WO2023038492A1 - Biological composition for preventing or treating cancer, comprising dendritic cells, natural killer cells and cytotoxic t cells - Google Patents
Biological composition for preventing or treating cancer, comprising dendritic cells, natural killer cells and cytotoxic t cells Download PDFInfo
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- WO2023038492A1 WO2023038492A1 PCT/KR2022/013616 KR2022013616W WO2023038492A1 WO 2023038492 A1 WO2023038492 A1 WO 2023038492A1 KR 2022013616 W KR2022013616 W KR 2022013616W WO 2023038492 A1 WO2023038492 A1 WO 2023038492A1
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- cells
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4613—Natural-killer cells [NK or NK-T]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4615—Dendritic cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
- A61K39/4622—Antigen presenting cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
Definitions
- the present invention relates to a pharmaceutical composition for preventing or treating cancer containing dendritic cells, natural killer cells and cytotoxic T cells.
- Cancer is a disease that ranks first in mortality in Koreans and has a steadily increasing incidence. It causes enormous losses to the Korean economy every year due to the suspension of economic activities, treatment costs, and deaths of cancer patients, so it is important to develop appropriate diagnosis and treatment methods. do.
- Conventional treatments for cancer include surgery, radiation therapy, and chemotherapy, but radiation therapy and chemotherapy have come to the fore with serious side effects.
- it is intended to develop an immune cell therapy, which is one of the therapies targeting the mechanism of avoiding immune action, which is one of the characteristics of cancer.
- Immune cell therapy currently under clinical study in various cancers includes natural killer cells, dendritic cells, and T cells.
- natural killer cells only a few patients respond to the above treatment and show complete remission, and in most cases, the cancer cell killing mechanism of immune cell therapy is avoided, so the success rate is not high.
- interactions between natural killer cells, dendritic cells, and T cells have been reported.
- Natural killer cells directly or indirectly regulate the response of T cells, and dendritic cells enhance the cytolytic function of natural killer cells, and natural killer cells secrete chemokines to assist the function of dendritic cells.
- cancer cells do not express 100% of the major histocompatibility complex, which is a self-recognition marker.
- T cell therapy of the adaptive immune system alone is not enough, and complex treatment of natural killer cells, an autoimmune system, is required.
- the need for combined treatment with immune cell therapy has emerged, and recently, new clinical trials and papers have reported the results of observing the effect of multiple cell therapies in parallel.
- treatment by a simple combination of natural killer cells or T cells has not been effective in the research results of the present inventors. A special culture of these cells gave good results.
- dendritic cells, natural killer cells, and T cells are concurrently treated to express anticancer effects.
- An object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer.
- An object of the present invention is to provide a method for preparing a pharmaceutical composition for preventing or treating cancer.
- a pharmaceutical composition for preventing or treating cancer containing dendritic cells, natural killer cells and cytotoxic T cells.
- cytokine is selected from the group consisting of IL-2 (Interleukin-2), IFN- ⁇ (Interferon- ⁇ ), IL-15, IL-7, IL-21 and 4-1BB ligand At least one pharmaceutical composition.
- IL-2 Interleukin-2
- IFN- ⁇ Interferon- ⁇
- IL-15 IL-15
- IL-7 Interferon- ⁇
- 4-1BB ligand At least one pharmaceutical composition.
- solid cancer is colorectal cancer, bile duct cancer, gastric cancer, lung cancer, liver cancer, rectal cancer, breast cancer, prostate cancer, skin cancer, head and neck cancer, pancreatic cancer, ovarian cancer, bladder cancer, kidney cancer, melanoma, small cell lung cancer, arsenic One selected from the group consisting of cell lung cancer, sarcoma, glioma, T-cell lymphoma and B-cell lymphoma, a pharmaceutical composition.
- PBMCs peripheral blood mononuclear cells
- step (b) isolating CD14+ mononuclear cells and CD14- mononuclear cells from the PBMCs isolated in step (a);
- step (c) amplifying and culturing natural killer cells from the PBMCs isolated in step (a);
- cytokine is selected from the group consisting of IL-2 (Interleukin-2), IFN- ⁇ (Interferon- ⁇ ), IL-15, IL-7, IL-21 and 4-1BB ligand At least one manufacturing method.
- IL-2 Interleukin-2
- IFN- ⁇ Interferon- ⁇
- IL-15 IL-15
- IL-7 Interferon- ⁇
- 4-1BB ligand At least one manufacturing method.
- step (g) a portion of the mature dendritic cells obtained in step (g) and the CD8+ naive T cells are co-cultured at a cell number ratio of 1:8 to 1:12. manufacturing method.
- step (h) the mature dendritic cells and the natural killer cells are co-cultured at a cell number ratio of 0.01:1 to 0.03:1.
- step (h) the cytotoxic T cells and the natural killer cells are co-cultured at a cell number ratio of 8:1 to 12:1.
- step (h) the mature dendritic cells and the cytotoxic T cells are co-cultured at a cell number ratio of 0.01:10 to 0.03:10.
- step (h) is cultured in a medium containing cytokines.
- cytokine is selected from the group consisting of Interleukin-2 (IL-2), Interferon- ⁇ (IFN- ⁇ ), IL-15, IL-7, IL-21, and 4-1BB ligand At least one manufacturing method.
- IL-2 Interleukin-2
- IFN- ⁇ Interferon- ⁇
- IL-15 Interleukin-15
- IL-7 Interferon- ⁇
- IL-21 Interleukin-21
- 4-1BB ligand At least one manufacturing method.
- step (i) is cultured in a medium containing cytokines.
- cytokine is selected from the group consisting of Interleukin-2 (IL-2), Interferon- ⁇ (IFN- ⁇ ), IL-15, IL-7, IL-21, and 4-1BB ligand At least one manufacturing method.
- IL-2 Interleukin-2
- IFN- ⁇ Interferon- ⁇
- IL-15 Interleukin-15
- IL-7 Interferon- ⁇
- IL-21 Interleukin-21
- 4-1BB ligand At least one manufacturing method.
- solid cancer is colorectal cancer, bile duct cancer, gastric cancer, lung cancer, liver cancer, rectal cancer, breast cancer, prostate cancer, skin cancer, head and neck cancer, pancreatic cancer, ovarian cancer, bladder cancer, kidney cancer, melanoma, small cell lung cancer, arsenic One selected from the group consisting of cell lung cancer, sarcoma, glioma, T-cell lymphoma and B-cell lymphoma, the manufacturing method.
- the tumor antigen is a protein extract derived from the cancer patient's tissue or a neoantigen peptide obtained through genome analysis.
- the pharmaceutical composition of the present invention including dendritic cells, natural killer cells and cytotoxic T cells, exhibits superior anticancer effects compared to the case of using each cell alone.
- the pharmaceutical composition of the present invention includes dendritic cells, natural killer cells, and cytotoxic T cells co-cultured with each other, it exhibits superior anticancer effects compared to the case of using each cell alone or in a simple mixture.
- Figure 1 is a schematic diagram of the manufacturing process of the preparation example.
- Figure 2 shows the results confirming that the amount of IL-12 (p70) secretion increased in mature dendritic cells compared to immature dendritic cells.
- Figure 3 is a result confirming that the degree of IFN- ⁇ expression is higher in CD8+ naive T cells co-cultured with mature dendritic cells than in non-co-cultured T cells.
- FIG. 4 shows the culture pattern of CD8+ naive T cells activated by co-culture with mature dendritic cells.
- 5a to 5d show cell populations expressing activating receptors or inhibitory receptors according to the natural killer cell culture process.
- Example 10 to 12 show apoptosis of cancer cells in Example 5 (FIG. 10 and FIG. 11) and Example 6 (FIG. 12) when the three cells were treated in combination compared to single dendritic cells, natural killer cells, or cytotoxic T cells. This is the result of confirming that the effect is excellent and that the cancer cell killing effect is excellent when cytokine pre-activation is performed compared to when it is not performed.
- the present invention relates to a pharmaceutical composition for preventing or treating cancer.
- the pharmaceutical composition of the present invention includes dendritic cells, natural killer cells and cytotoxic T cells.
- the composition of the present invention has superior cancer prevention or treatment effects compared to the case of using each cell alone or the case of using two types of cells.
- At least one of dendritic cells, natural killer cells, and cytotoxic T cells may be derived from a cancer prevention or treatment target.
- the ratio of the numbers of dendritic cells and natural killer cells may be, for example, 0.01:1 to 0.03:1, 0.015:1 to 0.025:1, or 0.018:1 to 0.022:1, but is not limited thereto.
- the ratio of the number of cytotoxic T cells and natural killer cells may be, for example, 8:1 to 12:1, 8.5:1 to 11.5:1, or 9:1 to 11:1, but is not limited thereto.
- At least one of dendritic cells, natural killer cells, and cytotoxic T cells may be cultured in a medium containing cytokines.
- the cytokine may be, for example, at least one selected from the group consisting of Interleukin-2 (IL-2), Interferon- ⁇ (IFN- ⁇ ), IL-15, IL-7, IL-21, and 4-1BB ligand. However, it is not limited thereto.
- the concentration of the cytokine is, for example, 40 U/mL to 5000 U/mL, 60 U/mL to 5000 U/mL, 80 U/mL to 5000 U/mL, 100 U/mL to 5000 U/mL, 200 U/mL to 4200 U/mL, 400 U/mL to 3200 U/mL, 600 U/mL to 2200 U/mL, 800 U/mL to 1200 U/mL, 3000 U/mL to 5000 U/mL, 3200 It may be U/mL to 4800 U/mL, 3500 U/mL to 4500 U/mL, or 3800 U/mL to 42000 U/mL, but is not limited thereto.
- the concentration of IL-2 is, for example, 100 U/mL to 5000 U/mL, 200 U/mL to 4200 U/mL, 400 U/mL to 3200 U/mL.
- the concentration of IFN- ⁇ is, for example, 40 U/mL to 4000 U/mL (2 ng/mL to 20 ng/mL), 60 U/mL to 3600 U/mL (3 ng/mL to 18 ng/mL), 80 U/mL to 3200 U/mL (4 ng/mL to 16 ng/mL), 100 U/mL to 2800 U/mL (5 ng/mL mL to 14 ng/mL) or 140 U/mL to 2400 U/mL (7 ng/mL to 12 ng/mL), but is not limited thereto.
- Dendritic cells, natural killer cells, and cytotoxic T cells may be co-cultured with each other.
- the effect of preventing or treating cancer is excellent compared to the case where each cell is cultured alone and then used in combination.
- Dendritic cells and natural killer cells may be co-cultured at a cell number ratio of, for example, 0.01:1 to 0.03:1, 0.015:1 to 0.025:1, or 0.018:1 to 0.022:1, but are limited thereto. It is not.
- Cytotoxic T cells and natural killer cells may be co-cultured at a cell number ratio of, for example, 8:1 to 12:1, 8.5:1 to 11.5:1, or 9:1 to 11:1. It is not limited. Dendritic cells and cytotoxic T cells may be co-cultured at a cell number ratio of, for example, 0.01:10 to 0.03:10, 0.015:10 to 0.025:10, or 0.018:10 to 0.022:10, but is limited thereto. it is not going to be
- Cancer is not limited as long as a preventive or therapeutic effect is expected when using immune cells, and may be, for example, solid cancer.
- Solid cancers include, for example, colorectal cancer, bile duct cancer, gastric cancer, lung cancer, liver cancer, rectal cancer, breast cancer, prostate cancer, skin cancer, head and neck cancer, pancreatic cancer, ovarian cancer, bladder cancer, kidney cancer, melanoma, small cell lung cancer, non-small cell lung cancer, and sarcoma.
- prevention refers to prophylactic measures that result in any degree of reduction in the likelihood of developing a condition to be prevented or a recurrence or recurrent condition, including minor, substantial or major reduction in the likelihood of developing or recurring the condition, as well as total prevention. Refers to a measure, and the degree of likelihood reduction is at least a minor reduction.
- treatment refers to treatment that results in a beneficial effect on a subject or patient suffering from the condition being treated, including not only cure but also relief of any degree, including minor, substantial, major relief, the degree of relief being At least a slight relief.
- the pharmaceutical composition of the present invention may be formulated and used in the form of a suspension, emulsion, and sterile injection solution according to a conventional method, respectively, but is not limited thereto.
- Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, dextrin, maltodextrin, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants, but is not limited thereto.
- Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, and lyophilized formulations.
- Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.
- the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.
- pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type, severity, drug activity, It may be determined according to factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field.
- the pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by a person skilled in the art.
- An effective amount in the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, and body weight. However, since it may increase or decrease according to the route of administration, severity of disease, sex, weight, age, etc., the dosage is not limited to the scope of the present invention in any way.
- the present invention relates to a method for preparing a pharmaceutical composition for preventing or treating cancer.
- the manufacturing method of the present invention includes the following steps (a) to (h).
- PBMCs peripheral blood mononuclear cells
- step (b) isolating CD14+ mononuclear cells and CD14- mononuclear cells from the PBMCs isolated in step (a);
- step (c) amplifying and culturing natural killer cells from PBMCs isolated in step (a);
- Dendritic cells, natural killer cells, cytotoxic T cells, and cancer may be within the above range, but are not limited thereto.
- the portion of mature dendritic cells and CD8+ naive T cells obtained in step (g) is 1:8 to 1:12, 1:8.5 to 1:11.5, 1:9 to 1:11 or 1:9.5 to 1 : It may be co-cultured at a cell number ratio of 10.5, but is not limited thereto.
- naive T cells can be activated to obtain cytotoxic T cells.
- mature dendritic cells and natural killer cells may be co-cultured at a cell number ratio of, for example, 0.01:1 to 0.03:1, 0.015:1 to 0.025:1, or 0.018:1 to 0.022:1. It may be, but is not limited thereto.
- the cytotoxic T cells and natural killer cells are co-cultured at a cell number ratio of, for example, 8:1 to 12:1, 8.5:1 to 11.5:1, or 9:1 to 11:1. It may be, but is not limited thereto.
- step (h) mature dendritic cells and cytotoxic T cells are co-cultured at a cell number ratio of, for example, 0.01:10 to 0.03:10, 0.015:10 to 0.025:10, or 0.018:10 to 0.022:10. It may be, but is not limited thereto.
- the co-cultivation in step (h) may be culturing in a medium containing cytokines.
- step (h) when the mature dendritic cells, cytotoxic T cells, and natural killer cells are co-cultured in a medium containing cytokines, an optimal condition for co-culture with cancer cells in step (i) can be created.
- the cytokine is not limited as long as it is used for co-cultivation of immune cells and cancer cells, but, for example, IL-2 (Interleukin-2), IFN- ⁇ (Interferon- ⁇ ), IL-15, IL- It may be at least one selected from the group consisting of 7, IL-21 and 4-1BB ligands.
- the concentration of the cytokine is, for example, 40 U/mL to 5000 U/mL, 60 U/mL to 5000 U/mL, 80 U/mL to 5000 U/mL, 100 U/mL to 5000 U/mL, 200 U/mL to 4200 U/mL, 400 U/mL to 3200 U/mL, 600 U/mL to 2200 U/mL, 800 U/mL to 1200 U/mL, 3000 U/mL to 5000 U/mL, 3200 It may be U/mL to 4800 U/mL, 3500 U/mL to 4500 U/mL, or 3800 U/mL to 42000 U/mL, but is not limited thereto.
- the concentration of IL-2 is, for example, 100 U/mL to 5000 U/mL, 200 U/mL to 4200 U/mL, 400 U/mL to 3200 U/mL.
- the concentration of IFN- ⁇ is, for example, 40 U/mL to 4000 U/mL (2 ng/mL to 20 ng/mL), 60 U/mL to 3600 U/mL (3 ng/mL to 18 ng/mL), 80 U/mL to 3200 U/mL (4 ng/mL to 16 ng/mL), 100 U/mL to 2800 U/mL (5 ng/mL mL to 14 ng/mL) or 140 U/mL to 2400 U/mL (7 ng/mL to 12 ng/mL), but is not limited thereto.
- the co-cultivation in step (i) may be culturing in a medium containing cytokines.
- the cytokine may be, for example, at least one selected from the group consisting of Interleukin-2 (IL-2), Interferon- ⁇ (IFN- ⁇ ), IL-15, IL-7, IL-21, and 4-1BB ligand. there is.
- the concentration of the cytokine is, for example, 200 U/mL to 400 U/mL, 220 U/mL to 380 U/mL, 240 U/mL to 360 U/mL, 260 U/mL to 340 U/mL or 280 U/mL. It may be U/mL to 320 U/mL, but is not limited thereto.
- the tumor antigen is not limited as long as it can mature immature dendritic cells, and may be, for example, a protein extract derived from a cancer patient's tissue or a neoantigen peptide obtained through genome analysis, but is not limited thereto.
- the present invention relates to a method for preventing or treating cancer.
- the treatment method of the present invention includes administering a pharmaceutical composition comprising dendritic cells, natural killer cells and cytotoxic T cells to a subject in need thereof.
- the method of the present invention has a superior cancer prevention or treatment effect compared to the case of using each cell alone or the case of using two types of cells by administering a pharmaceutical composition containing three types of immune cells to a subject in need thereof. .
- the pharmaceutical composition may be within the above range, but is not limited thereto.
- the subject may be a human and/or non-human animal.
- the subject may be diagnosed as a cancer patient or may include subjects at risk of cancer, but is not limited thereto.
- the cancer may be within the above range, but is not limited thereto.
- This preparation example was prepared according to the process of I to V below. 1 is a schematic diagram of the manufacturing process of this preparation example. In this preparation example, blood and cancer cells obtained from colorectal cancer patients were used.
- CD8 Microbeads (20 uL per 10 7 cells), mix gently and incubate in refrigerator (2-8°C) for 15 minutes.
- tubes containing the sample (ori tube), labeled cell fractions (pos tube), and tubes for collecting unlabeled cell fractions (neg tube) into the tube rack. Arrange the tubes as marked on the tube rack. Place the rack in the sample port.
- Peripheral blood mononuclear cells isolated for natural killer cell proliferation were co-cultured with irradiated K562 feeder cells in the presence of IL-2 and IL-21 in a 37°C incubator for 7 days.
- cytotoxicity using CCK-8 and surface receptor analysis of NK cells are performed using the K562 cell line as a target.
- CD14-positive monocytes were isolated from the blood of a colorectal cancer patient (SDH21028), differentiated into immature dendritic cells, and then loaded with peptides to mature dendritic cells.
- ELISA was performed to measure the amount of IL-12 (p70) secreted by mature dendritic cells in the group treated with the peptide in immature dendritic cells and the group not treated with the peptide. As a result, it was confirmed that the level of IL-12 (p70) secretion was higher in the group treated with the peptide (mDC in FIG. 2) than in the group not treated with the peptide (iDC in FIG. 2) (FIG. 2).
- CD8+ naive T cells were isolated from the blood of a patient (SDH21028), cultured, and then co-cultured with mature dendritic cells.
- the expression level of IFN- ⁇ an activated T cell marker, was measured in the group not co-cultured with mature dendritic cells and the group co-cultured. As a result, it was confirmed that the level of IFN- ⁇ expression was higher in the group co-cultured with mature dendritic cells (mDC/T cell in FIG. 3) than in the group not co-cultured with mature dendritic cells (T cell only in FIG. 3). (Fig. 3).
- CD8+ naive T cells and mDC mature dendritic cells were co-cultured to observe the culture pattern of activated T cells.
- mDC + TC in FIG. 4 when co-cultured (mDC + TC in FIG. 4) compared to the case where mature dendritic cells were not co-cultured (T cell only in FIG. 4), activated T cells due to mature dendritic cells clustered and grew more It was confirmed to do (FIG. 4).
- Peripheral blood mononuclear cells were isolated from the blood of a patient (SDH21028) and natural killer cells were cultured.
- the percentage of natural killer cells which initially accounted for 16.3% when the patient's peripheral blood mononuclear cells were cultured, increased to 98.8% over time, and the number increased approximately 251-fold.
- CCK-8 assay was performed to confirm the anticancer effect of the three immune cells obtained in the above preparation example on cancer cells.
- Primary tumor cells are seeded in a 96 well plate at 1x10 4 cells/100 ul/well, followed by incubation for 24 hours.
- Cytokine pre-activation was performed on the immune cells prior to co-cultivation of the prepared immune cells and target cells.
- the three immune cells were cultured alone for 24 hours in phenol red free RPMI supplemented with IL-2 at a concentration of 6000 units/ml.
- the three immune cells were co-cultured for 24 hours in phenol red free RPMI supplemented with IL-2 at a concentration of 4000 unit/ml or 6000 unit/ml.
- 100 ul of the existing culture medium was removed from the tumor cells obtained in (1) above, and 100 ul of the three immune cells of the above (2) were treated alone or jointly per well, and then cultured at 37 ° C for 24 hours.
- cytotoxicity of NK, T, and DC alone or in combination was confirmed as a target for primary cancer cells of SDH21029 colorectal cancer patients (FIG. 6).
- DC, T, and NK showed cytotoxicity of about 18%, 28%, and 40% (DC, T, and NK groups in FIG. 6).
- the combination group of the three immune cells pre-activated with 4000 U/ml IL-2 (4000 group in FIG. 6)
- about 57% of cytotoxicity was confirmed
- cytotoxicity of about 47% was confirmed.
- Example 1 The same as the method of Example 1 above, but the cytokine pre-activation in step 1-(2) of Example 1 was all performed using IL-2 at a concentration of 4000 units/ml.
- a cytotoxicity test on cancer cells was performed using DC, T, and NK cells alone or in combination of SDH21032 colorectal cancer patients pre-activated with IL-2 as effector cells (FIG. 7).
- T and NK showed cytotoxicity of about 67% and 91% (T and NK groups in FIG. 7).
- the group in which all three cells were pre-activated showed the highest cytotoxicity.
- step 1-(2) of Example 1 was performed using IL-2 at a concentration of 4000 units/ml and IFN- ⁇ at a concentration of 10 ng/ml. performed.
- a cytotoxicity test on cancer cells was performed using DC, T, and NK cells alone or in combination as effector cells of SDH21032 colorectal cancer patients pre-activated with IL-2 and IFN- ⁇ (FIG. 8).
- T and NK showed cytotoxicity of about 60% and 81% (T and NK groups in FIG. 8).
- the highest cytotoxicity was shown in the pre-activation group of the three cells (combination group in FIG. 8).
- step 1-(2) of Example 1 was performed using IL-2 at a concentration of 4000 units/ml and IFN- ⁇ at a concentration of 10 ng/ml. performed.
- a cytotoxicity test on cancer cells was performed using DC, T, and NK cells alone or in combination as effector cells of SDH21028 colorectal cancer patients pre-activated with IL-2 and IFN- ⁇ (FIG. 9).
- T and NK showed cytotoxicity of about 18% and 49% (T and NK groups in FIG. 9).
- the highest cytotoxicity was shown in the pre-activation group of the three cells (Combination group in FIG. 9).
- step 1-(2) of Example 1 was all performed using IL-2 at a concentration of 4000 units/ml, and the cytokine pre-activation was performed A combination group of three immune cells that were not tested was added.
- a combination therapy assay of DC, T, and NK cells was performed with cancer cells of SDH21054 colorectal cancer patients as targets.
- T and NK showed similar cytotoxicity
- the group pre-activated with three cells Pre-activated DCTNK group in FIG. 10)
- the highest cytotoxicity was confirmed.
- the combination group of cells without pre-activation confirmed cytotoxicity of about 20%.
- step 1-(2) of Example 1 was all performed using IL-2 at a concentration of 4000 units/ml, and the cytokine pre-activation was performed A combination group of three immune cells that were not tested was added.
- IL-2 was treated at a concentration of 300 units/ml and then cultured. The experiment was performed twice in total.
- a combination therapy assay of DC, T, and NK cells was performed with cancer cells of SDH21054 colorectal cancer patients as targets.
- T and NK showed similar cytotoxicity
- Pre-activated DCTNK group in FIG. 11 the group pre-activated with three cells
- the highest cytotoxicity of about 80% was confirmed.
- about 55% of cytotoxicity was confirmed in the combination group of non-pre-activated cells (Non DCTNK group in FIG. 11).
- a combination therapy assay of DC, T, and NK cells was performed with cancer cells of SDH21054 colorectal cancer patients as targets.
- T and NK showed similar cytotoxicity
- Pre-activated DCTNK group in FIG. 12 the group in which all three cells were pre-activated
- the highest cytotoxicity of about 80% was confirmed.
- about 55% of cytotoxicity was confirmed in the combination group of non-pre-activating cells (Non DCTNK group in FIG. 12).
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Abstract
The present invention relates to a pharmaceutical composition for preventing or treating cancer and, more specifically, the present invention comprises dendritic cells, natural killer cells and cytotoxic T cells, and thus exhibits anticancer effects that are better than those of when each type of cell is used alone. In addition, if a pharmaceutical composition of the present invention comprises co-cultured dendritic cells, natural killer cells and cytotoxic T cells, anticancer effects that are better than those of when each type of cell is used alone or used in a simple combination are exhibited.
Description
본 발명은 수지상 세포, 자연 살해 세포 및 세포 독성 T 세포를 포함하는 암의 예방 또는 치료용 약학 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating cancer containing dendritic cells, natural killer cells and cytotoxic T cells.
암은 한국인에서 사망률 1위를 차지하고 꾸준히 발생률이 증가하고 있는 질병으로 암 환자의 경제활동 중지, 치료비용, 사망 등으로 인해 해마다 한국 경제에 엄청난 손실을 초래하고 있어 적절한 진단 및 치료 방법의 개발이 중요하다. 암의 전통적인 치료법은 수술, 방사선 치료 및 화학적 항암 요법 등이 있으나 방사선 치료 및 화학적 항암 요법의 경우 심한 부작용을 야기하는 문제점이 대두되어 왔으며 암이 재발이 되어 진행된 경우에는 기존의 치료법으로는 치료가 힘들 수도 있다. 본 발명에서는 암의 특징 중의 하나인 면역 작용을 회피하는 기전을 타겟으로 하는 치료법 중 하나인 면역세포치료법을 개발하고자 한다.Cancer is a disease that ranks first in mortality in Koreans and has a steadily increasing incidence. It causes enormous losses to the Korean economy every year due to the suspension of economic activities, treatment costs, and deaths of cancer patients, so it is important to develop appropriate diagnosis and treatment methods. do. Conventional treatments for cancer include surgery, radiation therapy, and chemotherapy, but radiation therapy and chemotherapy have come to the fore with serious side effects. may be In the present invention, it is intended to develop an immune cell therapy, which is one of the therapies targeting the mechanism of avoiding immune action, which is one of the characteristics of cancer.
현재 다양한 암에서 임상 연구 진행 중인 면역세포치료제는 크게 자연 살해 세포, 수지상세포, T 세포 등이 있다. 하지만 극히 일부의 환자만이 위 치료에 반응하여 완전관해를 보이고 대부분의 경우 면역세포치료의 암세포 살상기전을 회피하여 성공률이 높지 않다. 최근 연구에서 자연 살해 세포, 수지상세포 및 T 세포의 각 세포간의 상호작용이 보고되고 있다. 자연 살해 세포는 직간접적인 방법으로 T 세포의 반응을 조절하며 수지상세포가 자연 살해 세포의 세포 융해 기능을 강화시키며 자연 살해 세포가 케모카인을 분비하여 수지상세포의 기능을 보조한다. 또한, 암세포는 자기 인식 마커인 주조직 적합성 복합체를 100% 발현하지는 않는다. 이런 경우는 적응 면역계의 T세포 요법만으로는 안되며 자가 면역계인 자연 살해 세포의 복합 치료가 필요하다. 이러한 상호작용을 살펴보았을 때, 면역세포치료의 병용 치료의 필요성이 대두되어 최근 새로운 임상시험이나 논문에서 복수의 세포치료를 병행하여 그 효과를 관찰한 결과를 보고하고 있다. 그러나 단순히 자연 살해 세포나 T 세포의 단순 배합에 의한 치료는 그 동안 본 발명자들의 연구 결과에는 효과가 없었다. 이 세포들의 특수 배양을 통해 좋은 결과를 얻었다. 본 발명에서는 수지상세포, 자연 살해 세포, T 세포를 동시에 병용 처리하여 항암 효과를 발현하고자 한다.Immune cell therapy currently under clinical study in various cancers includes natural killer cells, dendritic cells, and T cells. However, only a few patients respond to the above treatment and show complete remission, and in most cases, the cancer cell killing mechanism of immune cell therapy is avoided, so the success rate is not high. In recent studies, interactions between natural killer cells, dendritic cells, and T cells have been reported. Natural killer cells directly or indirectly regulate the response of T cells, and dendritic cells enhance the cytolytic function of natural killer cells, and natural killer cells secrete chemokines to assist the function of dendritic cells. In addition, cancer cells do not express 100% of the major histocompatibility complex, which is a self-recognition marker. In this case, T cell therapy of the adaptive immune system alone is not enough, and complex treatment of natural killer cells, an autoimmune system, is required. When looking at these interactions, the need for combined treatment with immune cell therapy has emerged, and recently, new clinical trials and papers have reported the results of observing the effect of multiple cell therapies in parallel. However, treatment by a simple combination of natural killer cells or T cells has not been effective in the research results of the present inventors. A special culture of these cells gave good results. In the present invention, dendritic cells, natural killer cells, and T cells are concurrently treated to express anticancer effects.
본 발명은 암의 예방 또는 치료용 약학 조성물을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer.
본 발명은 암의 예방 또는 치료용 약학 조성물의 제조 방법을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a method for preparing a pharmaceutical composition for preventing or treating cancer.
1. 수지상 세포, 자연 살해 세포 및 세포 독성 T 세포를 포함하는 암의 예방 또는 치료용 약학 조성물.1. A pharmaceutical composition for preventing or treating cancer containing dendritic cells, natural killer cells and cytotoxic T cells.
2. 위 1에 있어서, 상기 수지상 세포, 상기 자연 살해 세포 및 상기 세포 독성 T 세포 중 적어도 하나는 상기 암의 예방 또는 치료 대상으로부터 유래된 것인, 약학 조성물.2. The pharmaceutical composition according to 1 above, wherein at least one of the dendritic cells, the natural killer cells, and the cytotoxic T cells is derived from a subject for prevention or treatment of cancer.
3. 위 1에 있어서, 상기 수지상 세포 및 상기 자연 살해 세포의 수의 비율은 0.01:1 내지 0.03:1인 것인, 약학 조성물.3. The pharmaceutical composition according to 1 above, wherein the number ratio of the dendritic cells and the natural killer cells is 0.01:1 to 0.03:1.
4. 위 1에 있어서, 상기 세포 독성 T세포 및 상기 자연 살해 세포의 수의 비율은 8:1 내지 12:1인 것인, 약학 조성물.4. The pharmaceutical composition according to 1 above, wherein the ratio of the numbers of the cytotoxic T cells to the natural killer cells is 8:1 to 12:1.
5. 위 1에 있어서, 상기 수지상 세포, 상기 자연 살해 세포 및 상기 세포 독성 T 세포 중 적어도 하나는 사이토카인을 포함하는 배지에서 배양된 것인, 약학 조성물.5. The pharmaceutical composition according to 1 above, wherein at least one of the dendritic cells, the natural killer cells, and the cytotoxic T cells is cultured in a medium containing cytokines.
6. 위 5에 있어서, 상기 사이토카인은 IL-2(Interleukin-2), IFN-γ(Interferon-γ), IL-15, IL-7, IL-21 및 4-1BB 리간드로 이루어진 군에서 선택된 적어도 하나인, 약학 조성물.6. The method of 5 above, wherein the cytokine is selected from the group consisting of IL-2 (Interleukin-2), IFN-γ (Interferon-γ), IL-15, IL-7, IL-21 and 4-1BB ligand At least one pharmaceutical composition.
7. 위 5에 있어서, 상기 사이토카인의 농도는 40 U/mL 내지 5000 U/mL인, 약학 조성물.7. The pharmaceutical composition according to 5 above, wherein the concentration of the cytokine is 40 U/mL to 5000 U/mL.
8. 위 6에 있어서, 상기 IL-2(Interleukin-2)의 농도는 100 U/mL 내지 5000 U/mL인, 약학 조성물.8. The pharmaceutical composition according to 6 above, wherein the IL-2 (Interleukin-2) concentration is 100 U/mL to 5000 U/mL.
9. 위 6에 있어서, 상기 IFN-γ(Interferon-γ)의 농도는 40 U/mL 내지 4000 U/mL인, 약학 조성물.9. The pharmaceutical composition according to 6 above, wherein the concentration of IFN-γ (Interferon-γ) is 40 U/mL to 4000 U/mL.
10. 위 1에 있어서, 상기 수지상 세포, 상기 자연 살해 세포 및 상기 세포 독성 T 세포는 서로 공동 배양된 것인, 약학 조성물.10. The pharmaceutical composition according to 1 above, wherein the dendritic cells, the natural killer cells, and the cytotoxic T cells are co-cultured with each other.
11. 위 10에 있어서, 상기 수지상 세포 및 상기 자연 살해 세포는 0.01:1 내지 0.03:1의 세포 수의 비율로 공동 배양된 것인, 약학 조성물.11. The pharmaceutical composition according to 10 above, wherein the dendritic cells and the natural killer cells are co-cultured at a cell number ratio of 0.01:1 to 0.03:1.
12. 위 10에 있어서, 상기 세포 독성 T세포 및 상기 자연 살해 세포는 8:1 내지 12:1의 세포 수의 비율로 공동 배양된 것인, 약학 조성물.12. The pharmaceutical composition according to 10 above, wherein the cytotoxic T cells and the natural killer cells are co-cultured at a cell number ratio of 8:1 to 12:1.
13. 위 10에 있어서, 상기 수지상 세포 및 상기 세포 독성 T세포는 0.01:10 내지 0.03:10의 세포 수의 비율로 공동 배양된 것인, 약학 조성물.13. The pharmaceutical composition according to 10 above, wherein the dendritic cells and the cytotoxic T cells are co-cultured at a cell number ratio of 0.01:10 to 0.03:10.
14. 위 1에 있어서, 상기 암은 고형암인, 약학 조성물.14. The pharmaceutical composition according to 1 above, wherein the cancer is solid cancer.
15. 위 14에 있어서, 상기 고형암은 대장암, 담관암, 위암, 폐암, 간암, 직장암, 유방암, 전립선암, 피부암, 두경부암, 췌장암, 난소암, 방광암, 신장암, 흑색종, 소세포폐암, 비소세포폐암, 육종, 신경아교종, T-세포 림프종 및 B-세포 림프종으로 이루어진 군에서 선택된 하나인, 약학 조성물.15. The solid cancer according to 14 above, wherein the solid cancer is colorectal cancer, bile duct cancer, gastric cancer, lung cancer, liver cancer, rectal cancer, breast cancer, prostate cancer, skin cancer, head and neck cancer, pancreatic cancer, ovarian cancer, bladder cancer, kidney cancer, melanoma, small cell lung cancer, arsenic One selected from the group consisting of cell lung cancer, sarcoma, glioma, T-cell lymphoma and B-cell lymphoma, a pharmaceutical composition.
16. (a) 암 환자의 혈액으로부터 말초 혈액 단핵 세포(PBMC, peripheral blood mononuclear cell)를 분리하는 단계;16. (a) isolating peripheral blood mononuclear cells (PBMCs) from the blood of cancer patients;
(b) 상기 (a) 단계에서 분리된 PBMC로부터 CD14+ 단핵 세포 및 CD14- 단핵 세포를 분리하는 단계;(b) isolating CD14+ mononuclear cells and CD14- mononuclear cells from the PBMCs isolated in step (a);
(c) 상기 (a) 단계에서 분리된 PBMC로부터 자연 살해 세포를 증폭 배양하는 단계;(c) amplifying and culturing natural killer cells from the PBMCs isolated in step (a);
(d) 상기 CD14+ 단핵 세포로부터 미성숙 수지상 세포를 분화시키는 단계;(d) differentiating immature dendritic cells from the CD14+ mononuclear cells;
(e) 상기 CD14- 단핵 세포로부터 CD8+ 나이브(naive) T 세포를 분리하는 단계;(e) separating CD8+ naive T cells from the CD14- mononuclear cells;
(f) 상기 미성숙 수지상 세포에 상기 암 환자로부터 분리된 종양 항원을 처리하여 성숙 수지상 세포를 수득하는 단계;(f) obtaining mature dendritic cells by treating the immature dendritic cells with a tumor antigen isolated from the cancer patient;
(g) 상기 수득된 성숙 수지상 세포의 일부 및 상기 CD8+ 나이브(naive) T 세포를 공동 배양하여 세포 독성 T 세포를 수득하는 단계;(g) co-culturing a portion of the obtained mature dendritic cells and the CD8+ naive T cells to obtain cytotoxic T cells;
(h) 상기 세포 독성 T 세포를 수득하는 단계에 이용되지 않은 성숙 수지상 세포, 상기 세포 독성 T 세포 및 상기 자연 살해 세포를 공동 배양하여 사전 활성화(pre-activation)하는 단계; 및(h) co-cultivating and pre-activating mature dendritic cells, the cytotoxic T cells, and the natural killer cells, which are not used in the step of obtaining the cytotoxic T cells; and
(i) 상기 사전 활성화된 수지상 세포, 세포 독성 T 세포 및 자연 살해 세포를 상기 암의 예방 또는 치료 대상으로부터 유래된 암 세포와 공동 배양하여 활성화(activation)하는 단계;를 포함하는 암의 예방 또는 치료용 약학 조성물의 제조 방법.(i) co-cultivating and activating the pre-activated dendritic cells, cytotoxic T cells, and natural killer cells with cancer cells derived from a subject for prevention or treatment of cancer; A method for preparing a pharmaceutical composition for use.
17. 위 16에 있어서, 상기 수지상 세포, 상기 자연 살해 세포 및 상기 세포 독성 T 세포 중 적어도 하나는 사이토카인을 포함하는 배지에서 배양된 것인, 제조 방법.17. The method according to 16 above, wherein at least one of the dendritic cells, the natural killer cells, and the cytotoxic T cells is cultured in a medium containing cytokines.
18. 위 17에 있어서, 상기 사이토카인은 IL-2(Interleukin-2), IFN-γ(Interferon-γ), IL-15, IL-7, IL-21 및 4-1BB 리간드로 이루어진 군에서 선택된 적어도 하나인, 제조 방법.18. The method of 17 above, wherein the cytokine is selected from the group consisting of IL-2 (Interleukin-2), IFN-γ (Interferon-γ), IL-15, IL-7, IL-21 and 4-1BB ligand At least one manufacturing method.
19. 위 17에 있어서, 상기 사이토카인의 농도는 40 U/mL 내지 5000 U/mL인, 제조 방법.19. The method according to 17 above, wherein the concentration of the cytokine is 40 U/mL to 5000 U/mL.
20. 위 18에 있어서, 상기 IL-2(Interleukin-2)의 농도는 100 U/mL 내지 5000 U/mL인, 제조 방법.20. The method according to 18 above, wherein the IL-2 (Interleukin-2) concentration is 100 U/mL to 5000 U/mL.
21. 위 18에 있어서, 상기 IFN-γ(Interferon-γ)의 농도는 40 U/mL 내지 4000 U/mL인, 제조 방법.21. The method according to 18 above, wherein the concentration of IFN-γ (Interferon-γ) is 40 U/mL to 4000 U/mL.
22. 위 16에 있어서, 상기 (g) 단계에서 상기 수득된 성숙 수지상 세포의 일부 및 상기 CD8+ 나이브(naive) T 세포는 1:8 내지 1:12의 세포 수의 비율로 공동 배양된 것인, 제조 방법.22. The method according to 16 above, wherein a portion of the mature dendritic cells obtained in step (g) and the CD8+ naive T cells are co-cultured at a cell number ratio of 1:8 to 1:12. manufacturing method.
23. 위 16에 있어서, 상기 (h) 단계에서 상기 성숙 수지상 세포 및 상기 자연 살해 세포는 0.01:1 내지 0.03:1의 세포 수의 비율로 공동 배양된 것인, 제조 방법.23. The method according to 16 above, wherein in step (h), the mature dendritic cells and the natural killer cells are co-cultured at a cell number ratio of 0.01:1 to 0.03:1.
24. 위 16에 있어서, 상기 (h) 단계에서 상기 세포 독성 T세포 및 상기 자연 살해 세포는 8:1 내지 12:1의 세포 수의 비율로 공동 배양된 것인, 제조 방법.24. The method according to 16 above, wherein in step (h), the cytotoxic T cells and the natural killer cells are co-cultured at a cell number ratio of 8:1 to 12:1.
25. 위 16에 있어서, 상기 (h) 단계에서 상기 성숙 수지상 세포 및 상기 세포 독성 T세포는 0.01:10 내지 0.03:10의 세포 수의 비율로 공동 배양된 것인, 제조 방법.25. The method according to 16 above, wherein in step (h), the mature dendritic cells and the cytotoxic T cells are co-cultured at a cell number ratio of 0.01:10 to 0.03:10.
26. 위 16에 있어서, 상기 (h) 단계의 공동 배양은 사이토카인을 포함하는 배지에서 배양하는 것인, 제조 방법.26. The method according to 16 above, wherein the co-culture in step (h) is cultured in a medium containing cytokines.
27. 위 26에 있어서, 상기 사이토카인은 IL-2(Interleukin-2), IFN-γ(Interferon-γ), IL-15, IL-7, IL-21 및 4-1BB 리간드로 이루어진 군에서 선택된 적어도 하나인, 제조 방법.27. The method of 26 above, wherein the cytokine is selected from the group consisting of Interleukin-2 (IL-2), Interferon-γ (IFN-γ), IL-15, IL-7, IL-21, and 4-1BB ligand At least one manufacturing method.
28. 위 26에 있어서, 상기 사이토카인의 농도는 40 U/mL 내지 5000 U/mL인, 제조 방법.28. The method according to 26 above, wherein the concentration of the cytokine is 40 U/mL to 5000 U/mL.
29. 위 27에 있어서, 상기 IL-2(Interleukin-2)의 농도는 100 U/mL 내지 5000 U/mL인, 제조 방법.29. The method according to 27 above, wherein the IL-2 (Interleukin-2) concentration is 100 U/mL to 5000 U/mL.
30. 위 27에 있어서, 상기 IFN-γ(Interferon-γ)의 농도는 40 U/mL 내지 4000 U/mL인, 제조 방법.30. The method according to 27 above, wherein the concentration of Interferon-γ (IFN-γ) is 40 U/mL to 4000 U/mL.
31. 위 16에 있어서, 상기 (i) 단계의 공동 배양은 사이토카인을 포함하는 배지에서 배양하는 것인, 제조 방법.31. The method according to 16 above, wherein the co-culture in step (i) is cultured in a medium containing cytokines.
32. 위 31에 있어서, 상기 사이토카인은 IL-2(Interleukin-2), IFN-γ(Interferon-γ), IL-15, IL-7, IL-21 및 4-1BB 리간드로 이루어진 군에서 선택된 적어도 하나인, 제조 방법.32. The method of 31 above, wherein the cytokine is selected from the group consisting of Interleukin-2 (IL-2), Interferon-γ (IFN-γ), IL-15, IL-7, IL-21, and 4-1BB ligand At least one manufacturing method.
33. 위 31에 있어서, 상기 사이토카인의 농도는 200 U/mL 내지 400 U/mL인, 제조 방법.33. The method according to 31 above, wherein the concentration of the cytokine is 200 U/mL to 400 U/mL.
34. 위 16에 있어서, 상기 암은 고형암인, 제조 방법.34. The method according to 16 above, wherein the cancer is a solid cancer.
35. 위 34에 있어서, 상기 고형암은 대장암, 담관암, 위암, 폐암, 간암, 직장암, 유방암, 전립선암, 피부암, 두경부암, 췌장암, 난소암, 방광암, 신장암, 흑색종, 소세포폐암, 비소세포폐암, 육종, 신경아교종, T-세포 림프종 및 B-세포 림프종으로 이루어진 군에서 선택된 하나인, 제조 방법.35. The solid cancer according to 34 above, wherein the solid cancer is colorectal cancer, bile duct cancer, gastric cancer, lung cancer, liver cancer, rectal cancer, breast cancer, prostate cancer, skin cancer, head and neck cancer, pancreatic cancer, ovarian cancer, bladder cancer, kidney cancer, melanoma, small cell lung cancer, arsenic One selected from the group consisting of cell lung cancer, sarcoma, glioma, T-cell lymphoma and B-cell lymphoma, the manufacturing method.
36. 위 16에 있어서, 상기 종양 항원은 상기 암 환자 조직 유래 단백질 추출물 또는 유전체 분석을 통해 수득한 신생 항원 펩타이드인, 제조 방법.36. The method according to 16 above, wherein the tumor antigen is a protein extract derived from the cancer patient's tissue or a neoantigen peptide obtained through genome analysis.
본 발명의 약학 조성물은 수지상 세포, 자연 살해 세포 및 세포 독성 T 세포를 포함하여, 각 세포를 단독으로 이용하는 경우에 비해 우수한 항암 효과를 나타낸다.The pharmaceutical composition of the present invention, including dendritic cells, natural killer cells and cytotoxic T cells, exhibits superior anticancer effects compared to the case of using each cell alone.
또한, 본 발명의 약학 조성물이 서로 공동 배양된 수지상 세포, 자연 살해 세포 및 세포 독성 T 세포를 포함하는 경우, 각 세포를 단독 혹은 단순 혼합으로 이용하는 경우에 비해 우수한 항암 효과를 나타낸다.In addition, when the pharmaceutical composition of the present invention includes dendritic cells, natural killer cells, and cytotoxic T cells co-cultured with each other, it exhibits superior anticancer effects compared to the case of using each cell alone or in a simple mixture.
도 1은 제조예의 제조 과정을 도식화한 것이다.Figure 1 is a schematic diagram of the manufacturing process of the preparation example.
도 2는 성숙 수지상 세포에서 미성숙 수지상 세포에 비해 IL-12(p70) 분비량이 증가한 것을 확인한 결과이다.Figure 2 shows the results confirming that the amount of IL-12 (p70) secretion increased in mature dendritic cells compared to immature dendritic cells.
도 3은 성숙 수지상 세포와 공동 배양한 CD8+ naive T 세포에서 공동 배양하지 않은 T 세포에 비해 IFN-γ 발현 정도가 높은 것을 확인한 결과이다.Figure 3 is a result confirming that the degree of IFN-γ expression is higher in CD8+ naive T cells co-cultured with mature dendritic cells than in non-co-cultured T cells.
도 4는 성숙 수지상 세포와 공동 배양하여 활성화된 CD8+ naive T 세포의 배양 양상을 나타낸 것이다.4 shows the culture pattern of CD8+ naive T cells activated by co-culture with mature dendritic cells.
도 5a 내지 도 5d는 자연 살해 세포 배양 과정에 따른 activating receptor 또는 inhibitory receptor 발현 세포 군집을 나타낸 것이다.5a to 5d show cell populations expressing activating receptors or inhibitory receptors according to the natural killer cell culture process.
도 6 내지 10은 실시예 1 내지 실시예 4에서 단독의 수지상 세포, 자연 살해 세포 또는 세포 독성 T 세포를 처리한 경우에 비해 세 가지 세포를 병용 처리한 경우 암 세포 사멸 효과가 우수하다는 것을 확인한 결과이다.6 to 10 are results confirming that the cancer cell killing effect is excellent when the three cells are treated in combination compared to the case where dendritic cells, natural killer cells, or cytotoxic T cells are treated alone in Examples 1 to 4 am.
도 10 내지 12는 실시예 5(도 10 및 도 11) 및 실시예 6(도 12)에서 단독의 수지상 세포, 자연 살해 세포 또는 세포 독성 T 세포에 비해 세 가지 세포를 병용 처리한 경우 암 세포 사멸 효과가 우수하다는 것과 사이토카인 pre-activation을 수행하지 않은 경우에 비해 수행한 경우 암 세포 사멸 효과가 우수하다는 것을 확인한 결과이다.10 to 12 show apoptosis of cancer cells in Example 5 (FIG. 10 and FIG. 11) and Example 6 (FIG. 12) when the three cells were treated in combination compared to single dendritic cells, natural killer cells, or cytotoxic T cells. This is the result of confirming that the effect is excellent and that the cancer cell killing effect is excellent when cytokine pre-activation is performed compared to when it is not performed.
이하, 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 암의 예방 또는 치료용 약학 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating cancer.
본 발명의 약학 조성물은 수지상 세포, 자연 살해 세포 및 세포 독성 T 세포를 포함한다.The pharmaceutical composition of the present invention includes dendritic cells, natural killer cells and cytotoxic T cells.
본 발명의 조성물은 세 종류의 면역 세포를 포함함으로써 각 세포를 단독으로 이용하는 경우나 두 종류의 세포를 이용하는 경우에 비해 우수한 암의 예방 또는 치료 효과가 있다.By including three types of immune cells, the composition of the present invention has superior cancer prevention or treatment effects compared to the case of using each cell alone or the case of using two types of cells.
수지상 세포, 자연 살해 세포 및 세포 독성 T 세포 중 적어도 하나는 상기 암의 예방 또는 치료 대상으로부터 유래된 것일 수 있다.At least one of dendritic cells, natural killer cells, and cytotoxic T cells may be derived from a cancer prevention or treatment target.
수지상 세포 및 자연 살해 세포의 수의 비율은 예를 들면, 0.01:1 내지 0.03:1, 0.015:1 내지 0.025:1 또는 0.018:1 내지 0.022:1일 수 있으나, 이에 제한되는 것은 아니다.The ratio of the numbers of dendritic cells and natural killer cells may be, for example, 0.01:1 to 0.03:1, 0.015:1 to 0.025:1, or 0.018:1 to 0.022:1, but is not limited thereto.
세포 독성 T세포 및 자연 살해 세포의 수의 비율은 예를 들면, 8:1 내지 12:1, 8.5:1 내지 11.5:1 또는 9:1 내지 11:1일 수 있으나, 이에 제한되는 것은 아니다.The ratio of the number of cytotoxic T cells and natural killer cells may be, for example, 8:1 to 12:1, 8.5:1 to 11.5:1, or 9:1 to 11:1, but is not limited thereto.
수지상 세포, 자연 살해 세포 및 세포 독성 T 세포 중 적어도 하나는 사이토카인을 포함하는 배지에서 배양된 것일 수 있다. 사이토카인은 예를 들면, IL-2(Interleukin-2), IFN-γ(Interferon-γ), IL-15, IL-7, IL-21 및 4-1BB 리간드로 이루어진 군에서 선택된 적어도 하나일 수 있으나, 이에 제한되는 것은 아니다. 사이토카인의 농도는 예를 들면, 40 U/mL 내지 5000 U/mL, 60 U/mL 내지 5000 U/mL, 80 U/mL 내지 5000 U/mL, 100 U/mL 내지 5000 U/mL, 200 U/mL 내지 4200 U/mL, 400 U/mL 내지 3200 U/mL, 600 U/mL 내지 2200 U/mL, 800 U/mL 내지 1200 U/mL, 3000 U/mL 내지 5000 U/mL, 3200 U/mL 내지 4800 U/mL, 3500 U/mL 내지 4500 U/mL 또는 3800 U/mL 내지 42000 U/mL일 수 있으나, 이에 제한되는 것은 아니다. 사이토카인이 IL-2(Interleukin-2)인 경우, IL-2의 농도는 예를 들면, 100 U/mL 내지 5000 U/mL, 200 U/mL 내지 4200 U/mL, 400 U/mL 내지 3200 U/mL, 600 U/mL 내지 2200 U/mL, 800 U/mL 내지 1200 U/mL, 3000 U/mL 내지 5000 U/mL, 3200 U/mL 내지 4800 U/mL, 3500 U/mL 내지 4500 U/mL 또는 3800 U/mL 내지 42000 U/mL일 수 있으나, 이에 제한되는 것은 아니다. 사이토카인이 IFN-γ(Interferon-γ)인 경우, IFN-γ의 농도는 예를 들면, 40 U/mL 내지 4000 U/mL(2 ng/mL 내지 20 ng/mL), 60 U/mL 내지 3600 U/mL(3 ng/mL 내지 18 ng/mL), 80 U/mL 내지 3200 U/mL(4 ng/mL 내지 16 ng/mL), 100 U/mL 내지 2800 U/mL(5 ng/mL 내지 14 ng/mL) 또는 140 U/mL 내지 2400 U/mL(7 ng/mL 내지 12 ng/mL)일 수 있으나, 이에 제한되는 것은 아니다.At least one of dendritic cells, natural killer cells, and cytotoxic T cells may be cultured in a medium containing cytokines. The cytokine may be, for example, at least one selected from the group consisting of Interleukin-2 (IL-2), Interferon-γ (IFN-γ), IL-15, IL-7, IL-21, and 4-1BB ligand. However, it is not limited thereto. The concentration of the cytokine is, for example, 40 U/mL to 5000 U/mL, 60 U/mL to 5000 U/mL, 80 U/mL to 5000 U/mL, 100 U/mL to 5000 U/mL, 200 U/mL to 4200 U/mL, 400 U/mL to 3200 U/mL, 600 U/mL to 2200 U/mL, 800 U/mL to 1200 U/mL, 3000 U/mL to 5000 U/mL, 3200 It may be U/mL to 4800 U/mL, 3500 U/mL to 4500 U/mL, or 3800 U/mL to 42000 U/mL, but is not limited thereto. When the cytokine is IL-2 (Interleukin-2), the concentration of IL-2 is, for example, 100 U/mL to 5000 U/mL, 200 U/mL to 4200 U/mL, 400 U/mL to 3200 U/mL. U/mL, 600 U/mL to 2200 U/mL, 800 U/mL to 1200 U/mL, 3000 U/mL to 5000 U/mL, 3200 U/mL to 4800 U/mL, 3500 U/mL to 4500 It may be U/mL or 3800 U/mL to 42000 U/mL, but is not limited thereto. When the cytokine is IFN-γ (Interferon-γ), the concentration of IFN-γ is, for example, 40 U/mL to 4000 U/mL (2 ng/mL to 20 ng/mL), 60 U/mL to 3600 U/mL (3 ng/mL to 18 ng/mL), 80 U/mL to 3200 U/mL (4 ng/mL to 16 ng/mL), 100 U/mL to 2800 U/mL (5 ng/mL mL to 14 ng/mL) or 140 U/mL to 2400 U/mL (7 ng/mL to 12 ng/mL), but is not limited thereto.
수지상 세포, 자연 살해 세포 및 세포 독성 T 세포는 서로 공동 배양된 것일 수 있다. 수지상 세포, 자연 살해 세포 및 세포 독성 T 세포를 공동 배양하는 경우, 각 세포를 단독 배양 후 병용하여 이용하는 경우에 비해 암의 예방 또는 치료 효과가 우수하다. 수지상 세포 및 자연 살해 세포는 예를 들면, 0.01:1 내지 0.03:1, 0.015:1 내지 0.025:1 또는 0.018:1 내지 0.022:1의 세포 수의 비율로 공동 배양된 것일 수 있으나, 이에 제한되는 것은 아니다. 세포 독성 T세포 및 자연 살해 세포는 예를 들면, 8:1 내지 12:1, 8.5:1 내지 11.5:1 또는 9:1 내지 11:1의 세포 수의 비율로 공동 배양된 것일 수 있으나, 이에 제한되는 것은 아니다. 수지상 세포 및 세포 독성 T세포는 예를 들면, 0.01:10 내지 0.03:10, 0.015:10 내지 0.025:10 또는 0.018:10 내지 0.022:10의 세포 수의 비율로 공동 배양된 것일 수 있으나, 이에 제한되는 것은 아니다.Dendritic cells, natural killer cells, and cytotoxic T cells may be co-cultured with each other. In the case of co-culturing dendritic cells, natural killer cells, and cytotoxic T cells, the effect of preventing or treating cancer is excellent compared to the case where each cell is cultured alone and then used in combination. Dendritic cells and natural killer cells may be co-cultured at a cell number ratio of, for example, 0.01:1 to 0.03:1, 0.015:1 to 0.025:1, or 0.018:1 to 0.022:1, but are limited thereto. It is not. Cytotoxic T cells and natural killer cells may be co-cultured at a cell number ratio of, for example, 8:1 to 12:1, 8.5:1 to 11.5:1, or 9:1 to 11:1. It is not limited. Dendritic cells and cytotoxic T cells may be co-cultured at a cell number ratio of, for example, 0.01:10 to 0.03:10, 0.015:10 to 0.025:10, or 0.018:10 to 0.022:10, but is limited thereto. it is not going to be
암은 면역 세포를 이용하는 경우 예방 또는 치료 효과가 예상되는 것이라면 제한되지 않고, 예를 들면, 고형암일 수 있다. 고형암은 예를 들면, 대장암, 담관암, 위암, 폐암, 간암, 직장암, 유방암, 전립선암, 피부암, 두경부암, 췌장암, 난소암, 방광암, 신장암, 흑색종, 소세포폐암, 비소세포폐암, 육종, 신경아교종, T-세포 림프종 및 B-세포 림프종으로 이루어진 군에서 선택된 하나일 수 있으나, 이에 제한되는 것은 아니다.Cancer is not limited as long as a preventive or therapeutic effect is expected when using immune cells, and may be, for example, solid cancer. Solid cancers include, for example, colorectal cancer, bile duct cancer, gastric cancer, lung cancer, liver cancer, rectal cancer, breast cancer, prostate cancer, skin cancer, head and neck cancer, pancreatic cancer, ovarian cancer, bladder cancer, kidney cancer, melanoma, small cell lung cancer, non-small cell lung cancer, and sarcoma. , may be one selected from the group consisting of glioma, T-cell lymphoma, and B-cell lymphoma, but is not limited thereto.
용어 "예방"은 전체 예방 뿐만 아니라 병태의 발병 또는 재발병의 가능성의 경미한, 실질적인 또는 큰 감소를 포함하여 예방될 병태 또는 재발생 또는 재발하는 병태의 발병 가능성의 임의의 정도의 감소를 초래하는 예방적 조치를 지칭하고, 가능성 감소의 정도는 적어도 경미한 감소이다.The term “prevention” refers to prophylactic measures that result in any degree of reduction in the likelihood of developing a condition to be prevented or a recurrence or recurrent condition, including minor, substantial or major reduction in the likelihood of developing or recurring the condition, as well as total prevention. Refers to a measure, and the degree of likelihood reduction is at least a minor reduction.
용어 "치료"는 치유뿐만 아니라 경미한 완화, 실질적인 완화, 주요 완화를 포함하는 임의의 정도의 완화를 포함하여 치료될 병태를 앓고 있는 대상체 또는 환자에게 유리한 효과를 초래하는 처치를 지칭하고, 완화 정도는 적어도 경미한 완화이다.The term "treatment" refers to treatment that results in a beneficial effect on a subject or patient suffering from the condition being treated, including not only cure but also relief of any degree, including minor, substantial, major relief, the degree of relief being At least a slight relief.
본 발명의 약학적 조성물은, 각각 통상의 방법에 따라 현탁액, 에멀젼, 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있으나, 이에 제한되지 않는다.The pharmaceutical composition of the present invention may be formulated and used in the form of a suspension, emulsion, and sterile injection solution according to a conventional method, respectively, but is not limited thereto.
조성물에 함유될 수 있는 담체, 부형제 및 희석제로는 락토오즈, 덱스트로즈, 수크로스, 덱스트린, 말토덱스트린, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있으나, 이에 제한되지 않는다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면 활성제 등의 희석제 또는 부형제를 사용하여 조제되나, 이에 제한되지 않는다.Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, dextrin, maltodextrin, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants, but is not limited thereto.
비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다.Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, and lyophilized formulations. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.
본 발명의 약학적 조성물은 약제학적으로 유효한 양으로 투여한다. 본 발명에서, "약제학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와 순차적으로 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 통상의 기술자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type, severity, drug activity, It may be determined according to factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by a person skilled in the art.
본 발명의 약학적 조성물에서 유효량은 환자의 나이, 성별, 체중에 따라 달라질 수 있다. 그러나, 투여 경로, 질병의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.An effective amount in the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, and body weight. However, since it may increase or decrease according to the route of administration, severity of disease, sex, weight, age, etc., the dosage is not limited to the scope of the present invention in any way.
본 발명은 암의 예방 또는 치료용 약학 조성물의 제조 방법에 관한 것이다.The present invention relates to a method for preparing a pharmaceutical composition for preventing or treating cancer.
본 발명의 제조 방법은 하기 (a) 내지 (h) 단계를 포함한다.The manufacturing method of the present invention includes the following steps (a) to (h).
(a) 암 환자의 혈액으로부터 말초 혈액 단핵 세포(PBMC, peripheral blood mononuclear cell)를 분리하는 단계;(a) isolating peripheral blood mononuclear cells (PBMCs) from the blood of cancer patients;
(b) 상기 (a) 단계에서 분리된 PBMC로부터 CD14+ 단핵 세포 및 CD14- 단핵 세포를 분리하는 단계;(c) 상기 (a) 단계에서 분리된 PBMC로부터 자연 살해 세포를 증폭 배양하는 단계;(b) isolating CD14+ mononuclear cells and CD14- mononuclear cells from the PBMCs isolated in step (a); (c) amplifying and culturing natural killer cells from PBMCs isolated in step (a);
(d) 상기 CD14+ 단핵 세포로부터 미성숙 수지상 세포를 분화시키는 단계;(d) differentiating immature dendritic cells from the CD14+ mononuclear cells;
(e) 상기 CD14- 단핵 세포로부터 CD8+ 나이브(naive) T 세포를 분리하는 단계;(e) separating CD8+ naive T cells from the CD14- mononuclear cells;
(f) 상기 미성숙 수지상 세포에 상기 암 환자로부터 분리된 종양 항원을 처리하여 성숙 수지상 세포를 수득하는 단계;(f) obtaining mature dendritic cells by treating the immature dendritic cells with a tumor antigen isolated from the cancer patient;
(g) 상기 수득된 성숙 수지상 세포의 일부 및 상기 CD8+ 나이브(naive) T 세포를 공동 배양하여 세포 독성 T 세포를 수득하는 단계;(g) co-culturing a portion of the obtained mature dendritic cells and the CD8+ naive T cells to obtain cytotoxic T cells;
(h) 상기 세포 독성 T 세포를 수득하는 단계에 이용되지 않은 성숙 수지상 세포, 상기 세포 독성 T 세포 및 상기 자연 살해 세포를 공동 배양하여 사전 활성화(pre-activation)하는 단계; 및(h) co-cultivating and pre-activating mature dendritic cells, the cytotoxic T cells, and the natural killer cells, which are not used in the step of obtaining the cytotoxic T cells; and
(i) 상기 사전 활성화된 수지상 세포, 세포 독성 T 세포 및 자연 살해 세포를 상기 암의 예방 또는 치료 대상으로부터 유래된 암 세포와 공동 배양하여 활성화(activation)하는 단계.(i) co-cultivating and activating the pre-activated dendritic cells, cytotoxic T cells, and natural killer cells with cancer cells derived from a target for preventing or treating cancer.
수지상 세포, 자연 살해 세포, 세포 독성 T 세포 및 암은 전술한 범위 내의 것일 수 있으나, 이에 제한되는 것은 아니다.Dendritic cells, natural killer cells, cytotoxic T cells, and cancer may be within the above range, but are not limited thereto.
(g) 단계에서 수득된 성숙 수지상 세포의 일부 및 CD8+ 나이브(naive) T 세포는 1:8 내지 1:12, 1:8.5 내지 1:11.5, 1:9 내지 1:11 또는 1:9.5 내지 1:10.5의 세포 수의 비율로 공동 배양된 것일 수 있으나, 이에 제한되는 것은 아니다. (g) 단계에 의해 나이브 T 세포는 활성화되어 세포 독성 T 세포를 수득할 수 있다.The portion of mature dendritic cells and CD8+ naive T cells obtained in step (g) is 1:8 to 1:12, 1:8.5 to 1:11.5, 1:9 to 1:11 or 1:9.5 to 1 : It may be co-cultured at a cell number ratio of 10.5, but is not limited thereto. By step (g), naive T cells can be activated to obtain cytotoxic T cells.
(h) 단계에서 성숙 수지상 세포 및 자연 살해 세포는 예를 들면, 0.01:1 내지 0.03:1, 0.015:1 내지 0.025:1 또는 0.018:1 내지 0.022:1의 세포 수의 비율로 공동 배양된 것일 수 있으나, 이에 제한되는 것은 아니다. (h) 단계에서 세포 독성 T세포 및 자연 살해 세포는 예를 들면, 8:1 내지 12:1, 8.5:1 내지 11.5:1 또는 9:1 내지 11:1의 세포 수의 비율로 공동 배양된 것일 수 있으나, 이에 제한되는 것은 아니다. (h) 단계에서 성숙 수지상 세포 및 세포 독성 T세포는 예를 들면, 0.01:10 내지 0.03:10, 0.015:10 내지 0.025:10 또는 0.018:10 내지 0.022:10의 세포 수의 비율로 공동 배양된 것일 수 있으나, 이에 제한되는 것은 아니다.In step (h), mature dendritic cells and natural killer cells may be co-cultured at a cell number ratio of, for example, 0.01:1 to 0.03:1, 0.015:1 to 0.025:1, or 0.018:1 to 0.022:1. It may be, but is not limited thereto. In step (h), the cytotoxic T cells and natural killer cells are co-cultured at a cell number ratio of, for example, 8:1 to 12:1, 8.5:1 to 11.5:1, or 9:1 to 11:1. It may be, but is not limited thereto. In step (h), mature dendritic cells and cytotoxic T cells are co-cultured at a cell number ratio of, for example, 0.01:10 to 0.03:10, 0.015:10 to 0.025:10, or 0.018:10 to 0.022:10. It may be, but is not limited thereto.
(h) 단계의 공동 배양은 사이토카인을 포함하는 배지에서 배양하는 것일 수 있다. (h) 단계에서 성숙 수지상 세포, 세포 독성 T 세포 및 자연 살해 세포를 사이토카인을 포함하는 배지에서 공동 배양하는 경우, (i) 단계의 암 세포와의 공동 배양을 위한 최적의 상태를 만들 수 있다. 사이토카인은 면역 세포들과 암 세포와의 공동 배양을 위해 이용되는 것이라면 제한되지 않으나, 예를 들면, IL-2(Interleukin-2), IFN-γ(Interferon-γ), IL-15, IL-7, IL-21 및 4-1BB 리간드로 이루어진 군에서 선택된 적어도 하나일 수 있다. 사이토카인의 농도는 예를 들면, 40 U/mL 내지 5000 U/mL, 60 U/mL 내지 5000 U/mL, 80 U/mL 내지 5000 U/mL, 100 U/mL 내지 5000 U/mL, 200 U/mL 내지 4200 U/mL, 400 U/mL 내지 3200 U/mL, 600 U/mL 내지 2200 U/mL, 800 U/mL 내지 1200 U/mL, 3000 U/mL 내지 5000 U/mL, 3200 U/mL 내지 4800 U/mL, 3500 U/mL 내지 4500 U/mL 또는 3800 U/mL 내지 42000 U/mL일 수 있으나, 이에 제한되는 것은 아니다. 사이토카인이 IL-2(Interleukin-2)인 경우, IL-2의 농도는 예를 들면, 100 U/mL 내지 5000 U/mL, 200 U/mL 내지 4200 U/mL, 400 U/mL 내지 3200 U/mL, 600 U/mL 내지 2200 U/mL, 800 U/mL 내지 1200 U/mL, 3000 U/mL 내지 5000 U/mL, 3200 U/mL 내지 4800 U/mL, 3500 U/mL 내지 4500 U/mL 또는 3800 U/mL 내지 42000 U/mL일 수 있으나, 이에 제한되는 것은 아니다. 사이토카인이 IFN-γ(Interferon-γ)인 경우, IFN-γ의 농도는 예를 들면, 40 U/mL 내지 4000 U/mL(2 ng/mL 내지 20 ng/mL), 60 U/mL 내지 3600 U/mL(3 ng/mL 내지 18 ng/mL), 80 U/mL 내지 3200 U/mL(4 ng/mL 내지 16 ng/mL), 100 U/mL 내지 2800 U/mL(5 ng/mL 내지 14 ng/mL) 또는 140 U/mL 내지 2400 U/mL(7 ng/mL 내지 12 ng/mL)일 수 있으나, 이에 제한되는 것은 아니다.The co-cultivation in step (h) may be culturing in a medium containing cytokines. In step (h), when the mature dendritic cells, cytotoxic T cells, and natural killer cells are co-cultured in a medium containing cytokines, an optimal condition for co-culture with cancer cells in step (i) can be created. . The cytokine is not limited as long as it is used for co-cultivation of immune cells and cancer cells, but, for example, IL-2 (Interleukin-2), IFN-γ (Interferon-γ), IL-15, IL- It may be at least one selected from the group consisting of 7, IL-21 and 4-1BB ligands. The concentration of the cytokine is, for example, 40 U/mL to 5000 U/mL, 60 U/mL to 5000 U/mL, 80 U/mL to 5000 U/mL, 100 U/mL to 5000 U/mL, 200 U/mL to 4200 U/mL, 400 U/mL to 3200 U/mL, 600 U/mL to 2200 U/mL, 800 U/mL to 1200 U/mL, 3000 U/mL to 5000 U/mL, 3200 It may be U/mL to 4800 U/mL, 3500 U/mL to 4500 U/mL, or 3800 U/mL to 42000 U/mL, but is not limited thereto. When the cytokine is IL-2 (Interleukin-2), the concentration of IL-2 is, for example, 100 U/mL to 5000 U/mL, 200 U/mL to 4200 U/mL, 400 U/mL to 3200 U/mL. U/mL, 600 U/mL to 2200 U/mL, 800 U/mL to 1200 U/mL, 3000 U/mL to 5000 U/mL, 3200 U/mL to 4800 U/mL, 3500 U/mL to 4500 It may be U/mL or 3800 U/mL to 42000 U/mL, but is not limited thereto. When the cytokine is IFN-γ (Interferon-γ), the concentration of IFN-γ is, for example, 40 U/mL to 4000 U/mL (2 ng/mL to 20 ng/mL), 60 U/mL to 3600 U/mL (3 ng/mL to 18 ng/mL), 80 U/mL to 3200 U/mL (4 ng/mL to 16 ng/mL), 100 U/mL to 2800 U/mL (5 ng/mL mL to 14 ng/mL) or 140 U/mL to 2400 U/mL (7 ng/mL to 12 ng/mL), but is not limited thereto.
(i) 단계의 공동 배양은 사이토카인을 포함하는 배지에서 배양하는 것일 수 있다. 사이토카인은 예를 들면, IL-2(Interleukin-2), IFN-γ(Interferon-γ), IL-15, IL-7, IL-21 및 4-1BB 리간드로 이루어진 군에서 선택된 적어도 하나일 수 있다. 사이토카인의 농도는 예를 들면, 200 U/mL 내지 400 U/mL, 220 U/mL 내지 380 U/mL, 240 U/mL 내지 360 U/mL, 260 U/mL 내지 340 U/mL 또는 280 U/mL 내지 320 U/mL일 수 있으나, 이에 제한되는 것은 아니다.The co-cultivation in step (i) may be culturing in a medium containing cytokines. The cytokine may be, for example, at least one selected from the group consisting of Interleukin-2 (IL-2), Interferon-γ (IFN-γ), IL-15, IL-7, IL-21, and 4-1BB ligand. there is. The concentration of the cytokine is, for example, 200 U/mL to 400 U/mL, 220 U/mL to 380 U/mL, 240 U/mL to 360 U/mL, 260 U/mL to 340 U/mL or 280 U/mL. It may be U/mL to 320 U/mL, but is not limited thereto.
종양 항원은 미성숙 수지상 세포를 성숙시킬 수 있는 것이라면 제한되지 않고, 예를 들면, 암 환자 조직 유래 단백질 추출물 또는 유전체 분석을 통해 수득한 신생 항원 펩타이드일 수 있으나, 이에 제한되는 것은 아니다.The tumor antigen is not limited as long as it can mature immature dendritic cells, and may be, for example, a protein extract derived from a cancer patient's tissue or a neoantigen peptide obtained through genome analysis, but is not limited thereto.
본 발명은 암의 예방 또는 치료 방법에 관한 것이다.The present invention relates to a method for preventing or treating cancer.
본 발명의 치료 방법은 수지상 세포, 자연 살해 세포 및 세포 독성 T세포를 포함하는 약학 조성물을 이를 필요로 하는 대상체에게 투여하는 단계를 포함한다.The treatment method of the present invention includes administering a pharmaceutical composition comprising dendritic cells, natural killer cells and cytotoxic T cells to a subject in need thereof.
본 발명의 방법은 세 종류의 면역 세포를 포함하는 약학 조성물을 이를 필요로 하는 대상체에게 투여함으로써 각 세포를 단독으로 이용하는 경우나 두 종류의 세포를 이용하는 경우에 비해 우수한 암의 예방 또는 치료 효과가 있다.The method of the present invention has a superior cancer prevention or treatment effect compared to the case of using each cell alone or the case of using two types of cells by administering a pharmaceutical composition containing three types of immune cells to a subject in need thereof. .
상기 약학 조성물은 전술한 범위 내의 것일 수 있으나, 이에 제한되는 것은 아니다.The pharmaceutical composition may be within the above range, but is not limited thereto.
상기 대상체는 인간 및/또는 인간을 제외한 동물일 수 있다.The subject may be a human and/or non-human animal.
상기 대상체는 암 환자로 진단되거나 암의 위험이 있는 대상체들이 포함될 수 있으나, 이에 제한되지 않는다.The subject may be diagnosed as a cancer patient or may include subjects at risk of cancer, but is not limited thereto.
상기 암은 전술한 범위 내의 것일 수 있으나, 이에 제한되는 것은 아니다.The cancer may be within the above range, but is not limited thereto.
이하, 본 발명을 구체적으로 설명하기 위해 실시예를 들어 상세하게 설명하기로 한다.Hereinafter, examples will be described in detail to explain the present invention in detail.
제조예manufacturing example
본 제조예는 하기 Ⅰ 내지 Ⅴ의 과정에 따라 제조되었다. 도 1은 본 제조예의 제조 과정을 도식화한 것이다. 본 제조예에서는 대장암 환자로부터 수득된 혈액 및 암 세포를 이용하였다.This preparation example was prepared according to the process of I to V below. 1 is a schematic diagram of the manufacturing process of this preparation example. In this preparation example, blood and cancer cells obtained from colorectal cancer patients were used.
Ⅰ. 말초 혈액 단핵구 분리I. Isolation of peripheral blood mononuclear cells
1. 5개의 heparin tube(약 50 ml)에 혈액을 채취한다.1. Collect blood into 5 heparin tubes (about 50 ml).
2. 50 ml tube 3개를 준비한다.2. Prepare three 50 ml tubes.
3. 혈액을 50 ml tube로 옮기고 혈액의 양을 확인한다.3. Transfer the blood to a 50 ml tube and check the amount of blood.
4. tube 벽에 묻지 않도록 주의하면서 두개의 50 ml tube 중간에 각각 15 ml의 Ficoll-pague를 주입한다.4. Inject 15 ml of Ficoll-pague into the middle of each of the two 50 ml tubes, being careful not to get on the tube wall.
5. 혈액을 혼합하여 Ficoll-paque가 포함된 두 개의 50 ml tube에 각각 동일한 양의 혈액을 Ficoll-pague 솔루션 위에 천천히 띄운다.5. Mix the blood and slowly float the same amount of blood on the Ficoll-pague solution in two 50 ml tubes containing Ficoll-paque.
6. 2300 rpm, 25분, ACC/DERT-10에서 원심 분리한다.6. Centrifuge at 2300 rpm, 25 min, ACC/DERT-10.
7. (PBMC 셀 층을 방해하지 않고) 10 ml 내지 15 ml 의 analogous plasma 를 분리하여 15ml tube에 넣고 deep freezer(-80°C) 에 보관한다.7. Separate 10 ml to 15 ml of analogous plasma (without disturbing the PBMC cell layer), put it in a 15 ml tube, and store in a deep freezer (-80°C).
8. 남아 있는 plasma와 버피 코트 층을 세 번째 50ml tube에 넣는다. 가능한 많은 버피 코트 층을 수집하고 tube 안에 buffy ficoll-plague 층에 피펫이 닿지 않도록 해야한다. 이후 원심 분리 과정에 영향을 줄 수 있다.8. Add the remaining plasma and buffy coat layer to a third 50 ml tube. Collect as many layers of buffy coat as possible and avoid touching the layer of buffy ficoll-plague with the pipette in the tube. It can affect the subsequent centrifugation process.
9. tube의 45ml 눈금에 맞춰지도록 DPBS를 채워준다. 그리고 800 xg, 5분, ACC 44, RT 원심 분리한다.9. Fill DPBS to the 45ml scale of the tube. And centrifuged at 800 xg, 5 min, ACC 44, RT.
10. (선택 사항) 만일 tube에 RBC가 있는 경우, RBC를 1-2ml RBC lysis buffer로 5분 내지 10분동안 용해시켜 준다. Media로 중화시키고 800 xg, 5분, ACC 44, RT에서 원심 분리한다.10. (Optional) If there are RBCs in the tube, dissolve the RBCs with 1-2ml RBC lysis buffer for 5 to 10 minutes. Neutralize with Media and centrifuge at 800 xg, 5 minutes, ACC 44, RT.
11. 상층액을 제거하고 1 mL의 DPBS를 tube에 넣고 피펫으로 부드럽게 섞는다. 29 mL의 DPBS를 추가하여 총 부피가 30 mL이 되도록 한다.11. Remove the supernatant, add 1 mL of DPBS to the tube, and mix gently with a pipette. Add 29 mL of DPBS to bring the total volume to 30 mL.
12. 셀 카운팅을 한다.12. Perform cell counting.
Ⅱ. CD14+ 단핵구 분리 후 미성숙 수지상세포 배양II. Culture of immature dendritic cells after isolation of CD14+ monocytes
1. PBMC를 1500 rpm 이상에서 5분간 원심 분리 후 상층액을 제거한다.1. Centrifuge the PBMCs at 1500 rpm or higher for 5 minutes and remove the supernatant.
2. 15 mL tube에 총 10 세포 당 80 μL의 P.E 버퍼에 세포 펠렛을 현탁한다. tube에 'ori'로 표시한다.2. Suspend the cell pellet in 80 μL of P.E buffer per 10 cells in a 15 mL tube. Mark the tube as 'ori'.
3. 총 10 셀 당 20 μL의 CD14 마이크로 비드를 추가한다. 잘 섞어 냉장고 (2-8 ℃)에서 15 분 동안 정치한다.3. Add 20 μL of CD14 microbeads per 10 total cells. Mix well and set in the refrigerator (2-8 ° C) for 15 minutes.
4. autoMACS ™ Pro Separator 기기를 준비하고 프라이밍한다.4. Prepare and prime the autoMACS™ Pro Separator.
5. 15 mL tube 2개를 준비하고 'pos', 'neg'로 라벨링한다.5. Prepare two 15 mL tubes and label them with 'pos' and 'neg'.
6. 샘플이 들어 있는 tube(ori tube)와 라벨이 있는 cell fractions (pos tube), 라벨이 없는 세포 fractions (neg tube)을 수집하기 위한 tube를 tube 랙에 넣는다. tube는 tube 랙에 표시된 대로 배열한다. 샘플 포트에 랙을 놓는다.6. Put the tubes containing the sample (ori tube), labeled cell fractions (pos tube), and tubes for collecting unlabeled cell fractions (neg tube) into the tube rack. Arrange the tubes as marked on the tube rack. Place the rack in the sample port.
7. autoMACS™ Pro Separator에 CD14 비드 시약 코드를 입력한다.7. Enter the CD14 bead reagent code into autoMACS™ Pro Separator.
8. 시약 포트에 시약 병을 놓는다.8. Place the reagent bottle in the reagent port.
9. 분리 메뉴로 이동하여 분리할 샘플을 강조 표시한다.9. Go to the Separation menu and highlight the sample to be separated.
10. 원하는 라벨링 시약(CD14 비드)을 선택한다.10. Select the desired labeling reagent (CD14 beads).
11. 샘플의 총 부피를 입력한다(세포를 현탁하는데 사용되는 PE 버퍼의 양).11. Enter the total volume of the sample (amount of PE buffer used to suspend the cells).
12. 분리 프로그램 ‘Possel’을 선택한다.12. Select the separation program ‘Possel’.
13. 세척 프로그램을 선택하고 실행을 누른다.13. Select a wash program and press Run.
14. 분리 과정이 끝나면 랙의 positive fraction 위치에서 CD14+ 단핵세포를 수집한다.14. After the separation process, collect CD14+ monocytes from the positive fraction of the rack.
15. 세포를 세고 1500 rpm에서 5분 동안 원심 분리한다.15. Count the cells and centrifuge at 1500 rpm for 5 minutes.
16. 50 ng/mL의 IL-4와, 50 ng/mL의 GM-CSF가 첨가된 ImmunocultTM-ACF 수지상 세포 media에 펠릿을 현탁한다.16. Suspend the pellet in Immunocult ™ -ACF dendritic cell media supplemented with 50 ng/mL of IL-4 and 50 ng/mL of GM-CSF.
17. 세포를 12-wells (1-2x106 cells/well) 또는 6-well (3-4x106 cells/well) 플레이트에 시드하고 플레이트를 5% CO2, 37℃의 가습 인큐베이터에 넣는다.17. Seed cells into 12-wells (1-2x10 6 cells/well) or 6-well (3-4x10 6 cells/well) plates and place the plates in a 5% CO 2 , 37°C humidified incubator.
18. (day 3) 수지상 세포가 떨어지지 않도록 조심스럽게 인큐베이터에서 플레이트를 제거한다.18. (Day 3) Carefully remove the plate from the incubator so that the dendritic cells do not fall off.
19. 플레이트를 원형으로 부드럽게 흔들어 잔해물이 접시 중앙에 정렬되도록 한 다음 피펫으로 잔해물을 제거한다.19. Gently shake the plate in a circular motion to align the debris with the center of the dish, then remove the debris with a pipette.
20. 배지를 완전히 제거하고 50 ng/mL의 IL-4, 50 ng/mL의 GM-CSF가 첨가된 신선한 ImmunocultTM-ACF 수지상 세포 배지로 교체한다.20. Remove the medium completely and replace with fresh Immunocult ™ -ACF dendritic cell medium supplemented with 50 ng/mL of IL-4 and 50 ng/mL of GM-CSF.
21. 배양 플레이트를 인큐베이터에 다시 넣는다.21. Put the culture plate back into the incubator.
22. (day 5 - 종양세포에서 protein lysate 획득) 종양 세포를 카운팅한 후 RPMI 배지에서 6-well culture plate에 2% NaCl 또는 2% HOCl에 세포를 현탁하고 37℃에서 1시간 동안 세포를 배양한다.22. (Day 5 - Acquiring protein lysate from tumor cells) After counting the tumor cells, suspend the cells in 2% NaCl or 2% HOCl in a 6-well culture plate in RPMI medium and incubate the cells at 37°C for 1 hour. .
23. 세포를 eppendorf tube로 옮기고 5분 동안 1500 rpm에서 원심 분리한다.23. Transfer the cells to an eppendorf tube and centrifuge at 1500 rpm for 5 minutes.
24. 상층액을 버리고 DPBS에서 세포를 현탁한다.24. Discard the supernatant and suspend the cells in DPBS.
25. 1500 rpm에서 5분 간 원심 분리한다.25. Centrifuge at 1500 rpm for 5 minutes.
26. 총 3회 세척을 위해 4번과 5번을 반복한다.26. Repeat steps 4 and 5 for a total of 3 washes.
27. ImmunocultTM-ACF 수지상 세포 배지 100-200 ul에 세포를 현탁시킨다.27. Suspend the cells in 100-200 ul of Immunocult ™ -ACF dendritic cell medium.
28. -80℃ 냉장고 또는 액체 질소 가스 탱크에서 6회 동결-해동 주기를 수행한다.28. Perform 6 freeze-thaw cycles in a -80°C refrigerator or liquid nitrogen gas tank.
29. eppendorf tube의 세포를 30분 동안 초음파 처리한다.29. Sonicate the cells in the eppendorf tube for 30 minutes.
30. trypan blue exclusion을 통해 latent live tumor cells의 존재를 확인하고 생존 세포가 샘플에 존재하는 경우 생존율이 0%가 될 때까지 세포를 초음파 처리한다.30. Check the presence of latent live tumor cells by trypan blue exclusion, and if viable cells are present in the sample, sonicate the cells until viability is 0%.
31. tumor lysate를 사용할 때까지 -80℃에서 보관한다.31. Store tumor lysate at -80°C until use.
32. (day 5 - FFPE에서 protein lysate 획득) 10 um 두께의 환자 종양 조직은 병리학과에서 제공받았다(조각 3 ~ 4 개 / e-tube).32. (Day 5 - Protein lysate obtained from FFPE) 10 μm-thick patient tumor tissue was provided by the Department of Pathology (3 to 4 pieces/e-tube).
33. 탈 파라핀화 용액 (320 ul)을 샘플 tube에 추가한다.33. Add the deparaffinization solution (320 ul) to the sample tube.
34. 56 ℃에서 3분 동안 배양한다. 볼텍싱 후 스핀다운 해준다.34. Incubate at 56 °C for 3 minutes. Spin down after vortexing.
35. 상층액을 제거한다.35. Remove the supernatant.
36. (선택 사항) 조직에서 파라핀을 더 제거해야 하는 경우 2단계와 4단계를 반복한다.36. (Optional) Repeat steps 2 and 4 if more paraffin needs to be removed from the tissue.
37. 100 % 에탄올 (320 ul)을 추가하고 실온에서 3분 동안 배양한다. 11000 xg에서 1분 동안 원심 분리하고 한 번 더 반복한다. 상층액을 제거한다.37. Add 100% ethanol (320 ul) and incubate for 3 minutes at room temperature. Centrifuge at 11000 xg for 1 minute and repeat one more time. Remove the supernatant.
38. 95 % 에탄올 (320 ul)을 추가하고 실온에서 3분 동안 배양한다. 11000 xg에서 1분 동안 원심 분리하고 한 번 더 반복한다. 상층액을 제거한다.38. Add 95% ethanol (320 ul) and incubate for 3 minutes at room temperature. Centrifuge at 11000 xg for 1 minute and repeat one more time. Remove the supernatant.
39. 70 % 에탄올 (320 ul)을 추가하고 실온에서 3분 동안 배양한다. 11000 xg에서 1분 동안 원심 분리하고 한 번 더 반복한다. 상층액을 제거한다.39. Add 70% ethanol (320 ul) and incubate for 3 minutes at room temperature. Centrifuge at 11000 xg for 1 minute and repeat one more time. Remove the supernatant.
40. 0.01 M sodium citrate buffer, pH 6.0 (320 ul)을 tube에 넣고 99-100 ℃의 heating block에서 20분 간 배양한다.40. Add 0.01 M sodium citrate buffer, pH 6.0 (320 ul) to the tube and incubate for 20 minutes on a heating block at 99-100 °C.
41. 실온에서 20분 동안 식힌다.41. Cool at room temperature for 20 minutes.
42. 11000 xg에서 1 분 동안 원심 분리한다. 상층액을 제거한다.42. Centrifuge at 11000 xg for 1 minute. Remove the supernatant.
43. Tube에 DPBS를 추가하여 클린 벤치에서 다음 단계를 수행한다.43. Add DPBS to the tube and perform the next step on a clean bench.
44. 11000 xg에서 1 분 동안 원심 분리한다. 상층액을 제거한다.44. Centrifuge at 11000 xg for 1 minute. Remove the supernatant.
45. DPBS 1mL 에서 11000 xg, 5분 동안 원심 분리한다. 상층액을 제거한다. 이 과정을 총 3회 반복한다.45. Centrifuge in 1 mL of DPBS at 11000 xg for 5 minutes. Remove the supernatant. Repeat this process a total of 3 times.
46. 300-500 ul의 DPBS에 조직을 현탁하고 샘플을 Tube type M (Miltenyi)으로 옮긴다. GentleMAC 조직 해리기를 사용하여 분리하여 단백질 추출을 위한 “protein_01” 설정을 사용하여 단백질을 얻는다.46. Suspend the tissue in 300-500 ul of DPBS and transfer the sample to Tube type M (Miltenyi). Dissociate using the GentleMAC tissue dissociator to obtain the protein using the “protein_01” setting for protein extraction.
47. Tube 바닥에 있는 샘플을 수집하기 위해 3분 동안 1500 rpm에서 원심 분리한다.47. Centrifuge at 1500 rpm for 3 minutes to collect the sample at the bottom of the tube.
48. 샘플을 eppendorf tube로 옮기고 10000 rpm에서 10분 동안 원심 분리한다.48. Transfer the sample to an eppendorf tube and centrifuge at 10000 rpm for 10 minutes.
49. 종양 단백질이 포함된 상층액을 다른 tube에 수집한다.49. Collect the supernatant containing tumor proteins in another tube.
50. Buffer를 다음 과정에 따라 교체한다: ① spin column 을 2 ml collection tube에 끼운다. ② HiPPR detergent removal resin 을 부드럽게 흔들어 섞어 준 뒤 column에 200 ul를 넣어준다. ③ 1500 xg, 1분 동안 원심 분리 후 필터링된 잔여 버퍼를 제거한다. ④ column에 200 ul PBS를 넣고 1500 xg, 1분동안 원심 분리한다. 이 과정을 세 번 반복한다. ⑤ column 아랫부분에 plug를 끼우고 protein sample을 넣어 준 뒤 부드럽게 흔들어 주고, 10분간 상온에서 배양한다. ⑥ plug를 제거 하고 1500 xg, 2분 간 원심 분리한다.50. Replace the buffer according to the following procedure: ① Insert the spin column into a 2 ml collection tube. ② Mix the HiPPR detergent removal resin by gently shaking it, and then add 200 ul to the column. ③ After centrifugation at 1500 xg for 1 minute, remove the filtered remaining buffer. ④ Add 200 ul PBS to the column and centrifuge at 1500 xg for 1 minute. Repeat this process three times. ⑤ Insert a plug at the bottom of the column, add a protein sample, shake gently, and incubate at room temperature for 10 minutes. ⑥ Remove the plug and centrifuge at 1500 xg for 2 minutes.
51. 추출된 단백질의 총량을 측정한다. 단백질을 나누어 사용하고 사용할 때까지 -80℃에서 보관한다.51. Measure the total amount of extracted protein. Divide the protein and store at -80 °C until use.
52. 수지상 세포가 흔들리지 않도록 조심스럽게 인큐베이터에서 dish를 빼준다.52. Carefully remove the dish from the incubator so as not to shake the dendritic cells.
53. dish를 원형으로 부드럽게 흔들어 이물질이 dish의 중앙에 정렬되도록 한 다음 피펫으로 이물질을 제거한다.53. Gently shake the dish in a circular motion to align the foreign matter in the center of the dish, then remove the foreign matter with a pipette.
54. 배지를 완전히 제거하고 50 ng/mL의 IL-4 및 50 ng/mL의 GM-CSF가 보충 된 신선한 ImmunocultTM-ACF 수지상 세포 배지로 교체한다.54. Remove medium completely and replace with fresh Immunocult ™ -ACF Dendritic Cell Medium supplemented with 50 ng/mL IL-4 and 50 ng/mL GM-CSF.
55. 50 ug of FFPE tissue protein extract per 106cells을 세포 배양 배지에 추가한다. 또는 위의 'A'섹션에서 얻은 whole cell tumor lysate를 세포에 추가한다. 또는 tumor derived peptide(10 ug/mL)를 세포에 추가한다.55. Add 50 ug of FFPE tissue protein extract per 10 6 cells to the cell culture medium. Alternatively, add the whole cell tumor lysate from section 'A' above to the cells. Alternatively, tumor derived peptide (10 ug/mL) is added to the cells.
56. 배양 플레이트를 인큐베이터에 다시 넣어준다.56. Return the culture plate to the incubator.
57. 수지상 세포가 빠지지 않도록 조심스럽게 인큐베이터에서 접시를 제거한다.57. Carefully remove the dish from the incubator to avoid dislodging the dendritic cells.
58. whole tumor lysates of FFPE 또는 tumor specific peptide가 로딩된 수지상 세포를 포함하는 2000 ul의 DC 배지에 20 ul의 DC maturation supplement를 추가한다.58. Add 20 ul of DC maturation supplement to 2000 ul of DC medium containing DC cells loaded with whole tumor lysates of FFPE or tumor specific peptides.
59. 배양 플레이트를 부드럽게 흔든다.59. Gently shake the culture plate.
60. 배양 플레이트를 인큐베이터에 다시 넣어준다.60. Put the culture plate back into the incubator.
Ⅲ. CD14- 단핵구에서 CD8+ naive T 세포 분리 배양III. CD8+ naive T cell isolation and culture from CD14- monocytes
1. CD14 negative fraction 위치에서 얻은 CD14- PBMC를 수집한다.1. Collect CD14- PBMCs obtained from the CD14 negative fraction.
2. 세포 수를 센다.2. Count the cells.
3. 세포를 1500 rpm에서 5분 동안 원심 분리하고 상층액을 완전히 제거한다.3. Centrifuge the cells at 1500 rpm for 5 minutes and remove the supernatant completely.
4. PE buffer (40 uL per 107 cells)로 pellet을 현탁한다.4. Suspend the pellet with PE buffer (40 uL per 10 7 cells).
5. naive CD8+ T Cell Biotin-Antibody Cocktail(10 uL per 107cells)을 첨가하고, 부드럽게 섞어 냉장고(28 ℃)에서 10분 간 배양한다.5. Add naive CD8+ T Cell Biotin-Antibody Cocktail (10 uL per 10 7 cells), mix gently, and incubate in a refrigerator (28 °C) for 10 minutes.
6. PE 버퍼를 추가한다(30 uL of PE per 107 cells).6. Add PE buffer (30 uL of PE per 10 7 cells).
7. 즉시 anti-Biotin Microbeads (20 uL for 107 cells)를 첨가하고, 부드럽게 섞어준 뒤 냉장고 (2-8℃)에서 15분 동안 배양한다.7. Immediately add anti-Biotin Microbeads (20 uL for 10 7 cells), mix gently, and incubate in the refrigerator (2-8℃) for 15 minutes.
8. 2 mL PE buffer와 원심 분리기를 1500 rpm에서 5분 동안 추가한다.8. Add 2 mL PE buffer and centrifuge at 1500 rpm for 5 minutes.
9. 상층액을 완전히 흡입하고 500 μL의 PE buffer에 세포를 현탁한다. 15 ml tube에 세포를 옮기고 'ori'로 표시한다.9. Completely aspirate the supernatant and suspend the cells in 500 μL of PE buffer. Transfer cells to 15 ml tubes and mark with 'ori'.
10. 15 mL tube 2 개를 준비하고 'pos'및 'neg'로 라벨을 지정한다.10. Prepare two 15 mL tubes and label them 'pos' and 'neg'.
11. 샘플이 들어있는 tube (ori tube)와, 라벨이 있는 cell fractions (pos tube), 라벨이 없는 세포 fractions (neg tube)을 수집하기 위한 tube를 tube 랙에 넣는다. tube는 tube 랙에 표시된 대로 배열한다. 샘플 포트에 랙을 놓는다.11. Put the tubes containing the sample (ori tube), labeled cell fractions (pos tube), and tubes for collecting unlabeled cell fractions (neg tube) into the tube rack. Arrange the tubes as marked on the tube rack. Place the rack in the sample port.
12. 분리 메뉴로 이동하여 분리할 샘플을 강조 표시한다.12. Go to the Separation menu and highlight the sample to be separated.
13. 원하는 라벨링 시약을 선택한다(이 경우 비어 있어야 함).13. Select the desired labeling reagent (in this case it should be empty).
14. 샘플의 총 부피를 입력한다(세포를 현탁시키는 데 사용되는 PE buffer의 양(500 uL)).14. Enter the total volume of the sample (amount of PE buffer used to suspend the cells (500 uL)).
15. 분리 프로그램 'Depl05'를 선택한다.15. Select the separation program 'Depl05'.
16. 세척 프로그램을 선택하고 실행을 누른다.16. Select a wash program and press Run.
17. 분리 프로세스가 끝나면 랙의 negative fraction 위치에 네거티브 셀을 수집한다.17. After the separation process, collect the negative cells in the negative fraction position of the rack.
18. 1500 rpm에서 05분 동안 세포를 원심 분리하고 상층액을 완전히 흡입한다.18. Centrifuge the cells at 1500 rpm for 05 minutes and aspirate the supernatant completely.
19. PE buffer에 세포를 현탁한다(80 uL of PE per 107 cells).19. Suspend cells in PE buffer (80 uL of PE per 10 7 cells).
20. CD8 Microbeads (20 uL per 107 cells)를 추가하고 부드럽게 섞어 냉장고 (2-8℃)에서 15분 동안 배양한다.20. Add CD8 Microbeads (20 uL per 10 7 cells), mix gently and incubate in refrigerator (2-8℃) for 15 minutes.
21. 5분 동안 1500 rpm에서 2 mL PE 버퍼를 추가하여 원심 분리한다.21. Add 2 mL PE buffer and centrifuge at 1500 rpm for 5 minutes.
22. 상층액을 완전히 흡입하고 500 μL의 PE 버퍼에 세포를 현탁한다. 15 ml tube에 세포를 옮기고 'ori'로 표시한다.22. Aspirate the supernatant completely and suspend the cells in 500 μL of PE buffer. Transfer cells to 15 ml tubes and mark with 'ori'.
23. 15 mL tube 2개를 준비하고 'pos' 및 'neg'로 라벨을 지정한다.23. Prepare two 15 mL tubes and label them 'pos' and 'neg'.
24. 샘플이 들어 있는 tube (ori tube)와, 라벨이 있는 cell fractions (pos tube), 라벨이 없는 세포 fractions (neg tube)을 수집하기 위한 tube를 tube 랙에 넣는다. tube는 tube 랙에 표시된 대로 배열한다. 샘플 포트에 랙을 놓는다.24. Put the tubes containing the sample (ori tube), labeled cell fractions (pos tube), and tubes for collecting unlabeled cell fractions (neg tube) into the tube rack. Arrange the tubes as marked on the tube rack. Place the rack in the sample port.
25. 분리 메뉴로 이동하여 분리할 샘플을 강조 표시한다.25. Go to the Separation menu and highlight the sample to be separated.
26. 원하는 라벨링 시약을 선택한다(이 경우 공백이어야 함).26. Select the desired labeling reagent (should be blank in this case).
27. 샘플의 총 부피를 입력한다(세포를 현탁시키는 데 사용되는 PE 버퍼의 양(500 uL)).27. Enter the total volume of the sample (amount of PE buffer used to suspend the cells (500 uL)).
28. 분리 프로그램 ‘Possel’을 선택한다.28. Select the separation program ‘Possel’.
29. 세척 프로그램을 선택하고 실행을 누른다.29. Select a wash program and press Run.
30. 분리 과정이 끝나면 랙의 positive fraction 위치에 양성 세포를 수집한다. 이것은 naive CD8+ T cells이다.30. After the separation process, collect the positive cells in the positive fraction of the rack. These are naive CD8+ T cells.
31. 세포를 세고 2000 units/mL의 IL-2가 첨가된 T 세포 배양 배지에 세포를 현탁한다.31. Count the cells and suspend the cells in T cell culture medium supplemented with 2000 units/mL of IL-2.
32. 세포를 12-wells(1-2x106 cells/well)에 넣고 37℃, 5% CO2, 가습된 인큐베이터에 플레이트를 넣는다.32. Put the cells into 12-wells (1-2x10 6 cells/well) and place the plate in a humidified incubator at 37℃, 5% CO 2 .
33. 조심스럽게 인큐베이터에서 플레이트를 꺼낸다.33. Carefully remove the plate from the incubator.
34. 조심스럽게 half media(세포 수집을 피하기 위해 맨 위 부분)를 제거하고 4000 unit/mL의 IL-2 (최종 농도는 2000 unit/mL)가 첨가된 새로운 T Cell Media로 교체한다.34. Carefully remove the half media (top part to avoid cell collection) and replace with fresh T Cell Media supplemented with 4000 units/mL of IL-2 (final concentration 2000 units/mL).
35. 배양 플레이트를 인큐베이터에 다시 넣는다.35. Put the culture plate back into the incubator.
Ⅳ. 성숙 수지상 세포와 CD8+ naive T 세포를 공동 배양하여 T 세포 활성화 후 증폭 배양IV. Co-culture of mature dendritic cells and CD8+ naive T cells to amplify after T cell activation
1. tumor protein를 처리하여 성숙시킨 수지상 세포와 CD8+ naive T 세포를 1X105 개의 세포를 U-bottom 96-well plate에 seeding 후 1X104 개의 성숙 수지상 세포를 넣어 준다.1. After seeding 1X10 5 cells of dendritic cells and CD8+ naive T cells matured by treating tumor protein in a U-bottom 96-well plate, insert 4 1X10 mature dendritic cells.
2. 위 세포들을 플레이트 상에서 250 g에서 3분 동안 원심 분리를 한다.2. Centrifuge the above cells on the plate at 250 g for 3 minutes.
3. 배양기에서 4일 동안 공동 배양한다.3. Co-culture in an incubator for 4 days.
4. 6000 units의 IL-2가 첨가된 나머지 세포 T 세포 배양 배지를 현탁한다.4. Suspend the remaining T cell culture medium with 6000 units of IL-2 added.
5. 피펫으로 세포를 혼합하고 250 g에서 3분 동안 원심 분리한다.5. Mix the cells with a pipette and centrifuge at 250 g for 3 minutes.
6. 배양기에서 세포를 배양한다.6. Cultivate the cells in an incubator.
7. 2일 마다 세포 상태를 확인하고 세포에 따라 half media change를 수행하거나 세포를 나눈다. PRE-REPEAT EXPANSION이 끝날 때까지 프로세스를 반복한다(14일).7. Check the cell condition every 2 days, and perform a half media change or divide the cells according to the cells. Repeat the process until the PRE-REPEAT EXPANSION is over (14 days).
8. (day 14) 배양기에서 세포를 부드럽게 제거하고 세포의 현미경 이미지를 찍는다.8. (Day 14) Gently remove the cells from the incubator and take microscopic images of the cells.
9. 세포의 양을 계산해서 보고서를 작성한다.9. Calculate the amount of cells and write a report.
10. CD8 및 CD4 양성 세포의 양을 측정하여 T 세포의 순도를 확인한다.10. Determine the purity of T cells by measuring the amount of CD8 and CD4 positive cells.
11. 세포와 감마선을 조사한 말초 혈액 단핵구를 1:200의 비율로 공동 배양을 시작한다.11. Start co-culture of cells and gamma-irradiated peripheral blood monocytes at a ratio of 1:200.
12. 2-3일 간격으로 약 14일 동안 half media change를 진행하거나 2배로 split한다.12. Conduct half media change or split twice for about 14 days at 2-3 day intervals.
Ⅴ. 말초 혈액 단핵구를 이용한 자연 살해 세포 배양V. Natural killer cell culture using peripheral blood monocytes
1. 자연 살해 세포 증식을 위해 분리된 말초 혈액 단핵구 세포는 방사선 조사된 K562 feeder cell과 IL-2, IL-21 존재 하에 37℃ incubator에서 7일 간 co-culture한다.1. Peripheral blood mononuclear cells isolated for natural killer cell proliferation were co-cultured with irradiated K562 feeder cells in the presence of IL-2 and IL-21 in a 37°C incubator for 7 days.
2. 배양한 세포의 증식을 위하여 2-3일 간격으로 IL-2와 IL-15를 포함한 배지를 이용하여 dividing이나 media change를 수행하면서 2주 간 배양한다.2. For the proliferation of cultured cells, culture for 2 weeks while performing division or media change using a medium containing IL-2 and IL-15 every 2-3 days.
3. 획득한 자연 살해 세포의 항암 활성을 확인하기 위하여 K562 cell line을 target으로 CCK-8을 이용한 cytotoxicity와 NK cell의 surface receptor 분석을 수행한다.3. To confirm the anticancer activity of the acquired natural killer cells, cytotoxicity using CCK-8 and surface receptor analysis of NK cells are performed using the K562 cell line as a target.
예비 실험preliminary experiment
위 제조예 제조 과정 중 또는 제조 후 예비 실험을 수행하였다.Preliminary experiments were performed during or after the preparation of the above Preparation Example.
Ⅰ. 환자의 조직에서 추출한 항원 또는 펩타이드를 탑재하여 성숙시킨 미성숙 수지상 세포의 IL-12(p70) 분비량 증가I. Increased secretion of IL-12 (p70) by immature dendritic cells loaded with antigens or peptides extracted from patients' tissues
대장암 환자(SDH21028)의 혈액으로부터 CD14 양성 단핵구를 분리 및 미성숙 수지상 세포로 분화 후 미성숙 수지상 세포에 펩타이드를 탑재하여 성숙시켰다.CD14-positive monocytes were isolated from the blood of a colorectal cancer patient (SDH21028), differentiated into immature dendritic cells, and then loaded with peptides to mature dendritic cells.
미성숙 수지상 세포에 펩타이드를 처리한 군 및 처리하지 않은 군에서 성숙 수지상 세포가 분비하는 IL-12(p70)의 분비량을 측정하기 위하여 ELISA를 실시하였다. 그 결과, 펩타이드를 처리하지 않은 군(도 2의 iDC)에 비해 펩타이드를 처리한 군(도 2의 mDC)의 IL-12(p70) 분비 수준이 높은 것을 확인하였다(도 2).ELISA was performed to measure the amount of IL-12 (p70) secreted by mature dendritic cells in the group treated with the peptide in immature dendritic cells and the group not treated with the peptide. As a result, it was confirmed that the level of IL-12 (p70) secretion was higher in the group treated with the peptide (mDC in FIG. 2) than in the group not treated with the peptide (iDC in FIG. 2) (FIG. 2).
Ⅱ. 성숙 수지상 세포와 공동 배양한 CD8+ naive T 세포의 IFN-γ 발현 정도 증가 및 세포 배양 양상II. Increased IFN-γ expression and cell culture pattern of CD8+ naive T cells co-cultured with mature dendritic cells
환자(SDH21028)의 혈액에서 CD8+ naive T 세포를 분리하여 배양 후 성숙 수지상 세포와 공동 배양하였다.CD8+ naive T cells were isolated from the blood of a patient (SDH21028), cultured, and then co-cultured with mature dendritic cells.
CD8+ naive T 세포 배양 후 성숙 수지상 세포와 공동 배양하지 않은 군 및 공동 배양한 군에서 활성화된 T 세포 마커인 IFN-γ의 발현 정도를 측정하였다. 그 결과, 성숙 수지상 세포와 공동 배양하지 않은 군(도 3의 T cell only)에 비해 성숙 수지상 세포와 공동 배양한 군(도 3의 mDC/T cell)의 IFN-γ 발현 정도가 높은 것을 확인하였다(도 3).After CD8+ naive T cell culture, the expression level of IFN-γ, an activated T cell marker, was measured in the group not co-cultured with mature dendritic cells and the group co-cultured. As a result, it was confirmed that the level of IFN-γ expression was higher in the group co-cultured with mature dendritic cells (mDC/T cell in FIG. 3) than in the group not co-cultured with mature dendritic cells (T cell only in FIG. 3). (Fig. 3).
CD8+ naive T 세포와 mDC(성숙 수지상 세포)를 공동 배양하여 활성화된 T 세포의 배양 양상을 관찰하였다. 그 결과 성숙 수지상 세포와 공동 배양하지 않은 경우(도 4의 T cell only)에 비해 공동 배양한 경우(도 4의 mDC+TC), 성숙 수지상 세포로 인해 활성화된 T 세포가 군집을 이루며 더 많은 성장을 하는 것을 확인하였다(도 4).CD8+ naive T cells and mDC (mature dendritic cells) were co-cultured to observe the culture pattern of activated T cells. As a result, when co-cultured (mDC + TC in FIG. 4) compared to the case where mature dendritic cells were not co-cultured (T cell only in FIG. 4), activated T cells due to mature dendritic cells clustered and grew more It was confirmed to do (FIG. 4).
Ⅲ. 자연 살해 세포 배양 양상III. Natural killer cell culture patterns
환자(SDH21028)의 혈액에서 말초 혈액 단핵 세포를 분리하여 자연 살해 세포를 배양하였다.Peripheral blood mononuclear cells were isolated from the blood of a patient (SDH21028) and natural killer cells were cultured.
환자의 말초 혈액 단핵 세포를 배양하여 처음 16.3%를 차지하였던 자연 살해 세포 비율이 시간이 지남에 따라 98.8%로 증가하였으며 그 수도 약 251배 증가하였다.The percentage of natural killer cells, which initially accounted for 16.3% when the patient's peripheral blood mononuclear cells were cultured, increased to 98.8% over time, and the number increased approximately 251-fold.
24 well24 well | DAY-0DAY-0 | DAY-14DAY-14 |
Purity (%)Purity (%) | 16.3%16.3% | 98.8%98.8% |
FoldFold | 1One | 251.51배251.51 times |
Converted NK cellsConverted NK cells | 2.45x10^62.45x10^6 | 6.14x10^86.14x10^8 |
또한, 자연 살해 세포 배양 과정을 거치면서 자연 살해 세포의 activating receptor를 발현하는 세포 군집이 증가하였고(도 5a 내지 도 5c) inhibitory receptor를 발현하는 세포 군집은 약간 증가한 것을 확인하였다(도 5d).In addition, it was confirmed that the cell population expressing activating receptors of natural killer cells increased during the natural killer cell culture process (FIGS. 5a to 5c), and the cell population expressing inhibitory receptors slightly increased (FIG. 5d).
실시예Example
위 제조예에서 수득한 3가지 면역 세포의 암 세포에 대한 항암 효과를 확인하기 위한 CCK-8 assay를 수행하였다.CCK-8 assay was performed to confirm the anticancer effect of the three immune cells obtained in the above preparation example on cancer cells.
Ⅰ. 실시예 1I. Example 1
1. 방법1. Method
(1) 세포 접종(cell seeding)(1) Cell seeding
Primary tumor cell을 96 well plate에 1x104 cells/100 ul/well로 seeding 후 24시간 incubation한다.Primary tumor cells are seeded in a 96 well plate at 1x10 4 cells/100 ul/well, followed by incubation for 24 hours.
(2) 면역 세포(Effector cell) 준비 및 사이토카인 pre-activation(2) Preparation of immune cells (Effector cell) and cytokine pre-activation
Effector cell과 target cell(primary tumor cell)의 개수 비율인 E:T ratio는 다음과 같다: NK cell : DC : T cell : target cell (1 x 104)= 1 : 0.02 : 10 : 1. target cell 1 x 104 cells을 기준으로 각 비율에 해당하는 NK cell, DC, T cell을 준비하였다.The E:T ratio, which is the ratio of the number of effector cells and target cells (primary tumor cells), is as follows: NK cell : DC : T cell : target cell (1 x 10 4 )= 1 : 0.02 : 10 : 1. target cell Based on 1 x 10 4 cells, NK cells, DCs, and T cells corresponding to each ratio were prepared.
준비된 면역 세포들과 target cell의 공동 배양 전 면역 세포들에 대해 사이토카인 pre-activation을 수행하였다. 세 가지 면역 세포를 6000 unit/ml 농도의 IL-2가 첨가된 phenol red free RPMI에서 24시간 단독 배양하였다. 또한, 세 가지 면역 세포를 함께 4000 unit/ml 또는 6000 unit/ml 농도의 IL-2가 첨가된 phenol red free RPMI에서 24시간 공동 배양하였다.Cytokine pre-activation was performed on the immune cells prior to co-cultivation of the prepared immune cells and target cells. The three immune cells were cultured alone for 24 hours in phenol red free RPMI supplemented with IL-2 at a concentration of 6000 units/ml. In addition, the three immune cells were co-cultured for 24 hours in phenol red free RPMI supplemented with IL-2 at a concentration of 4000 unit/ml or 6000 unit/ml.
(3) Effector cell 및 Target cell 공배양(co-culture)(3) Effector cell and target cell co-culture (co-culture)
위 (1)에서 수득된 tumor cell에 기존 배양액 100 ul를 제거하고 위 (2)의 세 가지 면역 세포를 단독 또는 공동으로 각 well 당 100 ul 처리한 뒤 37℃에서 24시간 동안 배양하였다.100 ul of the existing culture medium was removed from the tumor cells obtained in (1) above, and 100 ul of the three immune cells of the above (2) were treated alone or jointly per well, and then cultured at 37 ° C for 24 hours.
(4) CCK-8 detection(4) CCK-8 detection
위 (3)의 5가지 군(DC 단독, T cell 단독, NK cell 단독, 4000 unit/ml pre-activation 공동, 6000 unit/ml pre-activation 공동)에 CCK-8 용액을 10 ul 첨가한 후, 30분 동안 37℃의 배양기에서 정치 배양하였다. 그리고 부드럽게 파이펫팅 해준 뒤 2000 rpm에서 3분 동안 원심 분리 후 상층액 70 ul를 새로운 well에 옮겼다. 그리고 450 nm 파장에 대한 발색을 평가하여 세포의 사멸도를 측정하였다.After adding 10 ul of CCK-8 solution to the 5 groups (DC alone, T cell alone, NK cell alone, 4000 unit/ml pre-activation cavity, 6000 unit/ml pre-activation cavity) of the above (3), Static culture was performed in an incubator at 37° C. for 30 minutes. After gently pipetting, 70 ul of the supernatant was transferred to a new well after centrifugation at 2000 rpm for 3 minutes. In addition, the degree of apoptosis of cells was measured by evaluating color development at a wavelength of 450 nm.
2. 결과2. Results
SDH21029 대장암 환자의 primary cancer cell을 target으로 NK, T, DC의 단독 또는 조합의 세포 독성을 확인하였다(도 6). 각 cell을 단독으로 cancer cell과 co-culture하였을 때, DC, T, NK는 약 18%, 28%, 40%의 세포 독성이 나타났다(도 6의 DC, T 및 NK 군). 4000U/ml IL-2로 pre-activation한 세 가지 면역 세포의 조합 군(도 6의 4000 군)에서는 57%정도의 세포 독성을 확인하였고, 6000U/ml IL-2로 pre-activation한 세 가지 면역 세포의 조합 군(도 6의 6000 군)에서는 47%정도의 세포 독성을 확인하였다. 이 결과를 통해 세가지 cell을 함께 activation한 군에서 더 높은 세포 독성이 나타나는 것을 확인하였다. The cytotoxicity of NK, T, and DC alone or in combination was confirmed as a target for primary cancer cells of SDH21029 colorectal cancer patients (FIG. 6). When each cell was co-cultured with cancer cells alone, DC, T, and NK showed cytotoxicity of about 18%, 28%, and 40% (DC, T, and NK groups in FIG. 6). In the combination group of the three immune cells pre-activated with 4000 U/ml IL-2 (4000 group in FIG. 6), about 57% of cytotoxicity was confirmed, and In the cell combination group (6000 group in FIG. 6), cytotoxicity of about 47% was confirmed. Through this result, it was confirmed that higher cytotoxicity appeared in the group in which three cells were activated together.
Ⅱ. 실시예 2II. Example 2
1. 방법1. Method
위 실시예 1의 방법과 동일하되, 실시예 1의 1-(2) 단계의 사이토카인 pre-activation은 모두 4000 unit/ml 농도의 IL-2를 이용하여 수행하였다.The same as the method of Example 1 above, but the cytokine pre-activation in step 1-(2) of Example 1 was all performed using IL-2 at a concentration of 4000 units/ml.
2. 결과2. Results
IL-2로 pre-activation한 SDH21032 대장암 환자의 DC, T, NK cell의 단독 또는 조합을 effector cell로 하여 cancer cell에 대한 세포 독성 실험을 진행하였다(도 7). 각 cell을 단독으로 cancer cell과 co-culture하였을 때 T, NK는 약 67%, 91%의 세포 독성이 나타났다(도 7의 T 및 NK 군). 세 가지 세포를 함께 pre-activation한 군(도 7의 Combination 군)에서 가장 높은 세포 독성을 나타냈다.A cytotoxicity test on cancer cells was performed using DC, T, and NK cells alone or in combination of SDH21032 colorectal cancer patients pre-activated with IL-2 as effector cells (FIG. 7). When each cell was co-cultured with cancer cells alone, T and NK showed cytotoxicity of about 67% and 91% (T and NK groups in FIG. 7). The group in which all three cells were pre-activated (combination group in FIG. 7) showed the highest cytotoxicity.
Ⅲ. 실시예 3III. Example 3
1. 방법1. Method
위 실시예 1의 방법과 동일하되, 실시예 1의 1-(2) 단계의 사이토카인 pre-activation은 모두 4000 unit/ml 농도의 IL-2 및 10 ng/ml 농도의 IFN-γ를 이용하여 수행하였다.Same as the method of Example 1 above, but the cytokine pre-activation in step 1-(2) of Example 1 was performed using IL-2 at a concentration of 4000 units/ml and IFN-γ at a concentration of 10 ng/ml. performed.
2. 결과2. Results
IL-2 및 IFN-γ로 pre-activation한 SDH21032 대장암 환자의 DC, T, NK cell의 단독 또는 조합을 effector cell로 하여 cancer cell에 대한 세포 독성 실험을 진행하였다(도 8). 각 cell을 단독으로 cancer cell과 co-culture하였을 때 T, NK는 약 60%, 81%의 세포 독성이 나타났다(도 8의 T 및 NK 군). 세 가지 세포를 함께 pre-activation한 군(도 8의 Combination 군)에서 가장 높은 세포 독성을 나타냈다.A cytotoxicity test on cancer cells was performed using DC, T, and NK cells alone or in combination as effector cells of SDH21032 colorectal cancer patients pre-activated with IL-2 and IFN-γ (FIG. 8). When each cell was co-cultured with cancer cells alone, T and NK showed cytotoxicity of about 60% and 81% (T and NK groups in FIG. 8). The highest cytotoxicity was shown in the pre-activation group of the three cells (combination group in FIG. 8).
Ⅳ. 실시예 4IV. Example 4
1. 방법1. Method
위 실시예 1의 방법과 동일하되, 실시예 1의 1-(2) 단계의 사이토카인 pre-activation은 모두 4000 unit/ml 농도의 IL-2 및 10 ng/ml 농도의 IFN-γ를 이용하여 수행하였다.Same as the method of Example 1 above, but the cytokine pre-activation in step 1-(2) of Example 1 was performed using IL-2 at a concentration of 4000 units/ml and IFN-γ at a concentration of 10 ng/ml. performed.
2. 결과2. Results
IL-2와 IFN-γ로 pre-activation한 SDH21028 대장암 환자의 DC, T, NK cell의 단독 또는 조합을 effector cell로 하여 cancer cell에 대한 세포 독성 실험을 진행하였다(도 9). 각 cell을 단독으로 cancer cell과 co-culture하였을 때 T, NK는 약 18%, 49%의 세포 독성이 나타났다(도 9의 T 및 NK 군). 세 가지 세포를 함께 pre-activation한 군(도 9의 Combination 군)에서 가장 높은 세포 독성을 나타냈다.A cytotoxicity test on cancer cells was performed using DC, T, and NK cells alone or in combination as effector cells of SDH21028 colorectal cancer patients pre-activated with IL-2 and IFN-γ (FIG. 9). When each cell was co-cultured with cancer cells alone, T and NK showed cytotoxicity of about 18% and 49% (T and NK groups in FIG. 9). The highest cytotoxicity was shown in the pre-activation group of the three cells (Combination group in FIG. 9).
Ⅴ. 실시예 5V. Example 5
1. 방법1. Method
위 실시예 1의 방법과 동일하되, 실시예 1의 1-(2) 단계의 사이토카인 pre-activation은 모두 4000 unit/ml 농도의 IL-2를 이용하여 수행하였고, 사이토카인 pre-activation을 수행하지 않은 세 가지 면역 세포의 조합 군을 추가하였다.Same as the method of Example 1 above, but the cytokine pre-activation in step 1-(2) of Example 1 was all performed using IL-2 at a concentration of 4000 units/ml, and the cytokine pre-activation was performed A combination group of three immune cells that were not tested was added.
2. 결과2. Results
SDH21054 대장암 환자의 cancer cell을 target으로 DC, T, NK cell의 combination therapy assay를 수행하였다. 전날 pre-activated된 단독 cells(도 10의 Pre-activated DC, T 및 NK 군) 중 T와 NK가 비슷한 세포 독성을 보였고, 세가지 cell을 함께 pre-activation한 군(도 10의 Pre-activated DCTNK 군)에서 가장 높은 세포 독성을 확인하였다. 또한 pre-activation하지 않은 cell의 combination 군(도 10의 Non DCTNK 군)은 약 20%의 세포 독성을 확인하였다.A combination therapy assay of DC, T, and NK cells was performed with cancer cells of SDH21054 colorectal cancer patients as targets. Among the single cells pre-activated the previous day (Pre-activated DC, T, and NK group in FIG. 10), T and NK showed similar cytotoxicity, and the group pre-activated with three cells (Pre-activated DCTNK group in FIG. 10) ), the highest cytotoxicity was confirmed. In addition, the combination group of cells without pre-activation (Non DCTNK group in FIG. 10) confirmed cytotoxicity of about 20%.
Ⅵ. 실시예 6VI. Example 6
1. 방법1. Method
위 실시예 1의 방법과 동일하되, 실시예 1의 1-(2) 단계의 사이토카인 pre-activation은 모두 4000 unit/ml 농도의 IL-2를 이용하여 수행하였고, 사이토카인 pre-activation을 수행하지 않은 세 가지 면역 세포의 조합 군을 추가하였다. 또한 실시예 1의 1-(3) 단계의 target cell과의 공동 배양 시 300 unit/ml 농도의 IL-2를 처리한 뒤 배양하였다. 실험은 총 2회 수행하였다.Same as the method of Example 1 above, but the cytokine pre-activation in step 1-(2) of Example 1 was all performed using IL-2 at a concentration of 4000 units/ml, and the cytokine pre-activation was performed A combination group of three immune cells that were not tested was added. In addition, when co-cultivating with target cells in step 1-(3) of Example 1, IL-2 was treated at a concentration of 300 units/ml and then cultured. The experiment was performed twice in total.
2-1. 결과 12-1. result 1
SDH21054 대장암 환자의 cancer cell을 target으로 DC, T, NK cell의 combination therapy assay를 수행하였다. 전날 pre-activated된 단독 cells(도 11의 Pre-activated DC, T 및 NK 군) 중 T와 NK가 비슷한 세포 독성을 보였고, 세가지 cell을 함께 pre-activation한 군(도 11의 Pre-activated DCTNK 군)에서 약 80%의 가장 높은 세포 독성을 확인하였다. 또한 pre-activation하지 않은 cell의 combination 군(도 11의 Non DCTNK 군)에서 약 55%의 세포 독성을 확인하였다.A combination therapy assay of DC, T, and NK cells was performed with cancer cells of SDH21054 colorectal cancer patients as targets. Among the single cells pre-activated the previous day (Pre-activated DC, T and NK group in FIG. 11), T and NK showed similar cytotoxicity, and the group pre-activated with three cells (Pre-activated DCTNK group in FIG. 11) ), the highest cytotoxicity of about 80% was confirmed. In addition, about 55% of cytotoxicity was confirmed in the combination group of non-pre-activated cells (Non DCTNK group in FIG. 11).
2-2. 결과 22-2. result 2
SDH21054 대장암 환자의 cancer cell을 target으로 DC, T, NK cell의 combination therapy assay를 수행하였다. 전날 pre-activated된 단독 cells(도 12의 Pre-activated DC, T 및 NK 군) 중 T와 NK가 비슷한 세포 독성을 보였고, 세가지 cell을 함께 pre-activation한 군(도 12의 Pre-activated DCTNK 군)에서 약 80%의 가장 높은 세포 독성을 확인하였다. 또한 pre-activation하지 않은 cell의 combination 군(도 12의 Non DCTNK 군)에서 약 55%의 세포 독성을 확인하였다.A combination therapy assay of DC, T, and NK cells was performed with cancer cells of SDH21054 colorectal cancer patients as targets. Among the single cells pre-activated the previous day (Pre-activated DC, T, and NK group in FIG. 12), T and NK showed similar cytotoxicity, and the group in which all three cells were pre-activated (Pre-activated DCTNK group in FIG. 12) showed similar cytotoxicity. ), the highest cytotoxicity of about 80% was confirmed. In addition, about 55% of cytotoxicity was confirmed in the combination group of non-pre-activating cells (Non DCTNK group in FIG. 12).
Claims (36)
- 수지상 세포, 자연 살해 세포 및 세포 독성 T 세포를 포함하는 암의 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating cancer comprising dendritic cells, natural killer cells and cytotoxic T cells.
- 청구항 1에 있어서, 상기 수지상 세포, 상기 자연 살해 세포 및 상기 세포 독성 T 세포 중 적어도 하나는 상기 암의 예방 또는 치료 대상으로부터 유래된 것인, 약학 조성물.The pharmaceutical composition of claim 1, wherein at least one of the dendritic cells, the natural killer cells, and the cytotoxic T cells is derived from a subject for prevention or treatment of cancer.
- 청구항 1에 있어서, 상기 수지상 세포 및 상기 자연 살해 세포의 수의 비율은 0.01:1 내지 0.03:1인 것인, 약학 조성물.The pharmaceutical composition according to claim 1, wherein the ratio of the numbers of the dendritic cells and the natural killer cells is 0.01:1 to 0.03:1.
- 청구항 1에 있어서, 상기 세포 독성 T세포 및 상기 자연 살해 세포의 수의 비율은 8:1 내지 12:1인 것인, 약학 조성물.The pharmaceutical composition according to claim 1, wherein the ratio of the number of the cytotoxic T cells and the number of the natural killer cells is 8:1 to 12:1.
- 청구항 1에 있어서, 상기 수지상 세포, 상기 자연 살해 세포 및 상기 세포 독성 T 세포 중 적어도 하나는 사이토카인을 포함하는 배지에서 배양된 것인, 약학 조성물.The pharmaceutical composition according to claim 1, wherein at least one of the dendritic cells, the natural killer cells, and the cytotoxic T cells is cultured in a medium containing cytokines.
- 청구항 5에 있어서, 상기 사이토카인은 IL-2(Interleukin-2), IFN-γ(Interferon-γ), IL-15, IL-7, IL-21 및 4-1BB 리간드로 이루어진 군에서 선택된 적어도 하나인, 약학 조성물.The method according to claim 5, wherein the cytokine is at least one selected from the group consisting of IL-2 (Interleukin-2), IFN-γ (Interferon-γ), IL-15, IL-7, IL-21 and 4-1BB ligand Phosphorus, a pharmaceutical composition.
- 청구항 5에 있어서, 상기 사이토카인의 농도는 40 U/mL 내지 5000 U/mL인, 약학 조성물.The pharmaceutical composition according to claim 5, wherein the concentration of the cytokine is 40 U/mL to 5000 U/mL.
- 청구항 6에 있어서, 상기 IL-2(Interleukin-2)의 농도는 100 U/mL 내지 5000 U/mL인, 약학 조성물.The pharmaceutical composition according to claim 6, wherein the IL-2 (Interleukin-2) concentration is 100 U/mL to 5000 U/mL.
- 청구항 6에 있어서, 상기 IFN-γ(Interferon-γ)의 농도는 40 U/mL 내지 4000 U/mL인, 약학 조성물.The method according to claim 6, wherein the concentration of the IFN-γ (Interferon-γ) is 40 U / mL to 4000 U / mL, the pharmaceutical composition.
- 청구항 1에 있어서, 상기 수지상 세포, 상기 자연 살해 세포 및 상기 세포 독성 T 세포는 서로 공동 배양된 것인, 약학 조성물.The pharmaceutical composition of claim 1, wherein the dendritic cells, the natural killer cells, and the cytotoxic T cells are co-cultured with each other.
- 청구항 10에 있어서, 상기 수지상 세포 및 상기 자연 살해 세포는 0.01:1 내지 0.03:1의 세포 수의 비율로 공동 배양된 것인, 약학 조성물.The pharmaceutical composition of claim 10, wherein the dendritic cells and the natural killer cells are co-cultured at a cell number ratio of 0.01:1 to 0.03:1.
- 청구항 10에 있어서, 상기 세포 독성 T세포 및 상기 자연 살해 세포는 8:1 내지 12:1의 세포 수의 비율로 공동 배양된 것인, 약학 조성물.The pharmaceutical composition of claim 10, wherein the cytotoxic T cells and the natural killer cells are co-cultured at a cell number ratio of 8:1 to 12:1.
- 청구항 10에 있어서, 상기 수지상 세포 및 상기 세포 독성 T세포는 0.01:10 내지 0.03:10의 세포 수의 비율로 공동 배양된 것인, 약학 조성물.The pharmaceutical composition according to claim 10, wherein the dendritic cells and the cytotoxic T cells are co-cultured at a cell number ratio of 0.01:10 to 0.03:10.
- 청구항 1에 있어서, 상기 암은 고형암인, 약학 조성물.The pharmaceutical composition according to claim 1, wherein the cancer is a solid cancer.
- 청구항 14에 있어서, 상기 고형암은 대장암, 담관암, 위암, 폐암, 간암, 직장암, 유방암, 전립선암, 피부암, 두경부암, 췌장암, 난소암, 방광암, 신장암, 흑색종, 소세포폐암, 비소세포폐암, 육종, 신경아교종, T-세포 림프종 및 B-세포 림프종으로 이루어진 군에서 선택된 하나인, 약학 조성물.The method according to claim 14, wherein the solid cancer is colorectal cancer, bile duct cancer, gastric cancer, lung cancer, liver cancer, rectal cancer, breast cancer, prostate cancer, skin cancer, head and neck cancer, pancreatic cancer, ovarian cancer, bladder cancer, kidney cancer, melanoma, small cell lung cancer, and non-small cell lung cancer , Sarcoma, glioma, T-cell lymphoma and B-cell lymphoma, which is one selected from the group consisting of, a pharmaceutical composition.
- (a) 암 환자의 혈액으로부터 말초 혈액 단핵 세포(PBMC, peripheral blood mononuclear cell)를 분리하는 단계;(a) isolating peripheral blood mononuclear cells (PBMCs) from the blood of cancer patients;(b) 상기 (a) 단계에서 분리된 PBMC로부터 CD14+ 단핵 세포 및 CD14- 단핵 세포를 분리하는 단계;(b) isolating CD14+ mononuclear cells and CD14- mononuclear cells from the PBMCs isolated in step (a);(c) 상기 (a) 단계에서 분리된 PBMC로부터 자연 살해 세포를 증폭 배양하는 단계;(c) amplifying and culturing natural killer cells from the PBMCs isolated in step (a);(d) 상기 CD14+ 단핵 세포로부터 미성숙 수지상 세포를 분화시키는 단계;(d) differentiating immature dendritic cells from the CD14+ mononuclear cells;(e) 상기 CD14- 단핵 세포로부터 CD8+ 나이브(naive) T 세포를 분리하는 단계;(e) separating CD8+ naive T cells from the CD14- mononuclear cells;(f) 상기 미성숙 수지상 세포에 상기 암 환자로부터 분리된 종양 항원을 처리하여 성숙 수지상 세포를 수득하는 단계;(f) obtaining mature dendritic cells by treating the immature dendritic cells with a tumor antigen isolated from the cancer patient;(g) 상기 수득된 성숙 수지상 세포의 일부 및 상기 CD8+ 나이브(naive) T 세포를 공동 배양하여 세포 독성 T 세포를 수득하는 단계;(g) co-culturing a portion of the obtained mature dendritic cells and the CD8+ naive T cells to obtain cytotoxic T cells;(h) 상기 세포 독성 T 세포를 수득하는 단계에 이용되지 않은 성숙 수지상 세포, 상기 세포 독성 T 세포 및 상기 자연 살해 세포를 공동 배양하여 사전 활성화(pre-activation)하는 단계; 및(h) co-cultivating and pre-activating mature dendritic cells, the cytotoxic T cells, and the natural killer cells, which are not used in the step of obtaining the cytotoxic T cells; and(i) 상기 사전 활성화된 수지상 세포, 세포 독성 T 세포 및 자연 살해 세포를 상기 암의 예방 또는 치료 대상으로부터 유래된 암 세포와 공동 배양하여 활성화(activation)하는 단계;를 포함하는 암의 예방 또는 치료용 약학 조성물의 제조 방법.(i) co-cultivating and activating the pre-activated dendritic cells, cytotoxic T cells, and natural killer cells with cancer cells derived from a subject for prevention or treatment of cancer; A method for preparing a pharmaceutical composition for use.
- 청구항 16에 있어서, 상기 수지상 세포, 상기 자연 살해 세포 및 상기 세포 독성 T 세포 중 적어도 하나는 사이토카인을 포함하는 배지에서 배양된 것인, 제조 방법.The method according to claim 16, wherein at least one of the dendritic cells, the natural killer cells, and the cytotoxic T cells is cultured in a medium containing cytokines.
- 청구항 17에 있어서, 상기 사이토카인은 IL-2(Interleukin-2), IFN-γ(Interferon-γ), IL-15, IL-7, IL-21 및 4-1BB 리간드로 이루어진 군에서 선택된 적어도 하나인, 제조 방법.The method according to claim 17, wherein the cytokine is at least one selected from the group consisting of IL-2 (Interleukin-2), IFN-γ (Interferon-γ), IL-15, IL-7, IL-21 and 4-1BB ligand phosphorus, manufacturing method.
- 청구항 17에 있어서, 상기 사이토카인의 농도는 40 U/mL 내지 5000 U/mL인, 제조 방법.The method according to claim 17, wherein the concentration of the cytokine is 40 U/mL to 5000 U/mL.
- 청구항 18에 있어서, 상기 IL-2(Interleukin-2)의 농도는 100 U/mL 내지 5000 U/mL인, 제조 방법.The method according to claim 18, wherein the IL-2 (Interleukin-2) concentration is 100 U / mL to 5000 U / mL, the production method.
- 청구항 18에 있어서, 상기 IFN-γ(Interferon-γ)의 농도는 40 U/mL 내지 4000 U/mL 인, 제조 방법.The method according to claim 18, wherein the concentration of the IFN-γ (Interferon-γ) is 40 U / mL to 4000 U / mL, the manufacturing method.
- 청구항 16에 있어서, 상기 (g) 단계에서 상기 수득된 성숙 수지상 세포의 일부 및 상기 CD8+ 나이브(naive) T 세포는 1:8 내지 1:12의 세포 수의 비율로 공동 배양된 것인, 제조 방법.The method according to claim 16, wherein a portion of the mature dendritic cells obtained in step (g) and the CD8+ naive T cells are co-cultured at a cell number ratio of 1:8 to 1:12. .
- 청구항 16에 있어서, 상기 (h) 단계에서 상기 성숙 수지상 세포 및 상기 자연 살해 세포는 0.01:1 내지 0.03:1의 세포 수의 비율로 공동 배양된 것인, 제조 방법.The method according to claim 16, wherein in step (h), the mature dendritic cells and the natural killer cells are co-cultured at a cell number ratio of 0.01:1 to 0.03:1.
- 청구항 16에 있어서, 상기 (h) 단계에서 상기 세포 독성 T세포 및 상기 자연 살해 세포는 8:1 내지 12:1의 세포 수의 비율로 공동 배양된 것인, 제조 방법.The method according to claim 16, wherein in step (h), the cytotoxic T cells and the natural killer cells are co-cultured at a cell number ratio of 8:1 to 12:1.
- 청구항 16에 있어서, 상기 (h) 단계에서 상기 성숙 수지상 세포 및 상기 세포 독성 T세포는 0.01:10 내지 0.03:10의 세포 수의 비율로 공동 배양된 것인, 제조 방법.The method according to claim 16, wherein in step (h), the mature dendritic cells and the cytotoxic T cells are co-cultured at a cell number ratio of 0.01:10 to 0.03:10.
- 청구항 16에 있어서, 상기 (h) 단계의 공동 배양은 사이토카인을 포함하는 배지에서 배양하는 것인, 제조 방법.The method according to claim 16, wherein the co-culture of step (h) is cultured in a medium containing cytokines.
- 청구항 26에 있어서, 상기 사이토카인은 IL-2(Interleukin-2), IFN-γ(Interferon-γ), IL-15, IL-7, IL-21 및 4-1BB 리간드로 이루어진 군에서 선택된 적어도 하나인, 제조 방법.The method according to claim 26, wherein the cytokine is at least one selected from the group consisting of IL-2 (Interleukin-2), IFN-γ (Interferon-γ), IL-15, IL-7, IL-21 and 4-1BB ligand phosphorus, manufacturing method.
- 청구항 26에 있어서, 상기 사이토카인의 농도는 40 U/mL 내지 5000 U/mL인, 제조 방법.The method of claim 26, wherein the concentration of the cytokine is 40 U/mL to 5000 U/mL.
- 청구항 27에 있어서, 상기 IL-2(Interleukin-2)의 농도는 100 U/mL 내지 5000 U/mL인, 제조 방법.The method according to claim 27, wherein the IL-2 (Interleukin-2) concentration is 100 U / mL to 5000 U / mL, the production method.
- 청구항 27에 있어서, 상기 IFN-γ(Interferon-γ)의 농도는 40 U/mL 내지 4000 U/mL 인, 제조 방법.The method according to claim 27, wherein the concentration of the IFN-γ (Interferon-γ) is 40 U / mL to 4000 U / mL, the manufacturing method.
- 청구항 16에 있어서, 상기 (i) 단계의 공동 배양은 사이토카인을 포함하는 배지에서 배양하는 것인, 제조 방법.The method according to claim 16, wherein the co-culture of step (i) is cultured in a medium containing cytokines, the manufacturing method.
- 청구항 31에 있어서, 상기 사이토카인은 IL-2(Interleukin-2), IFN-γ(Interferon-γ), IL-15, IL-7, IL-21 및 4-1BB 리간드로 이루어진 군에서 선택된 적어도 하나인, 제조 방법.The method according to claim 31, wherein the cytokine is at least one selected from the group consisting of IL-2 (Interleukin-2), IFN-γ (Interferon-γ), IL-15, IL-7, IL-21 and 4-1BB ligand phosphorus, manufacturing method.
- 청구항 31에 있어서, 상기 사이토카인의 농도는 200 U/mL 내지 400 U/mL인, 제조 방법.The method of claim 31, wherein the concentration of the cytokine is 200 U/mL to 400 U/mL.
- 청구항 16에 있어서, 상기 암은 고형암인, 제조 방법.The method of claim 16 , wherein the cancer is a solid cancer.
- 청구항 34에 있어서, 상기 고형암은 대장암, 담관암, 위암, 폐암, 간암, 직장암, 유방암, 전립선암, 피부암, 두경부암, 췌장암, 난소암, 방광암, 신장암, 흑색종, 소세포폐암, 비소세포폐암, 육종, 신경아교종, T-세포 림프종 및 B-세포 림프종으로 이루어진 군에서 선택된 하나인, 제조 방법.35. The method of claim 34, wherein the solid cancer is colorectal cancer, cholangiocarcinoma, gastric cancer, lung cancer, liver cancer, rectal cancer, breast cancer, prostate cancer, skin cancer, head and neck cancer, pancreatic cancer, ovarian cancer, bladder cancer, kidney cancer, melanoma, small cell lung cancer, non-small cell lung cancer , sarcoma, glioma, T-cell lymphoma, and one selected from the group consisting of B-cell lymphoma, the manufacturing method.
- 청구항 16에 있어서, 상기 종양 항원은 상기 암 환자 조직 유래 단백질 추출물 또는 유전체 분석을 통해 수득한 신생 항원 펩타이드인, 제조 방법.The method according to claim 16, wherein the tumor antigen is a protein extract derived from the cancer patient's tissue or a neoantigen peptide obtained through genome analysis.
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WO2012175598A1 (en) * | 2011-06-21 | 2012-12-27 | Oncotyrol - Center For Personalized Cancer Medicine Gmbh | Method for activation of specific peripheral blood mononuclear cells |
CN106047810A (en) * | 2016-05-26 | 2016-10-26 | 深圳市金佳禾生物医药有限公司 | Novel DC-CTLs cell culture system and culture method thereof |
KR20190129046A (en) * | 2017-02-15 | 2019-11-19 | 더 리전트 오브 더 유니버시티 오브 캘리포니아 | Compositions and Methods for Activating NK Cells |
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WO2012175598A1 (en) * | 2011-06-21 | 2012-12-27 | Oncotyrol - Center For Personalized Cancer Medicine Gmbh | Method for activation of specific peripheral blood mononuclear cells |
CN106047810A (en) * | 2016-05-26 | 2016-10-26 | 深圳市金佳禾生物医药有限公司 | Novel DC-CTLs cell culture system and culture method thereof |
KR20190129046A (en) * | 2017-02-15 | 2019-11-19 | 더 리전트 오브 더 유니버시티 오브 캘리포니아 | Compositions and Methods for Activating NK Cells |
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