KR20160133087A - Anticancer Vaccine Adjuvant Comprising Inactivated Bacillus Subtilis Spore As Active Ingredient - Google Patents

Abstract

The present invention provides an anticancer vaccine adjuvant and anticancer pharmaceutical composition, both of which comprise inactivated Bacillus subtilis spores as an active ingredient, and induces and increases Th1 immune response. The present invention activates Th1 immune response by activating dendritic cells and particularly, achieves the effect of selectively killing cancer cells and cancer tissues by inducing the proliferation and activation of CD8^+T cells. The present invention is provided in a dry powder form which can be used in nasal and oral formulations capable of being administered through mucus membranes by being mixing mixed with water free of bacteria and heating materials prior to use, and also can be formulated in non-oral administration forms including hypodermic injection and intramuscular injection.

Description

(Anticancer Vaccine Adjuvant Comprising Inactivated Bacillus Subtilis Spore As Active Ingredient) containing an inactivated Bacillus subtilis spore as an active ingredient,

The present invention relates to an anticancer vaccine adjuvant and an anticancer pharmaceutical composition comprising inactivated Bacillus subtilis spore as an active ingredient.

Immune responses are largely divided into two types: humoral immunity and cell-mediated immunity. Humoral immunity involves the production of antibodies against foreign antigens. Antibodies are produced by B lymphocytes. Cell-mediated immunity involves activation of T lymphocytes that act on infected cells harboring foreign antigens or that stimulate other cells to act on infected cells. Both types of mammalian immune systems play an important role in combating disease.

Dendritic cells are known to be the most physiologically responsible cells for inducing immune responses. The dendritic cells of the tissue are activated by ingesting the antigen at the site of infection and then migrated to the lymphatic tissue and differentiate into highly efficient cells capable of presenting antigen to the circulating T cells. Activated dendritic cells induce the production of several cytokines such as IL (Interleukin) -6, IL-12, IL-18 and interferon-alpha. IL-12 is one of the most critical cytokines in the role of natural killer cells and Helper type I cell differentiation. IL-12 is composed of heterodimers of two proteins, p35 and p40. The gene expression of p35 occurs constitutively, whereas the expression of p40 is induced by stimulation. IL-12 is secreted mainly from dendritic cells and macrophages, which are specialized antigen presenting cells.

Immune supplements are essential for improving the efficacy of existing and next-generation vaccines. The adjuvant is added and / or formulated in immunization against the actual antigen (i. E., The substance that causes the desired immune response) to enhance the humoral and / or cell-mediated immune response. That is, the immunosuppressant has a characteristic of having immunity enhancing properties, particularly when co-administered with an antigen. For example, immunosuppressants such as aluminum hydroxide, emulsions of mineral oils, saponins, detergents, silicone compounds, thiourea, endotoxins of gram-positive bacteria, exotoxins of gram-negative bacteria, dead or attenuated live bacteria .

Chemotherapy has been used mainly in surgery, chemotherapy and radiation therapy. In the case of chemotherapy and radiotherapy, except for surgical operations, various preparations and radiation are used, but their application is limited to the extent that the human body can afford. In addition, radiotherapy is an anti-cancer therapy for treating various cancers occurring in the human body by irradiating an affected part with an appropriate amount of radiation. Such a radiation therapy lowers the hematopoietic function of the human body and suppresses the immune system, There is a problem of lowering.

Currently, immunoadjuvant compositions comprising a stable non-toxic diacyl lipoyl-oligosaccharide and an oligodeoxynucleotide with immunostimulatory activity (Korean Patent Laid-open Publication No. 10-2013-0113758), parnasoid-X (Korean Patent Laid-Open No. 10-2011-0081824) including a receptor (FXR) antagonist and an antigen, and an anticancer therapy adjuvant including salmonella expressing TNF-α (Korean Patent Registration No. 10-1285470) Various anti-cancer vaccine adjuvants have been continuously developed for anti-cancer therapy. However, anti-cancer vaccine adjuvants that can effectively deliver cancer vaccine to immune system cells and induce and activate immune responses to effectively inhibit cancer cells and cancer tissues have been developed It is not.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Disclosure of the Invention The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a method for inactivating Bacillus subtilis spore not only effectively delivering an anti-cancer vaccine (antigen) to an immune system cell, The present invention has been completed by confirming that cancer cells and cancer tissues can be killed by activating related immune cells (e.g., dendritic cells, CD8 ⁺ T cells, etc.).

The objects and advantages of the present invention will be more clearly understood from the following detailed description of the invention, claims and drawings.

In order to achieve the above object, the present invention provides an anticancer vaccine adjuvant which contains inactivated Bacillus subtilis spore as an active ingredient and induces and increases a Th1 immune response.

The present invention provides an anticancer pharmaceutical composition comprising an inactivated Bacillus subtilis spore as an active ingredient and inducing and increasing a Th1 immune response.

The present invention also provides a method of inducing and augmenting a Th1 immune response comprising injecting an inactivated Bacillus subtilis spore into a subject.

The present invention provides an anti-cancer vaccine adjuvant and an anticancer pharmaceutical composition comprising an inactivated Bacillus subtilis spore as an active ingredient and inducing and increasing a Th1 immune response.

The present invention is capable of specifically killing cancer cells and cancer tissues by activating dendritic cells, activating Th1 immune responses during the immune response, and particularly by proliferating and activating CD8 ⁺ T cells.

In addition, the present invention can be used as a nasal or oral dosage form which can be injected into mucous membranes in the form of a mixture with water in which germicidal or pyrogenic substances are removed before being used in the form of a dry powder, and parenteral It has an advantage that it can be formulated into a formulation.

FIG. 1 is a graph showing the amount of IL-12p40 cytokine secreted in a dendritic cell culture medium according to the ratio of dendritic cells derived from mouse bone marrow to Bacillus subtilis spore, by ELISA.
FIG. 2 is a graph showing the ratio of dividing T cells when co-cultured with OVA-specific OT-1 CD8 T cells after treatment of OVA protein and / or spore with dendritic cells derived from mouse bone marrow. Black bars indicate the results when co-cultured DC and T cells at 1: 5 and gray bars at 1:10.
Figure 3 is a graph showing the cleavage of OVA-specific CD8 T cells in the mouse body. The red graph is the PBS, the light blue graph is the OVA, and the yellow graph is the result graph when the OVA and the spore are inoculated.
Figure 4 shows that OVA-specific CD8 T cells that were inoculated three times with PBS, spore, OVA, OVA + spore, and OVA + MPL-A in mice, and the OVA-specific CD8 T cells that appeared after 7 days were injected into the spleen and lung, respectively, The results are shown in Fig.
FIG. 5 is a graph showing the foci of metastatic cancer when lung cancer cells expressing OVA protein were injected after three intranasal inoculations with PBS, OVA, Spore, and OVA + Spore.

According to one aspect of the present invention, there is provided an anticancer vaccine adjuvant which contains inactivated Bacillus subtilis spore as an active ingredient and induces and increases a Th1 immune response.

The inventors of the present invention have found that inactivation of Bacillus subtilis spore can not only effectively deliver an anti-cancer vaccine to immune system cells but can also kill cancer cells and cancer tissues by inducing and activating Th1 immune response.

Immune responses can be broadly divided into Th1 and Th2 immune responses. As used herein, the term " Th1 immune response " means a response that induces a cellular immune response, and " Th2 immune response " means a response that promotes a humoral immune response.

Disable containing as an active ingredient in the present invention, the Bacillus subtilis know in Spokane vivo And exhibits little side effects (see Embodiment 6 of the present invention). The term "inactivated " used in describing Bacillus subtilis spores in the present specification means that Bacillus subtilis spore does not proliferate or exhibit toxicity to an external subject, and in particular, in means non-proliferative or non-pathogenic in vivo .

Bacillus subtilis used in the present invention is a gram-positive bacterium having a length of 2-3 μm and is a catalase positive bacterium. It is a typical bacterium belonging to the genus Bacillus. It is widely distributed in the natural world such as soil, hay, and dust, and forms aerobic spore. Spore is resistant to heat, radiation and chemicals, and remains dormant for a long time.

In the context of the present invention, the term "MAMPs (microbe-associated molecular patterns )" is a microbial pathogen or - in the associated molecular patterns refers to microbial or pathogen-related molecules recognized by receptors of immune system cells in vivo , namely TLRs (Toll-like receptors) and PRRs (pattern recognition receptors), and include, for example, bacterial or bacterial pathogens such as bacterial flagellin, Lipoteichoic acid, peptidoglycan, and the like. Can be used in combination with the meaning of pathogen-associated molecular patterns (PAMPs) in the specification of the present invention.

Preferably, the inactivated Bacillus subtilis spore in the present invention comprises microbe-associated molecular patterns (MAMPs) on its surface.

In addition, the present invention may further include inactivated Bacillus subtilis spore-derived MAMPs in order to maximize the effect of delivering the antigenicity of the anti-cancer vaccine to the immune system cells, and MAMPs include fragments.

According to a preferred embodiment of the present invention, the present invention further comprises microbe-associated molecular patterns (MAMPs) derived from inactivated Bacillus subtilis.

When interleukin-12 is secreted from dendritic cells, T cells are differentiated into Th1 cells to induce Th1 immune response. When the Th1 immune response is induced, it acts to kill cancer cells as well as external pathogens. The present invention induces the secretion of interleukin-12 (IL-12) from dendritic cells and exerts the effect of killing cancer cells and cancer tissues through Th1 immune response.

According to a preferred embodiment of the present invention, the present invention is an anti-cancer vaccine adjuvant that induces the secretion of interleukin-12 (IL-12) from dendritic cells.

The present invention has the advantage of being able to effectively induce anticancer activity as well as vaccination against anticancer antigens as anticancer vaccine adjuvant.

In addition, the present invention can activate dendritic cells to induce proliferation of CD8 ⁺ T cells. The effect of the present invention is that the inactivated Bacillus subtilis spore, an active ingredient of the present invention, can induce antigen-presenting antigen (MHC class I) in dendritic cells, which are antigen-presenting cells, , Or to connect the body to the body.

According to a preferred embodiment of the present invention, the present invention is an anti-cancer vaccine adjuvant which can induce the proliferation of CD8 ⁺ T cells.

The present invention induces the proliferation of CD8 ⁺ T cells through dendritic cell activation, and the induced CD8 ⁺ T cells exert anti-cancer effects by killing cancer cells and cancer tissues.

The term "death" in the present invention is intended to kill cancer cells directly or to kill cancer cells by inhibiting the proliferation of cancer cells and inhibiting cancer cell metastasis. It means.

Preferably, the cancer that can be proliferated or killed in the present invention is selected from the group consisting of lung cancer, breast cancer, cervical cancer, ovarian cancer, endometrial cancer, melanoma, bladder cancer, pancreatic cancer, colon cancer, prostate cancer, leukemia, acute lymphocytic leukemia, (AML), chronic myelogenous leukemia, thyroid cancer, thyroid follicular cancer, renal cancer, non-small cell lung cancer, bladder cancer, head and neck cancer, gastric cancer, liver cancer, bone cancer, brain cancer, cholangiocarcinoma, small bowel cancer, uterine cancer And testicular cancer, and more preferably lung cancer.

According to one aspect of the present invention, there is provided an anticancer pharmaceutical composition comprising an inactivated Bacillus subtilis spore as an active ingredient and inducing and increasing a Th1 immune response.

The present invention has the effect of inducing or increasing the Th1 immune response and killing cancer cells or cancer tissues. More specifically, the composition of the present invention induces the proliferation and activity of CD8 ⁺ T cells by activating immune system cells such as dendritic cells, and the proliferated and activated CD8 ⁺ T cells exert the effect of killing cancer cells and cancer tissues.

The term "anti-cancer" in the present invention encompasses not only killing cancer cells or cancer tissues, but also means inhibiting the proliferation and metastasis of cancer cells or cancer tissues, or preventing cancer.

The term "pharmaceutically effective amount" as used herein means an amount sufficient to achieve the efficacy or activity (i. E. Inhibiting cancer cell growth or cancer cell killing activity) of the above anti-cancer drug. The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . The pharmaceutical composition of the present invention can be administered orally or parenterally. When administered parenterally, it can be administered by nasal injection, bolus injection, intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, , ≪ / RTI > most specifically into the nose.

In addition, the pharmaceutical composition of the present invention preferably determines the route of administration according to the kind of disease to be applied and the decision of prescription. For example, the concentration of the active ingredient contained in the composition of the present invention can be determined in consideration of the purpose of the treatment, the condition of the patient, the period of time required, and the like, and is not limited to a specific range of concentration. According to some embodiments of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-1,000 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. Here, the formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

Since the present invention is an anticancer pharmaceutical composition comprising inactivated Bacillus subtilis spore, which is an active ingredient of the anticancer vaccine adjuvant, the common description between them is omitted in order to avoid the excessive description of the present specification.

According to one aspect of the present invention, there is provided a method of inducing and augmenting a Th1 immune response comprising injecting an inactivated Bacillus subtilis spore into a subject.

The present invention includes the following steps.

(a) inactivation of Bacillus subtilis spore

Step (a) is a step of inactivating Bacillus subtilis spore, which comprises physically or chemically treating the Bacillus subtilis spore such that it does not show proliferation or pathogenicity.

In the present invention, "a step of physically or chemically treating Bacillus subtilis spore so as not to exhibit proliferation or pathogenicity" means that the Bacillus subtilisspore activity known in the art, that is, the cell proliferation or pathogenicity- Are included without limitation.

Preferably, the step (a) of the present invention comprises a step of heat-treating the culture medium containing Bacillus subtilis spore at 100 DEG C to 150 DEG C for 5 minutes to 60 minutes, more preferably, Bacillus subtilis spore And further comprising heat-treating the culture medium containing the Bacillus subtilis spore at a temperature of 110 ° C to 140 ° C for 15 minutes to 45 minutes. More preferably, the culture medium containing Bacillus subtilis spore is heat-treated at 115 ° C to 130 ° C for 25 minutes to 35 minutes And most preferably, the step of heat-treating the culture medium containing Bacillus subtilis spore at 120 ° C to 125 ° C for 28 minutes to 32 minutes.

(b) injecting into the subject

The step (b) comprises injecting the inactivated Bacillus subtilis spore prepared in the step (a) into a subject, orally or parenterally injecting the subject. When parenterally injected, it includes injecting by nasal injection, bolus injection, intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration, and the like.

Preferably, in the present invention, step (b) comprises the step of parenterally ingesting inactivated Bacillus subtilis spores, more preferably, inactivating Bacillus subtilis spores into the nasal cavity of the subject .

In the specification of the present invention, the term "subject" means a subject having cancer cells or cancer tissues, but also includes subjects to be prevented from developing cancer cells or cancer tissues. More specifically, the object refers to a mammal having cancer cells or prevention of cancer tissue development or cancer cells or cancer tissues. The subject is preferably a mammal other than a human.

(c) Immunization confirmation step

Step (c) is a step of confirming the immunization of the vaccinated subject according to steps (a) and (b), and confirming that cancer cells or cancer tissues are removed by the Th1 immune response.

Since the present invention is a method of using inactivated Bacillus subtilis spore, which is an active ingredient of the anticancer vaccine adjuvant, the description common to both of them is omitted in order to avoid the excessive description of the present specification.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  One : Bacillus From Subtilis Spoja  refine

A single colony of Bacillus subtilis HB3 strain (obtained from American Type Culture Collection, USA) was added to 25 ml of 3% TSB (Tryptic Soy Broth, "TSB-YE") liquid containing 0.5% yeast extract (YE) Respectively. After incubation at 37 ° C and 150 rpm for 4-5 hours, when the OD value was 0.4-0.6, the culture medium was added to 500 ml of TSB-YE medium. The cells were cultured at 37 ° C at 150 rpm for 3 days and then centrifuged at 6,000 rpm for 10 minutes to obtain bacteria. The obtained bacteria were washed twice with cold distilled water and stored at 4 ° C for 72 hours with distilled water. The distilled water was changed every 24 hours during storage.

Then, the microorganisms were obtained by centrifugation at 6,000 rpm for 10 minutes, followed by heat treatment at 68 ° C for 45 minutes. After heat treatment, the bacteria were separated into single units by sonication, and then spores were obtained by concentration gradient centrifugation using sugar or OptiPrap. In order to deactivate the obtained spore, it was sterilized in a sterilizer for 30 minutes at 121 ° C and then washed twice with cold distilled water. The washed bacteria were centrifuged at 13,000 rpm for 1 minute and stored in distilled water. The number of spores was counted and used in the following examples.

Example  2 : Non-active Bacillus Subtilis Spoja Dendritic cell  Mature effect

Bone marrow cells were isolated from C57 / BL6 mice (Orient Bio, Korea) and treated with 20 ng / ml mouse GM-CSF (mouse recombinant gm-csf, jw creagene, Korea) in RPMI 1640 Differentiated bone marrow-derived dendritic cells were plated in a 24-well plate at 1 × 10 ⁶ cells. Then, the unactivated Bacillus subtilis The supernatant was recovered after 24 hours. Interleukin-12 (cytokine secreted upon dendritic cell maturation) was measured from the recovered supernatant using an ELISA kit (R & D systems Co.) (Fig. 1).

A variety of MAMPs (microbe-associated molecular patterns) on the surface of the spore act on TLR2 (Toll-like receptor 2) and NOD2 (nucleotide-binding oligomerization domain-containing protein 2) And that IL-12 secretion, which is a cytokine that only occurs when matured dendritic cells, is induced by signal transduction.

In addition, this cytokine can be obtained by culturing T cells adjacent to dendritic cells with Th1 (Type 1 helper T cells, Zhu, J. et al. , Blood 112 (5): 1557 (2008)).

Example 3: Antigen delivery effect by MHC class I (cross-antigen presentation) in bone marrow-derived dendritic cells of unactivated Bacillus subtilis spore

Dendritic cells differentiated from mouse bone marrow were injected with 20 μg of the model vaccine antigen Endofit OVA (EndoFit ovalbumin, InvivoGen, USA) and unactivated Bacillus subtilis (100 and 500 per resin fine catcher) were mixed. Two hours later, the model vaccine antigen / unactivated Bacillus subtilis Spore-treated dendritic cells were washed with Complete RPMI 1640 medium (Gibco, USA).

CD8 ⁺ T cells were isolated using a magnetic separation method in a lymph node obtained from an OT-1 mouse (Jackson Laboratory) and then subjected to 2 μM CFSE-labeling. Then, the model vaccine antigen / unactivated Bacillus subtilis Spore-treated dendritic cells and CD8 ⁺ T cells were co-cultured at a ratio of 1: 5 and 1:10, respectively.

Unactivated Bacillus subtilis To measure the ability of Spore to deliver the OVA antigen to dendritic cells and present the SIINFEKL peptide of the OVA protein to MHC class I, dendritic cells and CD8 T cells were co-cultured. The proliferation of CD8 ⁺ T cells was confirmed by CFSE labeling at 60 hours after coculture. After 60 hours, untreated, model vaccine antigen alone and unactivated Bacillus subtilis We observed that the CD8 ⁺ T cells proliferated at a higher level in the model vaccine antigen / unactivated Bacillus subtilis spore treatment group than the control group treated alone with the spore. In addition, it has been found that the composition of unactivated Bacillus subtilis spore plays an important role in delivering the antigen presentation capability of dendritic cells to MHC class I (cross-antigen presentation).

Example 4: Effect of non-activated Bacillus subtilis spore Ability of cross-antigen presentation  In vivo in vivo ) evaluation

CD8 ⁺ T cells were isolated using a magnetic separation method in the lymph nodes obtained from OT-1 mice. It was then injected at ⁵ x 10 5 cells into the tail vein of one of the isolated CD8 ⁺ T cell labeling after 2 μM CFSE- group (obtained without the immune cells) mice. At 30 minutes after injection, the experimental mice were anesthetized with rumpun / ketamine. 20 μl of Endofit OVA / 1 × 10 ⁹ CFU of inactivated Bacillus subtilissposa were added to the anesthetized mouse in a volume of 20 μl each of 5 (5 μg / ml) of the previously prepared untreated and model vaccine antigen (20 μg of Endofit OVA) alone 10 [mu] L of each mouse was injected at intervals of minutes.

After 3 days, the mice were anesthetized, then the lymph nodes were harvested and the lungs were obtained after perfusion with heparin-containing PBS to obtain lungs from which RBCs were removed. The lymphocytes were stained with APC-labeled mouse CD8 antibody using an enzyme treatment method (Kerstin A Sauer, Nature protocols, 2007) in the acquired lymph nodes and lungs, and then stained with CFSE stained density using flow cytometry The cell proliferation was confirmed by the change. As a result, it was confirmed that high level of CD8 ⁺ T cells was proliferated in the model vaccine antigen / unactivated Bacillus subtilis spore group compared to the untreated and model vaccine antigen alone treatments.

Thus, it has been found that the composition of unactivated Bacillus subtilis spore plays an important role in inducing the ability of the model vaccine antigen to present a cross-antigen to dendritic cells without the aid of helper T cells.

Example  5: Non-active Bacillus Subtilis Spoa and  After immunization of model antigens with nasal passages, CD8 ⁺ T cell activity effect

(20 [mu] g Endofit OVA) alone, 1 x 10 < ⁹ > CFU unactivated Bacillus subtilis spore alone, 20 [mu] g Endofit OVA / 1 x 10 ⁹ CFU unactivated Bacillus subtilis spore and 20 μg of Endofit OVA / MPL ( - monophosphoryl lipid A, Invivogen, USA) were each prepared at 20 μl volume. C57 / BL6 mice (Orient Bio, Korea) were anesthetized with rumpun / ketamine, and then the prepared samples were injected into the nasal passages at intervals of 5 minutes in an amount of 10 占 퐇. The nasal vaccine and other treatments were administered three times at intervals of two weeks. On the 7th day after the last immunization, the antigen-specific CD8 ⁺ T cell response in the lungs and spleen was confirmed using a tetramer showing PE (Phycoerythrin) -SIINFEKL antigen.

Seven days after the last vaccination, mice were anesthetized and then harvested from the spleen. To obtain lungs from which RBCs were removed, lungs were obtained by perfusion with PBS containing Heparin. (APC-labeled) mouse CD8 antibody, FITC-labeled mouse CD44 antibody and PE (Phycoerythrin) -mediated antibody were obtained by obtaining lymphocytes from the obtained spleen and lungs by an enzyme treatment method. SIINFEKL antigen was stained with the proposed tetramer. Based on the changes in tetramer-positive cells, the stained immune cells were assayed for antigen-specific CD8 ⁺ T cell activity using a flow cytometry analyzer. As a result, in the model vaccine antigen / unactivated Bacillus subtilis spore immunization group compared to the non-treatment, non-active Bacillus subtilis spore alone and model vaccine antigen / MPL treatments in both spleen and lung, It was confirmed that a high level of antigen-specific CD8 ⁺ T cells was induced.

Thus, it was confirmed that the non-activated Bacillus subtilis spore has the ability to induce an antigen-specific CD8 ⁺ T cell immune response as a vaccine adjuvant.

Example 6: Cancer cell defense immunity induction efficacy test after immunization of non-activated Bacillus subtilis spore and model antigen to nasal cavity

1 × 10 ⁹ CFU unactivated Bacillus subtilis spore alone, 20 μg Endofit OVA / 1 × 10 ⁹ CFU unactivated Bacillus subtilis spore alone, 20 μg of Endophyte OVA alone, A sample of 20 [mu] l volume was prepared. The mice were then anesthetized with ruminal / ketamine, and 10 μl of the prepared samples were injected into the nasal passages at intervals of 5 minutes.

For immunization, nasal vaccine containing model vaccine antigen and non-activated Bacillus subtilis spore and other treatments were performed three times at 2-week intervals. B16-F10-OVA cells (Min Sook Ryu, Experimental Cell Research, 2014), a lung cancer metastatic cell line, were injected at 3 × 10 ⁵ cells through the tail vein of all groups of mice at 4 weeks after the final immunization. On the 15th day after injecting the lung cancer metastatic cell line into the mouse tail vein, mice were anesthetized with rumun / ketamine, and the lungs were obtained and the cancer cell metastasis sites (foci) present in the lungs were counted and compared in groups. As a result, it was confirmed that cancer cell metastasis sites did not appear in the model vaccine antigen / unactivated Bacillus subtilis spore immunized treatment group as compared with the non-treatment group, model vaccine antigen alone, and vaccine antigen adjuvant alone group.

Thus, immunization of mice with the model vaccine antigen and the inactivated Bacillus subtilis spore produced by the present invention as an active ingredient can induce CD8 ⁺ T cell immune response by inducing cross-presentation of the model vaccine antigen Induced cancer cell lines.

Claims (9)

An anti-cancer vaccine adjuvant which contains inactivated Bacillus subtilis spore as an active ingredient and induces and increases the Th1 immune response. 2. The anticancer vaccine adjuvant according to claim 1, wherein the inactivated Bacillus subtilis spore is spore having microbe-associated molecular patterns (MAMPs) on its surface. The anticancer vaccine adjuvant according to claim 1, wherein the anticancer vaccine adjuvant additionally includes inactivated Bacillus subtilis-derived MAMPs (Microbe-associated molecular patterns). The anti-cancer vaccine adjuvant according to claim 1, wherein the anti-cancer vaccine adjuvant induces secretion of interleukin-12 from dendritic cells. The anti-cancer vaccine adjuvant according to claim 1, wherein the anti-cancer vaccine adjuvant induces proliferation of CD8 ⁺ T cells. The method of claim 1, wherein the cancer is selected from the group consisting of lung cancer, breast cancer, cervical cancer, ovarian cancer, endometrial cancer, melanoma, bladder cancer, pancreatic cancer, colon cancer, prostate cancer, leukemia, acute lymphoblastic leukemia, Lymphoma, acute myelogenous leukemia (AML), chronic myelogenous leukemia, thyroid cancer, thyroid follicular cancer, kidney cancer, non-small cell lung cancer, bladder cancer, head and neck cancer, stomach cancer, liver cancer, brain cancer, cholangiocarcinoma, small bowel cancer, uterine cancer and testicular cancer Lt; RTI ID = 0.0 > 1, < / RTI > Inactivated Bacillus subtilis spore as an active ingredient and inducing and increasing the Th1 immune response. A method of inducing and augmenting a Th1 immune response comprising the step of inactivating Bacillus subtilis spore into a subject and immunizing. 9. The method of claim 8, wherein the method comprises injecting into the nasal cavity of the subject to be immunized.

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