Application of Src inhibitor in increasing cell differentiation number or cell amplification number

Abstract

The invention provides application of an Src inhibitor in increasing the cell differentiation number or the cell amplification number, and belongs to the technical field of biological medicine. When the Src inhibitor is applied to cell differentiation or cell amplification, the number of differentiated cells and the number of amplified cells can be effectively increased.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology. Specifically, the present application relates to the application of Src inhibitors in increasing the number of cell differentiation or cell proliferation. Background Art

[0002] NK cells, also known as natural killer cells, are an important immune cell in the human body. The ability to be used allogeneically is an important characteristic of NK cells. In recent years, the broad-spectrum tumor-killing activity and the ability to be used allogeneically have made NK cells a hot topic in the field of tumor treatment. However, the low number of differentiation and weak amplification ability have always been the pain points of the development of the NK cell industry. Summary of the invention

[0003] The present application aims to solve one of the technical problems in the related art at least to some extent. To this end, the present application proposes a method of using a Src inhibitor to induce cell differentiation or cell expansion culture to increase the number of cell differentiation or cell expansion.

[0004] In the first aspect, the present application proposes the use of Src inhibitors in increasing the number of cell differentiation or cell expansion. The use of Src inhibitors in cell differentiation or cell expansion can effectively increase the number of differentiated cells and the number of expanded cells.

[0005] In some examples of the present application, the aforementioned Src inhibitor is selected from at least one of Dasatinib, Ouercetin, UM-164, KX2-391, KX1-004 or its analogs. The aforementioned Src inhibitor can be used in cell differentiation or cell expansion applications to increase the number of differentiated cells or the number of expanded cells. In some preferred examples of the present application, the aforementioned Src inhibitor is selected from UM-164 or its analogs. It has been verified that the use of UM-164 for cell differentiation or cell expansion applications can effectively increase the number of differentiated cells or the number of expanded cells.

[0006] In the second aspect, the present application proposes a method for increasing the number of cell differentiation. According to an embodiment of the present application, the aforementioned method comprises: inducing differentiation treatment of cells to be differentiated using a differentiation medium containing a Src inhibitor. Inducing differentiation treatment of cells to be differentiated using a differentiation medium containing a Src inhibitor can effectively increase the number of proliferation of differentiated cells, and also has a significant effect on improving the cell phenotype.

[0007] In some examples of the present application, the aforementioned Src inhibitor is selected from at least one of Dasatinib, Ouercetin, UM-164, KX2-391, KX1-004 or its analogs. The aforementioned Src inhibitor can be used in cell differentiation applications to increase the number of differentiated cells. In some preferred examples of the present application, the aforementioned Src inhibitor is selected from UM-164 or its analogs. It has been verified that the use of UM-164 for cell differentiation applications can effectively increase the number of differentiated cells.

[0008] In some examples of the present application, the aforementioned induction differentiation treatment includes: in the differentiation stage, using a differentiation medium containing a first predetermined concentration of Src inhibitor to induce differentiation treatment on the differentiated cells, the aforementioned first predetermined concentration is selected from 1nM-100nM, optionally 1nM, 2nM, 3nM, 4nM, 5nM, 6nM, 7nM, 8nM, 9nM, 10nM, 15nM, 20nM, 25nM, 30nM, 35nM, 40nM, 45nM, 50nM, 55nM, 60nM, 65nM, 70nM, 75nM, 80nM, 85nM, 90nM, 95nM or 100nM. The induction differentiation treatment adopted in the present application is a routine laboratory step, and the detailed steps are not described here. The scheme of the present application is based on conventional steps. In the differentiation stage, a Src inhibitor (such as UM-164, etc.) containing a first predetermined concentration is used for differentiation culture. The results show that after the addition of UM-164, the number of differentiated cells is significantly increased, and the phenotype of the cells is also significantly improved.

[0009] Exemplarily, cell differentiation induction hematopoietic stem cells (iHSC) are used to induce differentiation of induced natural killer cells (iNK), and the induction differentiation method is selected from plating or suspension culture. During the induced differentiation culture process, UM-164 (1nM-100nM) is added, and the number of differentiated iNK cells and the proportion of iNK cells with CD56+ phenotype in all cells are effectively increased.

[0010] In some examples of the present application, the aforementioned differentiation medium is selected from NK differentiation medium. The inventors of the present application used different NK differentiation mediums (such as NK differentiation medium 1, NK differentiation medium 2 (5% Knockout serum replacement (KSR) added to NK differentiation medium 1), NK differentiation medium 3 (GTT-551H3 medium containing 5% fetal bovine serum, 1000 IU / mL IL-2 and 10 ng / mL IL-15)) to induce differentiation culture, and the amount of cells obtained by adding UM164 was significantly increased.

[0011] In some examples of the present application, the aforementioned NK differentiation medium includes: 1 nM-10 nM IL3, optionally 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM or 10 nM.

[0012] In some examples of the present application, the aforementioned differentiation stage includes: differentiation culture day 1 to day 21. It has been verified that adding UM-164 from day 1 to day 21 of induced differentiation culture can effectively increase the number of differentiated cells obtained and improve the cell phenotype of differentiated cells. At the end of differentiation, adding UM-164 not only fails to improve differentiation efficiency, but also causes cell apoptosis.

[0013] In some examples of the present application, the aforementioned Src inhibitor is used to increase the proportion of cells with CD56+ phenotype to all cells by more than 30%, 35%, 40%, 45%, 50% or 55%. It has been verified that during the induction of iNK cell differentiation, the use of Src inhibitors (such as UM-164, etc.) effectively increases the proportion of cells with CD56+ phenotype to all cells.

[0014] In some examples of the present application, the aforementioned cells to be differentiated are selected from induced hematopoietic stem cells. It should be understood that the aforementioned cells to be differentiated are only listed, not exhaustive. The concept of using Src inhibitors (such as UM-164, etc.) to induce differentiation of cells to be differentiated to increase the number of differentiated cells and the phenotype of differentiated cells is within the scope of protection of the present application.

[0015] In the third aspect, the present application proposes a method for increasing the number of cell amplification. According to an embodiment of the present application, the aforementioned method includes: using an amplification medium containing a Src inhibitor to amplify the cells to be amplified. Using an amplification medium containing a Src inhibitor to amplify the cells to be amplified can effectively improve the purity, survival rate and number of the cells to be amplified, and also has a significant effect on improving the cell phenotype.

[0016] In some examples of the present application, the aforementioned Src inhibitor is selected from UM-164 or its analogs. It has been verified that among the Src inhibitors, using an expansion medium containing UM-164 or its analogs to amplify the cells to be amplified can effectively improve the purity, survival rate and number of the cells to be amplified, and also has a significant effect on improving the cell phenotype.

[0017] In some examples of the present application, the aforementioned amplification treatment includes: using an amplification medium containing a second predetermined concentration of UM-164 or its analogues to amplify the cells to be amplified, wherein the second predetermined concentration is selected from 0.01nM-1000nM, and optionally 0.01nM, 0.1nM, 1nM, 2nM, 3nM, 4nM, 5nM, 6nM, 7nM, 8nM, 9nM, 10nM, 15nM, 20nM, 25nM, 26nM, 27nM, 28nM, 29nM, 30nM, 31nM, 32nM, 33nM, 34nM, 35nM, 36nM, 37nM, 38nM, 39nM, 40nM, 41nM, 42nM, 43nM, 44nM, 45nM, 46nM, 47nM, 48nM, 49nM, 50nM, 51nM, 52nM, 53nM, 54nM, 55nM, 56nM, 57nM, 58nM, 59nM, 61nM, 62nM, 63nM, 64nM, 65nM, 66nM, 67nM, 68nM, 69nM, 71nM, 72nM, 73nM, 74nM, 75nM, 76nM, 77nM, 78nM, 79nM, 80nM, 81nM, 82nM, 83nM, 84nM, 85nM, 86 In some preferred examples of the present application, the second predetermined concentration is selected from 0.5nM-100nM, more preferably 50nM. The amplification process used in the present application is a routine laboratory step, and detailed steps are not described herein. The scheme of the present application is based on conventional steps. In the amplification stage, a second predetermined concentration of Src inhibitor (such as UM-164, etc.) is used for amplification culture. The results show that after the addition of UM-164, the purity, survival rate and number of amplified cells are significantly improved, and it also has a significant effect on the improvement of cell phenotype.

[0018] In some examples of the present application, the aforementioned expansion medium is selected from GTT-551H3 medium.

[0019] In some examples of the present application, the aforementioned GTT-551H3 culture medium includes at least one of fetal bovine serum, IL-2 or IL-15.

[0020] In some examples of the present application, the concentration of the aforementioned IL-2 in the aforementioned GTT-551H3 culture medium is 800IU / mL-1200IU / mL, optionally 800IU / mL, 900IU / mL, 1000IU / mL, 1100IU / mL or 1200IU / mL. In some preferred examples of the present application, the concentration of the aforementioned IL-2 in the aforementioned GTT-551H3 culture medium is 1000IU / mL. This concentration of interleukin factors can maintain the growth of NK cells.

[0021] In some examples of the present application, the concentration of the aforementioned IL-15 in the aforementioned GTT-551H3 culture medium is 8IU / mL-12IU / mL, optionally 8IU / mL, 9IU / mL, 10IU / mL, 11IU / mL or 12IU / mL. In some preferred examples of the present application, the concentration of the aforementioned IL-15 in the aforementioned GTT-551H3 culture medium is 10IU / mL. This concentration of interleukin factors can maintain the growth of NK cells.

[0022] In some examples of the present application, the aforementioned Src inhibitor is used to increase the proportion of cells with CD337+ or CD335+ phenotype to all cells by more than 5%, 10%, 15%, 20%, 25% or 30%. It has been verified that during the expansion of NK cells, the use of Src inhibitors (such as UM-164, etc.) effectively increases the proportion of cells with CD337+ or CD335+ phenotype to all cells.

[0023] In some examples of the present application, the aforementioned cells to be expanded are selected from NK cells.

[0024] In some examples of the present application, the aforementioned NK cells include at least one selected from peripheral blood NK cells (PBNK), induced pluripotent cells (iPSC) derived NK cells, umbilical cord blood NK cells or NK-92 cells. It should be understood that the aforementioned cells to be amplified are only listed, not exhaustive. The use of Src inhibitors (such as UM-164, etc.) to amplify the cells to be amplified to increase the number of cells to be amplified and the concept of amplifying the cell phenotype is within the scope of protection of the present application.

[0025] In a fourth aspect, the present application proposes a composition for increasing the number of cell differentiation or cell expansion. According to an embodiment of the present application, the aforementioned composition includes a Src inhibitor. The aforementioned composition is used to induce cell differentiation or cell expansion culture, and can effectively increase the number of cell differentiation or cell expansion.

[0026] In some examples of the present application, the aforementioned Src inhibitor is selected from at least one of Dasatinib, Ouercetin, UM-164, KX2-391, KX1-004 or its analogs. In some preferred examples of the present application, the aforementioned Src inhibitor is selected from UM-164 or its analogs.

[0027] In some examples of the present application, the aforementioned composition further includes: at least one of differentiation medium, expansion medium, fetal bovine serum, IL-2 or IL-15.

[0028] In some examples of the present application, the concentration of the aforementioned Src inhibitor in the aforementioned differentiation medium is 1nM-100nM, optionally 1nM, 2nM, 3nM, 4nM, 5nM, 6nM, 7nM, 8nM, 9nM, 10nM, 15nM, 20nM, 25nM, 30nM, 35nM, 40nM, 45nM, 50nM, 55nM, 60nM, 65nM, 70nM, 75nM, 80nM, 85nM, 90nM, 95nM or 100nM.

[0029] In some examples of the present application, the aforementioned differentiation medium is selected from NK differentiation medium.

[0030] In some examples of the present application, the NK differentiation medium includes: 1 nM-10 nM IL3, optionally 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM or 10 nM.

[0031] In some examples of the present application, the concentration of the aforementioned Src inhibitor in the aforementioned expansion medium is 0.01nM-1000nM, optionally 0.01nM, 0.1nM, 1nM, 2nM, 3nM, 4nM, 5nM, 6nM, 7nM, 8nM, 9nM, 10nM, 15nM, 20nM, 25nM, 30nM, 35nM, 40nM, 45nM, 50nM, 55nM, 60nM, 65nM, 70nM, 75nM, 80nM, 85nM, 90nM, 95nM, 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM or 1000nM. In some preferred examples of the present application, the concentration of the aforementioned Src inhibitor in the aforementioned expansion medium is 0.5 nM-100 nM, more preferably 50 nM.

[0032] In some preferred examples of the present application, the aforementioned expansion medium is selected from GTT-551H3 medium.

[0033] In some examples of the present application, the concentration of the aforementioned IL-2 in the aforementioned GTT-551H3 culture medium is 800IU / mL-1200IU / mL, optionally 800IU / mL, 900IU / mL, 1000IU / mL, 1100IU / mL or 1200IU / mL. In some preferred examples of the present application, the concentration of the aforementioned IL-2 in the aforementioned GTT-551H3 culture medium is 1000IU / mL.

[0034] In some examples of the present application, the concentration of the aforementioned IL-15 in the aforementioned GTT-551H3 culture medium is 8IU / mL-12IU / mL, optionally 8IU / mL, 9IU / mL, 10IU / mL, 11IU / mL or 12IU / mL. In some preferred examples of the present application, the concentration of the aforementioned IL-15 in the aforementioned GTT-551H3 culture medium is 10IU / mL.

[0035] It can be understood that the characteristics and beneficial effects of other aspects are also applicable to this aspect and will not be elaborated here.

[0036] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0038] Figure 1 A schematic diagram of the results of increasing the amount of iNK differentiated cells by adding UM164 at D20 provided in the examples of the present application;

[0039] Figure 2 A schematic diagram showing the effect of adding UM164 to different differentiation mediums on the number of differentiated cells during iNK differentiation provided in the examples of the present application;

[0040] Figure 3 A schematic diagram showing the results of increasing the amount of iNK differentiated cells when UM164 provided in the examples of the present application is used in combination with different differentiation mediums;

[0041] Figure 4 / Figure 5 A schematic diagram of the results of adding UM164 on Day 10, Day 18, and Day 21 to increase the number of iNK differentiation provided in the examples of the present application;

[0042] Figure 6 A schematic diagram of iNK differentiation culture results obtained by adding UM164 to Culture Medium 1 provided in the embodiments of the present application;

[0043] Figure 7A schematic diagram of iNK differentiation culture results obtained by adding UM164 to the culture medium 2 provided in the examples of the present application;

[0044] Figure 8 A schematic diagram of iNK differentiation culture results obtained by adding UM164 to the culture medium 3 provided in the examples of the present application;

[0045] Fig. 9 A schematic diagram of the results of increasing the amount of iNK differentiated cells by UM164 at different concentrations (0.005 μM-0.1 μM) provided in the examples of this application;

[0046] Fig.10 This is a schematic diagram of the results of different concentrations (0.005 μM-0.1 μM) of UM164 provided in the examples of the present application for improving the CD56 positive rate of iNK differentiation;

[0047] Fig.11 This is a schematic diagram of the results of improving the CD56 positive rate of iNK differentiation in a shake flask system by 0.05 μM UM164 provided in the examples of the present application;

[0048] Fig.12 This is a schematic diagram of the results of the effect of 0.05 μM UM164 provided in the examples of the present application on the amount of iNK differentiated cells in the shake flask system;

[0049] Fig.13 A schematic diagram of the results of promoting the total cell multiplier of iNK cells and the NK cell multiplier of low-dose UM164 provided in the examples of the present application;

[0050] Fig.14 A schematic diagram showing the effect of using UM164 at the end of differentiation on differentiation efficiency provided in the examples of the present application;

[0051] Fig.15 A schematic diagram of the comparison results of iNK cell morphology provided in the examples of the present application;

[0052] Fig.16 A schematic diagram of the NK flow phenotype results of the Vehicle group provided in the examples of the present application;

[0053] Fig.17 A schematic diagram of the flow phenotyping results of iNK cells in the experimental group (UM-164) provided in the examples of this application;

[0054] Fig.18 A schematic diagram of the iNK growth curve results provided in the examples of the present application;

[0055] Fig.19A schematic diagram of iNK amplification results provided in the examples of the present application;

[0056] Fig. 20 A schematic diagram of iNK activity change results provided in the examples of the present application;

[0057] Fig.21 A schematic diagram of the growth curve results of iNK cells at different concentrations of UM-164 provided in the examples of the present application;

[0058] Fig. 22 A schematic diagram of the flow phenotyping results of iNK cells after UM-164 treatment provided in the examples of the present application;

[0059] Fig.23 A schematic diagram of the growth curve results of iNK in different concentrations of UM-164 provided in the examples of the present application;

[0060] Fig.24 A schematic diagram showing the summary results of the amplification multiples of iNK in different concentrations of UM-164 provided in the examples of the present application;

[0061] Fig.25 A schematic diagram of the activity curve results of iNK at different concentrations of UM-164 provided in the examples of the present application;

[0062] Fig.26 A schematic diagram of the amplification curve results of PBNK after UM-164 treatment provided in the examples of the present application;

[0063] Fig. 27 This is a schematic diagram of the effects of Dasatinib treatment on iNK cell proliferation provided in the examples of the present application. DETAILED DESCRIPTION

[0064] Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0065] In the process of describing the present invention, the relevant terms in this document are explained and illustrated. These explanations and illustrations are only for the convenience of understanding of the scheme and cannot be regarded as limitations on the protection scheme of the present invention.

[0066] 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.

[0067] 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.

[0068] The embodiments of the present application will be described in more detail below, and the examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, and should not be construed as limiting the present application. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained commercially.

[0069] Example 1: UM164 promotes cell differentiation

[0070] This example determines that UM164 promotes cell differentiation through the following test examples.

[0071] Commonly used cell culture reagents: cell culture grade DPBS, iHSC differentiation medium (manufacturer: CIB, catalog number: iHSCM-001), NK differentiation medium 1 (culture medium1, manufacturer: CIB, catalog number: iNKM-001), flow cytometry reagents;

[0072] Commonly used consumables: 15mL centrifuge tubes, 50mL centrifuge tubes, pipettes (10mL, 25mL), Aggrewell culture plates, sterile sample loading tanks, cell culture T bottles.

[0073] Experimental steps:

[0074] 1) When the confluence of induced pluripotent stem cells (iPSC) reached 80-90%, digestion and plating were performed; after counting, cells were taken out of the 3D culture system and cultured with iHSC differentiation medium. On the tenth day (D10), the cell differentiation into induced hematopoietic stem cells (iHSC) was completed, and the cell balls and culture medium were collected into a 50 mL centrifuge tube with a 1 mL pipette; after inversion and mixing, single cells were sampled and counted, and 5×10 5 -10×10 5 The cells were subjected to CD34 / CD43 flow cytometry detection;

[0075] 2) The remaining cells can be induced into iNK cells by plating or suspension culture;

[0076] 3) On the first day of differentiation culture, take 1x10 6The cells were resuspended in 5-10 mL of NK differentiation medium containing 5 ng / mL IL3 and placed in a T25 flask for mixing. The cells were observed under a microscope. The presence of a few aggregated cells was normal. The cells were then placed in a 5% CO 2 , static culture was carried out in a 37℃ constant temperature and humidity incubator;

[0077] 4) After 48 hours (D12), observe the cell status and add 5-10 mL of culture medium according to the cell proliferation situation;

[0078] 5) On day 5 of differentiation (D16), add 10-20 mL of NK differentiation medium;

[0079] 6) On day 7 of differentiation (D18), gently shake the cell culture flask to mix the cells evenly and count them. Take out 80%-100% of the cell suspension and place it in a centrifuge tube. Centrifuge at 500×g for 8 minutes and remove the supernatant. Resuspend the precipitated cells in 1 mL of NK differentiation medium and return them to the original culture flask. 5 / mL of cell density supplemented with NK differentiation medium; after gently mixing, the cells were placed in 5% CO 2 , static culture was carried out in a 37℃ constant temperature and humidity incubator;

[0080] 7) On day 11 of differentiation (D21), the cell flask was taken out and the medium was added to the flask in equal amounts according to the volume of medium in the flask. After the rehydration was completed, the flask was placed in a 5% CO 2 , cultured statically in a 37°C constant temperature and humidity incubator;

[0081] 8) On day 18 of differentiation (D28), gently shake the cell culture flask to mix the cells evenly and count them. Take out 80%-100% of the cell suspension and place it in a centrifuge tube. Centrifuge at 500×g for 8 minutes and remove the supernatant. Resuspend the precipitated cells in 1 mL of NK differentiation medium and return them to the original culture flask. 5 / mL of cell density supplemented with NK differentiation medium; after gently mixing, the cells were placed in 5% CO 2 , static culture was carried out in a 37℃ constant temperature and humidity incubator;

[0082] 9) At 20 days of differentiation (D30), the cell flask was taken out and the medium was added to the flask in equal amounts according to the volume of medium in the flask. After the rehydration was completed, the flask was placed in a 5% CO 2 , cultured statically in a 37°C constant temperature and humidity incubator;

[0083] 10) At D33+, the cells were taken out and the differentiation was observed under a microscope. The original iHSC differentiation morphology changed from round to hippocampal or sickle-shaped. At this time, the NK cells were immature NK cells and could be detected by flow cytometry.

[0084] Test Example 1

[0085] On D20, two equal portions of the iNK cells were taken and cultured in NK differentiation medium 1. 0.1 μM UM164 was added to one of the portions. After mixing, the cells were placed in an incubator and cultured until Day 30. Each time the culture medium was added or the medium was changed, an equal amount of UM164 was added. On Day 30, the cells were collected and counted with trypan blue. 5×10 5 Cell flow cytometry staining. Figure 1 As shown, after adding UM164, the amount of iNK obtained on Day 30 was 1.7 times that of the control group (Control).

[0086] Test Example 2

[0087] During the iNK differentiation process, the same amount of iNK as the control group was taken out and added to the induction medium containing UM164 for culture. The culture mediums were NK differentiation medium 1, NK differentiation medium 2 (culture medium 2, 5% Knockout serum replacement (KSR) added to NK differentiation medium 1), and NK differentiation medium 3 (culture medium 3, GTT-551H3 medium containing 5% fetal bovine serum, 1000 IU / mL IL-2 and 10 ng / mL IL-15). Each time the culture medium was supplemented or the medium was changed, an equal amount of UM164 was added. Figure 2 and Figure 3 It can be seen that the amount of iNK cells induced in different culture media with the addition of UM164 was significantly higher than that of the control group;

[0088] Test Example 3

[0089] During the iNK differentiation process, 4 groups of iNK cells with the same amount of cells were taken out and UM164 was added on Day 10, Day 18, and Day 21 of induction. The same amount of UM164 was added each time the culture medium was added or the medium was changed. Figure 4 and Figure 5 As shown, the amount of cells obtained in the groups supplemented with UM164 was 1.7-4 times that of the control group. Figure 6 , Figure 7 and Figure 8 As shown, when UM164 was added to NK differentiation medium 1, NK differentiation medium 2 (culture medium 2, 5% Knockout serum replacement (KSR) was added to NK differentiation medium 1), and NK differentiation medium 3 (culture medium 3, GTT-551H3 medium containing 5% fetal bovine serum, 1000 IU / mL IL-2 and 10 ng / mL IL-15), the iNK phenotype (CD56+) was significantly improved.

[0090] During the iNK differentiation process, four equal number of cells were taken and UM164 was added to three of the four portions at 0.005 μM, 0.05 μM, and 0.1 μM, respectively. Each time the culture medium was added or the medium was changed, an equal amount of UM164 was added. Fig. 9 and Fig.10 As shown, 0.005 μM, 0.05 μM and 0.1 μM UM164 all improved the cell yield and phenotype to varying degrees.

[0091] At D10, two iHSCs with the same cell volume were taken and the cells were differentiated in 125 mL shaking flasks on a shaker. 0.05 μM UM164 was added to one of the flasks. The rest of the differentiation steps were the same as those in Test Example 1. Each time the culture medium was added or the medium was changed, an equal amount of UM164 was added. Fig.11 and Fig.12 As shown, in the system without adding UM164, the phenotype expression of CD56 was extremely low, while in the system with adding UM164, CD56 increased to 57.85%, and the cell proliferation multiples reached 3.43 times that of the control group.

[0092] Test Example 4

[0093] like Fig.13 As shown in the figure, the addition of 1-2nM UM164 at the initial differentiation stage (Day 1) can slightly increase the CD56 positivity of iNK phenotype in the differentiation stage (from 75.49% to 78.72% and 76.1%); while the activation receptor CD69 positivity and cell viability did not change significantly. However, in the differentiation stage, 1nM and 2nM UM164 can significantly increase the total cell multiplication in the differentiation stage from 3985 times to 6050 times and 6320 times, respectively, and the total NK cell multiplication from 3008 times to 4763 times and 4080 times.

[0094] Test Example 5

[0095] like Fig.14 As shown in the figure, 10 μM UM164 was added at the end of differentiation (Day 28), and differentiation detection was performed on D30. The phenotype of differentiated iNK did not change significantly, but the viability decreased. The total differentiation multiple decreased from 2535 to 1538, and the total iNK multiple decreased from 1452 to 889. This indicates that adding high concentrations of UM164 at the end of differentiation cannot quickly improve differentiation efficiency and may also lead to apoptosis of differentiated cells.

[0096] Example 2: UM-164 promotes iNK cell expansion

[0097] This example determines that UM-164 promotes iNK cell expansion through the following experimental steps.

[0098] 1. Culture medium preparation

[0099] 5% fetal bovine serum (FBS) was added to the GTT-551H3 culture medium, followed by 1000 IU / mL IL-2 and 10 ng / mL IL-15. 0.1 μM UM-164 was added to the experimental group (UM-164). An equal amount of DMSO was added to the culture medium of the control group (Vehicle).

[0100] 2. Fluorescence activated cell sorting (FACS) identification

[0101] Take 1*10 6 The cells to be tested were tested for CD56+CD3, and 2.1*10 6 NK cells were expanded and cultured.

[0102] 3. Resuscitation of antigen presenting cells APC (mIL-21 / 4-1BBL-K562)

[0103] APC was taken out of liquid nitrogen, transferred to a 37°C water bath for rapid dissolution, and then the outer surface was wiped with alcohol and transferred to a biosafety cabinet. APC was quickly transferred to D-PBS and then centrifuged to remove the supernatant.

[0104] 4. Expansion and culture of induced NK cells (iNK)

[0105] The differentiated iNK cells were analyzed by flow cytometry to identify the proportion of CD56+CD3- iNK cells in the cell population. Then, 2.1*10 6 iNK cells were inoculated into T25 flasks. Then 3 mL of complete medium for the experimental group and the control group were added respectively. Then 3*10 6 After mixing well, place APC cells at 37°C with 5% CO 2 cultured in an incubator.

[0106] 5. Cell expansion by rehydration

[0107] On the third day (D3), slowly remove the cells from the incubator and add 3 mL of experimental group culture medium and 3 mL of control group culture medium respectively. When adding culture medium, slowly add it along the top of the culture bottle. The culture medium should be restored to room temperature in a light-proof environment before use. After adding the culture medium, put the T25 culture bottle back into the incubator for continued culture. Be careful to avoid violent shaking during the whole process.

[0108] On the sixth day (D6), the cells were taken out of the incubator, the cell culture medium was mixed evenly with a pipette, and then a sample was taken and stained with trypan blue and counted with a cell counter.6 Cells were rehydrated at a density of 10 cells / mL. The cells were aspirated from the T25 flask and transferred to a new T75 flask. A small amount of fresh culture medium was taken to rinse the bottom of the T25 flask. All the suspended cells were collected in a new T75 flask. Then, sufficient amounts of fresh culture medium for the experimental group and the control group were added according to the counting results. The cells were mixed by the cross method and returned to the incubator for continued culture. After that, the cells were taken out every 2 days for mixing and counting, and fresh culture medium was added according to the counting results. The cell density was controlled to be maintained at 0.7*10 after rehydration. 6 The cells were cultured at a density of 10 cells / mL until the 24th day.

[0109] On day 24 (D24), the cells were taken out of the incubator and the cell morphology was observed under a microscope ( Fig.15 ), then mixed evenly and counted, and then sufficient cells from the experimental group and the control group were taken out for flow cytometry detection, and the markers detected were CD56, CD3, CD16, and CD314.

[0110] Results Analysis

[0111] like Fig.16 , Fig.17 As shown in the figure, both the control group and the experimental group could amplify iNK cells with high purity. Fig.18 , Fig.19 As shown, the number of expanded NK cells in the experimental group far exceeded that in the control group; Fig.19 , Fig. 20 As shown in the figure, the total cell proliferation rate and cell viability of the experimental group were better than those of the control group. It can be seen that the addition of UM-164 can significantly promote the proliferation of NK cells.

[0112] Example 3: Effects of different concentrations of UM-164 on the expansion capacity and phenotype of iNK cells

[0113] In this example, the concentration of UM-164 was optimized through the following steps.

[0114] 1. Culture medium preparation

[0115] 5% FBS was added to the GTT-551H3 culture medium, followed by 1000 IU / mL IL-2 and 10 ng / mL IL-15. 0-750 nM UM-164 was added to the experimental group (UM-164). An equal amount of DMSO was added to the culture medium of the control group (Vehicle).

[0116] 2. FACS identification

[0117] Take 1*10 6 The cells to be tested were tested for CD56+CD3, and 2.1*10 6NK cells were expanded and cultured.

[0118] 3. Recovery of APC (mIL-21 / 4-1BBL-K562)

[0119] APC was taken out of liquid nitrogen, transferred to a 37°C water bath for rapid dissolution, and then the outer surface was wiped with alcohol and transferred to a biosafety cabinet. APC was quickly transferred to D-PBS and then centrifuged to remove the supernatant.

[0120] 4. iNK cell expansion and culture

[0121] The differentiated iNK cells were analyzed by flow cytometry to identify the proportion of CD56+CD3- iNK cells in the cell population. Then, 2.1*10 6 iNK cells were inoculated into T25 flasks. Then 3 mL of complete medium for the experimental group and the control group were added respectively. Then 3*10 6 After mixing well, place APC cells at 37°C with 5% CO 2 cultured in an incubator.

[0122] 5. Cell expansion by rehydration

[0123] On the third day (D3), slowly remove the cells from the incubator and add 3 mL of experimental group culture medium and 3 mL of control group culture medium respectively. When adding culture medium, slowly add it along the top of the culture bottle. The culture medium should be restored to room temperature in a light-proof environment before use. After adding the culture medium, put the T25 culture bottle back into the incubator for continued culture. Be careful to avoid violent shaking during the whole process.

[0124] On the sixth day (D6), the cells were taken out of the incubator, the cell culture medium was mixed evenly with a pipette, and then a sample was taken and stained with trypan blue and counted with a cell counter. 6 Cells were rehydrated at a density of 10 cells / mL. The cells were aspirated from the T25 flask and transferred to a new T75 flask. A small amount of fresh culture medium was taken to rinse the bottom of the T25 flask. All the suspended cells were collected in a new T75 flask. Then, sufficient amounts of fresh culture medium for the experimental group and the control group were added according to the counting results. The cells were mixed by the cross method and returned to the incubator for continued culture. After that, the cells were taken out every 2 days for mixing and counting, and fresh culture medium was added according to the counting results. The cell density was controlled to be maintained at 0.7*10 after rehydration. 6 The cells were cultured at a density of 10 cells / mL until day 16 (D16).

[0125] On D16, the cells were taken out of the incubator, mixed evenly and counted, and then sufficient cells from the experimental group and the control group were taken out for flow cytometry detection.

[0126] Results Analysis

[0127] like Fig.21 As shown in the figure, iNK cells showed different degrees of expansion advantages on different concentrations of UM-164. Its expansion ability further increased with the decrease of concentration. It showed the highest proliferation ability under the concentration of 50nM. Fig. 22 As shown, in flow cytometry, the expression level of CD337 / CD335 was higher after treatment with UM-164.

[0128] Example 4: Screening of the optimal concentration of UM-164

[0129] In this example, the optimal concentration of UM-164 for amplifying iNK cells was obtained through the following steps.

[0130] 1. Culture medium preparation

[0131] 5% FBS was added to the GTT-551H3 culture medium, followed by 1000 IU / mL IL-2 and 10 ng / mL IL-15. 0-50 nM UM-164 was added to the experimental group (UM-164). An equal amount of DMSO was added to the culture medium of the control group (Vehicle).

[0132] 2. FACS identification; take 1*10 6 The cells to be tested were tested for CD56+CD3, and 2.1*10 6 NK cells were expanded and cultured.

[0133] 3. Recovery of APC (mIL-21 / 4-1BBL-K562)

[0134] The APC was taken out of liquid nitrogen, transferred to a 37°C water bath for rapid dissolution, and then the outer surface was wiped with alcohol and transferred to a biosafety cabinet. The APC was quickly transferred to D-PBS and then centrifuged to remove the supernatant.

[0135] 4. iNK cell expansion and culture

[0136] The differentiated iNK cells were analyzed by flow cytometry to identify the proportion of CD56+CD3- iNK cells in the cell population. Then, 2.1*10 6 iNK cells were inoculated into T25 flasks. Then 3 mL of complete medium for the experimental group and the control group were added respectively. Then 3*10 6 After mixing well, place APC cells at 37°C with 5% CO 2 cultured in an incubator.

[0137] 5. Cell expansion by rehydration

[0138] On the third day (D3), slowly remove the cells from the incubator and add 3 mL of experimental group culture medium and 3 mL of control group culture medium respectively. When adding culture medium, slowly add it along the top of the culture bottle. The culture medium should be restored to room temperature in a light-proof environment before use. After adding the culture medium, put the T25 culture bottle back into the incubator for continued culture. Be careful to avoid violent shaking during the whole process.

[0139] On the sixth day (D6), the cells were taken out of the incubator, the cell culture medium was mixed evenly with a pipette, and then a sample was taken and stained with trypan blue and counted with a cell counter. 6 Cells were rehydrated at a density of 10 cells / mL. The cells were aspirated from the T25 flask and transferred to a new T75 flask. A small amount of fresh culture medium was taken to rinse the bottom of the T25 flask. All the suspended cells were collected in a new T75 flask. Then, sufficient amounts of fresh culture medium for the experimental group and the control group were added according to the counting results. The cells were mixed by the cross method and returned to the incubator for continued culture. After that, the cells were taken out every 2 days for mixing and counting, and fresh culture medium was added according to the counting results. The cell density was controlled to be maintained at 0.7*10 after rehydration. 6 The cells were cultured at a density of 10 cells / mL until day 12 (D12).

[0140] like Fig.23 As shown in the figure, further reducing the concentration of UM-164 did not show any better ability to promote amplification, indicating that 50 nM is the optimal concentration. Fig.24 As shown in the figure, all statistical results also showed that the expansion capacity of iNK was the highest at a concentration of 50 nM, which was significantly different from the control group without adding UM-164. Fig.25 As shown, the activity of iNK was the highest at a concentration of 50 nM.

[0141] Example 5: Effectiveness of UM-164 on the expansion of NK cells from various sources

[0142] This example verifies the effectiveness of UM-164 on the expansion of NK cells from various sources through the following steps.

[0143] 1. Culture medium preparation

[0144] 5% FBS was added to the GTT-551H3 culture medium, followed by 1000 IU / mL IL-2 and 10 ng / mL IL-15. 50 nM UM-164 was added to the experimental group (UM-164). An equal amount of DMSO was added to the culture medium of the control group (Vehicle).

[0145] 2. Recovery of APC (mIL-21 / 4-1BBL-K562)

[0146] APC was taken out of liquid nitrogen, transferred to a 37°C water bath for rapid dissolution, and then the outer surface was wiped with alcohol and transferred to a biosafety cabinet. APC was quickly transferred to D-PBS and then centrifuged to remove the supernatant.

[0147] 3. Expansion and culture of PBNK

[0148] The PBMCs in the blood were separated with lymphocyte separation fluid, and then the cells were counted. 1*10 6 PBMC cells were mixed with APC at a ratio of 1:1 and then inoculated into T25 flasks. 3 mL of complete medium for the experimental group and complete medium for the control group were added respectively, mixed well and placed at 37°C with 5% CO 2 cultured in an incubator.

[0149] 4. Cell expansion by rehydration

[0150] On the third day (D3), slowly remove the cells from the incubator and add 3 mL of experimental group culture medium and 3 mL of control group culture medium respectively. When adding culture medium, slowly add it along the top of the culture bottle. The culture medium should be restored to room temperature in a light-proof environment before use. After adding the culture medium, put the T25 culture bottle back into the incubator for continued culture. Be careful to avoid violent shaking during the whole process.

[0151] On the sixth day (D6), the cells were taken out of the incubator, the cell culture medium was mixed evenly with a pipette, and then a sample was taken and stained with trypan blue and counted with a cell counter. 6 Cells were rehydrated at a density of 10 cells / mL. The cells were aspirated from the T25 flask and transferred to a new T75 flask. A small amount of fresh culture medium was taken to rinse the bottom of the T25 flask, and all the suspended cells were collected in a new T75 flask. Then, sufficient fresh culture medium for the experimental group and the control group was added according to the counting results. The cells were mixed by the cross method and returned to the incubator for continued culture. After that, the cells were taken out every 2 days for mixing and counting, and fresh culture medium was added according to the counting results. The cell density was controlled to be maintained at 1*10 after rehydration. 6 The cells were cultured at a density of 10 cells / mL until day 14 (D14).

[0152] like Fig.26 As shown, UM-164 also showed an increase in the expansion capacity of PBNK, indicating that UM-164 can not only improve the expansion capacity of iPSC-NK, but also improve the expansion capacity of PBNK.

[0153] Example 6: Application of other Src inhibitors in cell expansion

[0154] 1. Culture medium preparation

[0155] 5% FBS was added to the GTT-551H3 culture medium, followed by 1000 IU / mL IL-2 and 10 ng / mL IL-15. The experimental group (Dasatinib) was supplemented with 0-50 nM Dasatinib. The control group (Vehicle) culture medium was supplemented with an equal amount of DMSO.

[0156] 2. iNK cell expansion and culture

[0157] The differentiated iNK cells were analyzed by flow cytometry to identify the proportion of CD56+CD3- iNK cells in the cell population. Then, 2.1*10 6 iNK cells were inoculated into T25 flasks. Then 3 mL of complete medium for the experimental group and the control group were added respectively. The cells were placed at 37°C with 5% CO 2 cultured in an incubator.

[0158] 3. Cell expansion by rehydration

[0159] On the third day (D3), slowly remove the cells from the incubator and add 3 mL of experimental group culture medium and 3 mL of control group culture medium respectively. When adding culture medium, slowly add it along the top of the culture bottle. The culture medium should be restored to room temperature in a light-proof environment before use. After adding the culture medium, put the T25 culture bottle back into the incubator for continued culture. Be careful to avoid violent shaking during the whole process.

[0160] On the sixth day (D6), the cells were taken out of the incubator, the cell culture medium was mixed evenly with a pipette, and then a sample was taken and stained with trypan blue and counted with a cell counter. 6 Cells were rehydrated at a density of 10 cells / mL. The cells were aspirated from the T25 flask and transferred to a new T75 flask. A small amount of fresh culture medium was taken to rinse the bottom of the T25 flask. All the suspended cells were collected in a new T75 flask. Then, sufficient amounts of fresh culture medium for the experimental group and the control group were added according to the counting results. The cells were mixed by the cross method and returned to the incubator for continued culture. After that, the cells were taken out every 2 days for mixing and counting, and fresh culture medium was added according to the counting results. The cell density was controlled to be maintained at 0.7*10 after rehydration. 6 The cells were cultured at a density of 10 cells / mL until day 12 (D12).

[0161] like Fig. 27As shown, the proliferation ability of iNK cells was not enhanced after the use of Dasatinib, indicating that the use of Dasatinib to treat iNK cells does not increase the proliferation ability of iNK cells.

[0162] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0163] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means 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 representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0164] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. Application of Src inhibitors in increasing the number of cell differentiation or cell proliferation.

2. The use according to claim 1, characterized in that: The Src inhibitor is selected from at least one of Dasatinib, Ouercetin, UM-164, KX2-391, KX1-004 or the like; Preferably, the Src inhibitor is selected from UM-164 or its analogs.

3. A method for increasing the number of cell differentiation, characterized in that: include: The cells to be differentiated were induced to differentiate using differentiation medium containing Src inhibitor.

4. The method according to claim 3, characterized in that The Src inhibitor is selected from at least one of Dasatinib, Ouercetin, UM-164, KX2-391, KX1-004 or the like; Preferably, the Src inhibitor is selected from UM-164 or its analogs.

5. The method according to claim 4, characterized in that The differentiation induction treatment comprises: In the differentiation stage, the cells to be differentiated are induced to differentiate using a differentiation medium containing a first predetermined concentration of a Src inhibitor, wherein the first predetermined concentration is selected from 1 nM to 100 nM; Preferably, the differentiation medium is selected from NK differentiation medium; Preferably, the NK differentiation medium comprises: 1 nM-10 nM IL3.

6. The method according to claim 5, characterized in that The differentiation stage includes: day 1 to day 21 of differentiation culture.

7. The method according to any one of claims 3 to 6, characterized in that: The use of the Src inhibitor increases the proportion of cells with CD56+ phenotype to more than 30%, 35%, 40%, 45%, 50% or 55% of all cells.

8. The method according to any one of claims 3 to 6, characterized in that: The cells to be differentiated are selected from induced hematopoietic stem cells.

9. A method for increasing the number of cell proliferation, characterized in that: include: The cells to be expanded are expanded using an expansion medium containing a Src inhibitor.

10. The method according to claim 9, characterized in that The Src inhibitor is selected from UM-164 or its analogs.

11. The method according to claim 10, characterized in that The amplification process comprises: Amplifying the cells to be amplified using an amplification medium containing a second predetermined concentration of UM-164 or an analog thereof, wherein the second predetermined concentration is selected from 0.01 nM to 1000 nM; Preferably, the predetermined concentration is selected from 0.5 nM-100 nM, more preferably 50 nM.

12. The method according to claim 11, characterized in that The expansion medium is selected from GTT-551H3 medium; Optionally, the GTT-551H3 culture medium includes at least one of fetal bovine serum, IL-2 or IL-15; Optionally, the concentration of IL-2 in the GTT-551H3 culture medium is 800 IU / mL-1200 IU / mL, preferably 1000 IU / mL; Optionally, the concentration of IL-15 in the GTT-551H3 culture medium is 8 IU / mL-12 IU / mL, preferably 10 IU / mL.

13. The method according to claim 12, characterized in that The use of the Src inhibitor increases the proportion of cells with CD337+ or CD335+ phenotype to more than 5%, 10%, 15%, 20%, 25% or 30% of all cells.

14. The method according to any one of claims 9 to 13, characterized in that: The cells to be expanded are selected from NK cells.

15. The method according to claim 14, characterized in that The NK cells include at least one selected from peripheral blood NK cells, induced pluripotent cell-derived NK cells, umbilical cord blood NK cells or NK-92 cells.

16. A composition for increasing the number of cell differentiation or cell expansion, characterized in that: include: Src inhibitors.

17. The composition according to claim 16, characterized in that The Src inhibitor is selected from at least one of Dasatinib, Ouercetin, UM-164, KX2-391, KX1-004 or the like; Preferably, the Src inhibitor is selected from UM-164 or its analogs; Optionally, the composition further comprises: at least one of differentiation medium, expansion medium, fetal bovine serum, IL-2 or IL-15; Optionally, the concentration of the Src inhibitor in the differentiation medium is 1 nM-100 nM; Preferably, the differentiation medium is selected from NK differentiation medium; Preferably, the NK differentiation medium comprises: 1nM-10nM IL3; Optionally, the concentration of the Src inhibitor in the expansion medium is 0.01 nM-1000 nM, preferably 0.5 nM-100 nM, more preferably 50 nM; Preferably, the expansion medium is selected from GTT-551H3 medium; Optionally, the concentration of IL-2 in the GTT-551H3 culture medium is 800 IU / mL-1200 IU / mL, preferably 1000 IU / mL; Optionally, the concentration of IL-15 in the GTT-551H3 culture medium is 8 IU / mL-12 IU / mL, preferably 10 IU / mL.

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