Salmonella strains for cancer prevention and treatment and their uses

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 the prevention and treatment of 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 or combinations thereof, corresponding to surgery, radiation therapy, and chemotherapy. Surgical removal, which removes most of the cancerous tissue, can be extremely effective in removing cancerous tissue located in specific areas, such as the breast, colon, and skin, but it is difficult to use to treat cancerous tissue in certain areas, such as the spine. In addition, systemic chemotherapy, which is commonly 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 the chemotherapy drugs.

[0003] On the other hand, when cancer develops in an individual, angiogenesis and cell growth proceed at a very rapid rate within the body, creating an environment within the cancerous tissue that is characterized by incomplete angiogenesis and oxygen deficiency, making it extremely suitable for the growth of anaerobic bacteria such as Salmonella strains or E. coli. As a result, cancer treatment using cancer-targeting bacteria such as Salmonella and Clostridium strains currently relies on the function of specific bacteria that can target solid tumors and grow within the tumor. However, by introducing oncolytic proteins or reporter proteins into these bacteria and administering the transformed bacteria to the individual, it is possible to specifically identify cancerous tissue and treat cancer while minimizing side effects that are toxic to normal cells.

[0004] Diseases in humans are triggered by toxins secreted by various bacterial pathogens that exist in nature. Among these diverse bacterial pathogens that can trigger disease, Salmonella enterica, which is closely related to our diet, is known to be a member of the Enterobacteriaceae family that inhabits the intestinal tract of primates, including humans, and secretes cytolysin, a known exotoxin. This secreted cytolysin is a cytotoxic protein with a molecular weight of approximately 34 kDa, and is known to cause hemolysis by destroying red blood cells in the intestines of primates, including humans, and to induce cell lysis by forming pores in the membranes of normal cells, leading to severe vascular inflammation and local tissue necrosis, and ultimately death. However, recent research has shown that cytolysin isolated and purified from Salmonella enterica specifically reacts with cancerous tissue in the intestinal tract of the body, inducing the death of cancerous tissue, and is attracting attention as a next-generation anti-cancer treatment. Therefore, bacteria transformed with genes that secrete cytotoxic substances such as cytolysin are considered to have very high potential for use as anti-cancer agents targeting cancer tissue.

[0005] Despite the possibility of diagnosing or treating cancer using bacteria, there is currently very little research on expression vectors that enable the specific expression of diagnostic and therapeutic proteins in cancer tissue. After bacteria are injected into the body, clearance occurs in the reticular endothelial system, such as the liver and spleen, for the first three days, and then they rapidly increase in cancer tissue after a certain period. Therefore, for safety reasons, it is necessary to ensure that therapeutic proteins are expressed after a certain period. For this reason, the use of inducible promoters is recommended for the expression of therapeutic proteins, but currently, inducible promoters such as the PBAD promoter used in experiments must use L-arabinose, which is not permitted for use in humans, as an inducer, 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 enabling bidirectional transcription of two genes using the TetA and TetR promoters. However, the difference in protein expression rates between the TetA and TetR promoters is more than 100:1, and its usability will only increase if this balance is achieved. Currently, there is a need to develop new technologies for transformed bacteria that have a clinically applicable expression system and whose protein expression levels are balanced. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] One objective of the present invention is to provide a DNA construct. Another object of the present invention is to provide a recombinant vector comprising 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 cancer diagnostic composition comprising the same. Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer, comprising the aforementioned bacterial strain as an active ingredient. A further object of the present invention is to provide an information-providing method for cancer diagnosis, which includes the step of processing the bacterial strain. However, the technical problems that this invention aims to solve are not limited to those mentioned above, and any other problems not mentioned will be clearly understood by those with ordinary skill in the art from the following description. [Means for solving the problem]

[0007] One embodiment of the present invention provides a DNA construct. The DNA construct of the present invention comprises genes that encode the first protein and the second protein; and corresponding first and second promoters, 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 further includes genes that encode regulatory proteins.

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

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

[0010] In the present invention, "regulation" or "expression regulation" can mean that the transcription and translation of a particular gene are activated or suppressed. The cis-acting factor of the present invention is a region of non-coding DNA that regulates the transcription of adjacent genes, is an essential component of the gene regulatory network, and controls gene expression. The cis-acting factor may be, but is not limited to, 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 terminators. In the present invention, the ribosome binding site (RBS) is also called the Shine-Dalgarno sequence (SD sequence). After the genetic information contained in DNA is transcribed into messenger RNA (mRNA), translation must occur in this mRNA. The RBS is a short sequence present on the mRNA that allows ribosomes to bind to it effectively.

[0011] In the present invention, the 5'-untranslated region (5'-UTR) is an untranslated region located on either side of the coding region, which is the part of mRNA that is translated into amino acids in the 5' region. It was previously thought to be junk that is wasted during the evolutionary process, but it has become known to play a significant role in regulating gene expression. In the present invention, the transcription factor binding site is a DNA region that plays a role in turning a specific nearby gene on or off. The transcription factor binding site may be, but is not limited to, at least one selected from the group consisting of promoters, enhancers, and silencers of the gene that encodes the regulatory protein.

[0012] The promoter of the gene encoding the regulatory protein of the present invention includes all promoters that can induce activity under the environmental conditions and developmental state of most host bacterial strains or cells, and preferably a weak promoter. The "weak promoter" of the present invention refers to a transcriptional expression level of a transcript transcribed from a downstreamly operably linked gene that is 1 × 10⁻¹⁶ -2 The following is preferably 1 × 10 -3 A promoter that induces the expression of the following, thereby increasing the expression level of the transcript to 1 × 10⁻⁶ -3 The following promoters are included, but are not limited to, any promoter expressed below, including, for example, the E. coli σ70 promoter; the E. coli σS promoter; the E. coli σ32 promoter; the B. subtilis σA promoter; the B. subtilis σB promoter; the Salmonella-derived promoters K112706 or K112707; the bacteriophage T7 promoter; the bacteriophage SP6 promoter; the yeast-derived promoter; the eukaryotic cell-derived promoters I712004 or K076017; and at least one selected from the group consisting of plant-derived promoters.

[0013] The E. coli σ70 promoter of the present invention corresponds to 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 may be selected from, but are not limited to.

[0014] The E. coli σS promoter of the present invention may be J45992 or J45993, but is not limited thereto. The E. coli σ32 promoter of the present invention may be J45504, K1895002, or K1895003, but is not limited thereto. 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 K143010, K143011 or K143013, but is not limited thereto. 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 J64998, but is not limited thereto.

[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 suppresses the regulatory protein is administered, compared to when it 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, but is not limited to, the base sequence at the -35 position relative to the gene encoding the regulatory protein being SEQ ID NO: 8, and the base sequence at the -10 position being SEQ ID NO: 9.

[0018] The enhancer of the present invention is a sequence found in both prokaryotes and eukaryotes, generally having a region of 50 to 1500 bp, and is located upstream or downstream of the origin of the nearby gene to induce the binding of the transcription factor. The silencer of the present invention maintains the same mechanism as the enhancer and acts as an antagonist to 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 they may be in the same region but in different regions of the same area.

[0019] The terminators of the present invention are also called transcription terminators, and they mediate the termination of gene or operon transcription using a dielectric material. In prokaryotes, there are Rho-dependent terminators and Rho-independent terminators. The trans-acting factor of the present invention is also called a trans-activating factor or 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, 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 a specific gene on or off. In the present invention, the aptamer is a part of a riboswitch and is generally referred to as an oligonucleotide or peptide substance capable of binding to a specific target molecule. The aptamer may also 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 riboswitches, FMN riboswitches, Cobalamin riboswitches, etc.

[0021] In this invention, the sRNA and antisense RNA (asRNA) refer to single-stranded RNA that can bind complementarily to a specific RNA. They bind complementarily to sense RNA, which is a messenger RNA (mRNA) that expresses a specific protein, and ultimately regulate the expression of that 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 is a promoter that transcribes a gene so that it can be specifically expressed downstream under specific chemical or physical conditions. For example, it may be a promoter for the LacZ gene that is expressed in the presence of galactose such as IPTG (isopropyl-beta-D-1-thiogalactopyranoside), an arabinose operon araBAD promoter that is expressed only in the presence of L-arabinose, or a 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 a tetA promoter and the second promoter may be a tetR promoter, but it 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 and regulates so that RNA polymerase cannot bind to them. For the purposes of the present invention, if the first promoter and the second promoter are tet promoters, the protein may be a TetR protein that binds to the regulatory site of the tet promoter and suppresses the activity of the tet promoter, but is not limited thereto.

[0023] The term "operably linked" in this invention means that one target nucleic acid fragment is functionally linked to another nucleic acid fragment, thereby influencing the function or expression of the target nucleic acid fragment. The "reporter protein" of the present invention is a protein that performs a function to enable the visual diagnosis of cancer, and may be, but is not limited to, at least one selected from the group consisting of, for example, fluorescent proteins, luciferases, and proteins used in nuclear medicine or MRI imaging.

[0024] The "fluorescent protein" of the present invention is a protein that emits fluorescence on its own so that cancer can be visually diagnosed, and may be, but is not limited to, at least one selected from the group consisting of, for example, 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). The protein used in nuclear medicine or MRI imaging according to the present invention may be, but is not limited to, at least one selected from the group consisting of, for example, herpes simplex virus thymidine kinsease, dopamine receptor, somatostatin receptor, sodium-iodide transporter, iron receptor, transferrin receptor, ferritin, and iron transporter (magA).

[0025] The term "cytokine" in this invention refers to a protein secreted by immune cells, and includes all cytokines that can be used in cancer immunotherapy to regulate the host immune response and induce the death of disease-related cells, such as cancer cells. Preferably, these include IFN-alpha2, IL-2, IL-15, IL-21, and IL-12, but are not limited thereto. The "chemokines" of the present invention include all those that play a role in regulating cell migration between tissues and the position and interaction of cells within tissues, and that can induce leukocytes into the tumor microenvironment to mediate the host response to disease, such as cancer. Preferably, these may be CXCR3, CCR5, etc., but are not limited thereto. The "immunomodulator" of the present invention refers to any substance that enables diverse treatments by utilizing the unique immune system of an individual, and that can activate immune cells to induce the death of disease-related cells, such as cancer cells.

[0026] The "anti-cancer protein" of the present invention is a peptide having the function of directly or indirectly inducing the death of cancer cells, and may be, but is not limited to, at least one selected from the group consisting of, for example, toxin proteins, antibodies or fragments of said antibodies, tumor suppressor proteins, angiogenesis inhibitors, cancer antigens, prodrug-converting enzymes, and pro-apoptotic proteins. The "toxin protein" of the present invention refers to a protein that has the function of directly or indirectly inducing the death of cancer cells, such as lysine, saporin, geronin, momordin, debouganin, diphtheria toxin, pseudomonas toxin, hemolysin (HlyA), FAS ligand (FASL), tumor necrosis factor-alpha (TNF-alpha), and TNF-related apoptosis-inducing ligand (TRAIL). , streptolysin O (SLO), pneumolysin (PLO), listeriolysin (LLO), It may be at least one selected from the group consisting of and cytolysin A (ClyA), and more preferably cytolysin A consisting of the amino acid sequence represented by Sequence 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, if its function is lost, induces indiscriminate cell division and growth in normal cells, thereby converting them into cancer cells. Examples include, but are not limited to, RB (Retinoblastoma protein) protein, p53 protein, APC (Adenomatous polyposis coli) protein, PTEN (Phosphatase and tensin homologue) protein, and CDKN2A (cyclin-dependent kinase inhibitor 2A) protein. The antibody or fragment of the antibody 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. For example, it may be an antibody specific to HER2, which is specifically expressed at a high level on 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 the antigenic site of a protein or peptide molecule. The form of the antibody is not particularly limited and includes polyclonal antibodies, monoclonal antibodies, or even parts of an antibody that have antigen-binding properties, and includes all types of immunoglobulin antibodies. It also includes special antibodies such as humanized antibodies, and 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. A functional fragment of an antibody molecule means a fragment that possesses at least 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 conventional methods after cloning the gene encoding the cancer antigen of the present invention into an expression vector by conventional methods to obtain the protein encoded by the gene. The "angiogenic inhibitor" of the present invention means a protein or compound that has the function of directly or indirectly inducing the death of cancer cells by suppressing the generation of new blood vessels around cancer cells. Preferably, the angiogenic inhibitor may be angiostatin, endostatin, thrombospondin, or protease inhibitory proteins, but is not limited thereto.

[0030] The term "cancer antigen" in this invention refers to a protein that is expressed in cancer cells but hardly expressed in normal cells, and which can induce an antitumor immune response, thereby directly or indirectly inducing the death of cancer cells. The cancer antigen in this invention may preferably be alpha-fetoprotein (AFP), vascular endothelial growth factor receptor 2 (VEGFR2), Survivin, Legmain, or prostate cancer specific antigen (PCSA), but is not limited thereto. The "precursor drug convertase" of the present invention is a protein that has the function of converting an inactive drug into an active drug through enzymatic metabolism. When such a precursor drug convertase is used, the inactive drug is metabolized and converted into an active drug that can directly or indirectly induce the death of cancer cells, making it extremely useful for the prevention or treatment of cancer. The precursor drug-converting enzyme of the present invention may preferably be 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., but is not limited thereto.

[0031] The "pro-apoptotic protein" of the present invention refers to a protein that induces the 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 preferably be L-ASNase, RNA-binding motif protein 5 (RBM5), etc., but is not limited thereto. The cancer antigen-specific oligonucleotide of the present invention is a nucleotide that can suppress the expression or function of the cancer antigen by binding complementarily to the gene or mRNA of the cancer antigen, and may be, but is not limited to, any one selected from the group consisting of antisense oligonucleotides, aptamers, siRNAs, and shRNAs.

[0032] The "antisense oligonucleotide" of the present invention means DNA, RNA, or derivatives thereof that contain a nucleic acid sequence complementary to a specific mRNA sequence, and can bind to the complementary sequence in mRNA and inhibit the translation of mRNA into protein. The antisense oligonucleotide can be synthesized in vitro using, for example, a conventional method using RNA polymerase I, and then administered into the body, or it can be synthesized in the body by a method such as using a vector whose recognition site (MCS) originates in the opposite direction. In this invention, the term "aptamer" refers to a small, single-stranded oligonucleotide capable of specifically recognizing a target substance with high affinity. For the purposes of this invention, the target substance may be a cancer antigen gene or mRNA.

[0033] The "siRNA" in this invention refers to a short double-stranded RNA capable of inducing RNA interference (RNAi) by cleaving a specific mRNA. It consists of a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a complementary sequence. For the purposes of this invention, the siRNA can specifically bind to mRNA transcribed from a gene encoding a cancer antigen, thereby effectively suppressing the expression of such a gene. The "shRNA" in this invention refers to short hairpin RNA, which has the advantages of a higher cell phenotype infection rate and the ability to maintain RNA interference for a long period of time compared to siRNA. RNA interference can be induced by the process of transforming cells with an RNA polymerase III promoter and then expressing adenovirus, lentivirus, or plasmid expression vector system, but is not limited to this. For the purposes of this invention, the shRNA can specifically bind to mRNA transcribed from genes encoding cancer antigens and effectively suppress the expression of such genes.

[0034] Another embodiment of the present invention provides a recombinant vector comprising the DNA construct of the present invention. The recombinant vector of the present invention, by comprising the DNA construct of the present invention, enables the expression of a regulatory protein by another promoter, thereby allowing a balanced expression of genes operably linked downstream of the first and second promoters specifically only when a substance that suppresses the regulatory protein is administered externally.

[0035] In the recombinant vector of the present invention, the contents relating to the DNA construct, anti-cancer protein, cytokine, chemokine, immunomodulator, oligonucleotide specific to cancer antigen, reporter protein, and promoter are the same as those described in the DNA construct and are omitted to avoid excessive complexity in this specification. The recombinant vector of the present invention can be a known recombinant vector such as a plasmid vector, cosmid vector, or bacteriophage vector, which can be introduced into a cell to express a protein. The recombinant vector can be easily produced by a person skilled in the art by 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, cosmid, lambda, lambdoid, M13, Mu, p1 P22, Qμ, T-even, T2, T3, T7 and other phages, and plant viruses, but is not limited thereto. For the purposes of the present invention, a recombinant vector suitable for the host cell can be selected depending on the properties of the host cell. In yet another embodiment of the present invention, a host cell or bacterial strain into which a recombinant vector comprising the DNA construct of the present invention has been introduced is provided.

[0037] The host cells of the present invention include, but are not limited to, at least one selected from the group consisting of, for example, bacterial cells such as Escherichia coli, Streptomyces or Salmonella strains; fungal cells such as yeast cells and Pichia pastris; insect cells such as Drosophila and 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. For the purposes of the present invention, the bacterial strain may be at least one selected from the group consisting of anaerobic bacterial strains, such as 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, and more preferably Salmonella typhimurium, but is not limited thereto.

[0038] The bacterial strain of the present invention may be a weakened form. In this invention, "attenuation" means modifying genes or other structures so that toxicity and other side effects can be reduced when the microorganism is administered to a patient. For the purposes of this invention, if the strain is a Salmonella strain, attenuation may involve 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. The method for modifying the gene of the present invention is carried out by various gene deletion or disruption methods known in the art, for example, such deletion and disruption methods are carried out by homologous recombination, chemical mutagenesis, irradiation mutagenesis, or transposon mutagenesis.

[0039] In the present invention, since the bacterial strain targets the inside of cancerous tissue, which is an environment with incomplete angiogenesis and oxygen deficiency that is extremely suitable for the proliferation of anaerobic bacterial strains, if a recombinant vector that can simultaneously and balancedly express a reporter protein and an anti-cancer protein that can be imaged in real time is introduced into such a bacterial strain, cancer can be diagnosed and treated very effectively. In the bacterial strain of the present invention, the details relating to the DNA construct, anti-cancer protein, cytokine, chemokine, immunomodulator, oligonucleotide specific to cancer antigen, reporter protein, promoter, and recombinant vector are the same as those described in the DNA construct and recombinant vector, and are omitted to avoid excessive complexity in this specification.

[0040] The recombinant vector of the present invention is introduced into host cells or bacterial strains by transformation (or phenotypic infection), but any transformation method can be used in the present invention and can be easily carried out by conventional methods of the art. Specifically, the recombinant vector can be introduced into the bacterial strain using, but is not limited to, conventional methods for transforming bacteria such as the Salmonella strain, CaCl2 precipitation, the Hanahan method which is more efficient by using the reducing agent DMSO (Dimethyl sulfoxide) in the CaCl2 method, electroporation, calcium phosphate precipitation, plasmofusion, stirring with silicon carbide fibers, agrobacteria-mediated transformation, PEG-mediated transformation, dextransphosphate, lipofectamine, and drying / inhibition-mediated transformation. In yet another embodiment of the present invention, a pharmaceutical composition for the prevention or treatment of cancer is provided. According to yet another aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising the aforementioned bacterial strain as an active ingredient.

[0041] The pharmaceutical composition of the present invention inhibits cancer growth or cancer metastasis. When the aforementioned bacterial strain of the present invention is transformed with the DNA construct according to the present invention and targets cancer in an organism, and then a substance that suppresses regulatory proteins is administered, a reporter protein and an anti-cancer protein that can be imaged in real time are simultaneously and in a balanced manner expressed within the strain. Therefore, cancer can be prevented or treated very effectively, and at the same time, cancer can be diagnosed in real time.

[0042] The "cancer" of the present invention is a disease characterized by the rapid and uncontrolled growth of mutated cells, including melanoma, fallopian tube cancer, brain cancer, small intestine cancer, esophageal cancer, lymph node cancer, gallbladder cancer, hematological cancer, thyroid cancer, endocrine cancer, oral cancer, liver cancer, biliary tract cancer, colorectal cancer, rectal cancer, cervical cancer, ovarian cancer, kidney cancer, gastric cancer, duodenal cancer, prostate cancer, breast cancer, brain tumor, lung cancer, anaplastic thyroid cancer, uterine cancer, colon cancer, bladder cancer, ureteral cancer, pancreatic cancer, bone / soft tissue sarcoma, skin cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, It 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, gastric cancer, duodenal cancer, prostate cancer, breast cancer, brain tumor, lung cancer, anaplastic thyroid carcinoma, uterine cancer, colon cancer, bladder cancer, ureteral cancer, pancreatic cancer, bone / soft tissue sarcoma and skin cancer, and more preferably colon cancer, but not limited thereto. The term "prevention" in this invention includes, without limitation, all actions that block, suppress, or delay symptoms caused by cancer using the active ingredient of this invention.

[0043] The term "treatment" in this invention means all actions using the active ingredient of this invention that result in an improvement in symptoms caused by cancer or an overall benefit to the individual, and means an attempt to obtain useful or favorable results, including clinical outcomes. Useful or favorable clinical outcomes may include, but are not limited to, alleviation or improvement of one or more symptoms or conditions, reduction of disease extent, stabilization of disease state, suppression of disease onset, suppression of disease spread, delay or prolongation of disease progression, delay or prolongation of disease onset, improvement or reduction of disease state, and reduction (in part or in whole), whether detectable or not. Furthermore, "treatment" may mean that the patient's survival is extended beyond what would be predicted in the absence of treatment. In addition, "treatment" may mean suppression of disease progression, temporary prolongation of disease progression, and more preferably, permanent cessation of disease progression. As is understood by those skilled in the art, if the treatment results in the opposite outcome, i.e., an outcome greater than all the benefits affected by the treatment, while improving a particular disease state, the outcome is not beneficial or favorable. In the present invention, the treatment may suppress cancer metastasis or cancer recurrence.

[0044] In this invention, "cancer metastasis" refers to the property of cancer to spread to another location geographically separated from the organ or part of the body where it originated. Metastatic cancer is cancer that has the property of metastasizing or has undergone metastasis. In particular, metastatic cancer may, but is not limited to, metastasis to the liver, lungs, bones, lymph nodes, or abdominal cavity. Metastatic cancer is difficult to treat, and its progression and treatment may be more complex than in the initial treatment process. In this invention, "cancer recurrence" means that cancer was not detected after treatment but was discovered again after a certain period of time. Recurrent cancer refers to cancer that results from the recurrence of cancer as described above. When cancer recurs, surgical removal is often difficult, and even when it is possible, it requires a fairly large-scale surgery. Furthermore, there may be limitations on anti-cancer treatment and radiation therapy. In the pharmaceutical composition of the present invention, the details relating to the DNA construct, anti-cancer protein, cytokine, chemokine, immunomodulator, oligonucleotide specific to cancer antigen, reporter protein, promoter recombinant vector, bacterial strain, and transformation are the same as those described for the DNA construct, recombinant vector, and bacterial strain, and are omitted to avoid excessive complexity in 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 beverage, and the pharmaceutical composition is characterized in that it is intended for human use. The pharmaceutical compositions of the present invention are not limited to these, but each can be prepared by conventional methods into oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, as well as topical preparations, suppositories, and sterile injection solutions. The pharmaceutical compositions of the present invention may contain pharmaceutically acceptable carriers. For oral administration, pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, and fragrances. For injections, buffers, preservatives, analgesics, solubilizers, isotonic agents, and stabilizers may be mixed and used. For topical administration, bases, excipients, lubricants, and preservatives may be used. The dosage forms of the pharmaceutical compositions of the present invention can be manufactured in a variety of ways by mixing them with the pharmaceutically acceptable carriers described above. For example, when administered orally, it can be manufactured in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injectable preparations, it can be manufactured in single-dose ampoules or in multi-dose formulations. In addition, it can be formulated into other dosage forms such as solutions, suspensions, tablets, capsules, and sustained-release formulations.

[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, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil. Additionally, fillers, anti-flocculants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives may be included. The administration routes of the pharmaceutical composition of the present invention are not limited to those listed above, but include oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal administration. Oral or parenteral administration is preferred.

[0047] The term "parenteral" in this invention includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-sacral, intrasternal, intradural, intrafocal, and intracranial injection or infusion techniques. The pharmaceutical compositions of this invention may further be administered in the form of suppositories for rectal administration. The pharmaceutical compositions of the present invention can vary considerably depending on various factors, including the activity of the specific compound used, age, weight, general health, sex, diet, administration time, route of administration, excretion rate, drug formulation, and the severity of the specific disease being prevented or treated. The dosage of the pharmaceutical compositions will vary depending on the patient's condition, weight, disease severity, drug form, route of administration, and duration, but can be appropriately selected by those skilled in the art, and can be administered at a dose of 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. The administration may be once a day or divided into several doses. The dosage does not limit the scope of the present invention in any way. The pharmaceutical compositions according to the present invention can be formulated into pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, and suspensions.

[0048] In yet another embodiment of the present invention, a composition for diagnosing cancer is provided. The diagnostic composition of the present invention contains the bacterial strain according to the present invention as an active ingredient. When the aforementioned bacterial strain of the present invention is transformed with the DNA construct according to the present invention and targets cancer cells in an organism, and then a substance that suppresses regulatory proteins is administered, a reporter protein and an anti-cancer protein that can be imaged in real time are simultaneously and in a balanced manner expressed within the strain. Therefore, cancer can be prevented or treated very effectively, and at the same time, cancer can be diagnosed in real time.

[0049] The term "diagnosis" in this invention means all actions to confirm cancerous tissue in vivo, including the ability to monitor the presence or absence of cancer in real time by a reporter protein expressed from a DNA construct introduced into the strain, when the strain is located targeting cancer. In the diagnostic composition of the present invention, the details relating to 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 are omitted to avoid excessive complexity in 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 includes the step of treating a biological sample isolated from a target individual with a bacterial strain into which the recombinant vector according to the present invention has been introduced. The method for providing information for the diagnosis of cancer according to the present invention may further include the step of diagnosing cancer if a reporter protein is expressed from the bacterial strain.

[0051] The term "biological sample" in this invention means any substance, tissue, or cell obtained from or derived from an individual, and may include, but is not limited to, tissue, cell, or cell extract. In the diagnostic information provision method of the present invention, the contents relating to the DNA construct, anti-cancer protein, cytokine, chemokine, immunomodulator, oligonucleotide specific to cancer antigen, 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 are omitted to avoid excessive complexity in this specification.

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

[0053] The present invention includes a method for preventing or treating cancer by administering an effective amount of a bacterial strain into which the recombinant vector, or a recombinant vector comprising flagellin and a toxin protein, has been introduced, to a target subject. In yet another embodiment of the present invention, a method for preventing or treating cancer is provided by co-administering effective amounts of a bacterial strain expressing exogenous flagellin and a bacterial strain expressing exogenous toxin protein to a subject. The "flagellin" of this invention, while not limited to this, is a granular protein that constitutes the spiral fibers of bacterial flagella. Its molecular weight varies greatly depending on the bacterial species (30,000 to 70,000), but cysteine ​​and tryptophan are absent from its amino acid composition, and in the case of Salmonella, about half of the lysine is methylated. There are various types of flagellin, such as flagellin A and B, and although their functions are diverse, they are known to have an immune-enhancing effect.

[0054] In this invention, "combined administration" refers to the use of various methods for cancer treatment (such as surgery, radiotherapy, chemotherapy, and immunotherapy) together or in a stepwise manner, but is not limited to this. It also refers to the administration of two or more anticancer drugs or drugs with different mechanisms of action, or the simultaneous use of physical therapy such as radiotherapy and chemotherapy such as anticancer drugs. When two drugs are used, they exhibit synergistic effects based on complementary mechanisms or drug dose adjustments. However, in actual clinical practice, the synergistic effect is significantly reduced, which also carries the risk of serious side effects such as immunosuppression and cardiotoxicity. According to yet another aspect of the present invention, the present invention provides a composition for the prevention or treatment of cancer comprising flagellin or a nucleotide encoding it; and a toxin protein or a nucleotide encoding it as an active ingredient.

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

[0056] The DNA construct according to the present invention can prevent and treat cancer by regulating the expression level of anti-oncogenes operably linked downstream of a first promoter and a second promoter within a host bacterial strain or cell. Furthermore, the DNA construct of the present invention allows for the expression of anti-cancer proteins at appropriate doses for cancer treatment by adjusting whether or not doxycycline treatment is performed. [Brief explanation of the drawing]

[0057] [Figure 1] This figure shows a schematic diagram of the DNA construct according to Preparation Example 1 of the present invention. [Figure 2]This figure shows the results of analyzing the growth pattern of a bacterial strain in which the DNA construct was recombinant according to Experimental Example 1 of the present invention. [Figure 3] This figure shows the results of the hemolytic activity of the bacterial strain on blood agar according to Experimental Example 1 of the present invention. [Figure 4] This figure shows the results of measuring TLR-5 signal activation according to Experimental Example 1 of the present invention. [Figure 5] This figure shows the difference in the expression level of recombinant bacterial strains depending on the doxycycline concentration, as shown in Experimental Example 1 of the present invention. [Figure 6] This figure shows the results of confirming the anticancer effect of recombinant bacterial strains using Experimental Example 2 of the present invention. [Figure 7] This figure shows the results of confirming the cancer targeting effect of recombinant bacterial strains according to Experimental Example 3 of the present invention. [Figure 8] This figure shows the results of confirming the cancer targeting effect of recombinant bacterial strains according to Experimental Example 3 of the present invention. [Figure 9] This figure shows the results of confirming the anticancer effect of recombinant bacterial strains according to Experimental Example 4 of the present invention. [Figure 10] This figure shows the results of confirming the anticancer effect of recombinant bacterial strains according to Experimental Example 4 of the present invention. [Figure 11] This figure shows an experimental method for confirming the anticancer effect of recombinant bacterial strains according to experimental examples of the present invention. [Figure 12] This figure shows the results of confirming the anticancer effect of recombinant bacterial strains according to Experimental Example 5 of the present invention. [Figure 13] This figure shows the results of confirming the anticancer effect of recombinant bacterial strains according to Experimental Example 5 of the present invention. [Figure 14] This figure shows the results of confirming the anticancer effect of recombinant bacterial strains according to Experimental Example 6 of the present invention. [Figure 15] This figure shows the results of confirming the anticancer effect of recombinant bacterial strains according to Experimental Example 6 of the present invention. [Figure 16] This figure shows the results of confirming the cancer recurrence suppression effect of recombinant bacterial strains according to Experimental Example 7 of the present invention. [Figure 17]This figure shows the results of confirming the cancer recurrence suppression effect of recombinant bacterial strains according to Experimental Example 7 of the present invention. [Figure 18] This figure shows the results of confirming the cancer metastasis inhibitory effect of recombinant bacterial strains according to Experimental Example 8 of the present invention. [Figure 19] This figure shows the results of confirming the cancer metastasis inhibitory effect of recombinant bacterial strains according to Experimental Example 8 of the present invention. [Figure 20] This figure shows the results of confirming the anticancer effect of recombinant bacterial strains according to Experimental Example 9 of the present invention. [Figure 21] This figure shows the results of confirming the anticancer effect of recombinant bacterial strains according to Experimental Example 9 of the present invention. [Figure 22] This figure shows the results of confirming the anticancer effect of recombinant bacterial strains according to Experimental Example 9 of the present invention. [Modes for carrying out the invention]

[0058] The present invention will be described in more detail below through the examples. These examples are merely for the purpose of illustrating the present invention in more detail, and it will be obvious to those with ordinary skill in the art that the scope of the present invention is not limited by these examples, as is the essence of the invention.

[0059] [Preparation Example 1] ● Preparation of a DNA construct regulated by doxycycline Using the pJL39 plasmid (Mol Ther., 21(11), p.1985-1995, (2013)) as a template, the tetR gene was amplified using forward primers (5'-CGGAATTCACCATGTCTAGATTAGATAAAAGTAAAGTGATTAACAG-3'; SEQ ID NO: 2) prepared to include the restriction enzyme EcoRI site, as shown in Figure 1A, and reverse primers (5'-GCTCTAGACAGCTGTTAAGACCCACTTTCACATTTAAGTTGTTTTTCT-3'; SEQ ID NO: 3) prepared to include the restriction enzyme PvuII-XbaI site. Subsequently, the amplified product was cleaved with the restriction enzymes EcoRI and XbaI, purified, and then processed into pBAD24 (catalog number ATCC(registered trademark) 87399). TM The pBAD-TetR plasmid was constructed by introducing it into a plasmid (ATCC, USA).

[0060] Subsequently, the pTetR-BAD plasmid was constructed by introducing a divergent promoter region containing multiple cloning sites into the pBAD-TetR plasmid using the PvuII and HindIII fragments of the pJL39 plasmid. The pTetII plasmid was constructed by removing the araC and araBAD promoters from the pTetR-BAD plasmid using NheI and Pcil restriction enzymes. Using pSF-OXB1 (Oxford Genetics, England) as a template, the constitutive promoter OXB1 (SEQ ID NO: 16), amplified with forward-facing primers (5'-CTACTCCGTCAAGCCGTCAAGCTGTTGTGACCGCTTGCT-3'; SEQ ID NO: 4) and reverse-facing primers (5'-TGAATTCCTCCTGCTAGCTAGTTGGTAACGAATCAGACGCCGGGTAATACCGGATAG-3'; SEQ ID NO: 5), was introduced into the pTetII plasmid using the Gibson assembly method to ultimately construct the pJH18 plasmid containing OXB1, tetA, and tetR promoters.

[0061] [Table 1]

[0062] [Preparation Example 2] ● Cancer cell line and culture conditions The CT26 colon cancer cell lines CRL-2638 and HB-8064 (ATCC, USA) and the mouse colorectal adenocarcinoma cell line MC38 (Massachusetts General Hospital and Harvard Medical School, USA, and Chonnam National University, South Korea) were used in the experiment. The cells were cultured in a 5% CO2 incubator at 37°C using high-glucose DMEM (Dulbecco's Modified Eagles Medium) medium (catalog number: #LM001-05, Wellgene, Korea) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

[0063] [Preparation Example 3] ● Preparation of a Salmonella strain into which a plasmid has been introduced The Salmonella strain used was SHJ2037 (relA::cat, spoT::kan), a Salmonella typhimurium (S. typhimurium) lacking ppGpp. After transforming the Salmonella strains with the plasmids prepared in Preparation Example 1 using electroporation, each transformed strain was cultured overnight in LB medium containing 100 μg / ml ampicillin. Subsequently, the culture solution was diluted 1:100 with fresh LB medium containing ampicillin, and further culture was performed to induce OD (Oral Dissociation). 600 When the value reached 0.5 to 0.7, doxycycline diluted with ethanol was added to the culture medium so that the final concentrations were 0, 10, 50, 100, 300, and 500 ng / ml, and the culture was performed 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) at 5 - 6 weeks of age corresponding to a weight of 20 - 30 g were used. MC38 or CT26 of Preparation Example 2 was subcutaneously injected into the flanks of the mice to construct a tumor animal model. For visualization of the tumor animal model and evaluation of tumor size, 2% isoflurane was used for anesthesia, and 200 mg / kg of ketamine and 10 mg / kg of xylazine were used during surgery. The evaluation of the tumor size (mm 3 ) can be calculated using (length × height × width) / 2. When the tumor size of the animal model was 1500 mm 3 or more, the animal model was euthanized.

[0065] [Preparation Example 4] ● Preparation of Experimental Animal Model C57BL / 6 and BALB / C mice (Orient Company, Korea) at 5 - 6 weeks of age corresponding to a weight of 20 - 30 g were used. MC38 or CT26 of Preparation Example 2 was subcutaneously injected into the flanks of the mice to construct a tumor animal model. For visualization of the tumor animal model and evaluation of tumor size, 2% isoflurane was used for anesthesia, and 200 mg / kg of ketamine and 10 mg / kg of xylazine were used during surgery. The evaluation of the tumor size (mm 3 ) can be calculated using (length × height × width) / 2. When the tumor size of the animal model was 1500 mm 3 or more, the animal model was euthanized.

[0066] [Experimental Example 1] Protein Expression and Activity Evaluation of Recombinant Strains [1 - 1] Comparison of Growth between Recombinant Strains and Existing Strains The recombinant strains SLpCR, SLpFR, and SLpFC prepared in Preparation Example 3, along with SLpEmpty used as a control group, were grown overnight in LB liquid medium containing ampicillin. After dilution at a ratio of 1:100 using fresh LB medium, additional culture was performed using OD. 600 When the value reached 0.5-0.7, doxycycline diluted with ethanol to a final concentration of 200 ng / ml was added to the culture medium, and the culture was performed in a shaking incubator at 200 rpm and 37°C. Depending on the culture time, OD 600 The values ​​were measured and the growth patterns of the bacterial strains were analyzed, and the results are shown in Figure 2. As shown in Figure 2, the recombinant SLpCR, SLpFR, and SLpFC strains showed almost no difference in growth rate compared to the control group, the SLpEmpty strain, and did not exhibit any particular growth inhibition during protein expression using doxycycline. Therefore, it was confirmed that the absence of pathogenicity genes in the strains created as described above did not affect the growth and gene expression of the strains.

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

[0068] [1-3] Confirmation of TLR-5 signaling activation by FlaB in recombinant bacterial strains To confirm FlaB expression in recombinant strains, we examined TLR-5 signal activation induced by FlaB. First, as a control group, we measured TLR-5 signal activation in FlaB 40ng and SLpEmpty strains. Then, we cultured the SLpFC recombinant strains produced in Preparation Example 3, separating them into groups without doxycycline (-) and with doxycycline administration (+), and measured TLR-5 signal activation, which is shown in Figure 4. As shown in Figure 4, the SLpFC recombinant strains were found to have activated TLR-5 signaling compared to the control group. In particular, the recombinant strains (+) cultured under doxycycline administration showed significantly higher TLR-5 signaling activation. This confirms that FlaB expression levels in recombinant strains are significantly higher when doxycycline is administered.

[0069] [1-4] Confirmation of FlaB and ClyA expression levels based on doxycycline concentration Western blotting was performed to confirm the FlaB and ClyA expression levels of recombinant strains based on doxycycline concentration. Doxycycline concentrations of 0, 10, 100, 200, 300, and 500 ng / ml were administered to the culture medium of the SLpJH18-FC recombinant strain, and ClyA (34 kDa) and FlaB (43 kDa) secreted by the recombinant strain up to 2 hours after subculturing, which are intermediate log phages, were performed by Western blotting and are shown in Figure 5. As shown in Figure 5, we confirmed that the expression levels of ClyA (34 kDa) and FlaB (43 kDa) increased as the administered doxycycline concentration increased from 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 adjusted by controlling the doxycycline concentration, and that a relatively balanced ratio of their expression and activity can be achieved.

[0070] [Experimental Example 2] Confirmation of the anticancer effect of recombinant bacterial strains (1) To confirm the anticancer effects of recombinant strains, cytotoxicity experiments against CT26 cell lines were conducted in vitro. After treating CT26 cells with the SLpCR, SLpFR, and SLpFC strains prepared in Preparation Example 3, and the SLpEmpty strain used as a control group, the cytoplasmic enzymes of necrotic cell death (LDH) released from damaged CT26 cells were analyzed and 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 remarkable anticancer effect of the SLpFC recombinant strain.

[0071] [Experimental Example 3] Confirmation of the cancer targeting effect of recombinant bacterial strains To confirm the cancer targeting effect of recombinant strains, in vivo experiments were conducted to confirm the number of recombinant strains in the tumor and the targeting image. Attenuated SLpJH18-FC and SLpJH18-FR recombinant strains were introduced into the CT26 mouse model of Preparation Example 4 at 1 × 10⁶ 7 In an experiment in which CFU was administered intravenously and doxycycline was administered orally at 3 dpi daily, while the control group did not receive doxycycline, the number of SLpJH18-FC strains in the tumors was measured and is shown in Figure 7, and the fluorescence image expressed by the SLpJH18-FR strains in the tumors was confirmed and is shown in Figure 8. As shown in Figures 7 and 8, recombinant strains were found in the tumor at a rate of 1 × 10⁶ regardless of doxycycline administration. 8 By confirming the presence of CFUs or more and their specific presence in tumors, we were able to confirm that the recombinant bacterial strain specifically targets cancer cells.

[0072] [Experimental Example 4] Confirmation of the anticancer effect of recombinant bacterial strains (2) To confirm the anticancer effect of the recombinant strain, in vivo experiments were conducted to determine the FlaB and ClyA expression levels of the SLpJH18-FC recombinant strain within tumors. A 1 × 10⁶ attenuated SLpJH18-FC recombinant strain was introduced into the CT26 mouse model of Preparation Example 4. 7 In an experiment in which CFU was administered intravenously and doxycycline was administered orally at 3 dpi daily, while the control group did not receive doxycycline, the amount of FlaB expression in the tumors is shown in Figure 9 and the amount of ClyA expression in the tumors is shown in Figure 10. As shown in Figures 9 and 10, we confirmed that FlaB and ClyA expression levels increased significantly when doxycycline was administered. Therefore, we were able to confirm that FlaB and ClyA expression levels can be adjusted by adjusting the doxycycline dosage after administering the SLpJH18-FC recombinant strain.

[0073] [Experimental Example 5] Confirmation of the anticancer effect of recombinant bacterial strains (3) To confirm the anticancer effect of the recombinant strain, in vivo experiments were conducted using the method shown in Figure 11 to confirm the growth inhibitory effect of the SLpJH18-FC recombinant strain on the CT26 cell line within tumors. A 1 × 10⁶ dose of the attenuated SLpJH18-FC recombinant strain was administered to the CT26 mouse model of Preparation Example 4. 7 In an experiment in which CFU was administered intravenously and doxycycline was administered orally at a dose of 1.7 mg / kg daily, while the control group did not receive doxycycline, tumor size is shown in Figure 12 and mouse survival rate is shown in Figure 13. As shown in Figures 12 and 13, administration of the SLpJH18-FC recombinant strain significantly increased tumor suppression ability and mouse survival rate compared to the control group, thus confirming the anticancer effect of the SLpJH18-FC recombinant strain.

[0074] [Experimental Example 6] Confirmation of the anticancer effect of recombinant bacterial strains (4) To confirm the anticancer effect of the recombinant strain, in vivo experiments were conducted using the method shown in Figure 11 to confirm the growth inhibitory effect of the SLpJH18-FC recombinant strain on the MC38 cell line within tumors. A 1 × 10⁶ dose of the attenuated SLpJH18-FC recombinant strain was administered to the CT26 mouse model of Preparation Example 4. 7 In an experiment in which CFU was administered intravenously and doxycycline was administered orally at a dose of 1.7 mg / kg daily, while the control group did not receive doxycycline, tumor size is shown in Figure 14 and mouse survival rate is shown in Figure 15. As shown in Figures 14 and 15, when the SLpJH18-FC recombinant strain was administered, the tumor suppression ability and survival rate of mice were significantly increased compared to the control group, thus confirming the anticancer effect of the SLpJH18-FC recombinant strain.

[0075] [Experimental Example 7] Confirmation of the cancer recurrence suppression effect of recombinant bacterial strains. To confirm the cancer recurrence-suppressing effect of recombinant bacterial strains, in vivo experiments were conducted using the method shown in Figure 11 to confirm the growth-inhibiting effect of the SLpJH18-FC recombinant bacterial strain on CT26 cell lines within tumors. A 1 × 10⁶ dose of the attenuated SLpJH18-FC recombinant bacterial strain was administered to the CT26 mouse model of Preparation Example 4. 7 After intravenous administration of CFU and daily oral administration of doxycycline at a dose of 1.7 mg / kg to completely treat the cancer, CT26 cells were re-administered 90 days later. The tumor size after re-administration is shown in Figure 16, and the survival rate of the mice is 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 compared to the control group, and the survival rate of the mice increased significantly. Thus, we were able to confirm the cancer recurrence inhibitory effect of the SLpJH18-FC recombinant strain.

[0076] [Experimental Example 8] Confirmation of the cancer metastasis inhibitory effect of recombinant bacterial strains To confirm the cancer metastasis-inhibiting effect of recombinant bacterial strains, in vivo experiments were conducted to confirm the growth inhibitory effect of the SLpJH18-FC recombinant strain on 4T1-Luc cell lines within tumors. A 4T1-Luc cell line expressing luciferase was prepared, and a mouse model was created in which the 4T1-Luc cell line was injected. Subsequently, the attenuated SLpJH18-FC recombinant strain was injected in 1 × 10⁻¹⁶ doses. 7 After intravenous administration of CFU and daily oral administration of doxycycline at a dose of 1.7 mg / kg, the location of the tumors was photographed and shown in Figure 18. The lungs were then removed, and the number of tumors that had metastasized to the lungs was measured and shown in Figure 19. As shown in Figures 18 and 19, when the SLpJH18-FC recombinant strain was administered, it was confirmed that tumors did not metastasize to the lungs compared to the control group, confirming that the SLpJH18-FC recombinant strain had a remarkable effect in suppressing tumor metastasis.

[0077] [Experimental Example 9] Confirmation of the anticancer effect of recombinant bacterial strains (5) To confirm the anticancer effect of recombinant bacterial strains, in vivo experiments were conducted to confirm the abstract effect of SLpJH18-FC recombinant strains on CT26 cell lines within tumors. Mouse models were prepared by injecting CT26 into both thighs. After treating only one side with attenuated SLpJH18-FC recombinant strains, doxycycline was administered orally daily. The size of both tumors was measured and is shown in Figure 20, tumor images were taken and are shown in Figure 21, and the Ki67 levels of mouse T cells were measured and are shown in Figure 22. As shown in Figures 20 and 21, it was found that administering the SLpJH18-FC recombinant strain to only one tumor also had an inhibitory effect on the remaining tumor. Furthermore, as shown in Figure 22, it was found that treatment with the SLpJH18-FC recombinant strain resulted in increased Ki67 levels and activation of epidemic cells, confirming the remarkable anticancer effect of the SLpJH18-FC recombinant strain.

[0078] Through the results described above, it can be seen that in the case of a plasmid containing the promoter of the present invention, the expression of regulatory proteins downstream of the promoter can be regulated, and ultimately the expression levels of anti-oncogenes downstream of the tetA and tetR promoters can be regulated. Although specific parts of the present invention have been described in detail above, it will be clear to those with ordinary skill in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the substantial scope of the invention is defined by the appended claims and their equivalents.

[0079] Through the results described above, it can be seen that in the case of a plasmid containing the promoter of the present invention, the expression of regulatory proteins downstream of the promoter can be regulated, and ultimately the expression levels of anti-oncogenes downstream of the tetA and tetR promoters can be regulated. Although specific parts of the present invention have been described in detail above, it will be clear to those with ordinary skill in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the substantial scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A nucleic acid encoding the TetR protein operably bound downstream of the OXB1 promoter; A nucleic acid encoding flagellin operably bound downstream of a first TetR-inducible promoter; and nucleic acids encoding a toxin protein operably bound downstream of a second TetR-inducible promoter. Includes, The first TetR-inducing promoter is the tetA promoter, and the second TetR-inducing promoter is the tetR promoter. DNA construct.

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

3. The aforementioned toxin proteins include lysine, saporin, geronin, momordin, debouganin, diphtheria toxin, pseudomonas toxin, hemolysin (HlyA), FAS ligand (FASL), tumor necrosis factor-alpha (TNF-alpha), TNF-related apoptosis-inducing ligand (TRAIL), and streptorysin O. The DNA construct according to claim 1, wherein at least one selected from the group consisting of O (SLO), pneumolysin (PLO), listeriolisin (LLO), and cytolysin A (ClyA).

4. A recombinant vector comprising the DNA construct according to any one of claims 1 to 3.

5. A bacterial strain into which the recombinant vector described in claim 4 has been introduced.

6. The strain according to claim 5, wherein the strain is at least one selected from the group consisting of strains of the genus Salmonella, Clostridium, Bifidobacterium, and Escherichia coli.

7. A pharmaceutical composition for the prevention or treatment of cancer, comprising the bacterial strain described in claim 5 as an active ingredient.

8. The pharmaceutical composition according to claim 7, wherein the cancer is at least one selected from the group consisting of melanoma, fallopian tube cancer, brain cancer, small intestine cancer, esophageal cancer, lymph node cancer, gallbladder cancer, hematological cancer, thyroid cancer, endocrine cancer, oral cancer, liver cancer, biliary tract cancer, colorectal cancer, rectal cancer, cervical cancer, ovarian cancer, kidney cancer, gastric cancer, duodenal cancer, prostate cancer, breast cancer, brain tumor, lung cancer, anaplastic thyroid cancer, uterine cancer, colon cancer, bladder cancer, ureteral cancer, pancreatic cancer, bone / soft tissue sarcoma, skin cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and solitary myeloma.

9. The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition inhibits the growth or metastasis of cancer.

10. The strain of bacteria according to claim 5, used for preventing or treating cancer.

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