DC-activated T lymphocyte loaded with MAGE-1 nonapeptide and its culture method and application

Abstract

The present invention provides a DC-activated T lymphocyte loaded with MAGE-1 nine peptides and a culture method and application thereof, belonging to the field of immunotherapy technology. The culture method comprises the following steps: (1) isolating dendritic cells of healthy people, co-incubating with MAGE-1 nine peptides in a first culture medium for 1 to 3 days, adding a second culture medium and continuing to incubate for 1 to 2 days, and obtaining a DC loaded with MAGE-1 nine peptides; (2) co-culturing the DC loaded with MAGE-1 nine peptides with T lymphocytes, activating the T lymphocytes, and obtaining T lymphocytes activated by DC loaded with MAGE-1 nine peptides. The present invention further improves the inhibitory effect of DC-activated T lymphocytes loaded with MAGE-1 nine peptides on the growth of liver cancer tumors by optimizing culture medium additives on the basis that MAGE-1 nine peptides and specific T cells have a certain growth inhibitory effect on liver cancer tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of immunotherapy, and in particular to a DC-activated T lymphocyte loaded with MAGE-1 nonapeptide, and a culture method and application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is a malignant tumor with high malignancy, low cure rate and poor prognosis. Western medicine mainly treats HCC with surgery, interventional therapy, chemotherapy, radiotherapy, etc. Although these treatments can achieve certain therapeutic effects, they also have serious destructive effects on normal cells and the immune system, so the side effects are prominent.

[0003] With the development of molecular immunology and cell biology, the advantages of gene therapy and immunotherapy have gradually become prominent. Tumor-specific killer T cells can effectively kill tumor cells. Therefore, more and more scholars are committed to establishing a treatment method based on tumor-specific killer T cells. For example, Lv Jianfeng et al. used MAGE-1 antigen nonapeptide NYKCRFPEI to incubate peripheral blood monolayers (PBMCs) as antigen presenting cells to stimulate lymphocytes and obtain liver cancer cell-specific killer T cells. (Lv Jianfeng, Leng Xisheng, Peng Jirun, et al. Application of MAGE-1 antigen peptide to induce specific cytotoxic T lymphocyte formation in vitro [J]. Chinese Journal of General Surgery, 2002, 17(4): 3. DOI: 10.3760 / j.issn: 1007-631X.2002.04.009.). For another example, Jin Hongjuan et al. studied the in vitro anti-tumor effect of dendritic cells loaded with MAGE-1 nonapeptide and found that the inhibition rate of DCs group on target cells was 76%, and the inhibition rate of DCs group sensitized with MAGE-1 nonapeptide was 87%. (Jin Hongjuan, Liu Xiaoli, Suo Jian. In vitro anti-tumor effect of dendritic cells loaded with MAGE-1 nonapeptide [J]. Chinese Journal of Gerontology, 2011, 31(9): 2. DOI: 10.3969 / j.issn.1005-9202.2011.09.045.). It can be seen that loading MAGE-1 nonapeptide on DC cells can improve the killing ability of specific T cells against liver cancer cells. However, the killing effect of specific T cells on liver cancer cells is limited by simply loading antigen peptides. Therefore, it is necessary to provide a tumor-specific killer T cell with stronger killing effect and better tumor growth inhibition effect.

[0004] Studies have found that in the process of culturing dendritic cells to induce T cells and in the process of inducing T cells using DCs loaded with tumor antigens, the growth factors in the culture medium have a significant effect on the activity of DCs and T cells. Therefore, starting from the culture medium for culturing DCs and T cells, it is expected to further improve the killing ability of specific T cells against liver cancer cells. Summary of the invention

[0005] The purpose of the present invention is to establish a specific killer T cell with stronger killing effect on liver cancer cells and better tumor inhibition effect.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for culturing T lymphocytes activated by DC loaded with MAGE-1 nonapeptide, comprising the following steps:

[0008] (1) Dendritic cells of healthy subjects were isolated and co-incubated with MAGE-1 nonapeptide in a first culture medium for 1 to 3 days, and then a second culture medium was added for further incubation for 1 to 2 days to obtain DC loaded with MAGE-1 nonapeptide;

[0009] (2) co-culturing the DC loaded with the MAGE-1 nonapeptide with T lymphocytes to activate the T lymphocytes, thereby obtaining T lymphocytes activated by the DC loaded with the MAGE-1 nonapeptide;

[0010] The first culture medium is a RPMI-1640 complete culture medium containing 700-900 U / mL of rhGM-CSF, 200-400 U / mL of TNF-α and 100-200 U / mL of rhIL-5;

[0011] The second culture medium is based on the first culture medium and contains 10-50 μg / mL of litchi peel extract.

[0012] Preferably, the concentration ratio of the dendritic cells to the MAGE-1 nonapeptide is (1-2)×10 5 ng / mL: 8~12ng / mL.

[0013] Preferably, the amount of the second culture medium added is 0.5 to 1 times the amount of the first culture medium added.

[0014] Preferably, the incubation temperature in step (1) is 35-37° C., and the CO 2 concentration is 4-5%.

[0015] Preferably, the cell number ratio of DC loaded with MAGE-1 nonapeptide to T lymphocyte is 8-10:1-2.

[0016] Preferably, the culture medium for co-culturing the DC loaded with MAGE-1 nonapeptide and T lymphocytes is RPMI-1640 complete culture medium containing 10-50 μg / mL litchi peel extract.

[0017] Preferably, the co-culture temperature of the DC loaded with MAGE-1 nonapeptide and T lymphocytes is 35-37° C., the CO 2 concentration is 4-5%, and the time is 3-4 days.

[0018] Preferably, the preparation method of the litchi peel extract is: adding water to the crushed litchi peel at a mass ratio of 1:3-5 and soaking for 5-10 hours, then boiling for 20-40 minutes, collecting the extract, and obtaining the litchi peel extract.

[0019] The present invention also provides T lymphocytes activated by DC loaded with MAGE-1 nonapeptide obtained by the culture method.

[0020] The present invention also provides a use of DC-activated T lymphocytes loaded with MAGE-1 nonapeptide in the preparation of a medicine for treating liver cancer.

[0021] The present invention provides a method for culturing T lymphocytes activated by DC loaded with MAGE-1 nonapeptide. On the basis that MAGE-1 nonapeptide and specific T cells have a certain growth inhibitory effect on liver cancer tumors, the inhibitory effect of T lymphocytes activated by DC loaded with MAGE-1 nonapeptide on liver cancer tumor growth is further improved by optimizing the culture medium additives. In in vitro experiments, the inhibition rate of MAGE-1-DC-T on BEL-7402 cells is 96%, and in in vivo experiments, the growth inhibition rate of BEL-7402 tumor inhibition is 89.8%. Therefore, the present invention establishes a specific killer T cell with stronger killing effect on liver cancer cells and better tumor inhibition effect. DETAILED DESCRIPTION

[0022] 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.

[0023] Example 1

[0024] Isolation of dendritic cells from healthy people. Take fresh peripheral blood from healthy people, use conventional Ficoll separation method to obtain mononuclear cells, and resuspend them in RPMI-1640 complete medium containing 10% fetal bovine serum to make the cell concentration 1×10 6 / mL, add 5mL of cell suspension into a T-25 culture flask, culture in a cell culture incubator at 37°C and 5% CO2 for 4 hours, then discard the culture medium and non-adherent cells, add the first culture medium and continue to culture in a cell culture incubator at 37°C and 5% CO2 for 4 days, change the medium every 2 days, and obtain dendritic cells (DC).

[0025] The first culture medium: RPMI-1640 complete culture medium containing 800 U / mL rhGM-CSF, 300 U / mL TNF-α and 150 U / mL rhIL-5.

[0026] Dendritic cells were incubated with MAGE-1 nonapeptide (NYKCRFPEI) at a concentration of 1×105 The cells were mixed at a concentration ratio of 10 ng / mL: 10 ng / mL, co-incubated in the first culture medium, and co-incubated in a cell culture incubator at 37°C and 5% CO2 for 2 days. The second culture medium equal to the first culture medium was added and cultured for another 2 days to obtain DC loaded with MAGE-1 nonapeptide.

[0027] The second culture medium: RPMI-1640 complete culture medium containing 800 U / mL rhGM-CSF, 300 U / mL TNF-α, 150 U / mL rhIL-5 and 30 μg / mL litchi peel extract.

[0028] Litchi peel extract: Add water to crushed (40 mesh) litchi peel in a mass ratio of 1:4 at room temperature (25°C) and soak for 8 hours, then boil for 30 minutes, filter with a 0.22 μm filter, collect the extract, and obtain the litchi peel extract.

[0029] Fresh peripheral blood was obtained from healthy subjects, and T lymphocytes were routinely collected using nylon wool columns. DC loaded with MAGE-1 nonapeptide and T lymphocytes were added to RPMI-1640 complete medium containing 30 μg / mL litchi peel extract at a cell number ratio of 10:1, and cultured in a cell culture incubator at 37°C and 5% CO2 for 4 days to obtain DC activated T lymphocytes loaded with MAGE-1 nonapeptide (MAGE-1-DC-T).

[0030] Comparative Example 1

[0031] The difference between this comparative example and Example 1 is that the culture medium is different.

[0032] The first culture medium composition of this comparative example is: RPMI-1640 complete culture medium containing 1000 U / mL of rhGM-CSF and 1000 U / mL of rhIL-4.

[0033] The second culture medium is the first culture medium of this comparative example with 200 U / mL of TNF-α added thereto.

[0034] The T lymphocyte culture medium was RPMI-1640 complete medium (without litchi peel extract).

[0035] The DC-activated T lymphocytes loaded with MAGE-1 nonapeptide were cultured and labeled as MAGE-1-DC-TP.

[0036] Comparative Example 2

[0037] The difference between this comparative example and Example 1 is that no litchi peel extract is added to the second culture medium and the T lymphocyte culture medium. DC activated T lymphocytes loaded with MAGE-1 nonapeptide are obtained by culture and are labeled as MAGE-1-DC-TL.

[0038] Comparative Example 3

[0039] The difference from Example 1 is that no MAGE-1 nonapeptide was loaded, and DC-activated T lymphocytes (DC-T) were obtained by culture.

[0040] Experimental Example 1

[0041] BEL-7402 cells in the logarithmic growth phase were taken and 1×10 6 Inoculate 96-well plates with 100 μg / mL and culture for 48 h for later use.

[0042] The MAGE-1-DC-T, MAGE-1-DC-TP, MAGE-1-DC-TL and DC-T cultured in Example 1 and Comparative Examples 1, 2 and 3 were mixed and incubated with BEL-7402 cells at an effector-target ratio of 10:1. After 18 hours of mixed culture, MTT assay was performed. Meanwhile, T lymphocytes (T cells) were used as controls. The inhibition rates of each group were calculated as shown in Table 1.

[0043] Inhibition rate = (1-(A value of experimental well-A value of effector cell control well) / A value of target cell control well) × 100%.

[0044] Among them, the A value of the test well refers to the A value measured after each group of T cells and BEL-7402 cells were mixed and incubated; the A value of the effector cell control well refers to the A value measured after each group of T cells were incubated alone; the A value of the target cell control well refers to the A value measured after BEL-7402 cells were incubated alone.

[0045] Table 1 Inhibitory rate of each group of T cells on BEL-7402 cells

[0046] Group Inhibition rate (%) MAGE-1-DC-T 96％ MAGE-1-DC-TP 79％ MAGE-1-DC-TL 85％ DC-T 74％ Tcell 5.6％

[0047] From the results of the assay in Table 1, it can be seen that the cytokines added to the culture medium play a very important role in the activation of T lymphocytes, and the added litchi peel extract can further enhance the activation effect of DC loaded with MAGE-1 nonapeptide on T lymphocytes. It is worth noting that DC not loaded with MAGE-1 nonapeptide can also activate the inhibitory effect of T lymphocytes on BEL-7402 cells to a great extent after being induced with the culture medium of the present invention.

[0048] Experimental Example 2

[0049] BEL-7402 cells were inoculated subcutaneously on the back side of BALB / C nude mice to prepare a nude mouse transplant tumor model. The longest diameter of the tumor was between 10 and 14 mm. A total of 20 mice were divided into 4 groups, with 5 mice in each group.

[0050] Experimental cells: MAGE-1-DC-T, MAGE-1-DC-TP, MAGE-1-DC-TL.

[0051] 1×10 5 The experimental cells were injected into the tumor-bearing mice next to the tumor site, and the mice were injected with an equal amount of normal saline as a control (CK). The tumor growth of the mice was observed and the changes in tumor size (measured as the longest tumor diameter (mm) × the shortest tumor diameter (mm)) were recorded. The results are shown in Table 2.

[0052] Table 2 Changes in tumor size in each group (mm 2 )

[0053]

[0054]

[0055] As can be seen from Table 2, MAGE-1-DC-T has a significant effect on inhibiting tumor growth, and compared with the CK group, the inhibition rate is 89.8%; while the inhibition rate of MAGE-1-DC-TP cultured in the common culture medium of Comparative Example 1 is 38.8%; and the inhibition rate of MAGE-1-DC-TL is 75.0%. It can be seen that the optimized induction culture medium of the present invention can greatly activate the ability of T lymphocytes to inhibit tumor growth.

[0056] As can be seen from the above examples, the present invention provides a method for culturing T lymphocytes activated by DC loaded with MAGE-1 nonapeptide. On the basis that MAGE-1 nonapeptide and specific T cells have a certain growth inhibitory effect on liver cancer tumors, the inhibitory effect of T lymphocytes activated by DC loaded with MAGE-1 nonapeptide on liver cancer tumor growth is further improved by optimizing the culture medium additives. In in vitro experiments, the inhibition rate of MAGE-1-DC-T on BEL-7402 cells was 96%, and in in vivo experiments, the growth inhibition rate of BEL-7402 tumor inhibition was 89.8%. It can be seen that the present invention establishes a specific killer T cell with stronger killing effect on liver cancer cells and better tumor inhibition effect.

[0057] 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 culturing T lymphocytes activated by DC loaded with MAGE-1 nonapeptide, characterized in that: The steps include: (1) Dendritic cells from healthy individuals were isolated and incubated with MAGE-1 nonapeptide in the first culture medium for 1 to 3 days, and then the second culture medium was added for further incubation for 1 to 2 days to obtain DC loaded with MAGE-1 nonapeptide; (2) co-culturing the DC loaded with the MAGE-1 nonapeptide with T lymphocytes to activate the T lymphocytes, thereby obtaining T lymphocytes activated by the DC loaded with the MAGE-1 nonapeptide; The first culture medium is a RPMI-1640 complete culture medium containing 700-900 U / mL of rhGM-CSF, 200-400 U / mL of TNF-α, and 100-200 U / mL of rhIL-5; The second culture medium is based on the first culture medium and adds 10-50 μg / mL litchi peel extract; The culture medium for co-culturing the DC loaded with MAGE-1 nonapeptide and T lymphocytes is RPMI-1640 complete culture medium containing 10-50 μg / mL litchi peel extract; The preparation method of the litchi peel extract is as follows: adding water to the crushed litchi peel in a mass ratio of 1:3-5 and soaking for 5-10 hours, then boiling for 20-40 minutes, collecting the extract, and obtaining the litchi peel extract; The concentration ratio of the dendritic cells to the MAGE-1 nonapeptide is (1-2)×10 5 ng / mL: 8~12ng / mL; The cell number ratio of DC loaded with MAGE-1 nonapeptide to T lymphocyte is 8-10:1-2.

2. The culture method according to claim 1, characterized in that The amount of the second culture medium added is 0.5 to 1 times the amount of the first culture medium added.

3. The culture method according to claim 2, characterized in that The incubation temperature in step (1) is 35-37°C and the CO2 concentration is 4-5%.

4. The culture method according to claim 3, characterized in that The co-culture temperature of the DC loaded with MAGE-1 nonapeptide and T lymphocytes is 35-37° C., the CO 2 concentration is 4-5%, and the time is 3-4 days.

5. T lymphocytes activated by DC loaded with MAGE-1 nonapeptide obtained by the culture method according to any one of claims 1 to 4.

6. Use of T lymphocytes activated by DC loaded with MAGE-1 nonapeptide according to claim 5 in the preparation of a drug for treating liver cancer.

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