WO2023038492A1 - Biological composition for preventing or treating cancer, comprising dendritic cells, natural killer cells and cytotoxic t cells - Google Patents

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

Biological composition for preventing or treating cancer comprising dendritic cells, natural killer cells and cytotoxic T cells

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. 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.

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.

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.

1. A pharmaceutical composition for preventing or treating cancer containing dendritic cells, natural killer cells and cytotoxic T cells.

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. 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. 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. 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. 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. The pharmaceutical composition according to 5 above, wherein the concentration of the cytokine is 40 U/mL to 5000 U/mL.

8. The pharmaceutical composition according to 6 above, wherein the IL-2 (Interleukin-2) concentration is 100 U/mL to 5000 U/mL.

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. 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. 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. 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. 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. The pharmaceutical composition according to 1 above, wherein the cancer is solid cancer.

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) isolating peripheral blood mononuclear cells (PBMCs) from the blood of cancer patients;

(b) isolating CD14+ mononuclear cells and CD14- mononuclear cells from the PBMCs isolated in step (a);

(c) amplifying and culturing natural killer cells from the PBMCs isolated in step (a);

(d) differentiating immature dendritic cells from the CD14+ mononuclear cells;

(e) separating CD8+ naive T cells from the CD14- mononuclear cells;

(f) obtaining mature dendritic cells by treating the immature dendritic cells with a tumor antigen isolated from the cancer patient;

(g) co-culturing a portion of the obtained mature dendritic cells and the CD8+ naive T cells to obtain cytotoxic T cells;

(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) 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. 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. 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. The method according to 17 above, wherein the concentration of the cytokine is 40 U/mL to 5000 U/mL.

20. The method according to 18 above, wherein the IL-2 (Interleukin-2) concentration is 100 U/mL to 5000 U/mL.

21. The method according to 18 above, wherein the concentration of IFN-γ (Interferon-γ) is 40 U/mL to 4000 U/mL.

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. 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. 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. 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. The method according to 16 above, wherein the co-culture in step (h) is cultured in a medium containing cytokines.

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. The method according to 26 above, wherein the concentration of the cytokine is 40 U/mL to 5000 U/mL.

29. The method according to 27 above, wherein the IL-2 (Interleukin-2) concentration is 100 U/mL to 5000 U/mL.

30. The method according to 27 above, wherein the concentration of Interferon-γ (IFN-γ) is 40 U/mL to 4000 U/mL.

31. The method according to 16 above, wherein the co-culture in step (i) is cultured in a medium containing cytokines.

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. The method according to 31 above, wherein the concentration of the cytokine is 200 U/mL to 400 U/mL.

34. The method according to 16 above, wherein the cancer is a solid cancer.

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. 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.

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.

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.

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-γ expression is higher in CD8+ naive T cells co-cultured with mature dendritic cells than in non-co-cultured T cells.

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.

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.

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.

Hereinafter, the present invention will be described in detail.

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.

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.

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-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.

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

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.

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.

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.

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. 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.

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).

(a) isolating peripheral blood mononuclear cells (PBMCs) from the blood of cancer patients;

(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) differentiating immature dendritic cells from the CD14+ mononuclear cells;

(e) separating CD8+ naive T cells from the CD14- mononuclear cells;

(f) obtaining mature dendritic cells by treating the immature dendritic cells with a tumor antigen isolated from the cancer patient;

(g) co-culturing a portion of the obtained mature dendritic cells and the CD8+ naive T cells to obtain cytotoxic T cells;

(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) 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.

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. By step (g), naive T cells can be activated to obtain cytotoxic T cells.

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.

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.

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.

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.

Hereinafter, examples will be described in detail to explain the present invention in detail.

manufacturing example

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. Isolation of peripheral blood mononuclear cells

1. Collect blood into 5 heparin tubes (about 50 ml).

2. Prepare three 50 ml tubes.

3. Transfer the blood to a 50 ml tube and check the amount of blood.

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. 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. Centrifuge at 2300 rpm, 25 min, ACC/DERT-10.

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. 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. Fill DPBS to the 45ml scale of the tube. And centrifuged at 800 xg, 5 min, ACC 44, RT.

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. 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. Perform cell counting.

II. Culture of immature dendritic cells after isolation of CD14+ monocytes

1. Centrifuge the PBMCs at 1500 rpm or higher for 5 minutes and remove the supernatant.

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. Add 20 μL of CD14 microbeads per 10 total cells. Mix well and set in the refrigerator (2-8 ° C) for 15 minutes.

4. Prepare and prime the autoMACS™ Pro Separator.

5. Prepare two 15 mL tubes and label them with 'pos' and 'neg'.

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. Enter the CD14 bead reagent code into autoMACS™ Pro Separator.

8. Place the reagent bottle in the reagent port.

9. Go to the Separation menu and highlight the sample to be separated.

10. Select the desired labeling reagent (CD14 beads).

11. Enter the total volume of the sample (amount of PE buffer used to suspend the cells).

12. Select the separation program ‘Possel’.

13. Select a wash program and press Run.

14. After the separation process, collect CD14+ monocytes from the positive fraction of the rack.

15. Count the cells and centrifuge at 1500 rpm for 5 minutes.

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. 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) Carefully remove the plate from the incubator so that the dendritic cells do not fall off.

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. 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. Put the culture plate back into the incubator.

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. Transfer the cells to an eppendorf tube and centrifuge at 1500 rpm for 5 minutes.

24. Discard the supernatant and suspend the cells in DPBS.

25. Centrifuge at 1500 rpm for 5 minutes.

26. Repeat steps 4 and 5 for a total of 3 washes.

27. Suspend the cells in 100-200 ul of Immunocult ^™ -ACF dendritic cell medium.

28. Perform 6 freeze-thaw cycles in a -80°C refrigerator or liquid nitrogen gas tank.

29. Sonicate the cells in the eppendorf tube for 30 minutes.

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. Store tumor lysate at -80°C until use.

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. Add the deparaffinization solution (320 ul) to the sample tube.

34. Incubate at 56 °C for 3 minutes. Spin down after vortexing.

35. Remove the supernatant.

36. (Optional) Repeat steps 2 and 4 if more paraffin needs to be removed from the tissue.

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. 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. 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. 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. Cool at room temperature for 20 minutes.

42. Centrifuge at 11000 xg for 1 minute. Remove the supernatant.

43. Add DPBS to the tube and perform the next step on a clean bench.

44. Centrifuge at 11000 xg for 1 minute. Remove the supernatant.

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. 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. Centrifuge at 1500 rpm for 3 minutes to collect the sample at the bottom of the tube.

48. Transfer the sample to an eppendorf tube and centrifuge at 10000 rpm for 10 minutes.

49. Collect the supernatant containing tumor proteins in another tube.

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. Measure the total amount of extracted protein. Divide the protein and store at -80 °C until use.

52. Carefully remove the dish from the incubator so as not to shake the dendritic cells.

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. 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. 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. Return the culture plate to the incubator.

57. Carefully remove the dish from the incubator to avoid dislodging the dendritic cells.

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. Gently shake the culture plate.

60. Put the culture plate back into the incubator.

III. CD8+ naive T cell isolation and culture from CD14- monocytes

1. Collect CD14- PBMCs obtained from the CD14 negative fraction.

2. Count the cells.

3. Centrifuge the cells at 1500 rpm for 5 minutes and remove the supernatant completely.

4. Suspend the pellet with PE buffer (40 uL per 10 ⁷ cells).

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. Add PE buffer (30 uL of PE per 10 ⁷ cells).

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. Add 2 mL PE buffer and centrifuge at 1500 rpm for 5 minutes.

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. Prepare two 15 mL tubes and label them 'pos' and 'neg'.

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. Go to the Separation menu and highlight the sample to be separated.

13. Select the desired labeling reagent (in this case it should be empty).

14. Enter the total volume of the sample (amount of PE buffer used to suspend the cells (500 uL)).

15. Select the separation program 'Depl05'.

16. Select a wash program and press Run.

17. After the separation process, collect the negative cells in the negative fraction position of the rack.

18. Centrifuge the cells at 1500 rpm for 05 minutes and aspirate the supernatant completely.

19. Suspend cells in PE buffer (80 uL of PE per 10 ⁷ cells).

20. Add CD8 Microbeads (20 uL per 10 ⁷ cells), mix gently and incubate in refrigerator (2-8℃) for 15 minutes.

21. Add 2 mL PE buffer and centrifuge at 1500 rpm for 5 minutes.

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. Prepare two 15 mL tubes and label them 'pos' and 'neg'.

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. Go to the Separation menu and highlight the sample to be separated.

26. Select the desired labeling reagent (should be blank in this case).

27. Enter the total volume of the sample (amount of PE buffer used to suspend the cells (500 uL)).

28. Select the separation program ‘Possel’.

29. Select a wash program and press Run.

30. After the separation process, collect the positive cells in the positive fraction of the rack. These are naive CD8+ T cells.

31. Count the cells and suspend the cells in T cell culture medium supplemented with 2000 units/mL of IL-2.

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. Carefully remove the plate from the incubator.

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. Put the culture plate back into the incubator.

IV. Co-culture of mature dendritic cells and CD8+ naive T cells to amplify after T cell activation

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. Centrifuge the above cells on the plate at 250 g for 3 minutes.

3. Co-culture in an incubator for 4 days.

4. Suspend the remaining T cell culture medium with 6000 units of IL-2 added.

5. Mix the cells with a pipette and centrifuge at 250 g for 3 minutes.

6. Cultivate the cells in an incubator.

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) Gently remove the cells from the incubator and take microscopic images of the cells.

9. Calculate the amount of cells and write a report.

10. Determine the purity of T cells by measuring the amount of CD8 and CD4 positive cells.

11. Start co-culture of cells and gamma-irradiated peripheral blood monocytes at a ratio of 1:200.

12. Conduct half media change or split twice for about 14 days at 2-3 day intervals.

V. Natural killer cell culture using peripheral blood monocytes

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. 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. 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.

preliminary experiment

Preliminary experiments were performed during or after the preparation of the above Preparation Example.

I. Increased secretion of IL-12 (p70) by immature dendritic cells loaded with antigens or peptides extracted from patients' tissues

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).

II. Increased IFN-γ expression and cell culture pattern of CD8+ naive T cells co-cultured with mature dendritic cells

CD8+ naive T cells were isolated from the blood of a patient (SDH21028), cultured, and then co-cultured with mature dendritic cells.

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 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. Natural killer cell culture patterns

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.

24 well	DAY-0	DAY-14
Purity (%)	16.3%	98.8%
Fold	One	251.51 times
Converted NK cells	2.45x10^6	6.14x10^8

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).

Example

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. Example 1

1. Method

(1) Cell seeding

Primary tumor cells are seeded in a 96 well plate at 1x10 ⁴ cells/100 ul/well, followed by incubation for 24 hours.

(2) Preparation of immune cells (Effector cell) and cytokine pre-activation

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.

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 and target cell co-culture (co-culture)

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

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. Results

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. Example 2

1. Method

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. Results

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. Example 3

1. Method

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. Results

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. Example 4

1. Method

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. Results

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. Example 5

1. Method

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. Results

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. Example 6

1. Method

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. result 1

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. result 2

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)

1. A pharmaceutical composition for preventing or treating cancer comprising dendritic cells, natural killer cells and cytotoxic T cells.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. The pharmaceutical composition according to claim 5, wherein the concentration of the cytokine is 40 U/mL to 5000 U/mL.
8. The pharmaceutical composition according to claim 6, wherein the IL-2 (Interleukin-2) concentration is 100 U/mL to 5000 U/mL.
9. 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.
10. 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.
11. 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.
12. 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.
13. 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.
14. The pharmaceutical composition according to claim 1, wherein the cancer is a solid cancer.
15. 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.
16. (a) isolating peripheral blood mononuclear cells (PBMCs) from the blood of cancer patients;

    (b) isolating CD14+ mononuclear cells and CD14- mononuclear cells from the PBMCs isolated in step (a);

    (c) amplifying and culturing natural killer cells from the PBMCs isolated in step (a);

    (d) differentiating immature dendritic cells from the CD14+ mononuclear cells;

    (e) separating CD8+ naive T cells from the CD14- mononuclear cells;

    (f) obtaining mature dendritic cells by treating the immature dendritic cells with a tumor antigen isolated from the cancer patient;

    (g) co-culturing a portion of the obtained mature dendritic cells and the CD8+ naive T cells to obtain cytotoxic T cells;

    (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) 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. 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.
18. 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.
19. The method according to claim 17, wherein the concentration of the cytokine is 40 U/mL to 5000 U/mL.
20. The method according to claim 18, wherein the IL-2 (Interleukin-2) concentration is 100 U / mL to 5000 U / mL, the production method.
21. 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.
22. 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. .
23. 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.
24. 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.
25. 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.
26. The method according to claim 16, wherein the co-culture of step (h) is cultured in a medium containing cytokines.
27. 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.
28. The method of claim 26, wherein the concentration of the cytokine is 40 U/mL to 5000 U/mL.
29. The method according to claim 27, wherein the IL-2 (Interleukin-2) concentration is 100 U / mL to 5000 U / mL, the production method.
30. 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.
31. The method according to claim 16, wherein the co-culture of step (i) is cultured in a medium containing cytokines, the manufacturing method.
32. 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.
33. The method of claim 31, wherein the concentration of the cytokine is 200 U/mL to 400 U/mL.
34. The method of claim 16 , wherein the cancer is a solid cancer.
35. 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.
36. 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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