KR20100109099A - Method for preparing dendritic cells having potential to induce cancer specific immune response, pharmaceutical composition and kit for treating or preventing cancer or inhibiting cancer metastasis comprising the same - Google Patents

Abstract

The present invention provides a method for producing mature dendritic cells with significantly improved ability to induce cancer specific immune responses, a pharmaceutical composition for preventing, treating or inhibiting metastasis of cancer, including the mature dendritic cells prepared by the above method, and the method A pharmaceutical kit comprising mature dendritic cells and imiquimod. According to the method of the present invention, the ability to induce cancer specific immune responses is remarkably improved, and safe mature dendritic cells with very low toxicity can be produced. Mature dendritic cells produced by the method of the present invention is very excellent in the treatment, prevention and metastasis inhibitory effect of cancer. Imiquimod used in the pharmaceutical kits of the present invention surprisingly enhances the immunotherapeutic, prophylactic and inhibitory effects of cancer metastasis by administering cancer specific mature dendritic cells.

Description

Method for preparing dendritic cells having potential to induce cancer specific immune response, a method for producing dendritic cells having a cancer specific immune response, a method for preventing, treating, or inhibiting metastasis of cancer comprising the dendritic cells Pharmaceutical Composition and Kit for Treating or Preventing Cancer or Inhibiting Cancer Metastasis Comprising the Same}

The present invention provides a method for producing mature dendritic cells with improved ability to induce cancer-specific immune responses, a pharmaceutical composition for preventing, treating or inhibiting cancer metastasis of cancer, including the mature dendritic cells prepared by the above method, and prepared by the method. A pharmaceutical kit comprising mature dendritic cells and an adjuvant.

Most of the cancer treatments known to date are surgery, chemotherapy, and radiation. However, such treatment is difficult to cure and involves many side effects. In particular, metastasis or recurrence is inoperable in most cases and shows the limitation of treatment by showing resistance to chemotherapy.

Recently, research and development are being conducted on cancer therapy using immune cells such as dendritic cells. Dendritic cells capture and activate foreign antigens through phagocytosis or pinocytosis to induce antigen-specific T-cell activity, and their own MHC (major histocompatibility complex) molecules when activated after foreign antigen capture. In addition, the expression of costimulatory factors (costimulatory factor) is known to induce cellular immunity is increased. The process by which dendritic cells induce cellular immunity is as follows. The mature dendritic cells sensitized to the antigen i) express a large amount of the receptive receptor (homing receptor) and thus migrate to the secondary immune organs, ii) expressing dendritic cell specific CC chemokine (DC-CK1) to inactivate T cells ( naive T cells), and iii) direct antigenic interactions between the antigens presented in MHC and the T cell receptor (TCR) to induce antigen-specific T cell activity.

Liver cancer, meanwhile, is one of the world's fifth most prognosis cancers. Current therapies are largely divided into surgical therapies such as hepatectomy and liver transplantation, and non-surgical therapies such as readiofrequency ablation, percutaneous ethanol injection, and transarterial chemoembolization. . Such treatment is selected according to the stage of liver cancer, liver function and activity of the patient. The causes of hepatocellular carcinoma include hepatitis viruses such as HBV and HCV, toxins such as aflatoxin, excessive intake of alcohol and abuse of drugs, and are common in East Asia including Korea, China, and Japan. Liver cancer progresses over 20-40 years, mainly due to chronic hepatitis due to viruses, alcohol, cirrhosis, and then liver cancer. Problems in the treatment of liver cancer are the occurrence of local recurrence, intrahepatic relapse and metastasis. As an alternative to solve these problems, systemic liver cancer treatment and immunotherapy are on the rise. In recent years, the development of liver cancer treatment mainly proceeds by expanding the indications after receiving approval from other cancers, mainly targeted drugs and humanized antibodies. Immunotherapy has been assessed primarily for advanced liver cancer in accordance with international guidelines. From 1990 to the present, clinical studies of immunotherapy for hepatocellular carcinoma can be classified into the following strategies (1). Iii) induction of nonspecific activation of immune cells through cytokine-based clinical trials, ii) application of autologous dendritic cells, cytokine-induced peripheral blood cells (cytokine induced PBL) and TIL (tumor infilterated lymphocyte) as cell-based clinical trials Et al., I) humanized antibody based clinical trials, i) autologous tumor cell vaccines, v) peptide vaccines.

In general, the process of making mature dendritic cell vaccines from progenitor cells is largely divided into two steps. The first stage culture is a culture that induces differentiation from various blood-derived cells into immature dendritic cells and is treated as a differentiation inducing substance (cytokine or other cell-differentiation promoter) and harvested as immature dendritic cells on the last day of culture. The second stage culture is a culture in which such immature dendritic cells are treated with cancer or pathogen-specific antigen, and the mature dendritic cells are harvested as a vaccine obtained by antigen-immune inducibility by adding dendritic cell maturation factor to the culture medium with the antigen. will be. The dendritic cell maturation factor generally varies according to the clinical researcher's choice, but a cytokine cocktail or a specific monocyte-conditioned medium (MCM) having a specific composition is generally used. In addition, dendritic cells may be directly isolated from living bodies without progenitor cell separation, and may be used as a vaccine through ex vivo conditioning through antigen sensitization.

In clinical studies of immunotherapy using dendritic cells, tumor lysates or cancer specific antigens are mainly used for sensitization of dendritic cells to activate cancer-specific immune responses (2). For more effective dendritic cell maturation by antigenic proteins, studies have been conducted to overcome these problems through cell surface targeting methods or fusion of membrane translocating peptides (3). In this respect, there has been a study of the PTD (YGRKKRRQRRR) of HIV-1 TAT protein (4). On the other hand, a series of CTP (cytoplasmic transduction peptide) candidates derived from PTD but having membrane permeability but do not migrate to the nucleus due to the altered structure have been produced (WO 2003-097671, 5).

Imiquimod (1- (2-methylpropyl) -1H-imidazo [4,5-c] quinolin-4-amine) is a compound used as an immune response modifier. The mechanism of action of imiquimod is not clear, but it is known that imiquimod binds to toll-like receptor (TLR) -7, a cell surface receptor involved in the recognition of pathogens, activating immune cells (6). Cells activated by imiquimod via TLR-7 secrete cytokines such as interferon-alpha, interleukin-6 and tumor necrosis factor-TN (7). The application of imiquimod to the skin activates Langerhans cells, which migrate to local lymph nodes to activate the acquired immune system. Other cells activated by imiquimod include natural killer cells, macrophages and B-lymphocytes (8). Recently, new research has shown that the anti-proliferation effect of imiquimod is caused by a mechanism completely different from the activation of the immune system. Occurs by increasing the level. Blocking OGFr function by siRNA technology also eliminates the anti-proliferative effect of imiquimod (9). Imiquimod is used primarily for topical administration in the form of a cream due to side effects from systemic administration, and the imiquimod 5% cream, marketed under the trade name Aldara, was developed by the US FDA in 1997 for actinic keratosis. It has been approved as a therapeutic drug for superficial basal cell carcinoma, external genital warts, etc.

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

The present inventors have tried to develop a dendritic cell therapy for cancer treatment. As a result, mature dendritic cells prepared by maturing immature dendritic cells in the presence of cancer-specific antigen protein fused with peptide YGRRARRRRRR have the ability to induce an excellent cancer-specific immune response, which has the effect of preventing, treating and inhibiting cancer metastasis. The present invention was completed by confirming that when imiquimod is used as an adjuvant when administering the mature dendritic cell therapeutic agent, it is possible to greatly increase the efficacy of immunotherapy, prophylaxis or inhibition of metastasis of cancer.

It is therefore an object of the present invention to provide a method for producing mature dendritic cells having excellent cancer specific immune response induction ability.

Another object of the present invention is to provide a pharmaceutical composition for treating or preventing cancer or inhibiting metastasis of cancer, including mature dendritic cells having the ability to induce cancer-specific immune responses.

Still another object of the present invention is to provide a pharmaceutical kit for treating or preventing cancer or inhibiting metastasis of cancer, including mature dendritic cells having a cancer-specific immune response and imiquimod.

Still another object of the present invention is to provide a method for treating or preventing cancer, or a method for inhibiting metastasis of cancer, comprising administering to a recipient suffering from cancer, a mature dendritic cell having cancer specific immune response inducing ability.

Still another object of the present invention is to provide a method for treating or preventing cancer, or a method for inhibiting metastasis of cancer, comprising the step of co-treating imiquimod with mature dendritic cells having cancer-specific immune response inducing ability.

The objects and advantages of the invention are more clearly illustrated by the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for producing a mature dendritic cell having the ability to induce cancer-specific immune response comprising the following steps: (a) a cancer-specific antigen in which immature dendritic cells are fused with peptide YGRRARRRRRR Sensitizing and maturing the protein; And (b) isolating the matured dendritic cells of step (a).

According to another aspect of the present invention, the present invention is (a) a pharmaceutically effective amount of mature dendritic cells having the ability to induce cancer-specific immune response prepared by the method for producing mature dendritic cells; And (b) provides a pharmaceutical composition for the treatment or prevention of cancer comprising a pharmaceutically acceptable carrier.

According to another aspect of the invention, the present invention (a) a pharmaceutical effective amount of mature dendritic cells having the ability to induce cancer-specific immune response prepared by the method of producing mature dendritic cells; And (b) provides a pharmaceutical composition for inhibiting cancer metastasis comprising a pharmaceutically acceptable carrier.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) a mature dendritic cell having the ability to induce cancer-specific immune response prepared by the method of producing mature dendritic cells; And (b) provides a pharmaceutical kit for the treatment or prevention of cancer comprising a composition containing a compound represented by the formula (1).

[Formula 1]

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) a mature dendritic cell having the ability to induce cancer-specific immune response prepared by the method of producing mature dendritic cells; And (b) provides a pharmaceutical kit for inhibiting metastasis of cancer comprising a composition containing a compound represented by the formula (1).

According to another aspect of the present invention, the present invention uses a dendritic cell comprising the step of administering to the recipient suffering from cancer the mature dendritic cells having the ability to induce cancer-specific immune response prepared by the method for producing a mature dendritic cells Provided are methods for treating or preventing cancer.

According to another aspect of the present invention, the present invention comprises the step of administering the dendritic cells comprising the steps of administering to the recipient suffering from cancer the mature dendritic cells having the ability to induce cancer-specific immune response prepared by the method of producing mature dendritic cells It provides a method for inhibiting metastasis of cancer used.

According to another aspect of the present invention, there is provided a method for treating or preventing cancer using dendritic cells, comprising the following steps: (a) Inducing the ability to induce cancer-specific immune responses prepared by the method of producing mature dendritic cells; Administering the mature dendritic cells having cancer to a recipient suffering from cancer; And (b) applying a composition containing the compound represented by Formula 1 to a site where mature dendritic cells have been administered; Or (a ') applying a composition containing the compound represented by Formula 1 to a site of administration of a recipient suffering from cancer to administer mature dendritic cells; And (b ′) administering a mature dendritic cell having a cancer specific immune response inducing capacity prepared by the method for producing mature dendritic cells to a recipient suffering from cancer.

According to another aspect of the invention, there is provided a method for inhibiting cancer metastasis using dendritic cells comprising the following steps: (a) to induce a cancer specific immune response prepared by the method of producing mature dendritic cells Administering the mature dendritic cells having cancer to a recipient suffering from cancer; And (b) applying a composition containing the compound represented by Formula 1 to a site where mature dendritic cells have been administered; Or (a ') applying a composition containing the compound represented by Formula 1 to a site of administration of a recipient suffering from cancer to administer mature dendritic cells; And (b ′) administering a mature dendritic cell having a cancer specific immune response inducing capacity prepared by the method for producing mature dendritic cells to a recipient suffering from cancer.

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

The present invention provides a method for producing a mature dendritic cell having the ability to induce cancer specific immune response, comprising the steps of: (a) sensitizing immature dendritic cells to cancer specific antigen proteins fused with peptide YGRRARRRRRR to mature; And (b) isolating the matured dendritic cells of step (a).

As used herein, the term "dendritic cell" refers to a specialized antigen presenting cell capable of presenting antigen to T cells through a major histocompatibility complex (MHC).

As used herein, the term "mature dendritic cell" refers to mature differentiation of immature dendritic cells with an appropriate cytokine or the like after antigen sensitization in ex vivo. In the following, dendritic cells are referred to as "DC" as needed, and mature dendritic cells are referred to as "mDC".

According to one embodiment of the present invention, self-derived mature dendritic cells having the ability to induce cancer-specific immune responses can be prepared by maturing induction culture of immature dendritic cells in the presence of cancer-specific antigens. Immature dendritic cells may be prepared by inducing differentiation of progenitor cells present in human organs, tissues, bone marrow or blood, or may be directly isolated from living bodies. Dendritic cells used in the present invention are preferably myeloid DCs (myeloid DC).

According to a preferred embodiment of the present invention, immature dendritic cells in the present invention are obtained by differentiating progenitor cells of aquatic cells to the immature stage in the presence of a suitable cytokine. Suitable cytokines used for immature differentiation include, but are not limited to, for example, Granulocyte macrophage colony stimulating factor (GM-CSF) and IL (Interleukin) -4, or GM-CSF and IL-13 or GM-CSF and IL-7. It is not limited. The most commonly used cytokine is a mixture of GM-CSF and IL-4. Appropriate amounts of cytokines added are sufficient to differentiate into dendritic cells and can be determined through optimization experiments in the laboratory of each investigator, and generally vary within concentration ranges of 100 U / mL-1000 U / mL, respectively. Is being applied.

As a medium used for inducing differentiation of dendritic cells into immature dendritic cells, a general medium used for culturing animal cells can be used. Preferably, the medium contains serum (eg fetal calf serum, horse serum and human serum). Medium that may be used in the present invention is, for example, RPMI series (e.g., RPMI 1640), Eagles's MEM (Eagle's minimum essential medium, Eagle, H. Science 130: 432 (1959)), α-MEM (Stanner, CP et al., Nat. New Biol. 230: 52 (1971), Iscove's MEM (Iscove, N. et al., J. Exp. Med. 147: 923 (1978)), 199 medium (Morgan et al. , Proc. Soc.Exp. Bio.Med . 73: 1 (1950)), CMRL 1066, RPMI 1640 (Moore et al., J. Amer. Med. Assoc. 199: 519 (1967)), F12 (Ham, Proc. Natl. Acad. Sci. USA 53: 288 (1965)), F10 (Ham, RG Exp. Cell Res. 29: 515 (1963)), DMEM (Dulbecco's modification of Eagle's medium, Dulbecco, R. et al. Virology 8: 396 (1959)), mixtures of DMEM and F12 (Barnes, D. et al., Anal. Biochem. 102: 255 (1980)), Way-mouth's MB752 / 1 (Waymouth, C. J. Natl Cancer Inst. 22: 1003 (1959)), McCoy's 5A (McCoy, TA, et al., Proc. Soc.Exp. Biol. Med. 100: 115 (1959)) and the MCDB series (Ham, RG et al. , In Vitro 14:11 (1978)). The medium may contain other components, such as antioxidants (eg β-mercaptoethanol). General descriptions of media and cultures are described in R. Ian Freshney, Culture of Animal Cells , Alan R. Liss, Inc., New York (1984), which is incorporated herein by reference.

Mature dendritic cells used in the present invention are obtained by inducing immature dendritic cells to be cultured and matured in the presence of a suitable cancer specific antigen protein and cytokine fused with peptide YGRRARRRRRR. Cytokines suitable for inducing maturation of immature dendritic cells in the present invention are, for example, TNF (Tumor necrosis factor) -α, IFN (Interferon) -γ, IL-1β, IL-6, PGE2 (Prostaglandin E2) or mixtures thereof It may be in the form, but is not limited thereto. In addition, poly I: C (polyinosinic: polycytidylic acid) may be further added to the medium to induce maturation of dendritic cells. Stimulation may also be enhanced with bacterial origin, such as CpG, inactivated Staphylococcus aureus (SAC), Staphylococcal enterotoxin B (SEB), or lipopolysaccharide (LPS).

As a medium that can be used for maturation, a medium used for inducing immature differentiation of progenitor cells of dendritic cells can be used.

According to a preferred embodiment of the present invention, the cancer antigen used in inducing maturation of the present invention is a liver cancer specific antigen.

The mature dendritic cells of the present invention are cells obtained by maturing immature dendritic cells in the presence of liver cancer specific antigen proteins fused with peptide YGRRARRRRRR, and have the ability to induce liver cancer specific immune responses.

Liver cancer specific antigen proteins that can be used in the present invention are, for example, AFP (Alpha-Fetoprotein) fused with peptide YGRRARRRRRR, GPC 3 (Glypican-3), MAGE 1 (Melanoma Associated Gene Family A1), NY-ESO-1 ( cancer / testis antigen), Aurora-A (serine / threonine protein kinase involved in centrosome formation during mitosis), and Gankyrin (liver-specific oncoprotein involved in cell cycle regulation).

According to a preferred embodiment of the invention, the liver cancer specific antigen protein is AFP, GPC 3 or MAGE 1 fused with peptide YGRRARRRRRR, more preferably 20- of the AFP amino acid sequence of SEQ ID NO: 1 sequence fused with peptide YGRRARRRRRR The 346 sequence, the 33-358 sequence in the GPC 3 amino acid sequence of SEQ ID NO: 2, or the MAGE1 amino acid sequence of SEQ ID NO: 3.

The peptide YGRRARRRRRR used in the present invention may be fused to the N terminus or C terminus of the cancer antigen protein.

According to a preferred embodiment of the present invention, a dendritic cell vaccine having improved titer can be prepared when the peptide is used in maturation culture of a fusion protein tagged at the N terminus of a cancer antigen protein.

The present invention (a) a pharmaceutically effective amount of mature dendritic cells having the ability to induce cancer-specific immune response; And (b) provides a pharmaceutical composition for the treatment or prevention of cancer comprising a pharmaceutically acceptable carrier.

According to a preferred embodiment of the present invention, the mature dendritic cells are cells produced by the method of producing mature dendritic cells of the present invention described above.

According to another preferred embodiment of the invention, said cancer is liver cancer.

Mature dendritic cells obtained by maturing immature dendritic cells in the presence of hepatocellular carcinoma specific antigen protein fused with peptide YGRRARRRRRR as described in the method for producing mature dendritic cells of the present invention is a state in which the hepatocellular carcinoma specific antigen is present on the surface of the cell and is at rest. Specific cell immunity to liver cancer cells can be induced by differentiating CD4 ⁺ T cells into Th1 cells and also by differentiating resting CD8 ⁺ T cells into activated cytotoxic cells (CTL). Thus, mature dendritic cells sensitized to liver cancer specific antigen proteins fused with peptide YGRRARRRRRR prepared by the method of the invention described above are very effective in the immunotherapy or prevention of liver cancer.

Mature dendritic cells that can be used in the pharmaceutical compositions of the present invention are autologous or allogenic dendritic cells derived from immature dendritic cells of the recipient. Most preferably, the dendritic cells of the present invention are autologous dendritic cells. Since autologous dendritic cells are derived from the cells of the recipient, there is an advantage that there is no problem of immune rejection reaction and is safe when administered to the recipient for treatment or prevention purposes.

The present invention (a) a pharmaceutically effective amount of mature dendritic cells having the ability to induce cancer-specific immune response; And (b) provides a pharmaceutical composition for inhibiting cancer metastasis comprising a pharmaceutically acceptable carrier.

Mature dendritic cells sensitized to a cancer specific antigen protein fused with the peptide YGRRARRRRRR prepared by the method of the present invention described above can surprisingly be very effectively used not only for the treatment or prevention of cancer but also for inhibiting cancer metastasis. In addition, the toxicity is very low to provide a safe pharmaceutical composition.

According to a preferred embodiment of the present invention, the present invention provides a pharmaceutical composition for inhibiting metastasis of liver cancer.

According to another preferred embodiment of the present invention, the present invention provides a pharmaceutical composition for inhibiting lung metastasis of liver cancer.

Mature dendritic cells that can be used in the pharmaceutical compositions of the present invention are autologous or allogenic dendritic cells derived from immature dendritic cells of the recipient. Most preferably, the dendritic cells of the present invention are autologous dendritic cells.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably parenterally. When administered parenterally, the pharmaceutical compositions of the present invention may be administered by intravenous infusion, subcutaneous infusion, intramuscular infusion, intraperitoneal infusion, and the like. It is preferable that the route of administration is determined according to the type of the disease to which the pharmaceutical composition of the present invention is applied.

Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to the recipient. Can be. The dose of the pharmaceutical composition of the present invention is preferably 1 x 10 ³ per 1 - a 1 x 10 ⁸ cells / kg (body weight).

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container.

The present invention (a) a pharmaceutical composition comprising mature dendritic cells having the ability to induce cancer-specific immune response; And (b) provides a pharmaceutical kit for the treatment or prevention of cancer comprising a composition containing a compound represented by the formula (1).

[Formula 1]

According to a preferred embodiment of the present invention, the mature dendritic cells are cells produced by the method of producing mature dendritic cells of the present invention described above.

According to another preferred embodiment of the invention, said cancer is liver cancer.

The compound of formula 1 is 1- (2-methylpropyl) -1H-imidazole [4,5-c] quinolin-4-amine, also called "imiquimod".

The composition containing "imiquimod" is administered in combination with a pharmaceutical composition comprising the mature dendritic cells of the present invention to significantly improve the immunotherapeutic or immunoprophylactic efficacy of cancer by dendritic cells.

The content of the compound in the compound-containing composition of Formula 1 is not particularly limited, but is preferably 1-10% by weight, more preferably 3-7% by weight, relative to the compound-containing composition of Formula 1.

According to a preferred embodiment of the invention, the compound of formula 1 has a formulation in the form of skin application, most preferably in the form of a cream. In addition to the imiquimod compound as an active ingredient, the cream-containing imiquimod compound contains isostearic acid, cetyl alcohol, stearyl alcohol, white petrolatum, polysorbate 60, sorbitan monostearate, glycerin, xanthan gum, purified water, Benzyl alcohol, methyl parabens, propyl parabens and the like may be included, but is not limited thereto.

In the present invention, the method of administering the compound containing the compound of Formula 1 is not particularly limited, but according to a preferred embodiment of the present invention, the method is applied by applying to the site of administration of dendritic cells.

The present invention (a) a pharmaceutical composition comprising mature dendritic cells having the ability to induce cancer-specific immune response; And (b) provides a pharmaceutical kit for inhibiting metastasis of cancer comprising a composition containing a compound represented by the formula (1).

Compound-containing compositions of formula (1) are co-administered with pharmaceutical compositions comprising mature dendritic cells described above, surprisingly significantly inhibiting metastasis of cancer.

According to a preferred embodiment of the present invention, the mature dendritic cells are cells produced by the method of producing mature dendritic cells of the present invention described above.

According to another preferred embodiment of the invention, the metastasis of cancer is metastasis of liver cancer.

According to another preferred embodiment of the invention, the metastasis of liver cancer is lung metastasis of liver cancer.

In the method of the present invention, the compound-containing composition of Formula 1 is applied to the administration site after or before administration of the self-derived mature dendritic cells sensitized to the cancer specific antigen to the recipient.

The method of applying the compound of formula 1 in the method of the present invention is not particularly limited and may be preferably applied to the skin to administer the self-derived mature dendritic cells, the number of application is not only once but twice as appropriate It can apply many times.

In the method of the present invention, the recipient to be used in combination of the autologous dendritic cell-containing composition and the compound of the formula (1) -containing composition is an animal, preferably a mammal, and more preferably a human.

The mature dendritic cells that can be used in the pharmaceutical kit for treating or preventing cancer of the present invention or the pharmaceutical kit for inhibiting cancer metastasis are autologous or allogenic dendritic cells derived from immature dendritic cells of the recipient. It is a cell. Most preferably, the dendritic cells of the present invention are autologous dendritic cells.

The present invention provides a method for treating or preventing cancer using dendritic cells, comprising the steps of: (a) administering mature dendritic cells having cancer-specific immune response to a recipient suffering from cancer; And (b) applying a composition containing the compound represented by Formula 1 to a site where mature dendritic cells have been administered; Or (a ') applying a composition containing the compound represented by Formula 1 to a site of administration of a recipient suffering from cancer to administer mature dendritic cells; And (b ′) administering mature dendritic cells having cancer specific immune response induction to a cancer suffering recipient.

According to a preferred embodiment of the present invention, the mature dendritic cells are cells produced by the method of producing mature dendritic cells of the present invention described above.

According to another preferred embodiment of the invention, said cancer is liver cancer.

The present invention provides a method for inhibiting cancer metastasis using dendritic cells, comprising the steps of: (a) administering mature dendritic cells having cancer specific immune response to a recipient suffering from cancer; And (b) applying a composition containing the compound represented by Formula 1 to a site where mature dendritic cells have been administered; Or (a ') applying a composition containing the compound represented by Formula 1 to a site of administration of a recipient suffering from cancer to administer mature dendritic cells; And (b ′) administering mature dendritic cells having cancer specific immune response induction to a cancer suffering recipient.

According to a preferred embodiment of the present invention, the mature dendritic cells are cells produced by the method of producing mature dendritic cells of the present invention described above.

According to another preferred embodiment of the invention, the metastasis of cancer is metastasis of liver cancer.

According to another preferred embodiment of the present invention, the metastasis of liver cancer is a lung metastasis of liver cancer.

The present invention provides a method for treating or preventing cancer using dendritic cells, comprising administering to the recipient suffering from cancer, mature dendritic cells having a cancer-specific immune response.

According to a preferred embodiment of the present invention, the mature dendritic cells are prepared by the method of producing mature dendritic cells of the present invention described above.

The present invention provides a method for inhibiting metastasis of cancer using dendritic cells, comprising administering mature dendritic cells having cancer specific immune response to a recipient suffering from cancer.

According to a preferred embodiment of the present invention, the mature dendritic cells are prepared by the method of producing mature dendritic cells of the present invention described above.

The mature dendritic cells used in the method for treating or preventing cancer of the present invention or the method for inhibiting metastasis of cancer are preferably autologous dendritic cells.

The features and advantages of the present invention are summarized as follows.

(i) By the method of producing mature dendritic cells of the present invention has a significantly improved ability to induce cancer specific immune response, very low toxicity can be produced safe dendritic cells.

(ii) The present invention provides the use of a dendritic cell having cancer specific immune response inducing ability to treat, prevent or inhibit cancer metastasis, in particular liver cancer.

(iii) The pharmaceutical kit of the present invention suggests the use of the combination treatment of imiquimod for immunotherapy with mature dendritic cells.

(iv) Concomitant treatment of imiquimod in the pharmaceutical kit of the present invention surprisingly enhances the immunotherapeutic, prophylactic and inhibitory effects of cancer metastasis by dendritic cell administration.

According to the method for producing mature dendritic cells of the present invention, it is possible to produce mature dendritic cells with significantly improved cancer specific immune response induction ability. The mature dendritic cells produced by the method of the present invention are not only excellent in the treatment, prophylaxis and metastasis suppression effect of cancer, but also very low in toxicity and safe. The combined administration of imiquimod used in the pharmaceutical kit of the present invention with the mature dendritic cells of the present invention surprisingly improves the immunotherapeutic, prophylactic efficacy of cancer and inhibition of metastasis of cancer.

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

Example

Materials and Methods

Example 1-1 Preparation of Recombinant Antigen Protein

CDNA was synthesized from human-derived liver cancer cell lines and breast cancer cell lines HepG2 and SK-BR-3 to clone the liver cancer specific antigens AFP (alpha-fetoprotein), MAGE 1 (melanoma associated gene family A1), and GPC 3 (glypican3).

The expression site was selected from the 20-346 amino acid sequence of the full-length amino acid sequence shown in SEQ ID NO: 1 for AFP, 33-358 amino acid sequence of the full-length amino acid sequence of SEQ ID NO: 2 for GPC 3, MAGE 1 The case selected the full length amino acid sequence set forth in SEQ ID NO: 3 sequence. In addition, an antigenic protein in which peptide YGRRARRRRRR was fused to the protein of choice was used.

After cloning the antigen cDNA synthesized in the YGRRARRRRRR peptide-fusion expression vector, E. coli expression strain BL21-DE3 was transformed, and transformants stably expressing the YGRRARRRRRR peptide-fusion antigen protein were selected. Transgenic strains expressing each fusion antigen were cultured in LB medium containing empicillin, while 0.1-0.5 mM IPTG (isopropylthio-β-D-galactoside; Sigma) was added to induce the expression of each fusion protein. After recovering the cells by centrifugation of the strain culture, the recombinant antigen protein in the form of an inclusion body was obtained from the cells through ultrasonic disruption and centrifugation. The obtained protein was washed several times with washing buffer (50 mM Tris, 100 mM NaCl, 0.05% Triton X-100, pH 8.0) to remove endotoxin as much as possible. Then 6M Gn-HCl solution (6M Guanidin-HCl, 100mM NaCl, 50mM Tris, pH8.0) was added to fully dissolve the inclusion body. Ni-NTA agarose affinity column was used to isolate proteins using His-tag present in the recombinant protein. The protein was eluted using an imidazole concentration gradient (100 mM-1M), and the separated protein was further purified through a Gel filteration column (Hiprep ^TM 16/60 sephacryl ^TM S-100 HR, Amersham-Pharmacia). Subsequently, imidazole and urea, which were included in the eluate, were removed by dialysis using PBS. The purified protein was concentrated to an appropriate concentration using Ultra-filteration (Vivaspin 20, Satorius co.), The concentrated protein was aliquoted and stored at -80 ℃. In order to confirm the purified protein, the corresponding protein band was confirmed by SDS-PAGE analysis, and the purified protein was specifically reacted with the anti-His antibody by Western blot using an anti-His antibody (Qiagene). It was confirmed that (see Fig. 1).

Example 1-2 Preparation of Tumor Cell Lines Expressing Human-Derived Liver Cancer Antigens

Tumor cell line used in liver cancer mouse model, MH134 cell line homologous to C3H mouse strain and homologous cell line transformed with eukaryotic cell expression vector (pcDNA3.1, Invitrogen) to stably express human AFP, MAGE 1, GPC 3 Was used.

Example 1-3 Isolation of Bone Marrow Cells (Progenitor Cells of Immature Dendritic Cells) for the Preparation of Dendritic Cells

Mouse (C3H) bone marrow cells were obtained from the femur and tibia. To obtain the femur and tibia, the mice were first sacrificed by cervical dislocation and the surgical site was sterilized using 70% ethanol. The femur and tibia were separated, soaked in 70% ethanol for about 30 seconds, sterilized, washed in phosphate buffered saline (PBS), and placed in RPMI-1640 medium. Both ends of the femur and tibia were cut using sterile surgical scissors, RPMI-1640 medium was placed in a 1 cc syringe, the needle tip was inserted into the cut region, and then pushed into a 50 ml tube to collect bone marrow cells. The collected cells were centrifuged to wash the bone marrow cells once and remove red blood cells contained in the bone marrow cells. After red blood cell removal, the cells were washed by centrifugation twice and stored on ice until used in the next experiment.

Example 1-4: Induction of Differentiation into Immature Dendritic Cells

10 ng / ml mouse recombinant interluekin-4 (mrIL-4) in RPMI-1640 medium containing 10% FBS (fetal bovine serum; Life technologies) as a medium for differentiation into immature dendritic cells; CreaGene INC.) And 10 ng / ml of the mouse recombinant macrophage / granulocyte colony growth promoter (mrGM-CSF; CreaGene INC.) In a medium containing 37 ° C CO ₂ incubator Bone marrow cells (progenitor cells of immature dendritic cells) isolated from above were cultured for 2 days. Since the progenitor cells of dendritic cells have plastic adherence that grows to the bottom, the progenitor cells of the dendritic cells were separated from the non-progenitor cells by culturing for 2 days and then separating the nonadsorbable cells from the adsorbent cells. After removing the non-adsorbent cells, again dendritic cell differentiation culture was performed after filling 2 ml of the same dendritic cell differentiation medium. At this time, a part of the medium was changed every 2-3 days so that the cytokines necessary for the differentiation of dendritic cells were not deficient. Immature dendritic cells were obtained at 6-7 days.

Example 1-5 Antigen Treatment and Maturation Induction Culture for Antigen Specific Mature Dendritic Cells

Immature dendritic cells were added with maturation medium 4 hours after treatment with the appropriate concentrations of each of the liver cancer specific antigen proteins YGRRARRRRRR peptide-fused. The medium used for maturation was mouse recombinant interferon (IFN) -endogen and mouse recombinant tumor necrosis factor (TNF) -α (Endogen) in dendritic cell differentiation medium. And polyI: C (Sigma) was added. On day 6-7, differentiation medium of mouse dendritic cells was replaced with maturation medium to which antigen was added at a constant concentration, and dendritic cell maturation culture was performed in a CO ₂ incubator. The final product, the mature dendritic cells were obtained by harvesting non-adsorbable suspended cells on the last day of production as non-adsorbable cells that do not adhere to the bottom. The mature dendritic cells were washed three times with phosphate solution and used for the next experiment.

The degree of maturation of the mature dendritic cells was determined by cell size, granularity and the expression of CD11c, CD40, CD54, CD80, CD86, Class II, and the purity of dendritic cells was confirmed by the expression of CD14. Surface antigen expression of dendritic cells was analyzed by flow cytometry.

Example 2-1 Experimental Animals

Experimental animals received 6-week-old female cut C3H mice from Orient-Bio, Korea, or Charles River, Japan, and used for the experiment. Breeding cages containing animals were used polycarbonate cages for mice, housed 4 per breeding cage. Rodent feed and autoclaved distilled water were freely ingested during the experiment. Breeding environment was maintained at a temperature of 20.0-24.3 ℃, humidity 36.9-67.4%, the number of ventilation 12 to 15 times per hour and 12 hours automatic lighting (light: 08:00-20:00, illuminance 150-300 lux). Individual identification of each animal was performed by the ear marker method, and each cage was attached to the individual identification card, and the test group and the animal number were marked.

Example 2-2 Induction of Solid Cancer Formation and Cancer Metastasis by Cell Lines Expressing Human-Derived Liver Cancer Antigens

Cell lines stably expressing human AFP or human MAGE 1, human GPC 3 (hereinafter referred to as MH134 / AFP or MH134 / MAGE 1, MH134 / GPC 3) were indicated by RPMI with 10% FBS and 400 μg / ml G418. Incubated / maintained in 100 mm plate using -1640 medium. After 2-3 passages, the cells were changed to fresh medium the day before the experiment to optimize cell viability. The number of cells suspended in a physiological saline injection solutions and living cells adjusted to 1.5 X 10 ⁵ / 100㎕ by inhalation to a 1 cc syringe was prepared liver cancer cell line expressing the antigen injection. Injection site of mouse subjects The skin was disinfected with alcohol swabs, and the needle was inserted into the subcutaneous tissue of the left back of the mouse at an angle of about 45 mm and the liver cancer cell line was injected. Solid cancer formation was observed at intervals of 2-3 days from the time when the liver cancer antigen-expressing cell line was injected, and the size of the solid cancer was measured using a caliper, and the size was converted according to the long axis X short axis ² X 0.52 formula. In the case of inducing cancer metastasis, the mouse was fixed using a calibrator and allowed to move to some extent to prevent asphyxia. The disinfection was performed by sweeping the entire tail down using an alcohol swab. Fixed to the portion of the vein stick the needle facing up liver cancer antigen expression cell line is maintained an angle within the injection (0.75 X 10 ⁵ / 100μl) containing 15 ° and inserted into a vein in the tail and parallel to the needle and the vein is maintained The contents were inserted as possible. Hemostasis was maintained for about 10 seconds after the injection was completed and the mouse was placed in a cage after checking the condition. Each experimental group and markers were checked and the next experiment was conducted.

Experimental Example 1 Preventive or Protective Immune Induction by Antigen-Specific Mature Dendritic Cells and / or Imiquimod Treatment

Example 1-5 was prepared the sensitizing antigen liver recombinant protein was suspended matured dendritic cells in sterile PBS solution in the number of live dendritic cells was adjusted to 1 X 10 ⁶ / 100㎕. According to the experimental group, 100 μl of the dendritic cell suspension or PBS solution was determined as a single dose and administered to the right flank of the mouse twice a week. At this time, mice were depilated using a razor 2-3 days before dendritic cell administration. When applying Aldala Cream (5% Imiquimod Cream, iNOVA Pharmaceuticals), a certain amount of Aldala Cream is taken on the day before or after the administration of dendritic cells or PBS, and the dendritic cells or PBS solution is administered. The site was rubbed and applied for 30 seconds. One week after the final administration of dendritic cells or PBS, a mixture of the appropriate amount of liver cancer antigen expressing cell lines (MH134 / AFP, MH134 / MAGE 1, MH134 / GPC 3) suspended in PBS as in Example 2-2 (1.5 X 10 ⁵ cells / 100 μl) was injected subcutaneously into the contralateral dorsal to which dendritic cells were administered. The anticancer effect of the antigen-specific mature dendritic cells and / or imiquimod combination in the solid cancer prevention or defense model constructed as above was investigated.

In the experimental group, the antigen-specific mature dendritic cell alone administration group (Ag-DC, n = 8), the Aldara cream alone administration group (PBS + Aldara cream, n = 8) and the antigen-specific mature dendritic cell + Aldara cream combination administration group (Ag-DC + Aldara cream, n = 8), and the control group is the PBS solution administration group.

Experimental Example 2: Induction of Treatment Immunity by Antigen-Specific Mature Dendritic Cells and / or Imiquimod Treatment

First, an appropriate amount of liver cancer antigen expressing cell lines (MH134 / AFP, MH134 / MAGE 1, MH134 / GPC 3) mixed solution (1.5 × 10 ⁵ cells / 100 μl) suspended in PBS as in Example 2-2 was left-backed (left flank) was injected subcutaneously or intravenously. After tumor implantation at the 2nd and 9, a point in time in Example 1-5 by sensitizing a liver cancer antigen recombinant protein produced in a suspended Province sukdoen dendritic cells in sterile PBS solution was alive number of dendritic cells, 1 X 10 ⁶ / 100㎕ that It was made. According to the experimental group, 100 μl of the dendritic cell suspension or PBS solution was determined as a single dose and administered to the right flank of the mouse. At this time, the mice were depilated using a razor 2-3 days before dendritic cell administration. When applying Aldala Cream (5% Imiquimod Cream, iNOVA Pharmaceuticals), a certain amount of Aldala Cream is taken on the day before or after the administration of dendritic cells or PBS, and the dendritic cells or PBS solution is administered. The site was rubbed and applied for 30 seconds. The anticancer immune effects of antigen-specific mature dendritic cells and / or imiquimod combinations in solid cancer treatment and cancer metastasis treatment models constructed as described above were investigated.

In the experimental group, the antigen-specific mature dendritic cell alone administration group (Ag-DC, n = 8), the Aldara cream alone administration group (PBS + Aldara cream, n = 8) and the antigen-specific mature dendritic cell + Aldara cream combination administration group (Ag-DC + Aldara cream, n = 8), and the control group is the PBS solution administration group.

Experimental Example 3 Observation of Pulmonary Metastases During Cancer Metastasis after Separation and Fixation of Lung Tissue

In the cancer metastasis treatment model constructed in Experimental Example 2, mice were sacrificed by cervical dislocation in the 18th day after induction of cancer metastasis by intravenous injection of cancer cell lines, and systemically sterilized and fixed with 70% ethanol. Subsequently, the skin was H-dissected and fixed to both sides based on the midline of the abdomen, and the peritoneum was cut into the midline again and then fixed to both sides. After confirming the abnormality of the intestinal organs, the diaphragm was cut and the sternum was cut along the rib cartilage. Lung tissue was collected, washed in phosphate buffer solution to remove blood coagulation tissue, and then immersed in Bouin's fixative (Sigma), a fixative solution, to observe the lung metastases caused by liver cancer antigen expressing cell lines.

Experimental Example 4 Liver Cancer Antigen-Specific T Cell Proliferation, Th1 Cytokine Secretion, and CTL Induction

Control and experimental mice used in Experimental Examples 1 and 2 were euthanized by cervical dislocation, and spleens were taken and stored in RPMI-1640 medium so that the separated spleens were not dried. Each spleen was taken, passed through a 70 μm mesh to remove suspended tissue, centrifuged once to harvest cells, and then suspended in 0.83% ammonium chloride solution to remove red blood cells. The prepared splenocytes were reacted with a nylon wool column for 1 hour to separate only T lymphocytes, and after receiving a non-adsorbable fraction, a flow cytometry analysis of CD3 antigen was performed. Confirmed and used in the next experiment. Efficacy T cells were mixed for 5 days with antigen presenting cells (APC) prepared for stimulation at 5: 1 ratio. APC was prepared by the following method two days before the experimental date. Spleens from normal mice were taken to remove erythrocytes and stimulated with 3 μg / ml Concanavalin A for 48 hours. After stimulation, an appropriate amount of liver cancer antigen recombinant protein (YGRRARRRRRR-AFP, YGRRARRRRRR-MAGE 1, YGRRARRRRRR-GPC 3) was treated and cultured for 24 hours. After 24 hours of incubation, Mitomycin C was treated for 40 minutes and washed three times to prepare APC. Efficacy T cells (Effector T cell) and co-culture with APC were taken on day 3 of 5 days, and T cell proliferation assay was performed by MTT analysis. In addition, a portion of the culture medium was taken on the 4th day of culture to confirm the secretion amount of Th1 type / Th2 type cytokines (IFN-gamma, IL-4). After 5 days of culture, dead cells were removed using Histopaque (Sigma), and effector T cells were isolated to express target cancer cells (MH134 / AFP, MH134 / GPC 3, and MH134 / MAGE 1 mixed cells). ) And effector T cells (E: T ratio) were mixed at 5: 1, 10: 1, 20: 1 and 40: 1 and incubated for 4 hours. Experiments were performed according to the instructions of Promega's Non-Radioactive Proliferation assay kit and the specific lysis rate was calculated.

Experimental Example 5: Safety Test of Mature Dendritic Cells of the Present Invention

To test the safety of the mature dendritic cells prepared in Example 1-5 according to the present invention, a single subcutaneous dose toxicity test, repeated subcutaneous dose toxicity test, and immunotoxicity test were performed using a mouse (Balb / C, male and female 6 weeks old). Was performed. This was done by Biotoxtec co., Ltd., which is certified as a GLP test institute. The single subcutaneous dose toxicity test was conducted in four test groups according to the dose. A total of 40 animals, 5 males and 5 males, were used for the experiment. The control group was treated with physiological saline, and the test group was administered with a subcutaneous route of 2.5 X 10 ⁵ , 7.5 X 10 ⁵ , and 2.5 X 10 ⁶ doses, respectively. Mortality, general symptoms and changes in body weight were observed for 14 days, and necropsy and histopathological examination were performed after 14 days of autopsy. For the repeated subcutaneous dose toxicity test and immunotoxicity test, three test groups were tested according to the dosage, and 10 females per group were used. The control group was treated with physiological saline solution, and the test group was administered with 5 × 10 ⁴ and 5 × 10 ⁵ doses subcutaneously. The control group and the 5 × 10 ⁵ dose group included the recovery group, which used 5 females per group. The administration cycle was repeated once a week for 5 weeks. The test group was necropsy 1 week after the final administration and the autopsy group was 4 weeks later. Test items included general symptoms, weight change, feed intake, ophthalmological test, urine test, hematological test, blood biochemical test, organ weight, autopsy and histopathological test, immunotoxicity test. The antibodies of CD4, CD8a and CD45R / B220 were used to test whether T cells and B cells were activated, and NK cell activation assay in the spleen was performed.

Experiment result

Experimental Example 1 and Experimental Example 4 Results: Solid Cancer Prevention or Protective Immune Induction

Experiment for confirming the prophylactic or protective immunity against solid cancer in mouse liver cancer model, the group of mice treated with PBS (n = 8) and the group of mice treated with dendritic cell treatment according to the present invention (n = 8) as a control group , Mouse group treated only with alda cream (n = 8), mouse group treated with the dendritic cell treatment agent and alda cream in combination (n = 8) according to the present invention (1.5 X 10 ⁵ cells / head) of liver cancer Antigen-expressing cell lines were administered subcutaneously and tumor growth was observed every 2-3 days. The size of the solid rock was calculated as the long axis X short axis ² X 0.52 using the caliper (Caliper). As a result, the mouse experimental group treated with the dendritic cell treatment, the dendritic cell treatment, and the Aldala cream according to the present invention showed a significant inhibitory effect of solid cancer growth than the PBS control group or the Aldala Cream-only experimental group. In particular, the three subgroups (CR: 43%) of the combination group treated with dendritic cell therapy and Aldara cream did not form a subcutaneous injection into solid cancer, and the other three (PR: 43%) had PBS control average solid cancer. Tumors were formed to a very small size compared to solid cancers of the other control and experimental groups with a size less than 70% of the size (see FIGS. 2A-2C).

In addition, the survival of the mice was continuously examined at intervals of 2-3 days to confirm the life extension effect of the mature dendritic cells according to the present invention on cancer-induced individuals. For tumor-induced mice, all died in the case of PBS control group 40 days after tumor induction, but 50% of the subjects receiving the dendritic cell therapeutic agent according to the present invention maintained the dendritic cells according to the present invention. The administration group maintained 80% or more, and especially in the case of the combination administration group, the life extension effect was continued even after 50 days (see FIG. 2D).

The inhibitory effect of solid cancer growth in the defense model is based on the proliferation of hepatic cancer antigen-specific T cells associated with cellular immune responses in experimental mouse subjects, ii) expression of IFN-γ, which is expected to be a Th1-driven immune response, and iii) CTL. It was expected to correlate with the activity results, and the following experiment was conducted to confirm this. One week, two weeks, or three weeks after the last dendritic cell treatment, the activity of spleen-derived T lymphocytes obtained by sacrifice of one mouse from the control group and each experimental group was investigated. In the case of splenic T lymphocytes isolated from the dendritic cell therapy according to the present invention and from mouse groups treated with a combination of dendritic cells and aldara cream, the cells proliferated at a higher rate than T lymphocytes derived from mouse groups treated with PBS. Was observed (see FIG. 3). The same results were also shown in the numerical comparison of IFN-γ and IL-4 in vitro, which can infer Th1-driven cellular immune responses (see FIGS. 4A and 4B). In addition, in the cytotoxic T lymphocyte activity against the target cell expressing the antigen, the mouse treated with the dendritic cell therapeutic agent according to the present invention and the mouse treated with the combination of the dendritic cell and the alda cream according to the present invention. Was found to be much higher than the control group, especially the combination administration group was the highest (see Fig. 5).

Experimental Example 2 and Experimental Example 4 Results: Induction of Immunity to Solid Cancer Treatment

In a mouse liver cancer model, a solid cancer was formed by subcutaneously injecting an appropriate amount of liver cancer antigen-expressing cell line into 28 mice in order to confirm therapeutic immunity against solid cancer. After solid cancer formation, the group of mice receiving PBS (n = 7), the group of mice receiving the dendritic cell treatment according to the present invention (n = 7), the group of mice treated with Aldara cream only (n = 7), the present invention The anticancer effect was investigated in the group of mice treated with dendritic cell treatment and alda cream. As an indicator, cancer cell density (FIG. 7A) and solid cancer growth (FIG. 7B) in cancer tissues were observed. In the mouse group treated with the dendritic cell therapeutic agent according to the present invention and the mouse group co-treated with the dendritic cell and aldara cream according to the present invention, it was confirmed that the density of cancer cells in the cancer tissues was lowered. It was confirmed that the density of cancer cells in the cancer tissue was significantly lower (FIG. 7A). In addition, in the growth of solid cancer, it was confirmed that the mouse group co-treated with the dendritic cells and aldara cream according to the present invention was very suppressed (FIG. 7B). These results show that the therapeutic effect by dendritic cells according to the present invention shows a change in the therapeutic properties in terms of the density of viable tumor cells constituting the tumor rather than inhibiting the size of solid cancer, combined treatment with Aldara cream In this case, it is very effective in suppressing tumor growth and decreasing parenchymal cell number in cancer tissues.

Dendritic cell therapeutic agent and Aldala according to the present invention through proliferation experiment of liver cancer antigen-specific T cells, CTL activity test, Th1 / Th2 cytokine analysis using spleen derived from each control and experimental group mouse subjects in the therapeutic immune model The therapeutic effect of the combination treatment group of the cream was further confirmed in relation to the cellular immune response associated with T cell activity. These experimental results were obtained by investigating the activity of spleen-derived T lymphocytes obtained at the expense of one mouse from each control group and one week after the last dendritic cell administration. Spleen T lymphocytes isolated from the dendritic cells administered mice according to the present invention was observed to proliferate at a higher rate than T lymphocytes derived from the mice received PBS (see Figure 8). In addition, it was confirmed that the Th1-directed cytotoxic effect on the target cells expressing liver cancer antigens in the mouse group co-administered with dendritic cell therapeutic agent + aldara cream (see FIGS. 9, 10A, and 10B). CTL induction of T lymphocytes isolated from Aldara cream-only mice was excluded due to lack of cell numbers. In addition, a single cell suspension was prepared by extracting adjacent lymph nodes of control and experimental groups, and when the proportion of immune cell groups was examined by flow cytometry, the combination group showed a slight increase in CD8 + T cells and significant T reg cells. A decrease was observed.

Experimental Example 3 Results: Induction of Immunity to Treat Metastatic Cancer

To confirm the therapeutic immunity against metastatic cancer in the liver cancer mouse model, cancer cell lines expressing liver cancer antigens are first injected by intravenous paths to induce cancer metastasis, and PBS administration to each mouse subject induced cancer metastasis, and the dendritic cell according to the present invention. Cell therapy alone administration, dendritic cell treatment according to the present invention and alda la cream combined administration and Alda la cream alone administration. Lung tissues were isolated 18-18 days after tumor cell administration and fixed with Bouin's fixative solution to observe lung metastases during cancer metastasis (FIGS. 6A and 6B, n = 8). MH134 Expressing Liver Cancer Specific Antigen The cancer metastasis model of tumor cells was characterized by a random range of metastatic nodules and a random range distribution (FIG. 6b). Therefore, rather than averaging the number of lung colonies generated in each individual, the range is randomly determined according to the distribution of lung colonies generated, and the number of colonies generated in the corresponding ranges is counted to prevent metastasis. Was determined (FIG. 6A). The colonies of MH134 expressing liver cancer specific antigens were smooth types, broad spots, and when the number of colonies reached 30 or more, they were fused to each other and difficult to count. In the case of treatment with dendritic cell treatment according to the present invention and in combination with aldara cream, both showed excellent inhibitory effect on lung metastasis, especially in the case of mouse group treated with dendritic cell treatment + aldaram cream according to the present invention. , 3 (37.5%) had no metastatic lesions, and the other 5 (62.5%) had no more than 5 colonies (see FIG. 6A).

Result of Experimental Example 5: Confirmation of Safety of Dendritic Cell Therapeutics

In the case of the single subcutaneous administration toxicity of mature dendritic cells according to the present invention, there were no deaths, abnormal symptoms and significant changes in body weight due to administration in the male and female test substance administration group. Necropsy showed no gross abnormalities in the male and female test groups. Therefore, histopathological examination was not performed on organ tissues. As a result, a single subcutaneous administration of the mature dendritic cells according to the present invention to Balb / C mice showed no mortality due to the test substance, and the approximate lethal dose was determined to be more than 2.5 X 10 ⁶ cells / head for each male and female. As a result of repeated subcutaneous dose toxicity, no change in test substance administration was found in general symptoms, body weight, feed intake, ophthalmological, urine, hematological, organ weight, and autopsy results. As a result of histopathological examination, cell infiltration at the administration site was observed in 5 X 10 ⁴ and 5 X 10 ⁵ cells / head in the test group, and it was determined that the test substance was caused by the test substance. However, only one case of the female 5 X 10 ⁵ cells / head group was observed in the recovery group. As a result of immunotoxicity test, it was judged that there was no effect of test substance in the male and female test substance administration group on lymphocyte activation and spontaneous killer cell activation in spleen. Therefore, the NOAEL of mouse creabox-etch seed is judged to be 5 X 10 ⁵ cells / head or more.

In general, in the clinical trials of liver cancer, dendritic cell dosages are at a level of 0.5-50 x 10 ⁶ cells / individual, and the maximum dosage generally used is 5 x 10 ⁷ cells / individual. Assuming a maximum dose of 5 X 10 ⁷ cells / individual and a 50 kg body weight of the ocular patient, the lethal dose of mature dendritic cells according to the present invention is at least 100 times the maximum expected clinical dose. It can be seen that the non-toxic amount is more than 20 times the maximum clinical trial scheduled dose, and thus the mature dendritic cells according to the present invention are very low in toxicity and are safe.

conclusion

This experiment demonstrated that mature dendritic cells sensitized with a peptide antigen YGRRARRRRRR fused to a solid cancer or cancer metastasis mouse model had very good tumor prevention and treatment effects and cancer metastasis suppression effects. It was demonstrated that the effects of tumor growth inhibition and cancer metastasis inhibition can be maximized.

A cancer cell line stably expressing human liver cancer antigens was constructed, and an animal model was selected to induce subcutaneous solid cancer and cancer metastasis through subcutaneous and tail vein injection by selecting a mouse system with the same histocompatibility, and the peptide YGRRARRRRRR When sensitized with hepatic cancer antigen protein fused with and combined with matured dendritic cell therapy and alda cream, it was found that the prevention and treatment of subcutaneous solid cancer was excellent and the number of lung metastases was excellently reduced. . In particular, the solid cancer prevention model showed not only tumor growth inhibition but also complete removal of the tumor, and as a result, it showed a remarkable effect on life extension. In other words, the combination therapy of dendritic cell therapy and imiquimod according to the present invention was able to induce not only an excellent tumor size reduction effect but also an ultimate life extension effect. More specifically, in the case of cancer treatment such as actual liver cancer, the initial progress after the first treatment by surgical and / or non-surgical treatment is good but the recurrence rate is very high, and after primary cancer treatment (such as removal of the tumor burden), results strongly suggest that the dendritic cell therapy administration and 2 recur in the primary cancer by fine tumor cells remaining at the time of co-administration of Al Dara cream according to the present invention, prevention or suppression (2 ^nd tumor prevention) effectively can function in to be.

In particular, in the cancer metastasis treatment model, it was confirmed that the treatment of mature dendritic cells according to the present invention effectively inhibited the induction of lung colony formation by tumor cells migrated into lung tissue after intravenous injection. This effect was also more pronounced when combined with imiquimod. As a result, the mature dendritic cell administration according to the present invention and the combination administration of alda cream are very effective in inhibiting metastasis of cancer.

The enhancement of the anticancer effect according to the present invention was further confirmed by liver cancer antigen specific T cell proliferation induction, CTL response test and Th1 / Th2 cytokine analysis in terms of T cell immunity.

In the subcutaneous solid cancer treatment model, as the tumor proliferation is aggressive, the inhibitory effect on the tumor size itself by the mature dendritic cell treatment according to the present invention was observed to be somewhat low. It was confirmed that the decrease. In conclusion, when the mature dendritic cells according to the present invention is treated to cancer patients after surgical or non-surgical treatment, anticancer effects can be obtained through anti-cancer immunity, which can suppress cancer metastasis and recurrence. The combination of Aldara cream with the administration of a cell therapy has experimentally demonstrated that dendritic cells can maximize the effect of immunotherapy against liver cancer.

In addition, the test of single subcutaneous administration toxicity, repeated subcutaneous administration toxicity and immunotoxicity of the mature dendritic cells according to the present invention was performed, it was confirmed that the safety is excellent in all items.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

references

Butterfield L.H. Recent advances in immunotherapy for hepatocellular carcinoma. SWISS MED WKLY. 2007 137: 83-90.

2.Sun K., Wang L., Zhang Y., Dendritic cells as therapeutic vaccines against tumors and its role in therapy for hepatocellular carcinoma. Cellular & Molecular immunity 2006; 3 (3): 197-203.

3. Pietesz GA et al. Generation of cellular immune responses to antigenic tumor peptides. Cell Mol Life Sci 2000; 57 (2): 290-310.

4. Morris MC et al. A peptide carrier for the delivery of biologically active proteins into mammalian cells, Nat. Biotechnol. 2001; 19: 1173-1176.

5. Kim D et al. Cytoplasmic transduction peptide (CTP): New approach for the delivery of biomolecules into cytoplasm in vitro and in vivo. Exp. Cell Res. 2006; 312: 1277-1288.

6. Hemmi, H., et al. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat Immunol .. 2002 3 (2): 196-200.

Sauder, D.N., Imiquimod: modes of action. British Journal of Dermatology. 2003 149 (Suppl. 66): 5-8.

8. Miller, R. L., et al. Imiquimod applied topically: a novel immune response modifier and a new class of drug. Int J Immunopharmacol. 1999 Jan; 21 (1): 1-14.

9.Zagon IS, Donahue RN, Rogosnitzky M, McLaughlin PJ (August 2008). "Imiquimod upregulates the opioid growth factor receptor to inhibit cell proliferation independent of immune function". Exp. Biol. Med. (Maywood) 233 (8): 968-79.

1 is a result of confirming by Western blot after SDS-PAGE analysis after purification of CTP-fused hepatocellular specific antigens (YGRRARRRRRR-AFP, YGRRARRRRRR-GPC 3, YGRRARRRRRR-MAGE 1) used for dendritic cell sensitization .

Figure 2a shows the effect of inhibiting solid cancer formation by combined administration of mature dendritic cells and alda cream according to the present invention in a mouse liver cancer defense model. In the defense model, mice treated with PBS (n = 8, PBS), mice treated with dendritic cell treatment (n = 8, Ag-DC), mice treated with Aldara cream only (n = 8, PBS + Aldara), a group of mice treated with dendritic cell therapy and Aldara cream (n = 8, Ag-DC + Aldara) were administered subcutaneously with an appropriate amount (1.5 X 10 ⁵ cells / head) of liver cancer antigen expressing cell line. Tumor growth was observed every 2-3 days. Figure 2b is a photograph showing the size of the tumor in each mouse of the experimental group and the control group in the mouse liver cancer defense model. Figure 2c shows the results of tumor formation inhibition in each of the experimental and control mice in the mouse liver cancer defense model. NR: control group (PBS treatment group) 70% or more of the mean tumor size, PR: control group less than 70% of the mean size, CR: no tumor at all. Figure 2d is a result of measuring the life of each mouse in the experimental group and the control group in the mouse liver cancer defense model.

3 is a result of MTT analysis of the proliferation analysis of liver cancer-specific T cells with respect to T cells isolated from splenocytes of each mouse in the control and test groups of the mouse liver cancer defense model. PBS: control group of PBS-treated mice, Ag-DC: group of mice treated with dendritic cell therapy, Ag-DC + aldara: group of mice treated with dendritic cell therapy and Aldara cream, PBS + Aldara: mice treated with Aldara cream only It's a military.

4A and 4B show IFN-γ and IL-4 from T cells after incubating T cells isolated from splenocytes of splenocytes of control and test mice of the mouse liver cancer defense model with liver cancer specific antigen-presenting cells. This is the result of measuring the amount of secretion. PBS: control group of PBS-treated mice, Ag-DC: group of mice treated with dendritic cell therapy, Ag-DC + aldara: group of mice treated with dendritic cell therapy and Aldara cream, PBS + Aldara: mice treated with Aldara cream only It's a military.

5 shows the results of measuring CTL activity in each individual mouse of the control and test groups of the mouse liver cancer defense model. PBS: control group of PBS-treated mice, Ag-DC: group of mice treated with dendritic cell therapy, Ag-DC + aldara: group of mice treated with dendritic cell therapy and Aldara cream, PBS + Aldara: mice treated with Aldara cream only group.

Figures 6a and 6b shows the effect of inhibiting lung metastasis by the combination of dendritic cells and aldara cream in each individual mouse in the control and test groups of the mouse lung metastasis treatment model. PBS: control group of PBS-treated mice, Ag-DC: group of mice treated with dendritic cell therapy, Ag-DC + aldara: group of mice treated with dendritic cell therapy and Aldara cream, PBS + Aldara: mice treated with Aldara cream only group.

Figure 7a is a result of histologically observed cancer cell density in the cancer tissue of each individual mouse in the control and test groups of the mouse liver cancer treatment model. PBS: PBS-treated mouse control group, Ag-DC: group of mice treated with dendritic cell therapy, Ag-DC + aldara: group of mice treated with dendritic cell therapy and Aldara cream, PBS + Aldara: mice treated with Aldara cream only group. Figure 7b is a result confirmed by the tumor size measurement in the control and test group mice of the mouse liver cancer treatment model of the combination treatment of dendritic cells and Aldara cream. PBS: control group of PBS-treated mice, Ag-DC: group of mice treated with dendritic cell therapy, Ag-DC + aldara: group of mice treated with dendritic cell therapy and Aldara cream, PBS + Aldara: mice treated with Aldara cream only group.

FIG. 8 shows the results of a T cell proliferation assay through MTT analysis on T cells isolated from splenocytes obtained from mice of control and test groups of mouse treatment models. PBS: control group of PBS-treated mice, Ag-DC: group of mice treated with dendritic cell therapy, Ag-DC + aldara: group of mice treated with dendritic cell therapy and Aldara cream, PBS + Aldara: mice treated with Aldara cream only group.

9 shows the results of measuring the CTL activity of splenic T lymphocytes obtained from each individual mouse in the control and test groups of the mouse treatment model. PBS: control group of PBS-treated mice, Ag-DC: group of mice treated with dendritic cell therapy, Ag-DC + aldara: group of mice treated with dendritic cell therapy and Aldara cream, PBS + Aldara: mice treated with Aldara cream only group.

10A and 10B show increased secretion of IFN-γ and IL-4 due to Th1-driven immune responses of T lymphocytes from each individual mouse in the control and test groups of the mouse treatment model. PBS: control group of PBS-treated mice, Ag-DC: group of mice treated with dendritic cell therapy, Ag-DC + aldara: group of mice treated with dendritic cell therapy and Aldara cream, PBS + Aldara: mice treated with Aldara cream only group.

<110> CHOONGWAE PHARM. Co. <120> Method for Preparing Dendritic Cells Having Potential to Induce          Cancer Specific Immune Response, Pharmaceutical Composition and          Kit for Treating or Preventing Cancer or Inhibiting Cancer          Metastasis Comprising the Same <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 327 <212> PRT <213> Homo sapiens <400> 1 Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu Asp Ser Tyr   1 5 10 15 Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr Ile Phe Phe              20 25 30 Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser Lys Met Val          35 40 45 Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp Glu Gln Ser      50 55 60 Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu Glu Leu Cys  65 70 75 80 His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser Asp Cys Cys Ser                  85 90 95 Gln Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His Lys Lys Pro             100 105 110 Thr Pro Ala Ser Ile Pro Leu Phe Gln Val Pro Glu Pro Val Thr Ser         115 120 125 Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn Lys Phe Ile     130 135 140 Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro Thr Ile Leu 145 150 155 160 Leu Trp Ala Ala Arg Tyr Asp Lys Ile Ile Pro Ser Cys Cys Lys Ala                 165 170 175 Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr Val Thr Lys             180 185 190 Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys Ala Val Met         195 200 205 Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile Thr Val Thr Lys Leu     210 215 220 Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Glu Ile Gln Lys Leu Val 225 230 235 240 Leu Asp Val Ala His Val His Glu His Cys Cys Arg Gly Asp Val Leu                 245 250 255 Asp Cys Leu Gln Asp Gly Glu Lys Ile Met Ser Tyr Ile Cys Ser Gln             260 265 270 Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys Leu Thr Thr         275 280 285 Leu Glu Arg Gly Gln Cys Ile His Ala Glu Asn Asp Glu Lys Pro     290 295 300 Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp Arg Asp Phe 305 310 315 320 Asn Gln Phe Ser Ser Gly Glu                 325 <210> 2 <211> 326 <212> PRT <213> Homo sapiens <400> 2 Ala Thr Cys His Gln Val Arg Ser Phe Phe Gln Arg Leu Gln Pro Gly   1 5 10 15 Leu Lys Trp Val Pro Glu Thr Pro Val Pro Gly Ser Asp Leu Gln Val              20 25 30 Cys Leu Pro Lys Gly Pro Thr Cys Cys Ser Arg Lys Met Glu Glu Lys          35 40 45 Tyr Gln Leu Thr Ala Arg Leu Asn Met Glu Gln Leu Leu Gln Ser Ala      50 55 60 Ser Met Glu Leu Lys Phe Leu Ile Ile Gln Asn Ala Ala Val Phe Gln  65 70 75 80 Glu Ala Phe Glu Ile Val Val Arg His Ala Lys Asn Tyr Thr Asn Ala                  85 90 95 Met Phe Lys Asn Asn Tyr Pro Ser Leu Thr Pro Gln Ala Phe Glu Phe             100 105 110 Val Gly Glu Phe Phe Thr Asp Val Ser Leu Tyr Ile Leu Gly Ser Asp         115 120 125 Ile Asn Val Asp Asp Met Val Asn Glu Leu Phe Asp Ser Leu Phe Pro     130 135 140 Val Ile Tyr Thr Gln Leu Met Asn Pro Gly Leu Pro Asp Ser Ala Leu 145 150 155 160 Asp Ile Asn Glu Cys Leu Arg Gly Ala Arg Arg Asp Leu Lys Val Phe                 165 170 175 Gly Asn Phe Pro Lys Leu Ile Met Thr Gln Val Ser Lys Ser Leu Gln             180 185 190 Val Thr Arg Ile Phe Leu Gln Ala Leu Asn Leu Gly Ile Glu Val Ile         195 200 205 Asn Thr Thr Asp His Leu Lys Phe Ser Lys Asp Cys Gly Arg Met Leu     210 215 220 Thr Arg Met Trp Tyr Cys Ser Tyr Cys Gln Gly Leu Met Met Val Lys 225 230 235 240 Pro Cys Gly Gly Tyr Cys Asn Val Val Met Gln Gly Cys Met Ala Gly                 245 250 255 Val Val Glu Ile Asp Lys Tyr Trp Arg Glu Tyr Ile Leu Ser Leu Glu             260 265 270 Glu Leu Val Asn Gly Met Tyr Arg Ile Tyr Asp Met Glu Asn Val Leu         275 280 285 Leu Gly Leu Phe Ser Thr Ile His Asp Ser Ile Gln Tyr Val Gln Lys     290 295 300 Asn Ala Gly Lys Leu Thr Thr Thr Ile Gly Lys Leu Cys Ala His Ser 305 310 315 320 Gln Gln Arg Gln Tyr Arg                 325 <210> 3 <211> 309 <212> PRT <213> Homo sapiens <400> 3 Met Ser Leu Glu Gln Arg Ser Leu His Cys Lys Pro Glu Glu Ala Leu   1 5 10 15 Glu Ala Gln Gln Glu Ala Leu Gly Leu Val Cys Val Gln Ala Ala Ala              20 25 30 Ser Ser Ser Ser Pro Leu Val Leu Gly Thr Leu Glu Glu Val Pro Thr          35 40 45 Ala Gly Ser Thr Asp Pro Pro Gln Ser Pro Gln Gly Ala Ser Ala Phe      50 55 60 Pro Thr Thr Ile Asn Phe Thr Arg Gln Arg Gln Pro Ser Glu Gly Ser  65 70 75 80 Ser Ser Arg Glu Glu Glu Gly Pro Ser Thr Ser Cys Ile Leu Glu Ser                  85 90 95 Leu Phe Arg Ala Val Ile Thr Lys Lys Val Ala Asp Leu Val Gly Phe             100 105 110 Leu Leu Leu Lys Tyr Arg Ala Arg Glu Pro Val Thr Lys Ala Glu Met         115 120 125 Leu Glu Ser Val Ile Lys Asn Tyr Lys His Cys Phe Pro Glu Ile Phe     130 135 140 Gly Lys Ala Ser Glu Ser Leu Gln Leu Val Phe Gly Ile Asp Val Lys 145 150 155 160 Glu Ala Asp Pro Thr Gly His Ser Tyr Val Leu Val Thr Cys Leu Gly                 165 170 175 Leu Ser Tyr Asp Gly Leu Leu Gly Asp Asn Gln Ile Met Pro Lys Thr             180 185 190 Gly Phe Leu Ile Ile Val Leu Val Met Ile Ala Met Glu Gly Gly His         195 200 205 Ala Pro Glu Glu Glu Ile Trp Glu Glu Leu Ser Val Met Glu Val Tyr     210 215 220 Asp Gly Arg Glu His Ser Ala Tyr Gly Glu Pro Arg Lys Leu Leu Thr 225 230 235 240 Gln Asp Leu Val Gln Glu Lys Tyr Leu Glu Tyr Arg Gln Val Pro Asp                 245 250 255 Ser Asp Pro Ala Arg Tyr Glu Phe Leu Trp Gly Pro Arg Ala Leu Ala             260 265 270 Glu Thr Ser Tyr Val Lys Val Leu Glu Tyr Val Ile Lys Val Ser Ala         275 280 285 Arg Val Arg Phe Phe Phe Pro Ser Leu Arg Glu Ala Ala Leu Arg Glu     290 295 300 Glu Glu Glu Gly Val 305  

Claims (16)

A method of producing mature dendritic cells having the ability to induce cancer-specific immune responses comprising the following steps: (a) sensitizing immature dendritic cells to cancer specific antigen proteins fused with peptide YGRRARRRRRR to mature; And (b) isolating the matured dendritic cells of step (a). According to claim 1, wherein step (a) is a maturation-inducing cytokine of dendritic cells, Tumor necrosis factor (TNF) -α, IFN (Interferon) -γ, IL (Interleukin) -1β, IL-6, PGE2 (Prostaglandin E2) or a mixture thereof. The method of claim 1, wherein the cancer specific antigen of step (a) is liver cancer specific antigen. According to claim 3, The liver cancer specific antigen is AFP (Alpha-Fetoprotein), GPC 3 (Glypican-3), MAGE 1 (Melanoma Associated Gene Family A1), NY-ESO-1 (cancer / testis antigen), Aurora- A (serine / threonine protein kinase involved in centrosome formation during mitosis), or Gankyrin (liver-specific oncoprotein involved in cell cycle regulation). The method of claim 4, wherein the liver cancer specific antigen is AFP (Alpha-Fetoprotein), GPC 3 (Glypican-3), or MAGE 1 (Melanoma Associated Gene Family A1). The method according to claim 5, wherein the liver cancer specific antigen is 20-346 sequences in the AFP amino acid sequence of SEQ ID NO: 1, 33-358 sequences of the GPC 3 amino acid sequence of SEQ ID NO: 2, or MAGE1 of SEQ ID NO: 3 And an amino acid sequence. (a) a pharmaceutically effective amount of a mature dendritic cell having the ability to induce cancer specific immune responses prepared by the method of any one of claims 1 to 6; And (b) a pharmaceutical composition for the treatment or prevention of cancer comprising a pharmaceutically acceptable carrier. 8. The composition of claim 7, wherein the cancer is liver cancer. (a) a pharmaceutically effective amount of a mature dendritic cell having the ability to induce cancer specific immune responses prepared by the method of any one of claims 1 to 6; And (b) a pharmaceutical composition for inhibiting cancer metastasis comprising a pharmaceutically acceptable carrier. 10. The composition of claim 9, wherein the cancer metastasis is metastasis of liver cancer. The composition of claim 10, wherein the metastasis of liver cancer is a lung metastasis of liver cancer. (a) a pharmaceutical composition comprising mature dendritic cells having the ability to induce cancer-specific immune responses prepared by the method of any one of claims 1 to 6; And (b) a pharmaceutical kit for the treatment or prevention of cancer comprising a composition containing a compound represented by the formula (1). [Formula 1]

13. The pharmaceutical kit of claim 12, wherein the cancer is liver cancer. (A) a pharmaceutical composition comprising mature dendritic cells having the ability to induce cancer-specific immune response prepared by the method of any one of claims 1 to 6; And (b) a pharmaceutical kit for inhibiting cancer metastasis comprising a composition containing a compound represented by the following formula (1). [Formula 1]

The pharmaceutical kit of claim 14, wherein the cancer metastasis is metastasis of liver cancer. The pharmaceutical kit of claim 15, wherein the metastasis of liver cancer is a lung metastasis of liver cancer.

Images