A composition for preventing or treating cancer, comprising a dual expression vector for simultaneously expressing a protein present in an intracellular environment and a protein secreted to the outside of the cell.

Abstract

The present invention relates to a composition for preventing or treating cancer, comprising a dual expression vector, and more specifically, to a method for effectively treating cancer by simultaneously inducing the action of a metabolic anti-cancer agent and an immune anti-cancer agent by using a dual expression vector that simultaneously expresses a HIF-1α inhibitor and an anti-cancer cytokine.

Description

[Technical field]

[0001] The present invention relates to a composition for preventing or treating cancer, comprising a dual expression vector, and more specifically, to a method for effectively treating cancer by simultaneously inducing the action of a metabolic anti-cancer agent and an immune anti-cancer agent by using a dual expression vector that simultaneously expresses an intracellular HIF-1α inhibitor and an anti-cancer cytokine that can be secreted outside the cell. [Background technology]

[0002] Despite the tireless efforts of humanity over the past several decades, cancer remains an intractable disease and one of the major unmet medical needs. Although various biochemical mechanisms related to the development of cancer have been elucidated and many anticancer drugs have been developed based on this knowledge, no effective treatment method has yet been presented. Recently, with the development and generalization of early diagnosis methods, when cancer is diagnosed at an early stage, treatment has shown a near-complete cure effect, but the number of cancer patients has been steadily increasing due to social and environmental factors, and by the time symptoms begin to be felt, cancer is often already quite advanced, and to date, the cancer treatment rate and survival rate are not high. In fact, according to a report by the World Health Organization in 2015, cancer is the first and second leading cause of death before the age of 70 in 91 of the 172 countries worldwide.

[0003] Cancer develops when cells proliferate indefinitely due to the accumulation of intracellular mutated genes caused by genetic and environmental factors, resulting in the formation of abnormal tissues. In particular, the most problematic mutations in recent years are those occurring in exons 18, 19, 20, and 21 of EGFR, with nucleotide deletions in exon 19 and L858R mutations in exon 21 being problematic. In addition, G12C mutations in KRAS, V900E mutations in BRAF, and mutations due to chromosomal rearrangements in ROS1 have also been identified as major causes.

[0004] Cancer treatment methods include not only surgical procedures such as surgery and radiation, but also many treatments using anticancer drugs. For example, first-generation chemical anticancer drugs such as Vinca Alkaloids (Vincristine, Vinblastine, Vinorelbine), Taxols (Paclitaxel, Docetaxel), Platinums (Cisplatin, Carboplatin), Doxorubicin, Etoposide, Gemcitabine, and Pemetrexed have been used as anticancer drugs that suppress cell division, but they have a significant effect on not only cancer cells but also normal cells, causing serious side effects such as general fatigue, hair loss, nausea, vomiting, loss of appetite, diarrhea, oral inflammation, decreased blood cells, and damage to certain organs.

[0005] To overcome these shortcomings, targeted anticancer drugs developed since the mid-1990s have been developed to treat cancer by targeting cancer-related genes such as specific mutated genes. Representative targeted anticancer drugs include Gefitinib, Erlotinib, Afatinib, Osimertinib, Crizotinib, Ceritinib, Alectinib, Brigatinib, and Dabrafenib, which target EGFR mutations, ALK mutations, and BRAF mutations. Although such targeted anticancer drugs have the advantage of having less toxic side effects than chemical anticancer drugs, it is known that resistant cancer cells that can avoid the mechanism of action of targeted anticancer drugs frequently appear within 8 to 14 months after administration, which not only reduces the effectiveness of cancer treatment but also increases the rate of cancer recurrence. For example, the appearance of drug-resistant EGFR T790M mutation against EGFR inhibitors can be cited as a representative example.

[0006] Subsequently, immune anticancer drugs, which help induce the death of cancer cells in the body by activating the immune system suppressed in cancer tissues, were presented as an alternative that could overcome the limitations of conventional anticancer drugs. Theoretically, immune anticancer drugs are applicable to most cancers regardless of specific gene mutations, and are expected to have fewer side effects than conventional anticancer drugs. However, the therapeutic effect of immune anticancer drugs was unexpectedly low. For example, Keytruda, an immune anticancer drug that targets the immune checkpoint inhibitor PD-1 / PD-L1, had very low reactivity when actually applied in treatment. In particular, in the case of non-small cell lung cancer, only 20-30% of patients showed therapeutic effects, and the remaining 70-80% of patients showed almost no response. Therefore, there is a pressing need to develop anticancer drugs that are effective against various cancers.

[0007] Recently, attempts have been made to develop metabolic anticancer drugs that target cancer cell-specific metabolic processes, particularly mitochondrial metabolism, activation of autophagy, and random absorption of nutrients from the outside in cancer cells under hypoxic conditions. These drugs are expected to overcome the high toxicity of first-generation chemical anticancer drugs, the frequent resistance of second-generation targeted anticancer drugs, and the low response rate of third-generation immune anticancer drugs. In particular, in relation to hypoxic conditions in cancer tissues, hypoxia-inducible factor-1 (HIF-1) is a transcription factor induced by hypoxia and is known to be the most important molecule in regulating the adaptation of cancer cells to hypoxic conditions. In particular, the amount of HIF-1α protein is known to have a close correlation with the prognosis of cancer patients. HIF-1 is activated by stimulation with cancer cell growth factors, hypoxic conditions, activation of oncogenes, or inactivation of tumor suppressor genes such as pVHL. Activated HIF-1 induces the expression of genes such as hexokinase 2, glucose transporter 1, erythropoietin, IGF-2, endoglin, VEGFA, MMP-2, uPAR and MDR1, resulting in the acquisition of traits such as resistance to apoptosis, improved angiogenesis, increased cell proliferation, cell migration, metastasis and invasion, ultimately causing cancer cells to become malignant. Therefore, although many attempts have been made to treat cancer by inhibiting HIF-1α, no effective therapeutic agent has been developed that has been commercially viable (Korean Patent Publication No. 10-2015-0039264).

[0008] Therefore, based on the results of intensive research into using HIF-1α inhibitors as effective cancer therapeutic agents, the inventors have developed an effective dual expression vector that can regulate cancer-specific metabolism by HIF-1α inhibitors while at the same time exerting immune anti-cancer effects by anti-cancer cytokines. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention has been made to solve the problems in the prior art as described above, and aims to provide a composition for preventing or treating cancer, which contains as an active ingredient a dual expression vector that simultaneously expresses an intracellular HIF-1α inhibitor and an anti-cancer cytokine that can be secreted outside the cell.

[0010] However, the technical problems that the present invention aims to solve are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those having ordinary skill in the art to which the present invention pertains from the following description. [Means for solving the problem]

[0011] The present invention provides a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient a recombinant vector including a polynucleotide encoding a hypoxia-inducible factor-1α inhibitor, a polynucleotide encoding a 2A peptide, and a polynucleotide encoding an anticancer cytokine.

[0012] The present invention also provides a food composition for preventing or ameliorating cancer, comprising as an active ingredient a recombinant vector including a polynucleotide encoding a hypoxia-inducible factor-1α inhibitor, a polynucleotide encoding a 2A peptide, and a polynucleotide encoding an anticancer cytokine.

[0013] In one embodiment of the present invention, the recombinant vector may be in a form in which a polynucleotide encoding a 2A peptide is inserted between a polynucleotide encoding a hypoxia inducible factor-1α inhibitor and a polynucleotide encoding an anti-cancer cytokine, and the recombinant vector may be in a form in which a polynucleotide encoding a hypoxia inducible factor-1α inhibitor, a polynucleotide encoding a 2A peptide, and a polynucleotide encoding an anti-cancer cytokine are operably linked in this order, or a polynucleotide encoding an anti-cancer cytokine, a polynucleotide encoding a 2A peptide, and a polynucleotide encoding a hypoxia inducible factor-1α inhibitor are operably linked in this order, but is not limited thereto as long as it is a dual expression vector in which a hypoxia inducible factor-1α inhibitor and an anti-cancer cytokine can be expressed simultaneously.

[0014] In another embodiment of the present invention, the hypoxia-inducible factor-1α inhibitor may be preferably a protein capable of suppressing the expression or activity of hypoxia-inducible factor-1α, more preferably cytochrome b5 reductase 3, von Hippel Lindau tumour suppressor protein (pVHL), arrest-defective-1 (ARD-1), hypoxia-inducible factor (HIF) Prolyl-Hydroxylase, Kelch-like ECH-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2), etc. Alternatively, it may be an antisense oligonucleotide (ASO), small interfering RNA (siRNA), microRNA (miRNA), aptamer, antibody, etc., which bind to HIF-1α and directly suppress its expression or activity. However, since the dual expression vector of the present invention acts as a mechanism for suppressing hypoxia inducible factor-1α in the body and metabolically suppressing the death, migration, metastasis, proliferation, etc. of cancer cells, the protein is not limited to these as long as it can suppress the expression or activity of hypoxia inducible factor-1α. The cytochrome b5 reductase 3 may most preferably be a protein containing the amino acid sequence represented by SEQ ID NO: 1, and may preferably contain an amino acid sequence having sequence homology of 80% or more, more preferably 90% or more, and even more preferably 95% or more with the amino acid sequence of SEQ ID NO: 1. The "sequence homology percentage" for an amino acid sequence is determined by comparing the optimally aligned sequence with a comparison region, and a part of the amino acid sequence in the comparison region may contain additions or deletions (i.e., gaps) compared to a reference sequence (not including additions or deletions) for the optimal alignment of the sequence.

[0015] In yet another embodiment of the invention, the 2A peptide may comprise the amino acid sequence set forth in SEQ ID NO:5.

[0016] In yet another embodiment of the present invention, the anti-cancer cytokine may be preferably interferon alpha, interferon beta, interferon gamma, interleukin 2, interleukin 7, interleukin 12, interleukin 15, interleukin 18, interleukin 21, interleukin 37, granulocyte-macrophage colony-stimulating factor, etc., and is not limited as long as it is an anti-cancer cytokine known to activate T cells, B cells, natural killer cells (NK cells), etc., and cause an immune response specific to cancer cells.

[0017] In yet another embodiment of the present invention, the recombinant vector may be a linear DNA, a plasmid DNA, or a viral vector, and the viral vector may be a retrovirus, an adenovirus, an adeno-associated virus, a vaccinia virus, a myxoma virus, a herpes simplex virus, a lentivirus, or the like, but is not limited thereto as long as it is a type that can be used as a dual expression vector that can simultaneously express a hypoxia inducible factor-1α inhibitor and an anti-cancer cytokine in the body.

[0018] In yet another embodiment of the present invention, the cancer may be, preferably, melanoma, small cell lung cancer, non-small cell lung cancer, glioma, liver cancer, thyroid tumor, gastric cancer, prostate cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, colon cancer, breast cancer, prostate cancer, glioblastoma, endometrial cancer, kidney cancer, colon cancer, pancreatic cancer, esophageal cancer, head and neck cancer, mesothelioma, sarcoma, bile duct cancer, small intestine adenocarcinoma, pediatric malignant tumor, epidermal cancer, etc., but is not limited thereto as long as the type of cancer can be treated by suppressing hypoxia inducible factor-1α.

[0019] The present invention also provides a method for preventing or treating cancer, comprising the step of administering to an individual a composition comprising, as an active ingredient, a recombinant vector comprising a polynucleotide encoding a hypoxia-inducible factor-1α inhibitor, a polynucleotide encoding a 2A peptide, and a polynucleotide encoding an anticancer cytokine.

[0020] The present invention also provides a use of a composition comprising, as an active ingredient, a recombinant vector including a polynucleotide encoding a hypoxia-inducible factor-1α inhibitor, a polynucleotide encoding a 2A peptide, and a polynucleotide encoding an anticancer cytokine, for the prevention or treatment of cancer.

[0021] The present invention also provides use of a recombinant vector comprising a polynucleotide encoding a hypoxia-inducible factor-1α inhibitor, a polynucleotide encoding a 2A peptide, and a polynucleotide encoding an anticancer cytokine, for producing a drug for use in cancer. Effect of the Invention

[0022] The dual expression vector simultaneously expressing the HIF-1α inhibitor and the anti-cancer cytokine according to the present invention effectively inhibits the proliferation, migration, metastasis, etc. of cancer by a substance capable of inhibiting HIF-1α, i.e., a metabolic anti-cancer drug, and at the same time, activates T cells, B cells, NK cells, etc. by an anti-cancer cytokine, i.e., an anti-cancer immunosuppressant, thereby inducing a cancer-specific immune response, thereby improving the therapeutic effect of the metabolic anti-cancer drug, and by administering one vector, the number of administrations can be reduced, thereby reducing the pain and costs of the patient. In addition, since the immune anti-cancer drug and the metabolic anti-cancer drug are simultaneously expressed, a high therapeutic effect can be exhibited even in a small amount, and side effects can be reduced, as well as the occurrence of resistance to the anti-cancer drug that occurs in the body can be reduced. Therefore, the dual expression vector of the present invention is expected to be effectively applied to the treatment of various cancers. In addition, since anti-cancer treatment must be performed according to the genetic cause or causative factor of cancer onset, the present invention is expected to provide various metabolic anti-cancer drug and immune anti-cancer drug treatment options, thereby significantly increasing the anti-cancer treatment rate. [Brief description of the drawings]

[0023] [Figure 1] FIG. 1 is a schematic diagram illustrating the structure of a dual expression vector according to one embodiment of the present invention, proteins produced by the dual expression vector, their locations, and their effects on cancer. [Diagram 2] FIG. 2 is a simplified schematic diagram of a dual expression vector according to one embodiment of the present invention. [Diagram 3] FIG. 3 shows the results of Western blotting to confirm whether or not a protein was expressed in a dual expression vector according to one embodiment of the present invention. [Figure 4] FIG. 4 shows the results of confirming the anti-cancer effect of a dual expression vector according to one embodiment of the present invention. [Diagram 5] FIG. 5 is a diagram showing the results of confirming the anti-cancer effect of a dual expression vector injected into an adenovirus according to one embodiment of the present invention. [Figure 6] FIG. 6 is a diagram showing the results of confirming activation of NK immune cells by cytokines secreted from a dual expression vector according to one embodiment of the present invention. [Figure 7] FIG. 7 is a diagram showing the results of confirming the immune activation effect of anti-cancer cytokines of a dual expression vector according to one embodiment of the present invention. BEST MODE FOR CARRYING OUT THEINVENTION

[0024] The dual expression vector of the present invention, which simultaneously expresses the HIF-1α inhibitor and the anti-cancer cytokine, can simultaneously express the HIF-1α inhibitor and the anti-cancer cytokine through a single administration, and it was confirmed that the expressed protein works normally and does not show toxicity in normal cells, but can effectively treat cancer by inducing apoptosis specifically in cancer cells. Therefore, the dual expression vector of the present invention, which simultaneously expresses the HIF-1α inhibitor and the anti-cancer cytokine, is expected to be applicable as an effective therapeutic agent for many cancers in which resistance has developed.

[0025] As used herein, the term "recombinant vector" refers to a vector capable of expressing a peptide or protein encoded by a heterologous nucleic acid inserted into the vector, and preferably refers to a vector manufactured to contain a HIF-1α inhibitor gene and an anti-cancer cytokine gene. The term "vector" refers to any medium for introducing and / or transferring bases into a host cell in vitro, ex vivo, or in vivo, and may be a replicating unit to which other DNA fragments can be bound and which can cause replication of the bound fragments, and the term "replicating unit" refers to any genetic unit (e.g., plasmid, phage, cosmid, chromosome, virus, etc.) that functions as an independent unit of DNA replication in vivo, i.e., that can replicate by its own regulation. The recombinant expression vector of the present invention may preferably include a promoter, which is a transcription initiation factor to which an RNA polymerase binds, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site and a sequence for regulating the termination of transcription and translation, a terminator, etc., and more preferably may further include a tagging gene for increasing the production amount of a recombinant protein, a tagging gene for maintaining the structural stability of a recombinant protein, a tagging gene for easy isolation of a recombinant protein, a selection marker gene such as an antibiotic resistance gene for selecting a transformant, etc. Representative examples of tags for easy isolation include an Avi tag, a Calmodulin tag, a polyglutamate tag, an E tag, a FLAG tag, an HA tag, a His tag, a Myc tag, an S tag, an SBP tag, an IgG-Fc tag, a CTB tag, a Softag 1 tag, a Softag 2 tag, a Softag 3 tag, a Softag 4 tag, a Softag 5 tag, a Softag 6 tag, a Softag 7 tag, a Softag 8 tag, a Softag 9 tag, a Softag 10 tag, a Softag 11 tag, a Softag 12 tag, a Softag 13 tag, a Softag 14 tag, a Softag 15 tag, a Softag 16 tag, a Softag 17 tag, a Softag 18 tag, a Softag 19 tag, a Softag 20 tag, a Softag 21 tag, a Softag 22 tag, a Softag 23 tag, a Softag 24 tag, a Softag 25 tag, a Softag 26 tag, a Softag 27 tag, a Softag 28 tag, a Softag 29 tag, a Softag 30 tag, a Softag 31 tag, a Softag 32 tag, a Softag 33 tag, a Softag 34 tag, a Softag 35 tag, a Softag 36 tag, a Softag 37 tag, a Softag 38 tag, a SoftagThe selection marker gene may include an antibiotic resistance gene such as kanamycin, G418, bleomycin, hygromycin, chloramphenicol, ampicillin, tetracycline, etc., and the promoter may include an antibiotic resistance gene such as pEMU promoter, MAS promoter, histone promoter, Clp promoter, cauliflower mosaic virus-derived 35S promoter, cauliflower mosaic virus-derived 19S RNA promoter, ubiquitin protein promoter, CMV (cytomegalovirus) promoter, SV40 (Simian virus 40) promoter, RSV (respiratory syncytial virus) promoter, EF-1α (elongation factor-1 The terminator may include a promoter such as a nopaline synthase (NOS), a patholin terminator, the octopine gene terminator of Agrobacterium tumefaciens, or the rrnB1 / B2 terminator of Escherichia coli, but there is no limitation on the type of gene to be added as long as it is a type that has been conventionally used in the production of recombinant proteins.

[0026] As used herein, "operably linked" refers to a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked to each other so as to perform their general functions. For example, a promoter and a nucleic acid sequence encoding a protein or RNA are operably linked to each other and can affect the expression of the coding sequence. The operably linked expression vector can be produced using recombinant gene technology well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art.

[0027] In this specification, "anticancer cytokine" refers to a collective term for cytokines that are known to activate T cells, B cells, NK cells, etc. in the body to induce cancer cell and cancer-specific immune responses, and play a role in killing cancer cells through autoimmunity. Examples include interferon alpha, interferon beta, interferon gamma, interleukin 2, interleukin 7, interleukin 12, interleukin 15, interleukin 18, interleukin 21, interleukin 37, granulocyte macrophage colony-stimulating factor, etc. IL-2 is known to regulate the growth and activity of T cells, NK cells, B cells, and monocytes, and to induce cancer cell-specific immune responses at high concentrations, and IL-7 acts on T cells, B cells, etc., and is known to be involved in T cell survival, B cell development, and the maintenance of the proliferation and homeostasis of these cells. In addition, CD56 BrightIt is known to increase the survival of NK cells and enhance immune action. IL-12 (p70) is known to induce the differentiation of naive T-helper cells into Th1 cells, thereby exerting an anti-cancer effect, increasing the activity of cytotoxic T cells, and increasing the survival of B cells. IL-15 is known to act on T cells and NK cells, regulating the proliferation and activity of these cells, and suppressing the induction of apoptosis in these immune cells. IL-21 is known to act on NK cells, T cells, and B cells, promoting the growth of these cells and increasing their activity. IL-37 is a cytokine belonging to the IL-1 family, and shows anti-tumor effects against various cancers depending on its concentration and interaction with other entities. In particular, it is known to act on cancer cells to increase the expression of p-STAT3 and VEGF, inducing the death of cancer cells, suppressing tumor angiogenesis, and acting on macrophages to suppress tumor-promoting inflammation, thereby regulating the tumor microenvironment. GM-CSF can exert anti-cancer effects by inducing the differentiation of myeloid cells, and in particular is known to be able to induce CTL-mediated tumor lysis by promoting the maturation of dendritic cells and the process of presenting tumor-specific antigens.

[0028] As used herein, "cancer" refers to malignant solid tumors predicted to grow indefinitely, as well as various blood cancers that arise from stem cells in hypoxic bone marrow, which can expand locally by infiltration and systematically through metastasis. Specific examples of cancer include, but are not limited to, adrenal gland cancer, bone cancer, brain cancer, breast cancer, bronchial cancer, colon and / or rectal cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, nervous tissue cancer, pancreatic cancer, prostate cancer, parathyroid cancer, skin cancer, stomach cancer, and thyroid cancer. Other examples of cancer include adenocarcinoma, adenoma, basal cell carcinoma, cervical dysplasia and carcinoma in situ, Ewing's sarcoma, squamous cell carcinoma, liquid cell carcinoma, malignant brain tumor, myeloma, intestinal ganglioneuroma, hyperplastic corneal nerve carcinoma, islet cell carcinoma, Kaposi's sarcoma, leiomyoma, leukemia, lymphoma, malignant carcinoma, malignant melanoma, malignant hypercalcemia, marfanoid habitus carcinoma, medullary carcinoma, metastatic skin cancer, mucosal neuroma, myelodysplastic syndrome, myeloma, mycosis fungoides, neuroblastoma, osteosarcoma, osteogenic and other sarcomas, ovarian cancer, pheochromocytoma, polycythemia vera, primary brain tumor, small cell lung cancer, ulcerative and papillary squamous cell carcinoma, seminoma, soft tissue sarcoma, retinoblastoma, rhabdomyoblastoma, reticulum cell sarcoma, and Wilms' tumor. Specific examples of cancer also include astrocytoma, gastrointestinal stromal tumor (GIST), glioma or glioblastoma, renal cell carcinoma (RCC), hepatocellular carcinoma (HCC), pancreatic neuroendocrine carcinoma, and the like.

[0029] As used herein, the term "prevention" refers to any action of suppressing or delaying the onset of a disease such as cancer by administering a composition according to the present invention.

[0030] As used herein, "treatment" refers to any action in which symptoms such as cancer are improved or beneficially altered by administration of a composition according to the present invention.

[0031] As used herein, "improvement" refers to an act of at least reducing a parameter associated with the condition being treated, e.g., the severity of a symptom. In this regard, the composition of the present invention may be used simultaneously or separately with a therapeutic drug for preventing or improving cancer.

[0032] As used herein, the term "individual" refers to a subject to which the composition of the present invention can be administered, and there is no limitation on the subject.

[0033] In the present specification, the term "pharmaceutical composition" refers to a form of capsule, tablet, granule, injection, ointment, powder or drink, and the pharmaceutical composition is intended for humans. The pharmaceutical composition of the present invention may include a pharmaceutical acceptable carrier. The pharmaceutical acceptable carrier may be a binder, lubricant, disintegrant, excipient, solubilizer, dispersant, stabilizer, suspending agent, dye, flavoring, etc., when administered orally, and may be a mixture of a buffer, preservative, soothing agent, solubilizer, isotonicity agent, stabilizer, etc., when administered as an injection, and may be a base, excipient, lubricant, preservative, etc., when administered topically. The dosage form of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with the pharmaceutical acceptable carrier as described above. For example, when administered orally, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and when administered by injection, it can be prepared in the form of unit dose ampoules or multiple doses. It can also be formulated into sugar-coated tablets, gels, pills, powders, granules, suppositories, external preparations, solutions, suspensions, sustained release preparations, slurries, etc. Examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoic acid, propylhydroxybenzoic acid, talc, magnesium stearate, mineral oil, etc. It may also contain fillers, anti-agglomerating agents, lubricants, wetting agents, flavorings, emulsifiers, preservatives, and the like.

[0034] The route of administration of the pharmaceutical composition according to the present invention is preferably oral or parenteral, but is not limited to these, and examples thereof include oral, intravenous, intramuscular, intra-articular, intrasynovial, intra-arterial, intramedullary, intradural, intracardiac, percutaneous, intradermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, rectal, intrasternal, intralesional, intracranial, and the like.

[0035] The dosage of the pharmaceutical composition of the present invention may vary depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated, and may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route, and period, but may be appropriately selected by those skilled in the art, and may be administered at 0.0001 to 500 mg / kg or 0.001 to 500 mg / kg per day. The dosage may be administered once a day or in several divided doses. Alternatively, when a pharmaceutical composition is prepared through a viral vector based on a dual expression vector, the dosage may be 1 to 10 based on the plaque forming unit (PFU), which is the basic unit of viral infectivity. 100 PFU or IFU or more can be administered in a single dose or in several divided doses. In addition, depending on the type of virus and the available titer, 1 to 10 100 and more infectious viral particles (VP ​​or VG) may be administered in a single dose or in separate doses. The above dosage amounts are not intended to limit the scope of the invention in any way.

[0036] In the present specification, the term "food composition" refers to various foods, such as beverages, gums, teas, vitamin complexes, dietary supplements, etc., and can be in the form of pills, powders, granules, infusions, tablets, capsules, or beverages. The food composition includes a functional health food composition. In this case, the amount of the dual expression vector in the food or beverage may generally be 0.01-30% by weight based on the total food weight in the case of the food composition of the present invention, and 0.02-10 g, preferably 0.3-1 g, based on 100 mL in the case of the health drink composition.

[0037] The food composition of the present invention may contain, as an essential ingredient, any of the ingredients added in addition to the dual expression vector, and may contain any of the usual food additives used in the art, such as natural carbohydrates, flavorings, flavorings, colorings, fillers, stabilizers, various nutrients, vitamins, minerals (electrolytes), etc. Examples of the natural carbohydrates include ordinary sugars such as monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; polysaccharides, e.g., dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, erythritol, etc. Examples of the flavorings include natural flavorings (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavorings (saccharin, aspartame, etc.). Examples of the flavorings include honey, D-mannitol, maltitol liquid, krill concentrate, etc. In addition, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, etc. may be contained. Such ingredients may be used independently or in combination. The proportion of such additives is not so important, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention. EXAMPLES

[0038] In the following, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided only to facilitate understanding of the present invention, and the contents of the present invention are not limited to the following examples.

[0039] [Example] Example 1: Construction of dual expression vectors that simultaneously express HIF-1α inhibitors and anti-cancer cytokines To create a dual expression vector that simultaneously expresses a metabolic anticancer drug and a cancer immunotherapy drug, the cytochrome b5 reductase 3 (CYB5R3, SEQ ID NO: 1) gene, which is known to suppress the expression of hypoxia-inducible factor-1α (HIF-1α), was used as an example of a metabolic anticancer drug. Examples of cytokines that are immunological anticancer agents include interleukin 2 (IL2, sequence number 9), interleukin 7 (IL7, sequence number 11), interleukin 12A (IL12A, sequence number 15), interleukin 12B (IL12B, sequence number 13), interleukin 15 (IL15, sequence number 17), interleukin 21 (IL21, sequence number 19), interleukin 37 (IL37, sequence number 21), and granulocyte-macrophage colony-stimulating factor (GM-CSF, sequence number 23). More specifically, the multi-cloning site (MCS) in the pDE1 vector containing the base sequence of SEQ ID NO:25 was cleaved using NheI and Xbal restriction enzymes, and genes encoding the CYB5R3 protein (SEQ ID NO:1) having an HA epitope (SEQ ID NO:3) bound to its C-terminus, the 2A peptide (porcine teschovirus-1 2A; CS peptide, SEQ ID NO:5), and a cytokine having a FLAG peptide (SEQ ID NO:7) bound to its C-terminus were inserted, in that order.

[0040] The dual expression vector constructed in this way transcribes the CYB5R3 protein with an HA epitope bound to its C-terminus-2A peptide-C-terminus of a cytokine expressed by a promoter in the vector during expression, and the transcribed mRNA is translated into a protein by ribosomes. At this time, the sequence before and the sequence after the 2A peptide sequence are translated separately into two proteins by ribosome skipping, and the CYB5R3 protein is present in the cytoplasm by binding to the plasma membrane or endoplasmic reticulum (ER) membrane by the transmembrane domain 1 (TM1) contained in the N-terminus of the protein, and the cytokine protein is secreted outside the cell in a mature form (final protein sequence in Table 1) by the signal peptide (SP) sequence contained in the protein. The structure of the dual expression vector of the present invention and the location of the protein produced by the dual expression vector are simply shown in FIG. 1.

[0041] A summary of the structures and nucleotide sequences of the constructed vectors is shown in FIG. 2 and Table 1.

[0042] [Table 1]

[0043] Example 2: Confirmation of protein expression from dual expression vector To confirm whether the dual expression vector prepared in the same manner as in Example 1 normally expresses the HIF-1α inhibitor and cytokines, HEK293T cells were cultured at 4 × 10 5The cells were seeded in DMEM (containing 4.5 g / L D-glucose) supplemented with 10% fetal bovine serum (FBS) to obtain 100 cells, and cultured for 24 hours. The medium was then removed, and the cells were washed once with phosphate buffered saline (PBS), and 2 mL of OPTI-MEM media without serum was added. Next, 3 μg of the dual expression vector and 0.1 μg of the pEGFP-C1 vector were transfected using Lipofectamine 2000 for 4 hours, and then 2 mL of OPTI-MEM media supplemented with 20% fetal bovine serum was added and cultured for 16 hours. Next, the existing medium was removed, and DMEM medium supplemented with 10% fetal bovine serum was added, and the cells were further cultured for 24 hours.

[0044] To perform Western blotting, the cell culture medium was centrifuged at 2,000xg at 4°C for 10 minutes, and the supernatant was transferred to a new tube. Next, 200μL of the supernatant was mixed with the same volume of 2X Laemmli sample buffer, boiled at 95°C for 5 minutes, and cooled to prepare a media sample (Growth media). Next, for the cell sample (Total cell extracts), the plate surface was scratched with a cell lifter to collect cells, and the plate was centrifuged at 3,000xg at 4°C for 5 minutes to remove the supernatant. 200μL of NP-40 Lysis supplemented with protease inhibitor cocktail was added, and the cells were disrupted using a sonicator. The disrupted cells were mixed with the same volume of 2X Laemmli sample buffer, boiled at 95°C for 5 minutes, and cooled to prepare a cell sample.

[0045] Western blotting was performed using the prepared cell samples and media samples. The cell samples were subjected to Western blotting using mouse anti-FLAG M2 antibody, mouse anti-HA antibody, mouse anti-GFP antibody, and mouse anti-β-actin antibody, respectively, while the media samples were subjected to Western blotting using mouse anti-FLAG M2 antibody and specific antibodies that can bind to each endogenous cytokine. All antibodies were purchased from Abcam, Sigma, and Cell Signaling Technology, respectively. The results are shown in Figure 3.

[0046] As shown in Figure 3, to check the transfection efficiency in the cell samples, GFP was expressed at similar levels in all samples by the GFP vector that was transfected together with the control group, confirming that transfection was normally induced. Next, cytokine expression was confirmed using FLAG antibody, and it was confirmed that no cytokine was observed in the pDE1-CYB5R3 vector, and that the cytokine drug bound to FLAG inserted in each of the remaining vectors was normally expressed. In addition, it was confirmed using HA antibody, and it was confirmed that CYB5R3 protein bound to HA was normally expressed in all vectors.

[0047] In addition, the media sample was confirmed to be normally expressed with FLAG antibody, and the cytokines bound to FLAG were also confirmed to be normally expressed using specific antibodies that can bind to each cytokine. In the case of GM-CSF-FLAG, a protein of 20 kDa was mainly observed in the cells, while in the media sample, a protein of 25 to 35 kDa was observed, confirming that post-translational modifications such as glycosylation were normally performed in the GM-CSF expressed by the dual expression vector. In the case of IL-2, the detection result using the FLAG antibody showed that it was weakly detected in the cells, but was detected at high concentrations in the cell culture medium, which means that the IL-2 produced in the cells was secreted outside the cells quickly and efficiently for the most part.

[0048] Example 3: Confirmation of anti-cancer effect of dual expression vector To confirm the anti-cancer effect of the dual expression vector prepared in the same manner as in Example 1, an experiment was carried out using a normal cell line, HEK293T, and a cancer cell line, A549. More specifically, each cell line was cultured at 4 × 10 5The cells were seeded in DMEM (containing 4.5 g / L D-glucose) supplemented with 10% fetal bovine serum or RPMI1640 (containing 4.5 g / L D-glucose) supplemented with 10% fetal bovine serum to obtain 100 cells, and cultured for 24 hours. The medium was then removed, the cells were washed once with phosphate buffered saline (PBS), and 2 mL of serum-free OPTI-MEM media was added. Next, 4 μg of dual expression vectors were transfected for 4 hours using Lipofectamine 3000 and P3000 reagent, after which the medium was completely removed, and 3 mL of DMEM (containing 1 g / L D-glucose) supplemented with 10% fetal bovine serum or RPMI1640 (containing 2 g / L D-glucose) supplemented with 10% fetal bovine serum was added, and the cells were cultured for 2 days. After 2 days, the cell culture medium was transferred to a new tube, the cells were washed with 3 mL of ice-cold PBS, the medium was completely removed, and the cells were transferred to a new tube, 1 mL of Trypsin-EDTA was added, and the reaction was allowed to proceed at 37°C for 5 minutes. Next, the cells were centrifuged at 200 x g for 5 minutes at 4°C to remove the supernatant, and the cell pellet was washed three times with ice-cold PBS. The cells were loosened with 400 μL of 1X Annexin V Binding Buffer, and the cell number was measured. Next, 1 × 10 5The cells were placed in a new tube, and the final volume was adjusted to 200μL using 1X Annexin V Binding Buffer, and propidium iodide and Annexin V-APC were added and reacted for 20 minutes in the dark at room temperature. Next, 300μL of 1X Annexin V Binding Buffer was added, and the degree of propidium iodide staining and the degree of Annexin V-APC staining were quantitatively analyzed using a Novocyte Cell Analyzer, and the degree of apoptosis induction was confirmed using this. For the analysis, 10,000 cells were counted, and the number of apoptotic cells among them was converted into a percentage ratio. Next, the percentage of apoptotic cells in the cells transfected with pDE1 used in the control group was calculated as 1, and the percentage of apoptotic cells in the other dual expression vectors was calculated relatively. The remaining cells were subjected to Western blotting in the same manner as in Example 2 to confirm whether the protein was normally expressed. All experiments below were performed three times, and statistical significance was confirmed by Student's t-test. * indicates P<0.05, and ** indicates P<0.01. The results are shown in FIG.

[0049] As shown in Figure 4, Western blotting confirmed that CYB5R3-HA and cytokine-FLAG proteins were normally expressed (left panel). Next, it was confirmed that each vector did not induce apoptosis in normal cells, thereby showing low cytotoxicity. In addition, it was confirmed that both the vector expressing CYB5R3 protein alone and the dual expression vector expressing cytokines together significantly increased apoptotic cells (right panel).

[0050] Example 4: Confirmation of anti-cancer effect of virus-based dual expression vector The dual expression vector prepared in the same manner as in Example 1 was injected into the virus, and the anti-cancer effect was confirmed. More specifically, the dual expression vector was first transfected into adenovirus (TaKaRa), and the virus was treated with a lung cancer cell line, H1299 cell line, at a concentration of 22, 50, 100, or 200 MOI, and then cultured for 72 hours. The cultured cells were then washed twice with phosphate buffer solution, 100% methanol was added, and the cells were stabilized at -20°C for 15 minutes. After that, the cells were washed three times with phosphate buffer solution, and the methanol was thoroughly removed. The cells were then treated with 0.4% Sulforhodamine B containing 1% Acetic Acid, and the cells were stained by reacting at room temperature for 30 minutes, and the remaining staining solution was thoroughly removed by washing four times with 1% Acetic Acid. Finally, 10 mM Tris solution was added to the cells, and the amount of remaining cells was measured by measuring the absorbance at 565 nm, and the anti-cancer effect was confirmed. The results are shown in FIG. 5.

[0051] As shown in Figure 5, in an experiment on anti-cancer effects using vectors injected into viruses, it was confirmed that the dual expression vectors containing both cyb5R3 and IL-21 genes showed significant anti-cancer effects even at low MOIs, compared to Ad-R3 containing only the cyb5R3 gene. From the above results, it was confirmed that the dual expression vectors of the present invention also showed anti-cancer effects when treated with viruses.

[0052] Example 5: Confirmation of the NK immune cell activation effect of the dual expression vector In order to confirm the NK immune cell activation effect of the dual expression vector prepared in the same manner as in Example 1, the dual expression vector was first transfected into adenovirus, and the virus was treated to a concentration of 50 MOI in HEK293T cell line, which was then cultured for 72 hours to obtain a culture medium (supernatant). The obtained culture medium was also treated with the same amount of H1299 cell line and NK-92 cell line, a human NK cell line, and further cultured for 4 hours. As a control, a medium containing cytokines secreted from a vector into which only the cyb5R3 gene was inserted was used. Next, the cells were stained in the same manner as in Example 4, and the amount of remaining cells was measured to confirm the anti-cancer effect of NK immune cells. The results are shown in Figure 6.

[0053] As shown in Figure 6, it was confirmed that IL-21 secreted from the dual expression vector increases the cancer cell-specific killing effect of NK cells compared to the control group. From the above results, it was confirmed that the anti-cancer cytokines expressed in the dual expression vector of the present invention increase the activation level of NK immune cells, thereby increasing the anti-cancer effect.

[0054] Example 6: Confirmation of anti-cancer immune activation effect of dual expression vector The anti-cancer immunity activating effect of the dual expression vector prepared in the same manner as in Example 1 was confirmed. More specifically, NK-92 cell lines were treated with adenovirus 50 MOI transfected with the dual expression vector and human rIL21 (Thermo Fisher), respectively, and the IFN-gamma secretion ability by IL21 was confirmed by ELISA at 24 and 48 hours. Next, the secretion amount of IL21 expressed by the dual expression vector was also measured by ELISA using the cell culture medium. The results are shown in Figure 7.

[0055] As shown in Figure 7, when rIL21 was directly treated, even when 10ng / mL was treated, a maximum of 3ng / mL of IFN-gamma was secreted for 48 hours, but when the dual expression vector was treated, even if the same amount of IL21 was expressed, 5ng / mL or more of IFN-gamma was secreted after 48 hours. From the above results, it was confirmed that the use of the dual expression vector in which the anti-cancer cytokine of the present invention and the metabolic anti-cancer drug HIF-1α inhibitor are both inserted can more significantly increase the immune activation effect of immune cells such as NK cells compared to the anti-cancer cytokine treated alone.

[0056] From the above results, it was confirmed that the dual expression vector of the present invention can effectively suppress cancer proliferation, migration, metastasis, etc. by simultaneously expressing a protein capable of suppressing HIF-1α and an anti-cancer cytokine, i.e., a metabolic anti-cancer drug, and at the same time, activate T cells, B cells, NK cells, etc. by the anti-cancer cytokine, i.e., an immune anti-cancer drug, to induce a cancer-specific immune response, thereby reducing side effects and improving the therapeutic effect. In addition, even if the same amount of anti-cancer cytokine is treated, the immune anti-cancer activation effect can be further improved when inserted into a vector and expressed, compared to when it is treated alone, so that it can be confirmed that the anti-cancer therapeutic effect can be more significantly improved compared to when the anti-cancer cytokine and the metabolic anti-cancer drug are simply administered in combination, and can be effectively applied to the treatment of various types of cancer.

[0057] The above description of the present invention is for illustrative purposes only, and a person skilled in the art to which the present invention pertains can easily understand that the present invention can be easily modified into other specific forms without changing the technical concept or essential features of the present invention. Therefore, the above-described embodiments should be understood to be illustrative in all respects and not limiting. [Industrial Applicability]

[0058] The dual expression vector according to the present invention, which simultaneously expresses the HIF-1α inhibitor and the anti-cancer cytokine, can not only significantly increase the anti-cancer treatment rate by simultaneously expressing the immune anti-cancer drug and the metabolic anti-cancer drug, but also provide various metabolic anti-cancer drug and immune anti-cancer drug treatment options, so that it can be effectively applied to the treatment of various types of cancer without restrictions.

Claims

1. A pharmaceutical composition for preventing or treating cancer, comprising a recombinant vector as an active ingredient, the recombinant vector comprising a polynucleotide encoding a hypoxia-inducible factor-1α inhibitor, a polynucleotide encoding a 2A peptide, and a polynucleotide encoding an anticancer cytokine, The hypoxia inducible factor-1α inhibitor is cytochrome b5 reductase 3; The composition, wherein the anti-cancer cytokine is secreted extracellularly in accordance with a signal peptide (SP) sequence contained in the anti-cancer cytokine, and is one or more selected from the group consisting of interleukin 7, interleukin 12, interleukin 21, interleukin 37, and granulocyte-macrophage colony-stimulating factor.

2. The pharmaceutical composition for preventing or treating cancer according to claim 1, characterized in that the recombinant vector is in a form in which a polynucleotide sequence encoding a 2A peptide is inserted between a polynucleotide sequence encoding a hypoxia-inducible factor-1α inhibitor and a polynucleotide encoding an anti-cancer cytokine.

3. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the cytochrome b5 reductase 3 comprises an amino acid sequence represented by SEQ ID NO:

1.

4. The pharmaceutical composition for preventing or treating cancer according to claim 1, characterized in that the 2A peptide comprises the amino acid sequence represented by SEQ ID NO:

5.

5. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the recombinant vector is a linear DNA, a plasmid DNA or a viral vector.

6. The pharmaceutical composition for preventing or treating cancer according to claim 5, characterized in that the viral vector is one or more selected from the group consisting of retrovirus, adenovirus, adeno-associated virus, vaccinia virus, myxoma virus, herpes simplex virus, and lentivirus.

7. The pharmaceutical composition for preventing or treating cancer according to claim 1, characterized in that the cancer is one or more selected from the group consisting of melanoma, small cell lung cancer, non-small cell lung cancer, glioma, liver cancer, thyroid tumor, gastric cancer, prostate cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, colon cancer, breast cancer, prostate cancer, glioblastoma, endometrial cancer, kidney cancer, colon cancer, pancreatic cancer, esophageal cancer, head and neck cancer, mesothelioma, sarcoma, bile duct cancer, small intestine adenocarcinoma, pediatric malignant tumor and epidermal cancer.

8. A food composition for preventing or improving cancer, comprising as an active ingredient a recombinant vector including a polynucleotide encoding a hypoxia-inducible factor-1α inhibitor, a polynucleotide encoding a 2A peptide, and a polynucleotide encoding an anticancer cytokine, The hypoxia inducible factor-1α inhibitor is cytochrome b5 reductase 3; The composition, wherein the anti-cancer cytokine is secreted extracellularly in accordance with a signal peptide (SP) sequence contained in the anti-cancer cytokine, and is one or more selected from the group consisting of interleukin 7, interleukin 12, interleukin 21, interleukin 37, and granulocyte-macrophage colony-stimulating factor.

9. Use of a recombinant vector comprising a polynucleotide encoding a hypoxia-inducible factor-1α inhibitor, a polynucleotide encoding a 2A peptide, and a polynucleotide encoding an anticancer cytokine for producing a drug for cancer, The hypoxia inducible factor-1α inhibitor is cytochrome b5 reductase 3; The anti-cancer cytokine is secreted extracellularly according to a signal peptide (SP) sequence contained in the anti-cancer cytokine, and is one or more selected from the group consisting of interleukin 7, interleukin 12, interleukin 21, interleukin 37, and granulocyte-macrophage colony-stimulating factor.

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