KR102015573B1 - Salmonella for cancer treatment and use thereof - Google Patents

Abstract

The present invention relates to Salmonella strains for cancer treatment and the use thereof, specifically, the strain according to the present invention is Salmonella typhimurium KST0650 strain resistant to radiation has excellent survival rate under oxidative stress and grow and proliferate in cancer cells This is possible and moves tumor-specifically in vivo. In addition, the KST0652 strain prepared by transducing a vector expressing a fusion protein of SspH1 protein and spliced ATF6 protein under the radiation response promoter to the tumor-derived mouse in combination with radiation therapy in tumor-induced mice By inhibiting and increasing the survival rate, the strain can be usefully used for the treatment of cancer.

Description

Salmonella strain for cancer treatment and use thereof {SALMONELLA FOR CANCER TREATMENT AND USE THEREOF}

The present invention relates to Salmonella strains and their use for the treatment of cancer.

The incidence of cancer is increasing by more than 5% annually worldwide due to the low birthrate and aging social structure, environmental deterioration due to industrial development, and westernization of food culture. Since cancer has been regarded as the object of human conquest, research from cell-level basic research to omni-directional chemotherapy has been promoted worldwide. The mechanism of cancer is yet to be clearly understood, and its demand for anticancer drugs is exploding due to the difficulty of preventing and cure of recurrence, and huge research funds are being invested in research institutes and companies. However, the cost of high-cost chemotherapy is not only a direct medical cost but also an indirect cost due to the contraction of rehabilitation and patient care after the onset of disease.

On the other hand, after the cancer suppression phenomenon reported by bacteria about 100 years ago, the development of anti-cancer strains have been made a lot, and some research groups are already in the clinical stage. The cellular environment of cancer tissue, represented by low oxygen partial pressure, rich nutrients, and lack of immune response, is also a characteristic habitat for pathogenic strains that grow in the body. In particular, the necrotic region is located in the center of the cancer tissue, and drug delivery is not efficiently performed, and it is known to cause cancer metastasis. In this connection, Clostridium (Clostridium), Bifidobacterium (Bifidobacterium) and Salmonella (Salmonella) strain proliferation and integrated in the rich nutrients of the necromancer tick areas such as and, in this process, inhibiting reduction and the growth of cancer tissue It has been observed to be used as a live strain for chemotherapy.

For example, Bifidobacterium longum is orally administered to construct an angiogenesis inhibitor, endostatin, to solid cancer tissues, and then to anti-cancer antibiotic adriamycin and adriamycin. Combination therapy has been studied (Xu YF, et. al ., Cancer Gene Ther. 2007, 14 (2): 151-7; Hu B, et al ., Cancer Gene Ther. 2009, 16 (8): 655-63).

In 1997, Dr. Bermudes of Yale University cited the possibility of Salmonella as a new anticancer carrier, and various research teams, including the National Institute of Cancer Research (NCI), have reported the results of Salmonella's cancer growth inhibition.

However, the anti-cancer treatment using Salmonella, which has weakened pathogenicity, is concerned about side effects due to the remaining Salmonella remaining after tumor cell death. In addition, patients chronically infected with residual Salmonella can infect their surroundings as Salmonella carriers. In particular, patients with weakened immune function or the elderly may develop lethal pulmonary disease by Salmonella with weakened pathogenicity. In addition, the antibodies produced by the remaining Salmonella can cause arthritis, ophthalmitis, urethritis, and the like, which are autoimmune diseases in the body. On the other hand, the conventional anti-cancer Salmonella can target cancer cells, but can not survive within the cell, effective treatment and delivery of anti-cancer substances into the cell is difficult. Therefore, there is a need for the development of Salmonella strains excellent in viability even in cells.

In this connection, the Republic of Korea Patent No. 10-0614220 discloses a Salmonella strain expressing FLT3 ligand gene that result in antitumor activity (Salmonella typhimurium ) KCCM-10604 and a composition for treating anticancer containing the same, Korean Patent Registration No. 10-0818144, Salmonella ( Salmonella typhimurium ) strain expressing the interferon gamma protein inducing anticancer effect and anticancer treatment containing the same The composition for this is disclosed.

Therefore, the present inventors are developing a Salmonella strain having excellent viability in cancer cells in cancer cells, Salmonella typhimurium KST0650 strain resistant to radiation has excellent survival rate under oxidative stress and can grow and proliferate in cancer cells. It was confirmed that the tumor specific migration in vivo. In addition, the KST0652 strain prepared by transducing a vector expressing a fusion protein of the SspH1 protein and the spliced ATF6 protein under the radiation response promoter to the KST0650 strain, the tumor size of the mouse by combination with radiation therapy in tumor-inducing mice The present invention was completed by suppressing and increasing the survival rate.

It is an object of the present invention to provide a Salmonella typhimurium strain resistant to radiation and the use of said strain.

In order to achieve the above object, the present invention provides a Salmonella typhimurium strain deposited with accession number KCTC13346BP.

The present invention also provides a Salmonella typhimurium strain transduced with an expression vector expressing a fusion protein of SspH1 protein and spliced ATF6 protein protein.

In addition, the present invention provides a composition for treating cancer, an anticancer adjuvant and a drug carrier for cancer, comprising the strain according to the present invention.

In the strain according to the present invention, Salmonella typhimurium KST0650 strain resistant to radiation has excellent survival rate under oxidative stress, is capable of growing and proliferating in cancer cells, and moves specifically in tumors in vivo. In addition, the KST0652 strain prepared by transducing a vector expressing a fusion protein of SspH1 protein and spliced ATF6 protein under the radiation response promoter to the tumor-derived mouse in combination with radiation therapy in tumor-induced mice By inhibiting and increasing the survival rate, the strain can be usefully used for the treatment of cancer.

Figure 1 is a schematic diagram showing the process of selecting the KST0650 strain resistant to oxidative stress among the mutations generated by irradiation with Salmonella typhimurium KST0649 strain.
2 is a graph showing the survival rate under oxidative stress of mutants generated by irradiation with KST0649 strain.
3 is a result confirming that the KST0650 strain has a higher oxidative stress resistance than the parent strains.
4 is a graph (A) and micrograph (B) confirming that the KST0650 strain exhibited a higher survival rate than the parent strain in cancer cells.
Figure 5 is a graph of the results confirming the degree of attenuation by administering to a mouse strain with KST0650 PFU of 0.5x10 ⁶ (A) or 1.0x10 ⁶ (B).
6 is a graph (A) confirming the number of KST0650 strains present in tumor cells after administering the KST0650 strain to tumor-inducing mice, and a photograph (B) confirming the location of the KST0650 strain in tumor cell tissues.
Figure 7 is a schematic diagram showing the production method of Salmonella typhimurium KST0652 strain in which a fusion protein of SspH1 protein and spliced ATF6 protein is introduced.
8 is a result of confirming the expression level of the fusion protein of the SspH1 protein and the spliced ATF6 protein according to the irradiation time (A) or irradiation dose (B) of the KST0652 strain by Western blot.
9 is a graph showing the results of the administration of the KST0652 strain to tumor-inducing mice, irradiation with radiation, and measurement of tumor size (A) or survival rate (B) of mice.

Hereinafter, the present invention will be described in detail.

The present invention provides a Salmonella typhimurium strain deposited with accession number KCTC13346BP.

The strain may be resistant to radioactivity and oxidative stress.

In a specific embodiment of the present invention, the present inventors were exposed to Salmonella typhimurium KST0649 strain 1.2 kGy radiation to improve the growth rate of about 80 times more than the parent strain KST0650 strain is resistant to radiation and oxidative stress Were screened and deposited as accession number KCTC13346BP (see FIGS. 1 and 2).

In addition, the KST0650 strain is four times better than the Salmonella typhimurium strain KST0651 or parent strain KST0649, which is conventionally used for cancer treatment under oxidative stress conditions (see FIG. 3), and is specific to tumors. It was confirmed that the cells survived for 28 days in cancer cells (see FIG. 6).

The present invention also provides a Salmonella typhimurium strain transduced with an expression vector expressing a fusion protein of SspH1 protein and spliced ATF6 protein.

SspH1 protein according to the present invention is a cell secretion protein of Salmonella typhimurium strain, the SspH1 protein may be a polypeptide consisting of any sequence known in the art. Specifically, the SspH1 protein may be a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, and may include a variant or fragment thereof. The SspH1 protein may be a polypeptide having at least 70%, 80%, 90%, 95% or 98% homology with the amino acid sequence set forth in SEQ ID NO: 1.

Spliced ATF6 (sATF) protein according to the present invention means a spliced form of ATF6 protein. The sATF6 protein may be a polypeptide consisting of any sequence known in the art, and may include variants or fragments thereof. In one example, the sATF6 protein may be derived from a mammal, eg, human, sheep, mouse, dog, rabbit, rat, cow, horse, and the like. Specifically, the sATF6 protein may be a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1. The sATF6 protein may be a polypeptide having at least 70%, 80%, 90%, 95% or 98% homology with the amino acid sequence set forth in SEQ ID NO: 2.

The fusion protein according to the present invention refers to a protein in a form in which SspH1 protein and sATF6 protein are fused. The fusion protein may be a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 3, and may include variants or fragments thereof. The fusion protein may be a polypeptide having at least 70%, 80%, 90%, 95% or 98% homology with the amino acid sequence set forth in SEQ ID NO: 3.

Polypeptides constituting the proteins according to the present invention may be variants or fragments of polypeptides having different sequences by deletion, insertion, substitution or combination of amino acid residues, to the extent that they do not affect the function of the protein. Amino acid exchange in proteins or peptides that do not alter the activity of the molecule as a whole is known in the art. In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, or the like.

The expression vector expressing a fusion protein of the SspH1 protein and the spliced ATF6 protein protein according to the present invention may further comprise a promoter. The promoter may be a promoter that is activated by radiation to increase the transcription of a lower gene, and promoters that are activated by radiation are well known in the art. The promoter activated by the radiation can be used by appropriate modification or selection by those skilled in the art. Specifically, the promoter may be any one or more selected from the group consisting of PepT, Hip-1, RecN, RecA, and RDRM.

The promoter may be operably linked to a gene encoding a protein to be expressed. The term "operably linked" means that a particular polynucleotide is linked to another polynucleotide to function. In other words, the operably linked gene encoding a particular protein means that the gene is transcribed into mRNA and translated into the protein by the action of the promoter.

Expression vectors according to the invention may further comprise a selection marker. Selection marker refers to a marker for the selection of transformed microorganisms or recombinant vectors, said selection marker imparting a selectable phenotype such as drug resistance, nutritional requirements, resistance to cytotoxic agents or expression of surface proteins. Can be used for When the expression vector including the selection marker is used, only cells expressing the selection marker survive in the environment in which the selection agent is treated, so that the transformed cells can be selected. Selective markers include herbicide resistance genes such as glyphosate, glufosinate ammonium or phosphinothricin, kanamycin, G418, bleomycin, hygromycin ( antibiotic resistance genes such as hygromycin) and chloramphenicol can be used. The selection marker may be used by appropriate modification or selection by those skilled in the art.

Expression vectors according to the invention can be transduced into strains by any method known in the art. In one example, the expression vector is a microinjection method, calcium phosphate precipitation method, CaCl ₂ method, electroporation method, liposome-mediated transfection method, Agrobacterium-mediated transfection method, DEAE-dextran treatment method and gene balm Transduction may be carried out using any one or more methods selected from the group consisting of.

In a specific embodiment of the present invention, we prepared a KST0652 strain by transducing the KST0650 strain with an expression vector prepared so that the fusion protein of the SspH1 protein and the spliced ATF6 protein could be expressed under the RecN promoter (FIG. 7). Reference).

The present invention also provides a composition for treating cancer comprising any one or more of the strains according to the present invention.

Strains that can be included in the composition for treating cancer, anticancer adjuvant and anticancer drug delivery according to the present invention are Salmonella typhimurium strain deposited with accession number KCTC13346BP and a fusion protein of SspH1 protein and spliced ATF6 protein in the strain. Expression vector expressing the may be any one or more selected from the group consisting of the transduced strain. The strain may have the characteristics as described above.

In a specific embodiment of the present invention, the inventors irradiated Salmonella typhimurium LT2 wild-type strains to select KST0650 strains resistant to radioactivity and oxidative stress.

In addition, the KST0650 strain is four times better than the Salmonella typhimurium strain KST0651 or parent strain KST0649, which is conventionally used for cancer treatment under oxidative stress conditions (see FIG. 3), and is specific to tumors. It was confirmed that they survived for 28 days in cancer cells (see FIG. 6).

Meanwhile, the KST0652 strain was prepared by transducing the KST0650 strain with an expression vector prepared so that the fusion protein of the SspH1 protein and the spliced ATF6 protein was expressed under the RecN promoter.

The KST0652 strain increased the expression level of SspH1 protein and spliced ATF6 protein inserted into the expression vector according to the radiation dose and radiation exposure time when exposed to radiation (see FIG. 8).

In addition, when the KST0652 strain was administered to the tumor-inducing mice and irradiated, the tumor size in the mouse was reduced, and the survival rate of the tumor-inducing mice was increased (FIG.

In addition, the tumor size of the mice injected with KST0652 strain was reduced by about 40% when the radiation was irradiated, and it was confirmed that the survival rate was maintained at 100% by 30 days (see FIG. 9).

Therefore, KST0650 and KST0652 strains according to the present invention from the above can be usefully used for cancer treatment.

The composition may include 10 to 95% by weight of the strain according to the present invention as an active ingredient based on the total weight of the composition. In addition, the composition of the present invention may further contain at least one active ingredient exhibiting the same or similar function in addition to the above-mentioned effective ingredient.

The compositions of the present invention may also include carriers, diluents, excipients or combinations of two or more commonly used in biological agents. Pharmaceutically acceptable carriers are not particularly limited so long as they are suitable for delivery of the composition in vivo, see, eg, Merck Index, 13th ed., Merck & Co. Inc. The compound, saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol or one or more of these components may be mixed. At this time, if necessary, other conventional additives such as antioxidants, buffers, bacteriostatic agents, and the like may be added.

When formulating the composition, it is prepared using commonly used diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants.

The composition of the present invention may be formulated as an oral or parenteral preparation. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, troches, and the like, which may comprise at least one excipient such as starch, calcium carbonate, sucrose, It may be prepared by mixing lactose and gelatin. In addition, lubricants such as magnesium styrate and talc may also be added. Meanwhile, liquid preparations include suspensions, solvents, emulsions, or syrups, and may include excipients such as wetting agents, sweeteners, fragrances, and preservatives.

Formulations for parenteral administration may include injections such as sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, and the like.

As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.

The composition of the present invention may be administered orally or parenterally according to a desired method, and parenteral administration may be external or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection. Can be selected.

The composition according to the invention is administered in a pharmaceutically effective amount. This may vary depending on the type of disease, the severity, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, the drug being used simultaneously, and the like. The composition of the present invention may be administered alone or in combination with other therapeutic agents. In combination administration, administration may be sequential or simultaneous.

However, for the desired effect, the amount of the active ingredient included in the composition according to the present invention may be 0.001 to 10,000 mg / kg, specifically 0.1 to 5 g / kg. The administration may be once a day or may be divided several times.

The present invention also provides an anticancer adjuvant comprising any one or more of the strains according to the present invention.

Strains that may be included in the composition for treating cancer, anticancer adjuvant and anticancer drug delivery according to the present invention include Salmonella typhimurium strain deposited with accession number KCTC13346BP and a fusion protein of SspH1 protein and spliced ATF6 protein in the strain. Expression vector expressing the may be any one or more selected from the group consisting of the transduced strain. The strain may have the characteristics as described above.

As used herein, the term "anticancer adjuvant" refers to an agent that can improve, enhance or enhance the anticancer effect in combination with radiotherapy, chemotherapy or surgical treatment. Thus, the anticancer adjuvant according to the present invention can be used to treat cancer by being treated in a patient simultaneously or sequentially with radiation therapy, chemotherapy or surgical treatment.

In a specific embodiment of the present invention, the inventors irradiated Salmonella typhimurium LT2 wild-type strains to select KST0650 strains resistant to radioactivity and oxidative stress.

In addition, the KST0650 strain is four times better than the Salmonella typhimurium strain KST0651 or parent strain KST0649, which is conventionally used for cancer treatment under oxidative stress conditions (see FIG. 3), and is specific to tumors. It was confirmed that they survived for 28 days in cancer cells (see FIG. 6).

Meanwhile, the KST0652 strain was prepared by transducing the KST0650 strain with an expression vector prepared so that the fusion protein of the SspH1 protein and the spliced ATF6 protein was expressed under the RecN promoter.

The KST0652 strain increased the expression level of SspH1 protein and spliced ATF6 protein inserted into the expression vector according to the radiation dose and radiation exposure time when exposed to radiation (see FIG. 8).

In addition, when the KST0652 strain was administered to the tumor-inducing mice and irradiated, the tumor size in the mouse was reduced, and the survival rate of the tumor-inducing mice was increased (FIG.

In addition, the tumor size of the mice injected with KST0652 strain was reduced by about 40% when the radiation was irradiated, and it was confirmed that the survival rate was maintained at 100% by 30 days (see FIG. 9).

Therefore, KST0650 and KST0652 strains according to the present invention from the above can be usefully used as an anticancer adjuvant.

The anticancer adjuvant of the present invention increases the anticancer effect by radiation therapy, chemotherapy or surgical treatment, and suppresses or ameliorates side effects caused by radiation therapy, chemotherapy or surgical treatment or the like. It can be used in combination with surgical treatment.

The route of administration of the anticancer adjuvant may be administered through any general route as long as it can reach the target tissue. The anticancer adjuvant of the present invention may be administered as intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, intranasal, pulmonary, rectal as desired. In addition, the anticancer adjuvant may be administered by any device that allows the active substance to migrate to the target cell.

The anticancer adjuvant may be formulated as a more effective and safe formulation.

The present invention also provides an anticancer drug carrier comprising any one or more of the strains according to the present invention.

Strains that can be included in the composition for treating cancer, anticancer adjuvant and anticancer drug delivery according to the present invention are Salmonella typhimurium strain deposited with accession number KCTC13346BP and a fusion protein of SspH1 protein and spliced ATF6 protein in the strain. Expression vector expressing the may be any one or more selected from the group consisting of the transduced strain. The strain may have the characteristics as described above.

In a specific embodiment of the present invention, the inventors irradiated Salmonella typhimurium LT2 wild-type strains to select KST0650 strains resistant to radioactivity and oxidative stress.

In addition, the KST0650 strain is four times better than the Salmonella typhimurium strain KST0651 or parent strain KST0649, which is conventionally used for cancer treatment under oxidative stress conditions (see FIG. 3), and is specific to tumors. It was confirmed that they survived for 28 days in cancer cells (see FIG. 6).

Therefore, the KST0652 strain and the KST0652 strain transduced with an expression vector expressing a fusion protein of SspH1 protein and spliced ATF6 protein protein in the KST0650 strain and the KST0650 strain can be used as an anticancer drug carrier.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are only for illustrating the present invention, and the contents of the present invention are not limited thereto.

Screening for strains with radiation resistance and confirming their effects

<1-1> Strain Selection

The Salmonella typhimurium KST0649 strain was irradiated with radiation to select a strain resistant to radiation in the following manner (Fig. 1).

Attenuated Salmonella Typhimurium thyphimurium ), KST0649 (Accession No. KCTC13345BP) strain derived from the strain KST0567, a bioprotective preserver of the Tokai University Medical Department, was inoculated into Luria Bertani (LB) broth and absorbed at a wavelength of 600 nm. Incubation was made until 0.5. After irradiating 1.2 kGy of radiation to the cultured KST0649 strain for 1 hour, the culture medium of the strain was plated and cultured in an LB solid medium containing 10 mM hydrogen peroxide. After 3 days of culture, 16 colonies were each cultured in LB liquid medium, and the cultured strains were added and cultured to the culture medium of CT26 cancer cells. A replication assay was performed using the cultured cells, and finally, the strains having the highest replication ability among the strains derived from the 16 colonies were finally selected (FIG. 2). The finally selected strain was named Salmonella typhimurium KST0650 strain and deposited on September 20, 2017 as accession number KCTC13346BP.

<1-2> Oxidative  Confirmation of strain viability against stress

About oxidative stress, the viability of Salmonella typhimurium KST0650 strain finally selected in Example <1-1> was confirmed by the following method.

Specifically, KST0650 strains were inoculated in LB liquid medium and incubated overnight. The next day the cells incubated in fresh LB liquid medium were reinoculated and incubated at a wavelength of 600 nm until the absorbance reached 0.5. The cultured cells were diluted 10 times five times, and 0.01 ml of diluted culture was spotted in LB solid medium containing 5 or 10 mM concentration of hydrogen peroxide. After culturing the medium at 37 ° C. for 24 hours, the number of colonies formed in each medium was measured to compare the viability of the strains, and the results of the photographing are shown in FIG. 3. At this time, as a control, a strain of KST0649, which is a parent strain, and a strain of KST0651 (KCTC13347BP), which is a Salmonella typhimurium strain used in conventional cancer treatment, were used.

As shown in FIG. 3, under the oxidative stress condition formed by hydrogen peroxide, the KST0650 strain exhibited excellent survival rate of 100 times or more than the KST0651 strain and 10 times or more than the KST0649 strain (FIG. 3).

From this, it was found that the KST0650 strain increased resistance to hydrogen oxide by radiation exposure, thereby increasing viability in cancer cells.

<1-3> Strains in Cancer Cells Viability  Confirm

The viability of Salmonella typhimurium KST0650 strain finally selected in Example <1-1> in cancer cells was confirmed by the following method.

First, CT26 cells, which are mouse colon cancer cell lines, were prepared by suspending them in a DMEM culture medium containing antibiotics to give ⁵ × 10 ⁵ cells / ml, and 0.3 ml of the cell suspension was placed in 48 well plates and cultured. After replacing the culture medium of the cultured cells with culture medium containing no antibiotics, the KST0650 strain was infected with the amount of 10 ⁶ CFU. At this time, KST0649, KST0651 and LT2 wild type strains were inoculated as a control. Cells infected with the strain were incubated at 37 ° C. for 1 hour and then washed with phosphate buffer to remove Salmonella strains that did not adhere to CT26 cells. In addition, Salmonella strains attached to the cell walls were also removed by treating the 48 well plate with 0.01 mg / ml gentamycin. 2 and 18 hours after infection with Salmonella strains, Salmonella strains outside the cells were removed with saline and cells were fixed with 1% formaldehyde. Salmonella was fluorescently stained by adding and reacting a Salmonella specific antibody (Abcam, UK) to which the fluorescent material FITC was bound. On the other hand, the position of the cells was confirmed by staining the intracellular DNA using DAPI (4,6-diamidino-2phenylindole, Sigma).

As a result, as shown in Figure 4A, KST0650 strain was significantly lower cell infectivity than the parent strain KST0649 strain, but showed a prominent growth rate in cancer cells by maintaining a similar level of intracellular proliferative capacity (Fig. 4A). In addition, as shown in Figure 4B, KST0650 strain increased the number of strains proliferated compared to the number of infected strains (Figure 4B).

Therefore, it can be seen from the above that the KST0650 strain according to the present invention shows excellent survival rate in cancer cells.

<1-4> of strain Attenuation  Check the degree

The attenuation degree of Salmonella typhimurium KST0650 strain selected in Example <1-1> was confirmed by the following method.

First, the KST0650 strain was cultured and suspended in PBS, and then the strain suspension was administered to the abdominal cavity of BALB / c mice to be 0.5x10 ⁶ or 1.0x10 ⁶ CFU (colony forming unit). At this time, KST0649 and LT2 wild type strains were used as a control. The result of observing the survival period while breeding the mice administered the strain is shown in FIG.

As shown in Figures 5A and 5B, all mice administered the KST0650 strain survived for at least 10 days. However, mice that received the LT2 wild-type strain all died after 3-4 days, and mice that received the KST0649 strain died about 9% after 9 days only when administered in an amount of 1.0x10 ⁶ CFU. (FIG. 5).

Therefore, it was confirmed from the above that the KST0650 strain is more attenuated than the parent strain, so that it can be used more safely for cancer treatment .

<1-5> Confirmation of Tumor Specific Migration of Strains

Tumor-specific migration of the Salmonella typhimurium KST0650 strain selected in Example <1-1> was confirmed by the following method.

First, tumor-inducing mice were prepared by administering 10 ⁶ CT26 cell lines subcutaneously to 6-week-old BALB / c mice (Orient Bio), and raising them until the size of tumors formed in the mice became 5 mm. It was. Meanwhile, the KST0650 strain was incubated overnight in LB liquid medium, and then the strain cultured the next day was reinoculated with fresh LB medium. Inoculated strains were incubated at a wavelength of 600 nm until the absorbance reached 0.5, centrifuged and washed with PBS. Washed strains were prepared by resuspending in PBS. The suspended strains were administered intraperitoneally of the tumor-inducing mice prepared above to make 10 ⁶ CFU. Tumor tissue was obtained by sacrifice of 5 mice every day after administration of the strain. The tumor tissue obtained was crushed and the crushed tumor tissue was suspended in PBS. The suspension was plated on an LB solid medium to measure the number of viable cells. At this time, by taking a portion of the tumor tissue and staining with Salmonella specific antibody according to a conventional method, Salmonella present in the tumor tissue was observed under a fluorescence microscope.

As a result, as shown in Figure 6, the KST0650 strain administered to the abdominal cavity of the mouse was present in a large number of tumor tissues, and survived for 28 days (Figure 6). Therefore, it can be seen from the above that the KST0650 strain is tumor-specifically moved in the body.

SspH1: sATF6  Preparation of the strain into which the gene is introduced and the antitumor activity of the strain

<2-1> Preparation of strain

To produce a strain capable of killing cancer cells more efficiently upon irradiation, the strain expresses the cell secreting protein SspH1 protein and the spliced ATF6 protein (sATF) in a fused form. Vectors were transduced.

Specifically, the radiation response promoter of Salmonella under the same conditions as described in Table 2 using the RecN-F (SEQ ID NO: 4) and RecN-R (SEQ ID NO: 5) primers shown in Table 1 as a template with the gene of the LT2 wild type strain as a template. Obtained. The obtained radiation response promoter was inserted into the pET28a vector using BglII and XbaI restriction enzymes. In addition, the SspH1 gene was obtained under the same conditions as described in Table 2 using the SspH1-F (SEQ ID NO: 6) and SspH1-R (SEQ ID NO: 7) primers described in Table 1 below as a template of the gene of the LT2 wild type strain. The SspH1 gene was inserted into the vector into which the radiation response promoter was inserted using NcoI and NotI restriction enzymes. On the other hand, in order to obtain the sATF6 gene, mRNA was obtained from CT26 cells, and cDNA was synthesized from the mRNA. Using the synthesized cDNA as a template, the sATF6 gene was obtained under the same conditions as described in Table 2 using the sATF6-F (SEQ ID NO: 8) and sATF6-R (SEQ ID NO: 9) primers described in Table 1 below. The sATF6 gene was inserted into a vector into which the radiation response promoter and the SspH1 gene were inserted using NotI and XhoI restriction enzymes to prepare a pET-RecN-SspH1: sATF6 vector.

name Sequence (5 '→ 3') SEQ ID NO: RecN-F AAA GAT CTG AGG TTT ATA TTG ATG SEQ ID NO: 4 RecN-R TTT CTA GAA GCT GTT TTC CTG TAT G SEQ ID NO: 5 SspH1-F TAC CAT GGT TAA TAT CCG CAA TAC SEQ ID NO: 6 SspH1-R TTG CGG CCG CTT CTG TGC ATT CCT CTC SEQ ID NO: 7 sATF6-F AAG CGG CCG CAA TGG AGT CGC CTT TTA G SEQ ID NO: 8 sATF6-R TTC TCG AGA CTT GGG ACT TTG AGC C SEQ ID NO: 9

Temperature time Repeat count 94 ℃ 5 minutes 1 cycle 94 ℃ 0.5 minutes 30 cycles 56.2 ℃ 0.5 minutes 72 ℃ 0.5 minutes 72 ℃ 10 minutes 1 cycle

The prepared pET-RecN-SspH1: sATF6 vector was transfected into the Salmonella typhimurium KST0650 strain selected in the above-described <1-1> strain to express a Salmonella typhimurium strain expressing the SspH1: sATF6 fusion protein. Obtained. The resulting strain was named KST0652 (FIG. 7).

<2-2> by irradiation SspH1: sATF6  Confirmation of expression level change of fusion protein

Irradiation of the strain prepared in Example <2-1> was confirmed by the following method to change the expression of the SspH1: sATF6 fusion protein.

Specifically, KST0652 strains were inoculated into LB liquid medium and incubated at a wavelength of 600 nm until the absorbance became 0.5. SspH1: sATF6 fusion with cultured KST0652 strains irradiated with 0.5, 1, 1.5 or 2 Gy of radiation for 1 hour or 2 Gy of radiation for 0, 0.5, 1.0, 1.5 or 2 hours The expression change of the protein was confirmed. The radiation was centrifuged at 10,000 g for 5 minutes to obtain cells and cultures, respectively. 30 μl of SDS sample buffer was added to the obtained cells and culture medium, and denatured at 100 ° C. for 10 minutes, followed by electrophoresis on 12% SDS polyacrylamide gel. After the electrophoresis, the protein present in the gel was transferred to the nitrocellulose membrane. The membrane was blocked with a buffer containing 3% bovine serum albumin and after 1 hour, rabbit anti-mouse ATF6 (Cat # .PRS3681, Sigma, USA) antibody was added as a primary antibody. The reaction was carried out for 1 hour. After the reaction, the membrane was washed three times at 10 minute intervals using a Tri-buffered saline with 0.1% Tween-20 (TBST) solution. HRP-bound IgG secondary antibody (horseradish peroxidase (HRP) -conjugated goat IgG secondary antibody; Bio-Rad, Berkeley, California, USA) was added to the washed membrane for 1 hour. After the reaction, the membrane was washed three times in the same manner as above, and then sensitized onto an X-ray film using a BM chemiluminescence blotting substrate (POD; Roche, Germany), followed by development to express the amount of sATF6 protein. The result of confirming is shown in FIG.

As shown in FIG. 8, the expression level of SspH1: sATF6 fusion protein increased in proportion to the irradiation dose and irradiation time (FIG. 8). Therefore, it was confirmed from the above that the KST0651 strain expressing the SspH1: sATF6 fusion protein can be used for cancer treatment in combination with radiation therapy of cancer patients.

<2-3> Confirmation of Survival and Tumor Size Changes in Mice Following Irradiation

Mouse survival rate and tumor size change when cancer strains were administered in Example <2-1> together with radiation therapy were confirmed by the following method.

First, the KST0650 strain was administered to tumor-inducing mice by the same methods and conditions as in Example <1-5> except that the KST0652 strain was used instead of the KST0650 strain. The tumor-inducing mice were irradiated with gamma rays of 2 Gy for 2 days after the administration of the strain. Gamma irradiation was performed using gamma cells, and a total of 6 Gy gamma rays were irradiated to mice. In this case, as a control group, the group irradiated with radiation alone without administration of the KST0650 strain was used. Tumor size was measured using a caliper every day from the day following injection of the strain, and the survival rate of the mice was calculated by counting the number of surviving mice for 30 days from the day following injection of the strain. The results of confirming the change in tumor size and survival rate are shown in FIG. 9.

As shown in FIG. 9A, the tumor size was reduced by about 80% in the experimental group administered with the KST0652 strain and irradiated with radiation, and the tumor killing effect was superior to the radiation-treated control group (FIG. 9A). In addition, as shown in FIG. 9B, the control group irradiated with radiation only survived about 60% of the mice for 30 days, whereas the experimental group administered with the KST0652 strain and irradiated with 100% mice survived for 30 days (FIG. 9B). ).

Therefore, it was confirmed from the above that the Salmonella strain according to the present invention can be used in the treatment of cancer in combination with radiation.

Korea Institute of Bioscience and Biotechnology Biological Resource Center KCTC13345BP 20170915 Korea Institute of Bioscience and Biotechnology Biological Resource Center KCTC13346BP 20170915 Korea Institute of Bioscience and Biotechnology Biological Resource Center KCTC13347BP 20170915 Korea Institute of Bioscience and Biotechnology Biological Resource Center KCTC13348BP 20170915

<110> KOREA ATOMIC ENERGY RESEARCH INSTITUTE <120> SALMONELLA FOR CANCER TREATMENT AND USE THEREOF <130> 2017P-07-044 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 140 <212> PRT <213> Salmonella typhimurium <400> 1 Met Val Asn Ile Arg Asn Thr Gln Pro Ser Val Ser Met Gln Ala Ile   1 5 10 15 Ala Gly Ala Ala Ala Pro Glu Ala Ser Pro Glu Glu Ile Val Trp Glu              20 25 30 Lys Ile Gln Val Phe Phe Pro Gln Glu Asn Tyr Glu Glu Ala Gln Gln          35 40 45 Cys Leu Ala Glu Leu Cys His Pro Ala Arg Gly Met Leu Pro Asp His      50 55 60 Ile Ser Ser Gln Phe Ala Arg Leu Lys Ala Leu Thr Phe Pro Ala Trp  65 70 75 80 Glu Glu Asn Ile Gln Cys Asn Arg Asp Gly Ile Asn Gln Phe Cys Ile                  85 90 95 Leu Asp Ala Gly Ser Lys Glu Ile Leu Ser Ile Thr Leu Asp Asp Ala             100 105 110 Gly Asn Tyr Thr Val Asn Cys Gln Gly Tyr Ser Glu Ala His Asp Phe         115 120 125 Ile Met Asp Thr Glu Pro Gly Glu Glu Cys Thr Glu     130 135 140 <210> 2 <211> 360 <212> PRT <213> Artificial Sequence <220> <223> spliced ATF6 <400> 2 Met Glu Ser Pro Phe Ser Pro Val Leu Pro His Gly Pro Asp Glu Asp   1 5 10 15 Trp Glu Ser Thr Leu Phe Ala Glu Leu Gly Tyr Phe Thr Asp Thr Asp              20 25 30 Asp Val His Phe Asp Ala Ala His Glu Ala Tyr Glu Asn Asn Phe Asp          35 40 45 His Leu Asn Phe Asp Leu Asp Leu Met Pro Trp Glu Ser Asp Leu Trp      50 55 60 Ser Pro Gly Ser His Phe Cys Ser Asp Met Lys Ala Glu Pro Gln Pro  65 70 75 80 Leu Ser Pro Ala Ser Ser Ser Cys Ser Ile Ser Ser Pro Arg Ser Thr                  85 90 95 Asp Ser Cys Ser Ser Thr Gln His Val Pro Glu Glu Leu Asp Leu Leu             100 105 110 Ser Ser Ser Gln Ser Pro Leu Ser Leu Tyr Gly Asp Ser Cys Asn Ser         115 120 125 Pro Ser Ser Val Glu Pro Leu Lys Glu Glu Lys Pro Val Thr Gly Pro     130 135 140 Gly Asn Lys Thr Glu His Gly Leu Thr Pro Lys Lys Lys Ile Gln Met 145 150 155 160 Ser Ser Lys Pro Ser Val Gln Pro Lys Pro Leu Leu Leu Pro Ala Ala                 165 170 175 Pro Lys Thr Gln Thr Asn Ala Ser Val Pro Ala Lys Ala Ile Ile             180 185 190 Gln Thr Leu Pro Ala Leu Met Pro Leu Ala Lys Gln Gln Ser Ile Ile         195 200 205 Ser Ile Gln Pro Ala Pro Thr Lys Gly Gln Thr Val Leu Leu Ser Gln     210 215 220 Pro Thr Val Val Gln Leu Gln Ser Pro Ala Val Leu Ser Ser Ala Gln 225 230 235 240 Pro Val Leu Ala Val Thr Gly Gly Ala Ala Gln Leu Pro Asn His Val                 245 250 255 Val Asn Val Leu Pro Ala Pro Val Val Ser Ser Pro Val Asn Gly Lys             260 265 270 Leu Ser Val Thr Lys Pro Val Leu Gln Ser Ala Thr Arg Ser Met Gly         275 280 285 Ser Asp Ile Ala Val Leu Arg Arg Gln Gln Arg Met Ile Lys Asn Arg     290 295 300 Glu Ser Ala Cys Gln Ser Arg Lys Lys Lys Lys Glu Tyr Met Leu Gly 305 310 315 320 Leu Glu Ala Arg Leu Lys Ala Ala Leu Ser Glu Asn Glu Gln Leu Lys                 325 330 335 Lys Glu Asn Gly Ser Leu Lys Arg Gln Leu Asp Glu Val Val Ser Glu             340 345 350 Asn Gln Arg Leu Lys Val Pro Ser         355 360 <210> 3 <211> 503 <212> PRT <213> Artificial Sequence <220> S223H: sATF6 fusion protein <400> 3 Met Val Asn Ile Arg Asn Thr Gln Pro Ser Val Ser Met Gln Ala Ile   1 5 10 15 Ala Gly Ala Ala Ala Pro Glu Ala Ser Pro Glu Glu Ile Val Trp Glu              20 25 30 Lys Ile Gln Val Phe Phe Pro Gln Glu Asn Tyr Glu Glu Ala Gln Gln          35 40 45 Cys Leu Ala Glu Leu Cys His Pro Ala Arg Gly Met Leu Pro Asp His      50 55 60 Ile Ser Ser Gln Phe Ala Arg Leu Lys Ala Leu Thr Phe Pro Ala Trp  65 70 75 80 Glu Glu Asn Ile Gln Cys Asn Arg Asp Gly Ile Asn Gln Phe Cys Ile                  85 90 95 Leu Asp Ala Gly Ser Lys Glu Ile Leu Ser Ile Thr Leu Asp Asp Ala             100 105 110 Gly Asn Tyr Thr Val Asn Cys Gln Gly Tyr Ser Glu Ala His Asp Phe         115 120 125 Ile Met Asp Thr Glu Pro Gly Glu Glu Cys Thr Glu Ala Ala Ala Met     130 135 140 Glu Ser Pro Phe Ser Pro Val Leu Pro His Gly Pro Asp Glu Asp Trp 145 150 155 160 Glu Ser Thr Leu Phe Ala Glu Leu Gly Tyr Phe Thr Asp Thr Asp Asp                 165 170 175 Val His Phe Asp Ala Ala His Glu Ala Tyr Glu Asn Asn Phe Asp His             180 185 190 Leu Asn Phe Asp Leu Asp Leu Met Pro Trp Glu Ser Asp Leu Trp Ser         195 200 205 Pro Gly Ser His Phe Cys Ser Asp Met Lys Ala Glu Pro Gln Pro Leu     210 215 220 Ser Pro Ala Ser Ser Ser Cys Ser Ile Ser Ser Pro Arg Ser Thr Asp 225 230 235 240 Ser Cys Ser Ser Thr Gln His Val Pro Glu Glu Leu Asp Leu Leu Ser                 245 250 255 Ser Ser Gln Ser Pro Leu Ser Leu Tyr Gly Asp Ser Cys Asn Ser Pro             260 265 270 Ser Ser Val Glu Pro Leu Lys Glu Glu Lys Pro Val Thr Gly Pro Gly         275 280 285 Asn Lys Thr Glu His Gly Leu Thr Pro Lys Lys Lys Ile Gln Met Ser     290 295 300 Ser Lys Pro Ser Val Gln Pro Lys Pro Leu Leu Leu Pro Ala Ala Pro 305 310 315 320 Lys Thr Gln Thr Asn Ala Ser Val Pro Ala Lys Ala Ile Ile Ile Gln                 325 330 335 Thr Leu Pro Ala Leu Met Pro Leu Ala Lys Gln Gln Ser Ile Ile Ser             340 345 350 Ile Gln Pro Ala Pro Thr Lys Gly Gln Thr Val Leu Leu Ser Gln Pro         355 360 365 Thr Val Val Gln Leu Gln Ser Pro Ala Val Leu Ser Ser Ala Gln Pro     370 375 380 Val Leu Ala Val Thr Gly Gly Ala Ala Gln Leu Pro Asn His Val Val 385 390 395 400 Asn Val Leu Pro Ala Pro Val Val Ser Ser Pro Val Asn Gly Lys Leu                 405 410 415 Ser Val Thr Lys Pro Val Leu Gln Ser Ala Thr Arg Ser Met Gly Ser             420 425 430 Asp Ile Ala Val Leu Arg Arg Gln Gln Arg Met Ile Lys Asn Arg Glu         435 440 445 Ser Ala Cys Gln Ser Arg Lys Lys Lys Lys Glu Tyr Met Leu Gly Leu     450 455 460 Glu Ala Arg Leu Lys Ala Ala Leu Ser Glu Asn Glu Gln Leu Lys Lys 465 470 475 480 Glu Asn Gly Ser Leu Lys Arg Gln Leu Asp Glu Val Val Ser Glu Asn                 485 490 495 Gln Arg Leu Lys Val Pro Ser             500 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> RecN-F <400> 4 aaagatctga ggtttatatt gatg 24 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> RecN-R <400> 5 tttctagaag ctgttttcct gtatg 25 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SspH1-F <400> 6 taccatggtt aatatccgca atac 24 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> SspH1-R <400> 7 ttgcggccgc ttctgtgcat tcctctc 27 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> sATF6-F <400> 8 aagcggccgc aatggagtcg ccttttag 28 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> sATF6-R <400> 9 ttctcgagac ttgggacttt gagcc 25

Claims (12)

Salmonella typhimurium KST0652 strain transfected with Salmonella thyphimurium KST0650 strain deposited with accession number KCTC13346BP expressing a fusion protein of SspH1 protein and spliced ATF6 (spliced ATF6, sATF) protein .
The strain of claim 1, wherein the KST0650 and KST0652 strains are resistant to radiation.
delete The strain of claim 1, wherein the SspH1 protein is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1.
The strain of claim 1, wherein the spliced ATF6 protein is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2.
The strain of claim 1, wherein the fusion protein is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 3.
The strain of claim 1, wherein the expression vector further comprises a promoter activated by radiation.
The strain of claim 7, wherein the radiation response promoter is any one or more selected from the group consisting of RecN, RecA, and RDRM.
The strain of claim 1, wherein the Salmonella typhimurium KST0652 strain was deposited with accession number KCTC13348BP.
Claim 1 Salmonella typhimurium cancer composition comprising the strain KST0652.
An anticancer adjuvant comprising the Salmonella typhimurium KST0652 strain of claim 1.
Claim 1 drug delivery drug for cancer comprising Salmonella typhimurium KST0652 strain.

Images