KR102683896B1 - Expression Vector for Secreting GM-CSF and Anticancer Recombinant Microbials Transformed thereby - Google Patents

Abstract

The present invention relates to a genetic construct capable of secreting GM-CSF with immune-inducing ability in the body, and an anti-cancer strain transformed therewith that has anti-cancer activity by stably secreting GM-CSF in the body, More specifically, a genetic construct characterized in that the anti-sigma factor flgM gene and the gene encoding the GM-CSF protein are linked to encode a fusion protein of flgM and GM-CSF, and are transformed thereby to produce GM-CSF in the body. It relates to an anti-cancer strain that has anti-cancer activity through stable secretion.

Description

Expression vector for secreting GM-CSF and anticancer recombinant strain transformed thereby {Expression Vector for Secreting GM-CSF and Anticancer Recombinant Microbials Transformed thereby}

The present invention relates to a genetic construct capable of secreting GM-CSF with immune-inducing ability in the body, and an anticancer strain transformed with the genetic construct to stably secrete GM-CSF in the body, thereby having anticancer activity.

Cancer is a disease that ultimately threatens life as the proliferation of cells does not stop, burrows into surrounding tissues and destroys normal cells. As the human body's regulatory ability decreases due to aging, it becomes more vulnerable to cancer. In an aging society, cancer has consistently ranked first among all causes of death for the past 10 years since 2007, exceeding twice the death rate from heart disease, which ranked second. .

The most effective response system to treat disease is to strengthen immunity, but since cancer cells are not foreign invaders, they avoid the body's immune response without triggering it. Certain viruses or bacteria infect cancer cells more than normal cells, and infected bacteria can become targets of attack by immune cells. Accordingly, anticancer treatment methods have been proposed that intentionally infect specific viruses or bacteria to stimulate the body's immune response to fight against cancer cells. Infectious bacteria such as Shigella, Vibrio cholera, and pathogenic E. coli only penetrate the cells of the intestinal tract, but do not reach the liver and spleen, which are important organs that trigger immune responses. In contrast, Salmonella can invade the spleen and liver through lymph nodes and stimulate a systemic immune response. Specifically, Leschner et al. (J. Mol. Med. 2010, 88, 763-773) infected CT26 tumor-bearing mice with fluorescent Salmonella intravenously and then tracked the infection route over time. . As a result, immediately after infection, the whole body of the mouse was infected through the blood, and 20 minutes after infection, Salmonella bacteria accumulated in the spleen and liver, and after 24 hours, Salmonella bacteria were observed to accumulate concentrated only in the tumor tissue.

However, Salmonella is a representative bacteria that causes food poisoning and can cause sepsis due to infection, which can be life-threatening. Therefore, it is too pathogenic to be used directly for cancer treatment. A research team at Yale University in the United States announced that by genetically engineering Salmonella, it can be attenuated by removing toxicity while maintaining its tumor-attacking properties, and that tumors can be suppressed by inducing immune stimulation through injection of the attenuated Salmonella. However, attenuated Salmonella not only has low survival and immune-inducing ability, but also has a risk of converting the attenuated strain to wild-type Salmonella due to mutation, causing sepsis. In addition, due to lack of research and development funds, most anti-cancer treatments using attenuated Salmonella have been stopped or put on hold in the first stage of the clinical process.

The induction of sepsis, which can occur along with cancer treatment using bacteria such as salmonella, is a very important problem to overcome, and in addition, stimulation of the immune response must also be actively achieved. Accordingly, research is mainly focused on developing strains that can additionally express or suppress substances helpful for anticancer treatment along with attenuation and immune activation, and use them for anticancer treatment. Registered Patent No. 10-0852687 discloses a Salmonella strain expressing tumor necrosis factor alpha by transducing a tumor necrosis factor alpha protein vector into an attenuated Salmonella strain and an anti-cancer treatment composition containing the same, Registered Patent No. 10 No. -1750007 published a treatment for solid tumors using an attenuated Salmonella strain in which L-asparaginase can be selectively expressed at the tumor site.

GM-CSF (Granulocyte-macrophage colony-stimulating factor) is a cytokine secreted by macrophages, T cells, mast cells, natural killer cells, endothelial cells, and fibroblasts. GM-CSF induces the differentiation of monocytes into M1 type macrophages, thereby inducing an innate immune response, while maturing them into dendritic cells and interacting with CD8 T cells to induce an acquired immune response, thereby playing a pivotal role in the immune response. plays a role GM-CSF expressed in bacteria has been proven to be bioequivalent in function and is used in various fields, but a series of processes are required to purify GM-CSF in the bacterial cytoplasm after expression.

Japanese Patent No. 6124460 discloses a pharmaceutical composition for preventing or treating cancer that specifically activates immune activity against cancer by using a fusion protein of a cancer-specific antigen and a cytokine containing GM-CSF.

If GM-CSF is targeted by Salmonella instead of cancer-specific antigen to express GM-CSF, the anticancer effect can be expected to be even better because the anticancer effect obtained from immune activation by Salmonella can be obtained in addition to the anticancer activity of GM-CSF. there is. However, according to preliminary experiments, when GM-CSF is expressed in Salmonella, the strain itself dies, so it was difficult to expect to simultaneously express the anticancer efficacy of Salmonella and the anticancer efficacy by expressing GM-CSF.

Republic of Korea Patent No. 10-0852687 Republic of Korea Patent No. 10-1750007 Japanese Patent No. 6124460

Leschner et al., J. Mol. Med. 2010, 88, 763-773.

The present invention does not kill the transformed strain, The purpose is to provide a genetic construct that allows GM-CSF, which has an immune-inducing ability, to be expressed in the body.

Another object of the present invention is to provide an anti-cancer recombinant strain transformed with the above genetic construct to stably express GM-CSF and secrete it into the host's body to exhibit anti-cancer activity.

Another object of the present invention is to provide an anticancer composition using the anticancer strain.

The present invention to achieve the above-mentioned object relates to a genetic construct characterized in that the anti-sigma factor flgM gene and the gene encoding the GM-CSF protein are linked to encode a fusion protein of flgM and GM-CSF. Hereinafter, the fusion protein of flgM and GM-CSF is referred to as "flgM-GMCSF".

The genetic construct of the present invention can be transformed into a strain to express flgM-GMCSF, which has the immune-inducing ability of GM-CSF, and secrete it out of the cell. Therefore, it is possible to prevent host cell death due to GM-CSF expression, and a fusion protein with immune-inducing ability can be easily obtained from the culture medium without going through a series of processes to purify GM-CSF present in the cytoplasm of the strain. .

In the genetic construct of the present invention, the flgM gene and the gene encoding the GM-CSF protein may be directly linked or connected through a linker. In the following examples, the structure of SEQ ID NO: 1 is illustrated in which a gene sequence encoding 14 amino acids is connected via a linker, but the structure is not limited thereto, and it will be easy for those skilled in the art to appropriately modify the length or sequence of the linker.

To improve expression efficiency, a shine-dalgarno sequence can be placed at the front of the gene construct of the present invention. In addition, it is natural that appropriate restriction enzymes can be placed at the front and rear ends so that the gene construct of the present invention can be introduced into the vector.

The present invention also relates to recombinant expression vectors containing the above genetic constructs. In the present invention, a “vector” is an operably linked DNA preparation capable of expressing the genetic construct of the present invention in a suitable host, and may be a plasmid, phage particle, or potential genomic insert. In the specification of the present invention, plasmid and vector are sometimes used interchangeably. It is natural that the expression vector can be selected and used with high efficiency depending on the sequence of the gene construct.

In addition, the present invention relates to a recombinant strain that is transformed with the above genetic construct and secretes the fusion protein flgM-GMCSF. The recombinant strain of the present invention can be transformed using a recombinant expression vector containing the above genetic construct. Transformation methods follow conventional methods, for example, calcium chloride method, electroporation, etc. can be used, but are not limited thereto.

The recombinant strain of the present invention is characterized by the fact that the flgM secretion system not only expresses flgM-GMCSF encoded by the gene construct of the present invention within the cell, but also secretes it to the outside of the cell. Due to these characteristics, the GM-CSF expressed within the cell allows the strain to stably and continuously express/secrete the fusion protein without being killed, and also confirms that the secreted fusion protein maintains the immune-inducing ability of GM-CSF. I was able to. Therefore, by using the strain of the present invention, a fusion protein with immune-inducing ability can be easily obtained from the culture medium without going through a complex series of processes of separating/purifying from the bacterial cytoplasm.

Accordingly, the present invention also provides flgM-GMCSF, a fusion protein of flgM and GM-CSF encoded by the above genetic construct. In the fusion protein of the present invention, flgM and GM-CSF may be linked directly or through a linker, depending on the sequence of the corresponding gene structure.

The recombinant strain of the present invention can be used as a strain to produce flgM-GMCSF, but can also be used on its own to secrete flgM-GMCSF in vivo, allowing GM-CSF to exhibit therapeutic effects, including cancer. It can be used in the treatment of diseases. The strain used at this time is harmless to the human body, and any strain that can secrete flgM-GMCSF can be used, for example, E. coli, attenuated Salmonella, Shigella, Acinetobacter, Pseudomonas aeruginosa, Listeria, and Clos. Tridium, intestinal microorganisms (beneficial bacteria), probiotics, etc. can be used.

In the strain of the present invention, the genetic construct is preferably operably linked to an inducible promoter. As used herein, “inducible promoter” refers to a promoter capable of switching the operation of a linked gene by an inducer, for example, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp. Promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, pBAD promoter, Tet promoter, trc promoter, pepT promoter, sulA promoter, pol 11(dinA) promoter, ruv promoter, uvrA promoter, uvrB promoter, uvrD promoter, umuDC promoter , lexA promoter, cea promoter, caa promoter, recN promoter, pagC promoter, hip promoter, ansB promoter, or pflE promoter.

In particular, the strain of the present invention may be an attenuated Salmonella strain. “Attenuated” refers to artificially weakening the toxicity of a living pathogen, mutating the genes involved in essential metabolism of the pathogen so that it cannot cause disease in the body and only stimulates the immune system to induce immunity. Salmonella attenuation-causing genes are well known in the art and include aroA, aroC, aroD, aroE, Rpur, htrA, ompR, ompF, ompC, galE, cya, crp, cyp, phoP, phoQ, rfaY, dksA, hupA, selected from the group consisting of sipC, clpB, clpP, clpX, pab, nadA, pncB, pmi, rpsL, hemA, rfc, poxA, galU, cdt, pur, ssa, guaA, guaB, fliD, flgK, flgL, relA and spoT This can be achieved by loss of function of one or more genes. When applied in vivo, the function of two or more of the above genes may be lost in order to prevent reduction to the wild type and further weaken toxicity.

It is preferable that the genetic construct for transforming the recombinant strain of the present invention includes the flhDC gene. The flhDC gene is a main regulator of the flagellin operon gene and is known to regulate the expression of the flgM gene. Accordingly, as a result of including the flhDC gene in the gene construct, it was confirmed that the expression level of flgM-GMCSF was significantly increased.

Attenuated Salmonella strains are known to have anticancer activity by targeting tumor sites and inducing an immune response. Therefore, when transformed with the genetic construct of the present invention, it is targeted to the tumor site and secretes the flgM-GMCSF fusion protein, thereby simultaneously inducing anticancer activity by attenuated Salmonella and anticancer activity by GM-CSF at the tumor site, resulting in a more powerful anticancer activity. indicates activity.

In the following examples, when the genetic construct consisting of SEQ ID NO: 1 was transformed, the secretion ability of the flgM-GMCSF fusion protein was the best in ppGpp-attenuated Salmonella. However, the optimal attenuated Salmonella may vary depending on the sequence or length of the linker, and it will be easy for those skilled in the art to select it. When the flhDC gene was included in the genetic construct for transforming the attenuated Salmonella, it was confirmed that the secretion amount of flgM-GMCSF of the strain was significantly increased. Accordingly, in the examples below, the strain with the best flgM-GMCSF secretion ability was flgM It was named -gmCSF-flhDC-pBAD33/ppGpp- and deposited at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center on January 19, 2021 (accession number KCTC14455BP).

The present invention also relates to an anticancer composition containing the above recombinant strain as an active ingredient. When the recombinant strain is an attenuated Salmonella strain and contains an inducible promoter, when the strain is targeted to cancer tissue after administration of the strain, a drug that induces the operation of the inducible promoter is administered to fuse flgM-GMCSF from the strain. It can be used to induce secretion of protein.

The composition of the present invention can be prepared by a method known in the pharmaceutical field for use as an anti-cancer drug, either by itself or by mixing with a pharmaceutically acceptable carrier, forming agent, diluent, etc. can be used

The composition of the present invention can be formulated and used as a preparation for oral or parenteral administration. The dosage of the active ingredient according to the present invention can be appropriately selected depending on the absorption of the active ingredient in the body, the form of the preparation, the patient's age, gender and condition, the severity of symptoms, etc., and the administration may be done once a day. , may be administered in several divided doses. The typical dosage is 0.001mg/kg·day to 10g/kg·day.

As described above, the genetic construct of the present invention can be transformed into a strain and stably secrete the extracellular flgM-GMCSF fusion protein while maintaining the immune-inducing ability of the GM-CSF protein, thereby separating GM-CSF. /Transformed strains without purification can be used to exert immune-inducing ability in the body.

Furthermore, the attenuated Salmonella strain transformed by the genetic construct of the present invention induces immunity by secreting GM-CSF in cancer tissue due to the characteristics of the Salmonella strain targeting the cancer site, while the immunity-inducing ability of Salmonella itself induces immunity. Since it exhibits superior anticancer activity, it can be usefully used as an anticancer composition.

Figure 1 is a diagram showing the base sequence of DNA designed to produce a plasmid for GM-CSF secretion.
Figure 2 is a cleavage map of flgM-gmCSF-pBAD33 plasmid.
Figure 3 is an electrophoresis photograph for detecting flgM-GMCSF in the culture medium of an attenuated Salmonella strain transformed with the flgM-gmCSF-pBAD33 plasmid.
Figure 4 is a microscopic observation image showing the immune-inducing ability of flgM-GMCSF secreted by the flgM-gmCSF-pBAD33/ppGpp- strain.
Figure 5 is a cleavage map of flgM-gmCSF-flhDC-pBAD33 plasmid.
Figure 6 shows flgM-gmCSF-pBAD33/ppGpp- and flgM-gmCSF-flhDC-pBAD33/ppGpp-
Figure 7 is a graph showing the tumor growth inhibition efficacy of flgM-gmCSF-pBAD33/ppGpp- and flgM-gmCSF-flhDC-pBAD33/ppGpp- strains in a tumor mouse model.
Figure 8 is a graph and photo showing the tumor growth inhibition efficacy of the flgM-gmCSF-flhDC-pBAD33/ppGpp- strain in a tumor mouse model.
Figure 9 is a graph showing changes in survival rate and body weight by the flgM-gmCSF-flhDC-pBAD33/ppGpp- strain in a tumor mouse model.

The present invention will be described in more detail below with reference to the attached examples. However, these embodiments are only examples to easily explain the content and scope of the technical idea of the present invention, and the technical scope of the present invention is not limited or changed thereby. Based on these examples, it will be obvious to those skilled in the art that various modifications and changes are possible within the scope of the technical idea of the present invention.

[Example]

Example 1: Construction of plasmid for GM-CSF secretion

A plasmid was created in which GM-CSF was fused with the flgM protein, which is secreted out of the cell through the basal body and hook.

First, we designed a gene that can express the fusion protein of flgM and GM-CSF. Recombinant Murine flgM DNA sequence of wild-type Salmonella (flgM anti-sigma-28 factor FlgM [Salmonella enterica subsp. enterica serovar Typhimurium str. LT2] Gene ID: 1252690, Locus tagSTM1172 NC_003197.2 (1257036..1257341, complement)) The amino acid sequence of GM-CSF (Pepro Tech Co., Ltd. 315-03 Accession Number: P01587 Gene ID: 12981) was linked to the DNA sequence converted through Salmonella codon optimization using a linker. The linker was constructed so that 4 amino acids were expressed before and after each of the 6 histidines. To increase the expression of the flgM gene, 14 shine-dalgarno sequences were inserted in front of the flgM gene. For cloning, a Nhel site was inserted at the front, and a HindIII site was inserted after the stop codon of GM-CSF. Figure 1 shows the base sequence designed through the above process. The gene synthesis of the corresponding sequence was commissioned and synthesized by Cosmogenetech Co., Ltd. Among these, the sequence excluding the restriction enzyme site and Shine-Dalgarno sequence corresponds to the flgM-GMCSF gene (SEQ ID NO: 1) encoding the fusion protein flgM-GMCSF.

The synthesized flgM-GMCSF was reacted at 37°C for 2 hours using 10 U each of NheI (Takara Co., Ltd.) and HindIII (Takara Co., Ltd.). The reaction product was subjected to electrophoresis, and 700 bp of DNA was extracted using a gel extraction kit (Qiagen) to obtain flgM-GMCSF insert DNA.

After reacting the pBad18, pBad24 or pBad33 plasmid with 10 U each of NheI and HindIII enzymes at 37°C for 2 hours, the reaction product was purified using a PCR purification kit (Qiagen) to obtain each vector DNA. Each vector DNA was ligated (Invitron) with flgM-GMCSF insert for 30 minutes at 25°C and transformed into DH5a competent cells. Transformed cells were plated on LB amp solid medium and cultured at 37°C to select colonies with antibiotic resistance. Six candidates from the selected colonies were selected and reacted with 10 U each of NheI and HindIII for 1 hour at 37°C, and the generation of a 700 bp band was confirmed through electrophoresis.

Among these, the plasmids introduced into the pBad18 and pBad24 vectors disappeared after 48 hours in an antibiotic-free medium when introduced into the attenuated Salmonella strain, and only the plasmid using the pBad33 vector continued to be maintained even in the attenuated Salmonella strain even without antibiotics. . Accordingly, we requested Cosmogenetech to analyze the base sequence of the candidate for the plasmid produced using pBad33, confirmed the insertion of the gene of SEQ ID NO: 1, and established it as a plasmid for secreting flgM-GMCSF (flgM-gmCSF-pBAD33). Figure 2 shows the cleavage map of flgM-gmCSF-pBAD33 plasmid.

Example 2: Preparation of attenuated Salmonella strain secreting flgM-GMCSF

1) Transformation of attenuated Salmonella strain by flgM-gmCSF-pBAD33 and confirmation of secretion of flgM-GMCSF

An attenuated Salmonella strain was transformed using the flgM-gmCSF-pBAD33 plasmid prepared in Example 1, and secretion of flgM-GMCSF was confirmed. As attenuated Salmonella strains, ppGpp- (distributed from Professor Jeong Jae-ho of Chonnam National University), aro A- (distributed from Professor Lee In-su of Hannam University), and galE flhDC- (self-produced, KCTC14455BP) strains were used.

A single colon of each transformed strain was inoculated into LB cm liquid medium and cultured with shaking at 37°C for 12 to 16 hours. Afterwards, it was diluted in fresh LB cm liquid medium to an OD ₆₀₀ of 0.05 and cultured with shaking at 37°C for 2 hours. When the OD ₆₀₀ reached 0.4 to 0.6 by shaking culture, arabinose was treated to a concentration of 0.2 w%, and the culture was shaken for an additional 5 hours at 37°C. For comparison, the control group was cultured with shaking under the same conditions without arabinose treatment. The culture was centrifuged at 3000 rpm to separate the supernatant, and then filtered through a 0.2 ㎛ filter to completely remove bacteria.

The amount of flgM-GMCSF in the culture medium was confirmed through Western blotting from the supernatant from which the bacteria were removed. Figure 3 is an electrophoresis photograph showing the results. Lanes 1, 3, 5, and 7 are the experimental group not treated with arabinose, and lanes 2, 4, 6, and 8 are the protein in the culture medium of the experimental group treated with arabinose. shows the detection results. In Figure 3, when the ppGpp- Salmonella strain was transformed with the flgM-gmCSF-pBAD33 plasmid, it was confirmed that flgM-GMCSF had a superior secretion ability compared to other attenuated strains.

2) Immune-inducing ability assay of flgM-GMCSF

To confirm whether the secreted flgM-GMCSF protein has the same immune-inducing activity as GM-CSF, macrophages were treated using the supernatant of the culture medium prepared to confirm the secretion of flgM-GMCSF in 1) and the changes were observed. observed. Specifically, RAW264.7 cells, a mouse macrophage cell line (distributed by Korea Cell Line Bank), were cultured at 37°C and 5% CO ₂ using DMEM medium containing 10% FBS and 1% streptomycin-penicillin. Cultured cells were dispensed at a concentration of 2×10 ⁵ cells/well, and 200 ㎕ of supernatant derived from the culture medium of the flgM-gmCSF-pBAD33/ppGpp- strain treated with or without PBS buffer or arabinose was treated and incubated for 24 hours. After further culturing, it was observed under a microscope at 200x magnification. Figure 4(a) shows that RAW264.7 cells are not differentiated and have a round shape as a result of treatment with only the PBS buffer. Figure 4(b) shows the results of treating the supernatant of the strain culture medium that was not treated with arabinose, and it can be seen that approximately 50% of the cells were transformed into an elongated shape. This is believed to be because immune activation was induced by substances secreted from the attenuated Salmonella itself. Figure 4 (c) shows the results of processing the supernatant of the strain culture medium treated with arabinose, showing that most cells showed a star shape and were differentiated into M1 type macrophages or dendritic cells.

From the above results, it can be seen that the flgM-gmCSF-pBAD33/ppGpp- strain expresses flgM-GMCSF by treatment with an expression inducer and secretes it into the culture medium, and that the secreted flgM-GMCSF protein exhibits immune-inducing activity like GM-CSF. You can.

3) Construction of an attenuated Salmonella strain with enhanced flgM-GMCSF secretion ability

Since the expression of flgM is dependent on the expression of flhDC, the flhDC gene was cloned into the flgM-gmCSF-pBAD33 plasmid to determine whether the expression of flgM-GMCSF could be enhanced.

A PCR product containing the flhDC gene was obtained by PCR using the genomic DNA of wild-type Salmonella LT2 as a template and the flhD-cla1-FOR and flhC-cla1-RE primers in Table 1 below. Specific PCR conditions are as follows; 5 μl of 10x buffer, 10 μl of 5x Q solution, 7.5 μl of 2 mM dNTPs, 2.5 μl of 20 μM flhD-cla1-FOR primer, 2.5 μl of 20 μM flhC-cla1-RE primer, 1 μl of Taq polymerase, 2 μl of 10 ng/μl genomic DNA. and 19.5 μl of water were mixed and PCR was performed using the Qiagen system at 95°C for 5 minutes, followed by 30 cycles of 94°C for 30 seconds, 49°C for 30 seconds, and 72°C for 1 minute, followed by 72°C for 10 minutes. 1 μg of PCR fragment purified with a PCR purification kit (Qiagen Co., Ltd.) and 10U of ClaI (Takara Co., Ltd.) were reacted at 37°C for 2 hours, and the reaction product was purified with a PCR purification kit (Qiagen Co., Ltd.) to prepare insert DNA.

Separately, the flgM-gmCSF-pBAD33 plasmid produced in 1) 1 ㎍ It was reacted with 10U of ClaI (Takara Co., Ltd.) at 37°C for 2 hours. The reaction product was purified using a PCR purification kit (Qiagen Co., Ltd.) to complete VECTOR DNA.

The insert DNA and VECTOR DNA obtained by the above method were ligated (Invitrogen Co., Ltd.) for 30 minutes at 25°C and then transformed into DH5a competent cells. Transformed cells were plated on LB cm solid medium and cultured at 37°C to select colonies with antibiotic resistance. Six candidates from the selected colonies were selected and reacted with 10U of ClaI (Takara Co., Ltd.) for 1 hour at 37°C, and the generation of a 1 kb band was confirmed through electrophoresis. Cosmogenetech was requested to analyze the base sequence of the candidate, and the insertion of the flhDCA gene was confirmed and established as the flgM-gmCSF-flhDC-pBAD33 plasmid.

After transforming the ppGpp-attenuated strain using the flgM-gmCSF-flhDC-pBAD33 plasmid, secretion of the flgM-GMCSF gene was confirmed by Western blot using the same method as in 1). Lanes 1 and 3 of Figure 6 are electrophoresis images showing the expression of flgM-GMCSF in the culture medium of the strain not treated with arabinose, and lanes 2 and 4 are the culture medium of the strain treated with arabinose. In the culture medium of the flgM-gmCSF-flhDC-pBAD33/ppGpp- strain treated with arabinose, a much larger amount of flgM-GMCSF was detected at the same concentration compared to the culture medium of the flgM-gmCSF-pBAD33/ppGpp- strain, and the flgM CSF of this example was detected at the same concentration. It can be seen that the ppGpp- strain transformed with the -gmCSF-flhDC-pBAD33 plasmid secretes flgM-GMCSF much better than the ppGpp- strain transformed with the flgM-gmCSF-pBAD33 plasmid.

The transformed strain was named flgM-gmCSF-flhDC-pBAD33/ppGpp-Salmonella, and was deposited at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center on January 19, 2021 (accession number KCTC14455BP).

Example 3: Evaluation of anticancer efficacy in tumor mouse model

As experimental animals, 6-week-old BALB/c female mice weighing approximately 20 g were purchased from OrientBio. Care, experimentation, and euthanasia of all animals were performed according to approved protocols (CNU IACUC-H-2020-7).

Murine CT26 colon carcinoma cells were purchased from the American Cell Line Bank (ATCC) and cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin. After collecting the CT26 tumor cells cultured in vitro, they were suspended in 20㎕ PBS, and then 1×10 ⁶ cells were subcutaneously injected into the right back of BALB/c mice to xenograft the tumor cells. About 2 weeks after transplantation of tumor cells, when the tumor size reaches 100 mm ³ , the flgM-gmCSF-flhDC-pBAD33/ppGpp- strain or the flgM-gmCSF-pBAD33/ppGpp- strain prepared in Example 2 is diluted with PBS. Infection was performed through the tail vein at a dose of approximately 5×10 ⁷ CFU/mice (0 day). As a control group, only the same amount of PBS buffer was injected instead of the strain, and as a comparison group, the attenuated strain ppGpp- Salmonella strain was infected at the same concentration. The transformed strain was injected intraperitoneally with 100 ㎕/mice of 40% L-arabinose every day to induce the expression of flgM-GMCSF from the 3rd day of administration. Afterwards, the weight of the mouse and the size of the tumor were measured and recorded at a certain time every day.

Tumor volume was calculated using the formula ⁽ tumor length

Figure 7 is a graph showing the size of the tumor as a result of the above experiment. As expected, the flgM-gmCSF-pBAD33-flhDC/ppGpp- strain significantly inhibits tumor growth. The ppGpp- strain also showed anticancer activity, which is explained to be due to immune induction by the attenuated Salmonella strain. The flgM-gmCSF-pBAD33/ppGpp- strain was also slightly less effective than the flgM-gmCSF-pBAD33-flhDC/ppGpp- strain, but showed twice as strong anticancer activity as the ppGpp- strain.

Figure 8 compares the tumor growth of the control group, the comparison group, the flgM-gmCSF-pBAD33-flhDC/ppGpp- strain treatment group without arabinose, and the flgM-gmCSF-pBAD33-flhDC/ppGpp- strain treatment group with arabinose administration. These are the graphs and photos shown. The group treated with the flgM-gmCSF-pBAD33-flhDC/ppGpp- strain treated with arabinose showed strong anticancer activity, but the group treated with the flgM-gmCSF-pBAD33-flhDC/ppGpp- strain treated with arabinose showed strong anticancer activity against the ppGpp- Salmonella strain. It showed a tumor growth pattern similar to that of the treatment group, confirming that the anticancer activity of the flgM-gmCSF-pBAD33-flhDC/ppGpp- strain was due to the anticancer activity of the Salmonella strain and the flgM-GMCSF protein expressed and secreted by the Salmonella strain. .

Figure 9 is a graph showing the survival rate and change in body weight over time after infection with a Salmonella strain. In the control group of mice that were not treated with the strain, the tumor volume reached 1,500 mm ³ in all mice after 23 days of the experiment, and the attenuated When infected with only Salmonella strains or when flgM-GMCSF was not expressed, the tumor exceeded the standard volume within 30 to 40 days, but when infected with flgM-gmCSF-pBAD33-flhDC/ppGpp- and treated with arabinose, the tumor exceeded the standard volume within 50 days. Even after this, the tumor size remained below the standard in 40% of mice. In all Salmonella treatment groups, a slight weight loss was observed immediately after infection, but the body weight gradually increased after 2 days, showing no difference in body weight from the control group after about 20 days.

Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC14455BP 20210119

Sequence list electronic file attached

Claims (10)

An attenuated Salmonella strain in which the anti-sigma factor flgM gene and the gene encoding the GM-CSF protein are linked and the gene construct encoding the fusion protein of flgM and GM-CSF is transformed by a recombinant expression vector contained in the pBad33 vector. A recombinant strain characterized by secreting a fusion protein of flgM and GM-CSF.
In claim 1,
A recombinant strain wherein the flgM gene and the gene encoding the GM-CSF protein are directly linked or connected through a linker.
In claim 1,
A recombinant strain, characterized in that the genetic construct encoding the fusion protein of flgM and GM-CSF consists of the sequence of SEQ ID NO: 1.
In claim 1,
The genetic construct is a recombinant strain comprising the flhDC gene.
delete In claim 1,
A recombinant strain, wherein the genetic construct is operably linked to an inducible promoter.
delete delete In claim 1,
The attenuated Salmonella strain is a recombinant strain, characterized in that the ppGpp- strain.
A pharmaceutical composition for treating cancer containing the recombinant strain according to any one of claims 1 to 4, 6, and 9.