Establishment method of robust humanized mouse tumor model system for evaluating NK targeted therapy

Abstract

The invention discloses an establishment method of a robust humanized mouse tumor model system for evaluating NK targeted therapy. Firstly, a stable monoclonal feeding cell line is constructed in vitro and used for amplifying NK cells separated from human peripheral blood mononuclear cells, and a magnetic bead separation method is adopted. By this method, the human-derived primary NK cells are robustly and consistently amplified while maintaining their purity and functionality. By comparing the amplified NK cells with primary NK cells in an in-vitro immune test, the amplified NK cells show a result equivalent to that of mother cells. An in-vivo experiment result shows that lasting and functional NK reconstruction can be established in a mouse which expresses the tumor of the hIL-15 and carries serious immunodeficiency. In addition, it is proved that the NK humanized tumor model system has response to the NK targeted drug of the SOC. The novel NK humanized tumor model system can be used for evaluating NK regulating drugs.

Description

Technical Field

[0001] The present invention relates to a method for establishing a robust humanized mouse tumor model system for evaluating NK targeted therapy. Background Art

[0002] With the development of a new wave of anti-tumor immunotherapy, new immunotherapies based on NK cells have emerged rapidly and made progress. Considering the inherent mechanism of NK cells, tumor immunotherapy has adopted various strategies to maximize the effectiveness of NK cells, including the following: 1) immune checkpoint inhibitors to relieve the inhibitory signals of NK cells; 2) activation of NK cell activity; 3) targeted antibody therapy to kill tumor cells through antibody-dependent cellular cytotoxicity (ADCC); and 4) cell therapy, such as CAR-NK. Although many immunotherapeutic drugs targeting NK cells or various NK regulation modes have been developed, and these drugs have made progress towards clinical trials. However, the NK cells in the robust humanized mouse tumor model system currently used to evaluate NK targeted therapy are mainly isolated from peripheral blood or PBMC directly, and then NK cells are expanded using cytokines. Or PBMC or sorted NK cells are expanded in antibody-coated culture flasks. Both the factor method and the antibody coating method are difficult to meet clinical needs in terms of the number and purity of NK cell expansion. Summary of the invention

[0003] The purpose of the present invention is to provide a method for establishing a robust humanized mouse tumor model system for evaluating NK targeted therapy, and the activation and expansion of NK cells are achieved only by two preparations, namely, feeder layer cells and IL-2 constructed by us.

[0004] The purpose of the present invention is achieved through the following technical solutions:

[0005] A method for establishing a robust humanized mouse tumor model system for evaluating NK targeted therapy, characterized in that the steps include:

[0006] (1) NK cells were isolated from hPBMC using CD56 magnetic beads;

[0007] (2) co-culturing the sorted NK cells with feeder cells and adding IL-2 during the culture process;

[0008] (3) ADCC assay and FACs were used to evaluate the function of expanded NK cells; ADCC assay, i.e., antibody-dependent cell-mediated cytotoxicity therapy, and FACs (i.e., flow cytometry validation) were used to validate NK cell function;

[0009] Specific method: Add different concentrations of rituximab to Raji-luc cells to allow the antibody to bind to the CD20 antigen on the surface of the target cells. Then add the NK cells prepared in step (2) to the culture system for co-culture. Mix NK cells and Raji-luc cells in a 96-well plate at different ratios of effector cells to target cells (5:1). Culture for 6 hours at 37°C and 5% CO2, and then detect cell lysis. Use flow cytometry and other methods to determine target cell toxicity. Calculate the percentage of specific lysis or cytotoxicity, and compare the differences between the experimental group and the control group (NK cells only, Raji-luc cells only, and rituximab only).

[0010] (4) Establishment of NK humanized mouse tumor model. Use the NK cells amplified in step (2) to reconstruct and humanize to obtain the SOC H-NK humanized model;

[0011] The specific method is as follows: resuspend the above 2*10^7 NK cells in 400μl culture medium, and infuse and implant them into mice by tail vein injection, so that the NK cells are reconstructed in the mice. Resuspend 2*10^6 A431 in 100μl culture medium, and inoculate mice by subcutaneous injection, so that the skin squamous cell carcinoma cells form tumors under the mouse skin;

[0012] (5) Validation of NK humanized mouse tumor model. Validation of SOC H-NK humanized model using NK targeted therapy positive drugs.

[0013] Specific method: Use NK targeted therapy positive drugs to perform SOC verification on the SOC H-NK humanized model. When the subcutaneous tumor volume reaches 100mm^3, group treatment is performed: G1: NDG-NK is the model control group, G2: IL15-NDG-NK is the model group, G3: IL15-NDG-NK-AFM24 is the dual antibody treatment group, and the therapeutic drug AFM24 is a humanized dual antibody targeting CD16a / EGFR. Administration method: intraperitoneal injection, frequency: twice a week, dosage: 5mg / kg. Observe the treatment results. The experimental results show that the NK humanized skin cancer animal model was successfully constructed, and the CD16a / EGFR dual antibody drug showed positive treatment results.

[0014] Preferably, the feeder cells in step (2) are K562 engineered cells.

[0015] Preferably, the K562 engineered cells in step (2) are irradiated before co-culturing.

[0016] Preferably, the dose of irradiation treatment of K562 engineered cells in step (2) is 300 Gy.

[0017] Preferably, the co-cultivation in step (2) adopts X-VIVO 15 culture medium.

[0018] Preferably, in step (2), the ratio of NK cells to feeder cells is 1:10.

[0019] The present invention discloses a method for in vitro culture and expansion of NK cells, specifically a method for in vitro culture and expansion of NK cells using autologous peripheral blood mononuclear cells, wherein the method uses a CD 56 magnetic bead sorting kit to sort out NK cells in hPBMC, activates NK cells by co-culture of feeder layer cells, and co-stimulates NK cells with cytokine IL-2, and by this method, humanized primary NK cells are robustly and consistently expanded (up to hundreds of times) while maintaining their purity and functionality. By comparison with primary NK cells in in vitro immune tests (including ADCC and FACS), the expanded NK cells show results comparable to those of the mother cells. NK cells obtained by the in vitro culture and expansion method of NK cells can specifically reconstruct functional and persistent human NK cells in a mouse system, providing a clinically relevant tumor microenvironment. In vivo experimental results show that persistent and functional NK reconstruction can be established in mice with severe immunodeficiency in tumors expressing hIL-15. In addition, it is demonstrated that this NK humanized tumor model system responds to NK targeted drugs of standard of care (SOC). This novel, robust humanized mouse tumor model system for evaluating NK-targeted therapies is an effective preclinical tool for evaluating NK-modulating drugs and can facilitate the development of novel immunotherapeutic agents that exploit both adaptive and innate immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 :Schematic diagram of h-NK tumor therapy model.

[0021] Figure 2 :Characteristics and functional verification of in vitro expanded NK cells, A: Representative images of in vitro expanded NK cells, on the first and seventh days; B: FACs results after 7 days of in vitro expanded NK cells; C: ADCC results showed that after rituximab treatment, there was no significant difference in the function of expanded NK cells and primary NK cells. ADCC and FACs tests proved that our in vitro culture system can effectively expand NK cells. At the same time, the expanded NK cells have complete NK cell functions.

[0022] Figure 3:Efficacy of in vitro expanded NK cells in humanized mouse tumor models and verification of NK cell transplantation effects. A: Growth curves of transplanted tumors under different treatment regimens; B: FACs results of h-NK cells in the peripheral blood of humanized mice, indicating that durable NK reconstruction was observed in B-NDG-hIL-15 mice; C: Mouse weight change curve; D: Tumor weight data at the end point of the transplanted tumor study.

[0023] Figure 4 : Streaming gate logic solution. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with embodiments, but the description of the embodiments does not impose any limitation on the protection scope of the present invention.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0026] The materials and instruments used in the following examples, unless otherwise specified, can be obtained from conventional commercial channels. Figure 1 shown.

[0027] Example 1

[0028] 1) PBMC recovery

[0029] Gently shake the cryopreserved samples in a 37°C water bath to thaw them. Try not to immerse the tube cap below the water surface to avoid cell contamination. Gently shake the cryopreserved tube during the thawing process until all the samples in the tube are melted. Generally, this process should be controlled within 3 minutes.

[0030] 2) NK cell sorting

[0031] NK cells in PBMC were isolated using CD56 magnetic bead isolation kit.

[0032] 3) Co-culture of PBMC and K562.41bbL.mbIL2 cells

[0033] 3.1) Feeder cells (feeder cells need to be irradiated with X-ray before co-culture, and the irradiation dose is: 300 Gy (100 Gy / h*3h)).

[0034] 3.2) Live feeder cells K562 and sorted NK cells were mixed at a ratio of 10:1 (NK 1.5M, K562 15M) and inoculated into a T75 flask with a total volume of 30 ml (28 ml culture medium + 2 ml mixed cells). X-VIVO 15 complete culture medium was used to control the cell density to 2E4 NK / cm 2 , add 3 μL of recombinant human interleukin-2 (125Ala) for injection (Shuanglu, Xingjier, National Medicine Standard No. S19991009) to each bottle and start culturing.

[0035] On the 2nd and 4th days, 3 μl of IL-2 was added to each bottle.

[0036] On the third day, centrifuge and change the medium: 250g, centrifuge for 5 minutes, discard the supernatant; add cell pellet + 30mL X-VIVO 15 complete medium, add 3μL IL-2 to each bottle, and do not change the bottle.

[0037] On the 4th day, 15 mL of culture medium and 2 μl of IL2 were added to each bottle, and on the 6th day, 10 mL of culture medium and 6 μl of IL2 were added to each bottle. (The actual amount of liquid added should be based on the color of the cell suspension or the state of the cells under the microscope. Generally, the amount of liquid added should be based on the color of the culture medium or when there are more cells).

[0038] On the 7th day, the cell viability and number were counted, NK cells were collected for inoculation, and some cells were taken out for flow cytometry detection of NK cell expansion ratio and purity. ADCC assay and FACs were used to evaluate the function of the expanded NK cells. Figure 2 As shown;

[0039] Specific method: Add different concentrations of rituximab to Raji-luc cells to allow the antibody to bind to the CD20 antigen on the surface of the target cells. Then add the NK cells prepared in step (2) to the culture system for co-culture. Mix NK cells and Raji-luc cells in a 96-well plate at different ratios of effector cells to target cells (5:1). Culture for 6 hours at 37°C and 5% CO2, and then detect cell lysis. Use flow cytometry and other methods to determine target cell toxicity. Calculate the percentage of specific lysis or cytotoxicity, and compare the differences between the experimental group and the control group (NK cells only, Raji-luc cells only, and rituximab only).

[0040] (4) Establishment of NK humanized mouse tumor model. Use the NK cells amplified in step (2) to reconstruct and humanize to obtain the SOC H-NK humanized model;

[0041] The specific method is as follows: resuspend the above 2*10^7 NK cells in 400μl culture medium, and infuse and implant them into mice by tail vein injection, so that the NK cells are reconstructed in the mice. Resuspend 2*10^6 A431 in 100μl culture medium, and inoculate mice by subcutaneous injection, so that the skin squamous cell carcinoma cells form tumors under the mouse skin;

[0042] (5) Validation of NK humanized mouse tumor model. Validation of SOC H-NK humanized model using NK targeted therapy positive drugs.

[0043] Specific method: Use NK targeted therapy positive drugs to perform SOC verification on the SOC H-NK humanized model. When the subcutaneous tumor volume reaches 100mm^3, group treatment is carried out: G1: NDG-NK is the model control group, G2: IL15-NDG-NK is the model group, G3: IL15-NDG-NK-AFM24 is the dual antibody treatment group, and the therapeutic drug AFM24 is a humanized dual antibody targeting CD16a / EGFR. Administration method: intraperitoneal injection, frequency: twice a week, dosage: 5mg / kg. The experimental results are as follows Figure 3 The experimental results showed that the NK humanized skin cancer animal model was successfully constructed, and the CD16a / EGFR dual antibody showed positive therapeutic results.

[0044] The mouse in vivo model proved that the NK cells expanded by our NK in vitro expansion culture system can be effectively expanded in the model mice. In addition, the NK cells expanded by our NK in vitro expansion culture system have complete NK cell function in the model mice.

[0045] The experimental reagents and instruments involved in the embodiments are as follows.

[0046] Experimental reagents

[0047]

[0048]

[0049] Experimental Instruments

[0050] Instrument brand Instrument Name Instrument Model Cytek Full spectrum flow cytometer Aurora / NL Xiangzhi Low speed refrigerated centrifuge TDL5B

[0051] Experimental steps:

[0052] Whole blood red blood cell lysis

[0053] Take 40μL of mouse whole blood and place it in a U-bottom 96-well plate. Add 280μL of red blood cell lysis buffer to each well and lyse at room temperature for 3 minutes. After lysis, centrifuge at 500g for 5 minutes at 4℃ and discard the supernatant. Repeat the lysis step once until the cell pellet is slightly reddish grayish white after centrifugation. After red blood cell lysis, add 300μL of PBS to each well, centrifuge at 500g for 5 minutes at 4℃, discard the supernatant, and the remaining cell pellet is the immune cell.

[0054] Flow staining

[0055] 1) Prepare the dead and live dye working solution at a ratio of 1 / 1000.

[0056] 2) Add 100 μL of dead-live dye working solution to the cell pellet and stain at 4°C for 15 min.

[0057] 3) After staining, add 200 μL FBS solution (2% serum in PBS solution) to each well to terminate the reaction, wash twice with FBS solution, centrifuge at 500 g for 5 min at 4°C, and discard the supernatant.

[0058] 4) Prepare FcR antibody blocking working solution at a ratio of 1:100.

[0059] 5) Add 100 μL of FcR antibody blocking solution to the cell pellet and incubate at 4°C for 5 min.

[0060] 6) Prepare the sample group antibody premix according to the instructions for use of each antibody.

[0061] 7) Add 100 μL of antibody premix to the cell suspension in the sample group; add antibodies to the non-sample wells according to the sample grouping table in 5.2.1. After adding antibodies, stain at 4°C for 30 minutes.

[0062] 8) After staining, wash twice with FBS and finally resuspend to 120 μL with FBS.

[0063] On-machine testing

[0064] Flow cytometry gating logic scheme is used to detect the proportion of NK cells in the expanded cells. Figure 4 shown.

[0065] In the flow cytometry experiment, the positive results and positive rates of each component were obtained through the flow cytometry analysis graph.

[0066] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for establishing a robust humanized mouse tumor model system for evaluating NK-targeted therapy, characterized in that The steps include: (1) NK cells were isolated from hPBMC using CD56 magnetic beads; (2) co-culturing the sorted NK cells with feeder cells and adding IL-2 during the culture process; (3) using ADCC assay, i.e. antibody-dependent cell-mediated cytotoxicity therapy and FACs, to verify NK cell function; (4) using the NK cells amplified in step (2) to infuse into mice, thereby reconstructing the NK cells in the mice to obtain a SOC H-NK humanized model; (5) Use NK targeted therapy positive drugs to perform SOC validation on the SOC H-NK humanized model.

2. The method according to claim 1, characterized in that: The feeder cells in step (2) are K562 engineered cells.

3. The establishment method according to claim 2, characterized in that: The K562 engineered cells in step (2) are irradiated before co-culturing.

4. The method according to claim 3, characterized in that: The dose of irradiation treatment of K562 engineered cells in step (2) is 300 Gy.

5. The establishment method according to claim 1, characterized in that: The co-culture in step (2) uses X-VIVO 15 culture medium.

6. The establishment method according to claim 1, characterized in that: In the step (2), the ratio of NK cells to feeder cells is 1:

10.

7. The establishment method according to claim 1, characterized in that: Step (3) is specifically as follows: adding different concentrations of rituximab to Raji-luc cells to allow the antibody to bind to the CD20 antigen on the surface of the target cells. Then, the NK cells prepared in step (2) are added to the culture system for co-culture, and the NK cells and Raji-luc cells are mixed in a 96-well plate at different ratios of effector cells to target cells, and cultured for 6 hours at 37° C. and 5% CO2, and then the cell lysis is detected, the target cell toxicity is determined, and the percentage of specific lysis or cytotoxicity is calculated.

8. The establishment method according to claim 1, characterized in that: Step (4) is specifically as follows: resuspending the NK cells prepared in step (2) in culture medium, and implanting them into mice by tail vein injection, so that the NK cells are reestablished in the mice, resuspending A431 cells in culture medium, and inoculating mice by subcutaneous injection, so that the skin squamous cell carcinoma cells form tumors subcutaneously in the mice.

9. The establishment method according to claim 1, characterized in that: Step (5) is specifically as follows: when the subcutaneous tumor volume of the mouse model established in step (4) reaches 100 mm^3, group treatment is performed: G1: NDG-NK is the model control group, G2: ILl5-NDG-NK is the model group, G3: ILl5-NDG-NK-AFM24 is the dual antibody treatment group, the therapeutic drug AFM24 is a human dual antibody targeting CD16a / EGFR, and the treatment results are observed.

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