CN116832062A - Application of small molecule inhibitor drug in enhancing NK cell anti-tumor effect and application thereof - Google Patents
Application of small molecule inhibitor drug in enhancing NK cell anti-tumor effect and application thereof Download PDFInfo
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Abstract
The present invention relates to an anti-tumor formulation comprising NK cells treated with Venetoclax. The invention also relates to a culture medium or a kit for improving the tumor killing activity of NK cells, application of Venetoclax in preparing NK cells with improved tumor killing activity and a method for preparing NK cells with improved tumor killing activity. NK cells cultured by Venetoclax show excellent killing performance while maintaining the high activity of the original NK cells, and can maintain the number and activity thereof in tumor microenvironment, thereby maintaining strong tumor killing activity.
Description
Technical Field
The invention relates to the field of cellular immunotherapy, in particular to application of a small molecule inhibitor drug for enhancing an NK cell anti-tumor effect and application thereof, wherein the small molecule inhibitor drug is Venetoclax, directly activates NK cells and remarkably promotes the effect of NK cell anti-tumor immunotherapy.
Background
Adoptive immune cell therapy is a promising new method for treating blood tumors and solid malignant tumors with limited clinical choices, and is one of the hot spots in the current tumor treatment field. In recent years, chimeric antigen receptor T cells (CAR-T cells) have achieved significant success in the treatment of hematological malignancies. However, the clinical application of the CAR-T cells has adverse reactions such as cytokine storm, neurotoxicity, graft versus host disease and the like, and the treatment effect on the solid tumor is not ideal, so that the clinical application of the CAR-T cells still faces challenges. Natural Killer (NK) cells are a unique group of congenital lymphocytes with the inherent ability to recognize and eliminate virally infected cells and tumor cells. Compared with T cells, NK cells have better safety and can not cause side effects such as cytokine storm, graft versus host disease and the like. In addition, NK cells do not require antigen presentation and are not MHC restricted, and can directly kill tumor cells. NK cells can identify and kill tumors through various identification mechanisms, and the anti-tumor spectrum is wide, so that the NK cells have wide application prospect in anti-tumor treatment and become hot spots in the field of development of cellular immunotherapy. However, one of the challenges in the development of NK cell-based immunotherapy is that NK cells have a weak antitumor activity in vivo, affecting their efficacy in vivo. Therefore, enhancing NK cell antitumor activity is critical for NK cell immune efficacy.
The BCL-2 protein is expressed by the BCL-2 gene coding translation, and is an important anti-apoptosis protein in the BCL-2 family. The research shows that the BCL-2 protein is highly expressed in various tumor cells and is closely related to the drug resistance of the chemotherapeutic drugs. Venetoclax, a specific small molecule inhibitor of BCL-2, in combination with the demethylating drug azacitidine, produces a rapid and sustained response in elderly acute myelogenous leukemia patients not suitable for chemotherapy, and is therefore FDA approved for treatment of acute myelogenous leukemia patients. However, vennetoclax has not been studied intensively in terms of NK cell function and immunotherapy thereof.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a small molecule inhibitor for enhancing the killing activity of NK cells on tumor cells and application thereof.
In order to solve the technical problems, the invention provides the following technical scheme:
in one aspect, the invention provides an anti-tumor formulation characterized in that the formulation comprises Venetoclax treated NK cells.
In some embodiments, the NK cells are derived from cord blood, bone marrow from patients with acute myeloid leukemia, peripheral blood such as normal donor peripheral blood, immortalized NK cell line NK92, iPSC-derived NK cells, or cord blood-derived CD34 + HSPCs induce differentiated NK cells.
In another aspect, the invention provides a culture medium or a kit for improving NK cell tumor killing activity, which is characterized in that the culture medium or the kit comprises a basal medium and Venetoclax, wherein the basal medium is selected from RPIM1640 culture medium, SCGM culture medium, IMDM culture medium and DMEM culture medium.
In some embodiments, the medium or kit further comprises two or more cytokines selected from the group consisting of IL-12, IL-15, IL-2 and IL-18.
In some embodiments, the Venetoclax is present in an amount of 200-1000nM, preferably 200-500nM, 200-600nM, 200-700nM, 200-800nM, 200-900nM, 200-1000nM, 200-1100nM, 200-1200nM, 200-1300nM, 200-1400nM, 200-1500nM, 200-1600nM, 200-1700nM, 200-1800nM, 200-1900nM, more preferably 400nM.
In some embodiments, the cytokines are IL-12 and IL-15, the IL-12 concentration is 5-20ng/mL, preferably 10ng/mL, and the IL-15 concentration is 5-100ng/mL, preferably 50ng/mL.
In another aspect, the invention provides the use of Venetoclax for the preparation of NK cells with increased tumor killing activity.
In another aspect, the invention provides the use of Venetoclx for the preparation of an agent for increasing NK cell tumor killing activity, optionally further comprising two or more cytokines selected from the group consisting of IL-12, IL-15, IL-2 and IL-18.
In another aspect, the invention provides a method of treating a tumor in a subject, the method comprising administering to the subject a therapeutically effective amount of Venetoclax-treated NK cells.
In another aspect, the present invention provides a method for preparing NK cells having improved tumor killing activity, the method comprising culturing the NK cells with the above-described medium and isolating the cultured cells.
In another aspect, the present invention provides a method for increasing the tumor killing activity of NK cells, which is characterized in that the method comprises culturing the NK cells with the above-mentioned medium.
In some embodiments, the NK cells are from a subject.
In some embodiments, the tumor is selected from acute myeloid leukemia, B-cell lymphoma, triple negative breast cancer, pancreatic cancer, ovarian cancer, lung cancer, and liver cancer.
Definition of the definition
Venetoclax, a specific inhibitor of BCL-2, having the formula C 45 H 50 ClN 7 O 7 S。
The structure is as follows
KG1a cells: KG1a is a human acute myeloid leukemia cell line, KG1a is a more primitive cell line with stem cell properties that induce differentiation into different cell types, such as myeloid, granulocyte, monocyte and erythroid. Almost all KG1a cells express CD34 and CD38, both of which are markers of myeloid cells and stem cells. Can be used for researching the basic and clinical problems related to acute myelogenous leukemia and other leukemia.
THP1 cells: THP1 is a human monocytic leukemia cell line, and THP1 is a relatively primitive cell line that can induce differentiation into macrophages or dendritic cells. The THP1 cell line surface highly expresses some marker molecules related to monocyte and/or macrophage differentiation and activation, such as CD11b, CD64, CD14, etc.
Effective target ratio: the invention relates to an effective target ratio of 0.5:1, 1:1, 2.5:1 and 5:1. Performing a plurality of effective target ratios can evaluate the killing effect of NK cells on different target cells, thereby determining the killing capacity and specificity of NK cells. This facilitates selection of the most suitable target cells, determination of the optimal cell proportion and optimal culture conditions, and evaluation of the effect of drugs or therapeutic methods on NK cell killing capacity. In addition, multiple effective target ratios can also evaluate differences between different samples, as well as evaluate the effect of different experimental conditions on the results. The effective target ratio of the invention is shown in the low effective target ratio of 0.5:1, the target ratio was close to the proportion of NK cells in human body, and enhancement of the killing effect of NK cells treated by Venetoclax can be observed.
NSG immunodeficient mice: NSG mice are a serious immunodeficiency mouse model, and are mainly characterized by comprising the following components: 1. systemic immunodeficiency: NSG mice are unable to develop an autoimmune response because of the lack of functional T, B and natural killer cells. This means that they are particularly susceptible to infection by a variety of pathogens, including infectious human viruses, parasites and bacteria, and the like, and need to be kept in a sterile environment. X-ray sensitivity: NSG mice are highly sensitive, mainly due to the lack of an intact DNA repair mechanism. This means that care is required to handle and maintain its health when modeling. 3. Specificity of human origin: NSG mice have good accommodations and similarities to human cells and tissues due to their lack of conservation in mRNA splicing and signaling. This makes NSG mice an effective mouse model for testing human tumors and immunotherapy. 4. High survival rate: NSG mice cannot reject donated cells due to imperfections in their immune system and are highly receptive to donations. This results in higher success rates and longevity than other mouse models in transplanting xenogeneic cells and tissues.
Advantageous effects
The invention discloses a small molecule inhibitor which can obviously enhance the killing capacity of NK cells from various sources to tumor cells, and the inhibitor can obviously enhance the killing activity of the NK cells while ensuring the high survival rate of the NK cells when being added into an NK cell culture medium. According to the invention, the NK cells cultured by the culture medium containing the small molecular inhibitor Venetoclax have excellent killing performance, and the killing rate of the cultured NK cells on KG1a cells can be improved by at least 20% under the condition of 1:1 effective target ratio while the high activity and the high purity of the NK cells are maintained, and the killing activity of the cultured NK cells on KG1a cells can reach 60% under the condition of 5:1 effective target ratio.
Drawings
FIG. 1 is a schematic of NK cell viability curve showing that Venetoclax treatment has no effect on NK cell viability. Using the statistical method of T-TEST, NS represents no statistical difference.
FIG. 2 is a graph of NK cell killing capacity in vitro, showing that Venetoclax treatment can significantly improve NK cell killing activity. Using statistical methods of T-TEST, P <0.05, P <0.01, P <0.001.
FIG. 3 shows the results of detection of NK cell killing factor expression levels, showing that Venetoclax treatment can significantly promote NK cell CD107a expression and IFN-gamma secretion. Using statistical methods of T-TEST, P <0.05, P <0.01, P <0.001.
FIG. 4 is a graph of the results of patient-derived NK cell killing assay showing that Venetoclax treatment can significantly increase NK cell killing activity. Using statistical methods of T-TEST, P <0.05, P <0.01, P <0.001.
FIG. 5 is a flow chart of the operation of NK cell treatment for HL60-Luc tumor-bearing mice. NSG mice were injected 5X 10 by tail vein 6 HL60-luc cells stably expressing luciferase were then infused with PBS, 5×10 via the tail vein 6 NK cells or 5X 10 cells 6 Venetoclax-treated NK cells were treated; AML load was monitored weekly by bioluminescence imaging for 2 weeks.
FIG. 6 is a test result of NK cells against tumor-bearing mice with HL60-Luc leukemia cells, showing that Venetoclax treatment can significantly improve the anti-tumor effect in NK cells and prolong survival. Figure a is an image of representative tumor burden for each group one week post-treatment and two weeks post-treatment prior to NK cell infusion treatment; panel b is a statistical plot of fluorescence intensity for images of tumor burden at different time periods of panel a, using statistical methods of T-TEST, representing P <0.05, representing P <0.01, representing P <0.001; panel c is a survival curve for three groups of mice. Using Kaplan-Meier survival analysis, P <0.05 is indicated, P <0.01 is indicated, and P <0.001 is indicated.
Detailed Description
The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.
In this specification, unless defined otherwise, all technical terms are defined conventionally as used and understood by those skilled in the art. All experimental methods are conventional, unless otherwise indicated, and reagents and materials are commercially available. These embodiments are merely examples, which are intended to better illustrate the principles and implementations of the invention and should not be taken in a limiting sense. Any simple modification or equivalent of the following embodiments is considered to be within the scope of the technical spirit of the present invention.
EXAMPLE 1NK cell preparation
1.1 isolation of umbilical cord blood mononuclear cells
Under sterile conditions, 150mL of cord blood from healthy volunteers was collected for the preparation of NK cells. 15mL of human lymphocyte separation liquid (Tianjin, ocean, LTS 1077) was added to a 50mL centrifuge tube, and 45mL of cord blood was slowly added along the tube wall, avoiding damaging the liquid level of the lymphocyte separation liquid. At room temperature, 450g, 0g up and 0 down, and centrifuged for 30 minutes. The conditions for 0-0 f rise are set so that shear, mechanical stress and other intense forces during centrifugation can be reduced or eliminated during cell separation, which helps to maintain cell integrity and viability and reduce adverse effects on cells. The pale yellow mononuclear cell layer was pipetted out into a new centrifuge tube and the cells were washed by adding sterile 1×pbs. And (3) performing 1800rpm, 9-9 and 10-9 centrifugation at 4 ℃ for 10 minutes, discarding the supernatant, washing the cells again with PBS, 1200rpm, 9-9 and 10-min centrifugation, discarding the supernatant, and obtaining the precipitation as the umbilical cord blood mononuclear cells.
1.2NK cell separation and purification
Sorting according to NK cell negative selection immunomagnetic beadsInstructions for the system (Miltenyi Biotec, 130-092-657) were operated to purify NK cells. PBS was resuspended and counted every 10 7 Adding 40 μl PBS to each cell to obtain single cell suspension, 10 times 7 Mu.l of NK cell biotin-antibody cocktail (Miltenyi Biotec, 130-092-657) was added to each cell, gently mixed and incubated at 4℃for 5min. Every 10 after antibody incubation was completed 7 Each cell was added with 30ul of PBS, 20. Mu. l NK cell MicroBead Cocktail (Miltenyi Biotec, 130-092-657), gently mixed, and incubated at 4℃for 10min. After incubation, PBS was added to 500. Mu.l, and the mixture was slowly passed through MACS chromatography column (Miltenyi Biotec, 130-042-401), and the column was washed with MACS Buffer to collect unbound cells, namely NK cells. Cells were resuspended in RPMI 1640 medium+10% FBS+1% diabodies (diabodies containing both penicillin and streptomycin antibiotics, biosharp, BL 505A) into 12-well plates, IL-12 (offshore protein, C158) was added to a final concentration of 10ng/mL and IL-15 (Peprotech, 200-15) to a final concentration of 50ng/mL. NK cells were cultured in vitro with the addition of IL-12 and IL-15 for the purpose of promoting NK cell survival, proliferation and activation. Simultaneously, the small molecule inhibitor Venetoclax is added, and cell activation is carried out in an incubator with 5% carbon dioxide at 37 ℃. The viability of NK cells was examined using a flow cytometer after treatment at different concentrations of vennetoclax. The specific steps are that after the treatment of Venetoclax with different concentrations is finished, cells are collected, 350g is centrifuged for 8min, supernatant is discarded, 1ml PBS buffer is added to resuspend and clean the cells, 350g is centrifuged for 8min, and supernatant is discarded. 100 μl PBS was used to resuspend cells, FITC-CD45 (Biolegend, 304308), APC-CY7-CD3 (Biolegend, 344818), BV421-CD56 (Biolegend, 362552), after 15min staining in the dark, 1ml PBS was added to resuspend to remove excess antibody, 100 μl l Annexin V binding buffer (following the holothurian, 40304ES 60) was used to resuspend cells, 5 μl Alex647-Annexin V (following the holothurian, 40304ES 60) and 10 μl PI (following the holothurian, 40304ES 60) dye were added, and the upflow cytometer was used to detect apoptosis of CD45+CD3-CD56+ NK cells in the dark for 10min.
FIG. 1 shows that the addition of the small molecule inhibitor Venetoclax of the present invention does not affect NK cell viability, and that NK cell viability in the control group is 90.2% and that the small molecule inhibitor Venetoclax200nM, 400nM, 1000nM treatment group viability is 90.8%, 90.7% and 88.5%, respectively.
Example 2Venetoclax promotes in vitro killing of tumor cells by cord blood-derived NK cells
Pretreatment of NK cells with the small molecule inhibitor Venetoclax: after the NK cells prepared in example 1 were prepared into single cell suspensions, a small molecule inhibitor Venetoclax (MedChemExpress, HY-15531), 10ng/mL of IL-12 and 50ng/mL of IL-15 was slowly added to the cell suspensions under aseptic conditions, and the mixture was then incubated in a water bath of a carbon dioxide incubator for 18 hours, the whole procedure requiring assurance of aseptic manipulation, wherein Venetoclax was mixed in the cell suspension at a final concentration of 400nM.
The following are the specific procedures for the Venetoclax-promoted in vitro killing experiments of NK cells against tumor cells:
2.1 pretreatment of target cells
The target cells used in the experiments were KG1a, THP1 cells (ATCC, TIB-202, CCL-246.1), respectively, and were labeled with CFSE (5, 6-carboxyfluorescein diacetate, biolegend, 423801), 5% CO at 37% 2 Incubate in incubator for 20min in dark, add complete medium (RPIM 1640 medium+10% foetal calf serum) to stop staining, 1000rpm, centrifuge for 5min, re-suspend cells with PBS, wash once, 1000rpm, centrifuge for 5min, discard supernatant, re-suspend cells with RPMI 1640 basal medium (Biosharp, BL 303A), count, plate, inoculate 20000-60000 cells per 100 μl per well in 96 well plate.
2.2 addition of effector cells-NK cells
NK cells pre-treated with the small molecule inhibitor Venetoclax in advance were collected, 350g, centrifuged for 10min, the supernatant discarded, the washed cells resuspended with PBS, 350g, centrifuged for 10min, and the supernatant discarded, i.e.Venetoclax together with medium was removed after pre-treatment. NK cells were formulated to the required concentration with RPIM1640 basal medium according to the pre-designed effective target ratio, plated according to the effective target ratio, 100 μl of NK cell suspension was added per well, and the mixture was gently shaken.
The experimental design was grouped as follows, 3 duplicate wells per group:
1) Pure target cell group: mu.l of the target cell suspension was inoculated into each well, and then 100. Mu.l of RPIM1640 basal medium was added to make up the volume.
2) NK cell group: inoculating target cells and NK cells according to different target effect ratios, wherein the specific ratio is as follows: 0.5:1, 1:1, 2.5:1, 5:1.
3) Venetoclax treatment of NK cell groups: target cells and NK cells pre-treated with Venetoclax were seeded at different effective target ratios: 0.5:1, 1:1, 2.5:1, 5:1.
2.3 Co-incubation and detection
After the inoculation according to the above group, the mixture was inoculated at 37℃with 5% CO 2 For 4 hours in the incubator of the culture apparatus,
after the co-incubation, 350g was centrifuged for 8min, the supernatant was discarded, alex647-Annexin V/PI (next holothurian, 40304ES 60) was added to label apoptotic cells, and the flow cytometer was used to detect apoptosis of CFSE+ target cells.
2.4 calculation of experimental results
Calculation formula of specific killing efficiency = [ (NK cell group% Annexin V-simple cell group% Annexin V)/100-simple cell group% Annexin V ]. Times.100, experimental result is expressed as mean ± SD.
From the results of FIG. 2, it can be seen that the killing efficiency of Venetoclax pretreated NK cells against target cells was significantly improved, and this effect was observed with a low effective target ratio of 0.5:1. In the KG1a cell line, the control group had killing efficiencies of 9.3125,9.963, 16.1775, 20.0125 at effective target ratios of 0.5:1, 1:1, 2.5:1, 5:1, respectively, whereas the Venentoclax-treated group had killing efficiencies of 21.45, 33.54, 41.97, 58.24, respectively. In the THP1 cell line, the control group had killing efficiencies of 10.75,13.18,36.37,52.60 at effective target ratios of 0.5:1, 1:1, 2.5:1, 5:1, respectively, while the Venentoclax treated group had killing efficiencies of 24.67,35.29,55.00,74.26, respectively. In an in vitro killing experiment, the killing efficiency of NK cells is generally increased along with the improvement of the effective target ratio, but the NK cell proportion in a normal organism does not reach the high proportion of the in vitro experiment, and in order to simulate the in vivo situation, we select 0.5:1, in the present invention at 5: there has been a relatively clear difference in the case of 1-effect target ratio and experiments to further increase the proportion of NK cells have not been performed.
Example 3Venetoclax promotes expression of cord blood-derived NK cell killing function molecules
Collecting Venetoclax pretreated NK cells and negative control NK cells, wherein the final concentration of Venetoclax mixed in cell suspension is 400nM,1200rpm, centrifuging for 10min, discarding supernatant, resuspending washed cells with PBS, 1200rpm, centrifuging for 10min, discarding supernatant, and adjusting cell density to 2X 10 with PBS 7 Per mL, 100 μl of cell suspension was added per tube, cells were incubated with flow antibody to PE-labeled CD107a (Biolegend, 328608) and FITC-labeled IFN- γ (Biolegend, 506504) and their expression was detected by flow cytometry.
In the process of killing target cells by NK cells, the NK cells are triggered by signals, cytotoxic particles are released through a degranulation process to reach the target cells, and the cytotoxic particles enter the target cells to trigger DNA fragmentation of the target cells so as to enable the target cells to apoptosis. CD107a is also an important factor involved in NK cell degranulation. Activated NK cells can synthesize and secrete a variety of cytokines, exerting the effects of regulating immunity and hematopoiesis and directly killing target cells. Thus, one skilled in the art evaluates NK cell killing ability by detecting secretion levels of CD107a and IFN-gamma.
As can be seen from fig. 3, the use of the small molecule inhibitors of the present invention can significantly promote NK cell killing, with the expression level of CD107a increasing from 4.7% to 20.4% and the expression level of IFN- γ increasing from 7.26% to 38.1%.
Example 4Venetoclax promotes patient-derived NK cell killing Activity
4.1 isolation of bone marrow mononuclear cells from patients
Under aseptic conditions, the marrow liquid of acute myeloid leukemia patients (from the first hospital blood department under the university of Chinese science and technology, all specimens were obtained with informed consent of the patients and approved by the hospital Rollers, with ethical number 2022-KY-089) was collected for preparing NK cells and primary tumor cells from the patients. 5mL of human lymphocyte separation liquid is added into a centrifuge tube, and 2mL of bone marrow liquid is slowly added along the tube wall, so as to avoid damaging the liquid level of the lymphocyte separation liquid. At room temperature, 450g, 0g up and 0 down, and centrifuged for 30 minutes. The pale yellow mononuclear cell layer was pipetted out into a new centrifuge tube and the cells were washed by adding sterile 1×pbs. And (3) performing 1800rpm, 9-9 and 10-9 centrifugation at 4 ℃ for 10 minutes, discarding the supernatant, washing the cells again with PBS, 1200rpm, 9-9 and 10-min centrifugation, discarding the supernatant, and obtaining the sediment, namely the bone marrow mononuclear cells.
4.2 isolation and purification of NK cells from patient sources and primary tumor cells
After isolation of the mononuclear cell suspension, the cells were washed with 1ml PBS, filtered through a 200 mesh gauze, and the cell pellet was resuspended in 250g centrifugation 10min,MACS Buffer (Miltenyi Biotec, 130-091-221), blocked with rat serum (Future, F001007), and incubated at about 4℃for about 30min. Flow-through antibodies to FITC-CD45 (bioleged, 304308), APC-CY7-CD3 (bioleged, 344818), BV421-CD56 (bioleged, 362552), APC-CD33 (bioleged, 303408) were added and incubated at 4℃for 30min in the absence of light. Cells were washed with PBS, MACS Buffer resuspended in cell pellet, filtered through 200 mesh white screen, transferred to sterile flow tubes and placed at 4 ℃. The BD flow sorter (Aria III) was opened and adjusted to perform cell grouping gating, followed by sorting out the target cell population, NK cells (CD45+CD3-CD56+), primary tumor cells (CD45+CD3-CD33+).
4.3NK cells co-incubation with target cells
After the prepared purified patient-derived NK cells were prepared into a single cell suspension, a small molecule inhibitor Venetoclax,10ng/mL IL-12 and 50ng/mL IL-15 was slowly added to the cell suspension under aseptic conditions and the mixture was then placed in a water bath of a carbon dioxide incubator for incubation for 18 hours, the whole procedure requiring a guaranteed aseptic technique, wherein Venetoclax was mixed in the cell suspension at a final concentration of 400nM. The target cells used in the experiment were KG1a cells and primary tumor cells, respectively, were labeled with CFSE at 37℃with 5% CO 2 Incubating in an incubator in the dark for 20min, and adding a complete culture mediumRPIM1640 medium+10% foetal calf serum), 1000rpm, centrifugation for 5min, re-suspending cells with PBS, washing once, 1000rpm, centrifugation for 5min, discarding supernatant, re-suspending cells with RPMI 1640 basal medium, counting, plating, and plating 20000-60000 cells/100 μl per well in 96-well plates. The effective target ratio is 2.5:1 (since patient-derived NK cell samples are precious and the number is small and no detection of killing efficacy of multiple effect target ratio is performed), 2.5:1 is selected because the target ratio is the one commonly used in vitro experiments), NK cells are added in proportion, and after inoculation is completed, at 37℃and 5% CO 2 After the co-incubation is finished, 350g is centrifuged for 8min, the supernatant is discarded, alex647-Annexin V/PI is added to mark apoptotic cells, and the apoptosis of CFSE+ target cells is detected in a flow-through manner. The experimental results were calculated as in example 2.
As can be seen from fig. 4, NK cells derived from patients can significantly improve the killing efficiency of target cells under Venetoclax pretreatment, and the killing efficiency of KG1a cells can be from 2.45% to 18.33%; the killing efficiency of the primary autologous tumor cells can be improved from 10.83% to 37.42%.
Example 5Venetoclax significantly promoted anti-tumor effects in NK cells
First, a single cell suspension of a human acute myeloid leukemia cell HL60-LUC (ATCC, CCL-240-LUC 2) in a good state of logarithmic growth phase was prepared for use. NSG immunodeficient mice (NM-NSG-001, southern model biological Co., ltd.) were selected, females, irradiated to clear the marrow at 2.5Gy for 4-6 weeks, and injected 5X 10 by tail vein 6 Tumor burden was examined in vivo on HL60-LUC cells/mouse, on about 2 weeks, and randomly divided into 3 groups by tumor burden: negative control, NK cell and vennoclax treated NK cell groups, 6 tumor-bearing mice per group. Tumor-bearing mice of NK cell group and NK cell group treated with Venetoclax were infused with NK cells 5X 10 by tail vein 6 /only.
The method of Venetoclax treatment of NK cells is as follows:
after the NK cells prepared in example 1 were prepared into single cell suspensions, a small molecule inhibitor Venetoclax (MedChemExpress, HY-15531), 10ng/mL of IL-12 and 50ng/mL of IL-15 was slowly added to the cell suspensions under aseptic conditions, and the mixture was then incubated in a water bath of a carbon dioxide incubator for 18 hours, the whole procedure requiring assurance of aseptic manipulation, wherein Venetoclax was mixed in the cell suspension at a final concentration of 400nM. NK cells pretreated with the small molecule inhibitor Venetoclax were collected, centrifuged for 10min at 350g, the supernatant was discarded, the washed cells were resuspended with PBS, centrifuged for 10min at 350g, and the supernatant was discarded.
During the experiment, in vivo imaging assays were performed once a week to assess tumor burden in each tumor-bearing mouse, while hIL-15 (Peprotech, 200-15) was injected every 3 days. hIL-15 was injected in order to maintain NK cell activity in mice. See fig. 5 for detailed experimental procedures.
As can be seen from fig. 6, the tumor burden of the Venetoclax treated NK cell group was significantly lower than that of the NK cell group alone and the negative control group, whereas the tumor burden of the NK cell group alone was slightly lower than that of the negative control group. After NK cell infusion treatment for two weeks, the fluorescence signal intensity of tumor burden in mice of control group is 1.06X10 10 Tumor burden fluorescence signal intensity in NK cell infusion treated mice group was 5.56X10 9 Whereas the intensity of fluorescent signal of the in vivo burden of the Venentoclax-treated NK cell infusion-treated mice was 1.22×10 9 . The lower the fluorescence signal intensity, the smaller the tumor burden. Experimental results show that compared with NK cells used alone, the NK cells treated by Venetoclax have obviously better therapeutic effect on acute myeloid leukemia, thereby proving the feasibility of the scheme in treating acute myeloid leukemia. In addition, the NK cells treated by Venetoclax can obviously prolong the survival time of tumor-bearing mice, the median survival time of a control group is 30 days, the median survival time of an NK cell group is 38 days, and the median survival time of an NK cell treatment group treated by Venetoclax is 57 days. This result further supports the safety of Venetoclax treated NK cells.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.
Claims (10)
1. An anti-tumor agent, characterized in that said agent comprises NK cells treated with Venetoclax.
2. The anti-tumor formulation according to claim 1, wherein the NK cells are derived from umbilical cord blood, bone marrow of acute myeloid leukemia patients, peripheral blood such as normal donor peripheral blood, immortalized NK cell line NK92, iPSC derived NK cells or umbilical cord blood derived CD34 + HSPCs induce differentiated NK cells.
3. A culture medium or kit for improving NK cell tumor killing activity, characterized in that the culture medium or kit comprises a basal medium and Venetoclax, wherein the basal medium is selected from the group consisting of RPIM1640 medium, SCGM medium, IMDM medium and DMEM medium.
4. The medium or kit of claim 3, further comprising two or more cytokines selected from the group consisting of IL-12, IL-15, IL-2 and IL-18.
5. The medium or kit according to claim 3 or 4, wherein the Venetoclax is present in an amount of 200-1000nM, preferably 200-500nM, 200-600nM, 200-700nM, 200-800nM, 200-900nM, 200-1000nM, 200-1100nM, 200-1200nM, 200-1300nM, 200-1400nM, 200-1500nM, 200-1600nM, 200-1700nM, 200-1800nM, 200-1900nM, more preferably 400nM.
6. The medium or kit according to claim 4, wherein the cytokines are IL-12 and IL-15, the concentration of IL-12 is 5-20ng/mL, preferably 10ng/mL, and the concentration of IL-15 is 5-100ng/mL, preferably 50ng/mL.
Use of vennetoclax for the preparation of NK cells with increased tumor killing activity.
8. A method of preparing NK cells having increased tumor killing activity, said method comprising culturing said NK cells with the medium of any one of claims 3-6 and isolating the cultured cells.
9. The method of claim 8, wherein the NK cells are from a subject.
10. The anti-tumor formulation according to claim 1 or 2, the culture medium or kit according to any one of claims 3-6, the use according to claim 7 or the method according to claim 8 or 9, characterized in that the tumor is selected from acute myeloid leukemia, B-cell lymphoma, triple negative breast cancer, pancreatic cancer, ovarian cancer, lung cancer and liver cancer.
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