Ag-DC-CIK cell and application thereof in preparation of antitumor drugs

Abstract

The invention relates to an Ag-DC-CIK cell and application of the Ag-DC-CIK cell in preparation of antitumor drugs, and belongs to the technical field of cell therapy. The Ag-DC-CIK cell is obtained by co-culturing an Ag-DC cell and a CIK cell; the Ag-DC is DC jointly loaded by a WT1 antigen, mucus glycoprotein 1 (MUC1) and Poly (I: C). A WT1 antigen, mucus glycoprotein 1 (MUC1) and Poly (I: C) are jointly loaded on a dendritic cell (DC) to obtain Ag-DC, the Ag-DC and a CIK cell are co-cultured to obtain an Ag-DC-CIK cell, and verification shows that the killing effect on various tumors including cervical cancer, colorectal cancer, gastric cancer and leukemia can be achieved. Moreover, compared with DC-CIK cells obtained by co-culture of non-loaded DC and CIK cells, the DC-CIK cells have the advantage that the killing effect on tumor cells is remarkably improved.

Description

Technical Field

[0001] The invention relates to Ag-DC-CIK cells and application thereof in preparing anti-tumor drugs, belonging to the technical field of cell therapy. Background Art

[0002] Cytokine induced killer cells (CIK cells) are a group of heterogeneous cells mainly CD3+CD56 that are cultured in vitro with cytokines from human peripheral blood mononuclear cells. They have both the powerful anti-tumor activity of other lymphocytes and the non-MIH (major tissue) restricted tumor killing ability of NK cells (natural killer cells), and are also called NK cell-like T lymphocytes. CIK cells have the advantages of rapid proliferation, high tumor killing activity, broad tumor killing spectrum, and low toxicity to normal tissues. They are currently widely used in clinical adoptive immunotherapy cells, which has also promoted the development of CIK cell in vitro development methods and the continuous progress and improvement of clinical applications.

[0003] The key to culturing CIK cells in vitro is to provide appropriate culture conditions and growth factors to promote their proliferation and increase their activity. In recent years, improvements in cell culture technology include optimizing culture medium components, the use of growth factors, and the control of cell density to increase the growth rate and killing activity of CIK cells. With the development of genetic engineering technology, people have used genetic engineering technology to enable CIK cells to have stronger tumor recognition and killing capabilities by transfecting antibody genes, etc. Given that different patients may have different responses to CIK cell therapy, personalized treatment strategies have gradually received attention. Personalized treatment can adjust CIK cell treatment plans based on the patient's immune characteristics and tumor characteristics to improve the targetedness and effectiveness of treatment.

[0004] With the development, CIK cell therapy has been shown to have certain efficacy in certain tumor types, but the effect may be limited in other types of tumors. Therefore, researchers have explored the possibility of combining CIK cell therapy with other treatments (such as chemotherapy, radiotherapy, immune checkpoint inhibitors, etc.) or other immune cells (such as dendritic cells (DC)), etc.) to improve the therapeutic effect. Among them, DC-CIK combined therapy has gradually become one of the more mature treatment options for tumor biotherapy. DC-CIK cell therapy is a cytokine-activated killer cell-dendritic cell mixed therapy, which contains both killer immune cells and dendritic cells. As a powerful professional antigen-presenting cell, DC plays an important role in the activation and differentiation of T cells. Combining CIK cells with DC to treat tumors not only helps to avoid the immune incompetence of T cells against some tumor cells, but also plays a synergistic anti-tumor effect. After DC and CIK are combined, CIK has significantly improved and increased cell proliferation activity, tumor killing activity and cytokine release. In the process of tumor treatment, DC first recognizes antigens and then activates the acquired immune system; the activated CIK cells efficiently kill tumor cells by exerting their own cytotoxicity and secreting cytokines. In addition to the characteristics of high tumor killing activity and wide tumor killing, DC-CIK cell therapy also has the characteristics of reducing immune tolerance, which can effectively prevent certain autoimmune diseases. The combination of DC and CIK cells plays an obvious role in biological immunotherapy, fully mobilizing autoimmunity, inhibiting the killing of tumor cells, and more importantly, without damaging normal tissues. In addition, with the continuous input of DC and CIK cells during treatment, the patient's immune system is rapidly enhanced, which can significantly improve the patient's quality of life and prolong the survival of tumor patients. Therefore, DC-CIK cell therapy is more clinically valuable than traditional treatment methods and is currently a more advanced, effective and mature biological treatment method.

[0005] In order to better exert the inhibitory activity of DC-CIK cells on tumor cells, researchers have conducted research through a variety of strategies. For example, combined with traditional treatments such as chemotherapy or radiotherapy, and combined with Chinese herbal extracts. Other researchers have modified DC cells at the genetic level, knocked down or overexpressed a certain gene to obtain a DC cell with stronger antigen presenting ability, and then cultured with CIK cells to obtain DC-CIK cells with stronger tumor cell inhibitory effects. In the study, people found that by selecting a suitable load to load DC cells and then co-culturing with CIK cells, the killing power of CIK cells against tumor cells can be effectively improved.

[0006] Wang et al. [1]The researchers pulsed DC with autologous tumor cell lysate (Ag) to produce Ag-DC. T lymphocytes were cultured to prepare CIK cells, and Ag-DC and CIK cells were co-cultured to prepare Ag-DC-CIK vaccine to explore the clinical efficacy of patients with advanced renal cell carcinoma and detect their immune function and adverse reactions. However, the DC-CIK cells prepared by them only have a significant specific killing effect on renal cell carcinoma, and there is a problem that they do not have a broad-spectrum tumor killing effect.

[0007] The Chinese invention patent with the announcement date of October 25, 2022 and the authorization announcement number of CN111996166B discloses a loaded DC cell, DC-CIK cell and its application in tumor cell therapy. It specifically discloses that DC cells loaded with 2'-fucosyllactose and CIK cells are co-cultured to obtain DC-CIK cells, and it is proved that compared with the control group, the DC-CIK cells prepared by it can effectively improve the killing power against liver cancer cells and inhibit the formation of tumors in mice. However, the DC-CIK cells prepared by it can only prove that it has a stronger killing power against liver cancer cells. Due to the strong heterogeneity of different tumors, it is unknown whether it has a broad spectrum of tumor killing. Summary of the invention

[0008] The first purpose of the present invention is to provide an Ag-DC-CIK cell, which provides an Ag-DC-CIK cell with strong killing ability and broad-spectrum tumor killing ability for the prior art.

[0009] The second purpose of the present invention is to provide the use of Ag-DC-CIK cells in the preparation of anti-tumor drugs, thereby providing a new available resource for the preparation of tumor drugs in the prior art.

[0010] In order to achieve the above object, a technical solution of Ag-DC-CIK cells in the present invention is: An Ag-DC-CIK cell, wherein the Ag-DC-CIK cell is obtained by co-culturing Ag-DC and CIK cells; the Ag-DC cell is a DC co-loaded with WT1 antigen, mucin glycoprotein 1 and Poly (I:C).

[0011] The beneficial effect of the above technical solution is that the Ag-DC-CIK cell of the present invention is a pioneering invention. The present invention loads WT1 antigen, mucin glycoprotein 1 and Poly (I: C) on dendritic cells (DC) to obtain Ag-DC, and co-cultures Ag-DC with CIK cells to obtain a DC-CIK (Ag-DC-CIK) cell. It has been verified that it can achieve a killing effect on a variety of tumors including cervical cancer, colorectal cancer, gastric cancer, and leukemia. Moreover, compared with the DC-CIK cells obtained by co-culturing unloaded DC and CIK cells, the killing effect on tumor cells is significantly improved.

[0012] Specifically, WT1 (Wilm's tumor gene) is a protein-coding gene that was originally discovered in childhood Wilms tumor (Wilms tumor) and is overexpressed in a variety of tumor types. WT1 has become a hot topic in cancer diagnosis and treatment research, and its overexpression is associated with tumor proliferation, invasion, and metastasis.

[0013] MUC1 (mucin glycoprotein 1) is a glycoprotein expressed on the surface of various epithelial cells. It has multiple biological functions, including cell adhesion, signal transduction and immune regulation. MUC1 is highly expressed in many tumors and is closely related to tumor invasion and metastasis.

[0014] Poly(I:C) is a synthetic double-stranded RNA molecule that simulates the natural double-stranded RNA produced during viral infection and can be considered an immune activator. Poly(I:C) stimulates the immune system's response by simulating viral infection, including activating the natural immune system and promoting the activation of antigen-presenting cells, thereby enhancing the body's immune response.

[0015] As a further improvement, the Ag-DC and CIK cells were obtained by inducing culture of peripheral blood mononuclear cells.

[0016] As a further improvement, the induction culture time is 7 to 8 days.

[0017] As a further improvement, the loading is to add WT1 antigen, mucin glycoprotein 1 and Poly (I:C) during the Ag-DC induction culture process.

[0018] As a further improvement, during the induction culture, the final concentrations of WT1, mucin glycoprotein 1, and Poly(I:C) are 30-75 ng / mL, 30-75 ng / mL, and 30-75 μg / mL, respectively.

[0019] As a further improvement, the ratio of the number of Ag-DCs to the number of CIK cells is 1:(10-15).

[0020] In order to achieve the above-mentioned object, the technical scheme of the application of Ag-DC-CIK cells in the preparation of anti-tumor drugs in the present invention is: The invention discloses an application of Ag-DC-CIK cells in the preparation of anti-tumor drugs.

[0021] The beneficial effect of the above technical solution is that: the present invention proves through in vitro experiments that Ag-DC-CIK cells co-cultured with CIK cells loaded with WT1 antigen, mucin glycoprotein 1 and Poly (I: C) can achieve a killing effect on a variety of tumors including cervical cancer, colorectal cancer, gastric cancer, and leukemia. Moreover, compared with DC-CIK cells obtained by co-culturing unloaded DC with CIK cells, the killing effect on tumor cells is significantly improved. This shows that the Ag-DC-CIK cells of the present invention can achieve a broad spectrum of anti-tumor effects, enhance immune response, and reduce immune tolerance, and are a potential effective anti-tumor drug.

[0022] As a further improvement, the tumor includes cervical cancer, colorectal cancer, gastric cancer, and leukemia.

[0023] Compared with the prior art, the present invention has the following beneficial effects: 1. Enhance immune response: Dendritic cells, as antigen presenting cells, can effectively activate T cell immune response. Loading antigens such as WT1, MUC1 and Poly (I: C) onto DCs can enhance the antigen presenting ability of DCs, thereby promoting the activation and proliferation of CIK cells and enhancing immune response.

[0024] 2. Enhance the killing effect on tumors: WT1 and MUC1 are common tumor-associated antigens that are overexpressed in many tumors. Therefore, loading them onto DCs can make it easier for CIK cells to recognize and kill tumor cells. Poly(I:C) as an immune activator can enhance the killing effect of CIK cells on tumors.

[0025] 3. Broad-spectrum anti-tumor effect: WT1 and MUC1 are overexpressed in many tumors, so this regimen has a broad-spectrum anti-tumor effect and can be used to treat various types of tumors. At the same time, the immune activation effect of Poly(I:C) can enhance the killing effect of CIK cells on different types of tumors, further expanding the scope of application of treatment.

[0026] 4. Reduce immune tolerance: Loading multiple antigens onto DCs can enhance antigen recognition and immune activation, thereby reducing the immune escape and tolerance of tumor cells. This helps to improve the efficacy and durability of treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1The morphological changes of DC cells and CIK cells during the induction process observed under an optical microscope in Example 2 of the present invention (40×); Figure 2 This is the identification of DC surface markers in Example 3 of the present invention (in the figure, A represents the original figure of flow cytometry identification, B represents the statistical analysis figure of the result of A, * represents P<0.05, ** represents P<0.01); Figure 3 Identification of CIK cell surface markers in Example 3 of the present invention (in the figure, A represents the identification of CIK cell surface markers CD3, CD4, and CD8 before and after induction without antigen loading by flow cytometry, B represents the identification of CIK cell surface markers CD8, CD4 and CD8, CD4 and CD56 before and after induction with antigen loading by flow cytometry, C represents the analysis chart of the experimental results of A and B, * is P<0.05, ** is P<0.01, *** is P<0.001); Figure 4 The CIK, DC-CIK and Ag-DC-CIK cell proliferation detection in Example 4 of the present invention; Figure 5 The killing effect of each effector cell on cervical cancer cells (Hela) detected by LDH in Example 5 of the present invention (in the figure, * is P<0.05, ** is P<0.01, *** is P<0.001, **** is P<0.0001, and ns is no significant difference); Figure 6 The killing effect of each effector cell on colorectal cancer cells (HCT116) detected by LDH in Example 5 of the present invention (in the figure, * is P<0.05, ** is P<0.01, *** is P<0.001, **** is P<0.0001); Figure 7 The killing effect of each effector cell on gastric cancer cells detected by LDH in Example 5 of the present invention (in the figure, A represents the gastric cancer cell line KATOⅢ as the target cell, B represents the gastric cancer cell line MKN45 as the target cell, C represents the gastric cancer cell line N87 as the target cell, * represents P<0.05, ** represents P<0.01, *** represents P<0.001, **** represents P<0.0001); Figure 8 The killing effect of each effector cell on leukemia cells (THP-1) detected by LDH in Example 5 of the present invention (in the figure, * is P<0.05, ** is P<0.01, *** is P<0.001, **** is P<0.0001); Fig. 9The effect of Ag-DC-CIK on tumor cell migration in Example 6 of the present invention (in the figure, the upper figure is a scratch test to detect the effect of each effector cell on MKN45 cell migration, and the lower figure represents the statistical analysis of the result A, * is P<0.05, ** is P<0.01, and ns represents no significant difference); Fig.10 The effect of Ag-DC-CIK on tumor organoids in Example 7 of the present invention (in the figure, A represents gastric cancer organoids cultured for 10 days, B represents the killing effect of each treatment group on gastric cancer organoids, C represents the apoptotic effect of each treatment group on gastric cancer organoids evaluated by AO-PI double staining for apoptosis, * is P<0.05, ** is P<0.01, *** is P<0.001, **** is P<0.0001, ns represents no significant difference); Fig.11 The effect of Ag-DC-CIK on xenograft tumor nude mice in Example 8 of the present invention (in the figure, A represents the tumor size of nude mice in each treatment group, and B represents the weight of nude mice in each treatment group); Fig.12 Graphs showing the HE staining results of different tissues (heart, spleen, lung, kidney and liver) of nude mice in each treatment group in Example 8 of the present invention. DETAILED DESCRIPTION

[0028] At present, in the prior art, DC-CIK cells are generally co-cultured with DC cells and CIK cells using tumor lysates to harvest DC-CIK cells, but this type of DC-CIK cells has strong specificity and does not have a broad-spectrum tumor killing effect. At the same time, the preparation cost is high. The DC-CIK cells obtained by some other methods also have the problem of not having a broad-spectrum tumor killing effect and having a weak ability to kill tumor cells. Based on this, the present invention provides an Ag-DC-CIK cell.

[0029] The present invention loads WT1 antigen, mucin glycoprotein 1 (MUC1) and Poly (I: C) on dendritic cells (DC) to obtain Ag-DC, and co-cultures Ag-DC with CIK cells to obtain Ag-DC-CIK cells. It has been verified that the Ag-DC-CIK cells can kill a variety of tumors including cervical cancer, colorectal cancer, gastric cancer, and leukemia. Moreover, compared with DC-CIK cells obtained by co-culturing unloaded DC with CIK cells, the killing effect on tumor cells is significantly improved.

[0030] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto; however, these embodiments are only examples and do not constitute any limitation on the scope of the present invention. The details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements fall within the protection scope of the present invention. The test methods in the following embodiments are conventional methods unless otherwise specified. The test materials used in the following embodiments are purchased from conventional biochemical reagent manufacturers unless otherwise specified.

[0031] Specific examples of Ag-DC-CIK cells and their use in preparing anti-tumor drugs of the present invention: The present invention adopts a strategy of combined stimulation of multiple antigens, loads antigens WT1, MUC1 and immune adjuvant Poly (I: C) into dendritic cells (DCs), and co-cultures with CIK cells to stimulate the proliferation and activation of CIK cells, thereby obtaining an Ag-DC-CIK cell with a broad-spectrum tumor-killing ability. The specific embodiment operation is as follows: Example 1 Extraction of PBMCs from umbilical cord blood In this embodiment, peripheral blood mononuclear cells (PBMCs) are extracted from umbilical cord blood. The specific implementation operation is as follows: Centrifuge fresh blood at 2500 rpm for 10 min, draw the upper light yellow plasma into a 50 mL centrifuge tube, inactivate it in a 56°C water bath for 30 min (8% calcium gluconate injection can be added and mixed for co-inactivation), then centrifuge at 2500 rpm for 10 min to remove the precipitate, transfer the upper plasma to a new 50 mL centrifuge tube and store it in a 4°C refrigerator for later use.

[0032] The lower blood cell layer was diluted 1:1 with physiological saline and mixed, and added to a centrifuge tube containing 12 mL of lymphocyte separation solution (the liquid interface should not be destroyed). Centrifuge at 400 g for 30 min, then aspirate the middle white blood cell layer, wash twice with PBS (including centrifugation at 350 g for 10 min) and count to obtain PBMCs.

[0033] Example 2 Induction and co-culture of DC and CIK cells In this example, the PBMCs cells separated in Example 1 were used to induce and culture DC and CIK cells respectively, and DC-CIK cells were co-cultured with CIK cells to obtain DC-CIK cells. The specific implementation steps are as follows: 1. Induction and culture of DC and CIK cells PBMCs were cultured in serum-free medium (Corning KBM581 serum-free medium) and the cell concentration was adjusted to 2 × 10 6 / mL, placed in a culture flask, incubated at 37℃, 5% CO2 incubator for 2h to allow monocytes to adhere to the wall, used to induce DC cells. The non-adherent suspension cells were collected and used to induce CIK cells.

[0034] Adjust the cell concentration of the suspended cells to 2×10 using 5% autologous serum medium (Corning KBM581 serum-free medium + 5% inactivated serum separated in the first step of PBMC extraction). 6 / mL; add 1000 U / mL of recombinant human IFN-γ for culture; 24 hours later, add 50 ng / mL of CD3 monoclonal antibody and 1000 U / mL of recombinant human IL-2 to stimulate the growth and proliferation of CIK cells; change half the volume of the medium or expand the bottle every 2 days, and add 500 U / ml of recombinant human IL-2; on the 7th day of culture, harvest the CIK cells, and then perform quality inspection on the induced CIK cells.

[0035] Adherent cells were added with 5% autologous serum medium containing recombinant human GM-CSF-1000 U / mL and recombinant human IL-4 1000 U / mL, and cultured in a 37°C, 5% CO2 incubator to induce monocytes to differentiate into DC cells; half the volume of the medium was replaced every 2 days, and cytokines were supplemented; on the 5th day of culture, antigens WT1 (final concentration of 50 ng / mL), MUC1 (final concentration of 50 ng / mL), and Poly(I:C) (final concentration of 50 μg / mL) were added to load DC with antigens (the loaded DCs are referred to as Ag-DCs below. If DCs are not loaded with antigens, this step can be omitted); on the 6th day of culture, recombinant human TNF-α (500 U / mL) was added to induce DC cell maturation; on the 7th or 8th day of culture, DC cells were harvested and the induced DC cells were quality inspected.

[0036] In other implementations, when the final concentration of WT1 is 30-75 ng / mL, the final concentration of MUC1 is 30-75 ng / mL, and the final concentration of Poly(I:C) is 30-75 μg / mL during induction culture, the same or similar effects as those in this embodiment can be obtained.

[0037] 2. Co-culture of DC and CIK cells to obtain DC-CIK cells The obtained mature DCs and Ag-DCs loaded with WT1, MUC1 and Poly(I:C) were co-cultured with mature CIK cells at a ratio of 1:10 (DC-CIK cells obtained by co-culturing loaded DCs with CIK cells are referred to as Ag-DC-CIK cells below). Recombinant human IL-2 (500 U / mL) was added to the culture medium (Corning KBM581 serum-free medium + 5% autologous serum + GM-CSF-1000U / mL and recombinant human IL-41000U / mL, and recombinant human TNF-α (500U / mL) was added on the 6th day of culture). The medium was replaced by half every 3 days, and recombinant human IL-2 (500 U / mL) was supplemented. The cells were collected on the 7th day, and the cells in each group were harvested, and the DC-CIK cells were subsequently subjected to quality inspection.

[0038] The DC and CIK cells in the induced culture process were observed under a microscope to observe the morphological changes of DC and CIK cells during the induction process. The specific results are as follows: Figure 1 shown.

[0039] As can be seen from the figure, under the directional stimulation and induction of cytokines, dendritic cells (DC) undergo obvious morphological changes during the culture process. Under the light microscope, the cells show an adherent growth state. After 1-2 days of culture, they begin to change to a semi-suspended and suspended growth state. By the 8th day, most cells are already in a suspended state. During the induction process, the cell volume gradually increases, and the morphology gradually changes from a round shape to an irregular shape similar to pseudopodia, with dendritic protrusions appearing on the surface. When these cells are subjected to antigen loading and TNF-α maturation stimulation, the dendritic cells further mature, increase in volume, increase in dendritic protrusions, and have more irregular contours.

[0040] Under the induction of corresponding cytokines, the suspended cells gradually expanded into CIK cells. Initially, the morphology of CIK cells was relatively consistent, scattered and evenly distributed, with small individuals and a relatively rounded shape. During the culture process, on the third day, the cells began to aggregate into clusters. Starting from the fifth day, the cells proliferated significantly, formed a colony state, and grew in suspension in the culture medium. As the culture time increased, the number of cell colonies gradually increased.

[0041] Example 3 Flow Cytometry Identification In this example, flow cytometry was performed on the cells induced and cultured in Example 2. The specific implementation steps are as follows: 1. Flow cytometry detection of DC and Ag-DC Collect the supernatant of induced DC and Ag-DC in a 50 mL centrifuge tube, wash the cells twice with PBS, add trypsin digestion, add culture medium to stop digestion when most DCs become round under the microscope, collect in the same 50 mL centrifuge tube, and centrifuge at 800 g for 10 min. Wash the cells twice with PBS and count. Resuspend in an appropriate volume of PBS (100 μL per sample) according to the number of antibody samples and cell numbers to be tested. Add the resuspended cell suspension to a 1.5 mL EP tube labeled with different antibodies as needed, add 5 μL of the corresponding antibody, and incubate in the dark for 15 min. DC detection index is CD80 + 、CD86 + and HLA-DR + .

[0042] Flow cytometry was used to identify the specific immune phenotype of DC cells before and after induction. The test results were as follows: Figure 2 As shown ( Figure 2 In the figure, before represents the group without antigen, which is the DC group (control), and after represents the group with antigen, which is the Ag-DC group). As can be seen from the figure, DC cultured from day 0 to day 7, its cell surface CD80 + From 20.7% to 71.2%, CD86 + Increased from 36.7% to 60.3%, HLA-DR + The percentage of DC cells with special immune phenotypes increased significantly from 25.4% to 34.1%. ＋ and CD86 ＋ There were significant differences in HLA-DR, indicating that DCs were successfully induced.

[0043] 2. Flow cytometry detection of CIK cells, DC-CIK cells and Ag-DC-CIK cells Collect CIK cells, DC-CIK cells and Ag-DC-CIK cells in 50 mL centrifuge tubes and centrifuge at 800 g for 10 min. Wash the cells twice with PBS and count them. Resuspend the cells in an appropriate volume of PBS (100 μL per sample) according to the number of antibody samples and cell numbers to be tested. Add the resuspended cell suspension to 1.5 mL EP tubes labeled with different antibodies as needed, add 5 μL of the corresponding antibody, and incubate in the dark for 15 min. The CIK detection index is CD3 + 、CD4 + 、CD8 + 、CD56 + 、CD3 + CD8 + and CD3 + CD56 +, DC-CIK, Ag-DC-CIK detection index is CD3 + CD8 + and CD3 + CD56 + .

[0044] Flow cytometry was used to identify the special immune phenotype of CIK cells before and after induction. The test results were as follows: Figure 3 As shown in Figure 1 (A is the analysis of the results before and after CIK induction without antigen loading, and B is the analysis of the results before and after DC-CIK induction with antigen loading). As can be seen from the figure, CIK cells were cultured from day 0 to day 7, and their cell surface CD3 + From 80.6% to 97%, CD4 + From 61.1% to 79.4%, CD8 + From 34.5% to 55.2%, CD56 + From 10.7% to 72.6%, CD3 + CD8 + From 31.4% to 52.6%, CD3 + CD56 + From 34.4 to 71.3%. After identification, the special immune phenotype of CIK cells showed a significant increase, CD3 ＋ 、CD4 ＋ 、CD8 ＋ 、CD56 ＋ 、CD3 ＋ CD8 ＋ 、CD3 ＋ CD56 ＋ There was a significant difference, indicating that CIK cells were successfully induced.

[0045] Example 4 Cell proliferation detection In this example, cell proliferation detection was performed on CIK cells, DC-CIK cells and Ag-DC-CIK cells induced and cultured in Example 2. The specific implementation operation was as follows: After co-culture, based on the number of CIK, DC-CIK, and Ag-DC-CIK cells on day 0 of co-culture, the three groups of cells were counted every 2 days until the end of co-culture, and the proliferation ratio of each group of cells was calculated for comparison. During the cell culture period, the number of suspended cells in each group was counted regularly, and the cell growth curve was prepared based on the number of cells on the starting counting day. Figure 4 It can be seen from the curve that the growth rate of DC-CIK and Ag-DC-CIK cells after co-culture is significantly greater than that of CIK cells.

[0046] Example 5 Lethality Detection In this example, Hela, HCT116, KATOⅢ, MKN45, N87 and THP-1 cells were used as target cells to verify the killing ability of Ag-DC-CIK cells on target cells. The specific implementation operations are as follows: The experimental groups were set up as follows: ① medium wells without cells (blank control wells); ② control cell wells without effector cell treatment (sample control wells). ③ cell wells without effector cell treatment for subsequent lysis (sample maximum enzyme activity control wells). ④ effector cell treatment wells (effector cells are: CIK, DC-CIK, Ag-DC-CIK, PBMC cells). Each well contains 1×10 target cells. 4 The final volume was 200 μL, and 5 replicate wells were set for each test group. Hela, HCT116, KATOⅢ, MKN45, N87 and THP-1 cells were used as target cells, and the effector cells and target cells of each group were seeded into 96-well plates at a ratio of 5:1, 10:1, 20:1, and 50:1.

[0047] After the two cells were co-cultured for 24 h, the culture supernatant was taken and the killing rate of effector cells against target cells was detected by lactate dehydrogenase (LDH) kit. The specific process was as follows: 1 h before the test (23 h of effector cells), 10% of the original culture medium volume of LDH release reagent (20 μL) was added to each well; LDH detection working solution was prepared according to the number of required detection wells, and each well plate was leveled after the detection time was reached. Centrifuge at 600 g for 5 min in a well plate centrifuge, and 120 μL of the supernatant from each well was taken and added to the corresponding well of a new 96-well plate. Under light-proof conditions, 60 μL of LDH working solution was added to each well and mixed, and the plate was placed on a shaker at room temperature (25°C) and slowly shaken and incubated in the dark for 30 min; after the incubation, the cell culture plate was placed on a microplate reader to measure the absorbance at 480 nm, and any wavelength greater than or equal to 600 nm was used as the reference wavelength for dual-wavelength detection. Calculation of cytotoxicity of each well: Cytotoxicity% = (OD value of the well treated with effector cells - OD value of the well treated with control cells without effector cells) / (OD value of the well treated with effector cells for subsequent lysis - OD value of the well treated with control cells without effector cells). Figures 5 to 8 shown.

[0048] As can be seen from the figure, compared with the control group, when the effector-target ratio was 5:1, there was no significant difference in the killing effect between the CIK group and the control group, and the killing effect of the DC-CIK group and the Ag-DC-CIK group was significantly higher than that of the control group. The killing effect of each effector group increased with the increase of the effector-target ratio. When the effector-target ratio was 10:1, 20:1, and 50:1, the killing effect of each effector group was significantly higher than that of the control group, and the Ag-DC-CIK group had a significant effect on Hela cells ( Figure 5 )、HCT116 cells( Figure 6 )、gastric cancer cells KATOⅢ, MKN45 and N87( Figure 7 )、Acute leukemia cell THP-1( Figure 8 ) was significantly higher than that of CIK group and DC-CIK group, which shows that Ag-DC-CIK cells have a broad-spectrum tumor killing ability.

[0049] Example 6 Effect of Ag-DC-CIK on Tumor Migration The effect of each effector cell on the migration of gastric cancer cell MKN45 was detected by scratch test. MKN45 was inoculated in a six-well cell culture plate. When the density reached about 70%, a 10μL pipette tip was used to draw a straight line on the cell surface to produce a regular wound area in the cell monolayer. The cells were rinsed with sterile PBS to remove cell debris and free cells in the cell culture medium. The wound area was photographed by the cell culture camera system, and the width of the scratch or the starting time point of cell migration was recorded. Effector cells (CIK, DC-CIK and Ag-DC-CIK) were added to each group and mixed cultured for 24h. The cell supernatant was removed and washed twice with sterile PBS. The microscope was used to take pictures and record in the same field of view. The cell migration rate was calculated using the formula: (scratch area at 0h - scratch area at 24h) / scratch area at 0h × 100% = migration rate. After analysis and comparison, compared with the control group, CIK, DC-CIK and Ag-DC-CIK group cells all inhibited tumor cell migration. There were differences between CIK and DC-CIK groups and the control group, and there was a significant difference between Ag-DC-CIK and the control group. There was no significant difference between CIK group and DC-CIK group, while Ag-DC-CIK had the strongest inhibitory effect on MKN45 migration, which was significantly different from CIK and DC-CIK groups ( Fig. 9 ).

[0050] Example 7 Effect of Ag-DC-CIK on Tumor Organoids Tumor organoids are a three-dimensional tumor model cultured in vitro that has certain tissue structures and morphological characteristics. The growth process of primary (P0) culture of gastric cancer organoids was observed under a microscope. The tumor tissue initially isolated was mainly single cells or cell clusters. Obvious 3D organoid formation was observed on the third day of growth. The overall structure of the organoids was spherical or elliptical, with a smooth appearance and a shiny surface, presenting a three-dimensional spherical or elliptical shape as a whole. After continuing to grow to the seventh day, the diameter of these cell spheres increased significantly and vacuolar morphology appeared. On the tenth day of growth, the diameter of some organoids could reach more than 300 μm. The surface usually showed a certain fine structure, which may have microprotrusions or microprotrusions. The interior of the tumor organoids showed a complex structure, including multiple levels of cell arrangement and tissue structure ( Fig.10Cells are often arranged in aggregates within tumor organoids, forming irregular masses or structures.

[0051] In order to detect the killing effect of CIK, DC-CIK and Ag-DC-CIK group cells on gastric cancer organoids, this example adopts CCK-8 cytotoxicity detection experiment analysis. CIK, DC-CIK and Ag-DC-CIK group cells all have significant killing effect on tumor organoids, while there is no obvious difference between CIK group and DC-CIK group, but Ag-DC-CIK has significant difference compared with other effector cell groups, and has the strongest killing effect on gastric cancer organoids ( Fig.10 The apoptotic effect of each group of effector cells on tumor organoids was evaluated by AO-PI double staining apoptosis detection experiment. After analysis, the survival rate of tumor cells in CIK, DC-CIK and Ag-DC-CIK groups was significantly lower than that in the control group, and they all had obvious killing effects on gastric cancer tumor organoids. There was no significant difference between CIK and DC-CIK. Among them, Ag-DC-CIK had significant differences with other effector groups and had the strongest killing effect on gastric cancer tumor organoids ( Fig.10 (as shown in C).

[0052] Example 8 Effects of Ag-DC-CIK on xenograft tumors in nude mice In order to further evaluate the therapeutic effect of DC-CIK loaded with antigens and DC-CIK and CIK on tumors, a nude mouse model of gastric cancer xenograft tumor was established in this example. MKN45 cells in good condition were collected and counted to determine the cell concentration; the counted tumor cells were divided into 1.5 mL EP tubes, 200 μL / 2 million tumor cells per tube; the cell suspension was subcutaneously injected into the left armpit of the nude mouse to establish a tumor model. The tumor volume was measured about ten days later. When the tumor volume was greater than 50mm 3 500 μL / 8 million effector cells (CIK, DC-CIK or Ag-DC-CIK cells) were injected into the tail vein for treatment, and the blank control group was injected with the same amount of PBS. Treatment was carried out three times a week for three weeks. The size of the tumor volume of nude mice was measured every day and statistically analyzed. After analysis, CIK, DC-CIK and Ag-DC-CIK had an inhibitory effect on the growth of nude mouse xenograft tumors, among which Ag-DC-CIK had the most obvious inhibitory effect ( Fig.11 The weight changes of nude mice were recorded every day during the experiment. After analysis, there was no significant difference in the weight of mice in CIK, DC-CIK and Ag-DC-CIK and the control group in the first 25 days. The weight of mice in the Ag-DC-CIK group was significantly higher than that in the other three groups on days 25-30 ( Fig.11 (shown in B).

[0053] In the process of evaluating the safety of each effector cell therapy, HE staining was used to observe the effects on the tissue structure and cell morphology of the five internal organs (heart, liver, spleen, lung, and kidney). Pathological changes such as inflammation, necrosis, fibrosis, and cell proliferation were detected to determine whether toxicity or adverse reactions occurred to these organs. After analysis, a mild inflammatory reaction may occur during the treatment, but its safety is controllable and no damage was caused to the five internal organs of nude mice ( Fig.12 ).

[0054] Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An Ag-DC-CIK cell, characterized in that: The Ag-DC-CIK cells are obtained by co-culturing Ag-DC and CIK cells; the Ag-DC is a DC co-loaded with WT1 antigen, mucin glycoprotein 1 and Poly (I:C).

2. The Ag-DC-CIK cell according to claim 1, characterized in that: The Ag-DC and CIK cells were obtained by inducing culture of peripheral blood mononuclear cells.

3. The Ag-DC-CIK cell according to claim 2, characterized in that: The induction culture time is 7 to 8 days.

4. The Ag-DC-CIK cell according to claim 2 or 3, characterized in that: The loading is to add WT1 antigen, mucin glycoprotein 1 and Poly (I: C) during the Ag-DC induction culture process.

5. The Ag-DC-CIK cell according to claim 4, characterized in that: During the induction culture, the final concentrations of WT1, mucin glycoprotein 1, and Poly(I:C) were 30-75 ng / mL, 30-75 ng / mL, and 30-75 μg / mL, respectively.

6. The Ag-DC-CIK cell according to any one of claims 1 to 3, characterized in that: During co-culture, the ratio of the number of Ag-DCs to the number of CIK cells is 1:(10-15).

7. Use of the Ag-DC-CIK cells according to any one of claims 1 to 6 in the preparation of anti-tumor drugs.

8. The use of Ag-DC-CIK cells in the preparation of anti-tumor drugs according to claim 7, characterized in that: The tumors include cervical cancer, colorectal cancer, gastric cancer, and leukemia.

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