Salmonella strains for cancer prevention and treatment and uses thereof

Abstract

The present invention relates to a DNA construct and a bacterial strain into which a recombinant vector containing the DNA construct has been introduced. The DNA construct of the present invention has the effect of preventing and treating cancer by expressing an anti-cancer gene operably linked downstream of a first promoter and a second promoter in a host bacterial strain or cell. Furthermore, the DNA construct of the present invention allows the expression of an anti-cancer protein at an appropriate dose for cancer treatment by controlling the presence or absence of doxycycline treatment.

Description

[Technical Field]

[0001] The present invention relates to a DNA construct for preventing and treating cancer and a bacterial strain into which a recombinant vector containing the DNA construct has been introduced. [Background technology]

[0002] Currently, most cancers are treated by individual methods, such as surgery, radiotherapy, and chemotherapy, or a combination of these.Surgery, which removes most of the cancerous tissue, can be very effective in removing cancerous tissue located in specific areas, such as the breast, colon, and skin, but is difficult to use in treating cancerous tissue in certain areas, such as the spine.In addition, systemic chemotherapy, which is often used for breast cancer, lung cancer, and testicular cancer, can induce side effects that disrupt the replication or metabolic processes of normal cells, and patients may develop resistance to chemotherapy-based therapeutic agents.

[0003] Meanwhile, when cancer develops in an individual, angiogenesis and cell growth progress at an extremely rapid rate within the body, creating an environment with incomplete angiogenesis and oxygen deficiency inside cancer tissue, which can be extremely suitable for the proliferation of anaerobic bacteria such as Salmonella or E. coli. Therefore, currently, cancer treatment using cancer-targeting bacteria such as Salmonella and Clostridium targets solid tumors and relies on the ability of specific bacteria to proliferate within tumors. However, by introducing an oncolytic protein or reporter protein into the bacteria and administering the transformed bacteria to an individual, it is possible to specifically identify cancer tissue and treat cancer while minimizing side effects that are toxic to normal cells.

[0004] Toxins secreted by various bacterial pathogens found in nature can cause human diseases. Among these disease-causing bacterial pathogens, Salmonella enterica, which is closely related to our diet, is a member of the Enterobacteriaceae family that inhabits the intestinal tract of primates, including humans, and secretes the exotoxin cytolysin. Cytolysin is a cytotoxic protein with a molecular weight of approximately 34 kDa. It is known to destroy red blood cells in the intestines of primates, including humans, by causing hemolysis and pore formation in the membranes of normal cells, leading to cell lysis and even death through severe vascular inflammation and local tissue necrosis. However, recent research has shown that cytolysin isolated and purified from Salmonella enterica specifically reacts with cancerous tissue in the intestinal tract, inducing its destruction, and is therefore attracting attention as a next-generation anticancer therapeutic agent. Therefore, bacteria transformed with a gene that secretes cytotoxic substance cytolysin are considered to have great potential for use as anti-cancer therapeutic agents targeting cancer tissues.

[0005] Despite the potential for cancer diagnosis and treatment using bacteria, little research has been conducted on expression vectors that enable the specific expression of proteins suitable for diagnosis and treatment in cancer tissue. After bacterial injection, clearance occurs within the reticuloendothelial system, such as the liver and spleen, over the first three days. After a certain period, the expression of therapeutic proteins increases rapidly in cancer tissue. For safety reasons, it is necessary to wait a certain period before allowing the therapeutic protein to be expressed. Therefore, the use of inducible promoters for therapeutic protein expression is recommended. However, inducible promoters, such as the PBAD promoter currently used in experiments, require the use of L-arabinose, which is not approved for human use, making clinical application difficult. The Ptet promoter, which uses doxycycline, an antibiotic approved for human use, is relatively easy to use clinically and has the advantage of allowing bidirectional transcription of two genes using the TetA and TetR promoters. However, the protein expression rate between the TetA and TetR promoters differs by more than 100:1, and its utility can only be enhanced by achieving a balance in this ratio. Thus, there is a need to develop new technologies for transformed bacteria that have a clinically applicable expression system and can achieve a balanced protein expression level. Summary of the Invention [Problem to be solved by the invention]

[0006] One object of the present invention is to provide a DNA construct. Another object of the present invention is to provide a recombinant vector containing the DNA construct. A further object of the present invention is to provide a bacterial strain into which the recombinant vector has been introduced, and a composition for diagnosing cancer containing the same. It is yet another object of the present invention to provide a pharmaceutical composition for preventing or treating cancer, which comprises said strain as an active ingredient. It is yet another object of the present invention to provide a method for providing information for the diagnosis of cancer, which comprises the step of treating said strain. However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0007] In one embodiment of the present invention, a DNA construct is provided. The DNA construct of the present invention comprises genes encoding the first protein and the second protein; and a first promoter and a second promoter corresponding thereto, wherein the first protein is flagellin and the second protein is a toxin protein. In the present invention, the flagellin is flagellin A or B. The DNA construct of the present invention additionally comprises a gene encoding a regulatory protein.

[0008] In another aspect, the present invention provides a recombinant vector comprising a DNA construct comprising a gene encoding a first protein and a corresponding first promoter; and a DNA construct comprising a gene encoding a second protein and a corresponding second promoter, respectively, wherein the first protein is flagellin and the second protein is a toxin protein, and a bacterial strain into which the recombinant vector has been introduced. Since the first and second promoters of the present invention can be simultaneously induced by a regulatory protein expressed by another promoter, the expression levels of the proteins encoded by the genes operably linked downstream of the first and second promoters in the host cell can be balanced compared to when a gene encoding the regulatory protein is operably linked downstream of the second promoter. Thus, when the DNA construct of the present invention is used, diagnosis and treatment can be performed simultaneously.

[0009] The "DNA construct" of the present invention is a structure that allows expression of a target protein or the like when introduced into a host strain or cell by transformation, and includes not only a gene encoding the target protein but also a base sequence corresponding to a promoter, which is an essential regulatory element operably linked to enable expression of the gene. The "promoter" of the present invention refers to a base sequence present in the upstream region of an operably linked gene in a host strain or cell, and a base sequence at a specific site of the DNA construct to which RNA polymerase can bind to initiate transcription. The expression of the regulatory protein of the present invention can be regulated by a cis-acting element (Cis-regulatory elements; CRE) or a trans-acting element (Trans-regulatory elements; TRE).

[0010] In the present invention, the terms "regulation" and "regulation of expression" can mean that the transcription and translation of a specific gene are activated or inhibited. The cis-acting element of the present invention is a non-coding DNA region that regulates the transcription of adjacent genes, is an essential component of a gene regulatory network, and controls gene expression. The cis-acting element may be at least one selected from the group consisting of a ribosome binding site (RBS), a 5'-untranslated region (5'-UTR), a transcription factor binding site, and a terminator, but is not limited thereto. In the present invention, the ribosome binding site (RBS) is also called the Shine-Dalgarno sequence (SD sequence), and refers to a short sequence present on mRNA that allows ribosomes to effectively bind to the mRNA after the genetic information contained in DNA is transcribed into messenger RNA (mRNA) and translation occurs.

[0011] In the present invention, the 5'-untranslated region (5'-UTR) is an untranslated region located on both sides of the coding region, which is the 5' region that is translated into amino acids in mRNA. It was thought to be a junk region discarded during evolution, but it has been discovered to play an important role in regulating gene expression. In the present invention, the transcription factor binding site is a DNA site that serves to turn on or off a specific nearby gene, and may be at least one selected from the group consisting of a promoter, an enhancer, and a silencer of the gene encoding the regulatory protein, but is not limited thereto.

[0012] The promoter of the gene encoding the regulatory protein of the present invention may be any promoter that can be induced to be active under most environmental conditions and developmental states of host strains or cells, and preferably may be a weak promoter. The "weak promoter" of the present invention is a promoter that produces a transcript transcribed from a gene operably linked downstream at an expression level of 1×10 -2 Less than 1 × 10 -3 a promoter that induces expression of the transcript at levels below 1×10 -3 The promoter may be at least one selected from the group consisting of: E. coli σ70 promoter; E. coli σ5 promoter; E. coli σ32 promoter; B. subtilis σA promoter; B. subtilis σB promoter; Salmonella-derived promoters K112706 or K112707; bacteriophage T7 promoter; bacteriophage SP6 promoter; yeast-derived promoter; eukaryotic cell-derived promoter I712004 or K076017; and plant-derived promoter, but is not limited to this.

[0013] The E. coli σ70 promoter of the present invention is selected from the group consisting of I14018, I14033, I14034, I732021, I742126, J01006, J23103, J23109, J23112, J23113, J23117, J23119, J23150, J23151, J44002, J48104, J56015, J64951, K088007, K119000, K119001, K1330002, K137029, K137030, K137031, K137032, K137085, K137086, K137087, K137088, K137089, K137090, K137091, K1585100, K1585101, K1585102, K158510 3, K1585104, K1585105, K1585106, K1585110, K1585113, K1585115, K1585116, K1585117 , K1585118, K1585119, K2486171, K256002, K256018, K256020, K256033, K292000, K82300 7, K823010, K823013, M13101, M13102, M13103, M13104, M13105, M13106, M13108, M13110, M31519, R1074, R1075 and S03331, but is not limited thereto.

[0014] The E. coli σS promoter of the present invention may be, but is not limited to, J45992 or J45993. The E. coli σ32 promoter of the present invention may be, but is not limited to, J45504, K1895002 or K1895003. The B. subtilis σA promoter of the present invention may be at least one selected from the group consisting of K143012, K143013, K823000, K823002 and K823003, but is not limited thereto.

[0015] The B. subtilis σB promoter of the present invention may be, but is not limited to, K143010, K143011 or K143013. The bacteriophage T7 promoter of the present invention may be at least one selected from the group consisting of I719005, J34814, J64997, K113010, K113011, K113012, K1614000, R0085, R0180, R0181, R0182, R0183, Z0251, Z0252 and Z0253, but is not limited thereto. The bacteriophage SP6 promoter of the present invention may be, but is not limited to, J64998.

[0016] The yeast-derived promoter of the present invention may be at least one selected from the group consisting of I766557, J63005, K105027, K105028, K105029, K105030, K105031, K122000, K124000, K124002, K319005, M31201, K2365040, K2365036, K2365041, K2365042, K2365032, K2365051, K2365514, K2365515 and K2365516, but is not limited thereto. The plant-derived promoter of the present invention may be at least one selected from the group consisting of PLPR0203, PLPR0210, PLPR0177, PLPR0193, PLPR0507, PLPR0422, PLPR0228, PLPR0226, PLPR0223, PLPR0040, PLPR0465, PLPR0232, PLPR0205, PLPR0247, PLPR0328, PLPR0525, AtREG383, AtREG415, AtREG416, OsREG438, OsREG443, OsREG501, PpREG186, PpREG194, and PpREG197, but is not limited thereto.

[0017] For the purposes of the present invention, when a gene encoding a regulatory protein is operably linked downstream of the weak promoter, the transcription of the gene located downstream of the first and second promoters can be regulated so that it occurs specifically only when a substance that inhibits the regulatory protein is administered, compared to when the gene is operably linked downstream of the first or second promoter. The promoter of the gene encoding the regulatory protein of the present invention may have a base sequence of SEQ ID NO: 8 at -35 position and SEQ ID NO: 9 at -10 position based on the gene encoding the regulatory protein, but is not limited thereto.

[0018] The enhancer of the present invention is a sequence found in both prokaryotes and eukaryotes, generally has a region of 50 to 1500 bp, and is located upstream or downstream from the start of the nearby gene to induce the binding of the transcription factor. The silencer of the present invention maintains the same mechanism as an enhancer and acts as an antagonism of the enhancer. The transcription factor that binds to the silencer is a repressor. The enhancer and the silencer may be located in adjacent regions, or may be in the same region but with different transcription factors.

[0019] The terminators of the present invention are also called transcription terminators, which mediate the termination of transcription of genes or operons in prokaryotes, and are classified as Rho-dependent and Rho-independent terminators. The trans-acting factor of the present invention is also called a transactivator or a trans-acting transcription factor, and is a factor that activates gene transcription in trans. The trans-acting factor may be at least one selected from the group consisting of the transcription factor, an aptamer, sRNA, and antisense RNA (asRNA), but is not limited thereto.

[0020] In the present invention, the transcription factor is a protein that binds to the transcription factor binding site and helps to turn on or off a specific gene. In the present invention, the aptamer is a part of a riboswitch and generally refers to an oligonucleotide or peptide substance capable of binding to a specific target molecule, and the aptamer may be a peptide aptamer or a nucleic acid aptamer. The riboswitch is a type of mRNA that regulates gene expression, and may include, but is not limited to, glmS riboswitch, FMN riboswitch, Cobalamin riboswitch, etc.

[0021] In the present invention, the term sRNA or antisense RNA (asRNA) refers to a single-stranded RNA that can complementarily bind to a specific RNA. It complementarily binds to sense RNA, which is messenger RNA (mRNA) that expresses a specific protein, ultimately regulating the expression of the protein. The first promoter and the second promoter of the present invention may be inducible promoters that are induced by the regulatory protein.

[0022] The "inducible promoter" of the present invention refers to a promoter that transcribes a gene linked downstream so that it can be specifically expressed only under specific chemical or physical conditions, and may be, for example, the promoter of the LacZ gene that is expressed in the presence of galactose such as IPTG (isopropyl-beta-D-1-thiogalactopyranoside), the araBAD promoter of the arabinose operon that is expressed only in the presence of L-arabinose, or the tet promoter whose expression is regulated by tetracycline. Preferably, the first promoter and the second promoter may be tet promoters, and more preferably, the first promoter may be the tetA promoter and the second promoter may be the tetR promoter, but is not limited thereto. The gene encoding the regulatory protein of the present invention is a protein that binds to the first promoter and the second promoter to regulate the binding of RNA polymerase. For the purposes of the present invention, when the first promoter and the second promoter are tet promoters, the gene may be, but is not limited to, a TetR protein that can bind to the regulatory site of the tet promoter to suppress the activity of the tet promoter.

[0023] The term "operably linked" as used herein means that one nucleic acid fragment of interest is functionally linked to another nucleic acid fragment, such that the function or expression of the one nucleic acid fragment of interest is affected by the other nucleic acid fragment. The "reporter protein" of the present invention is a protein that functions to enable visual diagnosis of cancer, and may be, for example, at least one selected from the group consisting of fluorescent proteins, luciferase, and proteins used in nuclear medicine or MRI imaging, but is not limited thereto.

[0024] The "fluorescent protein" of the present invention is a protein that exhibits fluorescence by itself to enable visual diagnosis of cancer, and may be, for example, at least one selected from the group consisting of green fluorescent protein (GFP), modified green fluorescent protein (MGFP), enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), and enhanced yellow fluorescent protein (EYFP), but is not limited thereto. The protein used in the nuclear medicine or MRI imaging of the present invention may be, but is not limited to, at least one selected from the group consisting of herpes simplex virus thymidine phosphate enzyme, dopamine receptor, somatostatin receptor, sodium-iodide transporter, iron receptor, transferrin receptor, ferritin, and iron transporter (magA).

[0025] The "cytokine" of the present invention is a protein secreted by immune cells, and includes all cytokines that can be used in cancer immunotherapy, as long as they can regulate the host immune response and induce the death of disease-related cells, such as cancer cells, and may preferably include, but are not limited to, IFN-alpha2, IL-2, IL-15, IL-21, and IL-12. The "chemokine" of the present invention refers to any chemokine that mediates the host response to disease, such as cancer, by inducing leukocytes into the tumor microenvironment, and is a chemokine that regulates cell migration between tissues and the location and interaction of cells within tissues. Preferably, the chemokine may be CXCR3, CCR5, etc., but is not limited thereto. The "immunomodulator" of the present invention is an agent that enables various treatments by utilizing the inherent immune system of an individual, and includes any agent that can activate immune cells and induce the death of disease-related cells, such as cancer cells.

[0026] The "anti-cancer protein" of the present invention refers to a peptide having the function of directly or indirectly inducing the death of cancer cells, and may be, for example, at least one selected from the group consisting of a toxic protein, an antibody specific to a cancer antigen or a fragment of the antibody, a tumor suppressor protein, an angiogenesis inhibitor, a cancer antigen, a prodrug-converting enzyme, and a pro-apoptotic protein, but is not limited thereto. The "toxic protein" of the present invention refers to a protein capable of directly or indirectly inducing the death of cancer cells, and may be, for example, at least one selected from the group consisting of ricin, saporin, gelonin, momordin, debouganin, diphtheria toxin, Pseudomonas toxin, hemolysin (HlyA), FAS ligand (FASL), tumor necrosis factor-alpha (TNF-alpha), TNF-related apoptosis-inducing ligand (TRAIL), and cytolysin A (ClyA), and more preferably, cytolysin A consisting of the amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto.

[0027] The "tumor suppressor protein" of the present invention refers to a gene that maintains its function while present in normal cells, but when its function is lost, induces indiscriminate cell division and growth in normal cells, causing them to transform into cancer cells. Examples of such a protein include, but are not limited to, RB (Retinoblastoma protein), p53, APC (Adenomatous polyposis coli), PTEN (Phosphatase and tensin homologue), and CDKN2A (cyclin dependent kinase inhibitor 2A). The antibody or antibody fragment specific to the cancer antigen of the present invention is an antibody that can specifically bind to an antigen, which is a protein that is specifically expressed at a high level on the surface or cytoplasm of cancer cells, and may be, for example, an antibody specific to HER2, which is specifically expressed at a high level in breast cancer or gastric cancer cells, but is not limited thereto.

[0028] The antibody of the present invention refers to a protein molecule that can specifically bind to an antigenic site of a protein or peptide molecule. The form of the antibody is not particularly limited, and includes all types of immunoglobulin antibodies, including polyclonal antibodies, monoclonal antibodies, and antibody fragments that have antigen-binding ability. Special antibodies such as humanized antibodies are also included. The antibody includes not only the complete form having two full-length light chains and two full-length heavy chains, but also functional fragments of the antibody molecule. The functional fragment of the antibody molecule refers to a fragment that retains at least the antigen-binding function, and may be, but is not limited to, Fab, F(ab'), F(ab')2, Fv, etc.

[0029] The "antibody" of the present invention can be produced by a conventional method after cloning a gene encoding the cancer antigen of the present invention into an expression vector by a conventional method to obtain a protein encoded by the gene. The "angiogenesis inhibitor" of the present invention refers to a protein or compound that has the function of directly or indirectly inducing the death of cancer cells by inhibiting the generation of new blood vessels around cancer cells. Preferably, the angiogenesis inhibitor may be, but is not limited to, angiostatin, endostatin, thrombospondin, protease inhibitor protein, etc.

[0030] The "cancer antigen" of the present invention refers to a protein that is expressed in cancer cells but is rarely expressed in normal cells, and that can directly or indirectly kill cancer cells by inducing an anti-tumor immune response. The cancer antigen of the present invention may be, but is not limited to, alpha-fetoprotein (AFP), vascular endothelial growth factor receptor 2 (VEGFR2), survivin, legumain, prostate cancer specific antigen (PCSA), etc. The "prodrug converting enzyme" of the present invention is a protein having the function of converting an inactive drug into an active drug through metabolic enzymatic reaction. When such a prodrug converting enzyme is used, an inactive drug is metabolized and converted into an active drug that can directly or indirectly induce the death of cancer cells, and thus the prodrug converting enzyme can be very useful for the prevention or treatment of cancer. The prodrug-converting enzyme of the present invention may be, but is not limited to, thymidine kinase, cytosine deaminase, nitroreductase, purine nucleoside phosphorylase, carboxypeptidase G2, chromate reductase YieF, herpes simplex virus type I thymidine kinase / ganciclovir (HSV1-TK / GCV), beta-glucuronidase, etc.

[0031] The "pro-apoptotic protein" of the present invention refers to a protein that induces direct or indirect death of cancer cells by depriving them of factors (proteins, nutrients, oligonucleotides, etc.) essential for the growth or maintenance of cancer cells. The pro-apoptotic protein of the present invention may be, but is not limited to, L-asnase, RNA-binding motif protein 5 (RBM5), etc. The cancer antigen-specific oligonucleotide of the present invention is a nucleotide that can suppress the expression or function of a cancer antigen by complementarily binding to the gene or mRNA of the cancer antigen, and may be any one selected from the group consisting of antisense oligonucleotides, aptamers, siRNAs, and shRNAs, but is not limited thereto.

[0032] The "antisense oligonucleotide" of the present invention refers to DNA, RNA, or a derivative thereof containing a nucleic acid sequence complementary to a specific mRNA sequence, and can inhibit translation of mRNA into protein by binding to the complementary sequence in the mRNA. The antisense oligonucleotide can be synthesized in vitro using a conventional method, for example, RNA polymerase I, and then administered to a living body, or can be synthesized in vivo using a vector in which the origin of the recognition site (MCS) is in the opposite direction. The term "aptamer" as used herein refers to a small single-stranded oligonucleotide capable of specifically recognizing a target substance with high affinity. For purposes of the present invention, the target substance may be a gene or mRNA of a cancer antigen.

[0033] The term "siRNA" as used herein refers to a short double-stranded RNA that can induce RNA interference (RNAi) by cleaving specific mRNA. It is composed of a sense RNA strand with a sequence homologous to the mRNA of a target gene and an antisense RNA strand with a sequence complementary to the sense RNA strand. For purposes of the present invention, the siRNA specifically binds to mRNA transcribed from a gene encoding a cancer antigen, thereby effectively suppressing the expression of such a gene. The "shRNA" of the present invention refers to a short hairpin RNA, which has the advantages of a higher cell transfection rate than siRNA and the ability to maintain RNA interference for a long period of time, and can induce RNA interference by transfecting cells with an RNA polymerase III promoter using an adenovirus, lentivirus, or plasmid expression vector system, followed by expression, but is not limited to this. For the purposes of the present invention, the shRNA can specifically bind to mRNA transcribed from a gene encoding a cancer antigen, thereby effectively suppressing the expression of such a gene.

[0034] In another embodiment of the present invention, there is provided a recombinant vector comprising the DNA construct of the present invention. The recombinant vector of the present invention contains the DNA construct of the present invention, and thereby allows the regulatory protein to be expressed by a separate promoter, thereby enabling the genes operably linked downstream of the first promoter and the second promoter to be expressed in a balanced manner specifically only when a substance that inhibits the regulatory protein is administered from the outside.

[0035] In the recombinant vector of the present invention, the details regarding the DNA construct, anti-cancer protein, cytokine, chemokine, immunomodulator, cancer antigen-specific oligonucleotide, reporter protein, promoter, etc. are the same as those described in the DNA construct, and will be omitted to avoid overcomplicating this specification. The recombinant vector of the present invention can be introduced into cells to express a protein using known recombinant vectors such as plasmid vectors, cosmid vectors, and bacteriophage vectors, and the recombinant vector can be easily produced by a person skilled in the art using any known method using DNA recombination technology.

[0036] In the present invention, specific examples of the recombinant vector can be selected from the group consisting of commercially widely used pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, Ti vectors, cosmids, phages such as lambda, lambdoid, M13, Mu, p1 P22, Qμ, T-even, T2, T3, and T7, and plant viruses, but are not limited thereto. For the purposes of the present invention, an appropriate recombinant vector can be selected depending on the properties of the host cell. In yet another embodiment of the present invention, there is provided a host cell or strain into which a recombinant vector containing the DNA construct of the present invention has been introduced.

[0037] The host cell of the present invention includes cells of mammalian, plant, insect, fungal or cellular origin, and may be at least one selected from the group consisting of bacterial cells such as Escherichia coli, Streptomyces or Salmonella strains, yeast cells, fungal cells such as Pichia pastoris; insect cells such as Drosophila or Spodoptera Sf9 cells; animal cells such as CHO (Chinese hamster ovary cells), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T cells, Bowes melanoma cells, HT-1080 cells, BHK cells (Baby Hamster Kidney cells), HEK cells (Human Embryonic Kidney cells) or PERC.6 cells (human retinal cells); and plant cells, but is not limited thereto. For the purposes of the present invention, the strain may be an anaerobic strain, for example, at least one selected from the group consisting of Salmonella strains, Clostridium strains, Bifidobacterium strains, and Escherichia coli strains, preferably at least one selected from the group consisting of Salmonella typhimurium, Salmonella choleraesuis, and Salmonella enteritidis, more preferably, but not limited to, Salmonella typhimurium.

[0038] The strains of the present invention may be attenuated. The term "attenuation" as used herein means that a gene or genes have been modified to reduce toxicity and other side effects when the microorganism is administered to a patient. For purposes of the present invention, when the strain is a Salmonella strain, the attenuation may be achieved by modifying at least one gene selected from the group consisting of aroA, aroC, aroD, aroE, Rpur, htrA, ompR, ompF, ompC, galE, cya, crp, cyp, phoP, phoQ, rfaY, dksA, hupA, sipC, clpB, clpP, clpX, pab, nadA, pncB, pmi, rpsL, hemA, rfc, poxA, galU, cdt, pur, ssa, guaA, guaB, fliD, flgK, flgL, relA, and spoA, but is not limited thereto. The gene of the present invention can be modified by various gene deletion or disruption methods known in the art, for example, by homologous recombination, chemical mutagenesis, irradiation mutagenesis, transposon mutagenesis, etc.

[0039] In the present invention, the strain targets the interior of cancer tissue, which is an oxygen-deficient environment with incomplete angiogenesis, which is extremely suitable for the growth of anaerobic strains. Therefore, when a recombinant vector capable of simultaneously expressing a reporter protein that can be imaged in real time and an anti-cancer protein in a balanced manner is introduced into such a strain, cancer can be diagnosed and treated simultaneously very effectively. In the strain of the present invention, the details regarding the DNA construct, anti-cancer protein, cytokine, chemokine, immunomodulator, cancer antigen-specific oligonucleotide, reporter protein, promoter, and recombinant vector are the same as those described in the DNA construct and recombinant vector, and will be omitted to avoid overcomplicating this specification.

[0040] The recombinant vectors of the present invention are introduced into host cells or bacterial strains by transformation (or transfection). The transformation method used in the present invention can be any transformation method commonly used in the art, and can be easily performed using methods commonly used in the art. Specifically, recombinant vectors can be introduced into the strains using commonly used methods for transforming bacteria such as the Salmonella strains, such as the CaCl2 precipitation method, the Hanahan method (in which the efficiency of the CaCl2 method is enhanced by using the reducing agent DMSO (dimethyl sulfoxide)), electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, PEG-mediated transformation, dextran sulfate, lipofectamine, and desiccation / repression-mediated transformation, but are not limited to these. In yet another embodiment of the present invention, there is provided a pharmaceutical composition for the prevention or treatment of cancer. In yet another aspect, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the strain of the present invention as an active ingredient.

[0041] The pharmaceutical composition of the present invention inhibits cancer growth or cancer metastasis. When the bacterial strain of the present invention is transformed with the DNA construct of the present invention and targets cancer in an individual, and then a substance that inhibits a regulatory protein is administered, a reporter protein that can be imaged in real time and an anti-cancer protein are simultaneously expressed in a balanced manner in the bacterial strain, making it possible to very effectively prevent or treat cancer and simultaneously diagnose cancer in real time.

[0042] The "cancer" of the present invention refers to a disease characterized by the rapid and uncontrolled growth of mutant cells, and includes melanoma, fallopian tube cancer, brain cancer, small intestine cancer, esophageal cancer, lymphatic gland cancer, gallbladder cancer, blood cancer, thyroid cancer, endocrine gland cancer, oral cancer, liver cancer, biliary tract cancer, colon cancer, rectal cancer, cervical cancer, ovarian cancer, kidney cancer, stomach cancer, duodenal cancer, prostate cancer, breast cancer, brain cancer, lung cancer, anaplastic thyroid cancer, uterine cancer, colon cancer, bladder cancer, ureter cancer, pancreatic cancer, bone / soft tissue sarcoma, skin cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, The cancer may be at least one selected from the group consisting of acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and solitary myeloma, and preferably at least one selected from the group consisting of liver cancer, biliary tract cancer, colorectal cancer, rectal cancer, cervical cancer, ovarian cancer, kidney cancer, stomach cancer, duodenal cancer, prostate cancer, breast cancer, brain tumor, lung cancer, undifferentiated thyroid cancer, uterine cancer, colon cancer, bladder cancer, ureter cancer, pancreatic cancer, bone / soft tissue sarcoma, and skin cancer, and more preferably colon cancer, but is not limited to this. The "prevention" of the present invention includes, without limitation, any action of blocking, suppressing or delaying symptoms caused by cancer using the active ingredient of the present invention.

[0043] The term "treatment" as used herein refers to any action taken to improve symptoms caused by cancer or benefit an individual using the active ingredient of the present invention, and refers to an attempt to achieve beneficial or desirable results, including clinical results. Beneficial or desirable clinical results may include, but are not limited to, the alleviation or improvement of one or more symptoms or conditions, reduction in the extent of disease, stabilization of the disease state, inhibition of disease occurrence, inhibition of disease spread, delay or prolongation of disease progression, delay or prolongation of disease onset, and improvement or alleviation, and attenuation (partial or total) of the disease state, whether detectable or not. Furthermore, "treatment" may refer to the prolongation of a patient's survival beyond that expected in the absence of treatment. Furthermore, "treatment" may refer to the inhibition of disease progression, temporary prolongation of disease progression, or, more preferably, permanent halt of disease progression. As understood by those skilled in the art, a result is not beneficial or desirable if it produces an opposite result in the treated patient while improving a particular disease state, i.e., a result that exceeds all the benefits of the treatment. In the present invention, the treatment may be one that suppresses cancer metastasis or cancer recurrence.

[0044] In the present invention, the term "cancer metastasis" refers to the tendency of cancer to spread to other locations separated by a distance from the organ or part where it originated. A metastatic cancer is a cancer that has the tendency to metastasize or has already metastasized. In particular, the metastatic cancer may metastasize to the liver, lung, bone, lymph nodes, or abdominal cavity, but is not limited thereto. The metastatic cancer may be difficult to treat, and the progression and treatment process may be more complicated than the initial treatment course. In the present invention, the term "cancer recurrence" refers to cancer that was not detected after treatment but is rediscovered after a certain period of time. Recurrent cancer refers to cancer that occurs as a result of cancer recurrence as described above. When cancer recurs, resection is often difficult, and even if resection is possible, a substantial surgical procedure is required. In addition, there may be limitations on anticancer treatment and radiation therapy. In the pharmaceutical composition of the present invention, the details regarding the DNA construct, anti-cancer protein, cytokine, chemokine, immunomodulator, cancer antigen-specific oligonucleotide, reporter protein, promoter recombinant vector, bacterial strain, and transformation are the same as those described above for the DNA construct, recombinant vector, and bacterial strain, and will be omitted to avoid overcomplicating this specification.

[0045] The pharmaceutical composition of the present invention is characterized in that it is in the form of a capsule, tablet, granule, injection, ointment, powder or drink, and is intended for humans. The pharmaceutical compositions of the present invention can be formulated into oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, as well as topical preparations, suppositories, and sterile injectable solutions, using conventional methods, but are not limited thereto. The pharmaceutical compositions of the present invention can also contain a pharmaceutically acceptable carrier. For oral administration, pharmaceutically acceptable carriers include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, and flavorings. For injections, buffers, preservatives, soothing agents, solubilizers, isotonicity agents, and stabilizers can be mixed together. For topical administration, bases, excipients, lubricants, and preservatives can be used. The pharmaceutical compositions of the present invention can be prepared in various dosage forms by mixing with the pharmaceutically acceptable carriers described above. For example, for oral administration, the compound may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it may be prepared in unit-dose ampoules or multi-dose forms. It may also be formulated into solutions, suspensions, tablets, capsules, sustained-release preparations, etc.

[0046] On the other hand, 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, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, fillers, anti-agglomerating agents, lubricants, wetting agents, flavorings, emulsifiers, preservatives, etc. may also be included. The routes of administration of the pharmaceutical composition of the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal, with oral or parenteral administration being preferred.

[0047] The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions of the present invention can also be administered in the form of suppositories for rectal administration. 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. The dosage of the pharmaceutical composition varies depending on the patient's condition, body weight, severity of the disease, drug form, administration route, and duration, but can be appropriately selected by those skilled in the art and may be administered at 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. The dosage may be administered once a day or in divided doses. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition of the present invention may be formulated into pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, or suspensions.

[0048] In yet another embodiment of the present invention, a composition for diagnosing cancer is provided. The diagnostic composition of the present invention comprises the strain of the present invention as an active ingredient. When the bacterial strain of the present invention is transformed with the DNA construct of the present invention and targets cancer cells in an individual, and then a substance that inhibits a regulatory protein is administered, a reporter protein that can be imaged in real time and an anti-cancer protein are simultaneously expressed in a balanced manner in the bacterial strain, making it possible to very effectively prevent or treat cancer and simultaneously diagnose cancer in real time.

[0049] The "diagnosis" of the present invention refers to all actions of identifying cancer tissue in vivo, including the ability to monitor the presence or absence of cancer in real time using a reporter protein expressed from a DNA construct introduced into the strain when the strain of the present invention targets and localizes to cancer. In the diagnostic composition of the present invention, the details regarding the DNA construct, anti-cancer protein, reporter protein, constitutive promoter, inducible promoter, recombinant vector, Salmonella strain, transformation, cancer, etc. are the same as those described in the DNA construct, recombinant vector, strain, and pharmaceutical composition, and will be omitted to avoid overcomplicating this specification.

[0050] In yet another embodiment of the present invention, a method for providing information for the diagnosis of cancer is provided. The method of the present invention comprises the step of treating a biological sample isolated from a target individual with a bacterial strain into which the recombinant vector of the present invention has been introduced. The method of the present invention for providing information for diagnosing cancer may further comprise the step of diagnosing cancer when the reporter protein is expressed from the bacterial strain.

[0051] The term "biological sample" as used herein refers to any substance, tissue, or cell obtained from or derived from an individual, and may include, but is not limited to, for example, tissue, cell, or cell extract. In the method for providing information for diagnosis of the present invention, the details regarding the DNA construct, anti-cancer protein, cytokine, chemokine, immunomodulator, cancer antigen-specific oligonucleotide, reporter protein, promoter, recombinant vector, bacterial strain, transformation, cancer, diagnosis, etc. are the same as those described in the DNA construct, recombinant vector, bacterial strain, pharmaceutical composition, and diagnostic composition, and will be omitted to avoid overcomplicating this specification.

[0052] In the present invention, flagellin can include, but is not limited to, flagellin A or B DNA constructs. The present invention includes a recombinant vector used to prevent or treat cancer, or a bacterial strain into which a recombinant vector that is a flagellin and a toxin protein has been introduced.

[0053] The present invention also includes a method for preventing or treating cancer, which comprises administering to a subject an effective amount of the recombinant vector of the present invention or a bacterial strain into which a recombinant vector comprising flagellin and a toxin protein has been introduced. In yet another embodiment of the present invention, there is provided a method for preventing or treating cancer by co-administering to a subject effective amounts of a bacterial strain expressing an exogenous flagellin and a bacterial strain expressing an exogenous toxin protein. The "flagellin" of the present invention refers to, but is not limited to, a granular protein that constitutes the helical filament of bacterial flagella. Its molecular weight varies greatly depending on the bacterial species (30,000-70,000), but its amino acid composition does not contain cysteine ​​or tryptophan, and in the case of Salmonella, approximately half of the lysines are methylated. Flagellins include flagellin A and B, which have diverse functions and are known to have immune-enhancing effects.

[0054] In the present invention, "combined administration" refers to the simultaneous or stepwise use of various cancer treatment methods (surgery, radiation therapy, chemotherapy, immunotherapy, etc.), including, but not limited to, the administration of two or more anticancer drugs or drugs with different mechanisms, or the simultaneous use of physical therapy such as radiation therapy and chemotherapy such as anticancer drugs. When two drugs are used, a synergistic effect is exhibited based on complementary mechanisms or drug dose adjustment. However, in actual clinical practice, the synergistic effect is significantly reduced, which can lead to serious side effects such as immunosuppression and cardiotoxicity. In yet another aspect, the present invention provides a composition for preventing or treating cancer, comprising, as active ingredients, flagellin or a nucleotide encoding the same; and a toxin protein or a nucleotide encoding the same.

[0055] The genes encoding flagellin or toxin proteins used in the present invention have already been described in detail, so their description will be omitted to avoid excessive duplication. The gene of the present invention may be delivered to a gene carrier, but it can also be administered in the form of a fully translated peptide to exert a similar pharmacological effect. [Effects of the Invention]

[0056] The DNA construct of the present invention can regulate the expression level of an anti-cancer gene operably linked downstream of the first promoter and the second promoter in a host strain or cell, thereby preventing and treating cancer. Furthermore, the DNA construct of the present invention allows the expression of anti-cancer proteins at appropriate doses for cancer treatment by controlling the presence or absence of doxycycline treatment. [Brief explanation of the drawings]

[0057] [Figure 1] FIG. 1 is a schematic diagram of a DNA construct according to Preparative Example 1 of the present invention. [Figure 2]FIG. 1 shows the results of analyzing the growth pattern of a strain recombined with a DNA construct according to Experimental Example 1 of the present invention. [Figure 3] FIG. 1 shows the hemolytic activity results of blood agar of bacterial strains according to Experimental Example 1 of the present invention. [Figure 4] FIG. 1 shows the results of measuring TLR-5 signal activation according to Experimental Example 1 of the present invention. [Figure 5] FIG. 1 is a diagram confirming the difference in expression level of recombinant strains depending on the concentration of doxycycline in Experimental Example 1 of the present invention. [Figure 6] FIG. 1 shows the results of confirming the anti-cancer effect of a recombinant strain according to Experimental Example 2 of the present invention. [Figure 7] FIG. 1 shows the results of confirming the cancer targeting effect of the recombinant strain according to Experimental Example 3 of the present invention. [Figure 8] FIG. 1 shows the results of confirming the cancer targeting effect of the recombinant strain according to Experimental Example 3 of the present invention. [Figure 9] FIG. 1 shows the results of confirming the anti-cancer effect of a recombinant strain according to Experimental Example 4 of the present invention. [Figure 10] FIG. 1 shows the results of confirming the anti-cancer effect of a recombinant strain according to Experimental Example 4 of the present invention. [Figure 11] FIG. 1 is a diagram showing an experimental method for confirming the anti-cancer effect of a recombinant strain according to an experimental example of the present invention. [Figure 12] FIG. 1 shows the results of confirming the anti-cancer effect of the recombinant strain according to Experimental Example 5 of the present invention. [Figure 13] FIG. 1 shows the results of confirming the anti-cancer effect of the recombinant strain according to Experimental Example 5 of the present invention. [Figure 14] FIG. 1 shows the results of confirming the anti-cancer effect of the recombinant strain according to Experimental Example 6 of the present invention. [Figure 15] FIG. 1 shows the results of confirming the anti-cancer effect of the recombinant strain according to Experimental Example 6 of the present invention. [Figure 16] FIG. 1 shows the results of confirming the effect of the recombinant strain in suppressing cancer recurrence according to Experimental Example 7 of the present invention. [Figure 17]FIG. 1 shows the results of confirming the effect of the recombinant strain in suppressing cancer recurrence according to Experimental Example 7 of the present invention. [Figure 18] FIG. 1 shows the results of confirming the inhibitory effect of the recombinant strain on cancer metastasis in Experimental Example 8 of the present invention. [Figure 19] FIG. 1 shows the results of confirming the inhibitory effect of the recombinant strain on cancer metastasis in Experimental Example 8 of the present invention. [Figure 20] FIG. 1 shows the results of confirming the anti-cancer effect of the recombinant strain according to Experimental Example 9 of the present invention. [Figure 21] FIG. 1 shows the results of confirming the anti-cancer effect of the recombinant strain according to Experimental Example 9 of the present invention. [Figure 22] FIG. 1 shows the results of confirming the anti-cancer effect of the recombinant strain according to Experimental Example 9 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0058] The present invention will be described in more detail with reference to the following examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of explaining the present invention in more detail, and that the scope of the present invention is not limited by these examples according to the gist of the present invention.

[0059] [Preparation Example 1]: Creation of a DNA construct regulatable by doxycycline Using the pJL39 plasmid (Mol Ther., 21(11), pp. 1985-1995, (2013)) as a template strand, the tetR gene was amplified using a forward primer (5'-CGGAATTCACCATGTCTAGATTAGATAAAAGTAAAGTGATTAACAG-3'; SEQ ID NO: 2) containing a restriction enzyme EcoRI site, as shown in Figure 1A, and a reverse primer (5'-GCTCTAGACAGCTGTTAAGACCCACTTTCACATTTAAGTTGTTTTTCT-3'; SEQ ID NO: 3) containing a restriction enzyme PvuII-XbaI site. The amplified product was then digested with the restriction enzymes EcoRI and XbaI and purified to obtain a tetR gene amplified product, which was then transformed into pBAD24 (catalog number ATCC® 87399). TM The pBAD-TetR plasmid was constructed by introducing the plasmid into the ATCC (Atlantic Press, USA).

[0060] The pTetR-BAD plasmid was then constructed by inserting the Divergent promoter region containing a multiple cloning site into the pBAD-TetR plasmid using the PvuII and HindIII fragment of the pJL39 plasmid. The araC and araBAD promoters were removed from the pTetR-BAD plasmid using NheI and Pcil restriction enzymes to construct the pTetII plasmid. Using pSF-OXB1 (Oxford Genetics, England) as a template, the constitutive promoter OXB1 (SEQ ID NO: 16) was amplified with the forward primer (5'-CTACTCCGTCAAGCCGTCAAGCTGTTGTGACCGCTTGCT-3'; SEQ ID NO: 4) and the reverse primer (5'-TGAATTCCTCCTGCTAGCTAGTTGGTAACGAATCAGACGCCGGGTAATACCGGATAG-3'; SEQ ID NO: 5). The amplified promoter was then introduced into the pTetII plasmid using the Gibson assembly method to construct the pJH18 plasmid, which contains the OXB1, tetA, and tetR promoters.

[0061] [Table 1]

[0062] [Preparation Example 2] Cancer cell lines and culture conditions CT26 colon cancer cell lines CRL-2638 and HB-8064 (ATCC, USA) and murine colon adenocarcinoma cell line MC38 (Massachusetts General Hospital and Harvard Medical School, USA, and Chonnam National University, Korea) were used in the experiments. The cells were cultured in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) medium (catalog number: #LM001-05, Wellgene, Korea) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a 5% CO incubator.

[0063] [Preparation Example 3] Preparation of Salmonella strains with introduced plasmids The Salmonella strain used was SHJ2037 (relA::cat, spoT::kan), a Salmonella typhimurium (S. typhimurium) strain deficient in ppGpp. The Salmonella strains were transformed with the plasmid prepared in Preparation Example 1 by electroporation, and each transformed strain was cultured overnight in LB medium containing 100 μg / ml ampicillin. The culture medium was then diluted 1:100 with fresh LB medium containing ampicillin, and further cultured until OD 600 When the value reached 0.5 to 0.7, doxycycline diluted with ethanol to final concentrations of 0, 10, 50, 100, 300, and 500 ng / ml was added to the culture medium, and the culture was then cultured in a shaking incubator at 200 rpm and 37°C.

[0064] [Preparation Example 4] Preparation of experimental animal model C57BL / 6 and BALB / C mice (Orient Company, Korea) aged 5 to 6 weeks, weighing 20 to 30 g, were used. MC38 or CT26 from Preparative Example 2 was subcutaneously injected into the flank of the mice to establish tumor animal models. For imaging of the tumor animal model and evaluation of tumor size, 2% isoflurane was used for anesthesia, and 200 mg / kg ketamine and 10 mg / kg xylazine were used during surgery. The tumor size (mm 3 The evaluation of the tumor size in the animal model can be calculated using (length × height × width) / 2. 3 In these cases, the animal model was euthanized.

[0065] [Preparation Example 4] Preparation of experimental animal model C57BL / 6 and BALB / C mice (Orient Company, Korea) aged 5 to 6 weeks, weighing 20 to 30 g, were used. MC38 or CT26 from Preparative Example 2 was subcutaneously injected into the flank of the mice to establish tumor animal models. For imaging of the tumor animal model and evaluation of tumor size, 2% isoflurane was used for anesthesia, and 200 mg / kg ketamine and 10 mg / kg xylazine were used during surgery. The tumor size (mm 3 The evaluation of the tumor size in the animal model can be calculated using (length × height × width) / 2. 3 In these cases, the animal model was euthanized.

[0066] [Experimental Example 1] Protein expression and activity evaluation of recombinant strains [1-1] Comparison of growth between recombinant and existing strains The recombinant strains SLpCR, SLpFR, and SLpFC prepared in Preparation Example 3, and SLpEmpty used as a control, were grown overnight in LB liquid medium containing ampicillin, and then diluted 1:100 with fresh LB medium. 600 When the OD value reached 0.5-0.7, doxycycline diluted with ethanol was added to the culture solution to a final concentration of 200 ng / ml, and the culture was incubated in a shaking incubator at 200 rpm and 37°C. 600 The values ​​were measured to analyze the growth patterns of the strains, and the results are shown in Figure 2. As shown in Figure 2, the SLpCR, SLpFR, and SLpFC recombinant strains showed little difference in growth rate compared to the control SLpEmpty strain, and did not show any significant growth inhibition during protein expression with doxycycline. This confirms that the lack of virulence genes in the strains constructed as described above does not affect their growth or gene expression.

[0067] [1-2] Confirmation of selective hemolytic activity of recombinant strains The recombinant strains cultured in Preparatory Example 3 were diluted with PBS and smeared on blood agar plates containing 0 or 20 ng / ml doxycycline. After incubation at 37°C overnight, the plates were photographed and the results are shown in Figure 3. As shown in Figure 3, it was confirmed that the hemolytic activity of the strain on blood agar was only observed when the gene encoding cytolysin A, doxycycline, was included (+).

[0068] [1-3] Confirmation of TLR-5 signal activation by FlaB in recombinant strains To confirm the expression of FlaB in the recombinant strain, we examined the activation of TLR-5 signaling by FlaB. First, we measured the activation of TLR-5 signaling in the control group (40 ng of FlaB and the SLpEmpty strain). Then, we cultured the SLpFC recombinant strain prepared in Preparatory Example 3 in the absence (-) and presence (+) of doxycycline, and measured the activation of TLR-5 signaling. The results are shown in Figure 4. As shown in Figure 4, the SLpFC recombinant strain was confirmed to activate TLR-5 signaling compared to the control group. In particular, the recombinant strain (+) cultured under doxycycline administration showed significantly higher TLR-5 signaling activation, confirming that the expression level of FlaB in the recombinant strain was significantly increased when doxycycline was administered.

[0069] [1-4] Confirmation of FlaB and ClyA expression levels by doxycycline concentration Western blot analysis was performed to confirm the expression levels of FlaB and ClyA in the recombinant strain as a function of doxycycline concentration. SLpJH18-FC recombinant strains were cultured in medium containing 0, 10, 100, 200, 300, and 500 ng / ml of doxycycline. Figure 5 shows the levels of ClyA (34 kDa) and FlaB (43 kDa) secreted by the recombinant strain up to 2 hours after subculture (mid-log phage). As shown in Figure 5, the expression levels of ClyA (34 KDa) and FlaB (43 KDa) increased as the concentration of doxycycline increased to 0, 10, 100, 200, 300, and 500 ng / ml. This confirms that the expression levels of ClyA and FlaB in the recombinant strain can be regulated by adjusting the concentration of doxycycline, and that the expression and activity ratios can be relatively balanced.

[0070] [Experimental Example 2] Confirmation of the anti-cancer effect of recombinant strains (1) To confirm the anti-cancer effect of the recombinant strains, an in vitro cytotoxicity experiment was conducted on the CT26 cell line. CT26 cells were treated with the SLpCR, SLpFR, and SLpFC strains prepared in Preparative Example 3, as well as the SLpEmpty strain used as a control. The cytoplasmic enzyme necrotic dehydrogenase (LDH) released from the damaged CT26 cells was analyzed, and the results are shown in Figure 6. As shown in Figure 6, the cytotoxic effect of the SLpFC recombinant strain expressing ClyA and FlaB was significantly greater than that of other recombinant strains and the control group, confirming the significant anti-cancer effect of the SLpFC recombinant strain.

[0071] [Experimental Example 3] Confirmation of the cancer targeting effect of the recombinant strain To confirm the cancer targeting effect of the recombinant strains, in vivo experiments were performed to confirm the number of recombinant strains in the tumor and the targeting image. The attenuated SLpJH18-FC and SLpJH18-FR recombinant strains were injected into the CT26 mouse model of Preparatory Example 4 at a dose of 1 × 10 7 An experiment was conducted in which SLpJH18-FC was administered intravenously at a dose of 100 CFU, doxycycline was administered orally daily 3 days after administration, and a control group was not administered doxycycline.The results were shown in Figure 7, and the fluorescent images expressed by the SLpJH18-FR strain in the tumor were confirmed and are shown in Figure 8. As shown in Figures 7 and 8 , the recombinant strain reached 1 × 10 in the tumor regardless of doxycycline administration. 8 It was confirmed that the recombinant strain was present at more than CFU and specifically present in tumors, confirming that it specifically targets cancer cells.

[0072] [Experimental Example 4] Confirmation of the anti-cancer effect of the recombinant strain (2) To confirm the anti-cancer effect of the recombinant strain, an in vivo experiment was performed to confirm the expression levels of FlaB and ClyA in the SLpJH18-FC recombinant strain in tumors. The attenuated SLpJH18-FC recombinant strain was injected into the CT26 mouse model of Preparatory Example 4 at a dose of 1 × 10 7 An experiment was conducted in which the tumors were intravenously administered at 100 CFU, doxycycline was administered orally every day at 3 dpi, and a control group was not administered doxycycline. The FlaB expression levels in the tumors are shown in Figure 9, and the ClyA expression levels in the tumors are shown in Figure 10. As shown in Figures 9 and 10, it was confirmed that the expression levels of FlaB and ClyA increased significantly when doxycycline was administered, confirming that the expression levels of FlaB and ClyA can be regulated by adjusting the dosage of doxycycline after administering the SLpJH18-FC recombinant strain.

[0073] [Experimental Example 5] Confirmation of the anti-cancer effect of recombinant strains (3) To confirm the anti-cancer effect of the recombinant strain, an in vivo experiment was performed as shown in Figure 11 to confirm the growth inhibitory effect of the SLpJH18-FC recombinant strain on the CT26 cell line in tumors. The attenuated SLpJH18-FC recombinant strain was injected into the CT26 mouse model of Preparatory Example 4 at a concentration of 1 x 10 7 An experiment was conducted in which the tumors were intravenously administered at 100 CFU, doxycycline was orally administered at 1.7 mg / kg daily, and a control group was not administered doxycycline. The tumor size is shown in FIG. 12, and the survival rate of the mice is shown in FIG. As shown in Figures 12 and 13, administration of the SLpJH18-FC recombinant strain significantly increased the tumor-suppressing ability and mouse survival rate compared to the control group, confirming the anti-cancer effect of the SLpJH18-FC recombinant strain.

[0074] [Experimental Example 6] Confirmation of the anti-cancer effect of the recombinant strain (4) To confirm the anti-cancer effect of the recombinant strain, an in vivo experiment was performed as shown in Figure 11 to confirm the growth inhibitory effect of the SLpJH18-FC recombinant strain on the MC38 cell line in tumors. The attenuated SLpJH18-FC recombinant strain was injected into the CT26 mouse model of Preparatory Example 4 at a concentration of 1 x 10 7 An experiment was conducted in which the mice were intravenously administered with 1.7 mg / kg of CFU and orally administered doxycycline daily, with a control group not receiving doxycycline. The tumor size is shown in FIG. 14, and the survival rate of the mice is shown in FIG. As shown in Figures 14 and 15, administration of the SLpJH18-FC recombinant strain significantly increased the tumor-suppressing ability and mouse survival rate compared to the control group, confirming the anti-cancer effect of the SLpJH18-FC recombinant strain.

[0075] [Experimental Example 7] Confirmation of the effect of recombinant strains in suppressing cancer recurrence To confirm the cancer recurrence suppression effect of the recombinant strain, an in vivo experiment was performed as shown in Figure 11 to confirm the growth suppression effect of the SLpJH18-FC recombinant strain on the CT26 cell line in tumors. The attenuated SLpJH18-FC recombinant strain was injected into the CT26 mouse model of Preparatory Example 4 at a dose of 1 x 10 7 CFU were administered intravenously, and doxycycline was administered orally at 1.7 mg / kg daily to completely treat the cancer, after which CT26 cells were re-administered 90 days later. The tumor sizes after re-administration are shown in Figure 16 , and the survival rates of the mice are shown in Figure 17 . As shown in Figures 16 and 17, when the SLpJH18-FC recombinant strain was administered, the tumor growth inhibitory effect was significantly greater than that of the control group, and the survival rate of mice was significantly increased, confirming the cancer recurrence inhibitory effect of the SLpJH18-FC recombinant strain.

[0076] [Experimental Example 8] Confirmation of the inhibitory effect of recombinant strains on cancer metastasis To confirm the inhibitory effect of the recombinant strain on cancer metastasis, an in vivo experiment was conducted to confirm the growth inhibitory effect of the SLpJH18-FC recombinant strain on the 4T1-Luc cell line in tumors. A luciferase-expressing 4T1-Luc cell line was prepared, and a mouse model was prepared by injecting the 4T1-Luc cell line. Subsequently, the attenuated SLpJH18-FC recombinant strain was injected into the mouse at 1 x 10 7 After intravenous administration of 1.7 mg / kg of doxycycline daily, the location of the tumor was photographed and shown in Figure 18. The lungs were removed and the number of tumors that had metastasized to the lungs was counted and shown in Figure 19. As shown in Figures 18 and 19, when the SLpJH18-FC recombinant strain was administered, it was confirmed that the tumor did not metastasize to the lungs compared to the control group, confirming that the SLpJH18-FC recombinant strain had a significant effect in inhibiting tumor metastasis.

[0077] [Experimental Example 9] Confirmation of the anti-cancer effect of recombinant strains (5) To confirm the anti-cancer effect of the recombinant strain, in vivo experiments were conducted to confirm the abscopal effect of the SLpJH18-FC recombinant strain in tumors on the CT26 cell line. A mouse model was prepared in which CT26 was injected into both thighs, and the attenuated SLpJH18-FC recombinant strain was administered unilaterally followed by daily oral administration of doxycycline. The size of both tumors was measured and shown in Figure 20, tumor images were taken and shown in Figure 21, and Ki67 levels in mouse T cells were measured and shown in Figure 22. As shown in Figures 20 and 21, administration of the SLpJH18-FC recombinant strain to only one tumor had an inhibitory effect on the remaining tumor. Furthermore, as shown in Figure 22, treatment with the SLpJH18-FC recombinant strain increased Ki67 levels and activated immune cells, confirming the significant anti-cancer effect of the SLpJH18-FC recombinant strain.

[0078] From the above results, it can be seen that in the case of a plasmid containing the promoter of the present invention, it is possible to regulate the expression of regulatory proteins located downstream of the promoter, and ultimately to regulate the expression levels of anti-cancer genes located downstream of the tetA and tetR promoters. Although certain parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

[0079] From the above results, it can be seen that in the case of a plasmid containing the promoter of the present invention, it is possible to regulate the expression of regulatory proteins located downstream of the promoter, and ultimately to regulate the expression levels of anti-cancer genes located downstream of the tetA and tetR promoters. Although certain parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A nucleic acid encoding a TetR protein operably linked downstream of an OXB1 promoter; a nucleic acid encoding flagellin operably linked downstream of a first TetR-inducible promoter; and a nucleic acid encoding a toxin protein operably linked downstream of a second TetR-inducible promoter; A DNA construct comprising:

2. The DNA construct of claim 1 , wherein the flagellin is flagellin A or B.

3. The toxic proteins include ricin, saporin, gelonin, momordin, debouganin, diphtheria toxin, Pseudomonas toxin, hemolysin (HlyA), FAS ligand (FASL), tumor necrosis factor-alpha (TNF-alpha), TNF-related apoptosis-inducing ligand (TRAIL), streptolysin O (STRAIL), and the like.

2. The DNA construct of claim 1, wherein the DNA construct is at least one selected from the group consisting of pneumolysin (PLO), listeriolysin (LLO), and cytolysin A (ClyA).

4. 2. The DNA construct of claim 1, wherein the first TetR-inducible promoter is a tetA promoter and the second TetR-inducible promoter is a tetR promoter.

5. A recombinant vector comprising the DNA construct according to any one of claims 1 to 4.

6. A strain into which the recombinant vector according to claim 5 has been introduced.

7. The bacterial strain according to claim 6, wherein the bacterial strain is at least one selected from the group consisting of a Salmonella strain, a Clostridium strain, a Bifidobacterium strain, and an Escherichia strain.

8. A pharmaceutical composition for preventing or treating cancer, comprising the strain of claim 6 as an active ingredient.

9. 9. The pharmaceutical composition of claim 8, wherein the cancer is at least one selected from the group consisting of melanoma, fallopian tube cancer, brain cancer, small intestine cancer, esophageal cancer, lymphatic gland cancer, gallbladder cancer, blood cancer, thyroid cancer, endocrine gland cancer, oral cancer, liver cancer, biliary tract cancer, colon cancer, rectal cancer, cervical cancer, ovarian cancer, kidney cancer, stomach cancer, duodenal cancer, prostate cancer, breast cancer, brain cancer, lung cancer, anaplastic thyroid cancer, uterine cancer, colon cancer, bladder cancer, ureter cancer, pancreatic cancer, bone / soft tissue sarcoma, skin cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and solitary myeloma.

10. The pharmaceutical composition according to claim 8, wherein the pharmaceutical composition inhibits cancer growth or cancer metastasis.

11. The strain of claim 6 for use in preventing or treating cancer.

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