KR102401077B1 - Pharmaceutical Composition for preventing or treating cancer comprising Squirrel fibroma virus and Reovirus - Google Patents

Abstract

The present invention relates to the use of Squirrel fibroma virus and reovirus for preventing or treating cancer. The pharmaceutical composition comprising the squirrel fibroma virus and reovirus of the present invention, or a biological sample treated with the two viruses, exhibits oncolytic activity by specifically infecting cancer cells without affecting normal human cells. has the effect of inhibiting the growth of

Description

TECHNICAL FIELD [0001] Pharmaceutical composition for preventing or treating cancer comprising Squirrel fibroma virus and Reovirus

The present invention relates to the use of Squirrel fibroma virus and reovirus for preventing or treating cancer.

The incidence of cancer continues to increase compared to the past due to industrial development, changes in the global ecosystem and diet, etc., and research on anticancer drugs continues to grow.

Pharmaceuticals currently used can be divided into chemotherapeutic agents and biological agents, and biological anticancer drugs using genes, enzymes, vaccines, and viruses are still insufficient at the commercialization stage, and chemical agents have various side effects due to pharmacological action and toxicity. Problems appearing are pointed out.

Accordingly, various treatment methods such as surgical therapy, radiation therapy, and chemotherapy have been researched to treat cancer, but the complete treatment of cancer has not yet been achieved. Of these, since surgical therapy and radiation therapy are useful methods in the early stages of cancer development, the dependence on chemotherapy is gradually increasing for cancers that are difficult to detect in the early stages, and chemotherapy has a history of more than 50 years. Hundreds of anticancer drugs have been developed and used in clinical practice so far, but there are still few cases in which clinically satisfactory effects are obtained.

Viruses are one of the biotherapeutic agents and have the concept of targeted therapeutics that attack using genetic mutations in tumor cells. Anticancer virus selectively proliferates in cancer cells and induces tumor necrosis and death.

An oncolytic virus is a virus used for the treatment of cancer. Cancer treatment research using wild type oncolytic virus is distinguished from gene therapy, which mainly aims to see the oncolytic effect of the expression of a therapeutic gene by inserting an existing therapeutic gene into the virus. As a result, research on wild-type tumor-destroying viruses began as it became known that some wild-type viruses have inherently powerful tumor-destroying abilities.

There have been reports for a long time that cancer is treated naturally due to natural infection by various types of viruses. Therapeutic studies have even reached phase 3 clinical application. In addition, adenovirus, poliovirus, herpes simplex virus, vesicular stomatitis virus, etc. have been developed, and methods to increase the efficacy and stability of the virus are being studied.

Republic of Korea Publication No. 10-2009-0125103

Therefore, while the present inventors were continuing their research to find an effective tumor-destroying virus, when squirrel fibroma virus and reovirus are used in combination, due to host specificity, normal human cells are not infected and cancer cells are not infected. The present invention was completed by confirming that it exhibits high stability and tumor destructive power for infecting only the squirrel fibroid virus when used in combination with reovirus and can exhibit a very superior 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 above-mentioned problems, 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 is (a) squirrel fibroma virus (Squirrel fibroma virus) and reovirus (Reovirus); Or (b) provides a pharmaceutical composition for preventing or treating cancer comprising the biological sample treated in (a) as an active ingredient.

The present invention also relates to (a) squirrel fibroma virus and reovirus; or (b) administering to the subject the biological sample treated in (a).

In addition, the present invention relates to (a) squirrel fibroma virus and reovirus; or (b) the use of the biological sample treated with (a) for preventing, ameliorating or treating 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, colorectal cancer, pancreatic cancer, brain cancer, ovarian cancer, and combinations thereof. may be, but is not limited thereto.

In another embodiment of the present invention, the squirrel fibroid virus or reovirus 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 an essential region or a non-essential region in the squirrel fibroma virus gene or reovirus gene to have anticancer activity can be increased, but is not limited thereto.

In another embodiment of the present invention, the squirrel fibroid virus or reovirus may be loaded in virus carrier cells and administered in vivo, but is not limited thereto.

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

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

In another embodiment of the present invention, the titer ratio of the squirrel fibroid virus and the reovirus may be 1:1 to 1:1000 (reovirus: 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 fibroma virus and reovirus to an ex vivo biological sample, but is limited thereto it is not going to be

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 fibroid virus may be administered simultaneously (simultaneous), separately (separate) or sequentially (sequential) with the reovirus, but is not limited thereto.

In another embodiment of the present invention, the squirrel fibroid virus or reovirus may be obtained using baby hamster kidney (BHK) 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 fibroid virus and reovirus of the present invention, or a biological sample treated with the two viruses, specifically infects cancer cells without affecting normal human cells and has oncolytic activity (oncolytic activity) ) to inhibit the growth of cancer cells.

In addition, since the combination of squirrel fibroid virus and reovirus exhibits a very superior synergistic effect than virus monotherapy, the present invention can be usefully used for the prevention and treatment of cancer.

1 shows the synergistic oncolytic effect of the combination of squirrel fibroma virus and reovirus in gastric cancer cells, and untreated (Mock), reovirus alone (REO), and squirrel fibroma virus alone on gastric cancer cells (MKN28, AGS, SNU668). (SFV) and the combination of reovirus and squirrel fibroma virus (REO + SFV) confirmed the death of cancer cells by CPE assay, and is a graph showing the WST assay.
Figure 2 shows the synergistic oncolytic effect of the combination of squirrel fibroma virus and reovirus in glioma cells, untreated (Mock), reovirus alone (REO), and squirrel fibroma virus alone on glioma cells (U87MG, SNU489). (SFV) and the combination of reovirus and squirrel fibroma virus (REO + SFV) confirmed the death of cancer cells by CPE assay, and is a graph showing the WST assay.
3 shows the synergistic oncolytic effect of the combination of squirrel fibroma virus and reovirus in lung cancer cells, and untreated (Mock), reovirus alone (REO), squirrel fibroma virus alone (SFV) and lung cancer cells (A549). It is a diagram showing cancer cell death by CPE assay and graphically through WST assay when infected with a combination of reovirus and squirrel fibroma virus (REO+SFV).
Figure 4 shows the synergistic oncolytic effect of the combination of squirrel fibroma virus and reovirus in liver cancer cells, untreated (Mock), reovirus alone (REO), squirrel fibroma virus alone (SFV) and liver cancer cells (Hep3B). It is a diagram showing cancer cell death by CPE assay and graphically through WST assay when infected with a combination of reovirus and squirrel fibroma virus (REO+SFV).
Figure 5 shows in vivo the synergistic oncolytic effect of the combination of squirrel fibroma virus and reovirus in melanoma. Reovirus alone (REO) and squirrel fibroma virus in C57BL/6 female mice induced with B16F10 melanoma allograft. It is a diagram showing the change in tumor size after infection alone (SFV) or in combination with reovirus and squirrel fibroma virus (REO+SFV).

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

While the present inventors were continuing their research to find an effective tumor-destroying (oncolytic activity) virus, when the squirrel fibroma virus and reovirus were used in combination, due to host specificity, the high rate of infecting only cancer cells without infecting normal human cells It was confirmed that not only stability and tumor destructive power were shown, but it was also confirmed that when the squirrel fibroma virus was used in combination with the reovirus, a very superior synergistic effect was exhibited than that of the virus monotherapy (see Examples 1 to 5).

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

The squirrel fibroid virus of the present invention can be prepared using known techniques such as in vivo passage using squirrel/woodchuck, primary squirrel kidney cells or sheep 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 “reovirus” refers to a virus having a double-stranded segmented RNA genome, and refers to any virus classified in the family Reoviridae. The virion of the reovirus has a diameter of 60-80 nm, and has two concentric capsid shells. This genome consists of double-stranded RNA, which is 10 to 12 discontinuous segments, and has a total genome size of 16 to 27 kbp, and each RNA segment has a different size.

In the present invention, the reovirus includes not only a naturally occurring reovirus, but also a modified or recombinant reovirus. The reovirus is said to be “naturally occurring” when it can be isolated from nature and has not been artificially altered by humans. For example, the reovirus may be from a “field source,” ie a person infected with the reovirus.

The reovirus can be modified, but can lytically infect mammalian cells with an active ras pathway. In addition, the reovirus may be pretreated chemically or biochemically (eg, by treatment with a protease such as chymotrypsin or trypsin) before administration to the proliferating cells. Pretreatment with a protease removes the virus' outer membrane or capsid, thereby increasing the infectivity of the virus. The reovirus can be coated with liposomes or micelles, for example, the virions can be treated with chymotrypsin in the presence of an alkyl sulfate detergent at a micelle-forming concentration to generate new infectious subviral particles. have.

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

The attenuated reovirus comprises an infectious replicable reovirus virion lacking a reovirus sigma 1 capsid protein detectable by the genome of a reovirus lacking the S1 gene of a wild-type reovirus. As such, attenuated reoviruses exhibit cytopathic effects on non-malignant cells while unexpectedly maintaining the ability of a mutated reovirus lacking a detectable reovirus sigma 1 capsid protein to infect target tumor cells productively. effect) is desirably avoided. As mentioned above, prior to the present disclosure, particles of a reovirus lacking sigma 1 were understood to be non-infectious.

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

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

The attenuated reovirus lacks a detectable sigma 1 capsid protein but is unexpectedly infectious. As described above, we show that sigma 1 is involved in the binding and attachment of reoviruses to cells through cell surface sialic acid residues in the early stages of viral replication infection. Despite lacking detectable sigma 1, the attenuated reovirus described herein is capable of host cell entry and replication of cytolytic viruses. In addition, an attenuated reovirus has a reduced (i.e., statistical It exhibits surprising properties inducing levels of reduced significance).

Attenuated reoviruses may be derived from any reovirus meaning a member of the reoviridae, and include reoviruses with varying affinities, which may be obtained from a variety of sources.

In certain embodiments, it may be a mammalian reovirus, and in other embodiments it may be a human reovirus. In other embodiments (eg, for use in animal models of relevance to human disease or for veterinary applications), the attenuated reovirus is administered to a non-human primate (eg, chimpanzee, gorilla, macaque, monkey, etc.). , cells of other mammalian species, including rodents (eg, mice, rats, gerbils, hamsters, rabbits, guinea pigs, etc.), dogs, cats, common domestic animals (eg, cattle, horses, pigs, goats), and the like. may be derived from one or more reoviruses exhibiting affinity for

Attenuated reoviruses involve 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-deficient and/or sigma 3-deficient and/or or generation and identification of sigma 3-deficient mutants), also isolation of naturally occurring sigma 1-deficient and/or sigma 1-deficient 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 reovirus genes in productively infected host cells). have.

The attenuated reovirus is an infectious, replicable reovirus virion that lacks the wild-type reovirus S1 gene and consequently the detectable reovirus sigma 1 capsid protein (i.e., contains the genome, core protein and protein envelope of the virus). virus particles).

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

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

The human reovirus can be composed of three serotypes: type 1 (Lang strain or T1L), type 2 (Jones strain, T2J) and type 3 (Dearing strain, T3D or Abney strain, T3A). The three serotypes can be easily identified based on a neutralization reaction and hemagglutinin inhibition assay.

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

As used herein, in the term “BHK-21 cells”, BHK is an abbreviation of Baby Hamster Kidney. BHK cells were originally isolated by polyoma transformation of hamster cells, and were added to the vaccine. It can be used as a substrate for viral propagation and virus-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 baby hamster kidneys of five 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 commonly known as the Syrian golden hamster (Mesocricetus auratus).

As used herein, the term “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 (adipose derived 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 a cancer patient.

In the present invention, the biological sample may be prepared to kill a plurality of cancer cells by applying effective amounts of squirrel fibroma virus and reovirus 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 fibroid virus or reovirus may include a heterologous gene for cancer treatment, but is not limited thereto.

The term “heterologous gene” is used to mean accepting any gene that is 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 an essential region or a non-essential region within the squirrel fibroma virus gene or reovirus gene to increase anticancer activity.

The foreign gene may be operably linked to a control sequence that allows expression of the electric foreign gene in a cell under in vivo conditions. Accordingly, the virus of the present invention can be used to deliver a foreign gene/genes to a cell in an in vivo condition in which the foreign gene can be expressed. The gene usually encodes a protein capable of enhancing the tumor-destructive properties of the virus. The gene itself may be a cytotoxin or may encode a protein capable of promoting/enhancing an anti-tumor immune response.

The foreign gene includes any gene for treating cancer by inserting it into a replication-competent tumor-destroying virus or an existing replication-defective virus vector.

As used herein, the term “therapeutic gene” is intended to describe all broad-spectrum genes whose expression influences desirable outcomes, for example, anticancer effects. The squirrel fibroid virus or reovirus of the present invention may comprise one or more desired sequences encoding a therapeutic gene. A therapeutic gene may have pharmacological or prophylactic activity when properly administered to a patient, particularly a patient suffering from a disease or condition or to be protected from such a disease or condition.

Such pharmacological or prophylactic activity is meant to be expected to be associated with a beneficial effect on the course or symptom of the disease or condition. Said sequence of interest may be homologous or heterologous to the target cell into which it is introduced, and encodes all or part of a polypeptide, in particular a therapeutic or prophylactic polypeptide that imparts therapeutic or prophylactic properties. Polypeptide is understood to be any translational product of a polynucleotide, regardless of size and glycosylated, and includes peptides and proteins. Therapeutic polypeptides include polypeptides capable of compensating for deficient or deficient proteins in an animal or human organism or those that act through toxic effects to limit or eliminate harmful cells from the body. They may also be immune conferring polypeptides that act as endogenous antigens to induce humoral or cellular responses, or both, for example, drug-sensitizing genes, proapoptotic genes, cells It includes, but is not limited to, a cytostatic gene, a cytotoxic gene, a tumor suppressor gene, an antigenic gene, an anti-angiogenic gene, a cytokine gene, etc. it is not going to be

The drug susceptibility gene is a gene for an enzyme that converts a non-toxic precursor (prodrug) into a toxic substance, and is also called a suicide gene because the transfected cells die. That is, when a precursor that is not toxic to normal cells is systemically administered, the precursor is converted into a toxic metabolite only in cancer cells, thereby destroying cancer cells by changing the sensitivity to drugs. Representatively, HSVtk (Herpes simplex virus-thymidine kinase) gene, ganciclovir, E. coli cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) are It is possible.

The apoptosis gene refers to a nucleotide sequence that is expressed and induces programmed cell death. Apoptosis genes known to those skilled in the art, encoding p53, adenovirus E3-11.6K (derived from Ad2 and Ad5) or adenovirus E3-10.5K (derived from Ad), adenovirus E4 gene, p53 pathway gene and caspase genes are included.

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, a retinoblastoma gene, an E2F-Rb fusion protein gene, a gene encoding a cyclin-dependent kinase repressor (eg, p16, p15, p18 and p19), a growth arrest specific homeobox (growth arrest specific homeobox) , GAX) gene and the like.

The cytotoxic gene refers to a nucleotide sequence that is expressed in a cell and exhibits a toxic effect. As an example, there is a nucleotide sequence encoding Pseudomonas exotoxin, ricin toxin, diphtheria toxin, and the like.

The tumor suppressor gene refers to a nucleotide sequence capable of suppressing a tumor phenotype or inducing apoptosis by being expressed in a target cell. Representatively, tumor necrosis factor-α, TNF-α, p53 gene, APC gene, DPC-4/Smad4 gene, BRCA-1 gene, BRCA-2 gene, WT-1 gene, retinoblastoma gene (Lee et al., Nature, 329,642, 1987), MMAC-1 gene, adenomatous polyposis coil protein, defective colon tumor (DCC) gene, MMSC-2 gene, NF-1 gene, chromosome 3p21 .3 located in the nasopharyngeal tumor suppressor gene, the MTS1 gene, the CDK4 gene, the NF-1 gene, the NF-2 gene and the VHL gene.

The antigenic gene refers to a nucleotide sequence that is expressed in a target cell to produce a cell surface antigenic protein that can be recognized by the immune system. Examples of antigenic genes known to those skilled in the art include carcinoembryonic antigen (CEA) and p53 (Levine, A., International Patent Application Publication No. WO 94/02167).

Examples of polypeptides encoded by more specific therapeutic genes include cytokines (alpha, beta or gamma interferon, interleukins, in particular IL-2, IL-6, IL-10 or IL-12, tumor necrosis factor (TNF), chemokines , Colony Stimulating Factor GM-CSF, C-CSF, M-CSF), Immunostimulatory Polypeptide (B7.1, B7.2, etc.), Coagulation Factor (FVIII, FIX), Growth Factor (Transforming Growth Factor TGF, Fiber blast growth factor FGF, etc.), enzymes (urease, renin, thrombin, metalloproteinase), nitric oxide synthase (NOS, SOD, catalase), enzyme inhibitors (alpha1-antitrypsin, antithrombin III, Viral protease inhibitors, plasminogen activator inhibitor PAI-1), CFTR (cystic fibrosis transmembrane conductance regulator) protein, insulin, dystrophin, MHC class I or II antigen, polypeptide capable of modulating/regulating the expression of cellular genes , a polypeptide capable of inhibiting bacterial, parasitic or viral infection or its occurrence (antigenic polypeptide, antigenic epitope, transdominant variant that competitively inhibits the action of a native protein), apoptosis inducers or inhibitors (Bax, Bcl2, BclX...), cytostatic agents (p21, p 16, Rb), apolipoprotein (ApoAI, ApoAIV, ApoE...), inhibitors of angiogenesis (angiostatin, endostatin), blood vessels Forming polypeptides (vascular endothelial growth factor VEGF family, FGF family, CCN family including CTGF, Cyr61 and Nov), oxygen radical scavengers, polypeptides with anti-tumor effects, antibodies, toxins, immunotoxins and markers (beta - comprises gene encoding galactosidase, luciferase) or any other gene of interest recognized in the art to be useful for treatment or prevention of clinical condition.Suitable anti-tumor gene includes tumor suppressor gene (e.g., Rb , p53, DCC, NF-1, Will Wilm's tumor, NM23, BRUSH-1, p16, p21, p56, p73 and their respective mutants), suicide gene products, antibodies, including those encoding polypeptides that inhibit cell division or transduction signals. , but not limited thereto. The suicide gene includes a gene encoding a protein having cytosine deaminase activity, thymidine kinase activity, uracil phosphoribosyl transferase activity, purinenucleoside phosphorylase activity and/or thymidylate kinase activity. , but not limited thereto. The therapeutic gene may be inserted into an essential region or a non-essential region in the squirrel fibroma virus or reovirus gene to increase its anticancer activity.

In the present invention, the squirrel fibroid virus or reovirus may be loaded in virus carrier cells (for example, Molecular Therapy. 2009 Vol 17 : 1667-1676) and administered in vivo, through this The therapeutic effect may 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 any action that inhibits cancer or delays the progression of cancer 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 changed beneficially by administration of the composition of the present invention, and refers to an attempt to obtain useful or desirable results, including clinical results. A useful or desirable clinical outcome, if not detectable or undetectable, may result in alleviation or amelioration of one or more symptoms or conditions, narrowing the scope of the disease, stabilizing the disease state, inhibiting the occurrence of the disease, inhibiting the spread of the disease, delaying or slowing the progression of the disease. , delay or delay onset of disease, amelioration or alleviation of disease state, and attenuation (in part or all). In addition, “treatment” may mean prolonging the survival of a patient beyond what would be expected in the absence of treatment. In addition, “treatment” may mean inhibiting disease progression, temporarily slowing the disease progression, and may refer to stopping the disease progression permanently.

Cancer diseases that can be treated in the present invention include blood cancer, colorectal cancer, brain cancer, glioma, neuroedema, anaplastic astrocytoma, medulloblastoma, gastric cancer, lung cancer, small cell lung carcinoma, cervical carcinoma, colon cancer, Rectal cancer, chordoma, throat cancer, Kaposi's sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, colorectal cancer, endometrial cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, liver carcinoma, cholangiocarcinoma, choriocarcinoma, seminothelioma, Testicular tumor, Wilms tumor, Ewing tumor, bladder carcinoma, angiosarcoma, endothelial sarcoma, adenocarcinoma, sweat gland carcinoma, sebaceous adenocarcinoma, papillary carcinoma, papillary adenosarcoma, cystadenosarcoma, bronchial carcinoma, medullary carcinoma, mastocytoma, mesothelioma, synovoma, melanoma tumor, leiomyoma, rhabdomyofibroma, neuroblastoma, retinoblastoma, oligodendroglioma, acoustic neuroma, hemangioblastoma, meningioma, pineal celloma, ependymoma, craniopharyngioma, epithelial carcinoma, embryonic carcinoma, squamous cell carcinoma, basal cell carcinoma, fibrosarcoma, myxoma , myxosarcoma, 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, colorectal cancer, pancreatic cancer, brain cancer, ovarian cancer, and combinations thereof, However, the present invention is not limited thereto.

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

Treatments 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 given composition of the present invention to a subject by any suitable method.

As used herein, the term “individual” refers to all animals, such as humans, monkeys, dogs, goats, pigs, or mice, having a disease that can be improved by administering the composition of the present invention.

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

As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit or risk ratio applicable to medical treatment, which includes the type, severity, and activity of the drug in the subject; Sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrently used drugs and other factors well known in the medical field may be determined.

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

The effective amount may vary depending on a number of factors such as pharmacokinetic properties of the virus, administration method, age, health and weight of the patient, the nature and range of the disease state, the number of treatments and the latest type of treatment, for example, the virulence of the virus and It may also vary depending on the potency. A person skilled in the art can adjust the appropriate amount based on the above factors. The virus can initially be administered in appropriate doses as needed, depending on the patient's clinical response. The effective amount of virus can be determined empirically and can depend on the maximum amount of virus that can be safely administered and the minimum amount of virus that produces the desired result.

When the virus is administered systemically, administration of a significantly higher dose of the virus may be required to induce a clinical effect similar to that achieved by injecting the virus into the diseased site. However, the appropriate dose level should be the minimum amount to achieve the desired results.

The concentration of the administered virus may vary depending on the virulence of the squirrel fibroid virus or reovirus strain to be administered and the characteristics of the cells being targeted.

In the present invention, the squirrel fibroid virus may be included in 10 ² to 10 ¹³ PFU (plaque forming units). For example, the squirrel fibroid 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.

Also, in the present invention, the squirrel fibroid virus may be included in an amount of 0.01 to 10 MOI (multiplicity of infection). For example, the squirrel fibroid virus may have an MOI of 0.01 to 9, 0.01 to 8, 0.01 to 7, 0.01 to 6, or 0.01 to 5; 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 reovirus may be included in 10 ² to 10 ¹⁵ PFU. For example, the reovirus 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 reovirus may be included in an MOI of 0.1 to 100. For example, the reovirus may be included in an MOI of 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 fibroid virus and the reovirus may be 1:1 to 1000:1 (reovirus: squirrel fibroma virus), but is not limited thereto. For example, the titer ratios of the squirrel fibroid virus and reovirus are 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 (reovirus: squirrel fibroma virus).

In addition, in the present invention, the titer ratio of the squirrel fibroma virus and reovirus may be 1:1 to 1000:1 (squirrel fibroma virus: reovirus), but is not limited thereto. For example, the titer ratios of the squirrel fibroid virus and reovirus are 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: reovirus).

An effective amount of virus may be administered repeatedly depending on the effectiveness of the initial treatment regimen. In general, dosing is administered periodically while monitoring for all responses. One of ordinary skill in the art will readily appreciate that, depending on the dosing schedule and route chosen, lower or higher doses than those indicated above may be administered.

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

On the other hand, the pharmaceutical composition according to the present invention may further include an appropriate carrier, excipient and/or diluent commonly used to prepare a pharmaceutical composition in addition to the active ingredient. In addition, according to a conventional method, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., oral preparations, external preparations, suppositories, and sterile injection solutions.

When formulating the composition, it can be prepared using a diluent or excipient such as a carrier, a stabilizer, an antibacterial agent, a buffer, a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant.

The pharmaceutical composition of the present invention may be administered to an individual by various routes. Any mode of administration can be envisaged, for example, by oral administration, intranasal administration, intratumoral administration, transbronchial administration, arterial injection, intravenous injection, subcutaneous injection, intramuscular injection or intraperitoneal injection. . The daily dose may be administered once or divided into several times a day.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Confirmation of apoptosis of gastric cancer cell lines

Gastric cancer cell lines (MKN28, AGS, SNU668) untreated (Mock), wild-type reovirus alone (REO, MKN28 1 MOI; AGS and SNU668 100 MOI), wild-type squirrel fibroid virus alone (SFV, MKN28 0.32 MOI; AGS and SNU668) 3.2 MOI) and a combination of reovirus and squirrel fibroma virus (REO+SFV), and the death of cancer cells was confirmed by CPE assay. Specifically, after 4 days of spreading cancer cells on a 96-well plate and infecting them with the virus, cell viability was measured through WST assay and converted into a graph is shown in FIG. 1 .

As the anticancer effect increases, the number of cells stained with crystal violet is small, and the purple color comes out. As shown in FIG. 1 , the relative cell viability was the lowest when infected with the reovirus alone or the squirrel fibroma virus alone (the rightmost bar graph in FIG. 1 ).

These results show that the combined use of reovirus and squirrel fibroma virus exhibits the best anticancer effect by showing a synergistic anticancer effect.

Example 2. Confirmation of death of glioma cell lines

Glioma cell lines (U87MG, SNU489) untreated (Mock), wild-type reovirus alone (REO, 10 MOI), wild-type squirrel fibroma virus alone (SFV, U87MG 0.16 MOI; SNU489, 1.6 MOI) and reovirus and squirrel fibroma It was infected with a combination of viruses (REO + SFV) and apoptosis of cancer cells was confirmed by CPE assay. Specifically, the results of cell viability were measured by WST assay 4 days after the cancer cells were spread on a 96-well plate and infected with the virus, and the result converted into a graph is shown in FIG. 2 .

As a result, as shown in FIG. 2 , the relative cell viability was the lowest when infected with the reovirus alone or when infected with the squirrel fibroma virus alone (the rightmost bar graph in FIG. 2 ).

These results show that the combined use of reovirus and squirrel fibroma virus exhibits the best anticancer effect by showing a synergistic anticancer effect.

Example 3. Confirmation of death of lung cancer cell lines

Infected lung cancer cell line (A549) with no treatment (Mock), wild-type reovirus alone (REO, 10 MOI), wild-type squirrel fibroma virus alone (SFV, 3.2 MOI), and a combination of reovirus and squirrel fibroma virus (REO+SFV) and apoptosis of cancer cells was confirmed by CPE assay. Specifically, the results of cell viability were measured through WST assay 4 days after the cancer cells were spread on a 96-well plate and infected with the virus, and the result converted into a graph is shown in FIG. 3 .

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

These results show that the combined use of reovirus and squirrel fibroma virus exhibits the best anticancer effect by showing a synergistic anticancer effect.

Example 4. Confirmation of apoptosis of liver cancer cell lines

Hepatocellular carcinoma cell line (Hep3B) was infected with untreated (Mock), wild-type reovirus alone (REO, 1 MOI), wild-type squirrel fibroma virus alone (SFV, 0.32 MOI), and reovirus and squirrel fibroma virus in combination (REO+SFV). and apoptosis of cancer cells was confirmed by CPE assay. Specifically, 4 days after the cancer cells were spread on a 96-well plate and infected with the virus, the cell viability was measured through WST assay, and the result converted into a graph is shown in FIG. 4 .

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

These results show that the combined use of reovirus and squirrel fibroma virus exhibits the best anticancer effect by showing a synergistic anticancer effect.

Example 5. Melanoma ( melanoma ) anticancer effect

C57BL/6 mice (female, DOB: 2019-, 5 weeks old) were injected with mouse melanoma cells (B16F10 cells) at 1 X 10 ⁵ cells/mice to prepare an allograft animal model to induce tumors. Thereafter, the animal model was infected with wild-type reovirus alone (1 X 10 ⁷ PFU/injection), wild-type squirrel fibroma virus alone (1 X 10 ⁴ PFU/injection), and reovirus and squirrel fibroma virus in combination, and then cancer cell death was achieved. It was confirmed by a reduction in tumor size. Virus infection was performed when the initial tumor size was 130 mm ³ , and virus infection was also performed the next day, and the size of the tumor was measured after injecting the virus twice in total.

As a result, as shown in FIG. 5 , the size of the tumor was reduced when each virus was injected alone compared to the negative control (Vehicle) CsCl, and in particular, when both viruses were injected in combination, the size of the tumor decreased, The anticancer effect was significantly increased (synergistic anticancer effect) (FIG. 5).

The results of Examples 1 to 5 above show that the highest anticancer effect appears when the reovirus or the squirrel fibroma virus is infected alone, compared to the case of infection with the reovirus alone or the squirrel fibroma virus. It shows that the combined use shows the best anticancer effect through the synergistic anticancer effect. In addition, it shows that the combined use of such reovirus and squirrel fibroma virus can exhibit anticancer effects against various carcinomas.

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it 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) containing wild-type squirrel fibroma virus (Squirrel fibroma virus) and reovirus (Reovirus) as an active ingredient,
A pharmaceutical composition for preventing or treating cancer, in which a gastric cancer patient group comprising MKN28 or AGS gastric cancer cells, a glioma patient group comprising U87MG or SNU489 glioma cells, or a lung cancer patient group comprising A549 lung cancer cells is a target patient group .
delete According to claim 1,
The squirrel fibroid virus or reovirus is characterized in that it comprises a foreign gene for the treatment of cancer, a pharmaceutical composition.
4. The method of claim 3,
The foreign gene for cancer treatment is inserted into an essential region or a non-essential region within the squirrel fibroma virus gene or reovirus gene, characterized in that it increases anticancer activity, pharmaceutical composition.
According to claim 1,
The squirrel fibroid virus or reovirus is loaded on virus carrier cells and administered in vivo, a pharmaceutical composition.
According to claim 1,
The squirrel fibroid virus is characterized in that it contains 10 ² to 10 ¹³ PFU, the pharmaceutical composition.
7. The method of claim 1 or 6,
The reovirus is a pharmaceutical composition, characterized in that it contains 10 ² to 10 ¹⁵ PFU.
According to claim 1,
The titer ratio of the squirrel fibroid virus and the reovirus is 1:1 to 1000:1 (reovirus: squirrel fibroma virus), a pharmaceutical composition.
delete delete According to claim 1,
The squirrel fibroid virus is a pharmaceutical composition, characterized in that administered simultaneously (simultaneous), separately (separate) or sequentially (sequential) with the reovirus.
According to claim 1,
The squirrel fibroid virus or reovirus is a pharmaceutical composition, characterized in that obtained using BHK (baby hamster kidney) cells.
According to claim 1,
The pharmaceutical composition, characterized in that it further comprises a chemotherapeutic agent or an immunotherapeutic agent.

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