Preparation method of DC-CIK co-culture cell, sensitization antigen thereof and application of DC-CIK co-culture cell

Abstract

The invention relates to a co-culture system based on dendritic cells (DC) and cytokine induced killer cells (CIK) and application of the co-culture system. According to the method, the DC cells are sensitized through combined application of two sensitization antigens, and the DC cells and the CIK cells are co-cultured, so that the specific anti-tumor activity of the cells is remarkably enhanced. In addition, by introducing a three-dimensional dynamic culture system, the antigen presentation capability of the DC cells and the killing activity of the CIK cells are effectively improved. The invention has obvious advantages in the aspect of improving the tumor immunotherapy effect, and can be widely applied to the treatment of various solid tumors and hematological malignant tumors.

Description

Technical Field

[0001] The present invention relates to the field of tumor immunotherapy, and in particular to a method for preparing a co-culture system based on dendritic cells (DC) and cytokine-induced killer cells (CIK), and a method for enhancing the specific anti-tumor activity of cells by sensitizing antigens. Background Art

[0002] DC-CIK co-culture cells are an immune cell therapy that combines dendritic cells (DC) and cytokine-induced killer cells (CIK). This approach aims to enhance anti-tumor immune responses and is often used in tumor immunotherapy research.

[0003] DC cells are powerful antigen-presenting cells that can effectively capture, process and present antigens to T cells, thereby initiating and regulating immune responses. In the DC-CIK co-culture system, DC cells are first sensitized with exogenous antigens (such as tumor cell lysates or specific tumor antigens) to enable them to effectively recognize and present tumor-associated antigens. CIK cells are a heterogeneous cell population, mainly composed of CD3+ and CD56+ cell subsets, and have high anti-tumor activity. CIK cells are characterized by having the dual characteristics of natural killer cells and T cells, and therefore exhibit strong cytotoxicity in anti-tumor immune responses.

[0004] In the DC-CIK co-culture process, mature DC cells and activated CIK cells are first obtained through in vitro culture. Then, sensitized DC cells are co-cultured with CIK cells to further activate CIK cells through the antigen presentation function of DC cells. This co-culture strategy can significantly enhance the specific killing ability of CIK cells against tumor cells.

[0005] DC-CIK cell combination therapy can significantly improve anti-tumor efficacy, has good immunological persistence and broad application prospects. Studies have shown that this treatment method has potential clinical application value in a variety of solid tumors and hematological malignancies. DC-CIK cell therapy has shown significant advantages in enhancing the immune system's recognition and elimination of tumor cells, becoming one of the important strategies in personalized tumor immunotherapy.

[0006] DC-CIK cell therapy has shown great potential in tumor immunotherapy. However, the traditional DC-CIK co-culture system still has certain limitations, such as the limited antigen presentation efficiency of DC cells, and the unsatisfactory proliferation ability and specific killing activity of CIK cells. Therefore, how to further optimize the DC-CIK co-culture system and improve its therapeutic effect has become a hot topic in current research. Summary of the invention

[0007] In order to make up for the deficiencies of the prior art, the object of the present invention is to provide a method for preparing DC-CIK co-cultured cells, a sensitizing antigen and an application thereof.

[0008] Therefore, the present invention discloses a method for preparing DC-CIK co-cultured cells, the method comprising the following steps:

[0009] (1) Isolation of PBMCs: PBMCs were isolated from blood using Ficoll-Paque solution;

[0010] (2) Preparation of DC cells: PBMCs isolated were divided into 1×10 6 DCs were inoculated at a density of 100 / ml in RPMI-1640 medium containing 10% FBS, 1000 IU / ml GM-CSF and 500 IU / ml IL-4, and then anti-CTLA-4 monoclonal antibody and AFP protein were added to induce DC maturation;

[0011] (3) Preparation of CIK cells: PBMCs were cultured at 1×10 6 The cells were inoculated at a density of 100 / ml into RPMI-1640 medium containing 10% FBS and 1000 U / ml IFN-γ, and then replaced with 10% FBS, 1000 U / ml IL-2 and anti-CD3 monoclonal antibody at a final concentration of 50 ng / ml to induce CIK cell generation;

[0012] (4) Co-culture of DC-CIK cells: Mature DC cells and CIK cells were uniformly inoculated on the surface of the three-dimensional scaffold in a certain proportion. The inoculated scaffold was placed in a dynamic culture device with a flow rate of 0.5 ml / min at 37°C and 5% CO. 2 DC-CIK cells were obtained by co-culturing for 48 to 72 hours under culture conditions.

[0013] Preferably, the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-CTLA-4 monoclonal antibody in the preparation of DC cells in step (2) of the present invention are as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0014] Preferably, the concentrations of the anti-CTLA-4 monoclonal antibody and AFP protein used in the preparation of DC cells in step (2) of the present invention are 10 μg / ml and 5 μg / ml, respectively.

[0015] Preferably, the amino acid sequences of the heavy chain and light chain variable regions of the anti-CD3 monoclonal antibody in the preparation of CIK cells in step (3) of the present invention are as shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0016] Preferably, in the step (4) of the present invention, the ratio of DC cells to CIK cells in the co-culture of DC-CIK cells is 1:5.

[0017] Preferably, in the step (4) of the present invention, the pore size of the three-dimensional scaffold in the co-culture of DC-CIK cells is 200-250 μm, and the porosity is about 90%.

[0018] In another aspect, the present invention also discloses a use of the prepared DC-CIK co-culture cells in the preparation of anti-tumor drugs.

[0019] The present invention adopts the combined application of two sensitizing antigens to activate the antigen presentation function of DC cells, and further enhances the proliferation and activation state of CIK cells by regulating the immune microenvironment. In addition, the present invention introduces a three-dimensional dynamic culture system. By co-culturing cells in a three-dimensional scaffold and enhancing signal transmission between cells in a dynamic flow culture environment, the antigen presentation ability of DC cells and the activity of CIK cells are improved.

[0020] The innovative preparation method of DC-CIK co-culture cells proposed in the present invention, combined with the application of specific sensitizing antigens, significantly improves the antigen presentation ability of DC cells and the killing activity of CIK cells. The method has obvious creativity and shows significant effects superior to the existing technology by optimizing culture conditions and using innovative antigens. It provides a good reference for the research and application of DC-CIK co-culture.

[0021] It should be noted that the anti-CTLA-4 monoclonal antibody and anti-CD3 monoclonal antibody prepared in this study were screened, prepared and identified using conventional mouse hybridoma cell technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the three-dimensional scaffold. DETAILED DESCRIPTION

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0024] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0025] Example 1: Isolation and storage of PBMCs

[0026] (1) Dilute the collected blood (in compliance with ethical requirements) by 2 times with PBS (e.g. 10 ml blood + 10 ml PBS).

[0027] (2) Use a 50 ml EP tube and add 15 ml of Ficoll-Paque solution for later use.

[0028] (3) Gently add 25 ml of the diluted blood to the upper layer of the Ficoll-Paque layer, maintain the liquid level, and centrifuge at 400 g for 30 minutes at room temperature, slowly ascending and descending.

[0029] (4) After centrifugation, a white film composed of mononuclear cells will be found on the upper layer of Ficoll-Paque. Use a Pasteur pipette to carefully collect the mononuclear cells and transfer them to a new centrifuge tube. Centrifuge at 400g for 10 minutes to wash the cells.

[0030] (5) Discard the supernatant, resuspend the cells, add PBS or serum-free culture medium, and centrifuge at 400g for 10 minutes to wash the cells.

[0031] (6) Discard the supernatant, resuspend the cells, add serum-free culture medium, and centrifuge at 400 g for 10 minutes to wash the cells.

[0032] (7) PBMCs were suspended in a cryopreservation solution containing 10% DMSO and 90% FBS at a concentration of 1 × 10 7 The cells were divided into aliquots at a concentration of 10 cells / ml, gradually cooled to -80°C, and then transferred to liquid nitrogen for long-term storage.

[0033] Example 2: Preparation of DC cells

[0034] (1) The isolated PBMCs were divided into 1×10 6 The cells were inoculated at a density of 100 / ml in RPMI-1640 medium containing 10% FBS, 1000 IU / ml GM-CSF and 500 IU / ml IL-4. After mixing, the cells were placed in a 37°C, 5% CO 2 The cells were cultured in an incubator for 5 days, and half of the culture medium was replaced every 2 days.

[0035] (2) On day 5, anti-CTLA-4 monoclonal antibody (10 μg / ml) and AFP protein (5 μg / ml) were added to sensitize the antigens and cultured for another 48 h to induce DC maturation.

[0036] The anti-CTLA-4 monoclonal antibody was prepared in this study, and the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO. 1 and SEQ ID NO. 2. Compared with the commercial monoclonal antibody (ab110650), the monoclonal antibody has higher activity, that is, when the indirect ELISA test is performed, at the same concentration, when both monoclonal antibodies are diluted to 10,000 times, the OD450nm value of the monoclonal antibody in this study is 1.78, while that of the commercial monoclonal antibody is 1.05.

[0037] At the same time, DC cells were prepared using the non-sensitized method for use in subsequent experiments.

[0038] (3) DC cells were suspended in a cryopreservation solution containing 10% DMSO and 90% FBS at a rate of 1×10 7 The cells were divided into aliquots at a concentration of 10 cells / ml, gradually cooled to -80°C, and then transferred to liquid nitrogen for long-term storage.

[0039] Example 3: CIK cell preparation

[0040] (1) PBMCs were cultured at 1×10 6 The cells were inoculated at a density of 1000 / ml into a culture bottle containing RPMI-1640 medium containing 10% FBS and 1000 U / ml IFN-γ, mixed and placed at 37°C and 5% CO 2 Incubate in an incubator for 24 hours.

[0041] (2) After 24 hours, replace the RPMI-1640 medium containing 10% FBS, 1000U / ml IL-2 and a final concentration of 50ng / ml anti-CD3 monoclonal antibody (prepared for this study, wherein the amino acid sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively) or a commercial anti-CD3 monoclonal antibody (Vokavi, T101), and continue to culture for 14 days, replacing half of the medium every 2 days. On the 14th day of culture, CIK cells reached a peak and showed significant killing activity. At this time, the cells can be collected for subsequent application, analysis or cryopreservation.

[0042] (3) CIK cells were suspended in a cryopreservation solution containing 10% DMSO and 90% FBS at a rate of 1×10 7 The cells were divided into aliquots at a concentration of 10 cells / ml, gradually cooled to -80°C, and then transferred to liquid nitrogen for long-term storage.

[0043] Example 4: Co-culture of DC-CIK cells

[0044] (1) The prepared three-dimensional scaffold (see Example 6 for details) was immersed in serum-free RPMI-1640 culture medium for 24 hours to remove the residual cross-linking agent.

[0045] (2) DC cells prepared in Example 2 and CIK cells prepared in Example 3 were mixed at a ratio of 1:5 and uniformly inoculated on the surface of the scaffold. For example, 1×10 5 DC cells and 5×10 5 The inoculated scaffolds were placed in a dynamic culture device with a flow rate of 0.5 ml / min at 37 °C and 5% CO. 2 The cells were cultured under the conditions of 48 to 72 hours.

[0046] (3) After the culture is completed, the cells are washed with PBS and the co-cultured cell population is collected. DC-CIK cells can be cryopreserved in a cryopreservation solution of 10% DMSO and 90% FBS, gradually frozen to -80°C and then transferred to liquid nitrogen for long-term storage.

[0047] At the same time, a conventional DC-CIK co-culture system without a three-dimensional scaffold was used to culture DC-CIK cells for subsequent experiments.

[0048] Example 5: Cell proliferation and functional detection

[0049] (1) Cell proliferation detection: Cell proliferation was detected according to the instructions of the CCK-8 kit (Dojindo Company). The results showed (as shown in Table 1) that the DC-CIK cells using the sensitized antigen and the three-dimensional dynamic culture system had the best proliferation rate (the higher the OD value, the better the cell proliferation), which also showed that the anti-CD3 monoclonal antibody prepared by the present invention was better than the commercial monoclonal antibody.

[0050] Table 1 CCK-8 kit test results

[0051]

[0052] (2) Cell activity detection: The cell activity was detected by MTT method. The results showed (Table 2) that the cell activity was significantly improved under the innovative culture system (the higher the A570 value, the better the cell activity).

[0053] Table 2 Results of cell activity detection by MTT method

[0054]

[0055] (3) Specific killing activity detection (LDH release method): Detect the killing activity of DC-CIK co-culture system on tumor cells. The results showed (Table 3) that the cell killing rate under the innovative culture system reached 85.6%, while that of the control group was only 44.5%.

[0056] Table 3 Specific lethality test results

[0057]

[0058] Example 6: Preparation of three-dimensional scaffolds

[0059] 1. Preparation of three-dimensional scaffold materials

[0060] (1) Preparation of gelatin solution: Dissolve 5 g of gelatin in 100 ml of sterile deionized water and place in a constant temperature water bath at 37°C and stir until completely dissolved.

[0061] (2) Preparation of sodium alginate solution: Dissolve 2 g of sodium alginate in 100 ml of sterile deionized water and stir until it is completely dissolved to form a uniform solution.

[0062] (3) Preparation of mixed solution: The gelatin solution and the sodium alginate solution were mixed in a volume ratio of 1:1 and stirred thoroughly to obtain a uniform mixed solution.

[0063] (4) Preparation of cross-linking solution: Prepare NHS (10 mM) and EDC (20 mM) solutions and mix them in a volume ratio of 1:1 to prepare the cross-linking agent solution.

[0064] 2. Scaffold formation and cross-linking

[0065] (1) Scaffold casting: Pour the mixed gelatin-sodium alginate solution into the pre-prepared mold, ensuring that the solution is evenly distributed in the mold.

[0066] (2) Cross-linking process: Place the mold in a 4°C refrigerator to cool. After the mixed solution solidifies, add the cross-linking agent solution evenly onto the surface of the stent to allow it to fully penetrate.

[0067] (3) CaCl 2 Cross-linking: Add 1.5% CaCl evenly on the surface of the stent. 2 The solution was allowed to stand for 1 hour to allow the sodium alginate to react with the Ca 2+ Cross-linking occurs to form a stable three-dimensional structure.

[0068] (4) Rinsing and curing: The cross-linked scaffold was removed and rinsed in PBS for 3 times, 10 minutes each time, to remove the unreacted cross-linker and CaCl 2 Subsequently, the scaffold was placed in a drying oven at 37°C for 24 hours.

[0069] 3. Characterization of 3D scaffolds

[0070] (1) Pore size and porosity analysis: A pore size analyzer was used to measure the pore size and porosity of the scaffold. The results showed that the pore size of the prepared scaffold was 200-250 μm and the porosity was about 90%, which was suitable for cell growth and interaction. The schematic diagram is shown in Figure 1 shown.

[0071] (2) Mechanical strength test: The scaffold was subjected to a mechanical strength test. The results showed that the cross-linked scaffold had high compressive strength and could maintain structural stability during the co-culture process.

[0072] Example 7: Anti-tumor effect of sensitized DC-CIK cells in animal models

[0073] 1. Establishment of tumor model

[0074] MCF-7 tumor cells were suspended in physiological saline and the concentration was adjusted to 1×10 7 Take an appropriate amount of tumor cell suspension and inoculate it subcutaneously on the right back of the mouse. Each mouse is inoculated with 100 μl (i.e. 1×10 6 The tumor volume was measured with a vernier caliper every 2 days to record the tumor growth.

[0075] 2. Cell Injection

[0076] Calculate and adjust the concentration of sensitized DC-CIK cells to 2×10 7 cells / ml. When the tumor volume reaches 100mm 3 Afterwards, sensitized DC-CIK cells were injected into the tail vein at a dose of 200 μl (i.e., 4×10 6 The positive control group was injected with anti-tumor drugs every 3 days, and the dosage was calculated according to the recommended dosage; the negative control group was injected with 200 μl of normal saline every 3 days. The treatment was repeated every 2 weeks, and the mice were observed continuously until the 28th day.

[0077] 3. Evaluation of tumor growth inhibition

[0078] (1) Tumor volume calculation: The long diameter (L) and short diameter (W) of the tumor were measured before and after treatment, and the formula (volume = L × W 2 / 2) Calculate the tumor volume. The results show (as shown in Table 4) that Group 3 has the best effect.

[0079] Table 4 Tumor volume tracking results

[0080]

[0081] Note: Group 1 to Group 4 are the same as Table 1 to Table 3.

[0082] (2) Calculation of tumor inhibition rate: On the last day of the experiment, the tumor volume of each group was compared with that of the negative control group (see Table 4 for specific data) to calculate the tumor inhibition rate. The results showed (as shown in Table 5) that Group 3 had the best effect.

[0083]

[0084] Table 5 Calculation results of tumor inhibition rate

[0085]

[0086] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing DC-CIK co-cultured cells, characterized in that: The method comprises the following steps: (1) Isolation of PBMCs: PBMCs were isolated from blood using Ficoll-Paque solution; (2) Preparation of DC cells: PBMCs isolated were divided into 1×10 6 DCs were inoculated at a density of 100 / ml in RPMI-1640 medium containing 10% FBS, 1000 IU / ml GM-CSF and 500 IU / ml IL-4, and then anti-CTLA-4 monoclonal antibody and AFP protein were added to induce DC maturation; (3) Preparation of CIK cells: PBMCs were cultured at 1×10 6 The cells were inoculated at a density of 100 / ml into RPMI-1640 medium containing 10% FBS and 1000 U / ml IFN-γ, and then replaced with 10% FBS, 1000 U / ml IL-2 and anti-CD3 monoclonal antibody at a final concentration of 50 ng / ml to induce CIK cell generation; (4) Co-culture of DC-CIK cells: Mature DC cells and CIK cells were uniformly inoculated on the surface of the three-dimensional scaffold in proportion, and the inoculated scaffold was placed in a dynamic culture device with a flow rate set to 0.5 ml / min. DC-CIK cells were obtained by co-culture at 37°C and 5% CO2 for 48 to 72 hours.

2. The method according to claim 1, characterized in that: The amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-CTLA-4 monoclonal antibody used in the preparation of DC cells in step (2) are shown in SEQ ID NO.1 and SEQ ID NO.

2.

3. The method according to claim 1, characterized in that The concentrations of the anti-CTLA-4 monoclonal antibody and AFP protein used in the preparation of DC cells in step (2) are 10 μg / ml and 5 μg / ml, respectively.

4. The method according to claim 1, characterized in that The amino acid sequences of the heavy chain and light chain variable regions of the anti-CD3 monoclonal antibody used in the preparation of CIK cells in step (3) are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

5. The method according to claim 1, characterized in that In the step (4) of DC-CIK cell co-culture, the ratio of DC cells to CIK cells is 1:

5.

6. The method according to claim 1, characterized in that The pore size of the three-dimensional scaffold in the co-culture of DC-CIK cells in step (4) is 200-250 μm, and the porosity is about 90%.

7. Use of the DC-CIK co-cultured cells prepared according to claim 1 in the preparation of anti-tumor drugs.

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