Composition for improving killing ability of NK (Natural Killer) cells and application of composition

Abstract

The invention discloses a composition for improving the killing ability of NK cells and application of the composition. The composition for improving the killing ability of the NK cells comprises a monoclonal antibody of a lymphocyte activation gene 3 and lenalidomide. According to the composition disclosed by the invention, all the components are mutually matched and synergistically play a role, so that the multiplication capacity of natural killer cells and the cytotoxicity marker positive rate are effectively improved, and in addition, the cytokine secretion level of the natural killer cells and the killing capacity of the natural killer cells on cancer cells are improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a composition for improving the killing ability of NK cells and an application thereof. Background Art

[0002] As a part of lymphocytes, natural killer cells (NK cells) mainly function to kill cells infected by microorganisms and malignant transformation. Unlike other lymphocytes, such as B cells and T cells, NK cells do not need to be sensitized in advance, but regulate the functions of cytotoxicity and lymphokine secretion through activating receptors and inhibitory receptors on the cell surface. The inhibitory signals of major histocompatibility complex class I (MHC-I) antigen-specific receptors are crucial to prevent hematopoietic cells from being attacked by NK cells. These NK cell surface receptors are divided into three families, namely KIRs, LIRs, and NKG2A. Among them, KIRs are members of the immunoglobulin superfamily, and their main function is to recognize classical human leukocyte antigen (HLA) A, B, and C molecules; LIRs and KIRs belong to the same Ig superfamily, and except for HLAⅠa class, they mainly recognize non-classical HLA-G molecules. NKG2A is a member of the NKG2 family, including A, B, C, D, E, F, and H, and belongs to the C-type lectin family, and its main ligand is non-classical HLA-EⅠ molecule.

[0003] Lymphocyte activation gene-3 (LAG-3), as a member of the immunoglobulin superfamily receptor, is upregulated on the surface of activated CD4+T cells, CD8+T cells, NK cells and other immune cells, and is an inhibitory receptor. It has been shown to play an important role in processes such as T cell dysfunction and T cell exhaustion. In addition to directly affecting the function and activity of effector T cells, LAG-3 can also affect immune function by promoting the inhibitory effect of regulatory T cells and affecting the maturation of dendritic cells (DC). The combination therapy of LAG-3 monoclonal antibody Relatlimab and PD-1 monoclonal antibody Nivolumab has been used to treat adult and pediatric patients over 12 years old with unresectable or metastatic melanoma.

[0004] Lenalidomide is an analog of thalidomide. Due to its immunomodulatory, anti-angiogenic and anti-tumor effects, it has been used to treat adult patients with multiple myeloma (MM). In the MM environment, the differentiation, activation and cytotoxic function of NK cells are severely impaired. Lenalidomide can stimulate the expression of co-stimulatory receptors such as CD16 and LFA-1 on the surface of NK cells, downregulate the expression of IKZF1, IKZF3, etc., promote the expression of NKG2 ligands on the surface of malignant transformed cells, and enable malignant transformed cells to interact with NK cells to complete cell killing. In addition, lenalidomide can also induce the secretion of IL-2 and IFN-γ, promote antibody-dependent cell contact cytotoxicity (ADCC) and NK cell cytotoxic activity. Lenalidomide can promote the proliferation, activation and cytotoxicity of NK cells, and enhance NK cell-mediated malignant cell killing and immune surveillance.

[0005] Therefore, there is an urgent need to provide a composition and method that can enhance the killing ability of NK cells. Summary of the invention

[0006] In view of the deficiencies in the prior art and actual needs, the present invention provides a composition for improving the killing ability of NK cells and its application. The components of the composition of the present invention cooperate with each other and work synergistically, effectively improving the proliferation ability of natural killer cells and the positive rate of cytotoxic markers, and also improving the cytokine secretion level of natural killer cells and their killing ability against cancer cells.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a composition for improving the killing ability of NK cells, wherein the composition for improving the killing ability of NK cells comprises lymphocyte activation gene 3 monoclonal antibody and lenalidomide.

[0009] The components of the composition of the present invention cooperate with each other and work synergistically, effectively improving the proliferation ability and cytotoxic marker positive rate of natural killer cells, and also improving the cytokine secretion level of natural killer cells and their killing ability to cancer cells.

[0010] Preferably, the mass ratio of the lymphocyte activation gene 3 monoclonal antibody to lenalidomide is 1:(600-1200).

[0011] The specific point values ​​among the above 600-1200 can be selected as 600, 630, 700, 800, 870, 900, 1000, 1100, 1200, etc.

[0012] In a second aspect, the present invention provides the use of the composition for improving the killing ability of NK cells described in the first aspect in the preparation of a drug for treating tumors.

[0013] In a third aspect, the present invention provides a kit for enhancing the killing ability of NK cells, wherein the kit for enhancing the killing ability of NK cells contains the composition for improving the killing ability of NK cells described in the first aspect.

[0014] In a fourth aspect, the present invention provides a method for enhancing the killing ability of NK cells, the method comprising: treating NK cells with the composition for improving the killing ability of NK cells described in the first aspect.

[0015] Preferably, the mass ratio of lymphocyte activation gene 3 monoclonal antibody to lenalidomide in the composition for improving the killing ability of NK cells is 1:(600-1200).

[0016] The specific point values ​​among the above 600-1200 can be selected as 600, 630, 700, 800, 870, 900, 1000, 1100, 1200, etc.

[0017] Preferably, the method further comprises: culturing NK cells in a culture medium containing CD3 monoclonal antibody, IL-5 and IL-2 for 5-8 days and then adding the composition for improving the killing ability of NK cells described in the first aspect to the culture medium.

[0018] Preferably, the final concentration of the CD3 monoclonal antibody in the culture medium is 1.8-3.2 μg / mL (e.g., 1.8 μg / mL, 2.0 μg / mL, 2.5 μg / mL, 2.8 μg / mL, 3.0 μg / mL, 3.2 μg / mL).

[0019] Preferably, the final concentration of IL-5 in the culture medium is 40-60 ng / mL (e.g., 40 ng / mL, 42 ng / mL, 44 ng / mL, 50 ng / mL, 55 ng / mL, 58 ng / mL, 60 ng / mL).

[0020] Preferably, the final concentration of IL-2 in the culture medium is 800-1200 IU / mL (e.g., 800 IU / mL, 820 IU / mL, 840 IU / mL, 900 IU / mL, 1000 IU / mL, 1100 IU / mL, 1200 IU / mL).

[0021] In a fifth aspect, the present invention provides a NK cell, wherein the NK cell is obtained by processing by the method described in the fourth aspect.

[0022] In a sixth aspect, the present invention provides the use of the NK cells described in the fifth aspect in the preparation of a product for treating tumors.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The components of the composition of the present invention cooperate with each other and work synergistically to effectively enhance the proliferation capacity of natural killer cells;

[0025] (2) The components of the composition of the present invention cooperate with each other and work synergistically, effectively increasing the positive rate of cytotoxic markers of natural killer cells;

[0026] (3) The components of the composition of the present invention cooperate with each other and work synergistically, effectively improving the killing ability of natural killer cells against tumor cells;

[0027] (4) The components of the composition of the present invention cooperate with each other and work synergistically to effectively increase the cytokine secretion level of natural killer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a microscopic observation of the natural killer cells obtained in Example 1;

[0029] Figure 2 This is a flow cytometry analysis of the purity of natural killer cells obtained in Example 1;

[0030] Figure 3 This is a diagram of the natural killer cell killing experiment. DETAILED DESCRIPTION

[0031] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0032] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0033] Human lymphocyte separation medium (Cat. No. 711012) and NK cell culture medium (Cat. No. 6113031) were purchased from Shenzhen Dakoway Biotechnology Co., Ltd. Gibco RPMI1640 culture medium (Cat. No. 11879020) and fetal bovine serum (Cat. No. A5256701) were purchased from Thermo Fisher SCIENTIFIC. Lactate dehydrogenase cytotoxicity detection kit (C0016) was purchased from Beyotime Biotechnology. Human IFN-γ ELISA detection kit (enzyme-linked immunosorbent assay) (Cat. No. EK180) was purchased from Lianke Biotechnology. LAG-3 monoclonal antibody (Cat. No. BMS-986016) and PD-1 monoclonal antibody (Cat. No. BMS-936558) were purchased from MCE with a purity of >95%. K562 cells (Cat. No. JY760) were purchased from Shanghai Jinyuan Biotechnology. Lenalidomide (CAS191732-72-6) and thalidomide (CAS 50-35-1) were purchased from Yisheng Biotechnology.

[0034] Example 1

[0035] This embodiment provides a composition for enhancing the killing ability of NK cells, which comprises the following components in parts by weight:

[0036] Lymphocyte activation gene 3 monoclonal antibody (1) and lenalidomide (1070).

[0037] Example 2

[0038] This embodiment provides a composition for enhancing the killing ability of NK cells, which comprises the following components in parts by weight:

[0039] Lymphocyte activation gene 3 monoclonal antibody (1) and lenalidomide (600).

[0040] Example 3

[0041] This embodiment provides a composition for enhancing the killing ability of NK cells, which comprises the following components in parts by weight:

[0042] Lymphocyte activation gene 3 monoclonal antibody (1) and lenalidomide (1200).

[0043] Comparative Example 1

[0044] This comparative example is different from Example 1 in that it does not contain lymphocyte activation gene 3 monoclonal antibody, and its mass fraction is allocated to the mass of lenalidomide.

[0045] Comparative Example 2

[0046] This comparative example is different from Example 1 in that lenalidomide is not contained, and the mass fraction thereof is allocated to the mass of the lymphocyte activation gene 3 monoclonal antibody.

[0047] Comparative Example 3

[0048] This comparative example is different from Example 1 in that lenalidomide is replaced by thalidomide in equal amounts.

[0049] Comparative Example 4

[0050] The difference between this comparative example and Example 1 is that the lymphocyte activation gene 3 monoclonal antibody is replaced by the programmed death receptor 1 monoclonal antibody in equal amounts.

[0051] Test Example 1

[0052] Detection of proliferation ability of NK cells derived from cord blood.

[0053] (1) Isolation of mononuclear cells from umbilical cord blood

[0054] Select suitable discarded public cord blood for NK cell preparation. Dilute the blood with physiological saline at a ratio of 1:1, add an appropriate amount of human lymphocyte separation solution to the centrifuge tube, slowly add the diluted cord blood along the tube wall, and there is a clear dividing line between the upper and lower layers. Centrifuge at 750×g for 25 minutes at room temperature. Aspirate the cells in the middle white film layer, centrifuge at 750×g for 8 minutes at room temperature, and the precipitate is the cord blood mononuclear cells.

[0055] (2) NK cell culture

[0056] Cell induction and culture were performed according to the NK cell culture medium operating instructions. Flow cytometry and cell counting were performed when the cells were harvested. The flow cytometry standard was CD3-CD56+CD16+. The NK cell flow cytometry results showed that the purity of CD3-CD56+CD16+ cells was higher than 92%, indicating that the NK cell culture was successful.

[0057] (3) NK cell grouping and antibody blocking

[0058] Control group: NK cells were cultured in NK cell culture medium at 37°C and 5% CO2 until the cells were harvested.

[0059] Experimental group: After culturing in NK cell culture medium (wherein the final concentration of CD3 monoclonal antibody is 2 μg / mL, the final concentration of IL-5 is 50 ng / mL, and the final concentration of IL-2 is 800-1200 IU / mL) for 8 days, the substances described in each embodiment and comparative example were added to the culture medium, and the cells were cultured at 37° C. and 5% CO2 until the cells were harvested. The natural killer cells (NK cells) obtained in Example 1 were observed under a microscope as shown in the figure below. Figure 1 As shown, the purity flow detection diagram is as follows Figure 2 shown.

[0060] 500 μL of samples were taken from the control group and the experimental group when the cells were harvested. After oscillation and mixing, the cells were counted using a SYSMEX XN-B4 counter, and TNC was taken as the cell density to calculate the number of harvested cells.

[0061] The results are shown in Table 1.

[0062] Table 1

[0063] Group Cell number Example 1 <![CDATA[10.31×10 9 ]]> Example 2 <![CDATA[9.68×10 9 ]]> Example 3 <![CDATA[9.03×10 9 ]]> Comparative Example 1 <![CDATA[8.64×10 9 ]]> Comparative Example 2 <![CDATA[8.53×10 9 ]]> Comparative Example 3 <![CDATA[8.57×10 9 ]]> Comparative Example 4 <![CDATA[8.41×10 9 ]]> Control group <![CDATA[8.29×10 9 ]]>

[0064] As shown in Table 1, the compositions of Examples 1-3 of the present invention effectively improve the proliferation capacity of natural killer cells. As shown in the data of Comparative Examples 1-4, the components of the compositions of the present invention cooperate with each other and work synergistically, and can effectively improve the proliferation capacity of natural killer cells under specific component combinations and ratios.

[0065] Test Example 2

[0066] Detection of cytotoxic marker positivity rate of NK cells derived from cord blood.

[0067] 1×10 7 The cell amount is 2×10 6 / mL were inoculated in RPMI1640 medium and K562 cells (4×10 5 / mL) for 6 h. After the co-culture, the cells were labeled with flow cytometry antibodies and the cytotoxic markers were detected by flow cytometry.

[0068] The results are shown in Table 2.

[0069] Table 2

[0070] Group Cytotoxic marker positive rate (%) Example 1 11.9 Example 2 9.87 Example 3 10.7 Comparative Example 1 7.06 Comparative Example 2 9.11 Comparative Example 3 9.25 Comparative Example 4 8.99 Control group 5.96

[0071] As shown in Table 2, the compositions of Examples 1-3 of the present invention effectively increase the positive rate of cytotoxic markers of natural killer cells. As shown in the data of Comparative Examples 1-4, the components of the composition of the present invention cooperate with each other and work synergistically, and can effectively increase the positive rate of cytotoxic markers of natural killer cells under specific component combinations and ratios.

[0072] Test Example 3

[0073] Detection of the killing ability of NK cells derived from cord blood on tumor cells.

[0074] K562, Raji, and Jurkat cell lines were cultured in 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2, and logarithmic phase cells were used as target cells for the experiment. NK cells in each embodiment and comparative example were used as effector cells. Target cells and effector cells were diluted to a concentration of 1×10 6 / mL and 5×10 6 Take 100 μL of the cell suspension of 100 μL / mL and inoculate it in a flat-bottom 96-well plate (effect-target ratio 5:1). Set up a blank control group, a single effector cell group, a single target cell group, and a maximum enzyme activity group at the same time, with 3 replicates in each group. Add and mix well and culture at 37°C and 5% CO2. Perform LDH concentration detection at 4h and 8h, respectively. Take out 20 μL of LDH release reagent to each well of the maximum enzyme activity group 1h before detection, and put it back to continue culturing after repeated blowing. Take it out after reaching the detection time point, centrifuge at 400×g for 5min, take 120 μL of supernatant, add 60 μL of LDH detection working solution, wrap it with aluminum foil, and incubate it on a horizontal shaker for 30min. After incubation, measure the absorbance at 490nm. The mortality rate was calculated using the following formula: mortality rate (%) = (absorbance of experimental group - absorbance of single effector cell group - absorbance of single target cell group + control group) / (absorbance of maximum enzyme activity group - absorbance of single effector cell group - absorbance of single target cell group + control group) × 100.

[0075] The results are shown in Table 3 and Figure 3 shown.

[0076] Table 3

[0077]

[0078]

[0079] From Table 3 and Figure 3 It can be seen that the compositions described in Examples 1-3 of the present invention effectively improve the killing ability of natural killer cells against tumor cells. From the data of Comparative Examples 1-4, it can be seen that the components of the composition of the present invention cooperate with each other and work synergistically, and can effectively improve the killing ability of natural killer cells against tumor cells under specific component combinations and ratios.

[0080] Test Example 4

[0081] Detection of cytokine secretion levels of NK cells derived from cord blood.

[0082] Take 1×10 6 The experimental and control group cells were placed in 1 mL of NK cell culture medium without additives and 2×10 5Raji cells were co-cultured and centrifuged at 300×g for 10 min after 4 h to remove the precipitate. 300 μL of washing solution was added to the ELISA plate and allowed to soak for 30 s, then patted dry on absorbent paper and used immediately. The reagents added were as follows:

[0083] Standard wells: add 100 μL of serially diluted standard;

[0084] Blank well: add 100 μL NK cell culture medium;

[0085] Sample wells: Add 100 μL of culture medium supernatant after centrifugation.

[0086] Add 50 μL of diluted detection antibody to each well, seal the plate, and incubate at room temperature for 2 hours at 100 rpm. After incubation, remove the liquid and add 300 μL of washing solution to each well for a total of 6 washes, patting dry on absorbent paper each time. Add 100 μL of diluted horseradish peroxidase-labeled streptavidin to each well, reseal the plate, and incubate at room temperature for 45 minutes at 100 rpm. Repeat the plate washing process, add 100 μL of chromogenic substrate TMB, and incubate at room temperature in the dark for 10 minutes. Add 100 μL of stop solution to each well and mix thoroughly until the color of the liquid changes from blue to yellow. Use an enzyme reader to detect the OD value at 450nm and 570 / 630nm wavelengths. The measured value at 450nm-570nm is the corrected OD value. Use the OD value of the standard well to establish a standard curve, and after the OD value of the sample well-OD value of the blank well is subtracted, the concentration of INF-γ is calculated by the standard curve.

[0087] The results are shown in Table 4.

[0088] Table 4

[0089] Group IFN-γ concentration (pg / mL) Example 1 482.1 Example 2 451.7 Example 3 447.5 Comparative Example 1 431.2 Comparative Example 2 425.3 Comparative Example 3 412.6 Comparative Example 4 429.8 Control group 395.8

[0090] As shown in Table 4, the compositions of Examples 1-3 of the present invention effectively increase the cytokine secretion level of natural killer cells. As shown in the data of Comparative Examples 1-4, the components of the composition of the present invention cooperate with each other and work synergistically, and can effectively increase the cytokine secretion level of natural killer cells under specific component combinations and ratios.

[0091] In summary, the components of the composition of the present invention cooperate with each other and work synergistically, effectively improving the proliferation ability of natural killer cells and the positive rate of cytotoxic markers, and also improving the cytokine secretion level of natural killer cells and their ability to kill cancer cells.

[0092] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A composition for improving the killing ability of NK cells, characterized in that: The composition for improving the killing ability of NK cells comprises lymphocyte activation gene 3 monoclonal antibody and lenalidomide.

2. The composition for improving the killing ability of NK cells according to claim 1, characterized in that: The mass ratio of the lymphocyte activation gene 3 monoclonal antibody and lenalidomide is 1:(600-1200).

3. Use of the composition for improving the killing ability of NK cells as claimed in claim 1 or 2 in the preparation of drugs for treating tumors.

4. A kit for enhancing the killing ability of NK cells, characterized in that: The kit for enhancing the killing ability of NK cells contains the composition for improving the killing ability of NK cells according to claim 1 or 2.

5. A method for enhancing the killing ability of NK cells, characterized in that: The method comprises: treating NK cells with the composition for improving the killing ability of NK cells according to claim 1 or 2.

6. The method according to claim 5, characterized in that The mass ratio of lymphocyte activation gene 3 monoclonal antibody to lenalidomide in the composition for improving the killing ability of NK cells is 1:(600-1200).

7. The method according to claim 5 or 6, characterized in that: The method further comprises: culturing NK cells in a culture medium containing CD3 monoclonal antibody, IL-5 and IL-2 for 5-8 days, and then adding the composition for improving the killing ability of NK cells according to claim 1 or 2 into the culture medium.

8. The method according to any one of claims 5 to 7, characterized in that: The final concentration of CD3 monoclonal antibody in the culture medium is 1.8-3.2 μg / mL; Preferably, the final concentration of IL-5 in the culture medium is 40-60 ng / mL; Preferably, the final concentration of IL-2 in the culture medium is 800-1200 IU / mL.

9. A NK cell, characterized in that: The NK cells are obtained by processing according to any one of claims 5-8.

10. Use of the NK cells according to claim 9 in the preparation of products for treating tumors.

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