WO2011099721A2

WO2011099721A2 - Composition for inducing the maturation of dendritic cells

Info

Publication number: WO2011099721A2
Application number: PCT/KR2011/000666
Authority: WO
Inventors: 이제중; 홍철이; 이준행; 이현주
Original assignee: Lee Je Jung; Hong Cheol Yi; Rhee Joon Haeng; Lee Hyun Ju
Priority date: 2010-02-12
Filing date: 2011-01-31
Publication date: 2011-08-18
Also published as: WO2011099721A3; KR100997753B1

Abstract

The present invention relates to a composition for inducing maturation of dendritic cells, and more particularly to a composition for inducing maturation of dendritic cells containing bacterial flagellin, tumor necrosis factor, interferone or mixtures thereof as active ingredients. Since the composition for inducing maturation of dendritic cells according to the present invention can effectively induce the maturation of immature dendritic cells into mature dendritic cells, the composition can be used as a maturation derivative of a noble dendritic cell and also used as a cellular therapeutic agent that establishes new conditions for tumor treatment.

Description

Specification

Title of the Invention: Composition for inducing maturation of dendritic cells

[1] The present invention relates to a composition for inducing maturation of dendritic cells.

Specifically, the present invention relates to a dendritic cell maturation-inducing composition containing bacterial flagellin, tumor necrosis factor, interferon or a combination thereof as an active ingredient.

Background

[2] Until now, most cancer treatments include surgery, chemotherapy and radiation therapy.

Done. However, these treatments have side effects and are difficult to cure. There is no fundamental treatment. In particular, metastatic or recurrent cancers cannot be operated in most cases and are often resistant to chemical treatments. Therefore, the development of new treatments for these cancer patients is urgently needed.

[3] The recent research and development of cancer treatments using dendritic cells is more patient-oriented than any of the existing treatments, and it is very effective in preventing the recurrence or recurrence of the same cancer because of its long-term efficacy by memory immunity. In addition, it is expected to be a safe and new anti-cancer immunotherapy and is currently being developed as a therapeutic vaccine against various types of cancer in developed countries. In particular, dendritic cell vaccine can be used as a therapeutic agent to prevent metastasis and recurrence after surgical removal of primary cancer. It will be a significant competitive advantage in the cancer treatment market. Dendritic cell cancer vaccines are produced in the form of customized treatments for each patient.They require expensive cytokines in the production process, which are more expensive than other treatments, but have sufficient commercial value in light of the increasing trend of cancer patients and international marketability. It is spurring development on developed countries.

[4] Dendritic cell vaccines are specific for cancer cells based on sophisticated intercellular cognitive action.

It is a method of inducing immune reaction and attacking and killing only cancer cells.

Treatment method (Barratt-Boyes SM and Figdor CG, Cytotherapy, 6: 105, 2004;

Grunebach F et al. Cancer Immunol Immunother, 54: 517, 2005). In particular, it is not necessary to be hospitalized during the treatment period, and it is a form of receiving regular vaccine visits to the hospital, so unlike other cancer treatments, it does not deteriorate the quality of life of patients. Are gathering. Existing clinical studies have reported that even if patients who did not show tumor response until the short-term evaluation time after vaccination, the response rate to other chemotherapy is greatly improved. Therefore, when combined with other chemotherapy as well as dendritic cell vaccine alone treatment, a greater synergistic effect can be expected in the treatment of refractory cancer.

[5] Dendritic cells can be divided into immature and mature dendritic cells by stages of differentiation. have. Dendritic cells are immature and do not induce T cell activity. In this case, almost no costimulatory factors are expressed in the dendritic cells, but the immature dendritic cells express a large amount of various receptor molecules necessary for antigen capture on the cell surface. Effective capture of the antigens plays an early stage in immunity. In addition, unlike other antigen-presenting cells, the expression of MHC (major histocompatibility antigen) is high, so a small amount of antigen can be effectively presented to induce T cell activity. In addition, immature dendritic cells can cross-present Thl and Th2 cells when sensitized to foreign antigens, effectively inducing cytotoxic T lymphocyte activity against dead microorganisms or dead cells as well as viral infections.

[6] After immature dendritic cells are sensitized by the antigen, they undergo a maturation step, which rapidly changes shape and activity, causing the molecules necessary for antigen capture to disappear. Instead, they are required for co-stimulation with T cells or for reaction with T cells. They become mature dendritic cells with significantly increased expression. In addition, dendritic cells in the maturation stage express a chemotactic receptor required for migration to lymph nodes, such as CCR7, through which they move to nearby lymph nodes or spleen to induce cellular immunity. They activate antigen-specific T cells in lymph nodes with a marked increase in the expression of co-stimulatory factors within 1-2 days after capturing the antigen.

[7] Cellular therapeutics for dendritic cells are an area of great research. Cell therapy for dendritic cells is known to have no side effects and is a safe therapeutic cancer vaccine. However, many problems have been raised as much research has been conducted. Recent studies have shown that when immature dendritic cells are used as cell therapeutics, they may activate cancer more. Cell therapy using dendritic cells should use mature mature dendritic cells.

10-2005-7016059). Dendritic cell therapies are also costly, tailored treatments tailored to each person _. It is also a problem.

Detailed description of the invention

Technical challenge

[8] As a result of intensive efforts to find a method for maturing immature dendritic cells into mature dendritic cells, the present inventors have treated immature dendritic cells more efficiently than conventional cytokines when the bacterium fullazelin and cytokines are treated with immature dendritic cells. It was confirmed that the maturation of mature dendritic cells to the present invention was completed the present invention.

[9] An object of the present invention is to provide immature dendritic cells with bacterial flagellin, tumor necrosis factor,

The present invention provides a method of inducing maturation of immature dendritic cells characterized by treatment with interferon or a combination thereof.

[10] Another object of the present invention is the maturation of mature using low-cost v-FlaB To provide dendritic cells.

Another object of the present invention is to provide a low-cost, high-performance cell therapy containing the mature dendritic cells.

Challenge solution

In order to achieve the above object, the present invention provides a dendritic cell maturation inducing composition containing a bacterial flagellin, tumor necrosis factor, interferon or a mixture thereof as an active ingredient.

[13] The method of inducing maturation of immature dendritic cells according to the present invention is characterized by v-FlaB protein derived from Vibrio bulnipicus (Vi & o vw / m jci), tumor necrosis factor, and TNF-α and interferon as IFN- It is characterized by processing with a mixture of a.

Hereinafter, the present invention will be described in detail.

[15] The present invention provides a dendritic cell maturation inducing composition containing a bacterial flagellin, tumor necrosis factor, interferon or a combination thereof as an active ingredient.

[16] The present invention is characterized in that the bacterial flagellin is a FlaB protein, and the bacterium is Vibrio bulnipicus (V n ^' o viiZ k ^).

[17] The present invention is characterized in that the tumor necrosis factor is TNF-a,

The interferon is IFN-a or IFN-γ, more preferably IFN-a.

[18] In the present invention, the flagellin as a dendritic cell maturation inducing composition may be used in combination with one or more bacteria-derived flagellins. In addition, all or part of the flagellin protein may be used, and when a part of flagellin is used, the FlaB protein is preferred.

[19] In addition, flagellin or a portion thereof used as a dendritic cell maturation inducing composition comprises an amino acid sequence variant thereof. Flageline variants are proteins that have deletions, insertions, or substitutions in the amino acid sequence, and any combination of deletions, insertions, and substitutions in the final structure of the protein may be possible, as long as the flageline variant maintains its function as an arthritis induction aid. . In some cases, in order to enhance the function as an induction adjuvant, it may be modified by phosphorylation, glycosylation, methylation, famesylation, or the like.

[2] this invention

[21] (a) differentiating monocytes isolated from blood into immature dendritic cells; And (b) treating the immature dendritic cells with bacterial flagellin, tumor necrosis factor, interferon or a combination thereof to mature into mature dendritic cells;

[23] A method of inducing maturation of immature dendritic cells is provided.

In the present invention, the step (a) is added by adding GM-CSF and interleukin-4

It is characterized by performing. In the present invention, the bacterial flagellin of step (b) is a v-FlaB protein derived from Vibrio vulnificus, characterized in that the treatment concentration of the v-FlaB protein is 0.1 to It is characterized by being 10 / ig /.

In the present invention, the interferon of step (b) is IFN-ot or IFN-γ, and more preferably IFN-ct.

[27] The present invention provides mature dendritic cells induced by the above method.

[28] In the present invention, the mature dendritic cells are characterized by showing positive immunological properties to CD80, CD83 or CD86.

[29] The present invention provides a cell therapy agent for treating cancer containing mature dendritic cells induced by the above method.

In the present invention, immature dendritic cells derived from monocytes were induced to mature dendritic cells using bacterial flagellin, tumor necrosis factor, interferon, or a combination thereof. It was confirmed that jelly, tumor necrosis factor, interferon or mixtures thereof induce maturation. Monocytes and dendritic cells used herein are cells extracted from blood provided from healthy donors.

[31] The method for confirming induction of maturation of dendritic cells of the present invention is characterized in that

In the process of inducing maturation of dendritic cells, the expression of protein surface factors, cytokine secretion and T lymphocyte activity are measured.

[32] In the present invention, the first step experimentally demonstrated that the bacterial flagellin, tumor necrosis factor, interferon or a combination thereof to induce maturation of immature dendritic cells derived from monocytes into mature dendritic cells. Experimentally demonstrated that mature dendritic cells induced by bacterial flagellin, tumor necrosis factor, interferon, or mixtures thereof induce T lymphocytes as a Thl type important for anticancer immunity. necrosis _factor. The function and cytotoxicity of apoptotic T cells induced by mature dendritic cells induced by interferon or a combination thereof were experimentally demonstrated.

[33] In the method for inducing maturation of immature dendritic cells of the present invention, the bacterial flagellin is preferably treated at a concentration of 0.1 to lO / ig / i, and the tumor necrosis factor is treated to a concentration of 10 to 100 ng / ng. Preferably, the interferon can be treated at a concentration of 500-1500 U /. Within the concentration range, the specific dendritic cell-specific, important for stimulating male τ cells, protein surface factor

The expression levels of CD80 and CD86 were high. See FIG. 1.

[34] As a result of confirming the cytokine secretion ability of mature dendritic cells induced by bacterial flagellin, tumor necrosis factor, interferon or a combination thereof, the secretion of interleukin-12p70 was significantly increased. . See FIG. 2.

[35] One of the important conditions for using mature dendritic cells as an immunocytotherapeutic agent is that mature dendritic cells migrate well to lymph nodes, which may cause antigen-specific induction of male T cells in lymph nodes. Apoptosis τ cells should be induced.

[36] In the present invention, the mobility of mature dendritic cells induced by bacterial flagellin, tumor necrosis factor, interferon, or a combination thereof to lymph nodes was investigated using chemokines that secrete lymph nodes.

It was confirmed that the cytokine alone increased significantly than mature dendritic cells. See FIG. 3.

[37] One of the most important features of mature dendritic cells is their ability to induce male T lymphocytes into Thl type with superior anticancer function or with Th2 or TM7 type necessary for inflammatory response or autoimmunity.

[38] In the present invention, the secretion of interferon-gamma in T lymphocytes activated by mature dendritic cells induced by bacterial flagellin, tumor necrosis factor, interferon or a combination thereof was significantly increased compared to that of interleukin-13. ， Compared with standard dendritic cells, it can be seen that the differentiation to TM7 type is significantly reduced, and proved to have excellent function as anticancer immune cell therapy. See FIGS. 5 and 6.

[39] Mature dendritic cells may also convert male T cells into antigen-specific cytotoxic T cells.

Have the ability to induce and are made of any combination of cytokines

The ability of apoptotic T cells depends on whether they are dendritic.

In the present invention, mature dendritic cells induced by bacterial flagellin, tumor necrosis factor, interferon, or a combination thereof were significantly superior to standard dendritic cells inducing antigen specific cytotoxic T cells (FIG. 7), it was confirmed that the secretion capacity of the cytotoxic enzymes secreted by the antigen-specific apoptotic T cells and the number of secretory cells were also excellent. See FIGS. 8 and 9.

[41] as well as antigen-specific killing target cells

The cell killing ability was remarkably excellent. See FIG. 10.

[42] A cell therapy agent for treating cancer containing mature dendritic cells of the present invention is conventional

Can be used with adjuvant. Immunostimulators are substances that promote the immune response to antigens in the specific process of initial activation of immune cells, which are not immunogens to the host, but are agents that strengthen immune cells by increasing the activity of cells of the immune system. These adjuvants increase the surface area of the antigen or extend the identity of the antigen in the body, allowing the immune system to access the antigen, delay antigen release, target the antigen to macrophages, or activate macrophages. It has been reported to act by a variety of mechanisms, including, but not limited to (Warren et al., 1986, Annu. Rev. Immunol., 4: 369-388). Typical immunopotentiators include Freund adjuvant, aluminum compound, mumurayl dipeptide, lipopolysaccharide (LPS), and the like.

[43] In addition, the cell therapy agent for treating cancer containing the mature dendritic cells of the present invention after surgery, In combination with radiation therapy or with anticancer drugs, the effect can be maximized.

Effects of the Invention

The bacterial flagellin, tumor necrosis factor, interferon or a combination thereof according to the present invention can efficiently induce maturation from immature dendritic cells to mature dendritic cells, and thus can be used as a maturation derivative of new dendritic cells. In addition, it can be used as a relatively inexpensive cell therapy agent for new conditions to treat tumors, thereby contributing to the popularization of anticancer immune cell therapy.

Brief description of the drawings

1 shows the maturation of mature dendritic cells against mature dendritic cells according to the present invention.

As a result of measuring the expression of specific protein surface factors,

2 is a result of examining the cytokine secretion ability of the mature dendritic cells according to the present invention,

3 shows the migration rate to lymph nodes of mature dendritic cells according to the present invention.

4 shows that matured dendritic cells of the present invention are involved in migration to lymph nodes.

It is a result of measuring the expression of a marker factor,

5 is a result of measuring expression of IFN- and IL-13 secreted from T cells differentiated into TH1 and TH2 induced by mature dendritic cells according to the present invention.

[50] Figure 6 is ΤΗΠ type T induced by matured dendritic cells according to the present invention.

As a result of measuring IL-17 secreted from the cells,

7 is a result of measuring the ratio of antigen-specific T cells of cytotoxic T cells induced by mature dendritic cells according to the present invention,

8 shows antigen specific CD8 + T in mature dendritic cells according to the present invention.

It is the result of measuring the expression rate of cytolytic enzyme in the cell,

9 is an absolute diagram present in the matured dendritic cells according to the present invention.

The number of antigen-specific CD8 + T cells and the number of T cells secreting cytolytic enzymes in the antigen-specific CD8 + T cells,

10 is a result of examining the antigen specific cytotoxicity of cytotoxic T cells against mature dendritic cells according to the present invention.

[55] (A: Number of IFNg-secreting cells B: Cytotoxicity to target cells)

Form for the implementation of the invention

The present invention will be described in more detail with reference to the following examples. However, the following examples are only intended to help the understanding of the present invention, and the scope of the present invention is not limited by these examples in any sense.

[57] At this time, unless there is another definition in the technical and scientific terms used,

Commonly understood by one of ordinary skill in the art The meaning of the present invention in the following description and attached drawings.

Descriptions of well-known functions and configurations that may be unnecessarily blurred are omitted.

Example 1 Induction of Differentiation of Immature Dendritic Cells and Mature Dendritic Cells from Monocytes

[59] Mononuclear cells were obtained by centrifugation using lymphoprep (Ficoll-Hypaque) from blood from healthy donors, and then CD14 positive monocytes using magnetic beads. Obtained. The obtained monocytes were prepared in a 6-well plate at a density of lx l ⁶ cells in a 37 ° C incubator supplied with 5% of 00 ₂ using IMDM medium added with 10% FBS, penicillin and streptomycin. Incubated for 6 days with GM-CSF (50 ng / m) and IL-4 (20 ng / m £) in order to induce differentiation into dendritic cells, and then differentiated into immature dendritic cells. standard 'cocktail (IL-lb (25 ng / mg), TNFa (50 ng / ι β), IL-6 (10 ng /) and PGE ₂ (1 ug / m)) or TNF-a (50 ng / mi ),

IFN- (1000 U / m £) and FlaB (lg /), respectively, or the combination of TNF-a (50 ng / mt), IFN-a (1000 U /) and ί¾Β (1 j¾g / m £) Alternatively, dendritic cells were matured by treating TNF-a and IFN-cc or TNF-a and FlaB, or IFN-a and FlaB, and incubating for 2 days.

Example 2 Changes in Cell Surface Protein Factors of Dendritic Cells Matured by Bacterial Flagellin, Tumor Necrosis Factor, and Interferon

[61] The method of Example 1 from immature dendritic cells induced differentiation from monocytes

In mature dendritic cells following treatment, such as CD80, CD83, CD86

Mature dendritic cell surface protein factor was measured by flow cytometry using a monoclonal antibody conjugated with PE- or FITC.

As a result, as can be seen in Figure 1, the mature dendritic cells are CD80 and

The expression of CD86 was increased, whereas the expression of the mature dendritic cell marker CD83 was TNF-α, TNF-a and IFN- for matured dendritic cells treated with a combination of IFN-α, FlaB, IFN-α and FlaB. Treatment of mixtures of a, mixtures of TNF-a and FlaB, and mixtures of TNF-ot, IFN-α and FlaB resulted in lower CD83 expression compared to mature dendritic cells, resulting in a weaker degree of induction of dendritic cell maturation. I could confirm that.

Example 3 Cytokine Secretion of Dendritic Cells Matured by Bacterial Flagellin, Tumor Necrosis Factor, and Interferon

The culture medium in which the cells were cultured is separated from the dendritic cells matured from Example 1 and secreted from the dendritic cells during maturation.

The amount of IL-12p70 and CD40L were expressed in mature dendritic cells for 48 hours.

After co-culture with J558 cells, the amount of IL-12p70 secreted during co-culture was measured using an ELISA kit specific for IL-12p70.

As can be seen from FIG. 2A, the amount of π-12ρ70 secreted during the maturation of dendritic cells was very low in most dendritic cells, and was matured by the combination of FlaB, TNF-α and IFN-a. Different amounts of IL-12p70 secreted from dendritic cells Increased over the amount secreted by dendritic cells. In addition, as can be seen in FIG. 2B, dendritic cells matured by the combination of FlaB, TNF-α and IFN-a differ from dendritic cells (especially TNF-a, IFN-a, standard dendritic cells) compared to the amount of IL-12p70 secreted was significantly increased.

Example 4 Dendritic Maturation by Bacterial Flagellin, Tumor Necrosis Factor, and Interferon

Go to the lymph nodes of the cells

[67] (I) CCR7 Lee? ^■ de. Cell Mobility Against CCL19 and CCL21

Secondary Lymphocytes to Various Dendritic Cells Matured from Example 1

In order to confirm the movement to the trachea, cell migration capacity of the CCR7 ligand, CCL19 and CCL21 was examined. 100 uL culture medium containing 5 X 10 ⁴ dendritic cells was applied to the upper chamber of the transwell plate and 600 ul of CCL19 or CCL21 at 250 ng / mL was applied to the bottom chamber for 3 hours at 37 ° C. Incubate 500 uL from the bottom chamber and measure the number of transferred dendritic cells using a flow cytometer within a fixed time of 60 seconds.

As a result, as can be seen from the results of FIG. 3, CCL19 and matured dendritic cells treated with TNF-cx, IFN- and FlaB, respectively, and a mixture of IFN-α and FlaB were treated with CCL19 and It was confirmed that the mobility of reaction to CCL21 was very low.

On the other hand, matured by the combination of TNF-α and IFN-a with the combination of IFN-a and FlaB.

Mobility in response to CCL19 and CCL21 in dendritic cells was markedly increased, and in particular, the mobility in response to CCL19 and CCL21 in dendritic cells matured by a combination of TNF-α, IFN-a and FlaB was observed in TNF-a and IFN-a. Compared to the dendritic cells' mobility, they were markedly increased.

[71] (2λ CCR7. Exposed for CD74 and CD38

[72] Secondary Lymphocytes of Various Dendritic Cells Matured from Example 1

Expression of CCR7, CD74, and CD38, cell surface proteins that affect mobility to organs, was confirmed.

As a result, as shown in the results of FIG. 4, the lymph nodes of mature dendritic cells

Expression of CCR7 involved in migration was dendritic cells matured by TNF-a, IFN-a and FlaB combinations, except for dendritic cells and standard dendritic cells matured by TNF-a or a combination of TNF-a and FlaB. Was significantly higher at. Meanwhile, mature

Expression of CD74, which inhibits migration to the lymph nodes of dendritic cells, can also be expressed by TNF-ot or

Dendritic cells matured by the combination of TNF-ct and FlaB were significantly higher in dendritic cells matured by a mixture of TNF-ce, IFN-α and FlaB.

On the other hand, the expression of CD38, which promotes migration to the lymph nodes of mature dendritic cells, is characterized by dendritic cells matured by other conditions including standard dendritic cells, except for dendritic cells matured by a combination of TNF-a and IFNa. It was significantly higher in dendritic cells matured by the combination of TNF-a, IFN-a and FlaB than cells. Therefore, FIGS. 3 and 4 In comparison, the degree of expression of CCR7 is important, but the degree of expression of CD74 and CD38 is also important in the second lymphoid organ of mature dendritic cells.

[75] [Example 5] Dendritic phase matured by bacterial flagellin, tumor necrosis factor, and interferon

T cell differentiation by cells

[76] Investigation of the effect of the TNF-ct, IFN-α and FlaB of Example 1 on the differentiation of THl, TH2, TH17 cells by coculture with male CD4 + T cells of dendritic cells matured by the mixture It was. Male CD4 + T cells were isolated using a negative selection kit, and standard dendritic cells were used as controls. The isolated 2 x 10 ⁴ male CD4 + T cells were co-cultured with an equal number of dendritic cells for 5 days at 37 ° C, followed by the addition of 10 ng / mL of IL-2 at 37 ° C for a total of 12 days. After incubation, 1 X 10 ⁶ cells were stimulated with PMA and Ionomycin for 24 hours, and the IFN-JL-13 and IL-17 secreted in the culture were measured using a specific ELISA kit.

As a result, as shown in FIGS. 5 and 6, IFN _Y promotes the differentiation of TH1 in dendritic cells matured by a combination of TNF-α, IFN-a and FlaB compared to standard dendritic cells. Although the expression was remarkably high, it was confirmed that the expression of IL-13 which promotes differentiation of TH2 and IL-17 which promotes differentiation of THI7 was remarkably low. This suggests that dendritic cells matured by a combination of TNF-α, IFN-a and FlaB are more resistant to anticancer immune responses than standard dendritic cells, which are more specific for autoimmune or inflammatory responses.

It suggests that the reaction is peculiar and powerful.

[78] [Example 6] Dendritic phase matured by bacterial flagellin, tumor necrosis factor, and interferon

Characteristics and Apoptosis of Apoptotic T Cells Induced by Cells

[79] The characteristics of antigen-specific apoptotic T cells induced by dendritic cells matured by the combination of TNF-α, IFN-a and FlaB of Example 1 were investigated.

[80] The test method was to isolate T cells using monoclonal antibodies specific for S. CD3 + T cells, co-culture with dendritic cells loaded with MART-1 peptides associated with melanoma for 3 days, and then 10 ng / mL. 7 days by adding IL-2 and 10 ng / mL of IL-7

Incubated. After 10 days, the same dendritic cells were stimulated with T cells cultured on day 0, and cultured for 10 days under culture medium containing lOng / mL of IL-2, followed by tetramer for MART-1. We analyzed the amount of apoptotic CD8 + T cells specific for antigen, and used monoclonal antibodies specific to IFN-, FasL, Granzyme B, and perforin to investigate the expression of cytolytic enzymes. The expression of these enzymes was examined.

In addition, in order to investigate the cytotoxicity of apoptotic T cells, IFN-expressing antigen-specific expression by antigen-specific apoptotic τ cells was measured using ELISPOT assay. In order to investigate the T2 cells loaded with MART-1, labeled with ⁵ ΐ ', were co-cultured with apoptotic T cells for 5 hours, and then the amount of ⁵¹ Cr released into the medium was measured. [79] As can be seen from the results, the antigens induced in the dendritic cells matured by the combination of TNF-a, IFN-α and FlaB of the present invention than in the matured dendritic cells treated with the 'gold standard' cocktail. It was confirmed that the amount of specific cell killing CD8 + T cells increased by 50%.

[83] Also, cytolytic enzymes. Expression of Granzyme B, IFNg, Perforin and FasL was also confirmed in 8, and the expression of cytolytic enzymes in dendritic dendritic cells matured by the combination of TNF-x, IFN-α and FlaB of the present invention is remarkable. The increase was confirmed to be high.

[84] In addition, the number of antigen-specific apoptotic CD8 + T cells, when compared with those induced by standard dendritic cells, can also be seen in 9, maturation by a mixture of TNF-a, IFN-a and I¾B of the present invention. It was confirmed that the number of antigen-specific cell-killing CD8 + T cells expressing cytolytic enzymes was also increased at least three to five times.

[85] Finally, the number of IFNg-secreting cells in antigen-specific apoptotic T-cells can be seen in Fig. 10, as well as the 'gold standard' cocktail in dendritic cells matured by a combination of TNF-a, IFN- and FlaB. At least 7-fold increase was observed in the matured dendritic cells treated with, and as a result of the cytotoxicity against the target cells, the mixture of TNF-a, IFN-α and FlaB of the present invention was found in mature dendritic cells. Induced cytotoxic T cells were found to be more than 8-fold higher.

Claims

Claim

The combination of bacterial flagellin, tumor necrosis factor and interferon

Dendritic cell maturation inducing composition containing as an active ingredient.

The method of claim 1,

The active ingredient is a dendritic cell maturation inducing composition, characterized in that the mixture of FlaB protein, TNF-ot, and IFN-γ.

The method of claim 2,

The FlaB protein is a dendritic cell maturation inducing composition, characterized in that the protein derived from Vibrio ulnipius bacillus vMZmj¾ci «).

(a) differentiating monocytes isolated from blood into immature dendritic cells; And

(b) treating the immature dendritic cells with a mixture of bacterial flagellin, tumor necrosis factor and interferon to mature into mature dendritic cells;

A method of inducing maturation of immature dendritic cells, comprising.

The method of claim 4,

Step (a) is the method of inducing maturation of immature dendritic cells, characterized in that the addition of GM-CSF and interleukin-4.

The method of claim 4,

The complex of step (b) is a mixture of FlaB protein, TNF-α, and IFN-γ, characterized in that the maturation of immature dendritic cells.