KR101209416B1 - Dendritic cell based cancer medicine using HBHA as an adjuvant - Google Patents

Abstract

The present invention relates to a dendritic cell cancer therapeutic agent. More specifically, the present invention relates to a dendritic cell cancer therapeutic agent using HBHA of Mycobacterium tuberculosis as an adjuvant. The agent for treating dendritic cell cancer using HBHA of Mycobacterium tuberculosis of the present invention as an adjuvant effectively suppresses tumor growth.

Description

Dendritic cell based cancer medicine using HBHA as an adjuvant}

The present invention relates to a dendritic cell cancer therapeutic agent. More specifically, the present invention relates to a dendritic cell cancer therapeutic agent using HBHA of Mycobacterium tuberculosis as an adjuvant.

Dendritic cells or dendritic cells (DC) are immune cells that form part of the mammalian immune system. These cells act as antigen-expressing cells that process the antigenic material and make it appear on the surface so that other cells in the immune system can recognize it. Dendritic cells are primarily present in small amounts in tissues that contact the external environment, such as the skin (cells in the skin, especially Langerhans cells), nose, lungs, stomach and intestinal lining. They can also be found immature in the blood. Once activated, they travel to lymphoid organs and interact with T and B cells to trigger an immune response. At certain stages of development they extend into processes called dendrites.

Dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells initially turn into immature dendritic cells. These cells are characterized by high endocytosis activity and T-cell activity capacity. Immature dendritic cells constantly devour pathogens such as viruses and bacteria in their surroundings. This is possible through a pattern recognition receptor (PRR), such as a toll-like receptor (TLR). TLRs recognize certain chemical characteristics found on a subset of pathogens. Immature dendritic cells also devour some cell membranes from a living autologous cell through a process called nibbling. Once they come into contact with existing antigens, they are activated into mature dendritic cells and migrate to lymph nodes. Immature dendritic cells devour pathogens and break down their proteins into small pieces that, when mature, appear on the cell surface using MHC molecules. At the same time, it increases cell surface receptors that act as co-receptors in T-cell activation, such as CD80 (B7.1), CD86 (B7.2), and CD40. They also increase CCR7, a chemotactic receptor that induces dendritic cells to go into the spleen through the bloodstream or through the lymphatic system to lymph nodes. Here these cells act as antigen-expressing cells: they activate helper T-cells, killer T-cells, as well as B cells by representing antigen-derived antigens with non-antigenic specific costimulatory signals.

All helper T-cells are specific for one particular antigen. Only specialized antigen expressing cells (macrophages, B lymphocytes and dendritic cells) activate the remaining helper T-cells when the right antigen is present. Macrophages and B lymphocytes, however, can only activate memory T-cells, while dendritic cells can activate both memory and naive T-cells, making them the most potent antigen-expressing cells.

Mature dendritic cells can be derived from monocytes, white blood cells that circulate in the body, which can be converted into dendritic cells or macrophages according to appropriate signals. Monocytes are derived from stem cells of bone marrow. Monocyte-derived dendritic cells can be produced from peripheral blood mononuclear cells (PBMC) in a laboratory. PBMCs can be planted in tissue culture flasks to allow monocytes to attach, which can be differentiated into immature dendritic cells by treatment with IL-4 and granulocyte-macrophage colony stimulating factor (GM-CSF). Subsequent treatment with TNF-α differentiates immature dendritic cells into mature dendritic cells.

Because dendritic cells are rare and difficult to isolate, only the approximate formation and development of dendritic cells of different types and subsets and their interrelationship are known. Dendritic cells are in constant communication with other cells in the body. This communication can take the form of direct cell-to-cell contact based on the interaction of cell surface proteins. For example, the interaction between the receptor B7 of dendritic cells and CD28 of lymphocytes is mentioned.

Intercellular interactions, however, can also occur remotely through cytokines. In vivo experiments stimulate dendritic cells with microbial extracts to allow dendritic cells to produce IL-12 rapidly. IL-12 causes virgin CD4 T cells to be Th1 phenotype. The best result is to activate the immune system so that dendritic cells attack the antigens shown on the surface.

The cytokines produced by dendritic cells depend on the type of cell. Lymphoblastic dendritic cells can produce large amounts of type 1 IFN, which collects more activated macrophages to enable phagocytosis. Lymphoblastic dendritic cells are involved in central and peripheral immune regulation, and myeloid dendritic cells are known to be involved in inducing immunity to foreign antigens or infections. Therefore, when the dendritic cells do not function normally, autoimmune diseases such as diabetes mellitus, rheumatoid arthritis, allergic hypersensitivity reactions may occur, or normal immune responses may not occur against infectious diseases or cancers.

In cancer patients, the function of dendritic cells is reduced and it is difficult to induce normal anticancer immunity. Until now, many substances (LPS, TNF-α, IL-1β) that regulate (activate) the differentiation of dendritic cells are known, but as a side effect of biotoxicity, there are many problems in direct application to the living body.

As described above, dendritic cells play an important role in enhancing the body's own immune function, so it is clear that the development of non-toxic immunomodulators that promote the differentiation of dendritic cells that give rise to a strong immune response and a clear understanding of the mechanism of action Cellular immune therapy using cells, especially cancer treatment has become an important challenge.

The present inventors conducted a study to meet the above requirements, found that HBHA derived from Mycobacterium tuberculosis effectively matures dendritic cells and developed a dendritic cell cancer treatment agent using the adjuvant.

Heparin-binding hemagglutin (HBHA) is a main 28-kDa protein of Mycobacterium tuberculosis antigen that attaches to epithelial cells through a C-terminal lysine rich domain that interacts with heparan sulfate (HS) containing proteoglycans on the surface of target cells. HBHA has recently been shown to be surface-exposed and secreted antigen, which induces the production of potent IFN-g by blood CD4 + and CD8 + T lymphocytes in healthy individuals with latent tuberculosis but not in active TB patients. . The present inventors thought that such HBHA could be used for the maturation of dendritic cells and found that it is possible to effectively mature dendritic cells with HBHA and to treat cancer.

Accordingly, the present invention provides a drug for treating dendritic cell cancer prepared by adding HBHA derived from Mycobacterium tuberculosis to an immature dendritic cell as an adjuvant.

Preferably the HBHA is expressed in M. smegmatis Recombinant HBHA (rMS-HBHA).

Preferably, the dendritic cell cancer therapeutic agent of the present invention is prepared by incubating for 24 hours with the HBHA.

Also preferably, the HBHA is added to the medium of dendritic cells at a dose of 0.5-1 μg / ml.

The dendritic cell cancer therapeutic agent of the present invention can be prepared by adding HBHA and incubating for 2 hours after adding OVA (ovalbumin) in order to enhance the therapeutic effect. Preferably the OVA is added at a concentration of 1 mM.

The dendritic cell cancer therapeutic agent of the present invention may be administered parenterally, for example, but not limited to subcutaneous, intraperitoneal, intramuscular, transdermal (transdermal / transcutaneous) injection. Cancer therapeutic agents of the invention may also be administered directly to the lesion site of a tumor. The dendritic cell cancer therapeutic agent of the present invention may be administered according to a single dose or multiple dose schedules, or may be administered with other immunomodulatory agents.

The cancer therapeutic agent of the present invention comprises the dendritic cells of the present invention together with one or more pharmaceutically acceptable carriers and / or diluents, which carriers are any carriers which do not cause a harmful reaction to the individual receiving the therapeutic agent. It is preferable. Suitable carriers are typically large and slow metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, lipid aggregates (eg oil droplets or liposomes) and inert virus particles. Including but not limited to. Such carriers are well known to those skilled in the art and can act as immunostimulants or adjuvant in addition to the adjuvant effect of dendritic cells themselves.

Cancer treatments of the invention may also include additional immunopotentiators that enhance the effectiveness of the treatment. Aluminum salts such as aluminum hydroxide, aluminum phosphate, aluminum sulfate and the like (alum); 5% Squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally varying amounts of N-acetylmuramil-L-alanyl-D-isoglutaminyl-L-alanine-2- MF59® (WO90 / 14837), including (1 ', 2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (MTP-PE)), submicron 10% squalene, 0.4% Tween 80, 5% Pluronic-Blocked Polymer and N-Acetyl-Muramil-L-Threonyl-D-Isoglutamine, microfluidized into particles or shaken to produce an emulsion of large particle size SAF®, including (thr-MDP), and one or more bacterial cell wall components selected from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM) and cell wall backbone (CWS), Oil-in-water emulsion formulations such as the Ribi ™ Adjuvant System (RAS®), including 2% squalene and 0.2% Tween 80; Saponin adjuvant; Freund's complete adjuvant (CFA) and incomplete adjuvant (IFA); Interleukins such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, interferons such as gamma interferon, macrophage colony stimulating factor (M-CSF) and tumor necrosis Cytokines such as factor (TNF); Cholera toxin (CT), pertussis toxin (PT), or heat labile toxin (LT) of E. coli , especially detoxified mutants of LT-R72, CT-S109, PT-K9 / G129 (WO93) / 13302 and WO92 / 19265) and the like ADP-ribosylated toxin of bacteria; And other substances acting as immunostimulating agents that enhance the effectiveness of cancer treatments.

The dendritic cell cancer therapeutic agent of the present invention may further include a diluent, for example, water, saline, glycerol, ethanol, and the like, and may further include auxiliary substances such as wetting agents, emulsifiers, pH buffers, and the like. have.

Suitable forms for injection of the cancer therapeutic agent of the present invention include sterile injectable solutions, or sterile aqueous solutions or dispersions for the immediate preparation of injectables and sterile powders. They must be stable under the conditions of manufacture and must be preserved against the contamination of microorganisms such as bacteria or fungi. Contamination of microorganisms can be prevented using various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, chimerosal and the like. It is preferable to include isotonic agents, such as sugars or sodium chloride, in order to reduce the pain of the patient unless it impairs the stability and efficacy of the cancer therapeutic agent.

Additives that delay absorption, such as aluminum monostearate or gelatin, can be added to allow the cancer therapeutic agent of the present invention to be absorbed for a long time.

In the solvent described above, HBHA is used as an adjuvant to combine an effective amount of pre-treated dendritic cells with various other components listed above, and other components except for dendritic cells and / or heat-sensitive immunoadjuvant cytokines, if necessary. For example, a sterile injectable solution is prepared by filtration sterilization of a buffer solution such as PBS. Dispersions are usually prepared by incorporating the various sterilized active ingredients into a sterile container containing the basic dispersion medium and the required other ingredients.

By 'effective amount' is meant an amount necessary to at least partially achieve the desired immune response or to delay or entirely stop the onset or progression of the particular disease to be treated. This amount depends on the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated, the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the assessment of the medical situation and other relevant factors. This amount is expected to fall within a relatively broad range that can be measured through routine trials.

The present inventors first conducted a toxicity test on dendritic cells of HBHA before conducting an efficacy test of HBHA to mature dendritic cells. As shown in Fig. 2, dendritic cells were treated with HbHA up to 1 μg / ml and stained with Annexin-V. As a result, 1 μg / ml of HBHA did not show cytotoxicity to dendritic cells. It can be said that it is not toxic.

In one embodiment of the present invention, an experiment was conducted to determine whether HBHA matures immature dendritic cells. As dendritic cells mature, the expression of co-stimulatory factors and molecules such as MHC class I and II is significantly increased, so HBHA is treated in dendritic cells at concentrations of 0.5 μg / ml and 1 μg / ml, respectively, Incubated. Here LPS was used as a positive control to compare the effects of HBHA. As can be seen in FIG. 3, HBHA dose-dependently increased the surface molecules of dendritic cells. Therefore, it can be seen that HBHA affects the surface molecular change of dendritic cells.

In another embodiment of the present invention, the experiment was conducted to determine the effect of HBHA on cytokine secretion of dendritic cells. As the dendritic cells mature, the secreted cytokines also change. Therefore, we tested whether dendritic cells treated with HBHA affected changes in pro-inflammatory or anti-inflammatory cytokine secretion that affect T-cells. IL-12 and IL-10 are major cytokines secreted from dendritic cells, and play an important role in the differentiation of T cells into Th1 and Th2, respectively. TNF-α, IL-6, IL-1b, IL-12p70, IL-10 in dendritic cells were measured by ELISA. As shown in FIG. 4, since IL-12 and IL-10 were increased and pro-inflammatory cytokines TNF-α, IL-6, and IL-1b were also increased, HBHA matured dendritic cells and changed the amount of cytokines. You can see that it gives.

In another embodiment of the present invention, the expression of CCL7 in HBHA-treated dendritic cells was examined. As dendritic cells mature, their ability to move increases, so receptors associated with migration can be used as an indicator of whether mature cells have matured. Of the dendritic cell migration-related receptors, CCR7 reacts with CCL19 at maturity, so HBHA-treated with dendritic cells, LPS-treated with positive controls, and no-treated negative controls were treated with CCR7-PE and CD11c. -Stained with FITC and measured by flow cytometry. As can be seen in Figure 5, when treated with HBHA CCR7 levels increased compared to untreated dendritic cells, it can be said that HBHA increases the ability of migration of dendritic cells.

In order to more clearly confirm this, another embodiment of the present invention was tested for chemotaxis of CCR19 of dendritic cells treated with HBHA. As a result of measuring the number of dendritic cells that migrate in response to 300ng / ml of CCR19, the binding ligand of CCR7 was found to be more mobile than HBHA-treated dendritic cells (Fig. 6). ). In conclusion, HBHA matures dendritic cells and increases their ability to move to secondary lymphoid organs and react with T cells.

Dendritic cells serve to differentiate virgin T cells into Th1 or Th2. Activated dendritic cells transmit antigenic information to T cells through costimulatory molecules, thereby activating the T cells. In another embodiment of the present invention, mixed-lymphocyte reaction experiments were performed to determine whether HBHA is involved in activation of virgin T cells by activating dendritic cells. Splenocytes of OT-1 mice were stained with CFSE and incubated with dendritic cells treated with HBHA. The number of T cells increased was measured by flow cytometry. As shown in FIG. 7, proliferation of CD8 + T cells was prominent in T-cells cultured with dendritic cells treated with HBHA compared to negative controls. These results confirm that HBHA increases the ability to activate T-cells, one of the important roles of dendritic cells.

In order to confirm whether dendritic cells using the HBHA of the present invention as an adjuvant are effective in inhibiting cancer cells, an exemplary antitumor treatment effect was tested in a mouse tumor model formed by EG7 cells. In the group receiving dendritic cells treated with HBHA and OVA, the tumor size was much smaller than in the other groups. In addition, as time passes after the dendritic cells are administered, it can be seen that the growth of tumor size is most markedly slowed down when the dendritic cell therapeutic agent of the present invention is administered (FIG. 9).

Regulatory T cells (Tregs), which express the transcription factor Foxp3, inhibit the activity of other immune cells. In the environment in which the tumor grows, a number of factors occur that form Treg cells in the tumor itself. In one embodiment of the present invention, the antitumor treatment effect of HBHA-treated dendritic cells was confirmed by measuring the number of Treg cells using antibodies to CD25 and CD4 molecules expressed in Treg cells. Tumor models using EG7 cell lines were injected with PBS, untreated dendritic cells, OVA-treated dendritic cells, and dendritic cells treated with HBHA and OVA, respectively, and splenocytes were isolated from each group of mice to obtain anti-CD25-Cy5. , Stained with anti CD4-FITC, anti FoxP3-PE and analyzed by flow cytometry. As a result, the Treg cells were significantly decreased in the group injected with HBHA-treated dendritic cells compared to other groups (FIG. 9). Therefore, it can be seen that dendritic cells treated with HBHA are effective anti-tumor therapeutics.

The present invention provides a drug for treating dendritic cell cancer using HBHA of Mycobacterium tuberculosis as an adjuvant, which effectively suppresses tumor growth.

1 is a diagram showing the results of A: CB staining and B: anti-HisHA antibody blotting on HBHA isolated according to Example 1, and confirming HBHA of about 28 kDa.
Figure 2 is a graph showing the results of the survival rate measurement experiment of dendritic cells treated with the HBHA of the present invention according to Example 2.
Figure 3 is a graph showing the results of the co-stimulatory factor and MHC class I, II expression analysis experiments of dendritic cells treated with the HBHA of the present invention according to Example 3.
Figure 4 is a graph showing the results of the cytokine (IL-6, IL-12, IL-1, TNF-α and IL-10) secretion test results of the dendritic cells treated with the HBHA of the present invention according to Example 4.
5 is a graph showing the results of the CCR7 expression of the dendritic cells treated with the HBHA of the present invention according to Example 5. CON: Control.
FIG. 6 is a graph showing the results of chemotaxis assay of dendritic cells treated with HBHA of the present invention according to Example 6. FIG.
7 is a graph showing cell migration-mixed lymphocyte responses of dendritic cells treated with HBHA of the present invention according to Example 7. FIG.
Figure 8 is a photograph comparing the tumor size 20 days after the cell therapy injection in the mouse according to Example 8. When the dendritic cell therapy treated with the HBHA of the present invention is used, it can be visually confirmed that the tumor size is much smaller than otherwise. PBS: phosphate buffered saline (negative control), iDC: immature dendritic cells, DC-OVA: OVA-treated dendritic cells, HBHA-OVA: HBHA-treated dendritic cells again.
9 is a graph showing the cancer cell inhibitory effect of the dendritic cell treatment treated with HBHA of the present invention according to Example 8. PBS: phosphate buffered saline (negative control), iDC: immature dendritic cells, DC-OVA: OVA-treated dendritic cells, HBHA-OVA: HBHA-treated dendritic cells again.
10 is a graph showing the results of a Treg cell inhibitory activity of the dendritic cells treated with the HBHA of the present invention according to Example 9. PBS: phosphate buffered saline (negative control), iDC: immature dendritic cells, DC-OVA: OVA-treated dendritic cells, HBHA-DC-OVA: HBHA-treated dendritic cells again.

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

≪ Example 1 >

Preparation of Dendritic Cells and Mycobacterium Tuberculosis HBHA

1.1 Isolation and Derivation of Dendritic Cells

Dendritic cells are derived from mouse bone marrow cells as previously known methods.

First, bone marrow was harvested using a bone marrow harvesting injection from C57BL / 6 mice. After the collected bone marrow was washed, red blood cells were removed using ammonium chloride. The isolated cells were placed in RPMI 1640 (10% heat-inactivated FBS, 2 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 5 × 10 ⁻⁵ M 2-ME, 10 mM in a 6-well plate). HEPES (pH 7.4), 10 ng / ml GM-CSF, 10 ng / ml IL-4) was added to incubate. On days 3 and 5 after incubation, suspended cells were removed and fresh medium was added.

Experiments to be described later used non-attached dendritic cells on day 6 of culture. In some experiments CD11c ⁺ dendritic cells were used after separation using anti-CD11c (N418) microbeads and magnetic cell sorting system (Vario MACS: Miltenyi Biotec, Auburn, CA). LPS ( E. coli 055: B type, Sigma) was used as a positive control for HBHA.

1.2 Preparation of Mycobacterium Tuberculosis HBHA

Recombinant HBHA (rMS-HBHA) was expressed in M. smegmatis .

A pMV3-38 plasmid (pMV261 vector) containing a full-length HBHA open reading frame (ORF) provided by G. Delogu of Sassari University, Italy, was inserted into M. smegmatis by electroporation. M. smegmatis (pMV3-38) recombinant strain expressing HBHA labeled with histidine was incubated at 37 ° C. for 4 days in Sauton's medium containing 50 μg / ml of hygromycin, followed by centrifugation. Cells were harvested by isolation.

Cells were resuspended in 10 mM potassium phosphate buffer (pH 7.0) containing 4 mM phenylmethylsulfonyl fluoride and 0.1 mg / ml lysozyme and sonicated on ice to lyse the cells. After centrifugation, the supernatant was placed in a phosphocellulose column (Whatman P11) equilibrated with 10 mM potassium phosphate buffer. The column was washed with 150 mM potassium phosphate buffer and the attached material eluted with 750 mM potassium phosphate buffer.

The eluate was concentrated and dialyzed with 20 mM Tris-HCl (pH 8.0) containing 500 mM NaCl and 20 mM imidazole, followed by nickel-nitrilotriacetic acid (Ni-NTA) agarose chromatography ( Invitrogen, Carlsbad, CA). SDS-PAGE and Coomassie blue staining were used to confirm the purification and immunoblotting was performed (FIG. 1).

<Example 2>

Survival Measurement of Dendritic Cells

To measure cell viability of dendritic cells stimulated with LPS or HBHA, dendritic cells were 200 ng / ml 5 μg ml ⁻¹ propidium iodide (PI) and Annexin- after treatment for 24 hours with concentrations of LPS (E. coli 055: B, Sigma) or 0.5 μg / ml and 1.0 μg / ml HBHA Double staining with V and analysis by flow cytometry. The results are shown in FIG.

<Example 3>

Analysis of Costimulatory Factors and Expression of MHC Class I and II

Dendritic cells were harvested after treatment of dendritic cells with HBHA at concentrations of 0.5 μg / ml and 1.0 μg / ml and LPS at 200 ng / ml for 24 hours, respectively. In order to inhibit nonspecific binding, 1 μg / ml of Fcγ I / III (BD bioscience) was treated at 4 ° C. for 20 minutes and then stained with CD11c-FITC (BD bioscience) for 20 minutes at 4 ° C. for dendritic cell analysis. . In addition, 1 μg of anti-CD80-PE (BD bioscience), anti-CD86-PE (BD bioscience), anti-MHC I and II-PE (BD bioscience) were used to determine the expression patterns of co-stimulatory factors and MHC molecules. / ml treatment and dyeing at 4 ℃ 30 minutes. Wash twice with PBS buffer (containing 2% FBS and 0.01% sodium azide) and analyze with FACScallibur (Beckson Dikinson, USA) after fixation of 1% paraformaldehyde. The results are shown in Fig.

<Example 4>

Effects of HBHA on Cytokine Secretion in Dendritic Cells: IL-6, IL-12, IL-1, TNF-α, and IL-10

each 1 x 10 ⁶ cells / ml One ml of media containing dendritic cells was divided into four groups, 0.5 μg / ml of HBHA, 1.0 μg / ml of HBHA and 200 ng / ml of LPS were added, and the other was not treated as a negative control. After 24 hours of incubation, the supernatants were subjected to ELISA kits (BD PharMingen, USA) for IL-6, IL-12, IL-1, TNF-α and IL-10 to measure cytokine secretion. The results are shown in FIG.

<Example 5>

CCR7 expression level measurement

each 1 x 10 ⁶ cells / ml One ml of media containing dendritic cells was divided into four groups, 0.5 μg / ml of HBHA, 1.0 μg / ml of HBHA and 200 ng / ml of LPS were added, and the other was not treated as a negative control. After incubation for 24 hours, cells were harvested. In order to inhibit nonspecific binding, 1 μg / ml of Fcγ I / III was treated at 4 ° C. for 20 minutes and then stained with CD11c-FITC for 20 minutes at 4 ° C. for dendritic cell analysis. Then, anti-CCR7-PE treatment and stained at 4 ℃ 30 minutes. Wash twice with PBS buffer (containing 2% FBS and 0.01% sodium azide) and analyze with FACScallibur (Beckson Dikinson, USA) after fixation of 1% paraformaldehyde. The results are shown in FIG.

<Example 6>

Chemotaxis Analysis

Purified dendritic cells were measured to migrate in response to chemokine CCL19 (300 ng / mL) with or without treatment with 1 μg / ml HBHA and 200 ng / ml LPS for 24 hours (negative control). The lower chamber of the transwell plate (pore size 8.0 μm; Corning, Acton, Mass.) Received serum-free medium with and without 500 μl of CCL19. Dendritic cells (1 × 10 ⁵ cells in 0.1 mL) resuspended in serum-free medium were placed in the upper chamber of the transwell plate and allowed to move for 3 hours at 37 ° C. under 5% CO ₂ . The number of migrated dendritic cells collected in the lower chamber was counted with 60-second counts (FACS). The results are shown in FIG.

&Lt; Example 7 >

Proliferation-Mixed Lymphocyte Response

From C57BL / 6 mice Take the spleen out Splenocytes were extracted by methods known to those skilled in the art and then transgenic ovalbumin (OVA) -specific CD8 ⁺ T-cells (negative selection from whole splenocytes using a mouse CD8 ⁺ T-cell kit (Miltenyi Biotec)). OT-1 T-cells). After staining with Cy5-binding anti-CD8 Ab, it was analyzed by flow cytometry to confirm that the purity is 93% or more. Lymphocytes were washed twice with PBS containing Ca ²⁺ and Mg ²⁺ and labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen).

OT-1 T-cells were resuspended in 1 μM CFSE PBS solution. After shaking for 8 minutes at 37 ℃ cells were washed once with pure fetal bovine serum and twice with PBS containing 10% fetal bovine serum. Dendritic cells (1 × 10 ⁴ cells per well) generated from bone marrow-derived monocytes of C57BL / 6 mice were incubated for 24 hours with 0.5 μg / ml and 1.0 μg / ml HBHA and 200 ng / ml LPS. Nothing was added to the negative control. Dendritic cells were activated with 1 μM / ml OVA peptide at 37 ° C. for 1 hour (not used for negative control) and washed thoroughly before use. CFSE-labeled OT-1 T-cells (1 × 10 ⁵ per well) with dendritic cells (1 × 10 ⁴ per well) 96-well microtiter culture plate triplicate wells (Nunc) at the U-shaped bottom Planted in. After 72 hours the cells were harvested and stained with Cy5-labeled anti-CD8 monoclonal Ab and analyzed by flow cytometry. The results are shown in FIG.

&Lt; Example 8 >

Cancer cell suppression experiment

3 E.G7-OVA cells in 8 C57BL / 6 mice The scanning increments as × 10 ⁵ was gae grow cancer cells for 7 days. After 24 hours of treatment with 1 mg / ml HBHA, Dendritic cells incubated for 2 hours with OVA were treated with 1 x 10 ⁶ cells / PBS for each C57BL / 6 mice that had tumors. 100 μl were injected intraperitoneally twice a week apart. For control, two mice each were injected with only the same number of dendritic cells without any treatment, OVA-treated dendritic cells and PBS. Tumor size was measured 20 times three times a week after the last injection. The results are shown in FIGS. 8 and 9.

&Lt; Example 9 >

Treg Cell Inhibitory Activity

T cells were isolated by removing the spleen 20 days after the last injection from the mouse tumor model prepared as in Example 8. The isolated cells were stained with CD4-FITC, CD25-Cy5 and Foxp3-PE and measured by FACS. The results are shown in FIG.

Although embodiments of the present invention have been described above with reference to the accompanying drawings, those skilled in the art to which the present invention pertains may be embodied in other specific forms without changing the technical spirit or essential features of the present invention. I can understand that. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (6)

A cancer therapeutic agent comprising dendritic cells prepared by adding HBHA (heparin-binding hemagglutin) of Mycobacterium tuberculosis to an immature dendritic cell as an adjuvant.
According to claim 1, wherein the HBHA is a cancer therapeutic agent, characterized in that the recombinant HBHA (rMS-HBHA) expressed in M. smegmatis .
According to claim 1, wherein the dendritic cell is a cancer therapeutic agent, characterized in that prepared by putting the HBHA of Mycobacterium tuberculosis in immature dendritic cells and cultured for 24 hours.
The cancer therapeutic agent according to claim 1, wherein the HBHA is added to the medium of dendritic cells at a dose of 0.5 to 1 µg / ml.
The method of claim 3, wherein the dendritic cells are cancer treatment, characterized in that the further OVA (ovalbumin) is added and incubated for 2 hours.
The cancer therapeutic agent according to claim 5, wherein the OVA is added at a concentration of 1 mM.

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