KR20160026034A - Pharmaceutical composition for preventing or treating cancers comprising dendritic cells with Foxp3 gene silenced - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer, and more particularly, to a pharmaceutical composition for preventing or treating cancer comprising dendritic cells knocked down with Foxp3 gene.
The composition according to the present invention is characterized in that the Foxp3 gene contains dendritic cells whose knock-down knock-out or Foxp3 protein activity is inhibited and the Foxp3 gene knockdown -down) Knock-out or inhibition of the activity of Foxp3 protein significantly decreased CD11b / CD11c / CD103 triple positive dendritic cells involved in immunoregulation, and the antigen-predominant and T cell-stimulated surface molecules were remarkably reduced (CTLs) capable of attacking cancer cells as well as a pharmaceutical composition or anticancer vaccine for preventing, ameliorating, or treating cancer are expected to be usefully used.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising Foxp3 gene knockdown dendritic cells,

The present invention relates to a pharmaceutical composition for preventing or treating cancer, and more particularly, to a pharmaceutical composition for preventing or treating cancer comprising dendritic cells knocked down with Foxp3 gene.

Dendritic cells (DCs) are important specialized antigen presenting cells (APCs) that initiate antigen-specific adaptive immune responses. Dendritic cells have various characteristics depending on their origin, shape, phenotype, function, and maturation process. In general, dendritic cells are differentiated in vitro in a medium supplemented with granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4 by extracting mouse bone marrow stem cells or human monocytes, Used in research. However, the in vivo mechanism of differentiation and maintenance of bone marrow stem cell (DC) into the body was scarcely known. Environmental or genetic factors are also important factors in dendritic cell (DC) differentiation, but they are not well known until now. Dendritic cells (DCs) are important cells that regulate immune homeostasis by promoting or inhibiting T cell responses depending on the situation. Local immunoreactive dendritic cells (DCs) play a role in immunosuppression or immune tolerance through the activation of regulatory T cells (Treg cells). In this case, interleukin 10 (IL-10) or transforming growth factor- Immunomodulation is progressed by the same cytokine expression. Treg cells expressing Foxp3 are responsible for the regulation of the immune response and play an important role in maintaining immune homeostasis.

Foxp3 protein is expressed specifically in regulatory T cells (Tregs), and Tregs, which are highly expressed in cancer tissues, play an important role in the immunosuppressive mechanism of cancer. However, analysis of cancer tissues shows that Treg as well as various immune cells And their roles and functions are not well known.

On the other hand, most of the cancer treatments so far have been performed mainly in surgery, chemotherapy, and radiation therapy. However, such treatment is accompanied by adverse effects, and it is difficult to cure the cancer. In particular, in the case of metastatic cancer or recurrent cancer, surgical treatment is not possible in most cases, and in many cases, the cancer therapeutic agent is resistant to chemotherapeutic agents. Therefore, a new therapeutic agent for cancer patients is urgently required. Among them, cancer treatment using dendritic cells (refer to Korean Patent No. 10-1169331) which is recently being researched and developed is more patient-oriented than conventional therapeutic agents and can be long-term efficacy by memory immunity. It is expected to be a new anti-cancer immunotherapy. However, over the past decade, the efficacy of dendritic cell cancer vaccines has not been as effective as expected, and there is no clear reason for this. Therefore, in recent research, strengthening the efficacy of dendritic cell vaccine has become the biggest problem.

DISCLOSURE OF THE INVENTION The present invention was conceived to solve the technical problems such as limitation of the therapeutic effect of the conventional dendritic cell anticancer vaccine as described above, and the present invention provides a dendritic cell suppressing Foxp3 gene expression or Foxp3 protein activity as an active ingredient And to provide a pharmaceutical composition for preventing or treating cancer.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The present invention provides a pharmaceutical composition for preventing or treating cancer comprising, as an active ingredient, dendritic cells inhibiting the expression of Foxp3 gene or the activity of Foxp3 protein.

In one embodiment of the present invention, the Foxp3 protein may have the amino acid sequence shown in SEQ ID NO: 1.

In another embodiment of the present invention, the dendritic cells may be differentiated from bone marrow stem cells or hematopoietic cells.

In another embodiment of the present invention, the dendritic cells may be separated directly from the blood.

In another embodiment of the present invention, expression of the Foxp3 gene can be suppressed by knocking-down or knocking out the Foxp3 gene.

In another embodiment of the present invention, the knock-down may be performed by an antisense nucleotide, siRNA, shRNA or ribozyme that binds complementarily to mRNA of the Foxp3 gene.

In another embodiment of the present invention, the knock-out may be accomplished by removing or damaging the DNA or mRNA encoding the Foxp3 gene.

In another embodiment of the present invention, the inhibition of the activity of the Foxp3 protein may be accomplished by a compound, a peptide, a peptide mimetic, a substrate analogue, an aptamer or an antibody that binds complementarily to the Foxp3 protein.

In another embodiment of the present invention, the cancer is selected from the group consisting of bladder cancer, osteosarcoma, blood cancer, breast cancer, melanoma, thyroid cancer, pituitary cancer, bone cancer, rectal cancer, Skin cancer, brain tumor, uterine cancer, head or neck cancer, gallbladder cancer, oral cancer, colon cancer, perianal cancer, central nervous system tumor, liver cancer and colon cancer.

The present invention also provides a cancer vaccine comprising as an active ingredient a dendritic cell in which the expression of Foxp3 gene or the activity of Foxp3 protein is inhibited.

Furthermore, the present invention provides a method for treating cancer comprising administering to a subject a dendritic cell in which the expression of Foxp3 gene or the activity of Foxp3 protein is inhibited.

In addition, the present invention provides a cancer preventive or therapeutic use of dendritic cells in which Foxp3 gene expression or Foxp3 protein activity is inhibited.

The composition according to the present invention is characterized in that the Foxp3 gene contains dendritic cells whose knock-down, knock-out or Foxp3 protein activity is inhibited, Foxp3 gene knockdown CD11c / CD103 triple-positive dendritic cells involved in immunomodulation were significantly reduced when the knock-down, knock-out, or Foxp3 protein activity was inhibited, and antigen-positive cells, lymph node mobility of cells, (CTLs) capable of attacking cancer cells and related T cells, as well as being useful as a pharmaceutical composition for preventing, ameliorating or treating cancer It is expected to be used.

Figure 1 shows the results of Western blot analysis of Foxp3 expression in bone marrow-derived dendritic cell differentiation.
FIG. 2 shows the results of confirmation of Foxp3 expression in a bone marrow-derived dendritic cell differentiation process using a confocal microscope.
FIG. 3 shows the results of FACS analysis of Foxp3 expression in bone marrow-derived dendritic cell differentiation.
FIG. 4 shows the results of FACS analysis of Foxp3 expression in immune cells that reside in the lymphatic organs.
FIG. 5 shows the results of confirming Foxp3 expression in dendritic cells present in peripheral blood mononuclear cells (PBMC).
FIG. 6 shows results of (A) FACS analysis of Foxp3 expression in dendritic cells present in lymphatic or peripheral blood of a tumor mouse model, and Foxp3 expression in dendritic cells (B, C) (B) and confocal microscope analysis (C).
FIG. 7 shows the results of confirming the change of dendritic cell population by Foxp3 knockdown.
FIG. 8 shows the phenotypic changes of immature dendritic cells (A) and (B) mature dendritic cells induced by Foxp3 knockdown.
FIG. 9 shows the results of confirming the migration ability of (A) CCL19 chemokine and (B) mVEGF by isolating dendritic cells expressing Foxp3 in peripheral blood mononuclear cells.
FIG. 10 shows the result of confirming the uptake capacity of the dendritic cells by Foxp3 knockdown.
FIG. 11 shows the results of analysis of the proliferative activity of (A) OT-I T cells by Foxp3 knockdown immature / mature dendritic cells, (B) Shows the results of analysis of the proliferative capacity of regulatory T cells.

The present inventors have found that the Foxp3 gene, which is known to be specifically expressed in regulatory T cells (Treg), is also expressed in dendritic cells, and that CD11b / CD11c / CD103 triple positive dendritic cells (hereinafter, ) For the first time confirmed that Foxp3 is highly expressed. These Foxp3 + dendritic cells were absent from the lymphoid organs, but only in the blood, and tumor models confirmed that the Foxp3 + dendritic cells in the blood were heavily infiltrated into the tumor tissue. This Foxp3 expression was thought to have an effect on the anti-cancer immunity induction activity of dendritic cells. Foxp3 gene knock-down experiments were performed to examine the in vitro immune inducing ability of dendritic cells. As a result, it was confirmed that the Foxp3 knockdown dendritic cells immunoreactive induction related cell surface antigen, lymph node and tumor tissue migration ability and T cell proliferation ability were consistently improved remarkably. These results suggest that Foxp3 + dendritic cells are a new target in the development of cancer drugs, and Foxp3 gene expression is a new target, since Foxp3 + dendritic cells that penetrate into the tumor may serve as a causative agent for promoting cancer growth by neutralizing the anti- The present inventors have newly found that the dendritic cell cancer vaccine can be developed by inhibiting or blocking the immune response.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating cancer comprising, as an active ingredient, dendritic cells in which Foxp3 gene expression or Foxp3 protein activity is inhibited. The Foxp3 protein preferably has the amino acid sequence of SEQ ID NO: 1, but is not limited thereto. The Foxp3 protein may be an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.

As used herein, the term "prophylactic " means any act that inhibits cancer or delays the onset of cancer by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" means any action that improves or alters the symptoms of cancer by administration of the pharmaceutical composition of the present invention.

"Cancer" as a disease to be improved, prevented or treated by the composition of the present invention is an aggressive characteristic that cells divide and grow by ignoring the normal growth limit, invasive characteristic penetrating into surrounding tissues And diseases caused by cells having metastatic characteristics spreading to other parts of the body. In the present specification, the cancer is also used in the same sense as a malignant tumor, including, but not limited to, solid tumors and blood born tumors. For example, in the present invention, the cancer may be selected from the group consisting of bladder cancer, bone cancer, blood cancer, breast cancer, melanoma, thyroid cancer, pituitary cancer, bone cancer, rectal cancer, , Head or neck cancer, gallbladder cancer, oral cancer, colon cancer, perineal cancer, central nervous system tumor, liver cancer, and colon cancer, but the present invention is not limited thereto.

In the present invention, the expression of the Foxp3 gene in dendritic cells is inhibited by knocking-down or knocking-out the Foxp3 gene, and more specifically, knock-down, Is made by an antisense nucleotide, siRNA, shRNA or ribozyme that binds complementarily to the mRNA of the Foxp3 gene and the knock-out is carried out by removing or damaging the DNA or mRNA encoding the Foxp3 gene But is not limited thereto.

In addition, in the present invention, the inhibition of Foxp3 protein activity is preferably selected from the group consisting of a compound that binds complementarily to the Foxp3 protein, a peptide, a peptide mimetic, a substrate analogue, an aptamer, and an antibody , But it is not limited thereto. Any drug that inhibits Foxp3 protein activity may be used.

In the present invention, "Peptide Minetics" is used to inhibit the activity of the Foxp3 protein by inhibiting the binding domain of the Foxp3 protein. The peptide mimetics may be a peptide or a nonpeptide, Or may be composed of amino acids joined by non-peptide bonds.

In the present invention, the term "aptamer" refers to a single-chain DNA or RNA molecule, which is obtained by an evolutionary method using an oligonucleotide library called SELEX (systematic evolution of ligands by exponential enrichment) Oligomers capable of binding with high affinity and selectivity to biological molecules can be obtained by isolation. Aptamers can specifically bind to a target and modulate the activity of the target, for example, by blocking the ability of the target to function through binding.

Moreover, in the present invention, "antibody" can specifically and directly bind to Foxp3 and effectively inhibit the activity of Foxp3. The antibody specifically binding to Foxp3 is preferably a polyclonal antibody or a monoclonal antibody. Antibodies specifically binding to Foxp3 may be prepared by known methods known to those skilled in the art, and commercially available Foxp3 antibodies can be purchased and used.

In the present invention, dendritic cells may be derived from human or mouse, and preferably dendritic cells can be isolated from human blood or differentiated from bone marrow stem cells separated from blood mononuclear or mouse bone marrow into dendritic cells.

In one embodiment of the present invention, Foxp3 was expressed in dendritic cells and Foxp3 was found to be highly expressed in CD11c / CD11b / CD103 triple positive dendritic cells, which are known to perform immunotolerance functions (see Example 3) .

In another embodiment of the present invention, the presence of CD11c + Foxp3 + dendritic cells in various lymphatic organs (Spleen, inguinal lymph node, mesenteric lymph node, peyer's patch and thymus) (See Example 4), and it was confirmed that Foxp3 expression was achieved only in dendritic cells circulating in blood (see Example 5).

In another embodiment of the present invention, Foxp3 expression in dendritic cells was not found in dendritic cells expressing Foxp3 in any lymphoid organs, whereas Foxp3 + dendritic cells were found in tumor tissues (See Example 6), and it was confirmed that triple positive dendritic cells were significantly reduced in Foxp3-knockdown (Foxp3-KD) compared to the normal control group (see Example 7).

In another embodiment of the present invention, the phenotype and function of dendritic cells induced by Foxp3 knockdown were examined. As a result, the expression of MHC II, CD86, CD80 and CD40, which are important for activating T cells, was increased 8).

In another embodiment of the present invention, dendritic cells expressing Foxp3 were observed to migrate into lymph nodes and tumor tissues. As a result, it was confirmed that dendritic cells expressing Foxp3 mostly migrated to tumor tissues and did not migrate to lymph nodes See Example 9).

In another embodiment of the present invention, the antigen uptake of Foxp3 knockdown dendritic cells was enhanced (see Example 10). As a result of the change in T cell immunity induction ability, OT-I T cell proliferation was greatly improved, while the proliferation of donor T cells and antigen nonspecific regulatory T cells was somewhat reduced (see Example 11).

Foxp3 + dendritic cells are a new target in the development of cancer drugs as Foxp3 + dendritic cells that penetrate into the tumor can serve as a causative agent for promoting cancer growth by neutralizing the in vivo anti-cancer immunity that attacks cancer. Therefore, Foxp3 + Dendritic cells in which the activity of Foxp3 protein is inhibited can be useful as anti-cancer vaccines.

Meanwhile, the pharmaceutical composition of the present invention can be applied to all kinds of dendritic cells in which Foxp3 gene knockdown, knock-out and Foxp3 protein activity are inhibited, together with known active ingredients having anticancer activity More than species.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method.

Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose , Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the age, sex, condition, body weight, absorbency, inactivation rate and excretion rate of the active ingredient in the body, the type of disease, May be administered in an amount of 0.1 mg / kg to 100 mg / kg per day, preferably 1 to 30 mg / kg per day, and may be administered once or several times a day.

The pharmaceutical compositions of the invention can be administered to a subject in a variety of routes. All modes of administration may be expected, for example, by subcutaneous, intravenous, intramuscular or intrauterine dural or intracerebral injection. The pharmaceutical composition of the present invention is determined depending on the kind of the active ingredient, together with various related factors such as the disease to be treated, the route of administration, the age, sex, and weight and disease severity of the patient.

In another aspect of the present invention, the present invention provides a method for treating cancer comprising administering the pharmaceutical composition to a subject. The term "individual" as used herein refers to a subject in need of treatment for a disease, and more specifically refers to a human or non-human primate, mouse, rat, dog, cat, It means mammals.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Experimental preparation

1-1. Dendritic cell separation

Dendritic cells (DCs) were cultured by differentiating bone marrow stem cells into dendritic cells. More specifically, bone marrow-derived dendritic cells (BM-DC) were obtained by separating bone marrow cells from the femur and tibia of 6-8 week old female C57BL / 6 or Foxp3 / gfp C57BL / 6 mice, The ACK lysis buffer (Lonza) was treated to remove. Cells were washed and cultured in cell culture medium (RPMI 1640 with 10% FBS and penicillin / streptomycin) containing 10 ng / ml mGM-CSF (Creagene Inc.). After 2 days, the supernatant of the cultured cells was removed and replaced with 2 ml of fresh culture medium containing mGM-CSF. On the fourth day of culture, 1 ml of a new culture medium containing mGM-CSF was added. On the 6th day of culture, non-adherent cells were collected and used as immature dendritic cells (immature DCs, imDC). Mature dendritic cells (mature DC, mDC) were prepared by immature dendritic cells (DCs) cultured for 24 hours in the presence of LPS (100-200 ng / ml).

1-2. Experimental Method

end. Western blot analysis

Cells were washed with cold PBS and lysed with 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM DTT, 30 mM NaF, 10 mM Na3VO4, 0.5% NP40 and protease inhibitor cocktail Lt; / RTI > Whole cell lysates were adjusted to the same concentration using Bradford reagent (Bio-Rad) and SDS-PAGE was performed on 8-12% acrylamide gel using 40-120 μg lysate and transferred to PVDF membrane (Milipore) . For immunoblotting, the membrane was blocked with 5% non-fat dry milk in TBS (TBST) containing 0.5% Tween 20 for one hour at room temperature. The membranes were washed 4 times with TBST, and the primary antibody diluted with 4% non-fat dry milk was treated at the optimal concentration and incubated overnight at 4 ° C. After washing 4 times with TBST, HRP-conjugated secondary antibody was incubated for 45 minutes. Binding antibodies were detected using a chemiluminescent HRP substrate (Millipore, USA) and autoradiographic film (Agfa Healthcare NV, Belgium).

I. Confocal microscope analysis

Cells were washed with cold PBS and stained with PE-labeled hamster anti-mouse CD11c (HL3) for 30 min. And washed 4 times with 500 μl PBS and floated on 20 μl Antifade mounting medium. Cells were then placed on the slide and coverslips were covered and confocal microscopy (Ziess) analysis was performed.

Example 2. Knock-down of the Foxp3 gene in dendritic cells [

Dendritic cells in which the Foxp3 gene knocked down were prepared by the following method.

SiRNA targeting Murine Foxp3 was designed and constructed using BLOCK-IT RNAi Designer (Invitrogen) and Dharmacon RNAi Technologies (Thermo Scientific) program. Two Foxp3 siRNAs were used for knock-down of Foxp3, and control siRNA was used as a control. The specific information of each siRNA is as follows:

5'-GCA CTG CCA AGC AGA TCA T-3 '(si-Foxp3) (SEQ ID NO: 3)

5'-GGG TAA CGG TGG ACC GTC T-3 '(control siRNA) (SEQ ID NO: 4)

Foxp3 si-RNA (siFoxp3) or control siRNA (sicon) were transfected into dendritic cell (DC) precursor cells on Day 4 or Day 5 using the method provided in Lipofectamine RNAiMAX / Gene porter transfection kit (Life Technologies) and kit. After incubation at room temperature for 20 minutes, 200 μl of the mixture was added to 2 ml of dendritic cell (DC) culture. After 4 hours of incubation, the same amount of RPMI1640 containing 10% FBS was added. After 24-48 hours, the cells were washed and used in subsequent experiments with Foxp3 knock-down immature dendritic cells (Foxp3-KD imDC).

Example 3 Expression of Foxp3 in Bone Marrow-Derived Dendritic Cell Differentiation

In order to confirm the expression of Foxp3 in the process of differentiation into dendritic cells, the following experiment was conducted.

First, Foxp3 protein expression was analyzed by Western blot analysis (Western blot analysis) during the differentiation of bone marrow-derived DC, BMDC by GM-CSF from mouse bone marrow cells in order to confirm the expression of Foxp3 gene at the protein level blot analysis, and the results are shown in Fig. As shown in Fig. 1, it was confirmed that Foxp3 expression was rapidly increased at the final stage of the dendritic cell differentiation process.

Next, Foxp3 + expression was confirmed using a confocal microscope, and the results are shown in FIG. As shown in Fig. 2, Foxp3 was expressed in CD11c + dendritic cells and confirmed to be located in the cytoplasm.

Next, Foxp3 + expression was reaffirmed through FACS analysis, and the results are shown in FIG. As shown in FIG. 3, CD11c expression was continuously increased in the process of differentiating mouse BMDC into dendritic cells, while Foxp3 was expressed with CD11c at the final stage of differentiation (FIG. 3A). A study of dendritic cell populations expressing Foxp3 in BMDC revealed that Foxp3 was significantly expressed in CD11c / CD11b / CD103 triple positive dendritic cells known to perform immune tolerance (FIG. 3B).

Example 4. Foxp3 expression in immune cells resident in lymphoid organs

The presence of CD11c + Foxp3 + dendritic cells was analyzed by FACS in several lymphatic organs (mesenteric lymph node, mesenteric lymph node, peyer's patch and thymus) in the mouse body, and the results are shown in Fig.

Foxp3 was not expressed in dendritic cells isolated from lymphoid organs and Foxp3 was highly expressed only in regulatory T cells of lymphoid organs.

Example 5. Expression of Foxp3 in Dendritic Cells Present in Peripheral Blood Mononuclear Cells (PBMC)

Dendritic cells circulating in the blood are known to be differentiated by GM-CSF. GM-CSF is a key cytokine used in BMDC differentiation, 3, Foxp3 expression was highly expressed in dendritic cells derived from GM-CSF. Based on this, Foxp3 expression was confirmed in dendritic cells present in mouse PBMC, and the results are shown in FIG.

As shown in FIG. 5, a significant amount of Foxp3 was expressed in a part of CD11c ^high dendritic cells as well as a part of T cells in PBMC. Foxp3 + dendritic cells were not contaminated with Foxp3 + regulatory T cells even when reanalyzed by reverse gating (Fig. 5A). On the other hand, Foxp3 + cells were not found in CD11c-CD11b + macrophages (Fig. 5B).

Example 6. Expression of Foxp3 in Dendritic Cells Present in Tumor Tissue

Dendritic cells circulating in the blood are important dendritic cells that are involved in defense in vivo and antitumor immunity induction. Since the dendritic cells present in PBMC express Foxp3 in Example 3, the possibility of penetrating some of them into tumor tissue was examined in a tumor mouse model, and the results are shown in Fig.

As shown in FIG. 6, Foxp3 + dendritic cells were not found in any lymphatic organs such as lymph nodes or spleens of tumor mice, whereas large amounts of Foxp3 + dendritic cells were present in peripheral blood mononuclear cells (Fig. 6A) Lt; RTI ID = 0.0 > Foxp3 + < / RTI > dendritic cells were also present (Figures 6B and C).

Foxp3 + dendritic cells were found to be present only in blood through the above Example 4 and Example 5. As a result, Foxp3 + dendritic cells present in tumor tissues were found to be infiltrated from the blood.

Example 7. Foxp3 knockdown to confirm the change of dendritic cell population

Foxp3 expression was very high in CD11b / CD11c / CD103 triple positive dendritic cells responsible for immunity tolerance in Example 3. Foxp3 was knocked down with si-RNA in the BMDC differentiation process, and the population of BMDC subgroups And the results are shown in FIG.

As shown in FIG. 7, in the Foxp3 knockdown (Foxp3-KD), the dendritic cell population expressing CD11c was greatly increased, but triple positive dendritic cells were significantly decreased compared with the normal control group. From the above results, it was found that Foxp3 expression plays an important role in the differentiation function of triple positive dendritic cells involved in immune tolerance.

Example 8. Confirmation of phenotype and function of dendritic cells by Foxp3 knockdown

To analyze the phenotype of the cells, direct immunofluorescence staining was performed. Cells were stained with FACS buffer for 20 min at 4 ° C with the following antibodies: FITC-labeled, anti-mouse CD86 (GL1), anti-mouse IA / IE (2G9) ^d ), PE-labeled hamster anti-mouse CD11c (HL3), anti-mouse CD80 (16-10A1), rat anti-mouse CD40 (3/23) (BD Pharmingen), FITC-labeled isotype control antibodies. After cell washing, cells were analyzed using FACS Calibur (BD) using CellQuest or FlowJo software.

As shown in Fig. 8, the expression of MHCII, CD80, CD86, and CD40, which are important for T cell immune induction in Foxp3 knockdown dendritic cells, was generally increased.

Example 9. Confirmation of migration ability of dendritic cells expressing Foxp3

In vitro chemotaxis assay using Transwell system was performed to determine whether dendritic cells expressing Foxp3 migrate to lymph nodes or tumor tissues. Chemotaxis of dendritic cells isolated from peripheral blood mononuclear cells (PBMC) was transferred to dendritic cells (DC) through a polycarbonate filter of 8 μM pore size in 24-well transwell chambers (SPL, Korea and Corning Costar, .

CCL19 (300 ng / ml) and mVEGF (100 ng / ml) diluted in 0.6 ml serum-free RPMI 1640 medium were added to the lower chambers of the Transwell plate and the upper chambers were separated from peripheral blood mononuclear cells (PBMC) One dendritic cell was filled and the transwell plate was incubated in a 37 ° C CO ₂ incubator for 3 hours. The dendritic cells (DCs) migrated in the lower chamber were harvested and Foxp3 expression was analyzed by FACS in the migrated dendritic cells (DC). The results are shown in FIG.

As shown in Fig. 9, it was confirmed that dendritic cells expressing Foxp3 do not migrate to the lymph node (CCL19) but migrate only to tumor tissue (mVEGF).

Example 10. Confirmation of antigen uptake capacity of dendritic cells by Foxp3 knockdown

Bone marrow-derived dendritic cells (BMDC) were generated from the bone marrow of C57BL / 6 mice. On day 4, Foxp3 si-RNA or control si-RNA was transfected into dendritic cell (DC) progenitor cells. After 48 hours, the cells were stimulated with LPS (100-200 ng / ml) for 24 hours. Cells were harvested and 2 × 10 ⁵ cells were stabilized in a FACS tube at 37 ° C. or 4 ° C. for 45 minutes and then uptaked for 1 hour with the addition of 1 mg / ml FITC-conjugated dextran. The reaction was terminated with cold staining buffer. The washed cells were stained with PE-conjugated anti-CD11 and analyzed using a FACS Calibur flow cytometer. The results are shown in FIG.

As shown in Fig. 10, it was confirmed that the antiprotozoal action of Foxp3 knockdown dendritic cells was also enhanced.

Example 11 Confirmation of T-cell proliferation inducing ability of Foxp3 knockdown DC (DC)

Foxp3 knockdown dendritic cells (DC / si-Foxp3) and Foxp3 normal dendritic cells (DC / si-con) were transfected with OT-1 T Or T cell proliferation after co-culturing with OT-II cells. More specifically Foxp3 knock-down dendritic cells and normal dendritic cells to Foxp3 _{257 -264} OVA (SIINFEKL) (SEQ ID NO: 5) and OVA _{323 -339} (ISQAVHAAHAEINEAGR) (SEQ ID NO: 6) were treated for 1 hour Fab Tide OT- 1 / OT-II T cells were incubated with CFSE (carboxy-fluorescein diacetate succinimidyl ester) for 1 h, and then incubated for 1 h with a T cell isolation kit (Miltenyi Biotech) The cells were co-cultured for 3 days, and the proliferated T cells were analyzed by a fluorescence flow cytometer. The results are shown in Figs. 11A and 11B. Foxp3 knockdown dendritic cells significantly enhanced CD8 + T cell proliferation compared to Foxp3 normal control dendritic cells, while OT-II T cell (CD4 + T cell) induction activity was somewhat reduced.

Foxp3 knockdown dendritic cells (DC / si-Foxp3) and Foxp3 normal dendritic cells (DC / si-con) were transiently transfected with Foxp3 / gfp C57BL / 6 mouse spleen. The cells were co-cultured with control T cells isolated with T cell isolation kit (Miltenyi Biotech) for 5 days, and the proliferated regulatory T cells were analyzed with a fluorescence flow cytometer. 11C. ≪ / RTI > As shown in FIG. 11, in the case of dendritic cells knockdown with Foxp3, it was confirmed that the proliferative capacity of antigen-nonspecific regulatory T cells was remarkably reduced. This suggests that Foxp3 DC plays an important role in the proliferative activity of regulatory T cells. These results indicate that Foxp3 + DC is involved in the proliferative activity of Foxp3 + regulatory T cells and also suppresses or regulates the CTL inducing ability of normal mDC.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> RESEARCH & BUSINESS FOUNDATION SUNGKYUNKWAN UNIVERSITY <120> Pharmaceutical composition for preventing or treating cancers          containing dendritic cells with Foxp3 gene silenced <130> PB14-12036 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 429 <212> PRT <213> Homo sapiens Foxp3 <400> 1 Met Pro Asn Pro Arg Pro Ala Lys Pro Met Ala Pro Ser Leu Ala Leu   1 5 10 15 Gly Pro Ser Pro Gly Val Leu Pro Ser Trp Lys Thr Ala Pro Lys Gly              20 25 30 Ser Glu Leu Leu Gly Thr Arg Gly Ser Gly Gly Pro Phe Gln Gly Arg          35 40 45 Asp Leu Arg Ser Gly Ala His Thr Ser Ser Leu Asn Pro Leu Pro      50 55 60 Pro Ser Gln Leu Gln Leu Pro Thr Val Pro Leu Val Met Val Ala Pro  65 70 75 80 Ser Gly Ala Arg Leu Gly Pro Ser Pro His Leu Gln Ala Leu Leu Gln                  85 90 95 Asp Arg Pro His Phe Met His Gln Leu Ser Thr Val Asp Ala His Ala             100 105 110 Gln Thr Pro Val Leu Gln Val Arg Pro Leu Asp Asn Pro Ala Met Ile         115 120 125 Ser Leu Pro Pro Ser Ala Ala Thr Gly Val     130 135 140 Arg Pro Gly Leu Pro Pro Gly Ile Asn Val Ala Ser Leu Glu Trp Val 145 150 155 160 Ser Arg Glu Pro Ala Leu Leu Cys Thr Phe Pro Arg Ser Gly Thr Pro                 165 170 175 Arg Lys Asp Ser Asn Leu Leu Ala Ala Pro Gln Gly Ser Tyr Pro Leu             180 185 190 Leu Ala Asn Gly Val Cys Lys Trp Pro Gly Cys Glu Lys Val Phe Glu         195 200 205 Glu Pro Glu Glu Phe Leu Lys His Cys Gln Ala Asp His Leu Leu Asp     210 215 220 Glu Lys Gly Lys Ala Gln Cys Leu Leu Gln Arg Glu Val Val Gln Ser 225 230 235 240 Leu Glu Gln Gln Leu Glu Leu Glu Lys Glu Lys Leu Gly Ala Met Gln                 245 250 255 Ala His Leu Ala Gly Lys Met Ala Leu Ala Lys Ala Pro Ser Val Ala             260 265 270 Ser Met Asp Lys Ser Ser Cys Cys Ile Val Ala Thr Ser Thr Gln Gly         275 280 285 Ser Val Leu Pro Ala Trp Ser Ala Pro Arg Glu Ala Pro Asp Gly Gly     290 295 300 Leu Phe Ala Val Arg Arg His Leu Trp Gly Ser His Gly Asn Ser Ser 305 310 315 320 Phe Pro Glu Phe Phe His Asn Met Asp Tyr Phe Lys Tyr His Asn Met                 325 330 335 Arg Pro Pro Phe Thr Tyr Ala Thr Leu Ile Arg Trp Ala Ile Leu Glu             340 345 350 Ala Pro Glu Arg Gln Arg Thr Leu Asn Glu Ile Tyr His Trp Phe Thr         355 360 365 Arg Met Phe Ala Tyr Phe Arg Asn His Pro Ala Thr Trp Lys Asn Ala     370 375 380 Ile Arg His His Leu Ser Leu His Lys Cys Phe Val Arg Val Glu Ser 385 390 395 400 Glu Lys Gly Ala Val Trp Thr Val Asp Glu Phe Glu Phe Arg Lys Lys                 405 410 415 Arg Ser Gln Arg Pro Asn Lys Cys Ser Asn Pro Cys Pro             420 425 <210> 2 <211> 1290 <212> DNA <213> Homo sapiens Foxp3 <400> 2 atgcccaacc ctaggccagc caagcctatg gctccttcct tggcccttgg cccatcccca 60 gagagtcttgc caagctggaa gactgcaccc aagggctcag aacttctagg gaccaggggc 120 tctgggggac ccttccaagg tcgggacctg cgaagtgggg cccacacctc ttcttccttg 180 aaccccctgc caccatccca gctgcagctg cctacagtgc ccctagtcat ggtggcaccg 240 tctggggccc gactaggtcc ctcaccccac ctacaggccc ttctccagga cagaccacac 300 ttcatgcatc agctctccac tgtggatgcc catgcccaga cccctgtgct ccaagtgcgt 360 ccactggaca acccagccat gatcagcctc ccaccacctt ctgctgccac tggggtcttc 420 tccctcaagg cccggcctgg cctgccacct gggatcaatg tggccagtct ggaatgggtg 480 tccagggagc cagctctact ctgcaccttc ccacgctcgg gtacacccag gaaagacagc 540 aaccttttgg ctgcacccca aggatcctac ccactgctgg caaatggagt ctgcaagtgg 600 cctggttgtg agaaggtctt cgaggagcca gaagagtttc tcaagcactg ccaagcagat 660 catctcctgg atgagaaagg caaggcccag tgcctcctcc agagagaagt ggtgcagtct 720 ctggagcagc agctggagct ggaaaaggag aagctgggag ctatgcaggc ccacctggct 780 gggaagatgg cgctggccaa ggctccatct gtggcctcaa tggacaagag ctcttgctgc 840 atcgtagcca ccagtactca gggcagtgtg ctcccggcct ggtctgctcc tcgggaggct 900 ccagacggcg gcctgtttgc agtgcggagg cacctctggg gaagccatgg caatagttcc 960 ttcccagagt tcttccacaa catggactac ttcaagtacc acaatatgcg accccctttc 1020 acctatgcca cccttatccg atgggccatc ctggaagccc cggagaggca gaggacactc 1080 aatgaaatct accattggtt tactcgcatg ttcgcctact tcagaaacca ccccgccacc 1140 tggaagaatg ccatccgcca caacctgagc ctgcacaagt gctttgtgcg agtggagagc 1200 gagaagggag cagtgtggac cgtagatgaa tttgagtttc gcaagaagag gagccaacgc 1260 cccaacaagt gctccaatcc ctgcccttga 1290 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-Foxp3 <400> 3 gcactgccaa gcagatcat 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> control siRNA <400> 4 gggtaacggt ggaccgtct 19 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> OVA peptide 257-264 <400> 5 Ser Ile Ile Asn Phe Glu Lys Leu   1 5 <210> 6 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> OVA peptide 323-339 <400> 6 Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly   1 5 10 15 Arg    

Claims (10)

A pharmaceutical composition for preventing or treating cancer comprising as an active ingredient a dendritic cell in which the expression of Foxp3 gene or the activity of Foxp3 protein is inhibited.
2. The pharmaceutical composition according to claim 1, wherein the Foxp3 protein has the amino acid sequence of SEQ ID NO: 1.
The pharmaceutical composition according to claim 1, wherein the dendritic cells are differentiated from bone marrow stem cells or hematopoietic cells.
The pharmaceutical composition according to claim 1, wherein the dendritic cells are directly separated from blood.
The pharmaceutical composition according to claim 1, wherein expression of the Foxp3 gene is suppressed by knock-down or knock-out of the Foxp3 gene.
6. The pharmaceutical composition according to claim 5, wherein the knock-down is an antisense nucleotide complementary to the mRNA of the Foxp3 gene, siRNA, shRNA or ribozyme. .
6. The pharmaceutical composition according to claim 5, wherein the knock-out is carried out by removing or damaging DNA or mRNA encoding the Foxp3 gene.
The pharmaceutical composition according to claim 1, wherein the inhibition of the activity of the Foxp3 protein is made by a compound, a peptide, a peptide mimetic, a substrate analogue, an aptamer or an antibody that binds complementarily to the Foxp3 protein.
The method of claim 1, wherein the cancer is selected from the group consisting of bladder cancer, bone cancer, blood cancer, breast cancer, melanoma, thyroid cancer, pituitary cancer, bone cancer, rectal cancer, throat cancer, larynx cancer, lung cancer, esophageal cancer, pancreatic cancer, Wherein the cancer is any one selected from the group consisting of cervical cancer, head or neck cancer, gallbladder cancer, oral cancer, colon cancer, perianal cancer, central nervous system tumor, liver cancer and colon cancer.
A cancer vaccine comprising, as an active ingredient, dendritic cells in which the expression of Foxp3 gene or the activity of Foxp3 protein is inhibited.

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