KR102555172B1 - Gene Construct 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 having immunity-inducing ability in the body, and to an anti-cancer strain transformed therewith and having anti-cancer activity by stably secreting GM-CSF in the body, More specifically, a gene 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 transformed by the gene construct to produce GM-CSF in vivo. It relates to an anti-cancer strain having anti-cancer activity by stably secreting it.

Description

Gene Construct for Secreting GM-CSF and Anticancer Recombinant Microbials Transformed thereby

The present invention relates to a genetic construct capable of secreting GM-CSF having immunity-inducing ability in the body, and to an anti-cancer strain transformed therewith and having anti-cancer activity by stably secreting GM-CSF in the body.

BACKGROUND OF THE INVENTION Cancer is a life-threatening disease in which the proliferative activity of cells does not stop, penetrates into surrounding tissues and destroys normal cells. As the human body's controllability decreases due to aging, it becomes vulnerable to cancer. In an aging society, cancer has been the undisputed number one cause of death for 10 years since 2007, surpassing twice the death rate from heart disease, which ranks second. .

The most effective response system for the treatment of disease is to strengthen the immune system, but since cancer cells are not foreign invaders, they do not trigger the body's immune response and avoid it. Certain viruses or bacteria infect cancer cells more than normal cells, and the infected bacteria can become targets of attack by immune cells. Accordingly, anti-cancer treatment methods have been suggested in which a specific virus or bacterium is deliberately infected to stimulate the body's immune response to fight 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 cause immune responses. In contrast, Salmonella can penetrate the spleen and liver through the lymph nodes and stimulate a systemic immune response. Specifically, Leschner et al. (J. Mol. Med. 2010, 88, 763-773) infected mice with CT26 tumors with fluorescent Salmonella by intravenous injection, and followed the infection route over time. . As a result, it was shown that the whole body was infected through the blood of the mouse immediately after infection, Salmonella bacteria accumulated in the spleen and liver 20 minutes after infection, and Salmonella bacteria accumulated intensively only in tumor tissues after 24 hours.

However, since Salmonella is a representative bacterium that causes food poisoning, it can cause sepsis by infection and threaten life, so its pathogenicity is too strong to be used directly for cancer treatment. A research team at Yale University in the United States announced that genetic manipulation of Salmonella can attenuate it by removing toxicity while maintaining its tumor-attacking properties, and suppressing tumors by inducing immune stimulation through injection of the attenuated Salmonella. However, attenuated Salmonella not only has low survival and reduced immunogenicity, but also may cause sepsis by converting the attenuated strain to wild-type Salmonella due to mutation. In addition, due to the lack of research and development funds, chemotherapy using attenuated Salmonella is mostly suspended or suspended in the first stage of the clinical process.

The induction of sepsis, which may be accompanied by cancer treatment using bacteria such as Salmonella, is a very important problem to be overcome, and in addition, stimulation of the immune response should be actively performed. Accordingly, studies to develop strains capable of additionally expressing or suppressing substances useful for anticancer treatment along with attenuation and activation of immunity, and to use them for anticancer treatment are mainly conducted. Registered Patent No. 10-0852687 discloses a Salmonella strain expressing tumor necrosis factor alpha transduced with a tumor necrosis factor alpha protein vector into an attenuated Salmonella strain and a composition for anti-cancer treatment containing the same, and Registered Patent No. 10 -1750007 published a treatment for solid cancer 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 innate immune response by inducing the differentiation of monocytes into M1 type macrophages, while maturing into dendritic cells to interact with CD8 T cells to induce the acquired immune response, which is pivotal for the immune response. play a role GM-CSF expressed in bacteria has been used in various fields because its bioequivalence has been demonstrated in its function, but a series of processes are required to purify GM-CSF in the bacterial cytoplasm after expression.

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

If GM-CSF is targeted by Salmonella instead of cancer-specific antigen and expressed, the anticancer effect can be expected to be more excellent 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, since the strain itself is killed when GM-CSF is expressed in Salmonella, it is difficult to expect the anticancer efficacy of Salmonella and the anticancer efficacy by expression of GM-CSF at the same time.

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

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

The present invention does not kill the transformed strain, It is an object of the present invention to provide a genetic construct capable of expressing GM-CSF having immunity-inducing ability in the body.

Another object of the present invention is to provide an anticancer recombinant strain transformed with the gene construct to stably express GM-CSF and secrete it into the body of the host to exhibit anticancer activity.

Another object of the present invention is to provide an anti-cancer composition using the anti-cancer strain.

In order to achieve the above object, the present invention relates to a genetic construct characterized in that an anti-sigma factor flgM gene and a gene encoding a 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 and secrete flgM-GMCSF, which has the immune inducing ability of GM-CSF, out of cells. Therefore, it is possible to prevent the death of host cells due to the expression of GM-CSF, and a fusion protein having immunity-inducing ability can be easily obtained from the culture medium without going through a series of processes to purify the GM-CSF present in the cytoplasm of the strain. .

In the gene construct of the present invention, the flgM gene and the gene encoding the GM-CSF protein may be directly linked or linked through a linker. In the following examples, the structure of SEQ ID NO: 1 connected via a linker having a gene sequence encoding 14 amino acids was exemplified, but it 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.

In order to improve expression efficiency, a shine-dalgarno sequence may be arranged at the front end 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 said genetic constructs. A "vector" in the present invention is a DNA preparation that is operably linked to allow expression of the genetic construct of the present invention in a suitable host, which may be a plasmid, phage particle or latent genomic insert. In the context of the present invention, plasmid and vector are sometimes used interchangeably. It goes without saying that the expression vector with high efficiency can be selected and used according to the sequence of the gene construct.

In addition, the present invention relates to a recombinant strain transformed by the gene construct and secreting the fusion protein flgM-GMCSF. The recombinant strain of the present invention can be transformed using a recombinant expression vector containing the gene construct. The transformation method follows a conventional method, and for example, a calcium chloride method, an electroporation method, etc. may be used, but is not limited thereto.

The recombinant strain of the present invention is characterized in that the secretion system of flgM operates and not only expresses flgM-GMCSF encoded by the genetic construct of the present invention inside the cell, but secretes it outside the cell. Due to these characteristics, the strain is not killed due to the GM-CSF expressed in the cell and can stably and continuously express/secrete the fusion protein, and it can be confirmed that the secreted fusion protein maintains the immune inducing ability of GM-CSF. could Therefore, by using the strain of the present invention, a fusion protein having an immune-inducing ability can be easily obtained from a culture medium without going through a series of complicated processes of isolation/purification from the bacterial cytoplasm.

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

The recombinant strain of the present invention may be used as a strain for producing flgM-GMCSF, but is used as such to secrete flgM-GMCSF in vivo, so that GM-CSF exhibits a therapeutic effect, including cancer, for example. It can be used for the treatment of ailments. Any strain used at this time can be used as long as it is harmless to the human body and can secrete flgM-GMCSF. Tridium or intestinal microbes (beneficial bacteria), probiotics, etc. can be used.

In the strain of the present invention, the gene construct is preferably operably linked to an inducible promoter. As used herein, "inducible promoter" is a promoter capable of switching the operation of a gene linked 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. "Attenuation" means artificially weakening the toxicity of living pathogens, mutating genes involved in essential metabolism of pathogens to induce immunity by stimulating only the immune system without causing disease in the body. Genes that cause attenuation of Salmonella are well known in the art, 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 It can be achieved by loss of function of one or more genes that become When applied in vivo, the functions 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 flhDC gene is included in the genetic construct transforming the recombinant strain of the present invention. The flhDC gene is known to regulate the expression of the flgM gene as a major regulator of the flagellin operon 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 greatly increased.

It is known that attenuated Salmonella strains have anticancer activity by being targeted to the tumor site and inducing an immune response. Therefore, when transforming with the gene construct of the present invention, it is targeted to the tumor site and secretes flgM-GMCSF fusion protein, which simultaneously induces anticancer activity by attenuated Salmonella and anticancer activity by GM-CSF at the tumor site, resulting in more powerful anticancer activity. indicates activity.

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

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

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

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

As described above, the genetic construct of the present invention can be transformed into a strain to stably secrete the flgM-GMCSF fusion protein to the outside of the cell while maintaining the immune inducing ability of the GM-CSF protein. / It is possible to exert the ability to induce immunity in the body by using the transformed strain without purification.

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 targeted to the cancer site, while the immunity inducing ability of Salmonella itself Since it exhibits more excellent anticancer activity, it can be usefully used as an anticancer composition.

1 is a view showing the nucleotide sequence of DNA designed for the construction of a plasmid for GM-CSF secretion.
Figure 2 is a cleavage map of the 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 microscope 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 the flgM-gmCSF-flhDC-pBAD33 plasmid.
6 shows flgM-gmCSF-pBAD33/ppGpp- and flgM-gmCSF-flhDC-pBAD33/ppGpp-
Figure 7 is a graph showing the tumor growth inhibition efficacy of the flgM-gmCSF-pBAD33 / ppGpp- and flgM-gmCSF-flhDC-pBAD33 / ppGpp- strains in a tumor mouse model.
Figure 8 is a graph and photos showing the tumor growth inhibitory efficacy of the flgM-gmCSF-flhDC-pBAD33/ppGpp- strain in a tumor mouse model.
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.

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

[Example]

Example 1: Construction of plasmid for GM-CSF secretion

A plasmid in which the flgM protein secreted outside the cell through the basosome and the hook was fused with GM-CSF was constructed.

First, a gene capable of expressing a fusion protein of flgM and GM-CSF was designed. The 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)) was recombinant Murine The amino acid sequence of GM-CSF (Co., Ltd. Pepro Tech 315-03 Accession Number: P01587 Gene ID: 12981) was combined with a DNA sequence converted through Salmonella codon optimization through a linker. The linker was constructed so that 4 amino acids in front and behind each of 6 histidines were expressed. To increase the expression of the flgM gene, 14 shine-dalgarno sequences were inserted in front of the flgM gene, an Nhel site was inserted at the front for cloning, and a HindIII site was inserted after the stop codon of GM-CSF. Figure 1 shows the nucleotide sequence designed through the above process. Gene synthesis of the sequence was synthesized by requesting Cosmogenetech Co., Ltd. Among them, sequences excluding the restriction enzyme site and the Shine-Dalgarno sequence correspond to the flgM-GMCSF gene (SEQ ID NO: 1) encoding the flgM-GMCSF fusion protein.

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

Among them, 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 was maintained in the attenuated Salmonella strain without antibiotics. . Accordingly, Cosmogenetech was requested to analyze the nucleotide sequence of the candidate for the plasmid prepared using pBad33, and the insertion of the gene of SEQ ID NO: 1 was confirmed and a plasmid (flgM-gmCSF-pBAD33) was established for secretion of flgM-GMCSF. Figure 2 shows the cleavage map of the flgM-gmCSF-pBAD33 plasmid.

Example 2: Construction of an attenuated Salmonella strain secreting flgM-GMCSF

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

The 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- (prepared from Professor Jeong Jae-ho, Chonnam National University), aro A- (prepared from Professor Lee In-soo, Hannam University), and galE flhDC- (self-produced, KCTC14455BP) strains were used.

A single colon of each transformed strain was inoculated into LB cm broth and cultured with shaking at 37°C for 12-16 hours. After diluting so that OD ₆₀₀ is 0.05 in fresh LB cm liquid medium, it was cultured with shaking at 37 ° C. for 2 hours. When the OD ₆₀₀ was 0.4 to 0.6 by shaking culture, arabinose was treated to a concentration of 0.2 w%, and shaking culture was further performed at 37 ° C. for 5 hours. For comparison, the control group was shaken and cultured under the same conditions without treatment with arabinose. The culture solution was centrifuged at 3000 rpm to separate the supernatant, and the bacteria were completely removed by filtering with a 0.2 μm filter.

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

2) Test of immune inducing ability of flgM-GMCSF

In order 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 solution prepared for the secretion of flgM-GMCSF in 1) and the change was observed. Observed. Specifically, RAW264.7 cells, a mouse macrophage cell line (available from Korea Cell Line Bank), were cultured at 37° C. and 5% CO ₂ using DMEM medium containing 10% FBS and 1% streptomycin-penicillin. The cultured cells were dispensed at a concentration of 2×10 ⁵ cells/well, and 200 μl of the supernatant derived from the culture medium of the flgM-gmCSF-pBAD33/ppGpp- strain treated with or without PBS buffer or arabinose was treated and treated for 24 hours. After further culturing, it was observed under a microscope at 200x magnification. Figure 4 (a) shows a round shape because RAW264.7 cells were not differentiated as a result of treatment with PBS buffer only. Figure 4 (b) is a result of treating the supernatant of the strain culture medium not treated with arabinose, it can be seen that about 50% of the cells are transformed into a long shape. It is believed that this is because immune activation is induced by substances secreted from the attenuated Salmonella itself. Figure 4 (c) shows that as a result of treating the supernatant of the strain culture medium treated with arabinose, most of the cells showed a star shape and 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 the secreted flgM-GMCSF protein exhibits immunity-inducing activity similar to GM-CSF. can

3) Preparation of 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 confirm whether the expression of flgM-GMCSF can 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 shown in Table 1 below. Specific PCR conditions are as follows; 10x buffer 5 μl, 5x Q solution 10 μl, 2 mM dNTPs 7.5 μl, 20 μM flhD-cla1-FOR primer 2.5 μl, 20 μM flhC-cla1-RE primer 2.5 μl, Taq polymerase 1 μl, 10 ng/μl genomic DNA 2 μl And 19.5 μl of water was mixed and PCR was performed using a Qiagen system at 95 ° C for 5 minutes, 30 cycles of 94 ° C for 30 seconds, 49 ° C for 30 seconds, 72 ° C for 1 minute and then 72 ° C for 10 minutes. 1 μg of PCR fragment purified with a PCR purification kit (Qiagen) and 10 U of ClaI (Takara) were reacted at 37 ° C for 2 hours, and the reaction product was purified with a PCR purification kit (Qiagen) to prepare insert DNA.

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

After ligation (Invitrogen Co., Ltd.) of insert DNA and VECTOR DNA obtained by the above method at 25° C. for 30 minutes, DH5a competent cells were transformed. Transformed cells were plated on LB cm solid medium and cultured at 37° C. to select antibiotic-resistant colonies. Six candidates of the selected colony were selected and reacted with 10U of ClaI (Takara Co., Ltd.) for 1 hour at 37 ° C, and the formation of a 1 kb band was confirmed through electrophoresis. Cosmogenetech requested an analysis of the candidate's nucleotide sequence, confirmed the insertion of the flhDCA gene, and established the flgM-gmCSF-flhDC-pBAD33 plasmid.

After transformation of the ppGpp-attenuated strain using the flgM-gmCSF-flhDC-pBAD33 plasmid, secretion of the flgM-GMCSF gene was confirmed by Western blotting in the same manner as in 1). Lanes 1 and 3 of FIG. 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. It can be confirmed that the ppGpp- strain transformed with the -gmCSF-flhDC-pBAD33 plasmid secreted 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 Resource Center on January 19, 2021 (accession number KCTC14455BP).

Example 3: Evaluation of anticancer efficacy in tumor mouse models

As experimental animals, 6-week-old BALB/c female mice weighing around 20 g were purchased from OrientBio and used. 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 harvesting CT26 tumor cells cultured in vitro, they were suspended in 20 μl PBS, and 1×10 ⁶ cells were subcutaneously injected into the right back of BALB/c mice to xenograft the tumor cells. After about 2 weeks of 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 was diluted with PBS. It was infected through the tail vein at a dose of about 5×10 ⁷ CFU/mice (day 0). As a control group, the same amount of PBS buffer was injected instead of the strain, and as a control group, the attenuated strain, ppGpp-Salmonella strain, was infected at the same concentration. The transformed strain was intraperitoneally injected with 100 μl/mice of 40% L-arabinose every day from day 3 of administration of the strain to induce the expression of flgM-GMCSF. Then, at a fixed time every day, the weight of the mouse and the size of the tumor were measured and recorded.

The tumor volume was calculated by the formula (tumor length × tumor width × tumor height)/2, and euthanized when the tumor volume reached 1,500 mm ³ or more according to the guidelines of the Ethics Committee for Animal Experiments at Chonnam National University.

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

Figure 8 compares the tumor growth of the control group, the control group, and the flgM-gmCSF-pBAD33-flhDC/ppGpp-strain treatment group not administered with arabinose and the flgM-gmCSF-pBAD33-flhDC/ppGpp-strain treatment group treated with arabinose. It is a graph and a picture to show. The flgM-gmCSF-pBAD33-flhDC/ppGpp- treated group treated with arabinose showed strong anticancer activity, but the flgM-gmCSF-pBAD33-flhDC/ppGpp- treated group not treated with arabinose showed ppGpp-Salmonella strain It can be confirmed that the anticancer activity of the flgM-gmCSF-pBAD33-flhDC/ppGpp- strain is due to the anticancer activity by the Salmonella strain and the flgM-GMCSF protein expressed and secreted by the Salmonella strain by showing a tumor growth pattern similar to that of the treated group. .

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

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Claims (12)

delete delete delete delete delete delete delete delete delete Anti-cancer recombinant strain with accession number KCTC14455BP.
delete A pharmaceutical composition for treating cancer containing the recombinant strain of claim 10.

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