Salmonella strains for cancer treatment and their uses

Abstract

The present invention relates to a Salmonella strain that selectively acts on cancer for the treatment of cancer, and a composition for preventing or treating cancer containing the same. In particular, the Salmonella strain according to the present invention has a tumor-suppressing effect but significantly low viability in normal organs, and therefore has a more pronounced anti-cancer effect than conventional inventions.

Description

Technical Field

[0001] The present invention relates to a Salmonella strain that selectively acts on cancer and a composition for preventing or treating cancer containing the same.

Background Art

[0002] Cancer is currently one of the diseases that cause the most deaths worldwide, and the cancer incidence rate continues to increase due to the extension of the average life expectancy and the decrease in the cancer onset age. According to the 2013 statistical data provided by the National Cancer Center in Korea, the number of cancer patients registered in the cancer registration statistics section in Korea in 2010 was 202,053 in total, and it shows a continuous increasing trend.

[0003] Therefore, regarding cancer, research on all-round anti-cancer treatments from basic research at the cellular level has been promoted worldwide. However, the cancer onset mechanism is still unclear, it is difficult to prevent recurrence and achieve complete cure, the demand for anti-cancer drugs has increased explosively, and a large amount of research funds have been invested in research institutes and companies. However, the high cost of anti-cancer treatment not only includes direct medical expenses, but also indirect expenses such as the atrophy of social and economic activities after the onset, rehabilitation, and patient care are added, which has become a great economic burden on the families of cancer patients and all members of society, and the introduction of new low-cost technologies is currently required.

[0004] In line with such a situation, since the cancer suppression phenomenon by bacteria was reported about 100 years ago, many researchers have developed strains with anti-cancer effects, and some research groups are already in the clinical stage.

[0005] The cellular environment of cancer tissues, characterized by low oxygen partial pressure, abundant nutrients, and lack of immune response, is also a characteristic of the habitats preferred by pathogenic strains that grow in the body. In particular, the necrotic region located at the center of cancer tissues is known to induce cancer metastasis and other problems as drug delivery is not efficient there. However, Clostridium, Bifidobacterium, Salmonella, etc. accumulate and grow in the abundant nutrients in the necrotic region at the center of cancer tissues. During this process, shrinkage and growth inhibition of cancer tissues have been observed, and they have been used as live strains for cancer treatment since ancient times.

[0006] The research team led by Dr. Xu in China has devised a system to deliver endostatin, an angiogenesis inhibitor, to solid cancer tissues by oral administration of Bifidobacterium longum. Subsequently, they reported on combination therapy research with adriamycin, an anticancer antibiotic (Xu YF, et al., Cancer Gene Ther. 2007, 14(2):1517; Hu B, et al., Cancer Gene Ther. 2009, 16(8):655 - 63).

[0007] In addition, various research teams including the National Cancer Institute (NCI) in the United States have reported the results of Salmonella inhibiting cancer growth, and reported the anticancer effects of attenuated Salmonella recombinant strains carrying anticancer therapeutic substances, including the development of mutant strains with weakened toxicity of Salmonella typhimurium (Nishikawa H, et al., J Clin Invest. 2006, 116(7):1946 - 54, Clairmont, C.et al.J.Infect.Dis.2000, 181:1996 - 2002.Nguyen VH, et al., Cancer Res. 2010, 1;70(1):18 - 23, Kim K, et al., PLoS One. 2013, 8(3):e60511.).

[0008] However, anticancer treatment using live bacterial strains has been a concern due to the long-term persistence in normal tissues outside the tumor tissue and the potential for side effects caused by the strains. In particular, the production of a potent anticancer therapeutic substance from recombinant Salmonella for tumor treatment may cause tissue damage by Salmonella remaining in normal tissues. Also, during chronic infection by residual Salmonella that remains without being removed after tumor cell death, there is a risk of infection to other people in the vicinity as carriers, and in patients with weakened immune function or the elderly, there is a possibility of fatal sepsis caused by Salmonella with weakened pathogenicity. Antibodies generated during chronic infection may cause autoimmune diseases, for example, arthritis, uveitis, urethritis, etc. Therefore, in the development of anticancer treatment technology using Salmonella, in order to enhance the specificity of Salmonella growth against tumor tissue and minimize excessive immune responses and resulting side effects in normal organs, it is necessary to develop a strain that can efficiently regulate the viability of Salmonella in normal tissues other than tumor tissue and can be rapidly removed during Salmonella infection in normal organs. Mutant strains lacking Salmonella pathogenicity genes have been reported to regulate the attenuated virulence and viability of Salmonella. Inactivation of SPI-1 (Salmonella pathogenicity island-1) or SPI-2 (Salmonella pathogenicity island-2), which are Salmonella pathogenicity gene clusters, has shown the effect of reducing the pathogenicity of Salmonella gallinarum (Patent 1015008050000), and additional deletion of a single gene belonging to the pathogenicity gene cluster in attenuated Salmonella rapidly decreased the viability of the mutant strain in the spleen. (Patent 1017735800000)

[0009] However, in Salmonella gallinarum, the lack of the SPI-1 or SPI-2 gene clusters results in a decrease in the invasion efficiency in avian epithelial cells and non-pathogenicity, making it impossible to confirm the viability of residual Salmonella in normal tissues. The decrease in the viability of Salmonella in the spleen due to the lack of a single gene belonging to the pathogenic gene cluster in attenuated Salmonella is considered to be an effect of increasing the immune response sensitivity to Salmonella by the spleen, which is an immune organ composed of lymphocytes and various immune cells, and it is difficult to think that the viability of Salmonella in all normal tissues in the body other than immune organs can be rapidly regulated.

[0010] Therefore, as a result of the efforts of the present researchers to efficiently regulate the viability of Salmonella in other normal tissues rather than tumor tissues and develop Salmonella strains with excellent anti-cancer effects, it was confirmed that by additionally lacking the Salmonella pathogenic gene cluster in previously known attenuated Salmonella strains, the viability in normal organs decreased and tumor growth was significantly suppressed, verifying that the strain of the present invention can be usefully used as an active ingredient of an anti-cancer composition, and thus the present invention was completed.

Summary of the Invention

Problems to be Solved by the Invention

[0011] One object of the present invention relates to a mutant strain of the genus Salmonella with a gene deletion and a method for producing the same.

[0012] Another object of the present invention is to provide a composition for preventing, improving or treating cancer using the mutant strain of the genus Salmonella provided by the present invention.

[0013] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those with ordinary knowledge in the art from the following description.

Means for Solving the Problems

[0014] Hereinafter, various embodiments described in the present application will be described with reference to the drawings. In the following description, for a complete understanding of the present invention, various specific details, such as specific forms, compositions, and processes, are described. However, a particular embodiment can be practiced without one or more of these specific details, or in combination with other known methods and forms. In other instances, well-known processes and manufacturing techniques are not described as specific details so as not to unnecessarily obscure the present invention. References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearances of "in one embodiment" or "an embodiment" in various places throughout this specification are not necessarily referring to the same embodiment of the present invention. Additionally, the particular features, forms, compositions, or characteristics are combined in any suitable manner in one or more embodiments.

[0015] Unless otherwise defined within the present invention, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0016] According to one embodiment of the present invention, it relates to a mutant strain of the genus Salmonella lacking Salmonella pathogenicity island-1 (SPI-1) and Salmonella pathogenicity island-2 (SPI-2).

[0017] In the present invention, the Salmonella pathogenicity island-1 (SPI-1) is a specific site within the Salmonella genome that has the entire group of genes of the type III secretion system and virulence proteins necessary for the invasion of intestinal epithelial cells at the early stage of Salmonella infection (Kimbrough TG et al, Microbes Infect, (2002); 4(1): 75-82).

[0018] In the present invention, the Salmonella pathogenicity island-1 (SPI-1) may consist of the nucleotide sequence represented by SEQ ID NO: 1.

[0019] In the present invention, Salmonella pathogenicity island-2 (SPI-2) is a specific genetic locus that includes response-phase proteins involved in the mechanism that enables Salmonella to survive without lysing within phagosomes after being phagocytosed by macrophages in immune organs such as intestinal immune organs, spleen, and liver after passing through epithelial cells, and a gene cluster of a type III secretion system that secretes these proteins (Waterman SR et al, Cell Microbiol, (2003); 5(8):501-511, Abrahams GL, Cell Microbiol, (2006); 8(5):728-737). The type III secretion system (TTSS) is a method of transmitting pathogenic substances developed in Gram-negative bacteria, and is a syringe-shaped instrument that injects pathogenic active proteins directly into the cytoplasm through the host cell membrane (Mota LJ et al, Ann Med. (2005); 37(4):234-249), and is a multi-protein complex structure. In the infection pathway of Salmonella, the process of transmitting pathogenic substances by the type III secretion system is known to be very important (Schlumberger MC et al, Curr Opin Microbiol, (2006); 9(1):46-54). Wild-type Salmonella adheres to and invades host intestinal epithelial cells using TTSS proteins, survives during the phagocytosis of macrophages, and in some cases, shows the phenomenon of causing systemic infection along the bloodstream. Therefore, when the TTSS proteins function normally, Salmonella infection progresses.

[0020] In the present invention, the Salmonella pathogenicity island-2 (SPI-2) may consist of the nucleotide sequence represented by SEQ ID NO: 2.

[0021] In the present invention, the mutant strain of the genus Salmonella may additionally have a gene encoding an anticancer protein introduced therein.

[0022] The "anticancer protein" of the present invention is a peptide having a function of directly or indirectly inducing the death of cancer cells, and is, for example, at least one selected from the group consisting of a toxin protein, an antibody specific for 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.

[0023] The "toxin protein" of the present invention is a protein having a function of directly or indirectly inducing the death of cancer cells, and is, for example, at least one selected from the group consisting of ricin, saporin, gelonin, momordin, debouganin, diphtheria toxin, pseudomonas toxin, HlyA, FAS ligand (FASL), tumor necrosis factor-α (TNF-α), TNF-related apoptosis-inducing ligand (TRAIL), and cytolysin A (ClyA), and more preferably may be cytolysin A, but is not limited thereto.

[0024] In the present invention, the introduction of the cytolysin A gene may be carried out by a recombinant vector, but is not limited thereto.

[0025] In the present invention, the recombinant vector may be prepared using the forward primer represented by SEQ ID NO: 13 and the reverse primer represented by SEQ ID NO: 14.

[0026] In the present invention, the cytolysin A (ClyA) gene encodes a protein that forms holes in the 34-kDa cell membrane and lyses cells, and is expressed in Escherichia coli, Salmonella, etc. The sequence of the cytolysin A is described in Wai SN, Lindmark B, Soderblom T, et al. Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin. Cell 2003;115:25-35, but is not limited thereto, and those derived from various prokaryotic cells and their functional variants can be used in the present application. In one embodiment, the protein accession number of cytolysin A (ClyA) is ABI83833.1, and the gene sequence is DQ910780.1. The cytolysin A gene may consist of the nucleotide sequence represented by SEQ ID NO: 15.

[0027] The "tumor suppressor protein" of the present invention is a gene that exists in normal cells and maintains its function, but when its function is lost, normal cells are converted into cancer cells by inducing undifferentiated cell division and growth. For example, it may be an RB (Retinoblastoma protein), p53 protein, APC (Adenomatous polyposis coli) protein, PTEN (Phosphatase and tensin homologue) protein, CDKN2A (cyclin dependent kinase inhibitor 2A) protein, etc., but is not limited thereto.

[0028] The antibody specific to the cancer antigen of the present invention or a fragment of the antibody is an antibody capable of specifically binding to an antigen which is a protein with a particularly high expression level specifically on the surface or in the cytoplasm of cancer cells. For example, it may be an antibody specific to HER2 or the like which has a particularly high expression level specifically in breast cancer or gastric cancer cells, but is not limited thereto.

[0029] The antibody of the present invention means a protein molecule capable of specifically binding to an antigenic site of a protein or peptide molecule. The form of the antibody is not particularly limited, and as long as it is a polyclonal antibody, a monoclonal antibody or one having antigen-binding properties, even in some cases of the antibody, it is included, and all types of immunoglobulin antibodies may be included. Further, special antibodies such as humanized antibodies are included, and the antibody includes not only a 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 means a fragment having at least an antigen-binding function, and may be Fab, F(ab’), F(ab’)2, Fv or the like, but is not limited thereto.

[0030] The “antibody” of the present invention can be produced by a conventional method after cloning the 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.

[0031] The “angiogenesis inhibitor” of the present invention means a protein or compound having a function of suppressing the generation of new blood vessels around cancer cells and directly or indirectly inducing the death of cancer cells. Preferably, the angiogenesis inhibitor may be angiostatin, endostatin, thrombospondin, a protease inhibitory protein or the like, but is not limited thereto.

[0032] The "cancer antigen" of the present invention refers to a protein that is expressed in cancer cells but hardly expressed in normal cells, and uses this as an antigen to induce an anti-tumor immune reaction, thereby inducing the direct or indirect death of cancer cells. The cancer antigen of the present invention is preferably alpha-fetoprotein (α-fetoprotein; AFP), vascular endothelial growth factor receptor 2 (Vascular endothelial growth factor receptor 2; VEGFR2), survivin, legumain, prostate cancer specific antigen (Prostate cancer specific antigen; PCSA), etc., but is not limited thereto.

[0033] The "prodrug-converting enzyme" of the present invention is a protein having a function of converting an inactive drug into an active drug by metabolism through an enzymatic reaction. When such a prodrug-converting enzyme is used, the inactive drug is metabolized and converted into an active drug that can directly or indirectly induce the death of cancer cells, so that it can be very usefully used for the prevention or treatment of cancer. The prodrug-converting enzyme of the present invention is preferably thymidine kinase, cytosine deaminase, nitroreductase, purine nucleoside phosphorylase, carboxypeptidase G2, chromate reductase YieF, herpes simplex virus type I thymidine kinase / ganciclovir (HSV1-TK / GCV), β-glucuronidase, etc., but is not limited thereto.

[0034] The "pro-apoptotic protein" of the present invention refers to a protein that induces the direct or indirect death of cancer cells by depriving cancer cells of essential factors (such as proteins, nutrients, oligonucleotides, etc.) for their growth or maintenance. The pro-apoptotic protein of the present invention may preferably be, but is not limited to, L-asparaginase (L-ASNase), RNA-binding motif protein 5 (RBM5), etc.

[0035] In the present invention, the mutant strain of the genus Salmonella further contains a reporter gene, and the reporter gene enables imaging of the mutant strain of the genus Salmonella.

[0036] The reporter gene encodes a protein capable of in vivo or ex vivo (in vitro) imaging, and includes bioluminescence genes, chemiluminescence genes, and fluorescence genes derived from various organisms.

[0037] In the present invention, the luminescence gene may be a luciferase gene derived from prokaryotic or eukaryotic cells. The luciferase gene derived from prokaryotic cells may be a bacterial luciferase gene (lux). The luciferase gene derived from eukaryotic cells may be a luminescence gene derived from firefly, but is not limited thereto. The gene may exist in the form of a plasmid or inserted into the genome of Salmonella.

[0038] In the present invention, the luminescence gene may exist in the form of a plasmid or inserted into the genome of Salmonella.

[0039] In the present invention, the mutant strain of the genus Salmonella may be derived from at least one selected from the group consisting of Salmonella typhimurium, Salmonella choleraesuis, Salmonella enteritidis, Salmonella infantis, Salmonella paratyphi, and Salmonella typhi. The listed Salmonella strains are Gram-negative facultative anaerobic bacilli. They are distinguished from Escherichia coli because they lack the ability to decompose lactose, do not form indole, and do not produce hydrogen sulfide. The Salmonella strains may have peritrichous flagella and be motile.

[0040] Salmonella typhimurium is a causative bacterium of enteric typhoid of the genus Salmonella. Salmonella typhimurium is a rod-shaped bacillus with a single flagellum and is Gram-negative. Salmonella typhimurium is heat-sensitive and dies at 60°C in 20 minutes. It can be primarily contaminated by livestock, wild animals, carriers, etc., as well as milk, eggs, etc., and can cause secondary infections due to contaminated growth, etc., such as salad, and may induce Salmonellosis, a type of food poisoning.

[0041] The Salmonella choleraesuis is a bacterium well-known as the swine cholera bacterium of the genus Salmonella, and it infects both humans and animals. The Salmonella choleraesuis is the main causative bacterium of acute septicemia caused by Salmonella. This bacterium is a Gram-negative facultative anaerobic bacillus with peritrichous flagella and motility. It is distinguished from Escherichia coli because it lacks the ability to decompose lactose, does not form indole, and does not produce hydrogen sulfide. The optimum temperature for growth is 35 - 37°C, the temperature range in which it can grow is 10 - 43°C, and it is killed by heating at 60°C for 20 minutes. The optimum pH is 7.2 - 7.4, and its size is 0.5 - 0.8 × 3 - 4 μm.

[0042] The Salmonella enteritidis is the causative bacterium of bacterial infectious food poisoning of the genus Salmonella, and is also called enteritis bacterium. The Salmonella enteritidis is a representative bacterium of Salmonella, and it may infect all animals and has a very high host adaptability. It is a Gram-negative facultative anaerobic bacillus with peritrichous flagella and motility. It is distinguished from Escherichia coli because it lacks the ability to decompose lactose, does not form indole, and does not produce hydrogen sulfide. The optimum temperature for growth is 35 - 37°C, the temperature range in which it can grow is 10 - 43°C, and it is killed by heating at 60°C for 20 minutes. The optimum pH is 7.2 - 7.4, and its size is 0.5 - 0.8 × 3 - 4 μm.

[0043] The Salmonella infantis is a strain that infects through eggs or poultry meat, and Salmonella paratyphi and Salmonella typhi are the causative strains of enteric typhoid.

[0044] In the present invention, the mutant strain of the genus Salmonella may be a mutant lacking the ability to synthesize guanosine polyphosphate. The guanosine polyphosphate may be guanosine-5'-diphosphate-3'-diphosphate (ppGpp).

[0045] In the present invention, the mutant strain of the genus Salmonella may be one in which at least one of the Salmonella relA gene (encoding ppGpp synthase) and the spoT gene (Salmonella-spoT) is inactivated.

[0046] In the present invention, the Salmonella genus strain lacking the gene having ppGpp synthase activity exhibits a tumor-suppressing effect, but also shows significant viability in normal organs and tissues, and there may be an infection caused by Salmonella. In addition, a strain in which only the pathogenic gene of Salmonella is deleted also maintains pathogenicity in normal organs and has little potential for use as a therapeutic strain. However, as in the present invention, a Salmonella genus strain in which the SPI-1 and SPI-2 genes are additionally deleted simultaneously in the Salmonella genus strain in which the ppGpp synthase activity gene is inactivated has extremely low viability of the strain in normal organs, and not only is there no infection caused by Salmonella, but also has the merit that the anti-cancer effect is significantly superior to that of the Salmonella genus strain lacking the gene having ppGpp synthase activity.

[0047] In the present invention, the mutant strain of the genus Salmonella may be derived from SHJ2037 (deposit number KCTC 10787BP).

[0048] In the present invention, the term "derived from" means an existing plasmid or strain before the recombinant plasmid or recombinant strain undergoes the gene recombination process.

[0049] For the deletion and inactivation of genes of the mutant strain of the genus Salmonella of the present invention, a recombinant vector can be used in a Salmonella host cell. The recombinant vector may be, but is not limited to, a plasmid. The plasmid may be introduced into a Salmonella strain by an electroporation method, but is not limited thereto. The electroporation method is a method of applying an electric shock to a protoplast so that the cell membrane can accept DNA. When an electric stimulus of a high voltage is applied to cells at regular intervals, small holes are opened in the cell membrane. At this time, DNA is inserted into the cells through these holes by the action of electrophoresis.

[0050] The mutant strain of the genus Salmonella of the present invention can be used for the prevention or treatment of cancer.

[0051] In the present invention, the "cancer" is a disease characterized by the rapid and uncontrolled growth of variant cells, and is at least one selected from the group consisting of melanoma, ovarian cancer, brain cancer, small intestine cancer, esophageal cancer, lymphoma, gallbladder cancer, blood cancer, thyroid cancer, endocrine adenocarcinoma, 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, undifferentiated 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, 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, undifferentiated thyroid cancer, uterine cancer, colon cancer, bladder cancer, ureteral cancer, pancreatic cancer, bone / soft tissue sarcoma, and skin cancer, and more preferably, it may be colorectal cancer, but is not limited thereto.

[0052] The mutant strain of the genus Salmonella of the present invention may have lower viability in normal tissues than in tumor tissues. Here, the normal tissue may be, but is not limited to, the tissue of an organ selected from the group consisting of the lung, liver, and spleen.

[0053] The mutant strain of the genus Salmonella of the present invention has extremely low viability in normal tissues, and not only is there no infection by Salmonella, but it also has the merit of having a significantly superior anti-cancer effect compared to conventional inventions.

[0054] In addition, if the mutant strain of the genus Salmonella is attenuated by two or more different mutations in the genome of the mutant strain of the genus Salmonella of the present invention, it is known that the probability of the mutant strain of the genus Salmonella reverting to the parental strain is extremely low and it is safe.

[0055] According to still another embodiment of the present invention, there is provided a method for producing a mutant strain of the genus Salmonella, comprising the step of obtaining a strain transformed by removing the Salmonella pathogenicity island-1 (SPI-1) and Salmonella pathogenicity island-2 (SPI-2) genes of a Salmonella genus strain.

[0056] The Salmonella pathogenicity island-1 (SPI-1) of the present invention may consist of the nucleotide sequence represented by SEQ ID NO: 1.

[0057] The Salmonella pathogenicity island-2 (SPI-2) of the present invention may consist of the nucleotide sequence represented by SEQ ID NO: 2.

[0058] In the present invention, the removal of the genes of Salmonella pathogenicity island-1 (SPI-1) and Salmonella pathogenicity island-2 (SPI-2) may be carried out by a recombinant vector, but is not limited thereto.

[0059] In the present invention, the recombinant vector may be prepared using the forward primer represented by SEQ ID NO: 7 and the reverse primer represented by SEQ ID NO: 8.

[0060] The recombinant vector may be derived from the pKD13, pCP20 or pJL39 plasmid, but is not limited thereto.

[0061] The pKD13 consists of 3434 bp and contains beta-lactamase, rrnB transcription terminator, priming site 1, natural FRT site, common priming site kt, Tn5 neomycin phosphotransferase, common priming site k2, common priming site k1, distal 35-nt of natural FRT site, priming site 4, lambda terminator and R6K gamma replication origin gene.

[0062] The pCP20 plasmid consists of 9497 bp and is composed of EcoRI, cat, Pstl, HindIII Ci857, flp, bamHi, beta-lactamase, mobA, mob2 and repA101ts genes.

[0063] The manufacturing method of the present invention can further include the step of introducing a gene encoding an anticancer protein.

[0064] In the present invention, the anticancer protein may be at least one selected from the group consisting of a toxin 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.

[0065] The toxin protein of the present invention may be at least one selected from the group consisting of ricin, saporin, gelonin, momordin, debouganin, diphtheria toxin, pseudomonas toxin, HlyA, FAS ligand (FASL), tumour necrosis factor-α (TNF-α), TNF-related apoptosis-inducing ligand (TRAIL), and cytolysin A (ClyA), and more preferably may be cytolysin A, but is not limited thereto.

[0066] In the present invention, the introduction of the cytolysin A gene may be performed by a recombinant vector, but is not limited thereto. The vector used for the introduction of the cytolysin A gene may be the same as or different from the vector used for the removal of the SP-1 and SP-2 genes.

[0067] In the present invention, the recombinant vector may be prepared using the forward primer represented by SEQ ID NO: 13 and the reverse primer represented by SEQ ID NO: 14.

[0068] In the present invention, the introduction of the plasmid among the recombinant vectors may be by electroporation, but is not limited thereto.

[0069] In the present invention, the step of introducing the cytolysin A (ClyA) gene may be performed before or after the removal of the genes of Salmonella pathogenicity island-1 (SPI-1) and Salmonella pathogenicity island-2 (SPI-2), and the order of execution is not particularly limited.

[0070] In the present invention, the Salmonella strain may be derived from at least one selected from the group consisting of Salmonella typhimurium, Salmonella choleraesuis, Salmonella enteritidis, Salmonella infantis, Salmonella paratyphi, and Salmonella typhi.

[0071] In the present invention, the Salmonella strain may be a mutant Salmonella strain lacking guanosine polyphosphate synthesis ability.

[0072] In the present invention, the Salmonella strain may be one in which the Salmonella-relA gene or spoT (Salmonella-spoT) gene encoding ppGpp synthase is inactivated.

[0073] The production method of the present invention can further include the step of culturing the strain transformed as described above.

[0074] In the present invention, the culturing step is carried out using LB medium (Lysogeny broth) containing an antibiotic. The LB medium was developed by Giuseppe Bertani to optimize the growth of Shigella - genus strains and plaque formation. General components include peptides, casein peptone, vitamins (including B vitamins), trace elements (nitrogen, sulfur, magnesium), and minerals. The osmotic pressure is adjusted with sodium chloride.

[0075] The production method of the present invention can further include the step of culturing the strain transformed as described above in an antibiotic medium to select mutant strains.

[0076] In the present invention, the antibiotic may be, but is not limited to, kanamycin or tetracycline.

[0077] Regarding the content of the Salmonella - genus mutant strain, the anti - cancer protein, the Salmonella - genus strain, and the recombinant vector of the present invention, it is the same as that described in the above composition and is omitted to avoid excessive complexity of the specification.

[0078] According to still another embodiment of the present invention, it relates to a pharmaceutical composition for preventing or treating cancer, which contains the Salmonella - genus mutant strain provided by the present invention as an active ingredient.

[0079] In the present invention, the "cancer" is a disease characterized by the rapid and uncontrolled growth of variant cells, and may be at least one selected from the group consisting of melanoma, fallopian tube cancer, brain cancer, small intestine cancer, esophageal cancer, adenocarcinoma of the lymph nodes, gallbladder cancer, blood cancer, thyroid cancer, endocrine adenocarcinoma, 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, undifferentiated 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. Preferably, it may be 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, undifferentiated thyroid cancer, uterine cancer, colon cancer, bladder cancer, ureteral cancer, pancreatic cancer, bone / soft tissue sarcoma, and skin cancer. More preferably, it may be colon cancer, but is not limited thereto.

[0080] The "prevention" of the present invention can include, without limitation, all acts of blocking the symptoms caused by cancer or suppressing or delaying those symptoms using the composition of the present invention.

[0081] The "improvement" or "treatment" of the present invention can include, without limitation, all acts in which the symptoms caused by cancer improve or become advantageous using the composition of the present invention.

[0082] The pharmaceutical composition of the present invention is characterized by being in the form of capsules, tablets, granules, injections, ointments, powders, or beverages, and is characterized by targeting humans.

[0083] The pharmaceutical composition of the present invention is not limited to these, but can be formulated into oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, etc., external preparations, suppositories, and sterile injection solutions by ordinary methods respectively. The pharmaceutical composition of the present invention can contain a pharmaceutically acceptable carrier. When orally administered, the pharmaceutically acceptable carrier can include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, etc. In the case of injections, buffers, preservatives, soothing agents, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used. In the case of topical administration, bases, excipients, lubricants, preservatives, etc. can be used. The dosage form of the pharmaceutical composition of the present invention can be variously manufactured by mixing with the pharmaceutically acceptable carrier as described above. For example, when orally administered, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. In the case of injections, it can be manufactured in unit-dose ampoules or multiple-dose forms. In addition, it can be formulated into solutions, suspensions, tablets, capsules, sustained-release preparations, etc.

[0084] Examples of carriers, excipients, and diluents suitable for the formulation of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil, etc. Additionally, fillers, anticoagulants, lubricants, wetting agents, flavors, emulsifiers, preservatives, etc. can be included.

[0085] The administration routes of the pharmaceutical composition of the present invention are not limited to these, but include oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal. Oral or parenteral administration is preferred.

[0086] As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intracapsular and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention can also be administered in the form of a suppository for rectal administration.

[0087] The pharmaceutical composition of the present invention can vary widely depending on various factors including the activity of the specific compound used, age, weight, general health, sex, formulation, time of administration, route of administration, excretion rate, drug combination, and severity of the particular disease to be prevented or treated. The dosage of the pharmaceutical composition varies depending on the patient's condition, weight, degree of disease, drug form, route of administration, and duration, but can be appropriately selected by those skilled in the art and can be administered at 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 times. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention can be formulated into tablets, coated tablets, capsules, solutions, gels, syrups, slurries, and suspensions.

[0088] In still another embodiment of the present invention, there is provided a composition for diagnosing cancer containing a mutant strain of the genus Salmonella as an active ingredient.

[0089] The diagnostic composition of the present invention contains the strain according to the present invention as an active ingredient.

[0090] In the strain of the present invention, after the mutant strain according to the present invention targets cancer cells in an individual, a reporter protein capable of real-time imaging is expressed in the strain, so that cancer can be diagnosed in real time without noise signals for normal tissues.

[0091] As used herein, the "reporter protein" 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.

[0092] The "fluorescent protein" of the present invention is a protein that exhibits fluorescence by itself so that cancer can be visually diagnosed, and is, 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.

[0093] The protein used in the nuclear medicine or MRI imaging of the present invention is, for example, at least one selected from the group consisting of Herpes simplex virus thymidine kinsease, Dopamine receptor, Somatostatin receptor, Sodium-iodide transporter, iron receptor, Transferrin receptor, Ferritin, and iron transporter (magA), but is not limited thereto.

[0094] The "diagnosis" of the present invention means all acts of confirming an in-vivo cancer tissue, including the ability to monitor in real time the presence or absence of cancer by a reporter protein introduced into a vector introduced into the strain or into a chromosome when the strain of the present invention is located targeting cancer.

[0095] In the diagnostic composition of the present invention, the content related to the mutant strain, vector, transformation, cancer, etc. is the same as that described above, and is omitted to avoid excessive complexity of this specification.

[0096] In still another embodiment of the present invention, there is provided a method for providing information for diagnosing cancer, including the step of treating a mutant strain of the genus Salmonella.

[0097] The method of the present invention includes the step of treating a biological sample isolated from a target individual with the mutant strain according to the present invention.

[0098] The method for providing information for diagnosing the cancer of the present invention may further include the step of diagnosing cancer when a reporter protein is expressed from the strain.

[0099] The "biological sample" of the present invention means any substance, tissue or cell obtained from an individual or derived from an individual, and can include, for example, tissue, cell, or cell extract, but is not limited thereto.

[0100] In the method for providing information for the diagnosis of the present invention, the content related to the mutant strain, anti-cancer protein, reporter protein, vector, strain, transformation, cancer, diagnosis, etc. is the same as that described in the pharmaceutical composition for preventing or treating the reporter protein, vector, strain, cancer, and is omitted to avoid excessive complexity of this specification.

[0101] In still other embodiments of the present invention, it relates to a method for preventing or treating cancer, which includes the step of administering to an individual an effective amount of a mutant strain of the genus Salmonella provided by the present invention.

[0102] In the present invention, the "individual" refers to an individual in need of cancer prevention or treatment, and can include all mammals and non-mammals. Here, examples of the mammals can include, but are not limited to, humans, non-human primates, such as chimpanzees, other apes or monkey species; livestock animals, such as cows, horses, sheep, goats, pigs; domestic animals, such as rabbits, dogs or cats; laboratory animals, such as rodents, such as rats, mice or guinea pigs, etc. Also, in the present invention, examples of the non-mammals can include, but are not limited to, birds or fish.

[0103] In the present invention, the "administration" means the process of introducing the active ingredient of the present invention into an individual by any suitable method. The preparation of the mutant strain of the genus Salmonella administered as described above is not particularly limited, and it may be administered in a solid-form preparation, a liquid-form preparation or an aerosol preparation for inhalation, or it may be administered in a solid-form preparation intended to be converted into a liquid-form preparation for oral or parenteral administration immediately before use. For example, it may be formulated and administered in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories and sterile injection solutions, but is not limited thereto.

[0104] In addition, in the present invention, at the time of administration, a pharmaceutically acceptable carrier can be additionally administered together with the mutant strain of the genus Salmonella of the present invention. Here, as the pharmaceutically acceptable carrier, when administered orally, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment, a flavor, etc. can be used. In the case of an injection, a buffer, a preservative, a soothing agent, a solubilizer, an isotonic agent, a stabilizer, etc. can be mixed and used. In the case of topical administration, a base, an excipient, a lubricant, a preservative, etc. can be used. The dosage form of the mutant strain of the genus Salmonella of the present invention can be variously produced by mixing with the pharmaceutically acceptable carrier as described above. For example, when administered orally, it can be produced in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. In the case of an injection, it can be produced in a unit dosage ampoule or a multiple dosage form. In addition, it can be formulated into solutions, suspensions, tablets, capsules, sustained release preparations, etc.

[0105] 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, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil, etc. can be used. In addition, a filler, an anticoagulant, a lubricant, a wetting agent, a flavor, an emulsifier, a preservative, etc. can be additionally included.

[0106] The route of administration of the mutant strain of the genus Salmonella according to the present invention is not limited to these, but includes oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal. Oral or parenteral administration is preferred.

[0107] In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention can further be administered in the form of suppositories for rectal administration.

[0108] In the present invention, "effective amount" refers to a sufficient amount of an agent to provide a preferred biological result. The said result may be a reduction and / or alleviation of the signs, symptoms or causes of a disease, or any other preferred change in the biological world. For example, an "effective amount" for therapeutic use is the amount of the mutant strain of the genus Salmonella disclosed in the present invention required to provide a clinically significant reduction in a disease. The appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation. Accordingly, the expression "effective amount" generally refers to the amount at which an active substance has a therapeutic effect. In the case of the present invention, the active substance is an agent for cancer prevention, improvement or treatment.

[0109] The mutant strain of the genus Salmonella of the present invention can vary widely depending on various factors including the activity of the specific compound used in combination, age, body weight, general health, sex, formulation, administration time, administration route, excretion rate, drug combination and the severity of the specific disease to be prevented or treated. The dosage of the mutant strain of the genus Salmonella varies depending on the patient's condition, body weight, degree of disease, drug form, administration route and duration, but can be appropriately selected by one of ordinary skill in the art and can be administered at 0.0001 to 100 mg / kg or 0.001 to 100 mg / kg per day. The administration may be once a day or divided into several times. The said dosage does not limit the scope of the present invention in any way. The mutant strain of the genus Salmonella according to the present invention can be formulated into tablets, dragees, capsules, solutions, gels, syrups, slurries, suspensions.

[0110] In the present invention, the description regarding the cancer overlaps with that described above, and in order to avoid excessive complexity of the specification, the detailed description thereof is omitted below.

[0111] The mutant strain of the genus Salmonella of the present invention can be used alone or in combination with methods using surgery, radiotherapy, hormone therapy, chemotherapy, and biological response modifiers.

Advantages of the Invention

[0112] The mutant strain of the genus Salmonella lacking the gene according to the present invention has very low viability in normal tissues, but effectively induces the death of cancer cells while surviving in cancer tissues, and has the merit of having no side effects of infection by the genus Salmonella together with an excellent anti-cancer effect.

Brief Description of the Drawings

[0113]

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Mode for Carrying Out the Invention

[0114] According to one embodiment of the present invention, it relates to a mutant strain of the genus Salmonella lacking Salmonella pathogenicity island-1 (SPI-1) and Salmonella pathogenicity island-2 (SPI-2).

Example

[0115] Hereinafter, the present invention will be described in more detail through examples. These examples are merely for explaining the present invention more specifically, and it will be apparent to those with ordinary knowledge in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

[0116] [Preparation Example 1] Preparation of Attenuated Salmonella The attenuated Salmonella of the present invention used the SHJ2037 strain as a template, and pathogenicity-related genes or gene groups were deleted by homologous recombination using lambda phage to prepare an attenuated Salmonella strain.

[0117] Specifically, a DNA fragment carrying a Kan cassette at the position of the open reading frame of each gene was PCR amplified using the pKD13 plasmid. The PCR primer sequences used for the removal of a total of three genes (groups) are as shown in Table 1 below. The PCR product, pKD46, was introduced into a suspension of Salmonella typhimurium SMR2130 (SHJ2037, DrelA, DspoT) containing pKD46 intracellularly by electroporation and cultured in a kanamycin medium to select colonies. Thereafter, the pCP20 plasmid (Datsenko KA, et al., Proc Natl Acad Sci US A. 2000, 6; 97(12):6640-5) was introduced into the transformed strain by electroporation, and the kan cassette was removed by FLP recombinase to prepare a strain.

[0118]

Table 1

[0119] [Preparation Example 2] Preparation of Recombinant Salmonella for Gene Expression Figure 1 schematically shows a method for preparing the pJH18 plasmid. Using the pJL39 plasmid (Mol Ther., 21(11), p. 1985 - 1995, (2013)) as a template strand, a forward primer (5’-CGGAATTCACCATGTCTAGATTAGATAAAAGTAAAGTGATTAACAG-3’; SEQ ID NO: 9) prepared to contain a restriction enzyme EcoRI site and a reverse primer (5’-GCTCTAGACAGCTGTTAAGACCCACTTTCACATTTAAGTTGTTTTTCT-3’; SEQ ID NO: 10) prepared to contain a restriction enzyme PvuII - XbaI site were used to amplify the tetR gene. Thereafter, restriction enzymes EcoRI and XbaI were added to the amplification product for cleavage, which was purified to obtain a tetR gene amplification product, and then a pBAD - TetR plasmid was prepared through the process of introducing this into the pBAD24 (catalog number, ATCC® 87399, ATCC, USA) plasmid.

[0120] Thereafter, through the PvuII and HindIII fragments of the pJL39 plasmid, a Divergent promoter region containing a multiple cloning site was introduced into the pBAD - TetR plasmid to prepare a pTetR - BAD plasmid. The araC and araBAD promoters were removed from the pTetR - BAD plasmid using NheI and Pcil restriction enzymes to prepare a pTetII plasmid.

[0121] Also, using pSF-OXB1 (Oxford Genetics, England) as a template, the constitutive promoter OXB1 amplified with a forward primer (5’-CTACTCCGTCAAGCCGTCAAGCTGTTGTGACCGCTTGCT-3’; SEQ ID NO: 11) and a reverse primer (5’-TGAATTCCTCCTGCTAGCTAGTTGGTAACGAATCAGACGCCGGGTAATACCGGATAG-3’; SEQ ID NO: 12) was introduced into the pTetII plasmid using the Gibson assembly method, finally producing the pJH18 plasmid containing OXB1, tetA, and the tetR promoter.

[0122] Using the pJH18 plasmid as a template, the pJH18-CR plasmid was produced by introducing genes encoding Rluc8 and cytolysin A (ClyA) in the combination as shown in Figure 1 downstream of the promoter. At this time, the forward primer used for introducing the cytolysin A (ClyA) gene was 5’-AGTCCATGGTTATGACCGGAATATTTGC-3’ (SEQ ID NO: 13), and the reverse primer was 5’-GATGTTTAAACTCAGACGTCAGGAACCTC-3’ (SEQ ID NO: 14).

[0123] [Preparation Example 3] Preparation of Transformed Salmonella Mutant Strains The Salmonella strains prepared in Preparation Example 1 were transformed with the plasmids prepared in Preparation Example 2 using the electroporation method, and then each of the transformed strains was cultured overnight using an LB solid medium containing 100 μg / ml of ampicillin. Subsequently, the generated colonies were cultured in an LB liquid medium containing ampicillin and used for the experiment. Summarizing the transformed contents in the thus-prepared Salmonella mutant strains is as shown in Table 2 below.

[0124]

Table 2

[0125] [Experimental Example 1] Expression and Activity Evaluation of Recombinant Salmonella Proteins [1-1] Growth Comparison between Salmonella Mutant Strains and Existing Strains After culturing the recombinant SHJ2037 and CNC18 colonies prepared in Comparative Example 1 and the Production Example overnight in an LB liquid medium containing ampicillin, they were diluted at a ratio of 1:100 using a new LB medium, and by additional culturing, when the OD 600 value reached 0.5 to 0.7, doxycycline diluted with ethanol to a final concentration of 200 ng / ml was added to the culture solution, and it was cultured in a shaking incubator under the conditions of 200 rpm and 37°C. The OD 600 value was measured to analyze the growth pattern of the strain, and the results are shown in Figure 2.

[0126] On the other hand, non-recombinant existing Salmonella colonies were treated in the same manner, and the results are shown in Figure 3.

[0127] As shown in Figures 2 and 3, the CNC18 recombinant Salmonella had almost no difference in growth rate from the existing attenuated Salmonella SHJ2037 and the wild-type Salmonella strain, and also had no special growth inhibition during protein expression using doxycycline. Therefore, it was confirmed that the lack of pathogenic genes in the Salmonella strain prepared as described above did not affect the growth and gene expression of the Salmonella strain.

[0128] [1-2] Comparison of Protein Expression Levels by Western Blot Analysis To evaluate the expression levels of the cytolysin A (ClyA) gene between the recombinant SHJ2037 and CNC18 Salmonella strains prepared in Comparative Example 1 and the Production Example, Western blot analysis was performed on the Rluc8 protein expressed in the strains cultured as in Experimental Example [1-1] using specific antibodies.

[0129] Specifically, the culture solution of the strain in Experimental Example [1-1] was at 4×10 7Dilute with PBS to 10⁶ CFU / ml, centrifuge at 13,000 rpm for 5 minutes to collect the pellet. Wash the pellet fraction with PBS and mix with SDS sample buffer containing 0.2% β-mercaptoethanol (catalog number: EBA-1052, ELPIS BIOTECH) to obtain a bacterial lysate. Then, electrophorese the bacterial lysate on 12% SDS-PAGE, transfer the proteins from the gel to a nitrocellulose membrane, and block at room temperature with 5% non-fat milk. Thereafter, use the Rluc8 antibody (catalog number: AB3256, Millipore, USA) to confirm the expression level of the Rluc8 protein, and the results are shown in Figure 4.

[0130] As shown in Figure 4, it was confirmed that the two recombinant SHJ2037 and CNC18 Salmonella strains overexpressed the Rluc8 gene and there was no significant difference in the expression levels between the strains.

[0131] [1-3] Comparison of functional expression levels by protein activity analysis For the measurement of luciferase activity in the strains cultured as in the above Experimental Example [1-1], resuspend the strains in 1 ml of PBS as shown in Figure 5. Then, add 1 μg / ml coelenterazine diluted with ethanol as a substrate to the resuspended strains, and measure the luciferase activity value under the condition of a 1-second exposure time using a NightOWL II LB 983 In Vivo imaging system (Berthold technologies, GmbH&Co.KG, Germany) or a Bio-Rad imager ChemoDocTM XRS+ system. The values measured in this way were standardized by the CFU of each strain and calculated in relative luminescence units (RLU), and the results are shown in Figure 6.

[0132] As shown in Figure 6, the luciferase activity value was confirmed only in the presence of doxycycline, and it was confirmed that the derivative sensitivity and the maximum activity value were similar.

[0133] Also, the recombinant Salmonella strain selected by the above Preparation Example 3 diluted with PBS was spread on a blood agar plate containing 0 or 20 ng / ml of doxycycline, cultured overnight at 37°C, and then a plate photograph was taken, and the results are shown in Fig. 7.

[0134] As shown in Fig. 7, it was confirmed that the hemolytic activity of the strain on blood agar appears only when doxycycline of the gene encoding cytolysin A is included (+).

[0135] Based on the above results, it was confirmed that the recombinant CNC18 Salmonella strain according to the present invention can regulate the expression of the cytolysin A (ClyA) gene and has a function as an anti-cancer gene delivery vector.

[0136] [Experimental Example 2] Confirmation of DAMP signal secretion of tumor cells by recombinant Salmonella treatment To confirm the effect of the recombinant Salmonella strain on tumor cells, changes in damage-associated molecular patterns (DAMPs) that can cause an immune reaction in tumor cells treated with the SHJ2037 and CNC18 strains were confirmed. ATP that is extracellularly secreted among DAMPs was measured. Specifically, CRL-2638 and HB-8064 (ATCC, USA), which are mouse CT26 colon cancer cell lines, were cultured in a high-glucose DMEM (Dulbecco's Modified Eagles Medium) medium (catalog number: #LM 001-05, Welgene, Korea) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator. After that, the recombinant Salmonella was treated with the tumor cells and co-cultured. The culture solution was measured for ATP using an assay kit over time, and the results are shown in Fig. 8.

[0137] As shown in Fig. 8, it was confirmed that the ATP value secreted by tumor cells increased when treated with Salmonella, and that the Salmonella strain itself according to the present invention can be used for the treatment of tumors.

[0138] [Experimental Example 3] Analysis of the distribution of recombinant Salmonella in a small animal tumor model When a live-attenuated Salmonella strain was injected into a mouse model, which is a small animal used in research for cancer treatment, the growth rate and distribution of the strain in the host were confirmed.

[0139] Specifically, four strains, SHJ2037, CNC16, CNC17, and CNC18, were each cultured in an LB liquid medium, washed and diluted with PBS. Each of the strains was injected into the tail vein of a mouse so that the final strain concentration was 2×10 7 CFU / mouse. After euthanizing three mice for each strain, the organs of the host were removed, crushed and diluted, and then spread and cultured on an LB solid medium to measure the viable cell count. The results are shown in Fig. 9. Also, mice containing tumors were treated in the same manner, the tumors of the host were removed, crushed and diluted, and then spread and cultured on an LB solid medium to measure the viable cell count. The results are shown in Fig. 10.

[0140] As a result, as shown in Figs. 9 and 10, it was confirmed that the CNC18 strain showed very low viability in the liver and rapidly died, but remained in the tumor and had specific and selective targeting ability.

[0141] In contrast, as shown in Fig. 9, the CNC16 strain had higher viability in the liver than the SHJ2037 strain and more than 100 times higher viability than CNC18. That is, it was confirmed that the CNC16 strain had non-specific targeting ability in organs other than tumors.

[0142] Also, CNC17 also showed higher viability in the liver than the SHJ2037 strain. That is, it was confirmed that the CNC17 strain also had non-specific targeting ability.

[0143] As shown in Fig. 10, in tumors, the viability of the CNC17 strain is more than 10 times lower than that of the CNC18 strain. That is, it was confirmed that the CNC17 strain has a lower effect on tumors compared to the CNC18 strain.

[0144] [Experimental Example 4] Analysis of Hyperinflammatory Reaction by Injection of Salmonella (1) First, to confirm the hyperinflammatory reaction by injection of Salmonella, after injecting SHJ2037, CNC18 strain, and PBS into 3 mice each out of 9 CT26 tumor model mice, the spleen weights of each mouse over time were measured and compared. As shown in Fig. 11, the mice were laparotomized, the spleens were excised, and the size of the spleen was measured for each mouse, and the results are shown in Fig. 12.

[0145] As shown in Fig. 12, it was found that the spleen size increased in the strain treatment group compared to the control group treated with PBS, but the spleen size in the CNC18 strain treatment group was smaller than that in the SHJ2037 strain treatment group, confirming that the Salmonella strain according to the present invention minimizes the hyperinflammatory reaction.

[0146] [Experimental Example 5] Analysis of Hyperinflammatory Reaction by Injection of Salmonella (2) Additionally, to confirm the hyperinflammatory reaction by injection of Salmonella, after injecting SHJ2037, CNC16, CNC17, and CNC18 strains into mice in the same manner as in Experimental Example 4, the spleen weights were measured and compared on the 1st, 3rd, and 5th days.

[0147] As a result, as shown in Fig. 13, the spleen weights in the SHJ2037, CNC16, and CNC17 strain treatment groups increased compared to the control group treated with PBS, but CNC18 had the smallest increase among the strain treatment groups.

[0148] From the above results, it was confirmed that the attenuated Salmonella strain according to the present invention minimizes the side effects of the strain on the host by maintaining a strong targeting ability to tumors while significantly reducing viability and rapidly dying in all normal organs from the early stage of infection.

[0149] [Experimental Example 6] Analysis of the anti-cancer effect of attenuated Salmonella in a small animal tumor model (1) The tumor cells (CT26 1×10 6 cell / mice) cultured as in Experimental Example 2 were subcutaneously injected into the flanks of mice (BALB / C, n = 7) to construct a tumor animal model. The SHJ2037 and CNC18 Salmonella strains were injected into the tail vein of this model. To evaluate the anti-cancer effect of the tumor animal model, after anesthesia with 2% isoflurane, the size of the tumor (mm 3 ) was measured using (length × height × width) / 2, and the results are shown in Figures 14 and 15.

[0150] As shown in Figures 14 and 15, it was confirmed that the Salmonella-treated group inhibited cancer growth and increased the survival rate compared to the control group. In particular, it was confirmed that the CNC18 strain was significantly superior to the SHJ2037 strain in terms of tumor growth inhibitory effect.

[0151] Therefore, it was confirmed that the CNC18 strain, which maintains a strong targeting ability to tumors while having a greatly reduced viability and rapidly dying in all normal organs from the early stage of infection, has excellent tumor treatment efficacy while minimizing side effects due to in-host infection.

[0152] [Experimental Example 7] Analysis of the anti-cancer effect of attenuated Salmonella in a small animal tumor model (2) The cultured tumor cells (MC38, mouse colon adenocarcinoma cells, 1×10 6 cell / mice) were subcutaneously injected into the flanks of mice (C57BL / 6, n = 6) to construct a tumor animal model. The SHJ2037 and CNC18 Salmonella strains were injected into the tail vein of this model. To evaluate the anti-cancer effect of the tumor animal model, after anesthesia with 2% isoflurane, the size of the tumor (mm 3 ) was measured using (length × height × width) / 2, and the results are shown in Figures 16 and 17.

[0153] As shown in FIGS. 16 and 17, it was confirmed that the Salmonella-treated group inhibited cancer growth and increased the survival rate compared to the control group. In particular, it was confirmed that the CNC18 strain suppressed tumor growth more than the SHJ2037 strain.

[0154] [Experimental Example 8] Confirmation of Immunostimulatory Effect by Injection of Salmonella To confirm the immunostimulatory effect by injection of Salmonella, immune cells were measured and compared. The SHJ2037 and CNC18 Salmonella strains were injected into the tumor animal model constructed as in Experimental Example 6, and immune cells were collected on the third day. The immune cells were collected from the tumor and the lymph nodes around the tumor. After measuring the amount of the collected immune cells, the results were shown as in FIGS. 18 to 24.

[0155] As shown in FIGS. 18 to 22, the proportion of white blood cells, neutrophils, natural killer cells, and CD8+ T cells increased in the CNC18 strain-treated group compared to the PBS control group and the SHJ2037 control group, and dendritic cells in the lymph nodes also increased. In contrast, as shown in FIGS. 23 and 24, M2 macrophages and regulatory T cells (Treg cells) that have an immunosuppressive effect decreased in the CNC18 strain-treated group compared to the PBS control group and the SHJ2037 control group.

[0156] From the above results, it was confirmed that the attenuated Salmonella strain CNC18 according to the present invention significantly enhanced the immune function compared to the PBS control group and the SHJ2037 control group.

[0157] [Experimental Example 9] Imaging Analysis of Tumors by Salmonella (1) The tumor cells cultured as in Experimental Example 2 were subcutaneously injected into the flanks of mice to construct a tumor animal model. The SHJ2037lux and CNC18lux Salmonella strains were injected into this model. Here, the SHJ2037lux and CNC18lux strains are strains in which a luminescent gene has been introduced into the SHJ2037 strain and the CNC18 strain. The luminescent gene used was the bacterial luciferase gene (lux).

[0158] To evaluate the imaging effect of the strain in a tumor animal model, after removing the organs and tumors at each time point using an In Vivo Imaging System (IVIS), images were taken, and the results are shown in Figures 25 to 27.

[0159] As shown in Figures 25 to 27, when the CNC18lux strain was injected like the SHJ2037lux strain, it was confirmed that it showed viability specifically and selectively in the tumor and could be imaged in real time.

[0160] [Experimental Example 10] Imaging analysis of tumors by Salmonella (2) After constructing a multiple myeloma model by injecting cultured MOPC cells into the tibia of a mouse, the SHJ2037lux and CNC18lux Salmonella strains were injected as in Experimental Example 9. Next, images were taken at each time point using an in vivo imaging system (In Vivo Imaging System; IVIS), and the results are shown in Figures 28 and 29.

[0161] As shown in Figures 28 and 29, when the CNC18lux strain was injected, it was confirmed that it showed viability specifically and selectively in the tumor and could be imaged in real time. In particular, unlike SHJ2037lux where the image signal was dispersed and remained in the peripheral part of the tumor, in the case of the CNC18lux strain, it reached the deep part of the tumor and a clearer image could be confirmed in real time.

[0162] As can be seen from the above Experimental Examples 1 to 10, the CNC18 strain was confirmed to have excellent tumor treatment efficacy while minimizing the side effects caused by in-host infection, as it maintained a strong targeting ability to the tumor while its viability significantly decreased and it rapidly died in all normal organs from the early stage of infection.

[0163] As described above in detail for specific parts of the present invention, it is obvious to those with ordinary knowledge in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention thereby. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Industrial Applicability

[0164] The present invention relates to a Salmonella strain that selectively acts on cancer and a composition for preventing or treating cancer containing the same.

Claims

1. Salmonella pathogenicity island-1 (SPI-1); and Salmonella pathogenicity island-2 (SPI-2); are deleted, A mutant strain of the genus Salmonella for anti-cancer, in which the gene encoding ppGpp synthase is additionally deleted, The gene encoding the ppGpp synthase is the Salmonella-relA gene and the Salmonella-spoT gene, The mutant strain of the genus Salmonella has a specific and selective targeting ability against cancer, suppresses tumor growth, but has low viability in normal liver, The mutant strain of the genus Salmonella.

2. The Salmonella pathogenicity island-1 (SPI-1) consists of the nucleotide sequence represented by SEQ ID NO: 1, and the mutant strain of the genus Salmonella according to Claim 1.

3. The Salmonella pathogenicity island-2 (SPI-2) consists of the nucleotide sequence represented by SEQ ID NO: 2, and the mutant strain of the genus Salmonella according to Claim 1.

4. The mutant strain of the genus Salmonella according to Claim 1, into which a gene encoding an anti-cancer protein is additionally introduced.

5. The anti-cancer protein is at least one selected from the group consisting of a toxin protein, an antibody specific for a cancer antigen or a fragment of the antibody capable of binding to a cancer antigen, a tumor suppressor protein, an angiogenesis inhibitor, a cancer antigen, a prodrug-converting enzyme, and a pro-apoptotic protein. The mutant strain of the genus Salmonella according to Claim 4.

6. The mutant strain of the genus Salmonella according to claim 5, wherein the toxin protein is 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-α (TNF-α), TNF-related apoptosis-inducing ligand (TRAIL), and cytolysin A (ClyA).

7. The mutant strain of the genus Salmonella according to claim 6, wherein the gene encoding the cytolysin A consists of the nucleotide sequence represented by SEQ ID NO:

15.

8. The tumor suppressor protein is at least one selected from the group consisting of retinoblastoma (RB) protein, p53 protein, adenomatous polyposis coli (APC) protein, phosphatase and tensin homolog (PTEN) protein, and cyclin-dependent kinase inhibitor 2A (CDKN2A) protein, and the mutant strain of the genus Salmonella according to claim 5.

9. The mutant strain of the genus Salmonella according to claim 5, wherein the angiogenesis inhibitor is at least one selected from the group consisting of angiostatin, endostatin, thrombospondin, and protease inhibitor protein.

10. The cancer antigen is alpha-fetoprotein (α-fetoprotein; AFP), vascular endothelial growth factor receptor 2 (Vascular endothelial growth factor receptor 2; VEGFR2), survivin, legumain, and prostate-specific antigen (Prostate The mutant strain of the genus Salmonella according to claim 5, which is at least one selected from the group consisting of cancer specific antigen; PSA).

11. The mutant strain of the genus Salmonella according to claim 5, wherein the prodrug converting enzyme is at least one selected from the group consisting of thymidine kinase, cytosine deaminase, nitroreductase, purine nucleoside phosphorylase, carboxypeptidase G2, chromate reductase YieF, herpes simplex virus type I thymidine kinase / ganciclovir; HSV1-TK / GCV) and β-glucuronidase.

12. The mutant strain of the genus Salmonella according to claim 5, wherein the pro-apoptotic protein is L-asnase or RNA-binding motif protein 5; RBM5).

13. The mutant strain of the genus Salmonella according to claim 1, which is derived from at least one selected from the group consisting of Salmonella typhimurium, Salmonella choleraesuis, Salmonella enteritidis, Salmonella infantis, Salmonella paratyphi, Salmonella gallinarum, and Salmonella typhi.

14. The mutant strain of the genus Salmonella according to claim 1, wherein the mutant strain of the genus Salmonella is a mutant lacking the ability to synthesize guanosine polyphosphate.

15. A step of removing the Salmonella pathogenicity island-1 (SPI-1) and Salmonella pathogenicity island-2 (SPI-2) genes of the Salmonella genus strain, and additionally including a step of obtaining a transformed strain by deleting the gene encoding the ppGpp synthase. The gene encoding the ppGpp synthase is the Salmonella relA gene and the Salmonella spoT gene. A method for producing a mutant strain of the genus Salmonella for anti-cancer use according to any one of claims 1 to 14.

16. The method for producing a mutant strain of the genus Salmonella according to claim 15, wherein the removal of the genes of the Salmonella pathogenicity island-1 (SPI-1) and Salmonella pathogenicity island-2 (SPI-2) is performed by a recombinant vector.

17. The method for producing a mutant strain of the genus Salmonella according to claim 15, wherein the Salmonella pathogenicity island-1 (SPI-1) consists of the nucleotide sequence represented by SEQ ID NO:

1.

18. The method for producing a mutant strain of the genus Salmonella according to claim 15, wherein the Salmonella pathogenicity island-2 (SPI-2) consists of the nucleotide sequence represented by SEQ ID NO:

2.

19. The method for producing a mutant strain of the genus Salmonella according to claim 15, further including a step of introducing a gene encoding an anti-cancer protein.

20. The anti-cancer protein is at least one selected from the group consisting of ricin, saporin, gelonin, momordin, debouganin, diphtheria toxin, Pseudomonas toxin, hemolysin (HlyA), FAS ligand (FAS ligand; FasL), tumor necrosis factor-α (Tumor necrosis factor-α; TNF-α), TNF-related apoptosis-inducing ligand (TNF-related apoptosis-inducing ligand; TRAIL), and cytolysin A (Cytolysin A; ClyA). The method for producing a mutant strain of the genus Salmonella according to claim 19.

21.

21. The gene encoding the cytolysin A consists of the nucleotide sequence represented by SEQ ID NO:

15. The method for producing a mutant strain of the genus Salmonella according to claim 20.

22. The Salmonella genus strain is at least one selected from the group consisting of Salmonella typhimurium, Salmonella choleraesuis, Salmonella enteritidis, Salmonella infantis, Salmonella paratyphi, Salmonella gallinarum, and Salmonella typhi. The method for producing a mutant strain of the genus Salmonella according to claim 15.

23. The Salmonella genus strain is a mutant lacking guanosine polyphosphate synthesis ability. The method for producing a mutant strain of the genus Salmonella according to claim 15.

24. The method for producing a mutant strain of the genus Salmonella according to claim 15, further comprising the step of culturing the transformed strain.

25. The method for producing a mutant strain of the genus Salmonella according to claim 15, further comprising the step of culturing the transformed strain in an antibiotic medium to select a mutant strain.

26. A pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, a mutant strain of the genus Salmonella according to any one of claims 1 to 14.

27. The cancer is selected from the group consisting of melanoma, fallopian tube cancer, brain cancer, small intestine cancer, esophageal cancer, adenocarcinoma of the lymph nodes, gallbladder cancer, blood cancer, thyroid cancer, endocrine adenocarcinoma, 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, undifferentiated 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. The pharmaceutical composition according to claim 26.

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