Activity-enhanced CIK cell, preparation method thereof and application of activity-enhanced CIK cell as medical preparation

Abstract

The invention provides an activity-enhanced CIK (cytokine-induced killer) cell, a preparation method thereof and application of the activity-enhanced CIK cell as a medical preparation, and belongs to the technical field of medical preparations. The invention provides a preparation method of an activity enhanced CIK (cytokine-induced killer) cell. The preparation method comprises the following steps: (1) separating mononuclear cells in peripheral blood of healthy people; (2) inoculating the mononuclear cells into an initial complete medium for culturing, and counting as the 0th day; (3) respectively adding IL-2 on the first day of culture and adding a 4-1BB antibody on the third day of culture, and carrying out activation culture; and (4) supplementing liquid from the fourth day of the culture, then supplementing liquid once every other day, and harvesting the activity enhanced CIK cells after the culture is finished. The activity enhanced CIK cell provided by the invention has the characteristics of strong in-vitro proliferation ability and high killing activity.

Description

Technical Field

[0001] The present invention relates to the technical field of medical preparations, and in particular to an activity-enhanced CIK cell and a preparation method thereof and an application thereof as a medical preparation. Background Art

[0002] CIK cells are killer cells induced by multiple cytokines and are a new type of immune active cells. CIK cells have strong proliferation ability, strong cytotoxicity, and certain immune characteristics. CIK cells have a strong ability to recognize tumor cells and have a significant effect on patients after surgery or chemotherapy. They can eliminate extremely small residual metastatic lesions, prevent the spread and recurrence of cancer cells, and improve the body's immunity. Therefore, enhancing the in vitro proliferation and killing activity of CIK cells is of great significance for tumor treatment. Summary of the invention

[0003] The purpose of the present invention is to obtain CIK cells with strong in vitro proliferation ability and high killing activity, so as to provide effective medical preparations for tumor treatment.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for preparing activity-enhanced CIK cells, comprising the following steps: (1) Isolate mononuclear cells from healthy human peripheral blood; (2) inoculating the mononuclear cells into an initial complete culture medium for culture, which is counted as day 0; (3) adding IL-2 on the first day of the culture and adding 4-1BB antibody on the third day to perform activation culture; (4) Starting from the 4th day of the culture, the fluid is replenished, and then replenished every other day. After the culture is completed, the activity-enhanced CIK cells are harvested.

[0005] Preferably, the initial complete culture medium contains 10 ng / ml to 80 ng / ml of CD3 monoclonal antibody and 50 ng / ml to 120 ng / ml of IFN-γ.

[0006] Preferably, the final concentration of IL-2 after addition is 500 IU / ml to 1000 IU / ml.

[0007] Preferably, the final concentration of the 4-1BB antibody after addition is 10 ng / ml to 80 ng / ml.

[0008] Preferably, in step (3), X-VIVO medium containing 500 IU / ml to 600 IU / ml of IL-2 is used for fluid replenishment, and the cell concentration is adjusted to (6 to 7)×10 5 Pieces / ml.

[0009] Preferably, the culturing period is 13 to 15 days.

[0010] The present invention also provides an activity-enhanced CIK cell obtained by the preparation method.

[0011] The present invention also provides an application of the activity-enhanced CIK cells in preparing a medical preparation for killing tumors.

[0012] Preferably, the tumor is nasopharyngeal carcinoma.

[0013] CIK cells, or cytokine-induced killer cells (CIK), are a type of T lymphocytes, killer cells activated by cytokines, and a heterogeneous group of cells. They belong to specific immune cells (acquired immune cells). The main effector cells of CIK cells are CD3+ / CD56+ cells, also known as NK cell-like T lymphocytes, which have the powerful anti-tumor activity of T lymphocytes and the non-MHC restricted tumor killing advantages of NK cells.

[0014] The activity-enhanced CIK cells provided by the present invention are induced and cultured by adding CD3 monoclonal antibody and IFN-γ, stimulated by adding IL-2, and activated by adding 4-1BB antibody. In particular, after activation by adding 4-1BB antibody, cell activity is significantly enhanced, the expression of CD3+ and CD8+ is increased, and the tumor cell killing ability is increased. It can be used as a medical preparation for the treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the effect of 4-1BB antibody on the proliferation activity of CIK cells.

[0016] Figure 2 This is the effect of 4-1BB antibody on the expression of CD3+ and CD8+ in CIK cells.

[0017] Figure 3 The killing activity of CIK cells cultured and activated by 4-1BB antibody on tumor cells. DETAILED DESCRIPTION

[0018] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0019] Example 1 1. Peripheral Blood Collection and PBMC Isolation 1.1 Sample centrifugation: Collect 50 ml of peripheral blood from NPC patients and transfer the peripheral blood in the collection tube to a new 50 ml disposable sterile centrifuge tube using a 10 ml disposable sterile pipette. Centrifuge at 2300 rpm for 10 min (9 for ascending and 7 for descending) at room temperature (25°C).

[0020] 1.2 Plasma separation: Use a 10mL disposable sterile pipette to carefully aspirate the upper plasma layer after centrifugation in 1.1, transfer it into a new disposable 50mL sterile centrifuge tube, and then place it in a 56℃ water bath for 30min. After inactivation, cool it in a 4℃ refrigerator overnight, centrifuge it at 2700rpm for 10min at room temperature (25℃), collect the plasma supernatant into a new 50mL disposable sterile centrifuge tube with a 10mL disposable sterile pipette, and use it as autologous plasma for later use, and store it in a 4℃ refrigerator.

[0021] 1.3 PBMC separation: Separation by Ficoll density gradient centrifugation. Remove the plasma from the cell layer in 1.1, and dilute the peripheral blood cells to Ficoll in a volume ratio of 2:1 using a 10mL disposable sterile pipette with physiological saline to 30ml. Then use a 10mL disposable sterile pipette to add 15mL of lymphocyte separation solution to a new 50mL centrifuge tube, and then use a new 10mL disposable sterile pipette to mix the diluted blood cells and add them to the centrifuge tube (note that when adding to the liquid surface at the beginning, it should be very slow, and be careful to add the blood cells above the liquid surface of Ficoll, and do not damage the contact surface between the separation solution and the cell suspension). Centrifuge at room temperature, 2000rpm, for 20min (0 rise, 0 drop).

[0022] 1.4 PBMC collection: After centrifugation, use a 10 mL disposable sterile pipette to remove the top layer of liquid layer by layer, then carefully aspirate the buffy coat layer to collect PBMCs, and transfer to a new 50 mL disposable sterile centrifuge tube. After sampling and counting, centrifuge at room temperature (25°C) at 1500 rpm for 5 minutes (9 for ascending and 7 for descending), and wash twice with 0.9% saline to obtain PBMCs.

[0023] 2. CIK Cell Inoculation The seeding cell density was 5×10 5 The cells were inoculated at a density of 100 cells / ml. The initial complete culture medium consisted of serum-free X-VIVO medium, CD3 monoclonal antibody and IFN-γ. The volume was 50 ml, containing 50 ng / ml CD3 monoclonal antibody and 100 ng / ml IFN-γ. The culture conditions were 37°C and 5% CO2. This was counted as day 0. The CD3 monoclonal antibody (6001T01) was purchased from Zhuhai Beso Cell Science and Technology Co., Ltd., and the IFN-γ (NBP2-34992) was purchased from NOVUS.

[0024] 3. CIK cell activation 3.1 On the first day of culture, 600 IU / ml IL-2 was added for stimulation, and on the third day, 50 ng / ml 4-1BB antibody was added for activation culture. Culture conditions: 37°C, 5% CO2.

[0025] 3.2 The live cells of the cultured CIK cells were counted and rehydrated on days D4, D6, D8, and D12, and the cell concentration was adjusted to 5×10 5 / ml, add X-VIVO medium containing 600IU / ml IL-2 as replenisher to maintain cell proliferation activity.

[0026] 3.3 Harvest cells after culture until D14.

[0027] Example 2 CIK cells harvested in Example 1 were used to count viable cells. At the same time, a comparative experiment without adding 4-1BB antibody was set up (other experimental steps were the same as in Example 1) to count viable cells. The results were as follows: Figure 1 As shown. Figure 1 The measurement results show that adding 4-1BB antibody to activate CIK cells can significantly enhance the proliferation activity of CIK cells.

[0028] The expression of CD3+ and CD8+ in the two groups of cells was measured. Figure 2 As shown. Figure 2 It can be seen that adding 4-1BB antibody to activate CIK cells can enhance the expression of CD3+ and CD8+.

[0029] Example 3 The CIK cell suspension collected in Example 1 and cultured on day D14 was placed in a 250 ml centrifuge tube and centrifuged at 1800 rpm for 5 min. After centrifugation, the cells were resuspended in 50 ml of physiological saline and samples were taken for endotoxin detection. The detection result was ≤0.25 EU / ml.

[0030] The CIK cells cultured to D14 in the comparative experiments of Example 1 and Example 2 were counted using a cell counter and the cell concentration was adjusted with a serum-free X-VIVO medium. HK1-EBV tumor cells were killed at an effector-target ratio of 10:1. The cells were cultured in a 37°C, 5% CO2 incubator. The killing results showed that the killing activity of D14 CIK cells cultured with 4-1BB was higher than that of CIK cells cultured without 4-1BB ( Figure 3 ).

[0031] It can be seen from the above examples that the present invention provides an activity-enhanced CIK cell having the characteristics of strong in vitro proliferation ability and high killing activity.

[0032] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing activity-enhanced CIK cells, characterized in that: The steps include: (1) Isolate mononuclear cells from healthy human peripheral blood; (2) inoculating the mononuclear cells into an initial complete culture medium for culture, which is counted as day 0; (3) adding IL-2 on the first day of the culture and adding 4-1BB antibody on the third day to perform activation culture; (4) Starting from the 4th day of the culture, the fluid is replenished, and then replenished every other day. After the culture is completed, the activity-enhanced CIK cells are harvested.

2. The method for preparing activity-enhanced CIK cells according to claim 1, characterized in that: The initial complete culture medium contains 10 ng / ml to 80 ng / ml of CD3 monoclonal antibody and 50 ng / ml to 120 ng / ml of IFN-γ.

3. The method for preparing activity-enhanced CIK cells according to claim 2, characterized in that: The final concentration of the IL-2 after addition is 500 IU / ml to 1000 IU / ml.

4. The method for preparing activity-enhanced CIK cells according to claim 3, characterized in that: The final concentration of the 4-1BB antibody after addition is 10 ng / ml to 80 ng / ml.

5. The method for preparing activity-enhanced CIK cells according to claim 4, characterized in that: In step (3), X-VIVO medium containing 500 IU / ml ~1000 IU / ml IL-2 was used for rehydration. During rehydration, the cell concentration was adjusted to (6~7)×10 5 Pieces / ml.

6. The method for preparing activity-enhanced CIK cells according to claim 5, characterized in that: The duration of the culture is 13 to 15 days.

7. An activity-enhanced CIK cell obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the activity-enhanced CIK cells according to claim 7 in the preparation of a medical preparation for tumor killing.

9. The use according to claim 8, characterized in that The tumor is nasopharyngeal carcinoma.

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