Immune cell activator and application thereof

Abstract

The invention belongs to the technical field of immune cells, and particularly relates to an immune cell activator and application thereof. The immune cell activator provided by the invention is prepared from the following components: concanavalin A, Shenmai injection, IL-2 (Interleukin-2), IL-18 (Interleukin-18), IL-1beta (Interleukin-1beta), a CD3 monoclonal antibody, L-glutamine, vitamin C, recombinant human transferrin, eudesmol and erioside C. The purity, the multiplication capacity and the tumor killing capacity of immune cells can be remarkably improved through the synergistic effect of the eucalyptol and the lanceolatoside.

Description

Technical Field

[0001] The invention belongs to the technical field of immune cells, and specifically relates to an immune cell activator and application thereof. Background Art

[0002] As an emerging method of tumor treatment, immune cell therapy has attracted much attention in the medical field. Immune cell therapy is to obtain the patient's immune cells, culture and expand the patient's immune cells in vitro, and then transport the in vitro cultured and expanded immune cells back to the patient's body, thereby enhancing the body's own immune function to achieve the purpose of treating tumors. As the research deepens, scholars have found that the in vitro culture and expansion process will affect the number, purity and activity of immune cells, thereby affecting the effect of immune cell therapy. Therefore, many scholars have improved the components of the culture medium used in the in vitro culture and expansion process, trying to increase the number, purity and activity of immune cells.

[0003] Traditional culture media often add extraneous serum. On the one hand, the composition and content of serum are unclear, which can easily cause cell contamination problems. On the other hand, the cost of serum is slightly high, which also greatly increases the cost of the experiment. The commonly used culture medium is serum-free culture medium. Since this type of culture medium does not add serum, it can greatly improve the accuracy and stability of the experimental results. In addition, the composition and dosage of the culture medium are clear, which can minimize damage to cells. However, the immune cells obtained by culturing with existing culture media often have problems such as poor cell proliferation ability and poor tumor killing ability, which in turn affects the clinical application of immune cells. Based on the above problems, the present invention provides an immune cell activator and its application, which aims to improve the proliferation ability and tumor killing ability of immune cells and promote the application of immune cells in clinical experiments. Summary of the invention

[0004] The first objective of the present invention is to provide an immune cell activator, which can significantly improve the purity, proliferation ability and tumor killing ability of immune cells.

[0005] The second objective of the present invention is to provide an application of an immune cell activator.

[0006] In order to achieve the above first purpose, the technical solution adopted by the present invention is: An immune cell activator for adding to immune cell culture medium, comprising the following ingredients: concanavalin A, shenmai injection, IL-2, IL-18, IL-1β, CD3 monoclonal antibody, L-glutamine, vitamin C, recombinant human transferrin, eucalyptol, and scutellaria baicalensis.

[0007] Preferably, based on the final volume of the culture medium, the contents of concanavalin A, shengmai injection, IL-2, IL-18, IL-1β, CD3 monoclonal antibody, L-glutamine, vitamin C, recombinant human transferrin, eucalyptol, and lanatoside A are: 14-26 ng / mL concanavalin A, 2-4 v / v% shengmai injection, 500-700 U / mL IL-2, 1000-1200 U / mL IL-18, 32-45 ng / mL IL-1β, 250-400 ng / mL CD3 monoclonal antibody, 90-120 μg / mL L-glutamine, 7-18 mg / mL vitamin C, 9-21 μg / mL recombinant human transferrin, 1-10 μg / mL eucalyptol, and 15-30 μg / mL lanatoside A.

[0008] Preferably, based on the final volume of the culture medium, the contents of concanavalin A, shengmai injection, IL-2, IL-18, IL-1β, CD3 monoclonal antibody, L-glutamine, vitamin C, recombinant human transferrin, eucalyptol, and guanidine are: 22 ng / mL concanavalin A, 3 v / v% shengmai injection, 600 U / mL IL-2, 1100 U / mL IL-18, 37 ng / mL IL-1β, 310 ng / mL CD3 monoclonal antibody, 106 μg / mL L-glutamine, 14 mg / mL vitamin C, 15 μg / mL recombinant human transferrin, 5 μg / mL eucalyptol, and 27 μg / mL guanidine.

[0009] Preferably, the culture medium is AIM-V serum-free medium.

[0010] In order to achieve the above second purpose, the technical solution adopted by the present invention is: An application of an immune cell activator, wherein the immune cell activator is added to an immune cell culture medium according to a certain content to culture immune cells.

[0011] Preferably, the immune cells are NKT cells.

[0012] Compared with the prior art, the beneficial effects of the present invention are mainly: The immune cell activator of the present invention comprises the following ingredients: concanavalin A, shenmai injection, IL-2, IL-18, IL-1β, CD3 monoclonal antibody, L-glutamine, vitamin C, recombinant human transferrin, eucalyptol, and guanosine. The synergistic effect of eucalyptol and guanosine can significantly improve the purity, proliferation ability and tumor killing ability of immune cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the NKT cell tumor killing rate of Examples 1-3 and Comparative Examples 1-5; Figure 2 This is a graph showing the results of flow cytometry phenotype detection of NKT cells in Example 1; Figure 3 This is a graph showing the results of flow cytometry phenotype detection of NKT cells in Comparative Example 1; Figure 4 This is a graph showing the results of flow cytometry phenotype detection of NKT cells in comparative example 2; Figure 5 This is a graph showing the results of flow cytometry phenotype detection of NKT cells in comparative example 3; Figure 6 This is a graph showing the results of flow cytometry phenotype detection of NKT cells in Comparative Example 4; Figure 7 This is a graph showing the flow cytometry phenotype detection results of NKT cells in comparative example 5. DETAILED DESCRIPTION

[0014] The technical scheme of the present invention is further described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0015] Example Example 1 A NKT cell activator and its application The composition of the NKT cell activator is: concanavalin A, Shenmai injection, IL-2, IL-18, IL-1β, CD3 monoclonal antibody, L-glutamine, vitamin C, recombinant human transferrin, eucalyptol, and lanatoside. The above activators are added to the AIM-V serum-free cell culture medium. The content of each component of the activator is shown in Table 1 based on the final volume of the culture medium: Table 1 The application process of NKT cell activator is as follows: (1) Obtaining PBMC cells Peripheral blood was collected from healthy donors, diluted with an equal amount of saline, and the diluted peripheral blood and lymphocyte separation solution were added to a centrifuge tube at a ratio of 2:1. The centrifuge speed was set to 1800 rpm / min and centrifuged for 25 minutes to collect the buffy coat cells, which were washed with saline and centrifuged at 1500 rpm / min for 10 minutes to obtain peripheral blood mononuclear cells (PBMCs). (2) Cell culture The obtained PBMCs were inoculated in AIM-V serum-free medium containing the above activator. The inoculation density of PBMCs in the medium was 2×106 The cells were cultured in a 37°C, 5% CO2 cell culture incubator for 10 days and replenished every other day with AIM-V serum-free medium containing the above activators to maintain a cell concentration of 2×10 6 After the culture, NKT cells were obtained.

[0016] Example 2 A NKT cell activator and its application The composition of the NKT cell activator is: concanavalin A, Shenmai injection, IL-2, IL-18, IL-1β, CD3 monoclonal antibody, L-glutamine, vitamin C, recombinant human transferrin, eucalyptol, and lanatoside A. The above activators are added to the AIM-V serum-free cell culture medium. The content of each component of the activator is shown in Table 2 based on the final volume of the culture medium: Table 2 The application process of NKT cell activator is as follows: (1) Obtaining PBMC cells Peripheral blood was collected from healthy donors, diluted with an equal amount of saline, and the diluted peripheral blood and lymphocyte separation solution were added to a centrifuge tube at a ratio of 2:1. The centrifuge speed was set to 1800 rpm / min and centrifuged for 25 minutes to collect the buffy coat cells, which were washed with saline and centrifuged at 1500 rpm / min for 10 minutes to obtain peripheral blood mononuclear cells (PBMCs). (2) Cell culture The obtained PBMCs were inoculated in AIM-V serum-free medium containing the above activator. The inoculation density of PBMCs in the medium was 2×10 6 The cells were cultured in a 37°C, 5% CO2 cell culture incubator for 10 days and replenished every other day with AIM-V serum-free medium containing the above activators to maintain a cell concentration of 2×10 6 After the culture, NKT cells were obtained.

[0017] Example 3 A NKT cell activator and its application The composition of the NKT cell activator is: concanavalin A, Shenmai injection, IL-2, IL-18, IL-1β, CD3 monoclonal antibody, L-glutamine, vitamin C, recombinant human transferrin, eucalyptol, and lanatoside. The above activators are added to the AIM-V serum-free cell culture medium. The content of each component of the activator is shown in Table 3 based on the final volume of the culture medium: Table 3 The application process of NKT cell activator is as follows: (1) Obtaining PBMC cells Peripheral blood was collected from healthy donors, diluted with an equal amount of saline, and the diluted peripheral blood and lymphocyte separation solution were added to a centrifuge tube at a ratio of 2:1. The centrifuge speed was set to 1800 rpm / min and centrifuged for 25 minutes to collect the buffy coat cells, which were washed with saline and centrifuged at 1500 rpm / min for 10 minutes to obtain peripheral blood mononuclear cells (PBMCs). (2) Cell culture The obtained PBMCs were inoculated in AIM-V serum-free medium containing the above activator. The inoculation density of PBMCs in the medium was 2×10 6 The cells were cultured in a 37°C, 5% CO2 cell culture incubator for 10 days and replenished every other day with AIM-V serum-free medium containing the above activators to maintain a cell concentration of 2×10 6 After the culture, NKT cells were obtained.

[0018] Comparative Example Comparative Example 1 A NKT cell activator and its application The difference from Example 1 is that no eucalyptol is added to the activator, and the rest is the same as Example 1.

[0019] Comparative Example 2 A NKT cell activator and its application The difference from Example 1 is that no eucalyptol is added to the activator, the content of lanatoside A is 32 μg / mL based on the final volume of the culture medium, and the rest is the same as Example 1.

[0020] Comparative Example 3 A NKT cell activator and its application The difference from Example 1 is that no lanatoside A is added to the activator, and the rest is the same as Example 1.

[0021] Comparative Example 4 A NKT cell activator and its application The difference from Example 1 is that no lanolin was added to the activator, the content of eucalyptol was 32 μg / mL based on the final volume of the culture medium, and the rest was the same as Example 1.

[0022] Comparative Example 5 A NKT cell activator and its application The difference from Example 1 is that no eucalyptol and lanatoside A are added to the activator, and the rest is the same as Example 1.

[0023] Experimental test results characterization 1. Detection of the growth number and expansion multiple of NKT cells The number of NKT cells in Examples 1-3 and Comparative Examples 1-5 was counted using a cell counter on the 0th, 3rd, 6th and 10th days of culture, respectively. The NKT cell expansion factor = the total number of cells obtained after culture / the number of PBMCs inoculated before culture. The number of NKT cell growth is shown in Table 4, and the NKT cell expansion factor is shown in Table 5.

[0024] Table 4 NKT cell growth number Table 5 NKT cell expansion multiples As shown in Tables 4-5, the growth number and expansion multiple of NKT cells obtained by using the NKT cell activator of the present invention are significantly higher than those of Comparative Examples 1-5.

[0025] In the activator of comparative example 1, eucalyptol was not added, in the activator of comparative example 2, eucalyptol was not added, and the content of lanthanoside A was increased, in the activator of comparative example 3, lanthanoside A was not added, in the activator of comparative example 4, the content of eucalyptol was increased, and in the activator of comparative example 5, eucalyptol and lanthanoside A were not added. It can be seen that the lack of eucalyptol or lanthanoside A in the activator will significantly affect the growth number and expansion multiple of NKT cells.

[0026] The above test results also show that the synergistic effect of eucalyptol and rutin can effectively enhance the proliferation ability of NKT cells.

[0027] 2. Detecting the Tumor-killing Ability of NKT Cells (1) Effector cell suspension Take the same number of NKT cells of Examples 1-3 and Comparative Examples 1-5 and transfer them to two groups of centrifuge tubes (each group of centrifuge tubes has 8 centrifuge tubes, and each centrifuge tube is added with NKT cells of Examples 1-3 and Comparative Examples 1-5), and adjust the cell concentrations of the two groups of centrifuge tubes to 1×10 6 / mL, 5×10 5 / mL, and two groups of effector cell suspensions were obtained.

[0028] (2) Setting up the test Experimental group: the effector cell NKT cell suspension and the K562 target cell suspension were plated at an effector-target ratio of 5:1 and 10:1 (100 μL of the effector cell NKT cell suspension and the K562 target cell suspension were added to each well, and 100 μL of the AIM-V serum-free medium containing the corresponding activators of Examples 1-3 and Comparative Examples 1-5 was added to each well), and 3 replicate wells were set for each group; Effector cell NKT natural release group: add 100 μL of the effector cell NKT cell suspension of the two groups respectively, and set up 3 replicate wells for each group; K562 target cell maximum release group: add 100 μL of 1×10 5 100 μL of AIM-V serum-free medium containing the corresponding activators of Examples 1-3 and Comparative Examples 1-5 was added to each well of a K562 target cell suspension of 100 μL / mL, and 3 replicate wells were set for each group; K562 target cell natural release group: 100 μL was added to each well at a density of 1×10 5 Each group was treated with a K562 target cell suspension of 100 cells / mL, and 3 replicate wells were set up in each group.

[0029] (3) Testing process The cells in the above groups were cultured in a cell culture incubator at 37°C and 5% CO2 for 1 day. The absorbance value of each group was detected by MTT method, and the tumor killing rate was statistically analyzed. The tumor killing rate = (experimental group - effector cell NKT natural release group - K562 target cell natural release group) / (K562 target cell maximum release group - K562 target cell natural release group) × 100%. The results are shown in Figure 1 shown.

[0030] Figure 1 It is the tumor killing rate of NKT cells in Examples 1-3 and Comparative Examples 1-5. Analysis Figure 1 It can be seen that the NKT cell tumor killing rate obtained by using the activator provided by the present invention is significantly higher than that of comparative examples 1 to 5. The above test results show that the activator of the present invention can significantly enhance the tumor killing ability of NKT cells.

[0031] 3. Detection of NKT cell flow cytometry phenotype The NKT cells of Examples 1-3 and Comparative Examples 1-5 were transferred to a centrifuge tube, and the cell pellets were obtained after centrifugation. The cell pellets were resuspended in PBS and the cell density was adjusted to 1×10 5 / mL, and the cell suspension was prepared; 20 μL of each cell suspension of Examples 1-3 and Comparative Examples 1-5 were placed in flow cytometry tubes, and labeled CD3 + CD56 + Monoclonal antibodies were incubated at room temperature in the dark for 20 min, excess antibodies were washed off, and flow cytometry was used for detection. The results are shown in Table 6. Figure 2-7 shown.

[0032] Table 6 NKT cell flow cytometry phenotype detection results Figure 2 This is the result of flow cytometry phenotype detection of NKT cells in Example 1. Figure 3 This is the result of flow cytometry phenotype detection of NKT cells in comparative example 1. Figure 4 This is the result of flow cytometry phenotype detection of NKT cells in comparative example 2. Figure 5 This is the result of flow cytometry phenotype detection of NKT cells in comparative example 3. Figure 6 This is the result of flow cytometry phenotype detection of NKT cells in comparative example 4. Figure 7 This is a graph showing the results of flow cytometry phenotype detection of NKT cells in comparative example 5. Figure 2-7 As shown in Table 6, the activator of Example 1 can increase the expression of CD3 in NKT cells. + CD56 + The above test results show that the activator of the present invention can improve the purity of NKT cells.

[0033] In summary, the synergistic effect of eucalyptol and rutin can significantly improve the purity, proliferation ability and tumor-killing ability of immune cells.

[0034] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. On the basis of the present invention, some modifications or replacements may be made thereto, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of protection claimed by the present invention.

Claims

1. An immune cell activator for adding to an immune cell culture medium, characterized in that: It includes the following ingredients: concanavalin A, ginseng and wheat injection, IL-2, IL-18, IL-1β, CD3 monoclonal antibody, L-glutamine, vitamin C, recombinant human transferrin, eucalyptol, and scutellaria baicalensis glycoside.

2. The immune cell activator according to claim 1, characterized in that Based on the final volume of the culture medium, the contents of concanavalin A, shengmai injection, IL-2, IL-18, IL-1β, CD3 monoclonal antibody, L-glutamine, vitamin C, recombinant human transferrin, eucalyptol, and guanidine are: 14-26 ng / mL concanavalin A, 2-4 v / v% shengmai injection, 500-700 U / mL IL-2, 1000-1200 U / mL IL-18, 32-45 ng / mL IL-1β, 250-400 ng / mL CD3 monoclonal antibody, 90-120 μg / mL L-glutamine, 7-18 mg / mL vitamin C, 9-21 μg / mL recombinant human transferrin, 1-10 μg / mL eucalyptol, and 15-30 μg / mL guanidine.

3. The immune cell activator according to claim 1, characterized in that Based on the final volume of the culture medium, the contents of concanavalin A, shengmai injection, IL-2, IL-18, IL-1β, CD3 monoclonal antibody, L-glutamine, vitamin C, recombinant human transferrin, eucalyptol, and guanidine are: 22 ng / mL concanavalin A, 3 v / v% shengmai injection, 600 U / mL IL-2, 1100 U / mL IL-18, 37 ng / mL IL-1β, 310 ng / mL CD3 monoclonal antibody, 106 μg / mL L-glutamine, 14 mg / mL vitamin C, 15 μg / mL recombinant human transferrin, 5 μg / mL eucalyptol, and 27 μg / mL guanidine.

4. The immune cell activator according to claim 1, characterized in that The culture medium is AIM-V serum-free culture medium.

5. The use of the immune cell activator according to any one of claims 1 to 4, characterized in that: The immune cell activating agent is added to the immune cell culture medium according to the content to culture the immune cells.

6. The use of the immune cell activator according to claim 5, characterized in that: The immune cells are NKT cells.

Images