KR102092469B1 - Composition for inducing maturation of dendritic cell comprising Ribosomal Protein S3 - Google Patents

Abstract

The present invention relates to the use of the ribosomal protein S3 (Ribosomal Protein S3; RPS3) for inducing the maturation of immature dendritic cells, and more specifically, a composition for inducing maturation of dendritic cells containing RPS3 as an active ingredient and maturation through the same It relates to a cancer vaccine or cancer treatment comprising dendritic cells.
The composition for inducing maturation of dendritic cells of the present invention can effectively induce maturation of dendritic cells by TLR4 binding of ribosome protein S3 and immature dendritic cells. Accordingly, when the matured dendritic cell is stimulated with a cancer antigen, it can generate a specific T cell for the cancer antigen, and can exhibit tumor growth inhibition and cancer treatment effects. Ribosome protein S3-mediated mature dendritic cells have a higher maturation efficiency than LPS used for inducing maturation of dendritic cells, and thus can exhibit high levels of cancer treatment effects and cancer prevention effects exhibited by mature dendritic cells. In addition, LPS may cause side effects such as inducing an inflammatory reaction in vivo, but ribosome protein S3 may be more effective because it can have biosafety using recombinant proteins derived from humans.

Description

Composition for inducing maturation of dendritic cell comprising Ribosomal Protein S3}

The present invention relates to the use of the ribosomal protein S3 (Ribosomal Protein S3; RPS3) for inducing the maturation of immature dendritic cells, and more specifically, a composition for inducing maturation of dendritic cells containing RPS3 as an active ingredient and maturation through the same It relates to a composition for preventing or treating cancer comprising dendritic cells.

Antigen-presenting cells (APCs) refer to cells that activate a protein antigen by endocytosis and then present the antigen-derived peptide fragment to T cells together with MHC type I molecules. In charge of cellular immunity. Of these, dendritic cells (DCs) are the strongest antigen presenting cells that serve as bridges for the innate and adaptive immune systems. Dendritic cells are mainly present in small amounts in tissues that come into contact with the external environment, such as the inner walls of the skin, nose, lungs, stomach and intestines. In particular, cells in the skin are called Langerhans cells. Dendritic cells can be found immature in the blood, and when activated, they migrate to the lymphoid organs and interact with T cells and B cells to initiate an immune response. At certain stages of development, the cells stretch a process called dendrites.

Dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells initially differentiate into immature dendritic cells, and are characterized by high endocytosis activity and T-cell activation ability. Immature dendritic cells constantly phagocytose pathogens such as viruses and bacteria in the surroundings. This is possible through pattern recognition receptors (PRRs) such as toll-like receptors (TLRs).

In particular, Toll-like receptor 4 (TLR4) is the first receptor identified in the TLR family, and is amplified through MyD88 (myeloid differentiation 88) -dependent signaling process and MyD88-independent signaling process. Activates immune signaling. LPS recognized through accessory molecules such as lipopolysaccharide (LPS) -binding protein (LBP), cluster of differentiation 14 (CD 14) and myeloid differentiation factor 2 (MD2) activates TLR4. Activated TLR4 induces initial activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and migration to the nucleus through Myd88-dependent signaling, and activation of mitogen-activated protein kinases (MAPKs) Induces Activation of the NFκB and MAPKs includes tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). Secretes inflammatory cytokines. The MyD88-independent signaling process is known to secrete type 1 interferon by being induced by the activation of tram / trip (TRAM / TRIF), interferon-regulatory factors (IRFs) and NFκB.

TLR recognizes certain chemical features found on a subset of pathogens, and immature dendritic cells phagocytose cell membranes through a process called nibbling from living autologous cells. When they come into contact with existing antigens, they are activated into mature dendritic cells and migrate to the lymph nodes. Immature dendritic cells phagocytize pathogens, break down their proteins into small pieces, and when mature, these pieces appear on the cell's surface using the Major Histocompatibility Complex (MHC) molecule. At the same time, it increases cell surface receptors that act as co-receptors in T-cell activation, such as Cluster of Differentiation (CD) 80, CD86, and CD40. They activate helper T-cells, killer T-cells, as well as B cells by expressing antigens derived from pathogens along with non-antigenic specific costimulatory signals.

All helper T-cells are specific for one specific antigen. Only specialized antigen-presenting cells (eg macrophages, B lymphocytes, dendritic cells) activate antigen-specific helper T-cells when the correct antigen is present. However, macrophages and B lymphocytes can only activate memory T-cells, while dendritic cells are the most potent antigen-presenting cells because they can activate both memory and naive T-cells.

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

Fully mature dendritic cells are qualitatively and quantitatively different from immature dendritic cells. Fully mature dendritic cells express high levels of Major Histocompatibility Complex (MHC) type I and II antigens, and high levels of T cell co-stimulatory molecules, CD80 and CD86. These changes increase the ability of dendritic cells to activate T cells, which not only increases the density of antigens on the cell surface, but also increases the amount of T cell activation through counterparts of co-stimulatory molecules on T cells such as CD28. Because it increases. Additionally, mature dendritic cells produce large amounts of cytokines, which stimulate and induce T cell responses. Two of these cytokines are interleukin-10 (IL-10) and interleukin-12 (IL-12). These cytokines have the opposite effect on the direction of the induced T cell response. When IL-10 is generated, a Th-2 type response is induced, whereas when IL-12 is generated, a Th-1 type response is induced. The latter reaction is particularly preferred in cases where a cellular immune response is desired, for example in cancer immunotherapy. The Th-1 type response induces and polarizes cytotoxic T lymphocytes (CTLs), a means of attacking the cellular immune system. This effector attack method is most effective in inhibiting tumor growth. IL-12 also induces the growth of natural killer (NK) cells and has anti-angiogenic activity, which are all effective anti-tumor attack means. Thus, the use of dendritic cells that produce IL-12 is theoretically optimal for use in immunostimulation.

On the other hand, in cancer patients, the function of dendritic cells is reduced, so it is difficult to induce normal anti-cancer immunity. So far, many studies have been conducted on substances that control (activate) dendritic cell activity, but as a side effect of biotoxicity, there have been many problems in direct application to a living body. However, as described above, dendritic cells play an important role in enhancing the immune function of the body itself, thereby promoting the activity of dendritic cells to mature them, and can be applied to cell immunotherapy using mature dendritic cells that cause the strong immune response. Research continues to see if it exists.

Ribosomal Protein S3 (RPS3) is a component of the ribosome and is located on the outer surface of the 40S subunit and is cross-linked to the initiator factors eIF2 and eIF3. RPS3 has a nuclear localization signal at the N-terminal site, which means that RPS3 has a function in the cytoplasm and a repair function in the nucleus. In addition, many ribosomal proteins have secondary functions such as replication, transcription, RNA processing, DNA repair, and malignant transformation.

RPS3 has been observed to move into the nuclear membrane or nucleus when inducing apoptosis in immune cells, and it is known that when the RPS3 protein is overexpressed, it activates caspase-8 / caspase-3, which promotes apoptosis, and increases cytokine secretion. (Chang-Young Janga et al. , FEBS Letters , 560: 81, 2004). In addition, RPS3 is an essential part of the NFκB complex, interacts with p65 to increase the DNA binding ability of the NFκB complex, and RPS3 induces a transcriptional complex to the κ site, inducing immunoglobulin κ light chain expression in B cells, It is known to induce proliferation and cytokine secretion of T cells (Mona Johannessen et. Al. , Microbiology , 159: 2001, 2013). Moreover, the RPS3 or RPS3 gene is known to be overexpressed in various cancers (Pogue-Geile et. Al. , Mol Cell Biol , 11: 3842-3849, 1991), in this regard, methods of diagnosis of cancer and expression of RPS3 protein A lot of research has been conducted on the suppression method (Republic of Korea Patent No. 10-1654244 and Korea Patent No. 10-1480523).

As such, RPS3 may have a correlation with cancer by increasing the expression level in cancer cells, and it has been reported that it induces T cell proliferation and cytokine secretion, but RPS3 induces dendritic cell maturation. No research has been done on whether it can be done.

Accordingly, the present inventors tried to find a substance that can be usefully used as a vaccine for preventing and treating cancer by inducing maturation of dendritic cells. As a result, ribosomal protein S3 is incorporated into toll-like receptor 4 (TLR4) of immature dendritic cells. It binds to induce the process of maturation of dendritic cells, and thus it was confirmed that matured dendritic cells can generate CD8 + cytotoxic T cells specific for the cancer antigen when the cancer antigen is recognized. In addition, the present inventors by confirming that the dendritic cells matured by the ribosomal protein S3, when stimulated by a cancer antigen, exhibit not only a therapeutic effect on cancer, but also form a memory T cell to exhibit a preventive effect. , The present invention was completed.

An object of the present invention is to provide a composition for inducing maturation of dendritic cells comprising ribosomal protein S3 as an active ingredient.

Another object of the present invention is to provide a method for inducing maturation of dendritic cells, characterized by treating ribosome protein S3.

 Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising dendritic cells matured by the above method as an active ingredient.

In order to achieve the above object, the present invention provides a composition for inducing maturation of dendritic cells comprising ribosomal protein S3 (RPS3) as an active ingredient.

In addition, the present invention provides a method for inducing maturation of dendritic cells, including; culturing immature dendritic cells by processing ribosome protein S3.

In a preferred embodiment of the present invention, the ribosomal protein S3 may be composed of the amino acid sequence of SEQ ID NO: 1.

In another preferred embodiment of the present invention, the dendritic cells may be to express a toll-like receptor 4 (TLR4).

In another preferred embodiment of the present invention, the ribosome protein S3 may specifically bind to toll-like receptor 4 (TLR4) expressed in dendritic cells to induce maturation of dendritic cells.

In another preferred embodiment of the present invention, by the TLR4 binding of the ribosomal protein S3 and dendritic cells, (i) tumor necrosis factor α (TNF-α), interleukin-1β (interleukin-1β; IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12p70 (interleukin-12p70; IL-12p70) and interferon-β (interferon- β; increased expression of any one or more cytokines selected from the group consisting of INF-β);

(Ii) increased expression of any one or more surface factors selected from the group consisting of CD40, CD80, CD83, CD86, CCR7 (C-C chemokine receptor type 7) and MHC 1 (major histocompatibility complex 1) on dendritic cell surfaces; And

(Iii) TLR4 lower signaling, MAPK (mitogen-activated protein kinases) and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activity may be increased.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising mature dendritic cells prepared by the method for inducing maturation of dendritic cells as an active ingredient.

In a preferred embodiment of the present invention, the cancer is colorectal cancer, hemorrhoidal cancer, blood cancer, laryngeal cancer, esophageal cancer, oral cancer, basal cell cancer, biliary cancer, thyroid cancer, rectal cancer, stomach cancer, prostate cancer, breast cancer, kidney cancer, liver cancer, It may be any one or more selected from the group consisting of brain tumor, lung cancer, uterine cancer, colon cancer, bladder cancer and pancreatic cancer.

In another preferred embodiment of the present invention, the composition may further treat a cancer antigen peptide.

In another preferred embodiment of the present invention, the cancer antigen peptide may be OVA consisting of amino acids of SEQ ID NO: 14 or E7 consisting of amino acids of SEQ ID NO: 15.

In another preferred embodiment of the present invention, the production of T cells may be increased by the composition, and the T cells may be CD8 + IFN-γ T cells.

The present invention provides a composition for inducing maturation of dendritic cells containing ribosomal protein S3 as an active ingredient, and it was confirmed that ribosome protein S3 and immature dendritic cell TLR4 binding effectively induce maturation of dendritic cells. Accordingly, when the matured dendritic cell is stimulated with a cancer antigen, it can generate a specific T cell for the cancer antigen, and can exhibit tumor growth inhibition and cancer treatment effects.

The ribosome protein S3-mediated mature dendritic cells have a higher maturation efficiency than LPS used for inducing maturation of dendritic cells, and thus can exhibit high levels of cancer treatment effects and cancer prevention effects exhibited by mature dendritic cells. In addition, LPS may cause side effects such as inducing an inflammatory reaction in vivo, but ribosome protein S3 may be more effective because it can have biosafety using recombinant proteins derived from humans.

1 is a result of confirming the over-expression of purified RPS3 by SDS-PAGE.
2 is a result confirming the relationship between RPS3 and TLR4 through luciferase analysis.
3 is a result of measuring the expression level of cytokines secreted from dendritic cells when RPS3 is treated by concentration in dendritic cells derived from mouse bone marrow.
4 is a result of measuring the expression level of the maturation marker factor expressed on the surface of dendritic cells when RPS3 is treated by concentration in dendritic cells derived from mouse bone marrow.
5 is a result of measuring the activity of MAPK and NF-κB, the lower signaling of TLR4, in dendritic cells when RPS3 is treated by concentration in dendritic cells derived from mouse bone marrow.
6 is a result of measuring the expression level of cytokines secreted from dendritic cells when RPS3 is treated by concentration in a human-derived mononuclear cell line.
7 is a result of measuring the expression level of the maturation marker factor expressed on the surface of dendritic cells when RPS3 is treated by concentration in a human-derived mononuclear cell line.
8 is a result of measuring the expression level of cytokines secreted from dendritic cells when human-derived dendritic cells are treated with RPS3 by concentration.
9 is a result of measuring the expression level of the maturation marker factor expressed on the surface of dendritic cells when RPS3 is treated by concentration in human-derived dendritic cells.
10 is a schematic diagram (A) of 1-95 RPS3 and 91-245 RPS3 production and the result of confirming the purified mutant RPS3 by SDS-PAGE (B).
11 is a result confirming the association between the mutant RPS3 and TLR4 through luciferase analysis.
12 is a result of measuring the expression level of cytokines secreted from dendritic cells when the mutant RPS3 is treated.
13 is a result of measuring the expression level of the maturation marker factor expressed on the surface of dendritic cells when the mutant RPS3 is treated.
14 is a result of confirming the production level of CD8 + IFN-γT cells specific for cancer antigens in mice inoculated by stimulating RPS3 induced mature dendritic cells with cancer antigens: (A) OVA peptide as cancer antigen; (B) It is the result of treating E7 peptide as a cancer antigen.
15 is a result of measuring whether tumor formation, when stimulating RPS3 induced mature dendritic cells with cancer antigens and injecting cancer cells into inoculated mice: (A) OVA peptide as a cancer antigen; (B) It is the result of treating E7 peptide as a cancer antigen.
FIG. 16 shows the results of confirming tumor size and survival time when a mouse injected with cancer cells was inoculated with an RPS3-induced mature dendritic cell vaccine.
17 is a result of measuring the expression level of cytokines secreted from dendritic cells when RPS3 is treated on dendritic cells derived from TLR4 knocked-out mouse bone marrow.
18 is a result of measuring the expression level of the maturation marker factor expressed on the surface of dendritic cells when RPS3 is treated on dendritic cells derived from TLR4 knockout mouse bone marrow.
19 is a result of measuring the activity of MAPK and NF-κB, which are lower signaling of TLR4, in dendritic cells when RPS3 is treated on dendritic cells derived from mouse bone marrow from TLR4 knocked out.
Figure 20 is a result confirming the production level of cancer antigen-specific CD8 + IFN-γ T cells in mice inoculated with mature dendritic cell vaccine prepared by RPS3 treatment of TLR4 knockout mouse bone marrow-derived dendritic cells
FIG. 21 shows the results of confirming tumor size and survival time when inoculated into a mouse injected with cancer cells with a mature dendritic cell vaccine prepared by treating RPS3 with dendritic cells derived from TLR4 knockout mouse bone marrow.

Hereinafter, the present invention will be described in detail.

As described above, mature dendritic cells are used as cell therapeutic agents for diseases including cancer, but research has been continued to discover substances that can more effectively induce the maturation process of immature dendritic cells.

The present invention sought to solve the above-mentioned problem by confirming that induction of maturation of dendritic cells is effectively induced by the binding of ribosomal protein S3 and TLR4 (toll-like receptor 4) of immature dendritic cells. Dendritic cells matured with ribosomal protein S3, when stimulated with a cancer antigen, can generate specific T cells for the cancer antigen, and can exhibit tumor growth inhibition and cancer treatment effects.

Accordingly, the present invention provides a composition for inducing maturation of dendritic cells comprising ribosomal protein S3 (RPS3) as an active ingredient.

In addition, the present invention provides a method of inducing maturation of dendritic cells, including; culturing immature dendritic cells by processing ribosomal protein S3.

The ribosomal protein S3 (hereinafter referred to as 'RPS3' or 'RPS3 protein') is characterized by comprising the amino acid sequence of SEQ ID NO: 1, specifically, the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 2 It is preferred. More specifically, RPS3 of the present invention is most preferably a human-derived protein.

However, RPS3 of the present invention is not limited only to the amino acid sequence set forth in SEQ ID NO: 1, and may include a functional equivalent thereof. The functional equivalent is at least 70%, preferably at least 80%, more preferably at least 90%, more preferably at least 70% of the amino acid sequence of the novel antibiotic peptide, due to the amino acid addition, substitution or deletion of the peptide. Refers to a protein having a sequence homology of% or more, and the RPS3 protein of the present invention exhibits substantially the same / similar structure or sequence of a region recognizable by TLR4.

In the present invention, the dendritic cells can express dendritic cells toll-like receptor 4 (TLR4), and the ribosome protein S3 specifically binds TLR4 expressed in dendritic cells to induce maturation of dendritic cells. It is characterized by.

The dendritic cell is one of the immune cells of the antigen presentation of the immune system, and when a specific antigen is recognized and activated, the recognized antigen-specific T cell can be generated. From the viewpoint that RPS3 of the present invention can induce the maturation process of dendritic cells by binding to TLR4 on the surface of immature dendritic cells, it is preferable that the dendritic cells express TLR4. Specifically, among the immune cells known as antigen-presenting cells, cells expressing TLR4 as a surface receptor may include macrophage, etc., in addition to dendritic cells, but it is common for a person skilled in the art to express macrophages through TLR4. There is a case that induces immune suppression, and it is not appropriate because there is a fear that it may have an effect contrary to the purpose of enhancing immune activity. In addition, the TLR4 receptor may also be expressed on the surface of cancer cells. At this time, when the cancer cells bind to RPS3, there is a fear that cell death of the cancer cells is suppressed.

Therefore, the method for inducing dendritic cell maturation of the present invention using RPS3 is preferably performed in vitro or ex vivo blocked as not containing other immune cells. As it is known in the art that RPS3 can be used as a target for cancer treatment by increasing the expression level in cancer cells, when RPS3 is administered to the body to show activity, there is a fear that a result contrary to the cancer treatment effect may appear. .

In a specific embodiment of the present invention, the recombinant RPS3 protein was purified (FIG. 1), and when the recombinant RPS3 protein was treated with HEK293 cells transformed to overexpress TLR4, it was confirmed that the RPS3 protein concentration was dependently related to TLR4. (Figure 2).

In the present invention, by TLR4 binding of the RPS3 protein and dendritic cells,

(Ⅰ) Tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 -10; IL-10), increased expression of any one or more cytokines selected from the group consisting of interleukin-12p70 (IL-12p70) and interferon-β (INF-β);

(Ii) increased expression of any one or more surface factors selected from the group consisting of CD40, CD80, CD83, CD86, CCR7 (C-C chemokine receptor type 7) and MHC 1 (major histocompatibility complex 1) on dendritic cell surfaces; And

(Iv) It is characterized by increased activity of mitogen-activated protein kinases (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (MAPK), which are lower signaling of TLR4.

In a specific embodiment of the present invention, as a result of treating the RPS3 protein on immature dendritic cells derived from mice and confirming a maturation marker, cytokine production increases depending on the concentration of RPS3 protein (FIG. 3), and the surface factor, which is an indicator of maturation of dendritic cells, It was confirmed that the expression level increased (Fig. 4). In addition, when RPS3 protein was treated with immature dendritic cells in 10 minute increments, TLR4 sub-signals, mitogen-activated protein kinases (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). It was confirmed that the activity was increased (Fig. 5).

In another specific embodiment of the present invention, as a result of treating the RPS3 protein in a human-derived mononuclear cell line and confirming a maturation marker, cytokine production increases depending on the concentration of the RPS3 protein (FIG. 6), and a surface factor that is an indicator of maturation of dendritic cells. It was confirmed that the expression level of was increased (Fig. 7). In addition, as a result of processing the RPS3 protein on human-derived immature dendritic cells and confirming the maturation marker, it was found that cytokine production increased depending on the concentration of RPS3 protein (FIG. 8) and the expression level of the surface factor, which is an indicator of maturation of dendritic cells, increased. It was confirmed (Fig. 9).

That is, in the present invention, it was confirmed that the RPS3 protein specifically binds to TLR4 of immature dendritic cells, thereby effectively inducing the maturation of dendritic cells.

In the present invention, 1-95 RPS3 and 91-245 RPS3 proteins were produced based on the KH domain present in the RPS3 protein in order to confirm which part of the RPS3 protein binds to TLR4 and affects the lower signal transduction pathway (FIG. 10) ), 91-245 RPS3 protein was confirmed to be associated with TLR4 (Fig. 11). In addition, when the 1-95 RPS3 and 91-245 RPS3 proteins were treated with immature dendritic cells derived from mice, it was confirmed that the surface factor and cytokine secretion of dendritic cells increased by the 91-245 RPS3 protein (FIG. 12 and FIG. 13). That is, it was confirmed that the rear portion of RPS3 corresponding to amino acids 91-243 affects the maturation and activity of dendritic cells.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising mature dendritic cells prepared by the method for inducing maturation of dendritic cells as an active ingredient.

In the present invention, the dendritic cells are characterized in that dendritic cells that specifically respond to cancer antigens by treating cancer antigen peptide in addition to dendritic cells matured with RPS3.

In the present invention, the composition is characterized in that the cancer antigen peptide is further processed, preferably, the cancer antigen peptide is characterized in that OVA consisting of the amino acid of SEQ ID NO: 14 or E7 consisting of the amino acid of SEQ ID NO: 15 You can. In addition, in the present invention, the production of cancer antigen-specific T cells is increased by mature dendritic cells stimulated with the cancer antigen peptide, and preferably, the T cells are CD8 + IFN-γ T cells.

In the pharmaceutical composition of the present invention, the cancer can be applied without limitation as long as it is a cancer understood by those skilled in the art that dendritic cells can be applied as a preventive vaccine or a cell therapeutic agent. Specifically, the cancer is colon cancer, tumor cancer, blood cancer, larynx cancer, esophageal cancer, oral cancer, basal cell cancer, biliary tract cancer, thyroid cancer, rectal cancer, stomach cancer, prostate cancer, breast cancer, kidney cancer, liver cancer, brain tumor, lung cancer, uterine cancer, It may be any one or more selected from the group consisting of colon cancer, bladder cancer and pancreatic cancer.

In the pharmaceutical composition of the present invention, the mature dendritic cells are dendritic cells matured through a method for inducing maturation of dendritic cells with RPS3 or a maturation composition of dendritic cells comprising RPS3 of the present invention. The dendritic cells matured by the RPS3 are further stimulated by the antigen of the target cancer, and can be used as a vaccine for preventing cancer and / or a cell therapeutic for treating cancer.

In another specific embodiment of the present invention, the present inventors provide cancer of an OVA peptide (egg albumin; ovalbumin) or E7 peptide (E7 peptide derived from type 16 human papilloma virus) to dendritic cells derived from mouse bone marrow induced by RPS3 protein or LPS. A dendritic cell vaccine was prepared by stimulation with an antigenic peptide, and as a result of inoculating the vaccine with the prepared vaccine, it was confirmed that the most CD8 + IFN-γ T cells were generated in the group administered with the vaccine prepared using the RPS3 protein. (Figure 14). That is, it was confirmed that dendritic cells activated with a cancer antigen can generate CD8 + T cells specific to the cancer antigen. In addition, after inoculating mice with the dendritic cell vaccine prepared in the same manner as above, when cancer cells were injected, it was confirmed that the tumor size did not increase due to the cancer prevention effect (FIG. 15). This can be seen as the result that the activated dendritic cells produced memory T cells.

In another specific embodiment of the present invention, it was intended to confirm the cancer treatment effect of the dendritic cell vaccine prepared by the same method as above. As a result of injecting cancer cells into the mice first and then administering the dendritic cell vaccine 3 days later, it was confirmed that the tumor generated in the group administered with the vaccine prepared using the RPS3 protein had the smallest tumor size and the longest survival time of the mouse. (Fig. 15). In addition, RPS3-mediated mature dendritic cells have a higher maturation efficiency than LPS used for inducing maturation of dendritic cells in the prior art, and thus can exhibit high levels of cancer treatment effects and cancer prevention effects exhibited by mature dendritic cells. Confirmed.

In the present invention, in order to clarify the relationship between RPS3 protein and TLR4, the activity of dendritic cells was compared by treating RPS3 protein on wild type dendritic cells derived from mouse bone marrow and dendritic cells knocked out of TLR4. As a result, unlike wild-type mouse-derived dendritic cells, it was confirmed that cytokine secretion (FIG. 17) and surface factor production (FIG. 18) did not increase in TLR4 knockout dendritic cells, and MAPK and NF-, which are lower signal transductions of TLR4. It was confirmed that κB activity did not appear (Fig. 19).

In another specific embodiment of the present invention, wild type and TLR4 knockout dendritic cells were treated with RPS3 protein or LPS to induce maturation of dendritic cells, and stimulation of the dendritic cells with cancer antigen peptides (E7 peptides) Vaccines were prepared. When the prepared dendritic cell vaccine was inoculated into a mouse, it was confirmed that unlike the wild-type dendritic cell vaccine, in the group inoculated with the TLR4 knockout dendritic cell vaccine, almost no T cells were generated (FIG. 20). In addition, as a result of injecting cancer cells into the mice and administering the dendritic cell vaccine 3 days later, the group inoculated with the TLR4 knockout dendritic cell vaccine had a much larger tumor size than the wild type dendritic cell vaccination group, and The survival period was also confirmed to be short (FIG. 21).

That is, RPS3 protein alone does not induce maturation of immature dendritic cells. When RLR3 protein and TLR4 of dendritic cells specifically bind, maturation of dendritic cells is effectively induced, and dendritic cells derived from maturation with RPS3 protein are used. It was confirmed that the cancer treatment effect of the prepared dendritic cell vaccine was also excellent.

In addition, the present invention provides an anti-cancer adjuvant comprising ribosome protein S3 as an active ingredient.

In addition, the present invention provides an immune enhancer comprising ribosomal protein S3 as an active ingredient.

The anti-cancer adjuvant of the present invention, when using dendritic cells as a vaccine or therapeutic agent for cancer, preferably serves to increase the prophylactic or therapeutic effect, but is not limited thereto.

The immune enhancer of the present invention is a substance that promotes an immune response to an antigen, and is not an immunogen to a host, but means an agent, a molecule, etc. that enhances immunity by increasing the cellular activity of the immune system.

The pharmaceutical composition for preventing or treating cancer of the present invention may be various oral or parenteral formulations. When formulating the composition, one or more buffers (e.g. saline or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g. EDTA or glutathione), fillers, extenders, binders, adjuvants (e.g., Aluminum hydroxide), suspending agents, thickener wetting agents, disintegrating agents or surfactants, diluents or excipients.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient in one or more compounds such as starch (corn starch, wheat starch, rice starch, potato Starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose, methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl -It is prepared by mixing cellulose or gelatin. For example, tablets or dragees can be obtained by mixing the active ingredient with a solid excipient and then grinding it and adding a suitable adjuvant to the granule mixture.

In addition, lubricants such as magnesium stearate, talc and the like are used in addition to simple excipients. Liquid preparations for oral administration include suspending agents, intravenous solutions, emulsions or syrups, etc. In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents or preservatives are included. You can. In addition, if necessary, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant, and may further include an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, and a preservative. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspension solutions, emulsions, lyophilized preparations or suppositories. As the non-aqueous solvent and suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, etc. may be used.

The pharmaceutical composition for preventing or treating cancer of the present invention may be administered orally or parenterally, and externally applied to the skin when administered parenterally; It can be formulated according to methods known in the art in the form of injections for intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine dura mater or intracranial injection.

The injections must be sterile and protected from contamination of microorganisms such as bacteria and fungi. For injections, examples of suitable carriers include, but are not limited to, solvents or dispersion media comprising water, ethanol, polyols (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.), mixtures thereof and / or vegetable oils. You can. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) with triethanol amine or sterile water for injection, isotonic solutions such as 10% ethanol, 40% propylene glycol and 5% dextrose. Etc. can be used. In order to protect the injection from microbial contamination, it may further include various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In addition, the injection may additionally include an isotonic agent such as sugar or sodium chloride in most cases.

The pharmaceutical composition for preventing or treating cancer of the present invention is administered in a pharmaceutically effective amount. In the present invention, "a 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 is the type of patient's disease, severity, and activity of the drug , Sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including co-drugs and other factors well known in the medical field. The pharmaceutical composition for preventing or treating cancer of 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 a conventional therapeutic agent, and may be administered single or multiple. That is, the total effective amount of the pharmaceutical composition for preventing or treating cancer of the present invention may be administered to a patient in a single dose, and a fractionated treatment protocol administered for a long time in multiple doses ). Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, which can be easily determined by those skilled in the art.

The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and disease severity. As a daily dose, when administered parenterally, it is preferably administered in an amount of 0.01 to 50 mg, more preferably 0.1 to 30 mg per kg of body weight per day, based on the pulse extract. It can be divided into 1 to several times to be administered in an amount of preferably 0.01 to 100 mg, more preferably 0.01 to 10 mg per 1 kg of body weight per day based on the soybean extract. However, since the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, and age, the dosage is not limited to the scope of the present invention in any way.

The pharmaceutical composition for preventing or treating cancer of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormonal therapy, chemotherapy, and biological response modifiers.

Hereinafter, the present invention will be described in more detail through examples.

These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Preparation of recombinant RPS3 protein

In the present invention, as a maturation inducer for use in inducing maturation of dendritic cells, recombinant RPS3 protein was overexpressed and purified.

The RPS3 expression vector was constructed from cDNA of the human pancreatic adenocarcinoma CFPAC-1 cell line (CRL-1918, ATCC). First, the gene encoding the RPS3 protein (SEQ ID NO: 1; NP_000996.2) in CFPAC-1 cDNA (SEQ ID NO: 2; NM_001256802.1) can be specifically amplified, so that the amplified gene can be inserted into a vector. Primers were prepared to include NdeI and NotI restriction enzyme sites. The underscore of the primer sequence below represents the restriction site.

Forward primer (SEQ ID NO: 3): 5'- CATATG GCAGTGCAAATATCCAAGAAG-3 '

Reverse primer (SEQ ID NO: 4): 5'- GCGGCCGC TTATGCTGTGGGGACTGG-3 '

The DNA containing the RPS3 protein coding gene (SEQ ID NO: 5) amplified with the primer was inserted into a pET28b bacterial expression vector (Cat.No. 69865-3CN, Novagen) to produce a pET28b-RPS3 vector. The vector contains a Hig-tag site, located at the front of the NotI restriction enzyme site, and when the inserted RPS3 gene is expressed, the His-tag is expressed in front of the RPS3 protein (N-terminal).

The pET28b-RPS3 vector was transformed into Escherichia coli Rosetta competent cells. The transformed E. coli was inoculated in 5 ml LB broth medium containing kanamycin and cultured for 16 hours, followed by pre-culture, and then transferred to 250 ml LB broth medium for main culture. Is disclosed. After the main culture for 3 hours, 1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside, BioBasic) was added to the medium, and further cultured at 25 ° C. for 12 hours to induce RPS3 protein expression.

After the incubation, E. coli was centrifuged at 6000 RPM for 10 minutes to obtain cells, followed by SoluLyse pH 7.4 (Genlantis), 5 U / mL deoxynuclease I (BioBasic), 50 μg / mL Lysozyme (BioBasic) and 5 mM DTT (BioBasic) was added to dissolve at room temperature for 2 hours. The lysed E. coli was centrifuged at 12000 RPM for 15 minutes to obtain a cell pellet, and 6M urea buffer was added to the pellet to dissolve on ice for 1 hour, and then centrifuged at 13000 RPM for 15 minutes. The supernatant was obtained, filtered through a 0.45 μm filter, and Ni-NTA agarose (Qiagen) was added and incubated at 4 ° C. overnight. RPS3 was then purified with PBS containing imidazole and 6M urea.

The purified RPS3 protein was removed by dialysis to remove imidazole and urea, and until the amount of endotoxin reached 0.1 EU / mg or less as measured by the LAL assay kit (Lonza), Triton X- It was removed using 114 Surfact-Amps solution (Thermo Scientific).

The finally obtained recombinant RPS3 protein was electrophoresed with 12% SDS-gel and then transferred with a PVDF membrane (Roche). Blocking was performed for 1 hour at 25 ° C with 5% skim milk (BD), and the primary antibodies RPS3 antibody (Novu) and His antibody (MBL) were treated after blocking, respectively. All primary antibodies were diluted 1: 1000 with 5% BSA, treated on the membrane, reacted for 1 hour at 25 ° C, and washed three times at 10 minute intervals using TBS-T buffer.

The secondary antibody (Anti-mouse, Merk) was diluted 1: 5000 with 5% skim milk, treated on the membrane, and reacted at 25 ° C for 1 hour. Washing was performed three times at 10 minute intervals using TBS-T buffer. Thereafter, the membrane was treated with ECL solution (ECL, Merck Millipore), and the target band was identified using S-4000 (GE healthcare).

As a result, as shown in Figure 1, it was confirmed that the recombinant RPS3 protein (SEQ ID NO: 6) was purified to a high purity of about 27 kDa.

As a control, the pET28a-GFP vector (Novagen) was transformed into Escherichia coli BL21 (DE3) competent cell (NEB). E. coli cells expressing GFP were cultured at 37 ° C. for 5 hours, and then 1 mM IPTG was added to culture at 37 ° C. for 5 hours, and GFP (SEQ ID NO: 7) was purified in the same manner as RPS3 protein.

Confirmation of association with RPS3 protein and TRL4

2-1: RPS3 protein and TLR4 association confirmation

Luciferase analysis was performed using a Dual-Glo Luciferase Assay System (Promega, Cat.No. E2920).

First, 293 / hTLR4A-MD2-CD14 cells transformed to overexpress TLR4 (Cat code No .: 293-htlr4md2cd14, Invivogen) are dispensed into 96 well white plates (SPL) so that the number of cells is 2Ⅹ10 ⁴ per well. Then, it was transformed with pGL4.32 [luc2P / NF-κB vector (Cat.No. E849A, Promega) and pRL-TK vector (Cat.No. E2241, Promega). The transformed cells were cultured at 37 ° C. for 16 hours, followed by RPS3 (1 ng / ml, 10 ng / ml, 100 ng / ml), GFP (5 μg / ml) and LPS (100 ng / ml), respectively. Treated and incubated at 37 ° C. for 2 hours. Firefly luminescence as the NF-κB activity value, and Renilla luminescence as the correction value were measured according to the protocol provided by the manufacturer (Promega). Luciferase activity was calculated as the ratio of firefly-renilla luminescence in each well.

As a result, as shown in FIG. 2, the relationship between RPS3 protein and TLR4 was confirmed by confirming that NF-κB activity, a TLR4 sub-signal, is increased depending on the concentration of RPS3 protein.

Confirmation of induction of maturation of immature dendritic cells derived from mice by RPS3 protein

3-1: Isolation of immature dendritic cells from mice

The present inventors isolated immature dendritic cells from the bone marrow of mice to determine whether RPS3 protein can induce maturation of dendritic cells.

First, femoral bone marrow was collected using a bone marrow injection from C57BL / 6 mice 6 to 8 weeks old. After washing the collected bone marrow, red blood cells were removed using ammonium chloride, and blood cells in plasma were separated.

The isolated cells were 50 U / mL penicillin streptomycin, 1000 μM 2-mercaptomethanol, recombinant mouse IL-4 and granulocyte microphagy colony stimulating factor; Inoculated in RPMA1640 medium to which GM-CSF;) was added, and cultured at 37 ° C and 5% CO ₂ conditions, immature dendritic cells derived from mice were obtained. At this time, GM-CSF was used to induce differentiation into dendritic cells.

3-2: Confirmation of changes in the level of dendritic cell cytokine secretion by RPS3 protein

To confirm that dendritic cells can be induced to mature by RPS3 protein, the secretion level of cytokine markers secreted from the matured dendritic cells was confirmed.

First, the immature dendritic cells isolated in Example 3-1 were cultured in a 24-well plate to be 1 × 10 ⁶ cells / ml per well, and then the RPS3 protein purified in Example 1 was 0.01 μg / ml, 0.1 μg / Ml, treated to 1 µg / ml, and incubated for 16 hours at 37 ° C and 5% CO ₂ . The group not treated with anything and the group treated with the purified GFP protein in Example 1 were used as a negative control, and as a positive control, LPS (Lipopolysaccharide), which is well known to promote dendritic cell maturation, was 100 ng / ml. Incubated by treatment.

After the end of the culture, the culture supernatant was obtained, and the total amount of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-10 and IL-12p70) and interferon-beta (IFN-β) ELISA kits for TNF-α, IL-1β, IL-6, IL-10, and IL-12p70 were measured in ebioscience, ELISA kits for IFN-β measurement It was purchased from Assay Science and was measured according to the manufacturer's manual.

As a result, as shown in FIG. 3, the untreated control group and the GFP protein-treated group not treated with RPS3 protein did not show cytokine secretion, whereas the concentration of RPS3 protein-treated dendritic cells was pro-inflammatory cytotoxicity. It was confirmed that the secretion of kin and IFN-β increased. In particular, in the case of the 1 μg / ml RPS3 protein-treated group, it was confirmed that cytokines are secreted at a higher level than the positive control LPS-treated group.

3-3: Confirmation of the change in the expression level of dendritic cell surface factors by RPS3 protein

In order to confirm that the maturation of dendritic cells is induced by the RPS3 protein, the expression level of the surface factor, which is an indicator of maturation expressed on the surface of the matured dendritic cells, was confirmed. The surface factor is a co-stimulator (stimulatory checkpoint molecule) on T cells, which is responsible for activating antigen-specific adaptive immunity, particularly Th1 cells.

First, the immature dendritic cells isolated in Example 3-1 were cultured in a 24-well plate to be 1 × 10 ⁶ cells / ml per well, and then the RPS3 protein purified in Example 1 was 0.01 μg / ml, 0.1 μg / Ml, treated to 1 µg / ml, and incubated for 16 hours at 37 ° C and 5% CO ₂ . The group not treated with anything and the group treated with the purified GFP protein in Example 1 were used as a negative control, and as a positive control, LPS (Lipopolysaccharide), which is well known to promote dendritic cell maturation, was 100 ng / ml. Incubated by treatment.

After the incubation, dendritic cells were obtained, washed with PBS, and stained with FITC-CD11c and PE-CD40, PE-CD80, PE-CD86 and PE-MHCI, respectively. The fluorescent material was purchased from Bioligend, and was performed according to the manufacturer's manual. Then, flow cytometry was performed using FACSCalibur (Becton Dickinson) to confirm the expression levels of MHC I, CD40, CD80, and CD86.

As a result, as shown in FIG. 4, in the negative control group, it was confirmed that the expression of the surface factor of the dendritic cell increased depending on the concentration of the RPS3 protein, compared to the expression of the surface factor that is the indicator of maturation of the dendritic cell. In particular, in the case of the 1 μg / ml RPS3 protein treatment group, it was confirmed that the surface control factor was expressed at a level similar to that of the positive control LPS treatment group.

3-4: Confirmation of changes in the signaling system activity level in dendritic cells by RPS3 protein

In order to confirm whether the dendritic cells can be induced to mature by RPS3 protein, changes in the signaling system activation level in dendritic cells were confirmed over time after RPS3 protein treatment. To this end, the protein expression and phosphorylation levels of ERK, JNK, P38 and AKT, proteins of the MAPK (mitogenactivated protein kinases) signaling system, which are known as essential elements for the maturation of dendritic cells, were confirmed, and NF-κB was the expression of the activation indicator, IκBα. The level was confirmed.

First, the immature dendritic cells isolated in Example 3-1 were cultured in a 24-well plate to be 1 × 10 ⁶ cells / ml per well, and then treated with 1 μg / ml of RPS3 protein purified in Example 1 Incubated for 16 hours at 37 ° C and 5% CO ₂ conditions. The group not treated with anything and the group treated with the purified GFP protein in Example 1 were used as a negative control, and as a positive control, LPS (Lipopolysaccharide), which is well known to promote dendritic cell maturation, was 100 ng / ml. Incubated by treatment.

Dendritic cells were obtained at 10 minute intervals from the time of RPS3 protein treatment to the time of 60 minutes. The obtained cells were lysed for 1 hour on ice by treatment with RIPA buffer (0.5% NP-40, 1mM EDTA, 50mM Tris-Cl pH8.0, 120mM NaCl, protease inhibitor cocktail, 100mM PMSF, 0.5M NaF). The extracted protein was electrophoresed with 12% SDS-gel in the same amount, and then transferred with a PVDF membrane (Roche).

Western blot was performed in the same manner as in Example 1-1, and the primary antibodies p-ERK, total-ERK, p-P38, total-P38, p-JNK, total-JNK, p-AKT, and total-AKT And IκBα antibody was purchased from Cell Signaling, and β antibody was purchased from Santa Cruz. Secondary antibodies were purchased from Abbiotec and Enzo to react with each primary antibody.

As a result, as shown in Fig. 5, when the RPS3 protein was treated with dendritic cells, it was confirmed that phosphorylation of ERK, JNK, P38 and AKT was achieved within 1 hour. IκBα is an inhibitor of NF-κB, confirming that IκBα expression decreases, which means that the activation level of NF-κB is increased. That is, it was confirmed that the MAPK signaling system and NF-κB, which are lower signaling of TRL4, are activated by the RPS3 protein.

Confirmation of induction of maturation of human-derived mononuclear cell lines by RPS3 protein

In the present invention, in order to confirm whether the maturation of a human-derived mononuclear cell line is also induced by the RPS3 protein, GFP (5 µg / ml) and RPS3 protein (0.1 µg / ml and 1 µg / g) in human-derived mononuclear cell line THP-1 cell line. Ml) and LPS (100 ng / ml) were added to measure cytokine secretion and surface factor expression.

First, human monocytic THP-1 cells (Human monocytic THP-1 cells; TIB-202, ATCC) were prepared according to a known method (Tsuchiya S et al ., Int J Cancer , 26 (2): 171-176, 1980). Prepared by differentiation by treating 200 μg / ml of PMA (Sigma).

Measurement of cytokine secretion changes and the degree of surface factor expression were performed in the same manner as in Examples 3-2 and 3-3, and treated with THP-1 cells so that the RPS3 protein was 0.1 µg / ml and 1 µg / ml. In the case of surface factors, PE-CD80, PE-CD83, PE-CD86, and PE-CCR7 were stained, respectively, and the staining material was purchased from Bioligend and used.

As a result, as shown in Figure 6, it was confirmed that the secretion amount of TNF-α, IL-6, IL-10 and INF-β increases depending on the concentration of RPS3 protein. In addition, as shown in FIG. 7, it was confirmed that the expression of CD80, CD83, CD86, and CCR7, which are monocyte cell surface factors, is increased depending on the concentration of RPS3 protein. That is, it was confirmed that the maturation of the human mononuclear cell line is induced by the RPS3 protein.

Confirmation of induction of maturation of human-derived dendritic cells by RPS3 protein

In the present invention, in order to confirm that the maturation of human-derived dendritic cells is induced by RPS3 protein, GFP (5 µg / ml), RPS3 protein (0.1 µg / ml and 1 µg / ml) and LPS ( 100ng / ml) to measure cytokine secretion and surface factor expression.

First, human dendritic cells were separated from peripheral blood MNCs of healthy donors and then differentiated according to the protocol of the Institutional Reasearch Board of Hwasun Hospital of Chonnam National University.

The differentiated cells are sorted by CD14 ⁺ magnetic activating cell sorting system (MACs, Milenyi Biotec Inc. Auburn, CA, USA), and then 6-well plates (BD Falcon, to be 2Ⅹ10 ⁵ cells / well). San Jose, CA, USA) 10% fetal bovine serum (Hyclone; Logan UT, USA), 1% penicillin streptomycin, 50 ng / mL recombinant human granulocyte macrophage colony stimulator ( recombinant human granulocyte macrophage colony-stimulating factor; GM-CSF, LG Biochemical, Daejon, Korea) and RPMI1640 with 20 ng / ml recombinant mouse IL-4 (recombinant human IL-4, R & D Systems; Minneapolis, MN, USA) Cultured with medium. The culture medium was changed every 2 days, and differentiated after 6 days of culture.

Subsequently, the measurement of cytokine secretion changes and the degree of surface factor expression were performed in the same manner as in Examples 3-2 and 3-3, and the levels of cytokines (IL-10 and IL-12p70) secreted from human dendritic cells were It was measured according to the manual using a BD bioscience ELISA kit. In addition, human dendritic cells were stained with FITC-HLA-DR, PE-CD80 and FITC-CD86 (miltenyi) to measure the degree of surface factor expression of dendritic cells.

As a result, as shown in FIG. 8, it was confirmed that the secretion of IL-10 and IL-12 increased depending on the concentration of RPS3 protein, and as shown in FIG. 9, HLA, which is a surface factor of dendritic cells, was dependent on RPS3 protein concentration. It was confirmed that the expressions of -DR, CD80 and CD86 all increased. In particular, in the case of the 1 μg / ml RPS3 protein-treated group, it was confirmed that the cytokine secretion and surface factor expression were promoted in dendritic cells at a higher level than the LPS-treated group, which is a positive control group. It can be seen that the maturation of is effectively induced.

Purification and activity confirmation of mutant type RPS3 protein

6-1: Preparation and purification of mutant type RPS3 protein

In the present invention, to determine which part of the RPS3 protein specifically binds to TRL4 and induces maturation of dendritic cells, as shown in FIG. 10A, based on the KH domain (K Homology domain) present in the RPS3 protein. A mutant RPS3 protein was produced by dividing it into the front portion (amino acids 1-95; 1-95 RPS3) and the rear portion (amino acids 91-243; 91-243 RPS3) containing the KH domain.

In the CFPAC-1 cDNA constructed in Example 1, the RPS3 coding gene of SEQ ID NO: 2 can specifically amplify 1-95 RPS3 and 91-243 RPS3, respectively, and each amplified gene can be inserted into a vector. Primers were prepared to include NdeI and NotI restriction enzyme sites.

1-95 RPS3 coding gene amplification primer set

Forward primer (SEQ ID NO: 3): 5'- CATATG GCAGTGCAAATATCCAAGAAG-3 '

Reverse primer (SEQ ID NO: 8): 5'- GCGGCCGC TTAACCTCTAGTGGCCACCTT-3 '

91-243 RPS3 coding gene amplification primer set

Forward primer (SEQ ID NO: 9): 5'- CATATG GTGGCCACTAGAGGTCTGTGT-3 '

Reverse primer (SEQ ID NO: 4): 5'- GCGGCCGC TTATGCTGTGGGGACTGG-3 '

The primer-amplified 1-95 RPS3 coding gene (SEQ ID NO: 10) and 91-243 RPS3 coding gene (SEQ ID NO: 11) were inserted into the pET28b vector in the same manner as in Example 1, respectively, and the mutant type was 1-. 95 RPS3 (SEQ ID NO: 12) and 91-243 RPS3 (SEQ ID NO: 13) were purified, respectively. The purified recombinant protein is expressed by including the His-tag in front of the RPS3 protein (N-terminal) as in Example 1.

As a result, as shown in FIG. 10B, it was confirmed that 1-95 RPS3 and 91-243 RPS3 were purified to 13 kDa and 20 kDa with high purity, respectively.

6-2: Confirmation of association with TRL4 according to RPS3 protein location

Luciferase analysis was performed in the same manner as in Example 2-1 to confirm the association with TRL4 according to the location of the RPS3 protein, and the purified wild-type RPS3 (full length) in HEK293 cells transformed to overexpress TRL4 , 1-95 RPS3 and 91-243 RPS3 were treated at 100 ng / mL, respectively, and incubated at 37 ° C for 2 hours. The group not treated with anything and the group treated with the GFP protein purified in Example 1 were used as a negative control, and as a positive control, LPS was cultured by treating to 100 ng / ml.

As a result, as shown in FIG. 11, it was confirmed that although it is lower than the wild-type RPS3 protein in the rear portion (91-243 RPS3), not the front portion (1-95 RPS3) containing the KH domain, it is associated with TRL4.

6-3: Confirmation of the degree of maturation of dendritic cells according to the location of RPS3 protein

In order to confirm the cytokine secretion level of dendritic cells according to the location of the RPS3 protein, an experiment was performed in the same manner as in Example 3-2. The mouse-derived dendritic cells were treated with 100 ng / ml wild-type RPS3, 100 ng / ml 1-95 RPS3, 100 ng / ml 91-243 RPS3, 5 µg / ml GFP and 100 ng / ml LPS, respectively, for 16 hours. Cultured at 37 ° C and 5% CO ₂ conditions.

As a result of measuring the amount of TNF-α, IL-6 and IL-10 secreted by ELISA kit by obtaining the culture supernatant, as shown in FIG. 12, 91- corresponding to wild-type RPS3 protein and the rear part except 1-95 RPS3 It was confirmed that 243 RPS3 promotes cytokine secretion in dendritic cells.

In addition, experiments were performed in the same manner as in Example 3-3 to confirm the degree of surface factor expression of dendritic cells according to the location of the RPS3 protein. The mouse-derived dendritic cells were treated with 100 ng / ml wild-type RPS3, 100 ng / ml 1-95 RPS3, 100 ng / ml 91-243 RPS3, 5 µg / ml GFP and 100 ng / ml LPS, respectively, for 16 hours. Cultured at 37 ° C and 5% CO ₂ conditions. After obtaining dendritic cells and washing with PBS, they were stained with FITC-CD11c, PE-CD40, PE-CD80, PE-CD86, and PE-MHCI, respectively, and then subjected to flow cytometry to perform MHC I, CD40. , CD80 and CD86 expression levels were confirmed.

As a result, as shown in FIG. 13, it was confirmed that the wild-type RPS3 protein and the 91-243 RPS3 corresponding to the rear part promote expression of surface factors in dendritic cells, except for 1-95 RPS3. That is, it was confirmed that the portion corresponding to amino acids 91-243 of the RPS3 protein specifically reacts with TRL4 to affect the maturation and activity of dendritic cells.

Confirmation of cancer antigen specific T cell production effect of dendritic cell vaccine prepared using RPS3

To confirm that the dendritic cells induced by RPS3 maturation can be used as a cancer vaccine, it was confirmed that T cells specific for the cancer antigen can be generated in mice to which the dendritic cells have been administered.

First, the immature dendritic cells isolated in Example 3-1 were cultured in a 24-well plate to be 1 × 10 ⁶ cells / ml per well, and then 1 μg / ml of RPS3 protein purified in Example 1 and 100 ng of LPS After each treatment to be / ml, incubation for 16 hours at 37 ℃, 5% CO ₂ conditions to induce the maturation of dendritic cells. Then, OVA peptides known as cancer antigen peptides (egg albumin; ovalbumin) and E7 peptides (E7 peptides derived from type 16 human papilloma virus) were treated and incubated at 37 ° C for 1 hour. The cancer antigen was treated to be 1 µg / ml per 1 × 10 ⁶ cells, and after completion of the culture, washing was performed twice with PBS to prepare a dendritic cell vaccine.

OVA (Kb-restricted OVA 257-264 (SIINFEKL; SEQ ID NO: 14)) and E7 (Db-restricted E7 49-57 (RAHYNIVTE; SEQ ID NO: 15)) peptides were synthesized from Anygen (Jeollanam-do, Republic of Korea) Was used.

More specifically, no treatment control (CON); Immature dendritic cell administration group (imDC); OVA stimulated immature dendritic cell administration group (imDC + OVA); RPS3-mediated mature dendritic cell administration group (mDC (RPS3)); OVA-stimulated RPS3-mediated mature dendritic cell administration group (mDC (RPS3) + OVA); OVA-stimulated LPS-mediated mature dendritic cell administration group (mDC (LPS) + OVA); E7 stimulation immature dendritic cell administration group (imDC + E7); E7 stimulated RPS3-mediated mature dendritic cell administration group (mDC (RPS3) + E7); E7 stimulation LPS-mediated mature dendritic cell administration group (mDC (LPS) + E7); divided into a total of 10 groups, the experiment was conducted, the dendritic cell vaccine was also prepared accordingly prepared.

Female C57BL / 6 mice aged 6 to 8 weeks were purchased from OrientBio (Korea), and all mice had specific pathogens according to the Animal Care Regulations of the Institutional Animal Care and Use Committee (IACUC). It was managed without it.

Next, in order to confirm that the dendritic cell vaccine can generate T cells specific for the cancer antigen, C57BL / 6 mice were divided into 3 animals per group, and the dendritic cell vaccine prepared according to the group was 2 ×. Mice were inoculated twice at weekly intervals so as to be 10 ⁶ dogs / marital. On the 7th day after the last inoculation, the splenocytes of the mouse were isolated, and the splenocytes isolated in RPMI 1640 (including 10% FBS, 1% penicillin streptomycin, 0.5% 2-mercaptoethanol) medium were inoculated and inoculated at 37 ° C. , Cultured in 5% CO ₂ conditions. At this time, the culture medium was cultured with 1 μl / ml Golgi plug and 1 μl / ml cancer antigen (OVA or E7). After incubation for 16 hours, cells were obtained, washed with PBS, and stained with PE-CD8a antibody. Then, the cells stained with FITC-IFN-γ antibody after fixation and permeation were analyzed by flow cytometry (FACS) using FACSCallibur. As a result of flow cytometry analysis, cells positive for both CD8 and IFN-γ among splenocytes were defined as cancer antigen-specific CD8 + IFN-γ T cells, and the production rate of the corresponding CD8 + IFN-γ T cells in each group was confirmed.

As a result, as shown in FIG. 14, CD8 + at a significantly higher level than the other groups in the group (mDC (RPS3) + OVA and mDC (RPS3) + E7) administered with the dendritic cell vaccine prepared using the RPS3 protein It was confirmed to produce IFN-γ T cells. More specifically, in dendritic cells that did not induce stimulation with a cancer antigen, significant CD8 + IFN-γ T cells were not generated regardless of whether they were matured by RPS3, compared with dendritic cells that induced stimulation with a cancer antigen. In the inoculation group, it was confirmed that CD8 + IFN-γ T cells were generated when cancer antigens were re-administered from the extracted spleen cells.

In particular, it was confirmed that the RPS3 protein of the present invention shows higher CD8 + IFN-γ T cell production capacity than LPS, by showing a significantly higher level of CD8 + IFN-γ T cell production capacity in dendritic cells induced by RPS3 maturation than LPS. Did.

Confirmation of cancer prevention effect of dendritic cell vaccine prepared using RPS3

In the present invention, it was intended to confirm whether dendritic cells induced by maturation by RPS3 can effectively exhibit cancer prevention when used as a cancer vaccine.

In the same manner as in Example 7, a total of 10 mouse groups were inoculated with dendritic cells twice in a weekly interval. One week after the last inoculation, 5 × 10 ⁶ EG.7 cells in the OVA vaccine group using OVA peptide as an antigen; And 2.5 × 10 ⁵ TC-1 cells in the E7 vaccine group using the E7 peptide as an antigen; was injected subcutaneously in each mouse group. Then, through the Kaplan-Meier survival assay, the proportion of mice that did not show tumor growth was confirmed.

The EG.7 cells (CRL-2113, ATCC) are cells transformed to express OVA on mouse-derived EL4 cells, and TC-1 cells (prepared from the John Wu Hopkins School of Medicine and Pathology, TC Wu's laboratory) were used. ) Are transformed cells expressing HPV-16 E7 with mouse-derived lung epithelial cells, and prepared by culturing according to the protocol of each cell.

As a result, as shown in FIG. 15, it was confirmed that there was no mouse showing tumors produced by cancer cells injected subcutaneously in the group administered with the dendritic cell vaccine produced using the RPS3 protein and the cancer antigen peptide. That is, it was confirmed that dendritic cells matured by the RPS3 protein can have significantly higher cancer prevention effects than other mouse groups.

Confirmation of cancer treatment effect of dendritic cell vaccine prepared using RPS3

In the present invention, it was intended to confirm whether dendritic cells induced by maturation by RPS3 can effectively exhibit cancer treatment effects when used as a cancer vaccine.

First, normal C57BL / 6 mice were divided into a total of 10 groups as in Example 7, and then 2 × 10 ⁵ TC-1 cells and 1 × 10 ⁶ EG.7 cells were injected into the mice subcutaneously. Three days after the injection of cancer cells, dendritic cells were inoculated twice in a weekly interval to a total of 10 mouse groups in the same manner as in Example 7. In this case, mice injected with EG.7 cells were inoculated with OVA vaccine using OVA peptide as an antigen, and mice injected with TC-1 cells were vaccinated with 2 × 10 ⁶ / mari E7 vaccine using E7 peptide as antigen. Did.

After the last vaccination, mice were bred for a total of 60 days, and the size of tumor tissue formed from cancer cells was measured every 2-3 days. In addition, the survival rate of mice during the breeding period was confirmed through a Kaplan-Meier survival analysis experiment.

As a result, as shown in FIG. 16, the group administered with the dendritic cell vaccine produced using the RPS3 protein and the cancer antigen peptide had the smallest tumor size and the longest survival time of the mouse compared to other groups. Was confirmed.

More specifically, dendritic cells that did not induce stimulation with a cancer antigen did not show a significant cancer treatment effect irrespective of whether they were matured by RPS3, and immature dendritic cells among dendritic cell inoculation groups that induced stimulation with cancer antigens were not used. It was confirmed that the cancer treatment effect was not high even when inoculated.

In addition, LPS and RPS3 showed a significantly high level of cancer treatment effect in mature dendritic cells inducing maturation, and not only the tumor size was not significantly increased, but also the survival rate of mice was increased. It was confirmed that it showed the highest cancer treatment effect in the RPS3 treatment group.

Confirmation of dendritic cell activity with or without TLR4 expression

In the present invention, it was confirmed that RPS3 induces maturation of dendritic cells by binding with immature dendritic cell surface TLR4. To confirm the relationship between RPS3 and TLR4, the activity of dendritic cells was compared by treating RPS3 protein on mouse bone marrow-derived wild type dendritic cells and TLR4 knockout mouse-derived dendritic cells.

Separation of dendritic cells from mice was performed in the same manner as in Example 3-1, and TLR4 knockout mice (C57BL / 10ScNF) were purchased and used at the Jackson Laboratory (USA).

GFP (5 µg / ml), RPS3 protein (1 µg / ml) and LPS (100 ng / ml) were added to each dendritic cell isolated above to measure cytokine secretion, surface factor expression, and signaling system activation. It was performed in the same manner as in Examples 3-2 to 3-4.

As a result, as shown in FIGS. 17 to 18, unlike wild-type mouse-derived dendritic cells, cytokine secretion (FIG. 15) and surface factor production (FIG. 16) did not increase in dendritic cells derived from TLR4 knockout mice. It was confirmed that MAPK and NF-κB activity, which are lower signal transductions of TLR4, were not observed (FIG. 17).

That is, it was confirmed that maturation of dendritic cells is effectively induced when RPS3 protein alone does not induce maturation of immature dendritic cells, and when TLR4 of RPS3 protein and dendritic cells specifically bind.

Confirmation of T cell production effect and cancer treatment effect of dendritic cell vaccine according to the presence or absence of TLR4 expression

11-1: Confirmation of T cell generation in dendritic cell vaccine according to the presence or absence of TLR4 expression

In the present invention, in order to confirm the T cell generation effect and cancer treatment effect of the dendritic cell vaccine according to the presence or absence of TLR4 expression, dendritic cells using wild type mouse derived dendritic cells and TLR4 knockout mouse derived dendritic cells isolated in Example 10 above Vaccines were prepared.

First, dendritic cells were treated with 1 μg / ml RPS3 protein to induce maturation of dendritic cells, and the cancer antigen peptide E7 was treated to stimulate dendritic cells. A dendritic cell vaccine was prepared in the same manner as in Example 7, and the mice were inoculated twice a week at intervals of 1 week to confirm the production rate of cancer antigen-specific CD8 + IFN-γ T cells.

As a result, as shown in FIG. 20, dendritic cells that do not express TLR4 do not induce the maturation process by RPS3, so that the expression level of E7 antigen-specific CD8 + IFN-γ T cells does not appear, whereas RPS3 It was confirmed that E7 antigen-specific CD8 + IFN-γ T cells were significantly produced by the wild-type dendritic cell vaccine expressing TLR4 matured by (FIG. 16).

11-2: Confirmation of cancer treatment effect of dendritic cell vaccine according to the presence or absence of TLR4 expression

By confirming that dendritic cells matured by RPS3 can generate cancer antigen-specific T cells when expressing TLR4, it was intended to confirm whether the cancer treatment effect can also be significantly expressed.

First, normal C57BL / 6 mice were injected with 2 × 10 ⁵ TC-1 cells subcutaneously in each mouse group. Then, 3 days after the injection of the cancer cells, the wild type dendritic cell vaccine and the TLR4 knockout dendritic cell vaccine prepared in Example 11-1 were inoculated to be 2 × 10 ⁶ cells / piece, respectively, and the experiment was the same as in Example 9 above. Method. After the last vaccination, mice were bred for a total of 60 days, and the size of tumor tissue formed from cancer cells was measured every 2-3 days. In addition, the survival rate of mice during the breeding period was confirmed through a Kaplan-Meier survival analysis experiment.

As a result, as shown in FIG. 21, the tumor continued to grow in the same manner as the generation of E7 antigen-specific CD8 + IFN-γ T cells from dendritic cells not expressing TLR4, and 30 days after the injection of cancer cells. All mice were confirmed to die. In contrast, in the wild-type dendritic cell vaccination group expressing TLR4 matured by RPS3, tumor growth was not significantly observed, and the survival rate also persisted at a high level, confirming that it exhibits cancer treatment effects.

<110> Konkuk University Glocal Industry-Academic Collaboration Foundation <120> Composition for inducing maturation of dendritic cell comprising          Ribosomal Protein S3 <130> 1064381 <160> 15 <170> KoPatentIn 3.0 <210> 1 <211> 243 <212> PRT <213> Homo sapiens <400> 1 Met Ala Val Gln Ile Ser Lys Lys Arg Lys Phe Val Ala Asp Gly Ile   1 5 10 15 Phe Lys Ala Glu Leu Asn Glu Phe Leu Thr Arg Glu Leu Ala Glu Asp              20 25 30 Gly Tyr Ser Gly Val Glu Val Arg Val Thr Pro Thr Arg Thr Glu Ile          35 40 45 Ile Ile Leu Ala Thr Arg Thr Gln Asn Val Leu Gly Glu Lys Gly Arg      50 55 60 Arg Ile Arg Glu Leu Thr Ala Val Val Gln Lys Arg Phe Gly Phe Pro  65 70 75 80 Glu Gly Ser Val Glu Leu Tyr Ala Glu Lys Val Ala Thr Arg Gly Leu                  85 90 95 Cys Ala Ile Ala Gln Ala Glu Ser Leu Arg Tyr Lys Leu Leu Gly Gly             100 105 110 Leu Ala Val Arg Arg Ala Cys Tyr Gly Val Leu Arg Phe Ile Met Glu         115 120 125 Ser Gly Ala Lys Gly Cys Glu Val Val Val Ser Gly Lys Leu Arg Gly     130 135 140 Gln Arg Ala Lys Ser Met Lys Phe Val Asp Gly Leu Met Ile His Ser 145 150 155 160 Gly Asp Pro Val Asn Tyr Tyr Val Asp Thr Ala Val Arg His Val Leu                 165 170 175 Leu Arg Gln Gly Val Leu Gly Ile Lys Val Lys Ile Met Leu Pro Trp             180 185 190 Asp Pro Thr Gly Lys Ile Gly Pro Lys Lys Pro Leu Pro Asp His Val         195 200 205 Ser Ile Val Glu Pro Lys Asp Glu Ile Leu Pro Thr Thr Pro Ile Ser     210 215 220 Glu Gln Lys Gly Gly Lys Pro Glu Pro Pro Ala Met Pro Gln Pro Val 225 230 235 240 Pro Thr Ala             <210> 2 <211> 732 <212> DNA <213> Homo sapiens <400> 2 atggcagtgc aaatatccaa gaagaggaag tttgtcgctg atggcatctt caaagctgaa 60 ctgaatgagt ttcttactcg ggagctggct gaagatggct actctggagt tgaggtgcga 120 gttacaccaa ccaggacaga aatcattatc ttagccacca gaacacagaa tgttcttggt 180 gagaagggcc ggcggattcg ggaactgact gctgtagttc agaagaggtt tggctttcca 240 gagggcagtg tagagcttta tgctgaaaag gtggccacta gaggtctgtg tgccattgcc 300 caggcagagt ctctgcgtta caaactccta ggagggcttg ctgtgcggag ggcctgctat 360 ggtgtgctgc ggttcatcat ggagagtggg gccaaaggct gcgaggttgt ggtgtctggg 420 aaactccgag gacagagggc taaatccatg aagtttgtgg atggcctgat gatccacagc 480 ggagaccctg ttaactacta cgttgacact gctgtgcgcc acgtgttgct cagacagggt 540 gtgctgggca tcaaggtgaa gatcatgctg ccctgggacc caactggtaa gattggccct 600 aagaagcccc tgcctgacca cgtgagcatt gtggaaccca aagatgagat actgcccacc 660 acccccatct cagaacagaa gggtgggaag ccagagccgc ctgccatgcc ccagccagtc 720 cccacagcat aa 732 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> forward primer_RPS3 gene <400> 3 catatggcag tgcaaatatc caagaag 27 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer_RPS3 gene <400> 4 gcggccgctt atgctgtggg gactgg 26 <210> 5 <211> 743 <212> DNA <213> Artificial Sequence <220> <223> recombinant RPS3 gene <400> 5 catatggcag tgcaaatatc caagaagagg aagtttgtcg ctgatggcat cttcaaagct 60 gaactgaatg agtttcttac tcgggagctg gctgaagatg gctactctgg agttgaggtg 120 cgagttacac caaccaggac agaaatcatt atcttagcca ccagaacaca gaatgttctt 180 ggtgagaagg gccggcggat tcgggaactg actgctgtag ttcagaagag gtttggcttt 240 ccagagggca gtgtagagct ttatgctgaa aaggtggcca ctagaggtct gtgtgccatt 300 gcccaggcag agtctctgcg ttacaaactc ctaggagggc ttgctgtgcg gagggcctgc 360 tatggtgtgc tgcggttcat catggagagt ggggccaaag gctgcgaggt tgtggtgtct 420 gggaaactcc gaggacagag ggctaaatcc atgaagtttg tggatggcct gatgatccac 480 agcggagacc ctgttaacta ctacgttgac actgctgtgc gccacgtgtt gctcagacag 540 ggtgtgctgg gcatcaaggt gaagatcatg ctgccctggg acccaactgg taagattggc 600 cctaagaagc ccctgcctga ccacgtgagc attgtggaac ccaaagatga gatactgccc 660 accaccccca tctcagaaca gaagggtggg aagccagagc cgcctgccat gccccagcca 720 gtccccacag cataagcggc cgc 743 <210> 6 <211> 251 <212> PRT <213> Artificial Sequence <220> <223> recombinant RPS3 protein <400> 6 His His His His His His His Met Met Ala Val Gln Ile Ser Lys Lys   1 5 10 15 Arg Lys Phe Val Ala Asp Gly Ile Phe Lys Ala Glu Leu Asn Glu Phe              20 25 30 Leu Thr Arg Glu Leu Ala Glu Asp Gly Tyr Ser Gly Val Glu Val Arg          35 40 45 Val Thr Pro Thr Arg Thr Glu Ile Ile Ile Leu Ala Thr Arg Thr Gln      50 55 60 Asn Val Leu Gly Glu Lys Gly Arg Arg Ile Arg Glu Leu Thr Ala Val  65 70 75 80 Val Gln Lys Arg Phe Gly Phe Pro Glu Gly Ser Val Glu Leu Tyr Ala                  85 90 95 Glu Lys Val Ala Thr Arg Gly Leu Cys Ala Ile Ala Gln Ala Glu Ser             100 105 110 Leu Arg Tyr Lys Leu Leu Gly Gly Leu Ala Val Arg Arg Ala Cys Tyr         115 120 125 Gly Val Leu Arg Phe Ile Met Glu Ser Gly Ala Lys Gly Cys Glu Val     130 135 140 Val Val Ser Gly Lys Leu Arg Gly Gln Arg Ala Lys Ser Met Lys Phe 145 150 155 160 Val Asp Gly Leu Met Ile His Ser Gly Asp Pro Val Asn Tyr Tyr Val                 165 170 175 Asp Thr Ala Val Arg His Val Leu Leu Arg Gln Gly Val Leu Gly Ile             180 185 190 Lys Val Lys Ile Met Leu Pro Trp Asp Pro Thr Gly Lys Ile Gly Pro         195 200 205 Lys Lys Pro Leu Pro Asp His Val Ser Ile Val Glu Pro Lys Asp Glu     210 215 220 Ile Leu Pro Thr Thr Pro Ile Ser Glu Gln Lys Gly Gly Lys Pro Glu 225 230 235 240 Pro Pro Ala Met Pro Gln Pro Val Pro Thr Ala                 245 250 <210> 7 <211> 238 <212> PRT <213> Artificial Sequence <220> <223> GFP protein <400> 7 Met Ser Lys Gly Ala Glu Leu Phe Thr Gly Val Val Pro Ile Leu Ile   1 5 10 15 Glu Leu Asn Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu              20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys          35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe      50 55 60 Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln  65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu Arg                  85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Ser Arg Ala Glu Val             100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Thr Gly Thr         115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly Asn Lys Met Glu Tyr Asn     130 135 140 Tyr Asn Ala His Asn Val Tyr Ile Met Thr Asp Lys Ala Lys Asn Gly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val                 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro             180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Thr Leu Ser         195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Ile Tyr Phe Glu Phe Val     210 215 220 Thr Ala Ala Ala Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> reverse primer_1-95 RPS3 gene <400> 8 gcggccgctt aacctctagt ggccacctt 29 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> forward primer_91-243 RPS3 gene <400> 9 catatggtgg ccactagagg tctgtgt 27 <210> 10 <211> 299 <212> DNA <213> Artificial Sequence <220> <223> mutant 1-95 RPS3 gene <400> 10 catatggcag tgcaaatatc caagaagagg aagtttgtcg ctgatggcat cttcaaagct 60 gaactgaatg agtttcttac tcgggagctg gctgaagatg gctactctgg agttgaggtg 120 cgagttacac caaccaggac agaaatcatt atcttagcca ccagaacaca gaatgttctt 180 ggtgagaagg gccggcggat tcgggaactg actgctgtag ttcagaagag gtttggcttt 240 ccagagggca gtgtagagct ttatgctgaa aaggtggcca ctagaggtta agcggccgc 299 <210> 11 <211> 476 <212> DNA <213> Artificial Sequence <220> <223> mutant 91-243 RPS3 gene <400> 11 catatggtgg ccactagagg tctgtgtgcc attgcccagg cagagtctct gcgttacaaa 60 ctcctaggag ggcttgctgt gcggagggcc tgctatggtg tgctgcggtt catcatggag 120 agtggggcca aaggctgcga ggttgtggtg tctgggaaac tccgaggaca gagggctaaa 180 tccatgaagt ttgtggatgg cctgatgatc cacagcggag accctgttaa ctactacgtt 240 gacactgctg tgcgccacgt gttgctcaga cagggtgtgc tgggcatcaa ggtgaagatc 300 atgctgccct gggacccaac tggtaagatt ggccctaaga agcccctgcc tgaccacgtg 360 agcattgtgg aacccaaaga tgagatactg cccaccaccc ccatctcaga acagaagggt 420 gggaagccag agccgcctgc catgccccag ccagtcccca cagcataagc ggccgc 476 <210> 12 <211> 251 <212> PRT <213> Artificial Sequence <220> <223> mutant 1-95 RPS3 protein <400> 12 His His His His His His His Met Met Ala Val Gln Ile Ser Lys Lys   1 5 10 15 Arg Lys Phe Val Ala Asp Gly Ile Phe Lys Ala Glu Leu Asn Glu Phe              20 25 30 Leu Thr Arg Glu Leu Ala Glu Asp Gly Tyr Ser Gly Val Glu Val Arg          35 40 45 Val Thr Pro Thr Arg Thr Glu Ile Ile Ile Leu Ala Thr Arg Thr Gln      50 55 60 Asn Val Leu Gly Glu Lys Gly Arg Arg Ile Arg Glu Leu Thr Ala Val  65 70 75 80 Val Gln Lys Arg Phe Gly Phe Pro Glu Gly Ser Val Glu Leu Tyr Ala                  85 90 95 Glu Lys Val Ala Thr Arg Gly Leu Cys Ala Ile Ala Gln Ala Glu Ser             100 105 110 Leu Arg Tyr Lys Leu Leu Gly Gly Leu Ala Val Arg Arg Ala Cys Tyr         115 120 125 Gly Val Leu Arg Phe Ile Met Glu Ser Gly Ala Lys Gly Cys Glu Val     130 135 140 Val Val Ser Gly Lys Leu Arg Gly Gln Arg Ala Lys Ser Met Lys Phe 145 150 155 160 Val Asp Gly Leu Met Ile His Ser Gly Asp Pro Val Asn Tyr Tyr Val                 165 170 175 Asp Thr Ala Val Arg His Val Leu Leu Arg Gln Gly Val Leu Gly Ile             180 185 190 Lys Val Lys Ile Met Leu Pro Trp Asp Pro Thr Gly Lys Ile Gly Pro         195 200 205 Lys Lys Pro Leu Pro Asp His Val Ser Ile Val Glu Pro Lys Asp Glu     210 215 220 Ile Leu Pro Thr Thr Pro Ile Ser Glu Gln Lys Gly Gly Lys Pro Glu 225 230 235 240 Pro Pro Ala Met Pro Gln Pro Val Pro Thr Ala                 245 250 <210> 13 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> mutant 91-243 RPS3 protein <400> 13 His His His His His His His Met Met Glu Ser Gly Ala Lys Gly Cys   1 5 10 15 Glu Val Val Val Ser Gly Lys Leu Arg Gly Gln Arg Ala Lys Ser Met              20 25 30 Lys Phe Val Asp Gly Leu Met Ile His Ser Gly Asp Pro Val Asn Tyr          35 40 45 Tyr Val Asp Thr Ala Val Arg His Val Leu Leu Arg Gln Gly Val Leu      50 55 60 Gly Ile Lys Val Lys Ile Met Leu Pro Trp Asp Pro Thr Gly Lys Ile  65 70 75 80 Gly Pro Lys Lys Pro Leu Pro Asp His Val Ser Ile Val Glu Pro Lys                  85 90 95 Asp Glu Ile Leu Pro Thr Thr Pro Ile Ser Glu Gln Lys Gly Gly Lys             100 105 110 Pro Glu Pro Pro Ala Met Pro Gln Pro Val Pro Thr Ala         115 120 125 <210> 14 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Kb-restricted OVA 257-264 <400> 14 Ser Ile Ile Asn Phe Glu Lys Leu   1 5 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Db-restricted E7 49-57 <400> 15 Arg Ala His Tyr Asn Ile Val Thr Glu   1 5

Claims (12)

A composition for inducing maturation of dendritic cells comprising ribosomal protein S3 (RPS3) as an active ingredient.
According to claim 1, wherein the ribosome protein S3 is a composition for inducing maturation of dendritic cells, characterized in that consisting of the amino acid sequence of SEQ ID NO: 1.
The composition for inducing maturation of dendritic cells according to claim 1, wherein the dendritic cells express toll-like receptor 4 (TLR4).
The composition for inducing maturation of dendritic cells according to claim 1, wherein the ribosome protein S3 specifically binds TLR4 expressed in dendritic cells to induce maturation of dendritic cells.
According to claim 4, by the TLR4 binding of the ribosomal protein S3 and dendritic cells,
(Ⅰ) Tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 -10; IL-10), increased expression of any one or more cytokines selected from the group consisting of interleukin-12p70 (IL-12p70) and interferon-β (INF-β);
(Ii) increased expression of any one or more surface factors selected from the group consisting of CD40, CD80, CD83, CD86, CC chemokine receptor type 7 (CCR7) and major histocompatibility complex 1 (MHC 1) on the dendritic cell surface; And
(Iii) increased TLR4 signaling, mitogen-activated protein kinases (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB);
The composition for inducing maturation of dendritic cells, characterized in that made.
In vitro, immature dendritic cells treated with ribosomal protein S3 and culturing; Including the method of inducing maturation of dendritic cells.
The method for inducing maturation of dendritic cells according to claim 6, wherein the ribosome protein S3 is composed of the amino acid sequence of SEQ ID NO: 1.
The method of claim 6, wherein the dendritic cell expresses toll-like receptor 4 (TLR4).
The method of claim 6, wherein the ribosome protein S3 specifically binds TLR4 expressed in dendritic cells to induce maturation of dendritic cells.
The method according to claim 9, by the TLR4 binding of the ribosome protein S3 and dendritic cells,
(Ⅰ) Tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 -10; IL-10), increased expression of any one or more cytokines selected from the group consisting of interleukin-12p70 (IL-12p70) and interferon-β (INF-β);
(Ii) increased expression of any one or more surface factors selected from the group consisting of CD40, CD80, CD83, CD86, CC chemokine receptor type 7 (CCR7) and major histocompatibility complex 1 (MHC 1) on the dendritic cell surface; And
(Iii) increased TLR4 signaling, mitogen-activated protein kinases (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB);
Method of inducing maturation of dendritic cells, characterized in that is made.
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