 ² to 10 ¹⁵ PFU. For example, lyovirus is 10 ² to 10 ¹⁴ , 10 ² to 10 ¹³ , 10 ² to 10 ¹² , 10 ² to 10 ¹¹ , 10 ² to 10 ¹⁰ , 10 ² to 10 ⁹ , 10 ² to 10 ⁸ or 10 ² to 10 ⁷ PFU; 10 ³ to 10 ¹⁵ , 10 ³ to 10 ¹⁴ , 10 ³ to 10 ¹³ , 10 ³ to 10 ¹² , 10 ³ to 10 ¹¹ , 10 ³ to 10 ¹⁰ , 10 ³ to 10 ⁹ , 10 ³ to 10 ⁸ or 10 ³ to 10 ⁷ PFU; 10 ⁴ to 10 ¹⁵ , 10 ⁴ to 10 ¹⁴ , 10 ⁴ to 10 ¹³ , 10 ⁴ to 10 ¹² , 10 ⁴ to 10 ¹¹ , 10 ⁴ to 10 ¹⁰ , 10 ⁴ to 10 ⁹ , 10 ⁴ to 10 ⁸ or 10 ⁴ to 10 ⁷ PFU; Or 10 ⁵ to 10 ¹⁵ , 10 ⁵ to 10 ¹⁴ , 10 ⁵ to 10 ¹³ , 10 ⁵ to 10 ¹² , 10 ⁵ to 10 ¹¹ , 10 ⁵ to 10 ¹⁰ , 10 ⁵ to 10 ⁹ , 10 ⁵ to 10 ⁸ or 10 ⁵ to 10 ⁷ PFU may be included.

In addition, in the present invention, the lyovirus may be included in 0.1 to 100 MOI. For example, the lyovirus may be included in 0.2 to 100, 0.3 to 100, 0.4 to 100, 0.5 to 100, 0.6 to 100, 0.7 to 100, 0.8 to 100, 0.9 to 100, or 1 to 100 MOI.

In the present invention, the titer ratio of the squirrel fibroma virus and the lyovirus may be 1:1 to 1000:1 (riovirus: squirrel fibroma virus), but is not limited thereto. For example, the titer ratio of the squirrel fibroma virus and the lyovirus is 1:1 to 900:1, 1:1 to 800:1, 1:1 to 700:1, 1:1 to 600:1, 1:1 To 500:1, 1:1 to 400:1, 1:1 to 300:1, 1:1 to 200:1, 1:1 to 100:1, 1:1 to 90:1, 1:1 to 80 :1, 1:1 to 70:1, 1:1 to 60:1, 1:1 to 50:1, 1:1 to 40:1, 1:1 to 30:1, 1:1 to 20:1 Or 1:1 to 10:1 (riovirus: squirrel fibroma virus).

In addition, in the present invention, the titer ratio of the squirrel fibroma virus and the riovirus may be 1:1 to 1000:1 (squirrel fibroma virus: riovirus), but is not limited thereto. For example, the titer ratio of the squirrel fibroma virus and the lyovirus is 1:1 to 900:1, 1:1 to 800:1, 1:1 to 700:1, 1:1 to 600:1, 1:1 To 500:1, 1:1 to 400:1, 1:1 to 300:1, 1:1 to 200:1, 1:1 to 100:1, 1:1 to 90:1, 1:1 to 80 :1, 1:1 to 70:1, 1:1 to 60:1, 1:1 to 50:1, 1:1 to 40:1, 1:1 to 30:1, 1:1 to 20:1 Or 1:1 to 10:1 (squirrel fibroma virus: riovirus).

An effective amount of virus can be administered repeatedly depending on the effectiveness of the initial treatment regimen. Generally, administration is administered periodically while monitoring all responses. Those skilled in the art can readily appreciate that a lower or higher dose than indicated above can be administered depending on the dosing schedule and the route chosen.

In the present invention, the squirrel fibroadenoma virus may be administered simultaneously with a lyovirus (simultaneous), separately (separate) or sequentially, but is not limited thereto.

Meanwhile, the pharmaceutical composition according to the present invention may further include a suitable carrier, excipient, and/or diluent, which is usually used to prepare a pharmaceutical composition in addition to the active ingredient. In addition, it can be formulated and used in the form of oral formulations, external preparations, suppositories, and sterile injectable solutions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. according to a conventional method.

When formulating the composition, it can be prepared by using diluents or excipients, such as carriers, stabilizers, antibacterial agents, buffers, fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc., which are usually used.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example, oral administration, intranasal administration, intratumoral administration, bronchoscopy, arterial injection, intravenous injection, subcutaneous injection, intramuscular injection or intraperitoneal injection. . The daily dosage may be divided into once to several times a day.

Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Confirmation of death of gastric cancer cell line

Stomach cancer cell lines (MKN28, AGS, SNU668) untreated (Mock), wild type lyovirus only (REO, MKN28 1 MOI; AGS and SNU668 100 MOI), wild type squirrel fibroma virus alone (SFV, MKN28 0.32 MOI; AGS and SNU668) 3.2 MOI) and lyovirus and squirrel fibroma virus (REO+SFV), and the death of cancer cells was confirmed by CPE assay. Specifically, after 4 days of infection with a virus after spreading cancer cells on a 96-well plate, cell viability was measured through a WST assay, and the result of conversion to a graph is shown in FIG. 1.

The larger the anti-cancer effect, the smaller the number of cells dyed with crystal violet, resulting in a softer purple color. After all, if the cancer cells have a significantly reduced survival rate due to the anti-cancer effect, it can be considered that the anti-cancer effect is greater. As shown in FIG. 1, relative cell viability was the lowest when they were infected in combination than when the lyovirus alone or the squirrel fibroma virus was infected alone (rightmost bar graph in FIG. 1 ).

These results show that the combined use of lyovirus and squirrel fibroma virus shows the best anti-cancer effect by showing synergistic anti-cancer effect.

Example 2. Confirmation of glioma cell line death

Glioma cell lines (U87MG, SNU489) untreated (Mock), wild type lyovirus alone (REO, 10 MOI), wild type squirrel fibroadenoma virus alone (SFV, U87MG 0.16 MOI; SNU489, 1.6 MOI) and lyovirus and squirrel fibroma Infection with a combination of viruses (REO+SFV) and death of cancer cells was confirmed by CPE assay. Specifically, after 4 days of infection with a virus after spreading cancer cells on a 96-well plate, cell viability was measured through a WST assay and converted to a graph.

As a result, as shown in FIG. 2, the relative cell viability was the lowest when they were infected in combination, compared with the case of lyovirus alone or squirrel fibroma virus alone (rightmost bar graph in FIG. 2 ).

These results show that the combination use of lyovirus and squirrel fibroma virus shows the best anticancer effect by showing synergistic anticancer effect.

Example 3. Confirmation of lung cancer cell line death

Lung cancer cell line (A549) infected with no treatment (Mock), wild type lyovirus alone (REO, 10 MOI), wild type squirrel fibroma virus alone (SFV, 3.2 MOI) and a combination of riovirus and squirrel fibroma virus (REO+SFV) And the death of cancer cells was confirmed by CPE assay. Specifically, after 4 days of infection with a virus after spreading cancer cells on a 96-well plate, the results obtained by measuring the cell viability through a WST assay and converting it to a graph are shown in FIG. 3.

As a result, as shown in FIG. 3, the relative cell viability was the lowest when they were infected in combination, compared with the case of lyovirus alone or squirrel fibroma virus alone (rightmost bar graph in FIG. 3 ).

These results show that the combination use of lyovirus and squirrel fibroma virus shows the best anticancer effect by showing synergistic anticancer effect.

Example 4. Confirmation of death of liver cancer cell line

Liver cancer cell line (Hep3B) infected with no treatment (Mock), wild-type lyovirus alone (REO, 1 MOI), wild-type squirrel fibroma virus alone (SFV, 0.32 MOI), and a combination of riovirus and squirrel fibroma virus (REO+SFV) And the death of cancer cells was confirmed by CPE assay. Specifically, after 4 days of infection with a virus after spreading cancer cells on a 96-well plate, the results of measuring the cell viability through a WST assay and converting it to a graph are shown in FIG. 4.

As a result, as shown in FIG. 4, the relative cell viability was the lowest when they were infected in combination, compared with the case of lyovirus alone or squirrel fibroma virus alone (rightmost bar graph in FIG. 4 ).

These results show that the combination use of lyovirus and squirrel fibroma virus shows the best anticancer effect by showing synergistic anticancer effect.

Example 5. Melanoma ( melanoma Anticancer effect on)

C57BL/6 mice (female, DOB: 2019-, 5 weeks old) were injected with 1 x 10 ⁵ cells/mice of mouse melanoma cells (B16F10 cells) to produce tumor-induced allograft animal models. Afterwards, the animal model was infected with wild type lyovirus alone (1 X 10 ⁷ PFU/injection), wild type squirrel fibroma virus alone (1 X 10 ⁴ PFU/injection), and lyovirus and squirrel fibroma virus in combination, thereby killing cancer cells. It was confirmed by a reduction in tumor size. When the initial tumor size was 130 mm ³ , virus infection was performed, and the next day, virus infection was performed to inject the virus twice, and the tumor size was measured.

As a result, as shown in Figure 5, compared to the negative control (Vehicle) CsCl, the size of the tumor is reduced when each virus is injected alone, especially when the two viruses are injected in combination, the size of the tumor is reduced, that is The anticancer effect was significantly increased (synergistic anticancer effect) (FIG. 5 ).

The results of Examples 1 to 5 above show that the highest anti-cancer effect is exhibited when they are used in combination with lyovirus alone or squirrel fibroma virus alone, and these results show that lyovirus and squirrel fibroma virus. It shows that the combined use shows the best anticancer effect through synergistic anticancer effect. In addition, it is shown that the combination use of the lyovirus and the squirrel fibroma virus may have anti-cancer effects against various carcinomas.

The above description of the present invention is for illustration only, and those of ordinary skill in the art to which the present invention pertains can understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (13)

(a) Squirrel fibroma virus and Reovirus; Or (b) a pharmaceutical composition for preventing or treating cancer comprising the biological sample treated with (a) as an active ingredient.
According to claim 1,
The cancer is selected from the group consisting of gastric cancer, glioma, lung cancer, liver cancer, melanoma, prostate cancer, blood cancer, breast cancer, colon cancer, pancreatic cancer, brain cancer, ovarian cancer and combinations thereof. .
According to claim 1,
The squirrel fibroma virus or riovirus is characterized in that it contains a foreign gene for cancer treatment, pharmaceutical composition.
According to claim 3,
The foreign gene for cancer treatment is inserted into a squirrel fibroma virus gene or a lyovirus gene to be inserted into an essential region or a non-essential region required for replication, thereby increasing anticancer activity. Composition.
According to claim 1,
The squirrel fibroadenoma virus or lyovirus is mounted on virus carrier cells, characterized in that it is administered in vivo, pharmaceutical composition.
According to claim 1,
The squirrel fibroma virus is characterized in that contained in 10 ² to 10 ¹³ PFU, pharmaceutical composition.
The method of claim 1 or 6,
The lyovirus is characterized in that contained in 10 ² to 10 ¹⁵ PFU, pharmaceutical composition.
According to claim 1,
The titer ratio of the squirrel fibroma virus and the lyovirus is 1:1 to 1000:1 (riovirus: squirrel fibroma virus), the pharmaceutical composition.
According to claim 1,
The biological sample is manufactured to kill a plurality of cancer cells by applying effective amounts of squirrel fibroma virus and lyovirus to ex vivo biological sample.
The method of claim 1 or 9,
The biological sample is a bone marrow sample, characterized in that a fat-derived stem cell sample or blood sample, pharmaceutical composition.
According to claim 1,
The squirrel fibroma virus is characterized in that it is administered simultaneously (simultaneous), separately (separate) or sequential (sequential) with the lyovirus.
According to claim 1,
The squirrel fibroma virus or lyovirus is characterized in that it is obtained using BHK (baby hamster kidney) cells, pharmaceutical composition.
According to claim 1,
The pharmaceutical composition is characterized in that it further comprises a chemotherapeutic agent or an immunotherapeutic agent, pharmaceutical composition.

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