Animal model of car-t treatment for leukemia complicated with cytokine release syndrome, and preparation method therefor and use thereof

Abstract

The present invention relates to the field of leukemia research. Specifically disclosed are an animal model of CAR-T treatment for leukemia complicated with cytokine release syndrome, and a preparation method therefor and the use thereof. The method for preparing the animal model of CAR-T treatment for leukemia complicated with cytokine release syndrome comprises preparing CD19 CAR-T cells: separating a peripheral blood mononuclear cell, performing magnetic bead sorting of T cells, activating the T cells, and performing lentivirus infection and CAR-T cell amplification; and constructing CRS animal models with high-dose and low-dose CAR-T reinfusion: implanting a Nalm-6 cell line into a living animal body, loading a leukemia tumor, obtaining a living animal having a leukemia tumor burden, and injecting the CD19 CAR-T cell line into the living animal having the leukemia tumor burden. Provided is the new use of the animal model of CAR-T treatment for leukemia complicated with cytokine release syndrome. In the present invention, an in-vivo model capable of simulating clinical CRS is constructed by means of using mice having severe combined immunodeficiency, inoculating with high and low tumor cell burdens and reinfusing different doses of CAR-T cells, which provides guidance for scientific research on developing more effective clinical intervention measures.

Description

Animal model of CAR-T therapy for leukemia complicated with cytokine release syndrome, its preparation method, and application Technical Field

[0001] The present invention relates to the research field of leukemia, and in particular to an animal model for CAR-T treatment of leukemia complicated by cytokine release syndrome, as well as a preparation method and application thereof. Background Art

[0002] CAR-T cells have demonstrated promising therapeutic effects for leukemia, particularly lymphocytic leukemia. Chimeric antigen receptor T cells have become an effective treatment for relapsed / refractory hematologic malignancies. However, this remarkable efficacy is also accompanied by the development of 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 or higher. This syndrome manifests as significant elevation of IL-6, vascular leakage, hypotension, pulmonary edema, cardiac insufficiency, hepatic and renal failure, neurotoxicity, disseminated intravascular coagulation, and other multi-organ failure, even leading to death. This syndrome significantly impacts the efficacy and prognosis of CAR-T therapy. Current clinical interventions for CRS are limited, primarily cytokine antagonists and glucocorticoids, which cannot prevent the occurrence of severe adverse reactions. Therefore, it is urgent to identify key factors that contribute to CRS development through the development of CRS models and to promptly address 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, due to the large number of cells and cytokines involved in CRS, the in vitro cell model is difficult to truly simulate the occurrence of CRS and cannot meet the model requirements for the medical field to study inflammation induced by specific cytokines.

[0003] Summary of the Invention

[0004] In response to the above problems, the present invention provides an animal model for CAR-T treatment of leukemia complicated by cytokine release syndrome, as well as its preparation method and application, mainly to solve the problem that it is currently difficult to obtain an in vitro CAR-T treatment model for leukemia complicated by cytokine release syndrome.

[0005] In order to solve the above problems, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention relates to a method for preparing an animal model for CAR-T treatment of leukemia complicated with cytokine release syndrome, comprising the following steps:

[0007] Preparation of CD19 CAR-T cells: peripheral blood mononuclear cell isolation, T cell magnetic bead sorting, T cell activation, lentiviral infection, and CAR-T cell expansion;

[0008] Construction of CRS animal models after high and low doses of CAR-T infusion:

[0009] The Nalm-6 cell line is implanted into a living animal to perform leukemia tumor loading, thereby obtaining a living animal with leukemia tumor loading, and the CD19 CAR-T cell line is injected into the living animal with leukemia tumor loading.

[0010] In terms of each cell line, the number of CD19 CAR-T cell lines is 50 to 200 times that of Nalm-6 cells. 5 ~10 6 ; The number of CD19 CAR-T cells in the CD19 CAR-T cell line is 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 any of the above studies, the number of CD19 CAR-T cell lines was 100 times that of Nalm-6 cells.

[0011] The living animal is a SCID / Beige mouse, which has SCID / Beige severe combined immunodeficiency, lacks functional T cells and B cells, and has NK cell functional defects, but has healthy macrophage and dendritic cell functions.

[0012] The Nalm-6 cell line was prepared by the following method: taking a Nalm6 cell line in logarithmic growth phase, waiting for the target cells to grow to the logarithmic phase, adding lybrene to the culture medium, adding GFP-luc double-labeled lentivirus solution to the culture dish, shaking evenly and then culturing in an incubator, and culturing GFP-positive Nalm6 cells by flow cytometry. The obtained cells were expanded and cultured to obtain a GFP-luc double-labeled Nalm6 cell line.

[0013] Peripheral blood mononuclear cell separation: peripheral blood of the treated subject is collected and mononuclear cells are separated;

[0014] T cell activation: The culture flask was washed once with X-VIVO 15 medium pre-added with IL-2, sodium pyruvate, and glutamine. After aspiration, the culture medium was added and autologous inactivated plasma was added. The obtained T cells were transferred to the culture flask, mixed, and cultured in a CO2 incubator.

[0015] Lentivirus infection: Add the lentivirus solution to the culture flask to be infected, add reconstitution culture medium, mix gently, and place in a CO2 incubator for further culture. Centrifuge to obtain lentivirus-infected T cells.

[0016] CAR-T cell expansion: Transfer T cells into new culture flasks and culture until the cell number is greater than .

[0017] The second aspect of the present invention relates to an animal model for treating leukemia complicated by cytokine release syndrome using CAR-T prepared by the aforementioned method.

[0018] The third aspect of the present invention relates to a new use of CAR-T in the treatment of animal models of leukemia complicated by cytokine release syndrome.

[0019] First, the application of animal models of CAR-T therapy for leukemia complicated by cytokine release syndrome in the screening of drugs for leukemia treatment. This model can be directly used to screen for drugs that are effective in preventing and treating CAR-T therapy for leukemia complicated by cytokine release syndrome, thereby providing a basis for clinical screening of useful drugs. Of course, this application also frequently appears in some commercial research studies, whose main purpose is also to study the therapeutic effects of different drugs on CAR-T therapy for leukemia complicated by cytokine release syndrome.

[0020] Second, the application of animal models of CAR-T therapy for leukemia complicated by cytokine release syndrome in the development of drugs for the prevention and treatment of leukemia. This model is primarily used to screen for components with therapeutic effects on CAR-T therapy for leukemia complicated by cytokine release syndrome. These components can be used as active ingredients in drugs, thereby enabling corresponding drugs to treat CAR-T therapy for leukemia complicated by cytokine release syndrome.

[0021] The beneficial effects of the present invention are:

[0022] A simple and reliable CRS mouse model has been constructed. By using severe combined immunodeficient mice, inoculating them with high and low tumor cell loads, and infusing them with different doses of CAR-T cells, an in vivo model was constructed that maximally simulates clinical CRS, demonstrating excellent accuracy and consistency, providing scientific guidance for the discovery of more effective clinical interventions. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 shows in vivo imaging of mice with high and low tumor burdens before and after CAR-T cell infusion;

[0024] Figure 2 shows the weight changes of mice after CAR-T cell infusion;

[0025] Figure 3 shows the changes in IL-6 levels in mice after CAR-T cell infusion;

[0026] Figure 4 shows the changes in IL-1β levels in mice after CAR-T cell infusion;

[0027] Figure 5 shows the fibrinogen levels of the three groups of mice after CAR-T cell infusion;

[0028] Figure 6 shows HE staining of liver, lung, and brain tissues of three groups of mice after CAR-T transfusion;

[0029] Figure 7 shows the CAR-T cell infiltration in the liver, lung, brain, and spleen of each group of mice after reinfusion;

[0030] Figure 8 is 1*10 5 ,2*10 5 ,5*10 5 Tumor burden in three groups. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to the accompanying drawings.

[0032] Step 1: Preparation of CD19 CAR-T cells

[0033] A. Peripheral Blood Mononuclear Cell Isolation

[0034] 1) Take a 100ml bag of fresh single-collected blood from the patient, irradiate with UV light, disinfect with alcohol, place it in the center of the biosafety cabinet, and divide it into two 50ml centrifuge tubes A and B. Take 1ml of the blood sample and smear it on a plate for bacterial detection.

[0035] 2) Centrifuge tubes A and B at 400g for 8 minutes. Aspirate the upper plasma layer and transfer it to two 50ml centrifuge tubes C and D. Inactivate the plasma in a 56°C water bath for 30 minutes. Cool to room temperature and transfer to a biosafety cabinet. Add 20ml of normal saline to each tube A and B and gently pipette to mix thoroughly.

[0036] 3) Add 15 ml of Ficoll lymphocyte separation buffer to each of two 50 ml centrifuge tubes E and F. Tilt the tubes and slowly add the cell suspension from tubes A and B along the tube walls to tubes E and F. During the addition process, gradually upright the tubes to ensure that the mixture is above the Ficoll level and the interface is clear and stable.

[0037] 4) Centrifuge tubes E and F at 400 g for 18 minutes (speed increase: 1, speed decrease: 0). Carefully and steadily remove the tubes. Use a Pasteur pipette to collect the junctional cell layer from each tube and divide evenly between two 50 ml tubes G and H.

[0038] 5) Fill both tubes G and H to 50 ml with normal saline, tighten the caps, invert several times, and centrifuge at 130 g for 5 minutes at room temperature. After centrifugation, inspect the contents. If few residual red blood cells are present, proceed directly to the next step. If many remain, lyse the red blood cells once and centrifuge again at 130 g for 5 minutes at room temperature.

[0039] 6) Move centrifuge tubes G and H to the center of the safety cabinet, discard the supernatant, resuspend the pellet in X-VIVO 15 medium, and centrifuge at 130g for 5 minutes at room temperature.

[0040] BT cell magnetic bead sorting

[0041] 1) After the centrifugation in the previous step, discard the supernatant and resuspend the pellet with X-VIVO 15 medium. Take a small amount of cell suspension and count it with trypan blue. Centrifuge the remaining cell suspension at 300g for 10 minutes and discard the supernatant. Based on the cell count results, resuspend the pellet with buffer to a concentration of 2.5×10 8 / ml.

[0042] 2) Take 40 μl of cell suspension (containing 1×10 7 PBMC) were placed in a centrifuge tube, 10 μL of Pan T Cell Biotin Antibody Cocktail was added, mixed and incubated in a 4°C refrigerator for 5 min.

[0043] 3) Add 30 μL of buffer, then add 20 μL of Pan T Cell MicroBead Cocktail, mix well, and incubate in a 4°C refrigerator for 10 min.

[0044] 4) Mount an MS separation column on a magnetic rack and rinse once with 0.5 ml of buffer. After replacing the centrifuge tube, slowly add the magnetic bead-labeled cell suspension to the separation column for sorting. Collect the liquid after passing through the separation column and centrifuge at 130 g for 5 minutes at room temperature. Pour off the supernatant to obtain the T cell pellet.

[0045] CT cell activation

[0046] 1) Take a T25 cell culture flask pre-coated (at least 24 hours in advance) with CD3 and CD28, aspirate all the coating solution, wash once with 10 ml of X-VIVO 15 medium pre-added with IL-2, sodium pyruvate, and glutamine, aspirate and add 10 ml of culture medium and 1 ml of autologous inactivated plasma.

[0047] 2) Aspirate 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 After gentle mixing, the cells were placed in a CO2 incubator and cultured, which was designated as day 0 (D0).

[0048] D. Lentiviral infection

[0049] 1) D2, take 12×10 7 The lentivirus was thawed at room temperature. According to the T cell culture count results, 4×10 7T cells were added to a new culture flask for infection. The remaining cells were cultured and used as a control group for CAR-T cell function testing.

[0050] 2) After the lentiviral solution returns to room temperature, add it to the culture flask to be infected. Add reconstitution culture medium and adjust the cell density to 1×10 6 After gentle mixing, place the plate in a CO2 incubator for further culture.

[0051] 3) Observe and count cells every day, replenish fluids in time, and maintain the cell density at 2×10 6 cells / ml.

[0052] 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 pellet twice with an appropriate amount of physiological saline.

[0053] E.CAR-T cell expansion

[0054] 1) Transfer the washed T cells into a new culture flask and adjust the density to 5×10 5 to 2×10 6 / ml, mix well and take 1ml of cell fluid to spread on the plate to check bacteria, and put the rest into the carbon dioxide incubator to continue culturing.

[0055] 2) Observe and count the cells every day and transfer the bottles as needed. 8 , take appropriate amount of cells for killing experiment, factor release experiment, PCR detection and flow cytometry detection.

[0056] Step 2: Construct mouse models with high and low leukemia tumor burden

[0057] Fifteen specific pathogen-free SCID / Beige male mice, 8 weeks old and weighing 20-22 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. and randomly divided into 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 adaptive feeding in the isolation bag laboratory, 1*10 Nalm-6 cells were injected into the tail vein. 5 (low tumor burden group), 1*10 6 (High tumor burden group), in vivo imaging of small animals was taken on D7, D14, and D18 days after tumor implantation to evaluate tumor formation.

[0058] Step 3: Constructing CRS mouse models after high- and low-dose CAR-T infusion

[0059] On day 18 after tumor inoculation, the difference in tumor burden between high and low groups was clearly observed by small animal in vivo imaging technology. The low-dose CAR-T group was injected with 1*10 human CD19 CAR-T cells via tail vein. 7 The high-dose CAR-T group received tail vein injection of 1*10 human CD19 CAR-T cells. 8 Blood was collected from the retroorbital vein 12, 24, 48, 72, 96, 120, 144, and 168 hours after CAR-T cell infusion, and body weight was measured. Weight change trends were plotted. Blood samples were centrifuged at 3000 rpm for 15 minutes at 4°C, and the supernatant plasma was collected for analysis of IL-6 and IL-1β. At 168 hours after CAR-T cell infusion, mice were anesthetized with 1% sodium pentobarbital, and eyeballs were enucleated for blood collection. Organs such as the liver, kidney, lung, brain, and spleen were also harvested for HE staining to assess inflammatory infiltration. Immunohistochemical staining with a human CD8 antibody was also performed to assess CAR-T cell infiltration.

[0060] Figure 2 shows that the development of CRS in the body presents a hyperinflammatory state, which leads to high metabolic activity and thus weight loss. When the degree of CRS subsides, weight will increase again. Figures 3 and 4 show that IL-6 and IL-1β are typical cytokines in leukemia patients with CRS, both of which play a proinflammatory role. Figure 5 shows that elevated fibrinogen levels represent a hyperinflammatory state in the body. Figure 6 shows that in the hematoxylin and eosin (HE) staining, the arrows in the liver section indicate CAR-T cell infiltration, indicating the activation and expansion of CAR-T cells in vivo. Figure 7 shows that the distribution and infiltration of CAR-T cells in various organs were further detected by immunohistochemistry, and the arrows confirm the activation and expansion of CAR-T cells in vivo.

[0061] The occurrence of CRS is due to the activation and massive amplification of tumor cells in the body after the specific targeted CAR-T cells are reinfused, releasing a large number of cytokines and chemokines, such as IL-6, IFN-γ, GM-CSF, etc. These cytokines further activate bystander immune cells such as monocytes and macrophages, and further release cytokines such as IL-1β, IL-6, and IL-10. However, due to the limited clinical treatment of CRS and the difficulty in establishing in vitro models, the specific pathogenesis and occurrence process of CRS have not yet been elucidated. The SCID-Beige mouse used in the present invention is a severe combined immunodeficiency mouse that lacks functional T cells and B cells and NK cell function defects, but has sound macrophage and dendritic cell functions, has a high tumor formation rate and is suitable for xenotransplantation. CAR-T cell expansion and tumor burden are closely related to the occurrence of CRS. Therefore, the present invention constructs high and low leukemia tumor burden models and infuses high and low doses of CAR-T cells. After the CAR-T cells come into contact with tumor cells, they are activated and expanded to release different levels of cytokines, thereby activating macrophages in mice and causing CRS. On the 21st day after the tail vein injection of the corresponding number of Nalm6-luc cells, the tumor burden was detected by the small animal in vivo imaging system, and it was found that 1*10 5 ,2*10 5 ,5*10 5 Tumor burden in all three groups increased with increasing numbers of injected Nalm6-luc cells. Therefore, the high and low tumor burden models were positively correlated with the number of cells injected via the tail vein during modeling. Accordingly, further increasing the number of cells injected via the tail vein during modeling may help to increase the difference between the high and low tumor burden groups.

[0062] It will be apparent to those skilled in the art that various modifications to the above embodiments may be made without departing from the overall spirit and concept of the present invention. Such modifications fall within the scope of protection of the present invention. The protection scheme of the present invention shall be subject to the claims appended hereto.

Claims

1. A method for preparing an animal model of CAR-T for the treatment of leukemia complicated with cytokine release syndrome, characterized in that: The following steps are included Preparation of CD19 CAR-T cells: isolation of peripheral blood mononuclear cells, magnetic bead sorting of T cells, activation of T cells, lentiviral infection, and expansion of CAR-T cells; Construction of CRS animal model after high and low dose CAR-T infusion: The Nalm-6 cell line is implanted into living animals to perform leukemia tumor load, and living animals with leukemia tumor load are obtained, and the CD19 CAR-T cell line is injected into the living animals with leukemia tumor load.

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

3. The method for preparing an animal model of CAR-T for treating 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 is 10 5 ~10 6 ; The number of CD19 CAR-T cells in the CD19 CAR-T cell line is 10 7 ~10 8 .

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

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

7. The method for preparing an animal model of CAR-T for treating leukemia complicated with cytokine release syndrome according to claim 1, characterized in that: Peripheral blood mononuclear cell separation: Take peripheral blood from the subject and separate mononuclear cells; T cell activation: The culture flask was pre-added with X-VIVO 15 medium containing IL-2, sodium pyruvate, and glutamine, washed once, then the medium was added after aspiration, and autologous inactivated plasma was added. The obtained T cells were transferred to the culture flask, mixed, and cultured in a carbon dioxide incubator; Lentivirus infection: Add the lentivirus solution to the culture bottle to be infected, add the recombinant culture medium, mix gently, and place in a carbon dioxide incubator for further culture. Centrifuge to obtain the T cells infected with the lentivirus; CAR-T cell expansion: Transfer T cells into new culture flasks and culture until the cell number is greater than 10 8 .

8. An animal model for treating leukemia complicated with cytokine release syndrome using CAR-T prepared by any one of the preparation methods of claims 1 to 7.

9. Use of the animal model of CAR-T for treating leukemia complicated with cytokine release syndrome in claim 9 in screening drugs for treating leukemia.

10. Use of the animal model of CAR-T for treating leukemia complicated with cytokine release syndrome in claim 9 in the preparation of drugs for preventing and treating leukemia.

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