WO2021085853A1 - Composition for enhancing immune activity and method therefor - Google Patents
Composition for enhancing immune activity and method therefor Download PDFInfo
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- WO2021085853A1 WO2021085853A1 PCT/KR2020/012530 KR2020012530W WO2021085853A1 WO 2021085853 A1 WO2021085853 A1 WO 2021085853A1 KR 2020012530 W KR2020012530 W KR 2020012530W WO 2021085853 A1 WO2021085853 A1 WO 2021085853A1
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- cells
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Definitions
- the present invention relates to a substance for activating immune cells and a method for activating immune cells using the same. More specifically, it relates to a composition capable of enhancing the immune activity of the immune cells by culturing them together with the immune cells when culturing the immune cells.
- Pancreatic cancer is one of the common primary cancers, and less than 5% of all patients are malignant tumors with an average survival rate of 5 years after diagnosis. Compared to other carcinomas, it is known to be the only carcinoma that has not improved its average survival rate over several decades.
- a method known as an effective treatment for pancreatic cancer is surgical removal. Since the tumor mass of pancreatic cancer is mainly composed of fibrous tissue and only a part of cancer cells, it is difficult to evaluate the treatment response to cancer after chemotherapy, and pancreatic cancer is known as a cancer that does not work well with chemotherapy. Possible surgical resection is recommended rather than chemotherapy.
- An anticancer drug used clinically is gemcitabine (Eli Lilly and Company), but there are ongoing questions about the effectiveness of the treatment. However, there are no alternative substances and treatment methods available. Therefore, there is a demand for the development of an anticancer drug that can effectively treat pancreatic cancer and has a low side effect on use, or an adjuvant that can enhance the effect of existing anticancer drugs.
- Natural killer cells a cell commonly used as one of the active ingredients of cell therapy products, are activated by several cytokines to amplify and secrete cytolytic functions.
- the activated natural killer cells are interferon- It secretes gamma, tumor-necrosis factor (TNF), and autologous granulocyte/macrophage colony-stimulation factor (GM-CSF) together with chemokine to induce an inflammatory response. . It also affects adaptive immunity by inducing the differentiation and growth of monocytes, dendritic cells, and granulocytes (Prospects for the use of NK cells in immunotherapy of human cancer, NATURE REVIEWS, 2007).
- polysaccharides derived from plants in addition to proteins such as interferon gamma have various macrophage functions, i.e., cytotoxic activity against tumor cells and microorganisms, phagocytic activity alpha-zoyonecrosis factor, and specific cytokines such as interleukin. It activates the secretion of kine and promotes cell differentiation and proliferation.
- IFN- ⁇ interferon gamma
- An object of the present invention is to provide a method of manufacturing a cell therapy product capable of enhancing the immune activity of immune cells, which is an active ingredient of a cell therapy product.
- Another object of the present invention is to provide a composition for enhancing the therapeutic effect of a cell therapy agent for treatment or prevention of cancer.
- the compound represented by Formula 1 corresponds to 5-nitro-2-furaldehyde p-hydroxybenzoylhydrazone, and'nifuroxazide (for example, it may also be expressed as AMBATROLTM, ANTINALTM, BACIFURANETM, DIAFURYLTM)'.
- the compound represented by Formula 2 is 3-(2,4-dichloro-phenoxymethyl)-5-trichloromethyl-[1,2,4]oxadiazole (3-(2,4-dichloro Corresponds to -phenoxymethyl)-5-trichloromethyl-[1,2,4]oxadiazole).
- the culture medium may contain melatonin.
- melatonin is known to affect the circadian rhythm of physiological functions such as sleep timing, blood pressure control, and seasonal reproduction, among substances found in mammals, plants, bacteria, fungi, etc. Is one.
- Melatonin may be used as'melatonin alone or in combination with at least one selected from ginsenoside and ⁇ -glucan (hereinafter, MTAM1).
- MTAM1 can be used to cultivate lymphocytes, increases the proliferation rate of natural killer cells in immune cells obtained after cultivation, and further enhances the killing ability of the natural killer cells to cancer cells, so that cancer can be more effectively prevented or treated. .
- the lymphocytes When culturing lymphocytes in an environment in which MTAM1 is present, the lymphocytes may be first cultured with CD3.
- the MTAM1 may further include CD56, IL-18, or a combination thereof.
- the cultivation of MTAM1 with an additional immune activating factor, CD3, CD56, IL-18, or a combination thereof, can be performed as needed, simultaneously, sequentially or crossover.
- the "cellular therapeutic agent” refers to a drug (US FDA regulation) used for treatment, diagnosis, and prevention of cells and tissues manufactured through separation, cultivation and special manipulation from an individual. Or drugs used for treatment, diagnosis, and prevention through a series of actions such as proliferating and selecting live autologous, allogeneic, or xenogeneic cells in vitro to restore the function of tissues, or by changing the biological characteristics of cells in other ways. Means.
- the cell therapy agent may contain immune cells as an active ingredient.
- prevention refers to any action that inhibits or delays progression of cancer by administration of the composition of the present invention.
- treatment and “improvement” mean all actions in which symptoms of cancer are improved or beneficially changed by administration of the composition of the present invention.
- the "immune cells” may refer to all cells that are present in the body or have immune activity that can be separated from the body.
- the immune cells may be, for example, natural killer cells, T cells, B cells, dendritic cells, macrophages, or a combination thereof.
- the immune cells may be lymphocytes.
- the immune cells may be natural killer cells (NK cells).
- the compound of Formula 1 or 2 may contact isolated natural killer cells to enhance their immune activity.
- the immune cells obtained from the human body are a mixture of various types of immune cells. Therefore, it is common to induce differentiation into specific immune cells during the culture process or to activate only their proliferation.
- One embodiment of the present invention can successfully enhance its immune activity even when applied to isolated natural killer cells, as demonstrated in the following examples. This effect suggests that the expected immune activity can be obtained even in the final product of a cell therapy product containing pure natural killer cells as an active ingredient.
- the “culture” refers to maintaining or preserving cells in a viable state, and may be meant to include exposing or contacting immune cells to a specific environment.
- the compound represented by Formula 1 or 2, or a combination thereof may enhance the immune activity of immune cells by mixing and culturing them with immune cells.
- the immune activity may mean, for example, cytotoxicity against cancer cells or infected cells. Ultimately, this can further enhance the cancer treatment or prevention effect of a cell therapy product containing immune cells as an active ingredient.
- the cancer is, for example, pancreatic cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, colon cancer, cervical cancer, thyroid cancer, laryngeal cancer, leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer. , It may be selected from the group consisting of lymphoma, preferably pancreatic cancer.
- Separating the immune cells from the culture may mean substantially removing components remaining in the culture medium and the culture medium in addition to the immune cells.
- the separation method may use various methods such as membrane filtration, dialysis, precipitation method, cell adhesion and washing, etc., but is not limited thereto.
- the step of isolating immune cells may be employed, but the compound represented by Formula 1 or 2, melatonin, or a combination thereof is only used for immune activity of immune cells. Since it is used in a small amount to enhance immunity, the effect on the individual may be very insignificant even if it remains with the immune cells.
- the method of manufacturing the cell therapy agent may further include formulating the isolated immune cells as a cell therapy agent.
- the formulation includes all actions of processing a cell therapy product into a form suitable for administration to an individual in need thereof, and may mean, for example, mixing the isolated immune cells with a pharmaceutically acceptable carrier.
- Pharmaceutically acceptable carriers saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and one or more of these components may be mixed and used.
- Other conventional additives such as an agent, a buffer solution, and a bacteriostatic agent, may be added.
- diluents such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets.
- a target organ-specific antibody or other ligand may be used in combination with the carrier.
- it can be preferably formulated according to each disease or component by an appropriate method in the art or by using a method disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA. have.
- the cell therapy agent may be a solution, a suspension, a dispersion, an emulsion, a gel, an injectable solution, a sustained-release preparation of the active compound, and the like, preferably an injection.
- a buffer solution such as an acid aqueous solution or phosphate that can be used as an injection in order to ensure product stability according to the distribution of the injection formulation in the formulation step. It can be prepared as a very stable injection.
- composition for enhancing the therapeutic effect of a cell therapy agent comprising the compound represented by Formula 1 or 2, melatonin, or a combination thereof.
- composition for enhancing the therapeutic effect of the cell therapy agent may enhance the immune activity of immune cells, which is an active ingredient of the cell therapy agent, by contacting the cell therapy agent, and as a result, enhance the cancer treatment or prevention effect of the cell therapy agent.
- composition for enhancing the therapeutic effect of the cell therapy agent may be used as an additive to a culture medium for culturing immune cells to contact immune cells.
- the composition for enhancing the therapeutic effect of the cell therapy agent may be first mixed with the cell therapy agent before use in an individual in need of the cell therapy agent so that the cell therapy agent can exhibit rapid and efficient immune activity in an individual.
- the compound represented by Formula 1 or 2 may be included in 1 ⁇ M to 100 ⁇ M, 1 ⁇ M to 80 ⁇ M, 1 ⁇ M to 60 ⁇ M, or 1 ⁇ M to 40 ⁇ M.
- the composition for enhancing the therapeutic effect of the cell therapy agent may be removed from the mixture with the cell therapy agent before being administered to the subject to block unnecessary biological reactions in the subject.
- the compound represented by Formula 1 or 2 is used in a small amount enough to enhance the immune activity of immune cells, even if administered as a mixture or remains, the effect on the individual is insignificant.
- a first unit comprising a compound represented by Formula 1 or 2, melatonin, or a combination thereof; And it provides a kit comprising a second unit comprising a cell therapy product comprising immune cells as an active ingredient.
- the "unit” is merely a means for distinguishing the composition of the kit, and may exist in the form of a solid, powder, solution, suspension, gel, etc., and may mean that it is included in an appropriate container that can be separated from each other .
- the first unit and the second unit are separated in the kit and mixed with each other before use in an individual, thereby enhancing the immune activity of the immune cells of the second unit, thereby enhancing the therapeutic effect of the cell therapy product.
- the kit may further include a component capable of separating immune cells from a mixture of the first unit and the second unit.
- the configuration for separating the immune cells may be for substantially removing Formula 1 or 2, melatonin, or a mixture thereof from the mixture of the first unit and the second unit.
- the components of the first unit are only small enough to enhance the immune activity of immune cells, even if administered as a mixture or remain, the effect on the individual is insignificant.
- the description of the method of manufacturing the cell therapy agent may be interpreted in the same meaning as the description of the same configuration for the composition or kit for enhancing the therapeutic effect of the cell therapy agent.
- the method for producing a cell therapy product using the compound represented by Formula 1 or 2, or a combination thereof provided in the present invention, and a composition therefor remarkably enhance the cell killing ability of immune cells against cancer cells or infected cells, thereby effectively enhancing immune cells. It is possible to remarkably improve the efficacy of cancer treatment or prevention of the cell therapy product included as an ingredient.
- FIG. 3 to 6 are graphs showing cell surface antigens for each culture condition for test group 1, test group 2, and control group in Example 2, respectively, and FIG. 6 is a ratio of NK cells and NKT cells in cells harvested from each And the percentage of T cells.
- Example 7 shows cell growth curves for test group 1, test group 2, and control group in Example 2.
- Example 9 is a graph showing the results of a cell viability test for an immune cell activating substance according to the present invention in Example 2.
- ROS reactive oxygen species
- FIG. 12 is a graph showing the cell surface antigens of immune cells cultured and harvested after treatment with Test Group 1 on lymphocytes collected and isolated from blood in Example 2.
- FIG. 12 is a graph showing the cell surface antigens of immune cells cultured and harvested after treatment with Test Group 1 on lymphocytes collected and isolated from blood in Example 2.
- the present invention comprises the steps of culturing immune cells with a compound represented by Formula 1 or 2, melatonin, or a combination thereof; And it relates to a method for producing a cell therapy product comprising the immune cells as an active ingredient, comprising the step of isolating immune cells from the culture:
- Example 1 Confirmation of the ability to enhance immune activity of immune cells of Formulas 1 and 2
- Panc-1 a pancreatic cancer cell line
- DMEM Dulbecco's modified Eagle's medium
- NK-92 cells a natural killer cell line, were purchased from ATCC (Manassas, VA) and contain 20% FBS, 1% penicillin streptomycin, and 10 ng/ml recombinant human IL-2.
- ATCC Manassas, VA
- the NK-92 cells of Example 1-1 were each of Formulas 1 and 2 at a concentration of 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 20 ⁇ M, and 40 ⁇ M. Treated and incubated for 4 hours. Then, D-PlusTM cell viability assay kit (CCK-3000, Donginbiotech Co., Korea) was treated with 0.1X in a well-known manner, and absorbance at 450 nm was measured.
- D-PlusTM cell viability assay kit (CCK-3000, Donginbiotech Co., Korea) was treated with 0.1X in a well-known manner, and absorbance at 450 nm was measured.
- the survival rate was observed to be about 85% at all the tested concentrations, it was confirmed that there is no direct toxicity to NK-92 cells (Fig. 1).
- LDH cytotoxicity detection kit Takara, Japan
- NK-92 cells cultured with Formulas 1 and 2 had remarkably superior killing ability compared to the control group for Panc-1 cells (FIG. 2).
- Example 2 Confirmation of the ability of melatonin to increase immune activity of immune cells
- Lymphocytes were isolated from the patient's blood, suspended in a culture solution, and placed in a T-25 flask in which CD3 was solidified. Then, D56 5 ⁇ m and 10% of autologous plasma were added to the T-25 Flask and then cultured in a 37°C, 5% CO 2 incubator for 1-2 days. After being transferred to a T-75 Flask, it was incubated for 3-4 days in an incubator at 37°C and 5% CO 2.
- test groups 1 and 2 were treated with 10 ⁇ l or 20 ⁇ l of MTAM1, respectively, in a T-175 flask, and 5% of autologous plasma was added. In addition, 5% of autologous plasma was added to the control group for comparison, and then incubated in a CO 2 incubator for 48 hours.
- test groups 1 and 2 treatment with IL-18 and MTAM1 in the cell suspension cultivated in a T-175 flask, and after observing the activated state of the cells, the T-150 flask was incubated with a gas permeability of 1000 ml. After transferring to the culture medium containing the bag, it was cultured for 7 to 9 days to proliferate a large amount of cells.
- the T-150 Flask was transferred to a culture medium containing a 1000 ml of gas-permeable culture bag, and then cultured for 7 to 9 days to proliferate a large amount of cells.
- test groups 1 and 2 and the control group For each of the test groups 1 and 2 and the control group, cells containing a 1000 ml of CO 2 gas permeable culture bag and the culture solution were divided into 500 ml centrifuge tubes, and centrifuged for 17 minutes to harvest the cells. After the supernatant was carefully discarded so that the cells did not fall, the cells were washed with 250 ml of sterile physiological saline. The cells were harvested by rebalanced in the centrifuge, placed in a centrifuge tube, and centrifuged for 15 minutes. After carefully discarding the supernatant so that the cells do not fall off, 30 ml of a 100 ml sterile physiological saline bag for injection was collected using a syringe. The cells were suspended in 30 ml sterile physiological saline for injection. The suspended cells were poured into a cell strainer and filtered. The filtered cell suspension was placed in a sterile physiological saline bag for injection and suspended.
- FIGS. 3 to 6 are graphs of cell surface antigen tests for each culture condition for test group 1, test group 2, and control group, respectively, and FIG. 6 shows the ratio of NK cells and NKT cells, and T cell ratios for each. .
- Test group 1 Test group 2 Cell count Survival rate Cell count Survival rate Cell count Survival rate 2.26X10 9 83% 2.96X10 9 97% 3.86X10 9 95%
- Test group 2 Sample name IMBIO_NK+T cell_01 IMBIO_NK+T cell_04 IMBIO_NK+T cell_04 Cytotoxic activity (%) 31 68 59
- MTAM1 contains at least one of plant-derived melatonin, ginsenoside and ⁇ -glucan, preferably includes all of melatonin, ginsenoside and ⁇ -glucan.
- NK cells are regulatory lymphocytes of the innate immune system that control different types of tumors by limiting growth and proliferation, exert direct cellular cytotoxicity without other stimuli, and produce IFN- ⁇ and hypoimmune-promoting cytokines. It can be secreted to control both local tumor growth and metastasis.
- NK cells activated by IL-2 are similar to CTL, and after successively killing target cells by perforin and granzyme B, they survive. At this time, IL-2 Has the ability to accumulate granules and cytotoxicity of depleted NK cells.
- NK cells have antibody dependent cellular cytotoxicity (ADCC).
- ADCC antibody dependent cellular cytotoxicity
- Fc ⁇ RIII CD16
- Fc ⁇ RIII CD16
- the activity of the Fc receptor induces the activity of NK cells to induce the secretion of cytolytic cytokines, and the ADCC action of these NK cells is a characteristic that only NK cells different from CTL have.
- Melatonin has the effect of increasing the function of lymphocytes by increasing the production of glutathione by lymphocytes.
- IFN- ⁇ and cytokines such as Interleukin-1, 2, 6, 12 (Interleukin-1, 2, 6, 12) through the melatonin receptor on the surface of monocytes, it induces the activity of NK cells.
- MTAM1 according to the present invention increases the killing ability of NK cells against virus-infected cells and cancer cells, and increases cell viability by inhibiting free radicals in culture.
- Ginsenoside activates C-SRC kinase and increases intracellular calcium ion concentration by 2 to 3 times.
- the activation of the C-Src kinase-related pathway, which is an early stage of the intracellular signaling system, and the increase of intracellular calcium ions increase the expression of specific genes and promote cell proliferation.
- ginsenoside-Rp1 induces the production of immune cytokines at a specific concentration in dendritic cells and increases the activity of NK cells at low concentrations.
- beta-glucan increases the expression of TNF- ⁇ and IFN- ⁇ , and exhibits an anti-apoptosis effect in neutrophils.
- Picarine a glucan isolated from Laminaria digitata, increases phagocytic activity, increases the production and secretion of IL-1, IL-6, and TNF- ⁇ , as well as picarine is complementary. Increases cancer cell death by NK cells through the third receptor (CR3).
- FIG. 9 is a graph showing the results of a cell viability test for MTAM1, showing that MTAM1 according to the present invention has no intracellular toxicity, and further shows that it has an effect on proliferation.
- FIGS. 10 and 11 are graphs showing the measurement results of Reactive Oxygen Species (ROS), and it was confirmed through FIG. 10 that ROS was artificially increased in NK cells to decrease ROS after MTAM1 treatment, and FIG. 11 As a result of measuring ROS after culturing NK cells in a medium containing MTAM1 for 14 days, it was confirmed that melatonin blocks free radicals.
- ROS Reactive Oxygen Species
- H 2 0 2 represents a stimulator that increases active oxygen
- NAC represents a ROS scavenger.
- the present invention relates to a substance for activating immune cells and a method for activating immune cells using the same. More specifically, it relates to a composition capable of enhancing the immune activity of the immune cells by culturing them together with the immune cells when culturing the immune cells.
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Abstract
The present invention relates to a substance for activating immune cells by being added to a culture medium when the immune cells are cultured, and a method for activating immune cells using same, in which the ability of immune cells to kill cancer cells or infectious cells is significantly enhanced, and thus, the cancer-treating or preventing efficacy of a cell therapeutic agent comprising immune cells as an active ingredient can be remarkably enhanced.
Description
본 발명은 면역세포 활성화 물질 및 이를 이용한 면역세포의 활성화 방법에 관한 것이다. 보다 구체적으로는, 면역세포의 배양 시 상기 면역세포와 함께 배양함으로써 상기 면역세포의 면역 활성을 증진시킬 수 있는 조성물에 관한 것이다.The present invention relates to a substance for activating immune cells and a method for activating immune cells using the same. More specifically, it relates to a composition capable of enhancing the immune activity of the immune cells by culturing them together with the immune cells when culturing the immune cells.
췌장암(pancreatic cancer)은 흔한 원발성 암 중 한 종으로, 전체 환자의 5% 미만만이 진단 후 평균 5년의 생존율을 보이는 악성종양이다. 다른 암종들과 비교 시 수 십년 동안 그 평균 생존율이 향상되지 않은 유일한 암 종으로 알려져 있다. 현재 췌장암에 대한 효과적인 치료 방법으로 알려져 있는 방법은 수술을 통한 제거이다. 췌장암의 암 종괴 조직이 주로 섬유조직으로 이루어져 있고 암세포는 일부에 불과하여 항암 치료 후 암에 대한 치료 반응을 평가하는 데 어려움이 있는데다가, 췌장암은 비교적 항암 치료가 잘 듣지 않는 암이라고 알려져 있기 때문에 항암 화학요법 보다는 가능한 외과적인 절제가 권장된다. 임상적으로 사용되고 있는 항암제는 젬시타빈(Gemcitabine) (Eli Lilly and Company)이 있으나 치료 효과에 대한 지속적인 의문이 제기 되고 있는 실정이다. 하지만 이를 대체할 만한 물질 및 치료 방법이 전무한 상태다. 따라서 현재 췌장암을 효과적으로 치료할 수 있으면서 사용에 부작용이 적은 항암제 혹은 기존의 항암제의 효과를 증진시킬 수 있는 보조제의 개발이 요구되고 있는 실정이다.Pancreatic cancer is one of the common primary cancers, and less than 5% of all patients are malignant tumors with an average survival rate of 5 years after diagnosis. Compared to other carcinomas, it is known to be the only carcinoma that has not improved its average survival rate over several decades. Currently, a method known as an effective treatment for pancreatic cancer is surgical removal. Since the tumor mass of pancreatic cancer is mainly composed of fibrous tissue and only a part of cancer cells, it is difficult to evaluate the treatment response to cancer after chemotherapy, and pancreatic cancer is known as a cancer that does not work well with chemotherapy. Possible surgical resection is recommended rather than chemotherapy. An anticancer drug used clinically is gemcitabine (Eli Lilly and Company), but there are ongoing questions about the effectiveness of the treatment. However, there are no alternative substances and treatment methods available. Therefore, there is a demand for the development of an anticancer drug that can effectively treat pancreatic cancer and has a low side effect on use, or an adjuvant that can enhance the effect of existing anticancer drugs.
이러한 항암제의 보조제로서 세포치료제가 주목 받고 있다. 세포치료제의 유효성분 중 하나로 흔히 사용되는 세포인 자연살해세포는 여러 사이토카인에 의해 활성화(activation)되어 증폭하고 세포용해기능(cytolytic function)의 물질을 분비하게 되는데, 활성화된 자연살해세포는 인터페론-감마, 종양괴사인자(tumor-necrosis factor, TNF), 자가유래 과립세포-대식세포 집락자극인자(granulocyte/macrophage colony-stimulation factor; GM-CSF)를 케모카인(chemokine)과 함께 분비하여 염증반응을 일으킨다. 또한 단핵구(monocyte), 수지상 세포(dendritic cells), 과립구(granulocyte)의 분화와 성장을 유도함으로써 적응 면역에 영향을 미친다(Prospects for the use of NK cells in immunotherapy of human cancer, NATURE REVIEWS, 2007). Cell therapy is attracting attention as an adjuvant to these anticancer drugs. Natural killer cells, a cell commonly used as one of the active ingredients of cell therapy products, are activated by several cytokines to amplify and secrete cytolytic functions. The activated natural killer cells are interferon- It secretes gamma, tumor-necrosis factor (TNF), and autologous granulocyte/macrophage colony-stimulation factor (GM-CSF) together with chemokine to induce an inflammatory response. . It also affects adaptive immunity by inducing the differentiation and growth of monocytes, dendritic cells, and granulocytes (Prospects for the use of NK cells in immunotherapy of human cancer, NATURE REVIEWS, 2007).
한편, 면역 조절 관여에는 인터페론 감마(IFN- γ)와 같은 단백질 외에 식물에서 유래한 다당류가 다양한 대식세포 기능, 즉 종양세포와 미생물에 대한 세포독성 활성, 탐식활성알파조양괴사 인자, 인터루킨 등 특정 사이토카인의 분비를 활성화하며 세포의 분화 및 증식을 촉진한다.On the other hand, in the involvement of immune regulation, polysaccharides derived from plants in addition to proteins such as interferon gamma (IFN-γ) have various macrophage functions, i.e., cytotoxic activity against tumor cells and microorganisms, phagocytic activity alpha-zoyonecrosis factor, and specific cytokines such as interleukin. It activates the secretion of kine and promotes cell differentiation and proliferation.
이에 따라 특정 물질을 활용하여 위와 같은 면역세포의 면역 활성 증진효과를 높일 수 있다. 면역세포의 면역 활성을 효율적으로 증진시키고, 궁극적으로 이러한 면역세포를 이용한 암 치료 효과를 달성할 수 있는 방법이 다양하게 시도되고 있다. Accordingly, it is possible to increase the immune activity enhancing effect of the above immune cells by using a specific substance. Various attempts have been made to efficiently enhance the immune activity of immune cells and ultimately achieve cancer treatment effects using these immune cells.
본 발명의 일 목적은, 세포치료제의 유효성분인 면역세포의 면역 활성을 증진시킬 수 있는 세포치료제 제조 방법을 제공한다.An object of the present invention is to provide a method of manufacturing a cell therapy product capable of enhancing the immune activity of immune cells, which is an active ingredient of a cell therapy product.
본 발명의 다른 목적은, 암 치료 또는 예방용인 세포치료제의 치료 효과를 증진시키는 조성물을 제공한다.Another object of the present invention is to provide a composition for enhancing the therapeutic effect of a cell therapy agent for treatment or prevention of cancer.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.
본 발명의 일 구현 예에 따르면, 면역세포를 하기 화학식 1 또는 2로 표시되는 화합물, 멜라토닌 또는 이의 조합과 배양하는 단계; 및 상기 배양물로부터 면역세포를 분리하는 단계를 포함하는, 상기 면역세포를 유효성분으로 포함하는 세포치료제의 제조 방법에 관한 것이다:According to an embodiment of the present invention, the step of culturing immune cells with a compound represented by Formula 1 or 2, melatonin, or a combination thereof; And it relates to a method for producing a cell therapy product comprising the immune cells as an active ingredient, comprising the step of isolating immune cells from the culture:
[화학식 1][Formula 1]
[화학식 2][Formula 2]
본 발명에서 상기 화학식 1로 표시되는 화합물은 5-니트로-2-푸랄데하이드 p-하이드록시벤조일하이드라존(5-Nitro-2-furaldehyde p-hydroxybenzoylhydrazone)에 해당하며, '니푸록사지드(예, AMBATROL™, ANTINAL™, BACIFURANE™, DIAFURYL™)'로 나타낼 수도 있다. In the present invention, the compound represented by Formula 1 corresponds to 5-nitro-2-furaldehyde p-hydroxybenzoylhydrazone, and'nifuroxazide ( For example, it may also be expressed as AMBATROL™, ANTINAL™, BACIFURANE™, DIAFURYL™)'.
본 발명에서 상기 화학식 2로 표시되는 화합물은 3-(2,4-디클로로-페녹시메틸)-5-트리클로로메틸-[1,2,4]옥사디아졸(3-(2,4-dichloro-phenoxymethyl)-5-trichloromethyl-[1,2,4]oxadiazole)에 해당한다.In the present invention, the compound represented by Formula 2 is 3-(2,4-dichloro-phenoxymethyl)-5-trichloromethyl-[1,2,4]oxadiazole (3-(2,4-dichloro Corresponds to -phenoxymethyl)-5-trichloromethyl-[1,2,4]oxadiazole).
상기 면역세포를 배양하는 단계에서 배양액은 멜라토닌을 포함할 수 있다. 본 발명에서 멜라토닌(melatonin)은, 수면 타이밍, 혈압 조절, 계절에 따른 생식 등 생리적 기능의 활동일주기(circadian rhythm)에 영향을 미치는 것으로 알려진, 포유동물, 식물, 박테리아, 곰팡이 등에서 발견되는 물질 중 하나이다. 멜라토닌은 '멜라토닌 단독, 또는 진세노사이드(ginsenoside) 및 β-글루칸(β-glucan) 중 선택된 하나 이상과의 조합(이하, MTAM1)'으로 사용될 수 있다. MTAM1은 림프구를 배양하는데 사용될 수 있고, 배양 후 얻어지는 면역세포 내 자연살해세포의 증식 비율을 보다 높이고, 상기 자연살해세포의 암 세포에 대한 살상능 또한 더욱 증진시켜 암을 보다 효과적으로 예방 또는 치료할 수 있다. MTAM1이 존재하는 환경에서 림프구를 배양하는 경우, 상기 림프구는 CD3와 우선 배양된 것일 수 있다. 상기 MTAM1은 CD56, IL-18 또는 이의 조합을 더 포함할 수 있다. MTAM1과 추가적인 면역 활성화 인자인, CD3, CD56, IL-18 또는 이의 조합과의 배양은 필요에 따라, 동시에, 순차적으로 또는 교차적으로 수행될 수 있다.In the step of culturing the immune cells, the culture medium may contain melatonin. In the present invention, melatonin is known to affect the circadian rhythm of physiological functions such as sleep timing, blood pressure control, and seasonal reproduction, among substances found in mammals, plants, bacteria, fungi, etc. Is one. Melatonin may be used as'melatonin alone or in combination with at least one selected from ginsenoside and β-glucan (hereinafter, MTAM1). MTAM1 can be used to cultivate lymphocytes, increases the proliferation rate of natural killer cells in immune cells obtained after cultivation, and further enhances the killing ability of the natural killer cells to cancer cells, so that cancer can be more effectively prevented or treated. . When culturing lymphocytes in an environment in which MTAM1 is present, the lymphocytes may be first cultured with CD3. The MTAM1 may further include CD56, IL-18, or a combination thereof. The cultivation of MTAM1 with an additional immune activating factor, CD3, CD56, IL-18, or a combination thereof, can be performed as needed, simultaneously, sequentially or crossover.
본 발명에서 상기 "세포치료제(cellular therapeutic agent)"란, 개체로부터 분리, 배양 및 특수한 조작을 통해 제조된 세포 및 조직으로 치료, 진단 및 예방의 목적으로 사용되는 의약품(미국 FDA규정)으로서, 세포 혹은 조직의 기능을 복원시키기 위하여 살아있는 자가, 동종, 또는 이종세포를 체외에서 증식 선별하거나 다른 방법으로 세포의 생물학적 특성을 변화시키는 등의 일련의 행위를 통하여 치료, 진단 및 예방의 목적으로 사용되는 의약품을 의미한다. 일 구체예에서, 상기 세포치료제는 유효성분으로 면역세포를 포함할 수 있다.In the present invention, the "cellular therapeutic agent" refers to a drug (US FDA regulation) used for treatment, diagnosis, and prevention of cells and tissues manufactured through separation, cultivation and special manipulation from an individual. Or drugs used for treatment, diagnosis, and prevention through a series of actions such as proliferating and selecting live autologous, allogeneic, or xenogeneic cells in vitro to restore the function of tissues, or by changing the biological characteristics of cells in other ways. Means. In one embodiment, the cell therapy agent may contain immune cells as an active ingredient.
본 발명에서 "예방"은 본 발명의 조성물의 투여로 암을 억제하거나 진행을 지연시키는 모든 행위를 의미한다. In the present invention, "prevention" refers to any action that inhibits or delays progression of cancer by administration of the composition of the present invention.
본 발명에서 "치료" 및 "개선"은 본 발명의 조성물의 투여로 암의 증상이 호전 또는 이롭게 변경되는 모든 행위를 의미한다.In the present invention, "treatment" and "improvement" mean all actions in which symptoms of cancer are improved or beneficially changed by administration of the composition of the present invention.
본 발명에서 상기 “면역세포”는 체내에 존재하거나 체외로 분리할 수 있는 면역활성을 갖는 모든 세포를 의미할 수 있다. 면역세포는 예를 들어, 자연살해세포, T세포, B세포, 수지상세포, 대식세포, 또는 이들의 조합일 수 있다. 일 구체예에서, 상기 면역세포는 림프구일 수 있다. 다른 일 구체예에서, 상기 면역세포는 자연살해세포(natural killer cells, NK cells)일 수 있다. In the present invention, the "immune cells" may refer to all cells that are present in the body or have immune activity that can be separated from the body. The immune cells may be, for example, natural killer cells, T cells, B cells, dendritic cells, macrophages, or a combination thereof. In one embodiment, the immune cells may be lymphocytes. In another embodiment, the immune cells may be natural killer cells (NK cells).
일 구체예에서, 상기 화학식 1 또는 2의 화합물은 단리된 자연살해세포와 접촉하여 이의 면역 활성을 증진시킬 수 있다. 인체로부터 수득되는 면역세포는 다양한 면역세포 종류가 혼합물 형태이다. 따라서, 배양 과정에서 특정 면역세포로의 분화를 유도하거나, 이의 증식만 활성화하는 것이 일반적이다. 본 발명의 일 구체예는, 하기의 실시예에서도 입증된 바와 같이, 단리된 자연살해세포에 적용하여도 성공적으로 이의 면역 활성을 증진시킬 수 있다. 이러한 효과는, 유효 성분으로 순수한 자연살해세포를 포함하는 세포치료제의 최종 제품에 대해서도 기대하는 면역 활성을 얻을 수 있음을 시사한다.In one embodiment, the compound of Formula 1 or 2 may contact isolated natural killer cells to enhance their immune activity. The immune cells obtained from the human body are a mixture of various types of immune cells. Therefore, it is common to induce differentiation into specific immune cells during the culture process or to activate only their proliferation. One embodiment of the present invention can successfully enhance its immune activity even when applied to isolated natural killer cells, as demonstrated in the following examples. This effect suggests that the expected immune activity can be obtained even in the final product of a cell therapy product containing pure natural killer cells as an active ingredient.
본 발명에서, 상기 “배양”은 세포를 생존 가능한 상태로 유지 또는 보존하는 것으로서, 면역세포를 특정 환경에 노출 또는 접촉시키는 것을 포함하는 의미일 수 있다.In the present invention, the “culture” refers to maintaining or preserving cells in a viable state, and may be meant to include exposing or contacting immune cells to a specific environment.
일 구체예에서, 상기 화학식 1 또는 2로 표시되는 화합물, 또는 이의 조합은 면역세포와 혼합하여 배양함으로써 면역세포의 면역 활성을 증진시킬 수 있다. 상기 면역 활성은 예를 들어, 암세포 또는 감염세포에 대한 세포살상능(cytotoxicity)을 의미할 수 있다. 이는 궁극적으로, 면역세포를 유효성분으로 포함하는 세포치료제의 암 치료 또는 예방 효과를 더욱 증진시킬 수 있다. 본 발명에서, 상기 암은 예컨대 췌장암, 간암, 폐암, 대장암, 유방암, 전립선암, 난소암, 대장암, 자궁경부암, 갑상선암, 후두암, 백혈병, 뇌종양, 신경모세포종, 망막모세포종, 두경부암, 침샘암, 림프종으로 구성된 군으로부터 선택될 수 있고, 바람직하게는 췌장암일 수 있다.In one embodiment, the compound represented by Formula 1 or 2, or a combination thereof, may enhance the immune activity of immune cells by mixing and culturing them with immune cells. The immune activity may mean, for example, cytotoxicity against cancer cells or infected cells. Ultimately, this can further enhance the cancer treatment or prevention effect of a cell therapy product containing immune cells as an active ingredient. In the present invention, the cancer is, for example, pancreatic cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, colon cancer, cervical cancer, thyroid cancer, laryngeal cancer, leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer. , It may be selected from the group consisting of lymphoma, preferably pancreatic cancer.
상기 배양물로부터 면역세포를 분리하는 단계는, 면역세포 외에 배양액 및 배양액에 잔존하는 성분들을 실질적으로 제거하는 것을 의미할 수 있다. 분리하는 방법은, 예를 들어, 막 여과, 투석, 침전법, 세포 부착 및 세척 등 다양한 방법을 사용할 수 있고, 이에 제한되지 않는다. 면역세포가 개체에 적용되는 경우 발생할 수 있는 불필요한 생체 반응을 회피하기 위해 면역세포를 분리하는 단계를 채용할 수 있으나, 화학식 1 또는 2로 표시되는 화합물, 멜라토닌 또는 이의 조합은 단지 면역세포의 면역 활성을 증진시키기 위해 소량 사용되므로, 면역세포와 함께 잔존하더라도 개체에 미치는 효과는 매우 미미할 수 있다.Separating the immune cells from the culture may mean substantially removing components remaining in the culture medium and the culture medium in addition to the immune cells. The separation method may use various methods such as membrane filtration, dialysis, precipitation method, cell adhesion and washing, etc., but is not limited thereto. In order to avoid unnecessary biological reactions that may occur when immune cells are applied to an individual, the step of isolating immune cells may be employed, but the compound represented by Formula 1 or 2, melatonin, or a combination thereof is only used for immune activity of immune cells. Since it is used in a small amount to enhance immunity, the effect on the individual may be very insignificant even if it remains with the immune cells.
일 구체예에서, 상기 세포치료제의 제조 방법은, 분리된 면역세포를 세포치료제로 제형화하는 단계를 더 포함할 수 있다. 상기 제형화는 세포치료제를 이를 필요로 하는 개체에 투여하기에 적절한 형태로 가공하는 모든 행위를 포함하는 것으로서, 예를 들어, 분리된 면역세포를 약제학적으로 허용 가능한 담체와 혼합하는 것을 의미할 수 있다. 약제학적으로 허용 가능한 담체는, 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올, 리포좀 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있으며, 표적 기관에 특이적으로 작용할 수 있도록 표적 기관 특이적 항체 또는 기타 리간드를 상기 담체와 결합시켜 사용할 수 있다. 더 나아가 당해 기술 분야의 적정한 방법으로 또는 레밍턴의 문헌(Remington's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA)에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.In one embodiment, the method of manufacturing the cell therapy agent may further include formulating the isolated immune cells as a cell therapy agent. The formulation includes all actions of processing a cell therapy product into a form suitable for administration to an individual in need thereof, and may mean, for example, mixing the isolated immune cells with a pharmaceutically acceptable carrier. have. Pharmaceutically acceptable carriers, saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and one or more of these components may be mixed and used. Other conventional additives, such as an agent, a buffer solution, and a bacteriostatic agent, may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to form injection formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets. Thus, a target organ-specific antibody or other ligand may be used in combination with the carrier. Furthermore, it can be preferably formulated according to each disease or component by an appropriate method in the art or by using a method disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA. have.
상기 세포치료제는 액제, 현탁제, 분산액, 유제, 겔제, 주사 가능한 액제 및 활성 화합물의 서방출형 제제 등이 될 수 있으며, 바람직하게는 주사제가 될 수 있다. 상기 세포치료제가 주사제로 제형화되는 경우, 상기 제형화 단계에서 주사제 처방의 유통에 따른 제품 안정성을 확보하기 위하여 주사제로 사용 가능한 산수용액 또는 인산염 등의 완충용액을 사용하여 pH를 조절함으로써 물리적으로나 화학적으로 매우 안정한 주사제로 제조될 수 있다.The cell therapy agent may be a solution, a suspension, a dispersion, an emulsion, a gel, an injectable solution, a sustained-release preparation of the active compound, and the like, preferably an injection. When the cell therapy is formulated as an injection, physically or chemically by adjusting the pH using a buffer solution such as an acid aqueous solution or phosphate that can be used as an injection in order to ensure product stability according to the distribution of the injection formulation in the formulation step. It can be prepared as a very stable injection.
본 발명의 다른 구현 예에 따르면, 상기 화학식 1 또는 2로 표시되는 화합물, 멜라토닌 또는 이의 조합을 포함하는, 세포치료제의 치료 효과 증진용 조성물을 제공한다.According to another embodiment of the present invention, there is provided a composition for enhancing the therapeutic effect of a cell therapy agent, comprising the compound represented by Formula 1 or 2, melatonin, or a combination thereof.
상기 세포치료제의 치료 효과 증진용 조성물은, 세포치료제와 접촉함으로써, 상기 세포치료제의 유효성분인 면역세포의 면역 활성을 증진시키고, 결과적으로 세포치료제의 암 치료 또는 예방 효과를 증진시킬 수 있다.The composition for enhancing the therapeutic effect of the cell therapy agent may enhance the immune activity of immune cells, which is an active ingredient of the cell therapy agent, by contacting the cell therapy agent, and as a result, enhance the cancer treatment or prevention effect of the cell therapy agent.
상기 세포치료제의 치료 효과 증진용 조성물은, 면역세포와 접촉시키기 위해 면역세포를 배양하기 위한 배양액에 첨가물로서 사용할 수 있다.The composition for enhancing the therapeutic effect of the cell therapy agent may be used as an additive to a culture medium for culturing immune cells to contact immune cells.
일 구체예에서, 세포치료제가 개체 내에서 신속하고 효율적인 면역 활성을 나타낼 수 있도록, 상기 세포치료제의 치료 효과 증진용 조성물은 세포치료제를 필요로 하는 개체에 사용하기 전에 우선적으로 상기 세포치료제와 혼합될 수 있다. 상기 화학식 1 또는 2로 표시되는 화합물은 1 μM 내지 100 μM, 1 μM 내지 80 μM, 1 μM 내지 60 μM, 또는 1 μM 내지 40 μM으로 포함될 수 있다. 상기 세포치료제의 치료 효과 증진용 조성물은, 개체에 투여되기 전에 세포치료제와의 혼합물로부터 제거되어 개체 내에서 불필요한 생체 반응이 유발되는 것을 차단할 수 있다. 그러나, 화학식 1 또는 2로 표시되는 화합물은 면역세포의 면역 활성을 증진시킬 수 있을 정도로 소량 사용되므로, 혼합물 상태로 투여되거나 잔존하더라도 개체에 미치는 효과는 미미하다.In one embodiment, the composition for enhancing the therapeutic effect of the cell therapy agent may be first mixed with the cell therapy agent before use in an individual in need of the cell therapy agent so that the cell therapy agent can exhibit rapid and efficient immune activity in an individual. I can. The compound represented by Formula 1 or 2 may be included in 1 μM to 100 μM, 1 μM to 80 μM, 1 μM to 60 μM, or 1 μM to 40 μM. The composition for enhancing the therapeutic effect of the cell therapy agent may be removed from the mixture with the cell therapy agent before being administered to the subject to block unnecessary biological reactions in the subject. However, since the compound represented by Formula 1 or 2 is used in a small amount enough to enhance the immune activity of immune cells, even if administered as a mixture or remains, the effect on the individual is insignificant.
본 발명의 또 다른 구현 예에 따르면, 상기 화학식 1 또는 2로 표시되는 화합물, 멜라토닌 또는 이의 조합을 포함하는 제1 유닛; 및 면역세포를 유효성분으로 포함하는 세포치료제를 포함하는 제2 유닛을 포함하는, 키트를 제공한다.According to another embodiment of the present invention, a first unit comprising a compound represented by Formula 1 or 2, melatonin, or a combination thereof; And it provides a kit comprising a second unit comprising a cell therapy product comprising immune cells as an active ingredient.
본 발명에서, 상기 “유닛”은 단지 키트의 구성을 구분하기 위한 수단이고, 고체, 분말, 용액, 현탁액, 겔 등의 형태로 존재할 수 있고, 서로 분리 가능한 적절한 용기에 포함된 것을 의미할 수 있다.In the present invention, the "unit" is merely a means for distinguishing the composition of the kit, and may exist in the form of a solid, powder, solution, suspension, gel, etc., and may mean that it is included in an appropriate container that can be separated from each other .
상기 제1 유닛 및 제2 유닛은 상기 키트 내에서 분리되어 있고, 개체에 사용하기 전에 서로 혼합되어, 제2 유닛의 면역세포의 면역 활성을 증진시킴으로써 세포치료제의 치료 효과를 증진시킬 수 있다. The first unit and the second unit are separated in the kit and mixed with each other before use in an individual, thereby enhancing the immune activity of the immune cells of the second unit, thereby enhancing the therapeutic effect of the cell therapy product.
상기 키트는 제1 유닛 및 제2 유닛의 혼합물로부터 면역세포를 분리할 수 있는 구성을 더 포함할 수 있다. 상기 면역세포를 분리하는 구성은 실질적으로 상기 제1 유닛 및 제2 유닛의 혼합물로부터 화학식 1 또는 2, 멜라토닌 또는 이의 혼합물을 제거하기 위한 것일 수 있다. 그러나, 제1 유닛의 성분은 단지 면역세포의 면역 활성을 증진시킬 수 있을 정도로 소량이므로, 혼합물 상태로 투여되거나 잔존하더라도 개체에 미치는 효과는 미미하다.The kit may further include a component capable of separating immune cells from a mixture of the first unit and the second unit. The configuration for separating the immune cells may be for substantially removing Formula 1 or 2, melatonin, or a mixture thereof from the mixture of the first unit and the second unit. However, since the components of the first unit are only small enough to enhance the immune activity of immune cells, even if administered as a mixture or remain, the effect on the individual is insignificant.
상기, 세포치료제의 제조 방법에 대한 설명은, 세포치료제의 치료 효과 증진용 조성물 또는 키트에 대한 동일한 구성에 대한 설명과 같은 의미로 해석될 수 있다. The description of the method of manufacturing the cell therapy agent may be interpreted in the same meaning as the description of the same configuration for the composition or kit for enhancing the therapeutic effect of the cell therapy agent.
본 발명에서 제공하는 상기 화학식 1 또는 2로 표시되는 화합물, 또는 이의 조합을 이용한 세포치료제 제조 방법 및 이를 위한 조성물은 면역세포의 암세포 또는 감염세포에 대한 세포살상능을 현저히 증진시켜, 면역세포를 유효성분으로 포함하는 세포치료제의 암 치료 또는 예방 효능을 현저히 증진시킬 수 있다.The method for producing a cell therapy product using the compound represented by Formula 1 or 2, or a combination thereof provided in the present invention, and a composition therefor, remarkably enhance the cell killing ability of immune cells against cancer cells or infected cells, thereby effectively enhancing immune cells. It is possible to remarkably improve the efficacy of cancer treatment or prevention of the cell therapy product included as an ingredient.
도 1은, 화합물 1 및 2의 NK 세포에 대한 독성 시험 결과를 나타낸 것이다.1 shows the results of a toxicity test for NK cells of Compounds 1 and 2.
도 2는, 화합물 1 및 2와 각각 배양한 NK 세포의 Panc-1 세포에 대한 세포독성 시험 결과를 나타낸 것이다.2 shows the results of a cytotoxicity test for Panc-1 cells of NK cells cultured with Compounds 1 and 2, respectively.
도 3 내지 6은, 각각 실시예 2에서 시험군 1, 시험군 2 및 대조군에 대하여 배양조건 별로 세포표면항원을 검사한 그래프이고, 도 6은 각각으로부터 수확한 세포 내 NK 세포 및 NKT 세포의 비율 및 T 세포의 비율을 나타낸 것이다. 3 to 6 are graphs showing cell surface antigens for each culture condition for test group 1, test group 2, and control group in Example 2, respectively, and FIG. 6 is a ratio of NK cells and NKT cells in cells harvested from each And the percentage of T cells.
도 7은, 실시예 2에서 시험군 1, 시험군 2 및 대조군에 대한 세포성장곡선을 나타낸 것이다. 7 shows cell growth curves for test group 1, test group 2, and control group in Example 2.
도 8은, 실시예 2에서 시험군 1, 시험군 2 및 대조군에 대한 세포독성 시험 결과를 나타낸 것이다. 8 shows the cytotoxicity test results for test group 1, test group 2, and control group in Example 2.
도 9는 실시예 2에서 본 발명에 따른 면역세포 활성화 물질에 대한 세포생존시험(Cell viability test) 결과를 그래프로 나타낸 것이다. 9 is a graph showing the results of a cell viability test for an immune cell activating substance according to the present invention in Example 2.
도 10 및 11은 실시예 2에서 본 발명에 따른 면역세포 활성화 물질에 대한 활성산소(Reactive Oxygen Species, ROS) 측정 결과를 그래프로 나타낸 것이다. 10 and 11 are graphs showing the measurement results of reactive oxygen species (ROS) for the immune cell activating substance according to the present invention in Example 2.
도 12는 실시예 2에서 혈액으로부터 채취 및 분리한 림프구에 시험군 1의 처리 후 배양 및 수확한 면역세포의 세포표면항원을 검사한 그래프를 나타낸 것이다.12 is a graph showing the cell surface antigens of immune cells cultured and harvested after treatment with Test Group 1 on lymphocytes collected and isolated from blood in Example 2. FIG.
본 발명은 면역세포를 하기 화학식 1 또는 2로 표시되는 화합물, 멜라토닌 또는 이의 조합과 배양하는 단계; 및 상기 배양물로부터 면역세포를 분리하는 단계를 포함하는, 상기 면역세포를 유효성분으로 포함하는 세포치료제의 제조 방법에 관한 것이다:The present invention comprises the steps of culturing immune cells with a compound represented by Formula 1 or 2, melatonin, or a combination thereof; And it relates to a method for producing a cell therapy product comprising the immune cells as an active ingredient, comprising the step of isolating immune cells from the culture:
[화학식 1][Formula 1]
[화학식 2][Formula 2]
이하, 본 발명을 하기의 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples.
실시예 1: 화학식 1 및 2의 면역세포의 면역 활성 증진 능력 확인Example 1: Confirmation of the ability to enhance immune activity of immune cells of Formulas 1 and 2
1-1. 세포 배양1-1. Cell culture
췌장암세포주인 Panc-1는 한국세포주은행(서울대학교)에서 구매하였다. 세포는 10% FBS와 1% 페니실린 스트렙토마이신이 함유된 Dulbecco's modified Eagle's medium (DMEM, Hyclone, Logan, UT; Panc-1세포) 배지를 이용하여 37℃, 5% CO2 인큐베이터에서 배양하였다. 자연살해세포주인 NK-92세포는 ATCC (Manassas, VA) 에서 구매하였으며, 20% FBS와 1% 페니실린 스트렙토마이신, 10 ng/ml recombinant human IL-2가 함유된 Minimum Essential Medium Eagle alpha (MEM-α) 배지를 이용하여 37℃, 5% CO2 인큐베이터에서 배양하였다.Panc-1, a pancreatic cancer cell line, was purchased from Korea Cell Line Bank (Seoul National University). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Hyclone, Logan, UT; Panc-1 cells) medium containing 10% FBS and 1% penicillin streptomycin in a 5% CO2 incubator at 37°C. NK-92 cells, a natural killer cell line, were purchased from ATCC (Manassas, VA) and contain 20% FBS, 1% penicillin streptomycin, and 10 ng/ml recombinant human IL-2. ) Cultured in a 37°C, 5% CO2 incubator using a medium.
1-2. 화학식 1 및 2의 세포독성 확인1-2. Confirmation of cytotoxicity of Formulas 1 and 2
실험물질의 자연살해세포에 대한 독성을 평가하기 위해, 실시예 1-1의 NK-92세포에 대해 화학식 1 및 2 각각을 1 μM, 5 μM, 10 μM, 20 μM, 및 40 μM의 농도로 처리하여 4시간 동안 인큐베이션하였다. 그런 다음, D-Plus™ cell viability assay kit (CCK-3000, Donginbiotech Co., korea)를 잘 알려진 방식대로 0.1X 처리 후 450nm 흡광도 측정하였다.In order to evaluate the toxicity of the test substance to natural killer cells, the NK-92 cells of Example 1-1 were each of Formulas 1 and 2 at a concentration of 1 μM, 5 μM, 10 μM, 20 μM, and 40 μM. Treated and incubated for 4 hours. Then, D-Plus™ cell viability assay kit (CCK-3000, Donginbiotech Co., Korea) was treated with 0.1X in a well-known manner, and absorbance at 450 nm was measured.
그 결과, 시험된 모든 농도에서 생존률이 약 85%로 관찰되어, NK-92세포에 대한 직접적은 독성은 없는 것으로 확인되었다 (도 1).As a result, the survival rate was observed to be about 85% at all the tested concentrations, it was confirmed that there is no direct toxicity to NK-92 cells (Fig. 1).
1-3. 화학식 1 및 2의 면역세포의 면역 활성화 평가1-3. Evaluation of immune activation of immune cells of formulas 1 and 2
실시예 1-1의 NK-92세포를 각각 40 μM 의 화학식 1 및 2와 4시간 동안 배양한 후, NK-92 세포를 분리하고, 이를 암세포인 Panc-1 세포와 각 비율에 맞게 (E:T=2.5, 5, 10, 20) 공동 배양하였다. 4시간 후, 암세포 생존률에 대해 LDH cytotoxicity detection kit (Takara, Japan)로 잘 알려진 방식대로 실험을 진행하여, 490nm 흡광도 측정하였다.After culturing the NK-92 cells of Example 1-1 for 4 hours with 40 μM of Formulas 1 and 2, respectively, NK-92 cells were isolated, and these cells were separated from Panc-1 cells, which were cancer cells, in accordance with each ratio (E: T=2.5, 5, 10, 20) co-cultured. After 4 hours, the experiment was conducted in a manner well known as LDH cytotoxicity detection kit (Takara, Japan) for cancer cell viability, and absorbance at 490 nm was measured.
그 결과, 화학식 1 및 2와 배양된 NK-92 세포가 Panc-1 세포에 대해 대조군과 비교하여 현저히 우수한 살상능을 갖는 것으로 확인되었다 (도 2).As a result, it was confirmed that NK-92 cells cultured with Formulas 1 and 2 had remarkably superior killing ability compared to the control group for Panc-1 cells (FIG. 2).
실시예 2: 멜라토닌의 면역세포의 면역 활성 증진 능력 확인Example 2: Confirmation of the ability of melatonin to increase immune activity of immune cells
2-1. 림프구의 수득 및 배양2-1. Acquisition and culture of lymphocytes
환자의 혈액에서 림프구를 분리하고, 배양액에 현탁한 다음, 이를 CD3가 고상화되어 있는 T-25 플라스크(Flask)에 넣었다. 그런 다음, D56 5㎛ 및 자가유래 혈장 10%를 T-25 Flask에 첨가한 후 37℃, 5% CO
2 인큐베이터(incubator)에서 1~2일간 배양하였다. T-75 Flask에 옮긴 후, 37℃, 5% CO
2 인큐베이터에서 3~4일간 배양하였다. 이 때, 시험예 1 및 2 각각에는, T-75 Flask에서 CD56 5㎕, IL-18 및 면역세포 활성화 물질로 멜라토닌, 진세노사이드 및 β-글루칸의 혼합물(이하, MTAM1)을 11.5㎕ 또는 40㎕ 처리하였다. 그리고 비교를 위하여 대조군에서는 T-75 Flask에서 CD56 5㎕, IL-18를 처리한 후, CO
2 인큐베이터에서 3~4일간 배양하였다. 그 후, T-175 Flask에 옮기고, 37℃, 5% CO
2 인큐베이터에서 48시간 배양하였다.Lymphocytes were isolated from the patient's blood, suspended in a culture solution, and placed in a T-25 flask in which CD3 was solidified. Then, D56 5㎛ and 10% of autologous plasma were added to the T-25 Flask and then cultured in a 37°C, 5% CO 2 incubator for 1-2 days. After being transferred to a T-75 Flask, it was incubated for 3-4 days in an incubator at 37°C and 5% CO 2. At this time, in each of Test Examples 1 and 2, 11.5 µl or 40 of a mixture of melatonin, ginsenoside, and β-glucan (hereinafter, MTAM1) as a substance for activating CD56, IL-18, and immune cells was added in a T-75 flask. Ul was treated. And for comparison, in the control group, 5 µl of CD56 and IL-18 were treated in a T-75 flask, and then cultured in a CO 2 incubator for 3-4 days. Then, it was transferred to a T-175 Flask, and incubated for 48 hours in an incubator at 37° C. and 5% CO 2.
그 후, 시험군 1 및 2에는 T-175 Flask에서 MTAM1을 각각 10㎕ 또는 20㎕ 처리하고, 자가유래 혈장 5%를 첨가하였다. 그리고 비교를 위한 대조군에는 자가유래 혈장 5%를 첨가한 후, CO
2 인큐베이터에서 48시간 동안 배양하였다. Thereafter, test groups 1 and 2 were treated with 10 µl or 20 µl of MTAM1, respectively, in a T-175 flask, and 5% of autologous plasma was added. In addition, 5% of autologous plasma was added to the control group for comparison, and then incubated in a CO 2 incubator for 48 hours.
그 후, 시험군 1 및 2에 대하여, T-175 Flask에 배양 중인 세포현탁액에 IL-18과 MTAM1을 처리하고 세포의 활성화된 상태를 관찰한 후, T-150 Flask를 1000ml 용량의 가스투과성 배양백이 들어있는 배양액에 옮긴 후 7~9일간 더 배양하여 세포를 다량 증식시켰다. 대조군에 대하여는, T-150 Flask를 1000ml 용량의 가스투과성 배양백이 들어있는 배양액에 옮긴 후 7~9일간 더 배양하여 세포를 다량으로 증식시켰다.Thereafter, for test groups 1 and 2, treatment with IL-18 and MTAM1 in the cell suspension cultivated in a T-175 flask, and after observing the activated state of the cells, the T-150 flask was incubated with a gas permeability of 1000 ml. After transferring to the culture medium containing the bag, it was cultured for 7 to 9 days to proliferate a large amount of cells. For the control group, the T-150 Flask was transferred to a culture medium containing a 1000 ml of gas-permeable culture bag, and then cultured for 7 to 9 days to proliferate a large amount of cells.
2-2. 면역세포의 수득2-2. Acquisition of immune cells
시험군 1 및 2, 대조군 각각에 대하여, 1000ml 용량의 CO
2 가스투과성 배양백이 들어있는 세포와 배양액을 500ml 원심 분리 튜브에 나누어 옮기고, 17분(min)간 원심 분리하여 세포를 수확하였다. 상층액을 세포가 떨어지지 않도록 조심히 버린 후 멸균생리식염수 250ml로 세포를 세척하였다. 다시 원심 분리기 안에 밸런스를 맞추어 원심 분리관을 넣고 15분간 원심 분리하여 세포를 수확하였다. 상층액을 세포가 떨어지지 않도록 조심히 버린 후 100ml 주사용 멸균 생리식염수 백(bag)에서 주사기(syringe)를 사용하여 30ml 채취하였다. 30ml 주사용 멸균 생리식염수로 세포를 현탁하였다. 현탁한 세포를 셀스트레이너(cell strainer)에 부어 걸렀다. 거른 세포 현탁액을 주사용 멸균 생리식염수 백에 넣어 현탁시켰다.For each of the test groups 1 and 2 and the control group, cells containing a 1000 ml of CO 2 gas permeable culture bag and the culture solution were divided into 500 ml centrifuge tubes, and centrifuged for 17 minutes to harvest the cells. After the supernatant was carefully discarded so that the cells did not fall, the cells were washed with 250 ml of sterile physiological saline. The cells were harvested by rebalanced in the centrifuge, placed in a centrifuge tube, and centrifuged for 15 minutes. After carefully discarding the supernatant so that the cells do not fall off, 30 ml of a 100 ml sterile physiological saline bag for injection was collected using a syringe. The cells were suspended in 30 ml sterile physiological saline for injection. The suspended cells were poured into a cell strainer and filtered. The filtered cell suspension was placed in a sterile physiological saline bag for injection and suspended.
2-3. 면역세포의 면역 활성 평가2-3. Evaluation of immune activity of immune cells
실시예 2-2로부터 수득된 시험군 1 및 2, 그리고 대조군의 세포표면항원을 분석하여 림프구의 종류를 정량적으로 측정하기 위한 순도시험을 행하였고, 그 결과를 각각 도 3 내지 6에 나타내었다. 여기서, 도 3 내지 5는 각각 시험군 1, 시험군 2 및 대조군에 대하여 배양조건 별로 세포표면항원 검사한 그래프이고, 도 6은 각각에 대하여 NK 세포 및 NKT 세포의 비율, T 세포 비율을 나타내었다. A purity test for quantitatively measuring the type of lymphocytes was performed by analyzing the cell surface antigens of test groups 1 and 2 and the control group obtained from Example 2-2, and the results are shown in FIGS. 3 to 6, respectively. Here, FIGS. 3 to 5 are graphs of cell surface antigen tests for each culture condition for test group 1, test group 2, and control group, respectively, and FIG. 6 shows the ratio of NK cells and NKT cells, and T cell ratios for each. .
그 결과, 시험군 1 및 2는 대조군에 비하여 NK 세포 및 NKT 세포의 비율이 월등히 증가함을 알 수 있었다 (도 6). As a result, it was found that the ratio of NK cells and NKT cells significantly increased in test groups 1 and 2 compared to the control group (FIG. 6).
또한, 세포수 및 생존률에서 시험군 1 및 2에서는 대조군에 비하여 세포수 및 생존율이 향상됨을 알 수 있었다 (도 7 및 표 1).In addition, in terms of cell number and survival rate, it was found that the number of cells and the survival rate were improved in the test groups 1 and 2 compared to the control group (Fig. 7 and Table 1).
|
대조군 | 시험군 1Test group 1 | 시험군 2Test group 2 | |||||
| 세포수Cell count | 생존율Survival rate | 세포수Cell count | 생존율Survival rate | 세포수Cell count | 생존율Survival rate | ||
| 2.26X10 9 2.26X10 9 | 83%83% | 2.96X10 9 2.96X10 9 | 97%97% | 3.86X10 9 3.86X10 9 | 95%95% | ||
마지막으로 세포독성(Cytotoxicity) 시험 결과, 시험군 1 및 2의 세포상해활성능력(%)이 대조군의 세포상해활성능력에 비하여 월등히 향상됨을 알 수 있었다 (도 8 및 표 2).Finally, as a result of the cytotoxicity test, it was found that the cytotoxicity activity (%) of test groups 1 and 2 was significantly improved compared to the cytotoxicity activity of the control group (FIG. 8 and Table 2).
|
대조군 | 시험군 1Test group 1 | 시험군 2Test group 2 | ||
| 검체명Sample name | IMBIO_NK+T cell_01IMBIO_NK+T cell_01 | IMBIO_NK+T cell_04IMBIO_NK+T cell_04 | IMBIO_NK+T cell_04IMBIO_NK+T cell_04 | |
| 세포상해활성능(%)Cytotoxic activity (%) | 3131 | 6868 | 5959 |
이와 같은 결과는 MTAM1의 첨가에 따른 것으로, 여기서 MTAM1은 식물로부터 유래된 멜라토닌, 진세노사이드 및 β-글루칸 중 적어도 하나를 포함하고, 바람직하게는 멜라토닌, 진세노사이드 및 β-글루칸을 모두 포함하여 제조된다.NK 세포는 성장과 확산을 제한함으로써 종양의 여러 유형을 제어하는 타고난 면역 시스템의 조절 림프구로서, 다른 자극 없이 직접 세포의 세포 독성을 발휘하고, IFN-γ와 가은 면역촉진성의 사이토카인을 분비하여 국조 종양 성장(local tumor growth)과 전이(metastasis)를 모두 제어할 수 있다. This result is due to the addition of MTAM1, wherein MTAM1 contains at least one of plant-derived melatonin, ginsenoside and β-glucan, preferably includes all of melatonin, ginsenoside and β-glucan. NK cells are regulatory lymphocytes of the innate immune system that control different types of tumors by limiting growth and proliferation, exert direct cellular cytotoxicity without other stimuli, and produce IFN-γ and hypoimmune-promoting cytokines. It can be secreted to control both local tumor growth and metastasis.
특히 IL-2에 의해 활성화된 NK 세포는 CTL과 유사하며, 퍼포린(perforin)과 그란자임 B(granzyme B)에 의해 연속적으로 타겟(target) 세포를 살해 후 자신은 살아 남으며, 이때 IL-2는 고갈된 NK 세포의 세포독성과 과립을 축적시켜 주는 능력을 지니고 있다. 또한 NK 세포는 항체 의존적인 세포독성(antibody dependent cellular cytotoxicity, ADCC)을 가진다. NK 세포에서 발현되는 Fc 리셉터 중 FcγRIII(CD16)는 병원균(pathogen)에 감염된 세포의 표면에 결합되어 있는 IgG와 같은 항체의 Fc 부위를 인식하게 된다. Fc 리셉터의 활성은 NK 세포의 활성을 유도하여 세포용해성 사이토카인(cytolytic cytokine)을 분비하도록 유도하며, 이러한 NK 세포의 ADCC 작용은 CTL과는 다른 NK 세포만 가지고 있는 특징이다. In particular, NK cells activated by IL-2 are similar to CTL, and after successively killing target cells by perforin and granzyme B, they survive. At this time, IL-2 Has the ability to accumulate granules and cytotoxicity of depleted NK cells. In addition, NK cells have antibody dependent cellular cytotoxicity (ADCC). Among the Fc receptors expressed in NK cells, FcγRIII (CD16) recognizes the Fc region of antibodies such as IgG bound to the surface of cells infected with pathogens. The activity of the Fc receptor induces the activity of NK cells to induce the secretion of cytolytic cytokines, and the ADCC action of these NK cells is a characteristic that only NK cells different from CTL have.
멜라토닌은 림프구의 글루타티온의 생산을 늘려 림프구의 기능을 높이는 효과가 있다. 단핵구 표면에 있는 멜라토닌 수용체를 통해 IFN-γ와 인터루킨-1, 2, 6, 12(Interleukin-1, 2, 6, 12) 등의 사이토카인의 분비를 촉진시켜, NK 세포의 활성을 유도한다. 본 발명에 따른 MTAM1은 NK 세포의 바이러스 감염 세포 및 암 세포에 대한 살상능을 높여주고, 배양 중의 활성산소를 저해하여 세포 생존율을 증가시킨다. Melatonin has the effect of increasing the function of lymphocytes by increasing the production of glutathione by lymphocytes. By promoting the secretion of IFN-γ and cytokines such as Interleukin-1, 2, 6, 12 (Interleukin-1, 2, 6, 12) through the melatonin receptor on the surface of monocytes, it induces the activity of NK cells. MTAM1 according to the present invention increases the killing ability of NK cells against virus-infected cells and cancer cells, and increases cell viability by inhibiting free radicals in culture.
또한, 진세노이드(Ginsenoside)는 C-SRC 키나제(C-Src Kinase)를 활성화시키며, 세포 내 칼슘이온 농도를 2배~3배 증가시킨다. 세포 내 신호전달 체계의 초기단계인 C-Src 키나제 관련 경로의 활성화 및 세포 내 칼슘이온의 증가는 특정 유전자의 발현을 증가시키고 세포 증식을 촉진시킨다. 특히 진세노사이드-Rp1은 수지상 세포에 특이적으로 일정한 농도에서 면역 사이토카인의 생성반응을 유도하며 저농도에서 NK 세포의 활성도를 증가시킨다. In addition, Ginsenoside activates C-SRC kinase and increases intracellular calcium ion concentration by 2 to 3 times. The activation of the C-Src kinase-related pathway, which is an early stage of the intracellular signaling system, and the increase of intracellular calcium ions increase the expression of specific genes and promote cell proliferation. In particular, ginsenoside-Rp1 induces the production of immune cytokines at a specific concentration in dendritic cells and increases the activity of NK cells at low concentrations.
한편, 베타-글루칸은 TNF-α와 IFN-γ의 발현을 증가시키며, 호중구에서 항아포토시스(Anti-apoptosis) 효과를 나타낸다. 특히 라미나리아 디지타타(Laminaria digitata)에서 분리한 글루칸인 피카린(Phycarine)은 식세포 활성을 증가시키고, IL-1, IL-6, TNF-α의 생성 및 분비를 증가시킬 뿐만 아니라 피카린은 보체 제3 수용체(CR3)를 통한 NK 세포에 의한 암 세포 사멸작용을 증가시킨다.On the other hand, beta-glucan increases the expression of TNF-α and IFN-γ, and exhibits an anti-apoptosis effect in neutrophils. In particular, Picarine, a glucan isolated from Laminaria digitata, increases phagocytic activity, increases the production and secretion of IL-1, IL-6, and TNF-α, as well as picarine is complementary. Increases cancer cell death by NK cells through the third receptor (CR3).
도 9은 MTAM1에 대한 세포생사판별시험(Cell viability test) 결과를 나타낸 그래프로서, 본 발명에 따른 MTAM1이 세포 내의 독성이 없음을 보여주며, 나아가 증식에도 효과가 있음을 보여준다. 9 is a graph showing the results of a cell viability test for MTAM1, showing that MTAM1 according to the present invention has no intracellular toxicity, and further shows that it has an effect on proliferation.
또한, 도 10 및 도 11은 활성산소(Reactive Oxygen Species; ROS) 측정 결과를 나타낸 그래프로서, 도 10을 통해서 NK 세포에 인위적으로 ROS를 증가시켜 MTAM1 처리 후 ROS가 감소됨을 확인하였고, 도 11을 통해서 MTAM1이 들어있는 배지에서 NK 세포를 14일간 배양 후에 ROS를 측정한 결과 멜라토닌이 활성산소를 막아줌을 확인하였다. 여기서 H
20
2는 활성산소를 증가시키는 자극제를 나타내고, NAC는 ROS 스캐빈저(Scavenger)를 나타낸다.In addition, FIGS. 10 and 11 are graphs showing the measurement results of Reactive Oxygen Species (ROS), and it was confirmed through FIG. 10 that ROS was artificially increased in NK cells to decrease ROS after MTAM1 treatment, and FIG. 11 As a result of measuring ROS after culturing NK cells in a medium containing MTAM1 for 14 days, it was confirmed that melatonin blocks free radicals. Here, H 2 0 2 represents a stimulator that increases active oxygen, and NAC represents a ROS scavenger.
세포표면항원 분석에서는, 멜라토닌, 진세노사이드 및 β-글루칸으로 이루어진 면역세포 활성화 물질을 처리한 뒤 배양 및 수확된 세포는 CD3-CD56+CD16+의 표현형을 갖는 NK 세포가 74.2%, NKT 세포가 7.9%, T 세포가 12.7%의 비율로 구성된 것을 확인할 수 있었다 (도 12). 이는 도 6의 무처리한 대조군에서의 결과와 비교해 보더라도, 상기 면역세포 활성화 물질의 처리로 인해 수확된 세포 내 NK 세포 및 NKT 세포의 비율이 현저히 증가한 것을 알 수 있었다.In cell surface antigen analysis, cells cultured and harvested after treatment with an immune cell activating substance consisting of melatonin, ginsenoside and β-glucan were 74.2% of NK cells and 7.9 of NKT cells having a phenotype of CD3-CD56+CD16+. %, it was confirmed that the T cells consisted of 12.7% (FIG. 12). This can be seen that the ratio of NK cells and NKT cells in the harvested cells significantly increased due to the treatment of the immune cell activating substance, even when compared with the results in the untreated control group of FIG. 6.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.As described above, specific parts of the present invention have been described in detail, and it is clear that these specific techniques are only preferred embodiments for those of ordinary skill in the art, and the scope of the present invention is not limited thereto. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.
본 발명은 면역세포 활성화 물질 및 이를 이용한 면역세포의 활성화 방법에 관한 것이다. 보다 구체적으로는, 면역세포의 배양 시 상기 면역세포와 함께 배양함으로써 상기 면역세포의 면역 활성을 증진시킬 수 있는 조성물에 관한 것이다.The present invention relates to a substance for activating immune cells and a method for activating immune cells using the same. More specifically, it relates to a composition capable of enhancing the immune activity of the immune cells by culturing them together with the immune cells when culturing the immune cells.
Claims (14)
- 면역세포를 하기 화학식 1 또는 2로 표시되는 화합물, 멜라토닌 또는 이의 조합과 배양하는 단계; 및Culturing immune cells with a compound represented by the following Formula 1 or 2, melatonin, or a combination thereof; And상기 배양물로부터 면역세포를 분리하는 단계를 포함하는, 상기 면역세포를 유효성분으로 포함하는 세포치료제의 제조 방법:A method for producing a cell therapy product comprising the immune cells as an active ingredient, comprising the step of isolating immune cells from the culture:[화학식 1][Formula 1]
[화학식 2][Formula 2]
- 제1항에 있어서, 상기 면역세포는 림프구인 것인, 세포치료제의 제조 방법.The method of claim 1, wherein the immune cells are lymphocytes.
- 제1항에 있어서, 상기 면역세포는 자연살해세포(natural killer cells)인 것인, 세포치료제의 제조 방법.The method of claim 1, wherein the immune cells are natural killer cells.
- 제1항에 있어서, 상기 화학식 1 또는 2로 표시되는 화합물은 단리된 자연살해세포와 배양하여 이의 면역 활성을 증진시키는 것인, 세포치료제의 제조 방법.The method of claim 1, wherein the compound represented by Formula 1 or 2 is cultured with isolated natural killer cells to enhance their immune activity.
- 제1항에 있어서, 상기 면역세포의 면역 활성을 증진시키기 위한, 세포치료제의 제조 방법.The method for producing a cell therapy according to claim 1, for enhancing the immune activity of the immune cells.
- 제1항에 있어서, 상기 세포치료제는 암 치료 또는 예방용인 것인, 세포치료제의 제조 방법.The method of claim 1, wherein the cell therapy agent is for the treatment or prevention of cancer.
- 제5항에 있어서, 상기 면역 활성은 암세포 또는 감염세포에 대한 세포살상능(cytotoxicity)인 것인, 세포치료제의 제조 방법.The method of claim 5, wherein the immune activity is cytotoxicity against cancer cells or infected cells.
- 제6항 또는 제7항에 있어서, 상기 암은 췌장암, 간암, 폐암, 대장암, 유방암, 전립선암, 난소암, 대장암, 자궁경부암, 갑상선암, 후두암, 백혈병, 뇌종양, 신경모세포종, 망막모세포종, 두경부암, 침샘암, 림프종으로 구성된 군으로부터 선택되는, 세포치료제의 제조 방법.The method of claim 6 or 7, wherein the cancer is pancreatic cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, colon cancer, cervical cancer, thyroid cancer, laryngeal cancer, leukemia, brain tumor, neuroblastoma, retinoblastoma, A method for producing a cell therapy product selected from the group consisting of head and neck cancer, salivary gland cancer and lymphoma.
- 제1항에 있어서, 상기 분리된 면역세포를 세포치료제로 제형화하는 단계를 더 포함하는, 세포치료제의 제조 방법.The method of claim 1, further comprising formulating the isolated immune cells into a cell therapy agent.
- 하기 화학식 1 또는 2로 표시되는 화합물, 멜라토닌 또는 이의 조합을 포함하는, 세포치료제의 치료 효과 증진용 조성물로서,As a composition for enhancing the therapeutic effect of a cell therapy agent, comprising a compound represented by the following Formula 1 or 2, melatonin, or a combination thereof,상기 세포치료제는 면역세포를 유효성분으로 포함하고, 암 치료 또는 예방용이고,The cell therapy agent contains immune cells as an active ingredient, and is for cancer treatment or prevention,상기 세포치료제와 접촉함으로써, 상기 면역세포의 면역 활성을 증진시키는 것인, 세포치료제의 치료 효과 증진용 조성물:A composition for enhancing the therapeutic effect of a cell therapy agent, which enhances the immune activity of the immune cells by contacting the cell therapy agent:[화학식 1][Formula 1]
[화학식 2][Formula 2]
- 제10항에 있어서, 상기 세포치료제를 필요로 하는 개체에 사용하기 전에, 우선적으로 상기 세포치료제와 혼합되는 것인, 세포치료제의 치료 효과 증진용 조성물.The composition for enhancing the therapeutic effect of the cell therapy according to claim 10, wherein the cell therapy agent is first mixed with the cell therapy agent before use in an individual in need thereof.
- 제10항에 있어서, 상기 화학식 1 또는 2로 표시되는 화합물은 1 μM 내지 100 μM으로 포함된 것인, 세포치료제의 치료 효과 증진용 조성물.The composition of claim 10, wherein the compound represented by Formula 1 or 2 is contained in an amount of 1 μM to 100 μM.
- 하기 화학식 1 또는 2로 표시되는 화합물, 멜라토닌 또는 이의 조합을 포함하는 제1 유닛; 및 면역세포를 유효성분으로 포함하는 세포치료제를 포함하는 제2 유닛을 포함하는, 키트.A first unit comprising a compound represented by the following Formula 1 or 2, melatonin, or a combination thereof; And, a kit comprising a second unit comprising a cell therapy product comprising immune cells as an active ingredient.
- 제13항에 있어서, 상기 제1 유닛 및 제2 유닛은 분리되어 있고, 혼합되어 상기 제2 유닛의 면역세포의 면역 활성을 증진시키는 것인, 키트.The kit according to claim 13, wherein the first unit and the second unit are separated and mixed to enhance the immune activity of the immune cells of the second unit.
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