Compound for enhancing NK (Natural Killer) cytotoxic activity function and use method thereof

Abstract

The invention relates to the technical field of drug screening, in particular to a compound for enhancing the NK cytotoxic activity function and a use method. The inventor finds that the Astilbin screened by the invention is contacted with the NK cells, so that the cytotoxicity of the NK cells is greatly improved, amplification markers of the NK cells are also improved to a certain extent, phenotypes of the NK are not influenced, and the functions of the NK cells are improved. Moreover, the inventor also finds that after the NK cells are treated by the Astilbin, not only can the NK cells from iPSC be improved, but also the NK cells from cord blood have similar results. It is shown that the function of improving the cell function can be achieved on the NK from various sources through pretreatment of the Astilbin.

Description

Technical Field

[0001] The present invention relates to the technical field of drug screening, and in particular to a compound that enhances the cytotoxic activity of NK cells and a method for using the compound. Background Art

[0002] NK cells, also known as natural killer cells, are a crucial immune cell type in the human body. Their ability to be used allogeneically is a key characteristic of NK cells. In recent years, their broad-spectrum anti-tumor activity and allogeneic use have made NK cells a hot topic in the field of cancer treatment. While the effects of immunomodulatory drugs on NK cell function remain relatively unknown, natural polysaccharides, which often have immune-enhancing properties, have similarly been less studied.

[0003] NK cytotoxicity refers to the ability of natural killer cells (NK cells) to directly induce apoptosis or lysis of target cells (such as tumor cells or virus-infected cells) by releasing toxic granules or activating death receptor signals. Currently, there are relatively few compounds that have been published that enhance NK cytotoxicity, and further development is needed. Summary of the Invention

[0004] The present invention aims to at least partially address one of the technical problems in the related art. To this end, one object of the present invention is to provide the use of astilbin in enhancing NK cell cytotoxicity and methods for enhancing NK cell cytotoxicity. The inventors have discovered that contacting NK cells with astilbin screened for the present invention significantly improves the cytotoxicity of NK cells and also increases NK cell expansion markers to a certain extent, without affecting the NK cell phenotype, thereby enhancing NK cell function. Furthermore, the inventors have discovered that pretreatment with astilbin can enhance NK cell function in a variety of NK cell sources.

[0005] The first aspect of the present invention provides use of Astilbin in enhancing NK cell cytotoxic activity.

[0006] The inventors used high-throughput screening to screen a library of FDA-approved clinically used immunomodulatory compounds and natural polysaccharides, identifying astilbin and finding that it significantly enhances NK cell cytotoxicity. Astilbin, a natural flavonoid found in St. John's wort and traditional Chinese medicine, enhances NRF2 activation. Astilbin is found in various herbs and foods, such as sarsaparilla, astragalus, grapes, and red wine, and has a high safety profile.

[0007] The inventors found that adding the compound during the NK cell culture process to pretreat the NK cells improves the cytotoxic activity of the NK cells without affecting the NK cell surface markers.

[0008] In some embodiments of the present invention, the concentration of Astilbin is 0.01 uM to 50 uM.

[0009] In some preferred embodiments of the present invention, the concentration of Astilbin is 0.05uM to 15uM.

[0010] A second aspect of the present invention provides a drug or kit for enhancing NK cell cytotoxicity. According to some embodiments of the present invention, the drug or kit comprises Astilbin.

[0011] A third aspect of the present invention provides use of Astilbin in the preparation of a drug or a kit for enhancing NK cell cytotoxic activity.

[0012] A fourth aspect of the present invention provides a method for enhancing NK cell cytotoxicity. In some embodiments of the present invention, the method comprises:

[0013] Astilbin is brought into contact with the NK cells.

[0014] The inventors found that pre-treating NK cells with astilbin increased the cytotoxic activity of NK cells.

[0015] In some embodiments of the present invention, when the contact is performed, the concentration of Astilbin is 0.01 uM to 50 uM.

[0016] In some preferred embodiments of the present invention, the concentration of Astilbin is 0.05uM to 15uM.

[0017] A fifth aspect of the present invention provides a method for enhancing the proliferation capacity of NK cells. In some embodiments of the present invention, the method comprises:

[0018] Astilbin is brought into contact with the NK cells.

[0019] In some embodiments of the present invention, when the contact is performed, the concentration of Astilbin is 0.01 uM to 50 uM.

[0020] In some preferred embodiments of the present invention, the concentration of Astilbin is 0.05uM to 15uM.

[0021] A sixth aspect of the present invention provides a drug or kit for enhancing the proliferation ability of NK cells. In some embodiments of the present invention, the drug or kit includes Astilbin.

[0022] A seventh aspect of the present invention provides the use of Astilbin in the preparation of a drug or a kit for enhancing the proliferation ability of NK cells.

[0023] The inventors found that using Astilbin screened by the present invention and contacting it with NK cells greatly improved the cytotoxicity of NK cells, and also improved the expansion markers of NK cells to a certain extent, without affecting the phenotype of NK cells, thereby improving the function of NK cells. In addition, the inventors also found that after treating NK cells with Astilbin, not only did it have an improvement effect on iPSC-derived NK cells, but it also had similar results on umbilical cord blood-derived NK cells. This shows that pretreatment with Astilbin can improve the function of NK cells from various sources.

[0024] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0026] Figure 1 The compound screening process in the drug library in Example 1 is shown;

[0027] Figure 2 Shows the results of the initial screening of the compounds in Example 1;

[0028] Figure 3 Shows the results of rescreening of the compounds in Example 2;

[0029] Figure 4 The results show that the three candidate compounds in Example 3 have cytotoxic activity against K562 cells after pretreatment.

[0030] Figure 5 The figure shows the flow cytometry results of Ki-67 (cell proliferation marker) after iNK cells were treated with different concentrations of Astilbin in Example 4;

[0031] Figure 6 The CD56 flow cytometry phenotype of NK cells in Example 4 is shown;

[0032] Figure 7 The CD314 flow cytometry phenotype of NK cells in Example 4 is shown;

[0033] Figure 8 The CD335 flow cytometry phenotype of NK cells in Example 4 is shown;

[0034] Figure 9 The CD337 flow cytometry phenotype of NK cells in Example 4 is shown;

[0035] Figure 10 The cytotoxicity of NK cells to K562 cells after pretreatment with different concentrations of Astilbin in Example 4 is shown;

[0036] Figure 11 The cytotoxicity of different concentrations of Astilbin to K562 Luci cells after pre-treatment of CBNK cells in Example 5 is shown;

[0037] Figure 12 The amplification factor of CBNK cells pretreated with Astilbin at different concentrations in Example 5 is shown. DETAILED DESCRIPTION

[0038] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0039] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0040] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0041] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present invention belongs.

[0042] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0043] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0044] According to a specific embodiment of the present invention, the present invention provides a use of astilbin in enhancing NK cell cytotoxic activity. The concentration of astilbin is 0.01uM to 50uM. For example, the concentration of astilbin is 0.01uM, 0.05uM, 0.1uM, 0.15uM, 0.2uM, 0.5uM, 1uM, 5uM, 10uM, 15uM, 20uM, 25uM, 30uM, 35uM, 40uM, 45uM, 50uM, etc.

[0045] According to a preferred embodiment of the present invention, the concentration of Astilbin is 0.05uM to 15uM.

[0046] According to a specific embodiment of the present invention, the present invention provides a drug or a kit for enhancing the cytotoxic activity of NK cells, wherein the drug or the kit comprises Astilbin.

[0047] The drug or kit may contain other components besides astilbin, for example, other pharmaceutical excipients, or a diluent. The concentration of astilbin in the drug or kit may be adjusted based on actual needs.

[0048] According to a specific embodiment of the present invention, the present invention provides use of Astilbin in preparing a drug or a kit for enhancing NK cell cytotoxic activity.

[0049] The inventors found that Astilbin screened out in the present invention can be used as a drug or kit for enhancing the cytotoxic activity of NK cells.

[0050] According to a specific embodiment of the present invention, the present invention provides a method for enhancing NK cell cytotoxic activity, the method comprising:

[0051] Astilbin is brought into contact with the NK cells.

[0052] The inventors discovered that by adding astilbin during the culture of NK cells to pre-treat the NK cells, the toxicity of the NK cells against target cells (such as K562 cells) was enhanced.

[0053] There are no particular limitations on the concentration of Astilbin, contact time, and NK cell concentration during contact. According to a specific embodiment of the present invention, the concentration of Astilbin during contact is 0.01 μM to 50 μM. According to a preferred embodiment of the present invention, the concentration of Astilbin during contact is 0.05 μM to 15 μM.

[0054] According to a specific embodiment of the present invention, the present invention provides a method for enhancing the proliferation ability of NK cells, the method comprising:

[0055] Astilbin is brought into contact with the NK cells.

[0056] There are no particular limitations on the concentration of Astilbin, contact time, and NK cell concentration during contact. According to a specific embodiment of the present invention, the concentration of Astilbin during contact is 0.01 μM to 50 μM. According to a preferred embodiment of the present invention, the concentration of Astilbin during contact is 0.05 μM to 15 μM.

[0057] According to a specific embodiment of the present invention, the present invention provides a drug or a kit for enhancing the proliferation ability of NK cells, wherein the drug or the kit comprises Astilbin.

[0058] The inventors found that the screened Astilbin, when in contact with NK cells, can increase the expansion markers of NK cells to a certain extent, has no effect on the phenotype of NK cells, and improves the function of NK cells.

[0059] According to a specific embodiment of the present invention, the present invention provides use of Astilbin in preparing a drug or a kit for enhancing the proliferation ability of NK cells.

[0060] It should be noted that there is no particular limitation on the source of the "NK cells" used in this invention. Any source known in the art is encompassed. For example, the NK cells can be derived from peripheral blood, umbilical cord blood, iPS cells, bone marrow, and cell lines. Each source has its own characteristics in terms of maturity, expansion potential, and clinical application. The selection process should balance functional requirements, production costs, and therapeutic scenarios.

[0061] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0062] Example 1 Primary Drug Screening

[0063] We purchased existing FDA-approved clinical drug libraries and natural polysaccharide libraries for immune regulation, totaling 516 compounds for screening and testing.

[0064] Preparation of the drug screening library: Centrifuge the plate and add 30 μL / well of DMSO to dissolve the solution. Mix thoroughly and use as the stock solution. Prepare a new plate and dilute the drug library in NK cell culture medium (Corning KBM-581 medium supplemented with 10% FBS and 1000 U IL-2) to a working concentration of 10 μM.

[0065] iNK cell inoculation and culture: iPSC-derived NK cells were used for subsequent experiments. iNK cells were differentiated according to patent number CN118667760A. iNK cells were resuspended at a concentration of 0.7E6 / mL. 100 μL / well of the drug reservoir was added. The cells were then cultured in a 37°C incubator using the same NK cell culture medium described above. On the third day, 100 μL of culture medium was added and the cells were returned to the incubator for culture.

[0066] Cytotoxicity Assay: On day 6 of culture, remove the NK cell culture plate and carefully discard 100 μL of the upper culture medium. Resuspend K562 Luciferase at a concentration of 1E6 / mL and add 100 μL / well to the drug library plate containing NK cells. Incubate in an incubator for 4 hours, followed by the addition of 50 μL of Luciferase substrate. Incubate in the dark for 10 minutes before assaying using a multi-function microplate reader.

[0067] Drug screening process see Figure 1 , activity test results are shown in Figure 2 The results of the cytotoxicity test showed that the cytotoxicity of one drug after treatment was higher than that of the positive control group (APC activation group), and the cytotoxicity of four compounds was comparable to that of the positive group. There were 55 compounds with cytotoxicity higher than that of the control group.

[0068] Example 2 Drug Rescreening

[0069] The 60 compounds screened in Example 1 were taken out and re-formulated into a new drug screening plate for re-screening.

[0070] iNK cell inoculation and culture: Resuspend iPSC-derived NK cells at a concentration of 0.7E6 / mL. Add 100 μL / well of the drug reservoir. Culture in a 37°C incubator. On the third day, add 100 μL of culture medium and return the cells to the incubator.

[0071] Cytotoxicity assay: Remove the NK cell culture plate and carefully discard 100 μL of the upper culture medium. Resuspend K562 Luciferase at a concentration of 1E6 / mL and add 100 μL / well to the drug library plate containing NK cells. Incubate in an incubator for 4 hours, followed by the addition of 50 μL of Luciferase substrate. Incubate in the dark for 10 minutes before assaying using a multi-function microplate reader.

[0072] After rescreening, two compounds showed similar cytotoxicity to K562 cells as the positive control group, and one compound showed higher cytotoxicity to K562 cells than the positive control group ( Figure 3 ).

[0073] Example 3

[0074] The three compounds screened out in Example 2 were subjected to the next test.

[0075] iNK cells were pretreated with the three compounds: iPSC-derived NK cells were resuspended in NK cell culture medium at a density of 0.7E6 / mL. 10uM of each of the three compounds was then added and cultured in an incubator for 6 days.

[0076] Cytotoxicity Assay: Remove iPSC-NK cells from the incubator and count them. Based on the count, remove 1E6 iNK cells, centrifuge, remove the supernatant, and resuspend in 1 mL of 1640 medium for later use. Next, centrifuge 1E6 K562 Luci cells in the growth phase and resuspend in 1 mL of 1640 complete medium. Add the cells to a 96-well plate at a 1:1 effector-target ratio. Mix thoroughly and place in the incubator for co-culture. After 4 hours, add the Luci substrate enzyme and incubate in the dark for 10 minutes before assay.

[0077] Among the three compounds pre-treated, iNK cells pre-treated with Astilbin showed the strongest cytotoxicity to K562 Luci ( Figure 4 ), the results showed significant differences.

[0078] Example 4

[0079] To explore the effect of Astilbin on iPSC-NK, we pretreated iPSC-NK cells with different concentrations of Astilbin and detected the proliferation capacity and flow cytometry phenotype of iPSC-NK cells.

[0080] iPSC-NK cells were resuspended at a density of 0.7E6 / mL and then treated with various concentrations of Astilbin (0.05uM, 0.5uM, 5uM, 10uM, and 15uM). The cells were cultured in an incubator. On day 6, the iNK cells were removed and counted, and their phenotypes were analyzed using FACS. iNK cells treated with various concentrations were then co-cultured with K562 Luci to test their toxicity against K562 Luci.

[0081] The results of flow cytometry showed that the proliferation ability of iNK cells was improved to a certain extent after treatment with 0.5uM, 5uM, 10uM, and 15uM Astilbin. Figure 5 ). However, the cell phenotype (CD56) and toxicity-related indicators (CD335 / CD314 / CD337) showed no difference compared with the untreated group ( Figures 6-9 ).

[0082] Different concentrations of Astilbin pretreatment increased the toxicity of iNK cells to varying degrees ( Figure 10 ).

[0083] Example 5

[0084] To explore whether Astilbin has the same effect on NK cells from other sources, CBNK cells were pretreated with 1-10uM Astilbin, and the cytotoxic activity and proliferation capacity of CBNK were detected after 6 days.

[0085] Cord blood mononuclear cells were resuspended in NK cell culture medium and then expanded with aAPCs. Fresh NK cell culture medium was replenished every 3-4 days. After 12 days of expansion, 4E6 CBNK cells were centrifuged to remove the culture medium. The cells were resuspended in 4 mL of fresh NK cell culture medium and seeded into 12-well plates. The CBNK cells were then pretreated with various concentrations of astilbin and cultured in an incubator for a further 6 days.

[0086] Cytotoxicity assay: Remove CBNK cells from the incubator and count them. Based on the count, remove 0.5E6 of the CBNK cells, centrifuge, remove the supernatant, and resuspend in 1 mL of 1640 medium for later use. Next, centrifuge 1E6 of K562Luci cells in the growth phase and resuspend in 1 mL of 1640 complete medium. Add the cells to a 96-well plate at a 0.5:1 effector:target ratio. Mix thoroughly and place in the incubator for co-culture. After 4 hours, add the Luci substrate enzyme and incubate in the dark for 10 minutes before assaying.

[0087] After CBNK was treated with Astilbin, the treated CBNK had stronger killing activity against K562 Luci than the untreated CBNK ( Figure 11 ), and the proliferation ability of CBNK treated with Astilbin was also higher than that of the untreated group ( Figure 12 ).

[0088] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", "some implementation plans" or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0089] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. Use of Astilbin in enhancing NK cell cytotoxic activity.

2. The use according to claim 1, characterized in that The concentration of Astilbin is 0.01uM to 50uM; Optionally, the concentration of Astilbin is 0.05uM to 15uM.

3. A drug or kit for enhancing NK cell cytotoxic activity, characterized in that: Including Astilbin.

4. Use of Astilbin in the preparation of a drug or kit for enhancing NK cell cytotoxic activity.

5. A method for enhancing NK cell cytotoxic activity, characterized in that: include: Astilbin is brought into contact with the NK cells.

6. The method according to claim 5, characterized in that During the contacting, the concentration of Astilbin is 0.01uM to 50uM; Optionally, the concentration of Astilbin is 0.05uM to 15uM.

7. A method for enhancing the proliferation ability of NK cells, characterized in that: include: Astilbin is brought into contact with the NK cells.

8. The method according to claim 7, characterized in that During the contacting, the concentration of Astilbin is 0.01uM to 50uM; Optionally, the concentration of Astilbin is 0.05uM to 15uM.

9. A drug or kit for enhancing the proliferation ability of NK cells, characterized in that: Including Astilbin.

10. Use of Astilbin in the preparation of a drug or kit for enhancing the proliferation ability of NK cells.

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