Immune-enhanced salmonella strains for treatment of cancer and uses thereof

Abstract

The present invention relates to a DNA construct comprising: a gene encoding flagellin and a gene encoding an immunopotentiator (or adjuvant) protein. In order to achieve effective cancer treatment by selectively killing only cancer cells, the attenuated Salmonella strains of the present disclosure are designed to produce immunogenic substances in cancer tissue to induce a strong anti-cancer immune response to significantly inhibit tumor size in primary and metastatic cancers. Therefore, the strain can be advantageously used in a prophylactic or therapeutic composition for increasing survival rate.

Description

Technical Field

[0001] The present invention relates to an immune-enhanced Salmonella strain for treating cancer and its uses. Background Art

[0002] So far, most cancers are treated by a single means or a combination of means such as surgical resection, radiotherapy, or chemotherapy. For cancers located in a specific area (such as the breast, colon, or skin), surgical resection of most of the tumor may be very effective. However, for tumors located in special parts such as the spine, surgery is difficult to perform. In addition, systemic chemotherapy commonly used for cancers such as breast cancer, lung cancer, and testicular cancer may have side effects due to interfering with normal cell replication or metabolic processes. In addition, patients may develop drug resistance to the chemotherapy drugs used during the treatment process.

[0003] On the other hand, when cancer occurs in an organism, rapid angiogenesis and cell growth will form an environment of incomplete vascularization and hypoxia in the tumor tissue. Such conditions are very conducive to the proliferation of facultative anaerobes such as Salmonella or Escherichia coli. Therefore, current cancer therapies using tumor-targeting bacteria such as Salmonella or Clostridium rely on their ability to selectively proliferate within solid tumors. When a tumor-lysing protein or a reporter protein is introduced into such bacteria and the transformed strain is administered to an organism, it can specifically target tumor tissues, detect tumors, and reduce toxicity to normal cells, thereby enhancing the therapeutic effect.

[0004] In nature, a variety of bacterial pathogens secrete toxins that can cause human diseases. Among them, Salmonella enterica, as a bacterium closely related to human eating habits and known to parasitize in the gastrointestinal tract of primates (including humans), can secrete an exotoxin called cytolysin. Cytolysin is a cytotoxic protein with a molecular weight of about 34 kDa, which can exhibit hemolytic activity by destroying red blood cells in the gastrointestinal tract and form pores on the normal cell membrane, resulting in cell lysis. This may cause severe vascular inflammation and local tissue necrosis, and even death. However, recent studies have shown that cytolysin isolated and purified from Salmonella enterica can selectively react with tumor tissues in the gastrointestinal tract and induce tumor cell death, and thus is regarded as a new generation of anti-cancer drugs. Therefore, genetically engineered bacteria secreting the cytotoxic protein cytolysin have great application prospects as tumor-targeting anti-cancer therapies. Although bacteria have great potential in cancer diagnosis and treatment, research on expression vectors involving their selective expression of diagnostic or therapeutic proteins in tumor tissues is relatively limited.

[0005] Therefore, the present invention aims to develop an effective cancer prevention or treatment method by selectively targeting and killing cancer cells. Research has shown that engineered attenuated Salmonella can produce immunostimulatory molecules within tumor tissues, inducing a strong anti-tumor immune response, thereby effectively treating primary and metastatic cancers. The pharmaceutical composition of the present invention specifically targets and kills tumors in the body, thereby improving the survival rate of patients. Therefore, it is expected to be widely used in the field of cancer treatment. Summary of the Invention

[0006] Technical Problem

[0007] The inventors of the present invention have dedicated themselves to developing an effective cancer prevention or treatment drug by selectively killing only cancer cells. Therefore, they have confirmed that engineered attenuated Salmonella can produce immunostimulatory substances within tumor tissues, which can induce a strong anti-tumor immune response and can effectively treat not only primary tumors but also metastatic cancers. Through this discovery, they have verified the ability to specifically target and eliminate tumor cells in a living system, thereby improving the survival rate of patients and finally completing the present invention.

[0008] Therefore, an object of the present invention is to provide a DNA construct, which comprises: a gene encoding flagellin; and a gene encoding an adjuvant protein.

[0009] Other objects and advantages of the present invention will be more fully embodied in the following detailed description, claims and the content of the drawings.

[0010] However, the objects to be achieved by the present disclosure are not limited to the above-mentioned objects, and those skilled in the art can clearly understand other objects not listed herein through the following description.

[0011] Technical Solution

[0012] Hereinafter, various embodiments described herein will be described in conjunction with the drawings. To provide a more thorough understanding of the present disclosure, many specific details are set forth in the following description, such as specific configurations, compositions, and processes. However, some embodiments can be implemented without one or more of the above specific details, or in combination with other known methods and configurations. In other cases, well-known processes and preparation techniques are not described in detail to avoid unnecessarily obscuring the present disclosure. The phrase "in one embodiment" or "an embodiment" mentioned throughout this specification refers to a specific feature, configuration, composition, or characteristic described in connection with that embodiment being included in at least one embodiment of the present disclosure. Therefore, the phrases "in one embodiment" or "an embodiment" that appear in various places in this specification do not necessarily refer to the same embodiment of the present disclosure. In addition, in one or more embodiments, specific features, configurations, compositions, or characteristics can be combined in any suitable manner.

[0013] Unless otherwise indicated in the specification, all scientific and technical terms used in the specification have the same meanings as those commonly understood by those skilled in the technical field to which the present disclosure pertains.

[0014] In this specification, when a part is described as "comprising" a specific component, unless otherwise clearly stated, it should be understood that this term does not exclude the presence of other components and allows the inclusion of other components.

[0015] According to one aspect of the present invention, the present invention provides a DNA construct.

[0016] The DNA construct of the present invention comprises: a gene encoding flagellin; and a gene encoding an adjuvant protein.

[0017] The inventors of the present invention have developed, through extensive research, an effective cancer prevention or treatment drug that selectively kills only cancer cells. Thus, they have confirmed that an attenuated Salmonella strain engineered to produce an immune-stimulating substance that selectively acts on cancer can selectively enter and produce an immune-stimulating substance within tumor tissues, which can induce a strong anti-tumor immune response and effectively treat not only primary tumors but also metastatic cancers. The present invention has been completed by discovering a new and effective method and composition for treating cancer in a living system.

[0018] According to the present invention, flagellin and the adjuvant can be expressed as a fusion protein using an expression vector, but the present invention is not limited thereto.

[0019] In this specification, the term "fusion protein" refers to an artificially recombinant protein expressed by linking one or more genes of other proteins to a protein. By combining the functions of two or more proteins, the fusion protein is expected to exhibit a synergistic effect.

[0020] According to the present invention, in order to achieve the secretory expression of the fusion protein by genetically engineered bacteria, a signal peptide is added to the 5' end of the fusion protein. Specifically, the signal peptide can be selected from the following group: pectate lyase B (PelB), outer membrane protein A (OmpA), heat-stable enterotoxin 2 (StII), endo-xylanase, alkaline phosphatase (PhoA), outer membrane protein F (OmpF), and outer membrane porin E (PhoE).

[0021] In this specification, the term "Salmonella" refers to a genus in the phylum Proteobacteria of the family Enterobacteriaceae. These rod-shaped bacteria have a diameter of about 0.7 to 1.5 μm and a length of about 2 to 5 μm and are mainly present in the digestive tracts of humans and animals.

[0022] As used in the present invention, "flagellin" refers to (but is not limited to) the globular protein that constitutes the helical filament of bacterial flagella. The molecular weight of flagellin varies widely depending on the bacterial species (ranging from 30,000 to 70,000 Da). In terms of amino acid composition, it usually does not contain cysteine and tryptophan. Taking Salmonella as an example, approximately half of its lysine residues are methylated. There are types such as flagellin A and flagellin B, which, although having multiple functions, are known to have immunostimulatory properties.

[0023] In the present invention, the flagellin is flagellin A (FlaA) or flagellin B (FlaB).

[0024] In the specification of the present invention, the term "cancer" refers to a disease state characterized by uncontrolled cell proliferation, and such abnormal proliferation results in cell masses called tumors. These tumors may invade surrounding tissues and, in severe cases, metastasize to other organs in the body. Scientifically, cancer can also be called neoplasm. Although it can be treated by surgery, radiotherapy, or chemotherapy, in most cases, cancer is still incurable, causing great pain to patients and ultimately leading to death. Cancer is regarded as a refractory chronic disease. The causes of cancer can be roughly divided into internal and external factors. The exact mechanism of normal cell carcinogenesis has not been fully elucidated, but it is known that a considerable proportion of cancers are induced by environmental factors and other external causes. Internal factors include genetic susceptibility and immune status, and external factors cover chemical exposure, radiation, and viral infection. Genes related to cancer include oncogenes and tumor suppressor genes. The occurrence of cancer is due to the imbalance between these two types of genes caused by the above internal and external factors. In the present invention, the objects for prevention, improvement, or treatment may include but are not limited to: melanoma, fallopian tube cancer, brain cancer, small intestine cancer, esophageal cancer, lymphoma, gallbladder cancer, blood cancer, thyroid cancer, endocrine adenocarcinoma, oral cancer, liver cancer, cholangiocarcinoma, colon 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, colorectal 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 plasmacytoma.

[0025] According to the present invention, the first promoter and the second promoter of the present invention can be synchronously induced by a single regulatory protein whose expression is controlled by an independent promoter. Compared with the scheme of operably linking the gene encoding the regulatory protein downstream of the second promoter, the protein expression levels encoded by the genes operably linked downstream of the first promoter and the second promoter in or within the host cell can reach equilibrium. Therefore, the DNA construct described in the present invention enables the synchronization of diagnosis and treatment.

[0026] The term "DNA construct" of the present invention refers to a structural unit that is transformed and introduced into a host strain or cell to enable the expression of a target protein. It includes not only the gene encoding the target protein, but also basic regulatory elements such as promoter sequences, which are operably linked to allow gene expression.

[0027] The "promoter" described in the present invention refers to a nucleotide sequence located upstream of a gene operably linked within a host strain or cell. It is a specific DNA sequence of a DNA construct to which RNA polymerase binds and initiates transcription.

[0028] In the present invention, the 5′ untranslated region (5′-UTR) refers to the untranslated part of mRNA located adjacent to the coding region, which is translated into amino acids. Although once regarded as "redundant sequences", current studies have shown that it plays a key role in regulating gene expression.

[0029] In the present invention, a transcription factor binding site refers to a DNA region that has the function of turning on or off a specific adjacent gene. This site may include, but is not limited to, at least one element selected from the group consisting of a promoter, an enhancer, and a silencer encoding a regulatory protein gene.

[0030] According to a specific embodiment of the present invention, an immune adjuvant is a DNA construct comprising at least one selected from the group consisting of a gel-type adjuvant (metal salt compound), an emulsified oil adjuvant (lipid particle), a cytokine, a chemokine, a particulate adjuvant, and a microbial adjuvant. More specifically, the immune adjuvant is a DNA construct comprising at least one selected from the group consisting of IFN-α2, IL-2, IL-15, IL-21, IL-12, CXCR3, CCR5, T cells, and B cells. Even more specifically, the immune adjuvant is IL-15. Most specifically, the immune adjuvant is human IL-15, which is the gene or protein shown in Sequence 1 (SEQ ID NO: 1).

[0031] According to another specific embodiment of the present invention, flagellin is a DNA construct comprising at least one selected from the group consisting of flagellin A, B, C, D, and E. Specifically, the flagellin is flagellin A or flagellin B.

[0032] According to the present invention, the pBAD plasmid is used, and arabinose is used as an inducer.

[0033] The arabinose used in the present invention refers to a monosaccharide containing five carbon atoms, classified as an aldose with an aldehyde group, and its chemical formula is C5H 10 O5. Due to the biosynthetic pathway, in nature, most sugars mainly exist in the form of "D-type" or structurally similar to D-glyceraldehyde.

[0034] In the present invention, the promoter of the regulatory protein coding gene may include any promoter that can be activated under various environmental conditions or the developmental state of the host strain or cell. Preferably, it can be a weak promoter.

[0035] As used herein, the term "weak promoter" refers to a promoter capable of achieving a transcription level of the gene transcript operably linked downstream of 1×10 -2 or lower, preferably 1×10 -3 or lower. Any promoter capable of achieving a transcription level of 1×10 -3 or lower is included, such as: Escherichia coli σ70 promoter; Escherichia coli σS promoter; Escherichia coli σ32 promoter; Bacillus subtilis σA promoter; Bacillus subtilis σB promoter; promoters from Salmonella, such as K112706 or K112707; phage T7 promoter; phage SP6 promoter; promoters from yeast; promoters from eukaryotic cells. The term "DNA construct" in the present invention refers to a structural unit that is introduced into a host strain or cell by transformation to enable the expression of the target protein. It includes not only: the gene encoding the target protein, but also basic regulatory elements such as the promoter sequence, and these elements are operably linked to allow gene expression.

[0036] The "promoter" described in the present invention refers to a nucleotide sequence located upstream of the gene operably linked within the host strain or cell. It is a specific DNA sequence of the DNA construct to which RNA polymerase binds and initiates transcription.

[0037] In the present invention, the 5′ untranslated region (5′-UTR) refers to the untranslated part of the mRNA located adjacent to the coding region, which is translated into amino acids. Although once regarded as "redundant sequences", current research shows that it plays a key role in regulating gene expression.

[0038] In the present invention, the transcription factor binding site refers to a DNA region that has the function of turning on or off the adjacent specific gene. This site may include but is not limited to at least one element selected from the group consisting of: the promoter of the regulatory protein coding gene, enhancer, and silencer.

[0039] According to a specific embodiment of the present invention, the immunoadjuvant is a DNA construct comprising at least one selected from the group consisting of: gel-type adjuvant (metal salt compound), emulsified oil adjuvant (lipid particle), cytokine, chemokine, particulate adjuvant, and microbial adjuvant. More specifically, the immunoadjuvant is a DNA construct comprising at least one selected from the group consisting of: IFN-α2, IL-2, IL-15, IL-21, IL-12, CXCR3, CCR5, T cells, and B cells. Even more specifically, the immunoadjuvant is IL-15. Most specifically, the immunoadjuvant is human IL-15, which is the gene or protein shown in Sequence No. 1.

[0040] According to another specific embodiment of the present invention, the flagellin is a DNA construct comprising at least one selected from the group consisting of: flagellin A, B, C, D, and E. Specifically, the flagellin is flagellin A or flagellin B.

[0041] According to the present invention, the pBAD plasmid is used, and arabinose is used as an inducer.

[0042] The arabinose used in the present invention refers to a monosaccharide containing five carbon atoms, classified as an aldose with an aldehyde group, and its chemical formula is C5H 10 O5. Due to the biosynthetic pathway, in nature, most sugars mainly exist in the form of "D-type" or structurally similar to D-glyceraldehyde.

[0043] In the present invention, the promoter regulating the protein-coding gene may include any promoter that can be activated under various environmental conditions or the developmental state of the host strain or cell. Preferably, it can be a weak promoter.

[0044] As used herein, the term "weak promoter" refers to a promoter capable of achieving a transcription level of the gene transcript operably linked downstream of 1×10 -2 or lower, preferably 1×10 -3 or lower. Any promoter capable of achieving a transcription level of 1×10 -3 or lower is included, such as: Escherichia coli σ70 promoter; Escherichia coli σS promoter; Escherichia coli σ32 promoter; Bacillus subtilis σA promoter; Bacillus subtilis σB promoter; promoters from Salmonella, such as K112706 or K112707; bacteriophage T7 promoter; bacteriophage SP6 promoter; promoters from yeast; promoters from eukaryotic cells; and promoters from plants, but not limited thereto.

[0045] The Escherichia coli σ70 promoter according to the present invention may be at least one selected from the following group: 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, K1585103, K1585104, K1585105, K1585106, K1585110, K1585113, K1585115, K1585116, K1585117, K1585118, K1585119, K2486171, K256002, K256018, K256020, K256033, K292000, K823007, K823010, K823013, M13101, M13102, M13103, M13104, M13105, M13106, M13108, M13110, M31519, R1074, R1075, and S03331, but not limited thereto.

[0046] The Escherichia coli σS promoter according to the present invention may be J45992 or J45993, but not limited thereto.

[0047] The Escherichia coli σ32 promoter may be J45504, K1895002 or K1895003, but not limited thereto.

[0048] The Bacillus subtilis σA promoter may be at least one selected from the following group: K143012, K143013, K823000, K823002 and K823003, but not limited thereto.

[0049] The Bacillus subtilis σB promoter may be K143010, K143011 or K143013, but not limited thereto.

[0050] The phage T7 promoter can be at least one selected from the following group: I719005, J34814, J64997, K113010, K113011, K113012, K1614000, R0085, R0180, R0181, R0182, R0183, Z0251, Z0252, and Z0253, but not limited thereto.

[0051] The phage SP6 promoter can be J64998, but not limited thereto.

[0052] The yeast-derived promoter can be at least one selected from the following group: I766557, J63005, K105027, K105028, K105029, K105030, K105031, K122000, K124000, K124002, K319005, M31201, K2365040, K2365036, K2365041, K2365042, K2365032, K2365051, K2365514, K2365515, and K2365516, but not limited thereto.

[0053] The derivative promoter can be at least one selected from the following group: I712004 or K076017, and plant-derived promoters, but not limited thereto.

[0054] The Escherichia coli σ70 promoter according to the present invention may be at least one selected from the following group: 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, K1585103, K1585104, K1585105, K1585106, K1585110, K1585113, K1585115, K1585116, K1585117, K1585118, K1585119, K2486171, K256002, K256018, K256020, K256033, K292000, K823007, K823010, K823013, M13101, M13102, M13103, M13104, M13105, M13106, M13108, M13110, M31519, R1074, R1075, and S03331, but not limited thereto.

[0055] The Escherichia coli σS promoter according to the present invention may be J45992 or J45993, but not limited thereto.

[0056] The Escherichia coli σ32 promoter may be J45504, K1895002 or K1895003, but not limited thereto.

[0057] The Bacillus subtilis σA promoter may be at least one selected from the following group: K143012, K143013, K823000, K823002 and K823003, but not limited thereto.

[0058] The Bacillus subtilis σB promoter may be K143010, K143011 or K143013, but not limited thereto.

[0059] The phage T7 promoter can 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 not limited thereto.

[0060] The phage SP6 promoter can be J64998, but not limited thereto.

[0061] The yeast-derived promoter can 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 not limited thereto.

[0062] The plant-derived promoter of the present invention can 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 not limited thereto.

[0063] For the purposes of the present invention, when the coding gene of the regulatory protein is operably linked downstream of the weak promoter, compared to when the gene is operably linked downstream of the first promoter or the second promoter, the transcription of the gene present downstream of the first promoter and / or the second promoter can be specifically regulated to occur only when a substance that inhibits the regulatory protein is applied.

[0064] According to another aspect of the present invention, the present invention provides a recombinant vector comprising the DNA construct.

[0065] The recombinant vector of the present invention comprises the above DNA construct, wherein the regulatory protein is encoded and expressed by an independent promoter, so that gene-specific and co-expression operably linked downstream of the first promoter and the second promoter is allowed only when an external substance that inhibits the regulatory protein is administered.

[0066] The recombinant vector of the present invention can be used as a method for expressing proteins in cells and can be any known recombinant vector, such as a plasmid vector, a cosmid vector or a phage vector. Those skilled in the art can easily construct the recombinant vector using known methods based on recombinant DNA technology.

[0067] In the present invention, specific examples of the recombinant vector include, but are not limited to, vectors that are commercially available and widely used, such as pCDNA vectors, F, R1, RP1, Co1, pBR322, ToL, Ti vectors, cosmids, and phages, such as λ, λ-like, M13, Mu, p1, P22, Qμ and T-even, T2, T3, T7 phages, and plant viruses. The recombinant vector is appropriately selected according to the intended use of the present invention and in combination with the properties of the host cell.

[0068] In another embodiment of the present invention, a host cell or strain into which a recombinant vector containing the DNA construct of the present invention has been introduced is provided.

[0069] In the present invention, the host cell may include: cells derived from mammals, plants, insects, fungi or cells. Examples include bacteria such as Escherichia coli, Streptomyces or Salmonella; fungi such as yeast or Pichia pastoris; insect cells such as Drosophila or Sf9 (Spodoptera Sf9) cells of Spodoptera litura; and animal cells such as CHO (Chinese hamster ovary), SP2 / 0 (mouse myeloma), human lymphoblast, COS, NSO (mouse myeloma), 293T, B16 melanoma, HT-1080, BHK (baby hamster kidney), HEK (human embryonic kidney) or PER.C6 (human retina) cells, and plant cells. These host cells are not limited to these examples. For the purposes of the present invention, the strain may be an anaerobic strain, such as selected from Salmonella, Clostridium, Bifidobacterium and Escherichia coli. Preferably, it may be selected from the group consisting of Salmonella typhimurium, Salmonella choleraesuis and Salmonella enteritidis, more preferably Salmonella typhi, but not limited thereto.

[0070] The strain of the present invention may be attenuated.

[0071] The term "attenuated" as used in the present invention refers to the modification of a microorganism by means such as genetic engineering to reduce its toxicity or other adverse effects in the patient to whom it is administered. When the strain is of the genus Salmonella, attenuation can be achieved by introducing a mutation of 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, flgX, flgL, relA, and spoA, but not limited thereto.

[0072] In the present invention, the method for modifying the above genes can employ various gene deletion or gene disruption techniques known to those skilled in the art. For example, means such as homologous recombination, chemical mutagenesis, radiation mutagenesis, or transposon mutagenesis for gene deletion or gene disruption can be used.

[0073] In the present invention, the strain is designed to target the interior of tumor tissue - an environment that is very suitable for the proliferation of anaerobic strains and has the characteristics of incomplete vascularization and hypoxia. Therefore, when a recombinant vector capable of simultaneously and synergistically expressing a real-time imaging reporter protein and an anti-cancer protein is introduced into such a strain, efficient simultaneous diagnosis and treatment of cancer can be achieved.

[0074] The recombinant vector described in the present invention can be transformed (or transduced) into a host cell or strain, and any transformation method known in the art can be used, and these methods can be easily carried out using conventional techniques. Specifically, for example, transformation methods commonly used for bacteria such as Salmonella strains include: calcium chloride (CaCl2) precipitation method, Hanahan method - by using DMSO (dimethyl sulfoxide) and CaCl2 to improve transformation efficiency, electroporation method, calcium phosphate precipitation method, protoplast fusion method, silicon carbide fiber-mediated transformation method, Agrobacterium transformation method, PEG-mediated transformation, dextran sulfate method, liposome transfection method, and drying / inhibitor-mediated transformation method, etc., can all be used to introduce the recombinant vector into the strain; however, the present invention is not limited thereto.

[0075] According to a specific embodiment of the present invention, the strain can be a strain cell selected from the group consisting of: Salmonella, Clostridium tetani, Bifidobacterium, and Escherichia coli strains.

[0076] In another embodiment, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the above strain as an active ingredient.

[0077] As used herein, the term "prevention" refers to delaying the onset of a disease or disorder in a subject who has not been diagnosed but has a predisposition to the disease or disorder by means of an intervention.

[0078] As used herein, the term "treatment" refers to achieving an expected effective therapeutic effect by using the active ingredients described in the present invention, including but not limited to any action that relieves the symptoms caused by cancer or achieves clinical benefit. The expected effective therapeutic effects include but are not limited to: relieving or improving one or more disease symptoms, reducing the severity of the disease, stabilizing the disease state, delaying the onset or spread of the disease, delaying the progression or onset time of the disease, improving or alleviating the disease condition, and partial or complete remission of the disease. In addition, "treatment" means that the survival period of a patient is greater than the expected survival threshold in the case of not receiving the intervention through technical intervention. Moreover, the term "treatment" includes inhibiting or temporarily delaying the progression of the disease, and more preferably, "treatment" can permanently terminate the progression of the disease. Those skilled in the art should understand that if a treatment regimen improves a certain specific disease state but its accompanying side effects exceed the beneficial effects of the treatment, then the treatment regimen is not considered a clinically acceptable beneficial regimen.

[0079] Therefore, the pharmaceutical composition described in the present invention can be used alone to treat the disease or as an adjuvant treatment for treating the above diseases in combination with other anti-cancer drugs. Therefore, the term "treatment" or "therapeutic agent" used in this specification includes the meaning of "adjuvant treatment" or "adjuvant therapeutic agent".

[0080] As used herein, the term "pharmaceutical composition" includes, but is not limited to, dosage forms prepared by conventional methods, such as oral dosage forms (including powders, granules, capsules, tablets, and aqueous suspensions); topical preparations, suppositories, and sterile injections. The pharmaceutical composition described in the present invention contains one or more pharmaceutically acceptable carriers. The carriers in the case of oral administration include, but are not limited to: binders, lubricants, disintegrants, diluents, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavoring agents, etc. The carriers for injection preparations may include buffers, preservatives, analgesics, solubilizers, isotonicity regulators, stabilizers, etc. The carriers for topical preparations include: matrices, diluents, lubricants, and preservatives. The pharmaceutical composition described in the present invention can be made into various dosage forms by mixing with the above-mentioned pharmaceutically acceptable carriers. For example, oral preparations can be prepared into tablets, lozenges, capsules, elixirs, suspensions, syrups, or wafers, etc., and injection preparations can be prepared into single-dose or multi-dose vials, etc. Other dosage forms also include solutions, suspensions, tablets, capsules, and sustained-release preparations, etc. Examples of carriers, excipients, and diluents suitable for the preparations include, but are not limited to: lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone (povidone), water, methyl paraben, propyl paraben, talc, magnesium stearate, or mineral oil. In addition, other components such as fillers, anti-caking agents, lubricants, wetting agents, flavoring agents, emulsifiers, and preservatives are also included.

[0081] As used herein, the terms "administering" or "administration" refer to directly delivering the pharmaceutical composition described in the present invention into the body of a subject in a therapeutically effective amount to form an equivalent effective dose in the body of the subject. The administration method includes delivering the pharmaceutical composition into the body of a patient by any suitable method, and the administration route is any conventional route capable of reaching the target tissue. Specific examples include, but are not limited to: oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, pulmonary administration (inhalation administration), rectal administration, intracavitary administration, intraperitoneal administration, intrathecal administration. The administration dose of the pharmaceutical composition described in the present invention is adjusted according to various factors, including but not limited to: the type and severity of the disease, the nature and content of the active ingredient and other components in the composition, the type of formulation, the age, weight, general health status, gender, diet, administration time and route of the subject, the secretion rate of the composition, the duration of treatment, and the concomitant medications. For adults, the therapeutic pharmaceutical composition can be administered in a volume of 50 - 500 ml per dose. In the case of a compound, it can be administered at a dose of 0.1 ng / kg to 10 mg / kg, and the same dose range may be applicable for monoclonal antibodies. The administration frequency is 1 to 12 times per day. If administered 12 times a day, it is administered once every 2 hours. In addition, the pharmaceutical composition described in the present invention can be administered alone or in combination with other cancer treatment methods known to those skilled in the art, and the other cancer treatment methods include chemotherapy, radiotherapy, or surgery, etc. The pharmaceutical composition can also be used in combination with other therapies that can enhance the immune response, such as adjuvants or cytokines (or nucleic acids encoding such cytokines) known to those skilled in the art. Other conventional delivery methods can also be used, such as biolistic delivery or ex vivo treatment. Ex vivo treatment. For example, in ex vivo treatment, antigen-presenting cells (APCs), dendritic cells, peripheral blood mononuclear cells, or bone marrow cells are collected from a patient or a suitable donor and activated by applying the pharmaceutical composition in vitro, and then re-infused into the patient's body.

[0082] As used herein, the term "therapeutically effective amount" refers to the dose of the pharmaceutical composition described in the present invention administered to a subject in an amount capable of achieving a therapeutic or prophylactic effect, and the "therapeutically effective amount" includes a "prophylactically effective amount".

[0083] According to a specific embodiment of the present invention, the cancers of the present invention include at least one selected from the following group: melanoma, fallopian tube cancer, brain cancer, small intestine cancer, esophageal cancer, lymphoma, gallbladder cancer, blood cancer, thyroid cancer, endocrine adenocarcinoma, oral cancer, liver cancer, biliary tract cancer, colon 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, colorectal 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 plasmacytoma.

[0084] According to a specific embodiment of the present invention, the cancers of the present invention refer to cancers in which the proliferation or metastasis of tumor cells can be inhibited after administration of the pharmaceutical composition of the present invention.

[0085] In the present invention, the term "cancer metastasis" refers to the detachment of cancer cells from the primary organ or primary site and their migration to another site anatomically separated. Metastatic cancer refers to cancer with the potential for metastasis or that has already metastasized. The metastatic cancer may spread to sites including but not limited to the liver, lungs, bones, lymph nodes, or peritoneal cavity. Compared with the treatment of early-stage cancer, the treatment of metastatic cancer is more difficult, and its disease progression and treatment process are often more complex.

[0086] In the present invention, the term "cancer recurrence" refers to the reappearance of cancer after a period of undetectability after cancer treatment. Recurrent cancer is cancer caused by such recurrence. In the case of recurrence, surgical resection treatment is often difficult, and even if surgical resection can be performed, large-scale surgery needs to be carried out. In addition, in such cases, chemotherapy and radiotherapy may also be limited.

[0087] The pharmaceutical composition of the present invention can be formulated into dosage forms such as capsules, tablets, granules, injections, ointments, powders, or beverages, and can be used in humans.

[0088] The pharmaceutical composition of the present invention includes, but is not limited to, being prepared into oral dosage forms according to conventional methods, such as powders, granules, capsules, tablets, and aqueous suspensions, as well as being prepared into topical medicaments, suppositories, and sterile injections, etc. The pharmaceutical composition of the present invention may contain pharmaceutically acceptable carriers. For oral administration, pharmaceutically acceptable carriers may include binders, glidants, disintegrants, diluents, solubilizers, dispersants, stabilizers, suspending agents, colorants, and flavoring agents, etc. For injections, it includes buffers, preservatives, analgesics, solubilizers, isotonic agents, and stabilizers, etc. For topical medicaments, it includes matrices, excipients, lubricants, and preservatives, etc. The pharmaceutical preparations of the present invention can be prepared into various dosage forms by mixing with the above-mentioned pharmaceutically acceptable carriers. For example, for oral administration, it can be prepared into tablets, lozenges, capsules, elixirs, suspensions, syrups, or wafers, etc. For injections, it can be prepared into single-dose ampoules or multi-dose vials, etc. In addition, it can also be prepared into solutions, suspensions, tablets, capsules, or sustained-release agents, etc.

[0089] Examples of carriers, excipients, and diluents suitable for the preparation include, but are not limited to: lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone (povidone), water, methyl paraben, propyl paraben, talc, magnesium stearate, or mineral oil, etc. In addition, the pharmaceutical composition also includes fillers, anti-caking agents, lubricants, wetting agents, flavoring agents, emulsifiers, and preservatives, etc.

[0090] The administration routes of the pharmaceutical composition of the present invention include, but are not limited to, the following: oral administration, intravenous administration, intramuscular administration, intra-arterial administration, intraosseous administration, intradural administration, intracardiac administration, transdermal administration, subcutaneous administration, intraperitoneal administration, intranasal administration, enteral administration, topical administration, sublingual administration, or rectal administration, etc. Oral administration or parenteral administration routes are preferred.

[0091] As used herein, "parenteral administration" includes administration routes such as subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, intra-articular injection, intra-synovial injection, intrasternal injection, intradural injection, intralesional injection, and intracranial injection or infusion, etc. The pharmaceutical composition of the present invention can also be prepared into suppositories for rectal administration.

[0092] The dosage of the pharmaceutical composition according to the present invention is adjusted according to various factors, including but not limited to: the activity of the compound, the age, weight, overall health status, gender, diet, administration time, administration route, excretion rate, drug combination, and the severity of the disease to be prevented or treated. The dosage can be appropriately selected by those skilled in the art according to factors such as the patient's condition, weight, disease severity, formulation type, administration route, and treatment duration, and the daily dosage is in the range of 0.0001 mg / kg - 50 mg / kg or in the range of 0.001 mg / kg - 50 mg / kg. It can be administered once a day or in multiple doses per day. The dosage is not limited to the range described in the present invention. The pharmaceutical composition according to the present invention can be made into forms such as pills, sugar-coated tablets, capsules, liquid preparations, gels, syrups, suspension emulsions, or suspensions.

[0093] According to another aspect of the present invention, the present invention provides a strain transformed with a recombinant vector, wherein each recombinant vector respectively contains a DNA construct encoding flagellin and a DNA construct encoding an immune enhancing protein.

[0094] In a specific embodiment of the present invention, the immune enhancer is one or more selected from the group consisting of: metal salt compounds (gel-type adjuvants), lipid particles (emulsified oil adjuvants), cytokines, chemokines, particulate adjuvants, and microbial adjuvants. Specifically, the immune enhancer is selected from the group consisting of: IFN-α2, IL-2, IL-15, IL-21, IL-12, CXCR3, CCR5, T cells, and B cells. More specifically, the immune enhancer is IL-15. More specifically, the immune enhancer is human IL-15, which is the gene or protein shown in Sequence 1 (SEQ ID NO: 1).

[0095] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the above-mentioned strain as an active ingredient.

[0096] According to another aspect of the present invention, the present invention provides a composition for preventing or treating cancer, as active ingredients: flagellin or a nucleotide encoding the flagellin, and an immune enhancing protein or a nucleotide encoding the immune enhancing protein.

[0097] In a specific embodiment of the present invention, the immunopotentiator in the composition is selected from the group consisting of: metal salt compounds (gel-type adjuvants), lipid particles (emulsified oil adjuvants), cytokines, chemokines, particulate adjuvants, and microbial adjuvants. Specifically, the immunopotentiator is selected from the group consisting of: IFN-α2, IL-2, IL-15, IL-21, IL-12, CXCR3, CCR5, T cells, and B cells. More specifically, the immunopotentiator is IL-15. Even more specifically, the immunopotentiator is human IL-15, which is the gene or protein shown in Sequence 1 (SEQ ID NO: 1).

[0098] The gene of the present invention can be delivered by a gene delivery vector, but can also be administered in the form of a translated polypeptide to produce an equivalent pharmacological effect.

[0099] According to the present invention, the term "immunopotentiator (adjuvant)" refers to an immunoadjuvant used in vaccine development to enhance antigenicity or capable of enhancing the non-specific immune response against an antigen, which can be applied to cancer treatment and other fields. It can utilize the host's inherent immune system to achieve various therapeutic purposes, including any substance that can activate immune cells and induce the death of disease-related cells (such as cancer cells).

[0100] According to the present invention, the term "anticancer protein" refers to a polypeptide substance that can directly or indirectly induce the death of cancer cells, and its examples include at least one selected from the group consisting of: toxin proteins, specific antibodies against cancer antigens or fragments thereof, tumor suppressor proteins, angiogenesis inhibitors, cancer-related antigens, prodrug-converting enzymes, pro-apoptotic proteins, and flagellin, but are not limited thereto.

[0101] According to the present invention, the term "cytokine" refers to a protein secreted by immune cells. The cytokines of the present invention include cytokines used in cancer immunotherapy to regulate the host immune response and induce the death of disease-related cells (such as cancer cells). Preferably, these cytokines may include, but are not limited to: IFN-α2, IL-2, IL-15, IL-21, and IL-12.

[0102] According to the present invention, the term "chemokine" refers to a molecule that can regulate the migration of cells between tissues and the localization and interaction of cells within tissues, and it includes any substance that can direct leukocytes to the tumor microenvironment to mediate the host's immune response to diseases (such as cancer). Preferably, it may be CXCR3, CCR5, etc., but is not limited thereto.

[0103] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising a recombinant strain and an immune checkpoint inhibitor as active ingredients.

[0104] In a specific embodiment of the present invention, the composition is characterized by co - administration.

[0105] In a specific embodiment of the present invention, the immune checkpoint inhibitor of the present invention comprises at least one selected from the group consisting of: cytotoxic T lymphocyte - associated antigen - 4 (CTLA - 4), programmed death protein 1 (PD - 1), programmed death ligand 1 (PD - L1), KIR, LAG3, CD137, OX40, CD47, CD276, CD27, and GITR.

[0106] In a more specific embodiment of the present invention, the immune checkpoint inhibitor is programmed death ligand 1 (PD - L1).

[0107] In the present invention, the term "programmed death ligand 1 (PD - L1)" refers to a protein located on the surface of cancer cells or hematopoietic cells, and the PD - L1 is also known as CD274 or B7 - H1. PD - L1 or PD - L2 located on the surface of cancer cells binds to the PD - 1 protein located on the surface of T cells, thereby inhibiting the attack of T cells on cancer cells. The immune checkpoint inhibitor blocks the immune escape mechanism of cancer cells by binding to the PD - 1 receptor on the surface of T cells.

[0108] In a specific embodiment of the present invention, the cancer comprises at least one selected from the group consisting of: melanoma, fallopian tube cancer, brain cancer, small intestine cancer, esophageal cancer, lymphoma, gallbladder cancer, blood cancer, thyroid cancer, endocrine adenocarcinoma, oral cancer, liver cancer, cholangiocarcinoma, colon cancer, rectal cancer, cervical cancer, ovarian cancer, kidney cancer, gastric cancer, duodenal cancer, prostate cancer, breast cancer, brain tumor, lung cancer, undifferentiated thyroid cancer, endometrial 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 plasmacytoma.

[0109] In a specific embodiment of the present invention, the composition is a pharmaceutical composition for inhibiting the growth or metastasis of cancer.

[0110] According to another aspect of the present invention, the present invention provides a method for preventing or treating cancer, comprising the step of: administering a recombinant strain to a subject in need. The recombinant strain comprises a gene encoding flagellin and a gene encoding an adjuvant protein.

[0111] According to another aspect of the present invention, there is provided the use of a recombinant strain comprising a gene encoding flagellin and a gene encoding an adjuvant protein for preventing or treating cancer.

[0112] Beneficial effects

[0113] The technical features and advantages of the present invention are as follows:

[0114] (a) The present invention provides a DNA construct, which comprises a gene encoding flagellin; and

[0115] a gene encoding an adjuvant protein.

[0116] (b) The present invention designs an attenuated Salmonella strain to produce an immune stimulatory substance in tumor tissues, thereby inducing a strong anti-tumor immune response, selectively killing only cancer cells for effective cancer treatment. This method significantly inhibits the size of primary tumors and metastatic tumors and can effectively act on a prophylactic or therapeutic composition for improving survival rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] Figure 1a Shows a schematic diagram of the engineered plasmid pBAD IL15 / FlaB according to an embodiment of the present invention.

[0118] Figure 1b Shows the expression analysis of murine IL15 / FlaB by SDS-PAGE electrophoresis, anti-FlaB immunoblotting, and anti-mouse IL15 immunoblotting experiments according to an experimental embodiment of the present invention.

[0119] Figure 1c Shows the expression analysis of human IL15 / FlaB by SDS-PAGE electrophoresis, anti-FlaB immunoblotting, and anti-human IL15 (anti-hIL15) immunoblotting experiments according to an experimental embodiment of the present invention.

[0120] Figure 2a Shows a schematic diagram of the IL15 / FlaB activity detection system for analyzing the bioactivity of in vitro secreted IL15 / FlaB according to an experimental embodiment of the present invention.

[0121] Figure 2b Shows the expression of TLR5 after transfection with p3XFlag-hTLR5 (co-transfecting pCMV-b-gal and pNF-kB-luc simultaneously) according to an experimental embodiment of the present invention.

[0122] Figure 2c Shows that the signal intensity of IL15 / FlaB interacts with cells transfected with TLR5 and induces the transcription of TLR5-mediated NF-kB according to an experimental embodiment of the present invention.

[0123] Figure 2dShows the regulation of the NF-kB and p-NF-kB (p-NF-kB) signaling pathways after quantification of IL15 / FlaB and immunoblot signals according to an experimental embodiment of the present invention.

[0124] Figure 3 Shows the results of in vitro activity evaluation of IL15 / FlaB according to an experimental embodiment of the present invention.

[0125] Figure 4 Shows the colonization distribution of SL-Lux_pBAD-IL15 / FlaB in tumors after injection of MC38 cells (1×10 6 cells / mouse).

[0126] Figure 5 Shows the anti-tumor effect of the recombinant strain after transplantation of MC38 tumor cells according to an experimental embodiment of the present invention.

[0127] Figure 6 Shows the results of tumor size changes and survival rates caused by secondary tumor implantation after MC38 tumor transplantation according to an experimental embodiment of the present invention.

[0128] Figure 7 Shows the tumor size and survival rate after tumor colonization following injection of CT26 cells (1×10 7 cfu / mouse).

[0129] Figure 8 Shows the tumor size and survival rate results after re-implanting the tumor into Balb / c mice after CT26 tumor suppression according to an experimental embodiment of the present invention.

[0130] Figure 9 Shows the tumor size of the untreated Balb / c group after re-implanting the tumor after CT26 tumor transplantation according to an experimental embodiment of the present invention.

[0131] Figure 10 Shows the tumor size of the SLpFlaB-treated Balb / c mouse group after re-implanting the tumor after CT26 tumor transplantation according to an experimental embodiment of the present invention.

[0132] Figure 11 Shows the tumor size of the SLphIL15 / FlaB-treated Balb / c mouse group after re-implanting the tumor after CT26 tumor transplantation according to an experimental embodiment of the present invention.

[0133] Figure 12Shows the tumor size of the group of Balb / c mice treated with SLpmIL15 / FlaB after re-implantation of tumors following CT26 tumor transplantation, according to an experimental embodiment of the present invention.

[0134] Figure 13 Shows the results before and after treatment in a 4T1 tumor model, according to an experimental embodiment of the present invention.

[0135] Figure 14 Shows the effect of IL15 / FlaB expression on the metastasis of 4T1 tumors, according to an experimental embodiment of the present invention.

[0136] Figure 15a Shows the anti-tumor effect on metastatic malignant tumor mice after combined treatment with engineered bacteria and anti-PD-L1 antibody, and shows the PD-L1 expression induced by 4T1-Luc cells and B16F10 cells. Before flow cytometry detection, cells were stained with anti-PD-L1 antibody and isotype control antibody.

[0137] Figure 15b Shows a schematic diagram of the combined immunotherapy protocol of engineered bacteria and anti-PD-L1 antibody according to an embodiment of the present invention. The figure marks the time points of bacterial injection (SL), L-arabinose (L-ara) administration (+), and antibody injection. Among them, "isotype" is the control antibody.

[0138] Figure 15c Shows the in vitro bioluminescence intensity (BLI) of 4T1-Luc tumors and tumor weight data on the 15th day, according to an embodiment of the present invention.

[0139] Figure 15d Shows the change in the size of 4T1-Luc tumors after treatment with engineered bacteria, according to an embodiment of the present invention.

[0140] Figure 15e Shows the Kaplan-Meier survival curve and survival rate of 4T1 tumor-bearing mice, according to an embodiment of the present invention.

[0141] Figure 15f Shows the results of the change in the size of B16F10 tumors after treatment with engineered bacteria, according to an embodiment of the present invention.

[0142] Figure 15g Shows the Kaplan-Meier survival curve and survival rate of B16F10 tumor-bearing mice, according to an embodiment of the present invention.

[0143] Optimal Embodiment

[0144] To evaluate the anti-cancer effect of the recombinant strain described in the present invention, the attenuated recombinant strain was used at 1×10 7CFU / mouse were intravenously injected into the CT26 mouse model. The tumor growth inhibitory effects of the recombinant strains SLpBAD, SLphIL15, SLpmIL15, SLpFlaB, SLphIL15 / FlaB, and SLpmIL15 / FlaB on CT26 cell-derived xenografts were evaluated.

[0145] The results showed that compared with the control group, the administration of the recombinant strains SLphIL15 / FlaB and SLpmIL15 / FlaB inhibited tumor growth and improved the survival rate of mice, confirming the anti-cancer efficacy of the recombinant strains SLphIL15 / FlaB and SLpmIL15 / FlaB.

[0146] Examples of the invention

[0147] The technical solution of the present invention will be described in detail below in conjunction with specific examples. The following examples are only used to explain the present invention, but the protection scope of the present invention is not limited to the content described in the following examples. Examples

[0148] [Preparation Example 1] Cancer cell line and culture conditions

[0149] The CT26 colon cancer cell lines CRL-2638 and HB-8064 (ATCC, USA), and the mouse colorectal adenocarcinoma cell line MC38 (from Massachusetts General Hospital and Harvard Medical School, USA; Chonnam National University, Korea) were used in this experiment.

[0150] Cell culture was carried out using high-glucose Dulbecco's modified Eagle's medium (DMEM; product number: #LM 001-05, Welgene, Korea), which contained 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, under the conditions of 37 °C and 5% CO2.

[0151] [Preparation Example 2] Construction of Salmonella strains carrying plasmids

[0152] The Salmonella strains used in the present invention were Salmonella typhimurium SLΔppGpp (ΔrelA, ΔspoT) and CNC018 (ΔrelA, ΔspoT, ΔSPI1, ΔSPI2), and both strains lacked ppGpp.

[0153] The plasmid was introduced into the Salmonella strain by electroporation. After transformation, each strain was inoculated into LB medium containing 100 μg / mL ampicillin and cultured overnight. Subsequently, the overnight culture was diluted 1:100 into fresh LB medium containing ampicillin and cultured in a shaking incubator at 200 rpm and 37 °C. After incubation, the culture was centrifuged to collect bacterial pellets, washed with PBS buffer and used for subsequent experiments.

[0154] [Preparation Example 3] Preparation of Experimental Animal Model

[0155] C57BL / 6 and BALB / c mice aged 5 - 6 weeks and weighing 20 - 30 g (Orient Co., Ltd., Korea) were selected. A tumor animal model was constructed by subcutaneously injecting MC38 or CT26 cells described in Preparation Example 1 into the flank region of the mice as described in Preparation Example 1.

[0156] To image the tumor model and evaluate the tumor size, anesthesia was performed using 2% isoflurane. During the surgery, ketamine (200 mg / kg) and xylazine (10 mg / kg) were administered.

[0157] Tumor volume (mm 3 ) was calculated according to the following formula: (length × height × width) / 2. When the tumor volume exceeded 1500 mm 3 , the mice were euthanized.

[0158] [Experimental Example 1] Protein Expression and Activity Evaluation of Recombinant Strains

[0159] Comparison between Recombinant Strains and Existing Strains

[0160] The recombinant strains SLphIL15 / FlaB and SLpmIL15 / FlaB, and the control strain SLpEmpty, were cultured overnight in LB liquid medium containing ampicillin. Subsequently, the culture was diluted 1:100 into fresh LB medium and continued to be cultured until the OD600 value reached 0.5 - 0.7. At this time, L - arabinose was added to the culture to a final concentration of 0.2%, and then cultured in a shaking incubator at 200 rpm and 37°C. The growth pattern of the strains was analyzed by measuring the change in OD600 value over time.

[0161] [Experimental Example 2] Verification of Immunological Effects of Recombinant Strains

[0162] To evaluate the immune effect of the recombinant strains, an in vitro cytotoxicity test was performed using the CTLL - 2 cell line. CTLL - 2 cells were treated with the recombinant strains SLphIL15 / FlaB and SLpmIL15 / FlaB, and the control strains hIL15, mIL15, and SLpEmpty, and then the proliferation rate of CTLL - 2 cells was analyzed, as Figure 3 shown.

[0163] As Figure 3As shown, different from the SLpEmpty strain with no change in CTLL-2 proliferation, the recombinant strain SLpmIL15 / FlaB expressing IL15 / FlaB significantly enhanced CTLL-2 cell proliferation, thus confirming the immune-enhancing effect of the SLpmIL15 / FlaB recombinant strain. The sequences of human and murine IL-15 are shown in Table 1.

[0164] Table 1

[0165]

[0166]

[0167] [Experimental Example 3] Evaluation of the tumor-targeting effect of recombinant strains

[0168] To evaluate the tumor-targeting effect of recombinant strains, in vivo experiments were conducted to evaluate the number of recombinant strains in tumors and show their targeting by imaging. In the MC38 mouse model, 1×10 7 attenuated SLΔppGpp-Lux recombinant strains were intravenously injected. Then, the SLΔppGpp-Lux bacteria in tumors were quantified, and fluorescence images of the strains expressed in tumors were obtained, as Figure 4 shown.

[0169] As Figure 4 shown, it was found that the recombinant strains specifically existed in tumors, confirming that the recombinant strains selectively targeted tumor cells.

[0170] [Experimental Example 4] Evaluation of the anti-tumor effect of recombinant strains (1)

[0171] To evaluate the anti-tumor effect of recombinant strains, in vivo experiments were conducted according to the method shown in Figure 5 to evaluate the growth inhibitory effects of SLpBAD, SLphIL 15, SLpmIL 15, SLpFlaB, SLphIL 15 / FlaB, and SLpmIL 15 / FlaB recombinant strains on the MC38 cell line. In the MC38 mouse model, each attenuated recombinant strain was intravenously injected at a dose of 1×10 7 CFU, and the results of tumor volume and mouse survival rate are shown in Figure 5 shown.

[0172] As Figure 5 shown, compared with the control group, administration of SLphIL15 / FlaB and SLpmIL15 / FlaB recombinant strains could significantly increase the tumor inhibition rate and mouse survival rate, thus confirming the anti-tumor effects of SLphIL15 / FlaB and SLpmIL15 / FlaB recombinant strains.

[0173] [Experimental Example 5] Evaluation of the Inhibition of Tumor Recurrence by Recombinant Strains

[0174] To evaluate the inhibitory effect of recombinant strains on tumor recurrence, in vivo experiments were conducted to evaluate the tumor inhibitory effects of SLphIL15 / FlaB and SLpmIL15 / FlaB recombinant strains on the MC38 cell line. In the MC38 mouse model, each recombinant strain was intravenously injected at a dose of 1×10 7 CFU. After complete tumor regression, MC38 cells were re-inoculated 90 days later. The size of the recurrent tumors and the survival rate of the mice were evaluated, as Figure 6 shown.

[0175] As Figure 6 shown, compared with the control group, the mice treated with SLphIL15 / FlaB and SLpmIL15 / FlaB recombinant strains showed significant inhibition of tumor regeneration and a significant increase in the survival rate of the mice, confirming the tumor recurrence inhibitory effects of SLphIL15 / FlaB and SLpmIL15 / FlaB recombinant strains.

[0176] [Experimental Example 6] Evaluation of the Antitumor Effects of Recombinant Strains (2)

[0177] To evaluate the antitumor effects of recombinant strains, in vivo experiments were conducted as Figure 7 shown to examine the growth inhibitory effects of recombinant strains such as SLpBAD, SLphIL 15, SLpmIL 15, SLpFlaB, SLphIL 15 / FlaB, and SLpmIL 15 / FlaB on the CT26 cell line. In the CT26 mouse model, each attenuated recombinant strain was intravenously injected at a dose of 1×10 7 CFU. The tumor volume and the survival rate of the mice were evaluated, as Figure 7 shown.

[0178] As Figure 7 shown, compared with the control group, the administration of SLphIL15 / FlaB and SLpmIL15 / FlaB recombinant strains significantly enhanced the tumor inhibitory effect and increased the survival rate of the mice, confirming the antitumor efficacy of SLphIL15 / FlaB and SLpmIL15 / FlaB recombinant strains.

[0179] [Experimental Example 7] Evaluation of the Inhibition of Tumor Recurrence by Recombinant Strains

[0180] To evaluate the inhibitory effect of recombinant strains on tumor recurrence, in vivo experiments were conducted to evaluate the tumor inhibitory effects of SLphIL 15 / FlaB and SLpmIL 15 / FlaB recombinant strains on the CT26 cell line. In the CT26 mouse model, each attenuated recombinant strain was intravenously injected at a dose of 1×10 7The dose of CFU was intravenously injected. After complete tumor regression, 90 days later, CT26 cells were re-inoculated, and the tumor size and mouse survival rate after re-inoculation were evaluated, as Figure 8 shown.

[0181] As Figure 8 shown, compared with the control group, the inhibitory effect on tumor regeneration in mice treated with SLphIL15 / FlaB and SLpmIL15 / FlaB recombinant strains was significantly enhanced, and the survival rate was increased, confirming the inhibitory effect of SLphIL15 / FlaB and SLpmIL15 / FlaB recombinant strains on tumor recurrence. In the untreated group and the SLpFlaB-treated group, tumor regeneration occurred in all mice. In contrast, 8 out of 11 mice (72.7%) in the SLphIL15 / FlaB group had tumor eradication, while 12 out of 14 mice (85.7%) in the SLpmIL15 / FlaB group had tumor eradication ( Figures 9 - 12 ).

[0182] [Experimental Example 8] Evaluation of the anti-metastatic effect of recombinant strains

[0183] To evaluate the anti-metastatic effect of recombinant engineered strains, in vivo experiments were conducted to evaluate the tumor inhibitory effect of SLpBAD, SLphIL15, SLpmIL15, SLpFlaB, SLphIL15 / FlaB and SLpmIL15 / FlaB recombinant strains on the 4T1-Luc cell line. Mice were implanted with luciferase-expressing 4T1-Luc cells to establish a tumor model. Subsequently, each attenuated recombinant strain was intravenously injected at a dose of 1×10 7 CFU. The tumor localization was as Figure 13 shown; the number of tumor nodules metastasized to the lungs was as Figure 14 shown.

[0184] As Figure 13 and 14 shown, mice treated with SLphIL15 / FlaB and SLpmIL15 / FlaB recombinant strains did not show tumor metastasis to the lungs, indicating that SLphIL15 / FlaB and SLpmIL15 / FlaB strains have a significant inhibitory effect on tumor metastasis.

[0185] [Experimental Example 9] Evaluation of the anti-tumor effect of recombinant strains combined with immune checkpoint inhibitors

[0186] To evaluate the anti-tumor effect of the combined treatment of the recombinant strain and the immune checkpoint inhibitor anti-PD-L1 antibody, in vivo experiments were conducted in 4T1-Luc and B16F10 tumor models. Mice were treated with PBS, anti-PD-L1 antibody, SLphIF(+)+isotype control antibody, SLphIF(+)+anti-PD-L1 antibody, SLpmIF(+)+isotype control, and SLpmIF(+)+anti-PD-L1 antibody, and tumor size and survival rate were evaluated. The results showed that mice treated with SLphIF(+) and SLpmIF(+) strains showed significantly greater tumor inhibition compared to the control group treated with PBS. Notably, compared with treatment with the recombinant strain alone, the SLphIF(+)+anti-PD-L1 antibody treatment group and the SLpmIF(+)+anti-PD-L1 antibody treatment group had a more significant tumor inhibition effect. In 4T1-Luc tumor model mice, the combination therapy also significantly increased the survival rate of the mice ( Figure 15d and 15e ). In addition, a significant increase in the survival rate was also observed in mice bearing B16F10 tumors treated with the combination therapy ( Figure 15f and 15g ).

[0187] Although the specific aspects of the technical solution of the present invention have been described in detail above, those skilled in the art should understand that these detailed descriptions only show the preferred embodiments and do not limit the scope of the present invention. Therefore, the true protection scope of the present invention should be defined by the appended claims and their equivalent technical solutions.

[0188] Industrial Applicability

[0189] The present invention aims to selectively kill only cancer cells to develop effective cancer prevention or treatment drugs. By modifying attenuated Salmonella strains to produce immune-stimulating substances in tumor tissues, it induces a strong anti-tumor immune response, which not only proves effective treatment of primary tumors but also effective treatment of metastatic cancers. Since the pharmaceutical composition of the present invention specifically targets and kills cancer cells in vivo, thereby increasing the survival rate, it is expected to be widely used in the field of cancer treatment.

[0190] Sequence Listing

[0191] SEQ ID NO: 1. Human IL-15

[0192]

[0193]

[0194] SEQ ID NO: 2. Mouse IL-15

[0195]

[0196] SEQ ID NO: 3. PelB (pectate lyase B)

[0197]

Claims

1. A DNA construct, characterized in that, The DNA construct comprises: a gene encoding flagellin; and a gene encoding an adjuvant protein.

2. The DNA construct according to claim 1, wherein The adjuvant is selected from the group consisting of: gel-type adjuvants, emulsified oil adjuvants, cytokines, chemokines, particulate adjuvants, and microbial adjuvants.

3. The DNA construct according to claim 1, characterized in that, The flagellin is selected from the group consisting of: flagellin A, flagellin B, flagellin C, flagellin D, and flagellin E.

4. A recombinant vector, characterized in that, The vector comprises the DNA construct according to any one of claims 1 to 3.

5. A cell, characterized in that, The recombinant vector according to claim 4 has been introduced into the cell.

6. The cell according to claim 5, wherein The strain is at least one strain selected from the group consisting of: Salmonella, Clostridium, Bifidobacterium, Listeria, Enterococcus, Yersinia, and Escherichia coli.

7. A pharmaceutical composition for preventing or treating cancer, characterized in that, The pharmaceutical composition comprises the strain according to 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, lymphoma, gallbladder cancer, blood cancer, thyroid cancer, endocrine adenocarcinoma, oral cancer, liver cancer, biliary tract cancer, colon 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, colorectal 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 plasmacytoma.

9. The pharmaceutical composition according to claim 7, characterized in that, The cancer treatment is for inhibiting cancer growth or metastasis.

10. A strain transformed with a recombinant vector, the recombinant vector respectively comprising a DNA construct encoding flagellin and a DNA construct encoding an immune-enhancing protein.

11. The strain according to claim 10, characterized in that, The adjuvant includes: gel-type adjuvants, emulsified oil adjuvants, cytokines, chemokines, particulate adjuvants, and microbial adjuvants.

12. A pharmaceutical composition for preventing or treating cancer, characterized in that, The pharmaceutical composition comprises the strain according to claim 5 or claim 10 as an active ingredient.

13. A composition for preventing or treating cancer, characterized in that, The composition comprises, as an active ingredient: flagellin or a nucleotide encoding the flagellin; and an adjuvant protein or a nucleotide encoding the adjuvant protein.

14. The composition according to claim 13, wherein The adjuvant includes: gel-type adjuvants, emulsified oil adjuvants, cytokines, chemokines, particulate adjuvants, and microbial adjuvants.

15. A pharmaceutical composition for preventing or treating cancer, characterized in that, The pharmaceutical composition comprises the strain according to claim 5 and an immune checkpoint inhibitor as active ingredients.

16. The pharmaceutical composition according to claim 15, wherein, The pharmaceutical composition is administered in a combined administration manner.

17. The pharmaceutical composition according to claim 15, wherein, The immune checkpoint inhibitor comprises at least one selected from the group consisting of: cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), programmed death receptor protein 1 (PD-1), programmed death ligand protein 1 (PD-L1), KIR, LAG3, CD137, OX40, CD47, CD276, CD27, and GITR.

18. The pharmaceutical composition according to claim 15, wherein, The cancer is at least one selected from the group consisting of melanoma, fallopian tube cancer, brain cancer, small intestine cancer, esophageal cancer, lymphoma, gallbladder cancer, blood cancer, thyroid cancer, endocrine adenocarcinoma, oral cancer, liver cancer, biliary tract cancer, colon 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, colorectal 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 plasmacytoma.

19. The pharmaceutical composition according to claim 15, characterized in that, The pharmaceutical composition inhibits cancer growth or metastasis.

20. A method for preventing or treating cancer, characterized in that, The method includes administering a recombinant engineered strain to a subject, wherein the recombinant engineered strain comprises: a gene encoding flagellin; and a gene encoding an adjuvant protein.

21. Use of a recombinant engineering strain, characterized in that, The recombinant engineered strain comprises: a gene encoding flagellin; and a gene encoding an adjuvant protein, and the use is for preventing or treating cancer.

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