Animal models of CAR-T therapy for leukemia complicated by cytokine release syndrome, their preparation methods and applications

Abstract

This invention relates to the field of leukemia research, specifically disclosing an animal model of CAR-T therapy for leukemia complicated by cytokine release syndrome (CRS), its preparation method, and its application. The method for preparing the animal model of CAR-T therapy for leukemia complicated by CRS includes: obtaining CD19 CAR-T cells: peripheral blood mononuclear cell isolation, T cell magnetic bead sorting, T cell activation, lentiviral infection, and CAR-T cell expansion; constructing an animal model of CRS after high- and low-dose CAR-T infusion: implanting Nalm-6 cell line into living animals to induce leukemia tumor burden, obtaining living animals with leukemia tumor burden, and injecting CD19 CAR-T cell line into the living animals with leukemia tumor burden. This invention provides a novel application for the animal model of CAR-T therapy for leukemia complicated by cytokine release syndrome. By using severely combined immunodeficient mice and inoculating them with high- and low-burden tumor cells, and infusing different doses of CAR-T cells, this invention constructs an in vivo model that can simulate clinical CRS, providing research guidance for clinically exploring more effective interventions.

Description

Technical Field

[0001] This invention relates to the field of leukemia research, and more particularly to an animal model of CAR-T therapy for leukemia complicated with cytokine release syndrome, its preparation method, and its application. Background Technology

[0002] CAR-T therapy has shown good therapeutic effects on leukemia, especially lymphocytic leukemia. Chimeric antigen receptor T cells have become an effective treatment for relapsed / refractory hematologic malignancies; however, while achieving significant efficacy, they are also accompanied by cytokine release syndrome (CRS). CRS is the most common side effect of CAR-T therapy, occurring in approximately 50% to 100% of patients, with up to 48% experiencing grade ≥3 CRS. CRS manifests as significantly elevated IL-6, vascular leakage, hypotension, pulmonary edema, heart failure, liver and kidney failure, neurotoxicity, disseminated intravascular coagulation, and multiple organ failure, even death, severely impacting the efficacy and prognosis of CAR-T therapy. Currently, clinical interventions for CRS are limited, mainly involving cytokine antagonists and glucocorticoids, which cannot completely prevent serious adverse reactions. Therefore, it is urgent to construct CRS models to identify the key factors contributing to CRS and to promptly block its occurrence, thereby improving the safety and feasibility of CAR-T cell therapy. Currently, only CN114032208A has constructed an in vitro cytokine storm model. However, since CRS involves a large number of cells and cytokines, in vitro cell models cannot realistically simulate the occurrence of CRS and cannot meet the model requirements of the medical field for studying inflammation induced by specific cytokines. Summary of the Invention

[0003] To address the aforementioned issues, this invention provides an animal model of CAR-T therapy for leukemia complicated by cytokine release syndrome, its preparation method, and its application. The main purpose is to solve the current problem of the difficulty in obtaining an in vitro model of CAR-T therapy for leukemia complicated by cytokine release syndrome.

[0004] To solve the above problems, the present invention adopts the following technical solution:

[0005] The first aspect of the present invention relates to The method for preparing an animal model of CAR-T therapy for leukemia complicated by cytokine release syndrome includes the following steps.

[0006] Preparation of CD19 CAR-T cells: peripheral blood mononuclear cell isolation, T cell magnetic bead sorting, T cell activation, lentivirus infection, and CAR-T cell expansion;

[0007] Constructing animal models of CRS following high- and low-dose CAR-T infusion:

[0008] Nalm-6 cell line was implanted into living animals to induce leukemia tumor burden, and living animals with leukemia tumor burden were obtained. CD19 CAR-T cell line was then injected into the living animals with leukemia tumor burden.

[0009] Regarding each cell line, the number of CD19 CAR-T cell lines was 50 to 200 times that of Nalm-6 cells. Furthermore, the number of Nalm-6 cells in the Nalm-6 cell line was 10. 5 ~10 6 The number of CD19 CAR-T cells in the CD19 CAR-T cell line was 10. 7 ~10 8 More specifically, the number of Nalm-6 cells in the Nalm-6 cell line is 10. 5 2×10 5 5×10 5 10 6 In either case, the number of CD19 CAR-T cell lines was 100 times that of Nalm-6 cells.

[0010] The live animals were SCID / Beige mice, which have severe combined immunodeficiency of SCID / Beige, lacking functional T cells, B cells and NK cells, but with normal macrophage and dendritic cell function.

[0011] The Nalm-6 cell line was prepared by the following method: Nalm6 cells in the logarithmic growth phase were taken, and the target cells were grown to the logarithmic phase. Lybrene was added to the culture medium, and GFP-luc double-labeled lentivirus solution was added to the culture dish. After shaking well, the dish was placed in an incubator for culture. GFP-positive Nalm6 cells were sorted by flow cytometry, and the obtained cells were expanded to obtain the GFP-luc double-labeled Nalm6 cell line.

[0012] Peripheral blood mononuclear cell isolation: Peripheral blood of the treatment subject is collected for mononuclear cell isolation;

[0013] T cell activation: Wash the culture flask once with X-VIVO 15 medium pre-added with IL-2, sodium pyruvate and glutamine, discard the medium, add autologous inactivated plasma, transfer the obtained T cells to the culture flask, mix well and put it into a carbon dioxide incubator for culture.

[0014] Lentiviral infection: Add lentiviral solution to the culture flask to be infected, add recombinant culture medium, mix gently, and then place in a carbon dioxide incubator for further culture. Centrifuge to obtain T cells after lentiviral infection.

[0015] CAR-T cell expansion: T cells are transferred into new culture flasks and cultured until the cell number is greater than 100%.

[0016] The second aspect of the present invention relates to The aforementioned method was used to prepare an animal model of CAR-T therapy for leukemia complicated by cytokine release syndrome.

[0017] The third aspect of the present invention relates to Novel applications of CAR-T therapy in animal models of leukemia complicated by cytokine release syndrome.

[0018] Firstly, animal models of CAR-T therapy for leukemia complicated by cytokine release syndrome (CRTS) can be used to screen drugs for treating leukemia. These models can be directly used to screen drugs effective in preventing and treating CRTS in leukemia patients, thus providing a basis for clinical drug screening. Of course, this application also frequently appears in commercial studies, the main purpose of which is also to study the therapeutic effects of different drugs on CAR-T therapy for leukemia complicated by CRTS.

[0019] Secondly, the application of animal models of CAR-T therapy for leukemia complicated by cytokine release syndrome in the preparation of drugs for the prevention and treatment of leukemia. These models are mainly used to screen for components that have therapeutic effects on CAR-T therapy for leukemia complicated by cytokine release syndrome. These components can serve as active ingredients in drugs, thereby enabling the corresponding drugs to treat CAR-T therapy for leukemia complicated by cytokine release syndrome.

[0020] The beneficial effects of this invention are:

[0021] A simple and reliable CRS mouse model was constructed. By using severely combined immunodeficient mice, inoculating them with high and low tumor cell burdens, and reinfusing different doses of CAR-T cells, an in vivo model that can best simulate clinical CRS was constructed, with excellent accuracy and consistency, providing research guidance for discovering more effective interventions in clinical practice. Attached Figure Description

[0022] Figure 1 In vivo imaging of mice with high and low tumor burden before and after CAR-T cell infusion;

[0023] Figure 2 Changes in mouse body weight after CAR-T cell infusion;

[0024] Figure 3 Changes in IL-6 levels in mice after CAR-T cell reinfusion;

[0025] Figure 4 Changes in IL-1β levels in mice after CAR-T cell reinfusion;

[0026] Figure 5 The fibrinogen levels in three groups of mice after CAR-T cell infusion;

[0027] Figure 6 HE staining of liver, lung, and brain tissues from three groups of mice after CAR-T infusion;

[0028] Figure 7 The infiltration of CAR-T cells in the liver, lungs, brain, and spleen of mice in each group after reinfusion;

[0029] Figure 8 1*10 5 2*10 5 5*10 5 Tumor burden in three groups. Detailed Implementation

[0030] The invention will now be further described with reference to the accompanying drawings.

[0031] Step 1: Preparation of CD19 CAR-T cells

[0032] A. Isolation of peripheral blood mononuclear cells

[0033] 1) Take 100ml of fresh blood from the patient, disinfect it with ultraviolet light and alcohol, place it in the center of the biosafety cabinet, and divide it into two 50ml centrifuge tubes A and B. Take 1ml of blood sample and smear it on a plate for bacterial testing.

[0034] 2) Centrifuge tubes A and B at 400g for 8 minutes, collect the supernatant plasma, and transfer it to two 50ml centrifuge tubes C and D. Inactivate the plasma in a 56℃ water bath for 30 minutes, cool to room temperature, and then transfer to a biosafety cabinet. Add 20ml of physiological saline to each of centrifuge tubes A and B, and gently mix.

[0035] 3) Add 15ml of Ficoll lymphocyte separation medium to each of two 50ml centrifuge tubes E and F. Tilt the tubes and slowly add the cell suspension from centrifuge tubes A and B along the tube wall to centrifuge tubes E and F. Gradually straighten the centrifuge tubes during the addition process, always keeping the mixture on the Ficoll liquid surface, with a clear and stable interface.

[0036] 4) Centrifuge tubes E and F at 400g for 18 minutes (increase speed 1, decrease speed 0), carefully and steadily remove them, collect the junctional cell layer in each centrifuge tube with a Pasteur dropper, and distribute them evenly into two 50ml centrifuge tubes G and H.

[0037] 5) Add physiological saline to centrifuge tubes G and H to a final volume of 50 ml, tighten the caps, invert the tubes several times, and centrifuge at 130 g for 5 minutes at room temperature. After centrifugation, observe the contents. If there are few residual red blood cells, proceed directly to the next step. If there are many residual red blood cells, lyse the red blood cells once, and centrifuge again at 130 g for 5 minutes at room temperature.

[0038] 6) Move centrifuge tubes G and H to the center of the biosafety cabinet, discard the supernatant, resuspend the precipitate with X-VIVO 15 medium, and centrifuge at 130g for 5 minutes at room temperature.

[0039] BT Cell Magnetic Bead Sorting

[0040] 1) After centrifugation in the previous step, discard the supernatant, resuspend the pellet in X-VIVO 15 medium, take a small amount of cell suspension for trypan blue counting, and centrifuge the remaining cell suspension at 300g for 10 min, discarding the supernatant. Based on the cell count results, resuspend the pellet in buffer to a concentration of 2.5 × 10⁻⁶ cells / mL. 8 / ml.

[0041] 2) Take 40 μl of cell suspension (containing 1×10⁻⁶ cells / mL). 7 Add 10 μL of Pan T Cell Biotin Antibody Cocktail to a centrifuge tube, mix well, and incubate at 4°C for 5 min.

[0042] 3) Add 30 μL of buffer, then add 20 μL of PanT Cell MicroBead Cocktail, mix well, and incubate at 4°C for 10 min.

[0043] 4) Fix an MS separation column on a magnetic rack, rinse once with 0.5 ml buffer, replace the centrifuge tube below, slowly add the cell suspension labeled with magnetic beads into the separation column for separation, collect the liquid passing through the separation column, centrifuge at 130 g for 5 minutes at room temperature, pour off the supernatant, and the precipitate is T cells.

[0044] CT cell activation

[0045] 1) Take one T25 cell culture flask pre-coated (at least 24 hours in advance) with CD3 and CD28, aspirate all coating solution, wash once with 10 ml of X-VIVO 15 medium pre-added with IL-2, sodium pyruvate and glutamine, aspirate and then add 10 ml of medium and 1 ml of autologous inactivated plasma.

[0046] 2) Take a small amount of culture medium, resuspend the T cell pellet, and transfer it into a culture flask to a final concentration of 1×10⁻⁶. 6 / ml. Gently mix and place in a CO2 incubator to begin incubation, which is recorded as day 0 (D0).

[0047] D. Lentiviral infection

[0048] 1) D2, take 12×10 7 The lentivirus was thawed at room temperature. Based on the T cell culture count results, 4 × 10⁴ cells were collected. 7 T cells were added to a new culture flask for infection. The remaining cells were cultured further and used as a control group for CAR-T cell function testing.

[0049] 2) After the lentivirus solution has returned to room temperature, add it to the culture flasks to be infected. Add recombinant culture medium and adjust the cell density to 1×10⁻⁶. 6 / ml. Gently mix and then place in a CO2 incubator for further incubation.

[0050] 3) Observe and count the cells daily, and replenish fluids as needed to maintain a cell density of 2 × 10⁻⁶ cells / day. 6 cells / ml.

[0051] 4) D5, transfer the cell culture medium into a centrifuge tube, centrifuge at 130g for 5 minutes at room temperature, discard the supernatant, and wash the precipitate twice with an appropriate amount of physiological saline.

[0052] E.CAR-T cell expansion

[0053] 1) Transfer the washed T cells into a new culture flask and adjust the density to 5 × 10⁶ cells / year. 5 Up to 2×10 6 / ml, mix well, take 1ml of cell solution and plate it for bacterial inspection, and put the rest into a carbon dioxide incubator for further culture.

[0054] 2) Observe and count the cells daily, and rotate the bottles as needed. When the cell count is greater than 1×10⁻⁶, [the cells are ready for rotation]. 8 An appropriate amount of cells were taken for killing experiments, factor release experiments, PCR detection, and flow cytometry detection.

[0055] Step 2: Constructing mouse models with high and low leukemia tumor burden

[0056] Fifteen specific pathogen-free grade SCID / Beige male rats, 8 weeks old and weighing 20-22g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. They were randomly divided into four groups: a healthy control group (n=3), a low tumor burden group + low CAR-T dose group (low+low, n=4), a high tumor burden group + low CAR-T dose group (high+low, n=4), and a high tumor burden group + high CAR-T dose group (high+high, n=4). After one week of acclimatization in an isolated laboratory setting, they were injected with 1*10n Nalm-6 cell line via tail vein. 5 (Low tumor burden group), 1*10 6(High tumor burden group) Small animals were photographed on days 7, 14 and 18 after tumor implantation to assess tumor formation.

[0057] Step 3: Constructing CRS mouse models after high- and low-dose CAR-T infusion

[0058] On day 18 after tumor implantation, the difference in tumor burden between high and low doses was clearly observed using in vivo imaging technology in small animals. The low-CAR-T dose group received 1*10 human CD19 CAR-T cells via tail vein injection. 7 One high-CAR-T dose group received 10 human CD19 CAR-T cells via tail vein injection. 8 At 12h, 24h, 48h, 72h, 96h, 120h, 144h, and 168h after CAR-T cell infusion, blood samples were collected from the retroorbital vein, and body weight was measured and a weight change trend graph was plotted. Blood samples were centrifuged at 4℃, 3000 rpm for 15 minutes, and the supernatant plasma was collected to detect IL-6 and IL-1β. At 168h after CAR-T cell infusion, mice were anesthetized with 1% sodium pentobarbital, and blood was collected from the eyeballs. Liver, kidney, lung, brain, and spleen were collected for HE pathological staining to observe inflammatory infiltration. Immunohistochemical staining with human CD8 antibody was performed to observe CAR-T cell infiltration.

[0059] Figure 2 This indicates that the body has developed CRS, which presents a state of high inflammation. This leads to a high metabolic rate, resulting in weight loss. When the level of CRS decreases, the weight will increase again. Figures 3-4 This indicates that IL-6 and IL-1β are typical cytokines in CAR-T therapy for leukemia complicated with CRS, and both play a pro-inflammatory role. Figure 5 This indicates that elevated fibrinogen levels represent a state of high inflammation in the body. Figure 6 The results of HE staining show that the arrows on the liver sections indicate CAR-T cell infiltration, suggesting the activation and expansion of CAR-T cells in vivo. Figure 7 Further immunohistochemical analysis revealed the distribution and infiltration of CAR-T cells in various organs, with arrows confirming in vivo activation and expansion of CAR-T cells.

[0060] CRS occurs because the infusion of specifically targeted CAR-T cells leads to their activation and proliferation by tumor cells in vivo, releasing large amounts of cytokines and chemokines, such as IL-6, IFN-γ, and GM-CSF. These cytokines further activate bystander immune cells such as monocytes and macrophages, further releasing cytokines such as IL-1β, IL-6, and IL-10. However, due to the limited clinical treatment options for CRS and the difficulty in establishing in vitro models, the specific pathogenesis and process of CRS remain unclear. The SCID-Beige mouse used in this invention is a severely combined immunodeficient mouse, lacking functional T cells, B cells, and NK cells, but with intact macrophage and dendritic cell function, exhibiting a high tumorigenesis rate and suitability for xenotransplantation. CAR-T cell expansion and tumor burden are most closely related to the occurrence of CRS. Therefore, this invention constructs a high-low leukemia tumor burden model and infuses high- and low-dose CAR-T cells. This allows CAR-T cells to be massively activated and expanded upon contact with tumor cells, releasing different levels of cytokines, thereby activating macrophages in mice and inducing CRS. On day 21 after NSG mice were injected with the corresponding number of Nalm6-luc cells via the tail vein, tumor burden was detected using a small animal in vivo imaging system, revealing a tumor burden of 1*102. 5 2*10 5 5*10 5 Tumor burden increased with the number of Nalm6-luc cells injected, indicating a positive correlation between high and low tumor burden models and the number of cells injected via the tail vein at model establishment. Accordingly, further increasing the number of cells infused via the tail vein at model establishment may help increase the difference between the high and low tumor burden groups.

[0061] Those skilled in the art will appreciate that various modifications to the above embodiments can be made without departing from the overall spirit and concept of the present invention. All such modifications fall within the protection scope of the present invention. The protection scheme of the present invention is defined by the appended claims.

Claims

1. A method for preparing an animal model of CAR-T therapy for leukemia complicated by cytokine release syndrome, characterized in that, Includes the following steps Preparation of CD19 CAR-T cells: peripheral blood mononuclear cell isolation, T cell magnetic bead sorting, T cell activation, lentivirus infection, and CAR-T cell expansion; Constructing animal models of CRS following high- and low-dose CAR-T infusion: Nalm-6 cell line was implanted into living animals to induce leukemia tumor burden, and living animals with leukemia tumor burden were obtained. CD19 CAR-T cell line was then injected into the living animals with leukemia tumor burden.

2. The method for preparing an animal model of CAR-T therapy for leukemia complicated with cytokine release syndrome according to claim 1, characterized in that, The number of CD19 CAR-T cells in the CD19 CAR-T cell line is 50 to 200 times that of Nalm-6 cells in the Nalm-6 cell line.

3. The method for preparing an animal model of CAR-T therapy for leukemia complicated with cytokine release syndrome according to claim 2, characterized in that, The number of Nalm-6 cells in the Nalm-6 cell line was 10. 5 -10 6 The number of CD19 CAR-T cells in the CD19 CAR-T cell line was 10. 7 -10 8 .

4. The method for preparing an animal model of CAR-T therapy for leukemia complicated with cytokine release syndrome according to claim 3, characterized in that, The number of Nalm-6 cells in the Nalm-6 cell line was 10. 5 2×10 5 5×10 5 10 6 Any one of them.

5. The method for preparing an animal model of CAR-T therapy for leukemia complicated with cytokine release syndrome according to claim 4, characterized in that, The number of CD19 CAR-T cells in the CD19 CAR-T cell line was 100 times that of Nalm-6 cells in the Nalm-6 cell line.

6. The method for preparing an animal model of CAR-T therapy for leukemia complicated with cytokine release syndrome according to claim 1, characterized in that, The live animal was an SCID / Beige mouse; The steps for constructing the Nalm-6 cell line are as follows: Take Nalm6 cells in the logarithmic growth phase, wait for the target cells to grow to the logarithmic phase, add GFP-luc double-labeled lentivirus solution to the culture medium lybrene, shake well and place in an incubator for culture, sort GFP-positive Nalm6 cells by flow cytometry, and expand the obtained cells to obtain the GFP-luc double-labeled Nalm6 cell line.

7. The method for preparing an animal model of CAR-T therapy for leukemia complicated with cytokine release syndrome according to claim 1, characterized in that, Peripheral blood mononuclear cell isolation: Peripheral blood of the treatment subject is collected for mononuclear cell isolation; T cell activation: Wash the culture flask once with X-VIVO 15 medium pre-added with IL-2, sodium pyruvate and glutamine, discard the medium, add autologous inactivated plasma, transfer the obtained T cells to the culture flask, mix well and put it into a carbon dioxide incubator for culture. Lentiviral infection: Add lentiviral solution to the culture flask to be infected, add recombinant culture medium, mix gently, and then place in a carbon dioxide incubator for further culture. Centrifuge to obtain T cells after lentiviral infection. CAR-T cell expansion: T cells were transferred into new culture flasks and cultured until the cell count exceeded 102. 8 .

8. The application of the method for preparing an animal model of CAR-T therapy for leukemia complicated with cytokine release syndrome as described in claim 1 in screening drugs for the treatment of leukemia.

9. The application of the method for preparing an animal model of CAR-T therapy for leukemia complicated with cytokine release syndrome as described in claim 1 in the preparation of drugs for the prevention and treatment of leukemia.

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