KR-102960064-B1 - Anticancer Microbials Secreting Different Proteins and Anticancer Composition Comprising the Microbials

KR102960064B1KR 102960064 B1KR102960064 B1KR 102960064B1KR-102960064-B1

Abstract

The present invention relates to an anticancer strain capable of efficiently secreting heterologous proteins without mutually affecting secretion when secreting heterologous proteins by a secretion system of the same mechanism, and to an anticancer composition containing the same as an active ingredient. More specifically, the invention relates to an anticancer strain characterized by being transformed by a gene structure comprising (A) a gene encoding a ClyA protein operably linked to a first inducible promoter; and (B) a gene encoding a cytokine protein linked to an anti-sigma factor flgM gene operably linked to a second inducible promoter, thereby possessing a dual secretion system, and to an anticancer composition containing the same as an active ingredient.

Inventors

이효진
전흥진
김솔비
김은지
한민주
강소라

Assignees

충남대학교산학협력단

Dates

Publication Date: 20260508
Application Date: 20240320
Priority Date: 20230328

Claims (12)

(A) A gene encoding a ClyA protein operably linked to a first inducible promoter; and a gene construct comprising, (B) transformed by a gene construct comprising a gene encoding a cytokine protein linked to an anti-sigma factor flgM gene operably linked to a second inducible promoter, An anticancer strain characterized by being equipped with a dual secretion system.
In claim 1, The above strain is an anticancer strain characterized by being selected from attenuated Salmonella, Escherichia coli, Shigella, Acinetobacter, Pseudomonas aeruginosa, Listeria, Clostridium, or intestinal microorganisms and probiotics.
In claim 1, An anticancer strain characterized in that the first inducible promoter and the second inducible promoter are each independently selected from the group consisting of 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, and pflE promoter.
In claim 3, An anticancer strain characterized in that the first inducible promoter and the second inducible promoter are actuated by different inducing substances.
In any one of claims 1 to 4, An anticancer strain characterized in that the gene encoding the above ClyA protein has one or more tags attached to it.
In any one of claims 1 to 4, An anticancer strain characterized in that the above cytokine protein is selected from TNFa, INFa, INFβ, INFγ, IL-1β, IL2, IL12, and IL15.
In any one of claims 1 to 4, The above gene structure is an anticancer strain characterized by including the flhDC gene.
In any one of claims 1 to 4, An anticancer strain characterized by the connection between the above anti-sigma factor flgM gene and the gene encoding a cytokine protein being directly linked or connected through a linker.
In claim 8, An anticancer strain characterized by the cytokine protein being TNFa.
In claim 9, An anticancer strain characterized by a gene encoding a TNFa protein linked to an anti-sigma factor flgM gene, consisting of SEQ ID NO. 2.
In claim 10, The above strain is an anticancer strain characterized by being the strain with accession number KCTC19174P.
An anticancer composition containing an anticancer strain according to any one of claims 1 to 4 as an active ingredient.

Description

Anticancer strains secreting heterologous proteins and anticancer compositions containing the same {Anticancer Microbials Secreting Different Proteins and Anticancer Composition Comprising the Microbials} The present invention relates to an anticancer strain capable of efficiently secreting heterologous proteins without affecting mutual secretion when secreting heterologous proteins, and an anticancer composition containing the same as an active ingredient. Cancer is a life-threatening disease in which the proliferative activity of cells does not stop, invading surrounding tissues and destroying normal cells. As the body's regulatory capacity declines due to aging, it becomes more vulnerable to cancer; in an aging society, cancer has held the undisputed number one spot among all causes of death since 2007, exceeding twice the mortality rate of heart disease, which ranks second. While the most effective response system for treating diseases is to strengthen immunity, cancer cells evade the body's immune response because they are not external invaders. Certain viruses or bacteria infect cancer cells more frequently than normal cells, and infected bacteria can become targets of attack by immune cells. Accordingly, anticancer treatment methods have been proposed that intentionally infect the body with specific viruses or bacteria to stimulate the immune response and enable it to fight against cancer cells. Leschner et al. (J. Mol. Med. 2010, 88, 763-773) infected mice with CT26 tumors via intravenous injection with fluorescent Salmonella and tracked the infection pathways over time. The results showed that immediately after infection, the entire body was infected through the mice's blood; 20 minutes after infection, Salmonella accumulated in the spleen and liver; and 24 hours later, Salmonella was observed to be concentrated only in the tumor tissue. This suggested that by targeting cancer and activating immunity, it could be effectively used in anticancer immunotherapy. However, Salmonella is a representative bacterium that causes food poisoning and can induce sepsis through infection, which can be life-threatening; therefore, its pathogenicity is too strong to be used directly for cancer treatment. A research team at Yale University announced that genetically modifying Salmonella can attenuate it by removing only its toxicity while maintaining its tumor-attacking characteristics, and that injecting this attenuated Salmonella can induce immune stimulation to suppress tumors. Since attenuated Salmonella exhibits reduced toxicity as well as decreased immune-inducing capacity, recent research has focused on utilizing genetically modified strains for cancer treatment that express various effector substances capable of enhancing immune activity or possessing anticancer efficacy. Anticancer activity can be achieved through two pathways: one in which cytotoxic substances act directly on cancer cells to kill them, and another in which the host’s innate or adaptive immune response is induced to fight against cancer cells. Therefore, if anticancer strains are made to express substances that kill cancer cells and substances that induce immune responses, it is expected that the effectiveness of cancer treatment can be maximized. This is because the immune-inducing capacity of the attenuated strains can be complemented to induce anticancer activity through both of these pathways. In order for an anticancer strain expressing an effector substance to exhibit anticancer activity, in many cases, it must not merely stop at expressing the effector substance but must also secrete the expressed substance outside the cell. Accordingly, the inventors filed a patent application No. 10-2022-0142232 on October 31, 2022, for an anticancer strain capable of simultaneously secreting ClyA, which kills cancer cells, and GM-CSF, which differentiates monocytes and matures them into dendritic cells to induce an immune response, and for an anticancer composition containing the same as an active ingredient. ClyA (Cytolysin A) is a 34 kDa cytolytic protein expressed by E. coli , Salmonella, and some intestinal bacteria, which kills target cells by forming pores on the membrane. Attempts have been made to treat cancer by utilizing this cell-killing characteristic to target ClyA to cancer cells and induce its expression. The strain described above has the advantage of being convenient to operate because two proteins are simultaneously expressed by a single inducer, and efficient secretion is possible because ClyA and GM-CSF are secreted through a dual secretion system operating by independent secretion mechanisms. However, when the expression of heterogeneous proteins is simultaneously controlled by a single inducer, there was a problem in that the expression efficiency was inevitably reduced compared to when only one protein is expressed due to the limited amount of the inducer, and consequently, the amount secreted also decreased. In addition, as