Use of ULK1 inhibitors to reduce CAR-T cell exhaustion, decrease level of activation in vitro

Abstract

The application discloses application of a ULK1 inhibitor in reducing CAR-T cell exhaustion and reducing activation level in vitro. The application provides a CAR-T cell based on a ULK1 inhibitor and a preparation method, and application of the CAR-T cell pretreated by the ULK1 inhibitor in resisting tumors. The method is simple and easy to implement, the tumor is treated by adding the ULK1 inhibitor in a CAR-T cell culture process, the persistence of immune cells and the killing function can be obviously increased, and the refractory recurrence problem in the cell treatment process is solved to a great extent, and the survival period of a patient is prolonged.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of ULK1 inhibitors in reducing CAR-T cell depletion and lowering activation levels in vitro. Background Technology

[0002] Chimeric antigen receptor T-cell therapy (CAR-T) is used to treat refractory or relapsed leukemia and lymphoma. However, some patients still fail to achieve complete remission or relapse after receiving CAR-T immunotherapy. Current research suggests that CAR-T cell depletion and loss of effector function in vivo is one of the important reasons for relapse. Therefore, improving the persistence of CAR-T cells in vivo has become an urgent technical problem to be solved.

[0003] Some small molecule inhibitors, when co-cultured with CAR-T cells in vitro, can reduce CAR-T cell exhaustion in vivo. For example, patent application CN113462649A discloses the application of MEK inhibitors in reducing CAR-T cell exhaustion and terminal differentiation. The MEK inhibitor mentioned is trametinib. Adding MEK inhibitors during CAR-T cell culture can reduce CAR-T cell exhaustion and terminal differentiation, thereby improving the persistence of CAR-T cells in vivo and enhancing their anti-tumor effect. Another example is patent application CN110157680A, which discloses reducing CAR-T cell activation signal transduction by adding the tyrosine kinase inhibitor dasatinib, inhibiting CAR-T cell terminal differentiation, increasing the proportion of naive T cells and central memory T cells in CAR-T cell products, and simultaneously inhibiting the tendency of CAR-T cells to exhaust.

[0004] Autophagy is a highly conserved, lysosomal-based intracellular degradation pathway in eukaryotes, playing a crucial role in maintaining cellular and organismal homeostasis. Recent studies have increasingly demonstrated that autophagy is involved in various biological processes of immune cells, including survival, apoptosis, differentiation, activation, and effector function. When CD8-positive T cells encounter antigens, defects in autophagy facilitate the execution of effector functions, and the memory function of CD4-positive T cells also depends on changes in mitochondrial function and lipid metabolism induced by autophagy. Therefore, autophagy is a complex but promising target in immunotherapy.

[0005] The ULK1 inhibitor SBI-0206965, CAS number 1884220-36-3, has the structural formula shown in Formula I.

[0006] Summary of the Invention

[0007] To address the aforementioned shortcomings in the existing technology, this invention provides the application of ULK1 inhibitors in reducing CAR-T cell depletion and lowering activation levels in vitro.

[0008] This invention first provides the application of ULK1 inhibitors in reducing CAR-T cell exhaustion and lowering activation levels in vitro. Preferably, the ULK1 inhibitor is SBI-0206965. The concentration of SBI-0206965 used is 3 μM. Preferably, the ULK1 inhibitor is added to the culture medium on days 6 to 9 of CAR-T cell in vitro culture.

[0009] This invention also provides a method for in vitro CAR-T cell culture, wherein a ULK1 inhibitor is added to the culture medium on days 6 to 9 of CAR-T cell in vitro culture. Preferably, the ULK1 inhibitor is SBI-0206965. The concentration of SBI-0206965 used is 3 μM.

[0010] This invention also provides the application of ULK1 inhibitors in the preparation of anticancer drugs. The ULK1 inhibitor is used in combination with CAR-T cells, and the ULK1 inhibitor enhances the anticancer effect by reducing CAR-T cell depletion and lowering activation levels. The ULK1 inhibitor is SBI-0206965.

[0011] The beneficial effects of this invention are as follows: This invention provides a CAR-T cell based on a ULK1 inhibitor and a preparation method thereof, as well as the application of CAR-T cells pretreated with a ULK1 inhibitor in anti-tumor therapy. This method is simple and easy to implement. By adding a ULK1 inhibitor during the CAR-T cell culture process to treat tumors, the persistence and killing function of immune cells can be significantly increased, which largely solves the problem of refractory and relapsed treatment in the cell therapy process and prolongs the survival of patients. Attached Figure Description

[0012] Figure 1 Differentiation ratios and statistics of CAR-T cell subsets cultured for ULK1 inhibitors.

[0013] Figure 2 Activation rate and statistical graph of CAR-T cells cultured with ULK1 inhibitor.

[0014] Figure 3 Figure 1 shows the results of flow cytometry analysis of changes in the expression of PD1, LAG3, and TIM3 depletion markers and apoptosis levels in CAR-T cells cultured with ULK1 inhibitors.

[0015] Figure 4Figure 1. Flow cytometry results of CD19 target antigen expression on the surface of CD19 cells after treatment of B-cell acute lymphoblastic leukemia cell lines with ULK1 inhibitor.

[0016] Figure 5 A flow cytometry analysis of apoptosis results after treatment of the B-cell acute lymphoblastic leukemia cell line (Nalm-6) with a ULK1 inhibitor.

[0017] Figure 6 A flow cytometry analysis of apoptosis results after treatment of the B-cell acute lymphoblastic leukemia cell line (BALL-1) with a ULK1 inhibitor.

[0018] Figure 7 In vivo imaging results of CAR-T cells infused into mice 3 days after treatment with ULK1 inhibitor.

[0019] Figure 8 The curve showing the change in average fluorescence intensity in mice after 3 days of treatment with ULK1 inhibitors for CAR-T cells.

[0020] Figure 9 Survival curves of CAR-T cells treated with ULK1 inhibitor for 3 days before infusion into mice.

[0021] Figure 10 The proportion of CAR-T cells in vivo and the expression of differentiation and exhaustion indicators were determined after CAR-T cells were treated with ULK1 inhibitor for 3 days and then infused into mice.

[0022] In each figure, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. NS stands for No significance, meaning there was no statistically significant difference. Detailed Implementation

[0023] 1. Experimental Materials

[0024] HEK293T cells, ALL cell line Nalm-6, and ALL cell line BALL-1 were imported and preserved by the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Competent DH5α cells were purchased from Nanjing Novizan Biotechnology Co., Ltd. Lentiviral vectors were generated using a three-plasmid system: psPAX2, pMD2.G, and Lenti-EF1α. NSG mice, 6-8 weeks old, were purchased from Biocytogen Biotechnology Co., Ltd., and routinely housed in an SPF-grade environment at the Drug Safety Evaluation Research Center of Zhejiang University.

[0025] 2. Experimental apparatus

[0026] Cytoflex flow cytometer (Beckman Laboratories, USA); CytoFLEX LX flow cytometer (Beckman Laboratories, USA); flow cytometer (Beckman Laboratories, USA); small animal in vivo imaging system (PerkinElmer, USA).

[0027] 3. Main reagents

[0028] RPMI 1640 medium (Corning, USA); DMEM (High Glucose) medium (Corning, USA); Fetal bovine serum (FBS) (GIBCO, USA); Ficoll lymphocyte separation medium (Tianjin Haoyang Biological Products Technology Co., Ltd.); Plasmid extraction kit (Life Biotechnology, USA); Genomic DNA Purification Kit (Lifetech, CAT#K0512); PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara Corporation, Japan, CAT#6210A); Premix Ex Taq TM(Tli RNase HPlus), ROXplus (Takara Pharmaceuticals, Japan, CAT#RR42WR(LR×5)); IL-2 (Peprotech, USA); anti-CD3 / CD28 magnetic beads: clinical research grade Cat#40203D (Thermo Fisher Scientific, USA); Bright-Glo™ Luciferase Assay system (Promega, USA, Cat: E2620); D-Luciferin Firefly, potassium salt (PerkinElmer, USA, Cat: #122799); polybrene (Sigma-Adrich, USA); polyethyleneimine hydrochloride (PEI) (Polysciences, USA); SBI-0206965 powder (Selleck, USA); flow cytometry antibodies: anti-human CD3 (PE-cy7), anti-human CD19 (APC), anti-human CD4 (APC-cy7), anti-human CD8 (PE-cy7), anti-human CD62L (PE), anti-human CD45RO (APC), anti-human CD25 (APC), anti-human CD69 (PE-cy7), anti-human PD-1 (APC), anti-human TIM-3 (PE), anti-human LAG-3 (PE-cy7), anti-human FASL (PE), Annexin V (APC): PE, APC, PE-cy7, and APC-cy7 isotype controls were all purchased from Biolegend, Inc., USA. EasySep TM Human T-cell negative selection kit (Stem Cell, USA, CAT#17951); FITC Annexin V Apoptosis kit (BD Biosciences, USA, CAT 556547).

[0029] 4. Solution preparation

[0030] (1) RPMI 1640 complete medium: RPMI 1640 + 10% FBS + 1% penicillin and streptomycin;

[0031] (2) DMEM (high sugar) complete medium: DMEM (high sugar) medium + 10% FBS + penicillin and streptomycin;

[0032] (3) FBS: Before use, bathe in a 56℃ water bath for 30 min, cool to room temperature, and then dispense into 50ml centrifuge tubes and store at -20℃.

[0033] (4) Transfection reagent PEI solution: Accurately weigh 50 mg of linearized polyethyleneimine hydrochloride (Polyethylenimine, Linear, mw-25000), transfer it to a 50 ml centrifuge tube, add 50 mL of ddH2O, and place it in an 80 °C water bath to dissolve it completely. Adjust the pH to 7.0, filter it through a 0.22 μm filter membrane in a laminar flow hood for sterilization, aliquot it into sterile 1.5 mL EP tubes, and store at -20 °C;

[0034] (5) Preparation of IL-2 solution: Take one 500 μg vial of IL-2 lyophilized powder, dissolve it in 1 ml of 100 mM acetic acid, and further add 49 mL of PBS containing 0.1% BSA. The storage concentration is 1 × 10⁻⁶. 6 IU / ml, aliquoted and stored at -80℃;

[0035] (6) Bright-Glo TM Preparation of Luciferase Assay reaction solution: Mix 100ml of Bright-Glo at room temperature... TM Add Luciferase Assay Buffer to 1vial Bright-Glo™ Luciferase Assay Substrate, dissolve thoroughly, and then aliquot into 15ml centrifuge tubes for storage at -80℃.

[0036] (7) Preparation of ULK1 inhibitor SBI-0206965 solution for in vitro culture: Dissolve SBI-0206965 powder in DMSO to prepare a stock solution with a concentration of 10 mmol / L, aliquot and store at -80℃. Dilute with DMSO to the required concentration before use to ensure that the final concentration of DMSO is 0.1% in all experiments.

[0037] (8) Preparation of luciferin injection for small animal imaging: Before use, calculate the total required amount based on 3 mg / mouse, weigh the corresponding mass of D-Luciferin Firefly and potassium salt powder and dissolve them in the corresponding volume of DPBS to prepare a concentration of 15 mg / ml, and filter it through a 0.22 μm filter before use.

[0038] (9) 1×Annexin V binding buffer: Before use, take an appropriate volume of 10×Annexin V binding buffer, add 9 times the volume of ddH2O to dilute to 1× solution, and store in a refrigerator at 4℃.

[0039] Example 1: Effects of ULK1 inhibitors on subpopulation differentiation and activation of prepared CAR-T products

[0040] Peripheral blood was collected from healthy donors, and human peripheral blood mononuclear cells were isolated. T cells were isolated and activated using anti-CD3 / CD28 magnetic beads. Twenty-four hours after activation, the cells were infected with a CAR-T virus (structure: Lenti-EF1a-CD19-2nd-CAR(4-1BB), catalog number: LIC001A, purchased from Aikon Biotechnology Co., Ltd.). Infection efficiency was assessed three days post-infection. CAR-T cells cultured to day 9 were collected and processed at a concentration of 5 × 10⁶ cells / year. 5 The cells were seeded into 6-well plates and treated with the ULK1 inhibitor SBI-0206965 (3 μM). DMSO was used as a control. Cells were cultured at 37°C in a 5% CO2 incubator. Flow cytometry was performed on each group 72 hours after treatment to detect CAR-T cell subsets and activation markers. Cell subsets were defined as follows: naive T cells (Tn) (CD45RO-CD62L+), central memory T cells (Tcm) (CD45RO+CD62L+), effector memory T cells (Tem) (CD45RO+CD62L-), and effector T cells (Te) (CD45RO-CD62L-), all expressed as percentages. Activation-related molecules (CD25, CD69) were also expressed as percentages.

[0041] The results showed that the ULK1 inhibitor SBI-0206965 significantly increased the proportion of CD62L-positive CAR-T cells, that is, significantly increased the proportion of naive T cells and central memory T cells. Figure 1 Furthermore, it reduced the activation level of CAR-T cells, specifically by a significant decrease in the expression of both CD25 and CD69. Figure 2 ).

[0042] Example 2: Effects of ULK1 inhibitors on depletion and apoptosis of prepared CAR-T products

[0043] Collect CAR-T cells cultured in vitro to day 9, at a ratio of 5 × 10⁻⁶. 5Cells were seeded into 6-well plates and treated with the ULK1 inhibitor SBI-0206965 (3 μM). DMSO was used as a control. Cells were cultured at 37°C in a 5% CO2 incubator. Flow cytometry was performed 72 h after treatment to detect CAR-T cell exhaustion and apoptosis markers. Exhaustion-related inhibitory molecules (PD-1, TIM-3, LAG-3) are expressed as proportions. After collecting samples and adding them to flow cytometry tubes, residual culture medium was washed away by centrifugation with PBS. Cells were resuspended in 100 μL of 1×Annexin V binding buffer in each tube, and 1.5 μL of Annexin V APC was added to each tube. Cells were incubated at 4°C in the dark for 30 min before flow cytometry analysis.

[0044] The results showed that the ULK1 inhibitor SBI-0206965 significantly reduced CAR-T cell exhaustion, as evidenced by a significant decrease in the expression of PD-1, TIM-3, and LAG-3, and inhibited T cell apoptosis, as evidenced by a decrease in the Annexin-V positive rate. Figure 3 ).

[0045] Example 3: Effects of ULK1 inhibitors on apoptosis and antigen expression in B-cell acute lymphoblastic leukemia cells

[0046] Two B-cell acute lymphoblastic leukemia cell lines (Nalm-6 and BALL-1) cultured in vitro were collected and analyzed at a concentration of 5 × 10⁻⁶ cells / mL. 5 The cells were seeded into 6-well plates and treated with the ULK1 inhibitor SBI-0206965 (3 μM). DMSO was used as a control. Cells were cultured at 37°C in a 5% CO2 incubator. Flow cytometry was performed at 24 h, 48 h, and 72 h post-treatment to detect CD19 target antigen expression on the surface of tumor cells, expressed as mean fluorescence intensity. At 72 h post-treatment, flow cytometry was performed to detect tumor cell apoptosis. Samples were collected and added to flow cytometry tubes. After centrifugation with PBS to remove residual culture medium, 100 μL of 1×Annexin V binding buffer was added to each tube to resuspend the cells. 1.5 μL of Annexin V APC was added to each tube, and the cells were incubated at 4°C in the dark for 30 min. Then, 300 μL of 1×Annexin V binding buffer was added to resuspend the cells before flow cytometry. 1 μL of PI dye was added to each tube before flow cytometry. Apoptosis is defined as follows: early apoptotic cells (Annexin-V+PI-), late apoptotic cells (Annexin-V+PI+), necrotic cells (Annexin-V-PI+), and normal cells (Annexin-V-PI-).

[0047] The results showed that the ULK1 inhibitor SBI-0206965 significantly increased the expression of CD19 target antigen in B-type acute lymphoblastic leukemia cells. Figure 4 It can also significantly increase tumor cell apoptosis. Figure 5 and Figure 6 ).

[0048] Example 4: The tumor-killing effect of CAR-T cells treated with ULK1 inhibitor on mice.

[0049] (1) Collect CAR-T cells cultured for 9 days, and administer at a dose of 1×10⁻⁶. 6 Cells were seeded into 6-well plates and treated with ULK1 inhibitor SBI-0206965 (3 μM) and DMSO, respectively. The cells were cultured at 37°C in a 5% CO2 incubator and collected after 72 h for in vivo infusion in mice.

[0050] (2) Preparation of the ALL-NSG mouse model: 6-8 week old NCG mice were housed in an SPF-grade animal research center. Nalm-6 cell lines (with luciferase) in the logarithmic growth phase were used to prepare a cell concentration of 1×10⁻⁶. 7 / ml, at 1×10 6 Administer 100 μL via tail vein injection to each mouse. Five days later, tumor burden was assessed using a small animal in vivo imaging system. The mice were randomly divided into three groups based on fluorescence intensity. The following day, each group received 1×10⁻⁶ μL of the injected drug. 6 Cell dosage: CAR-T cells and T cells were injected via tail vein, with 5 mice in each group. CAR-T cells or MockT cells / mouse. Flow cytometry was used to detect the number, differentiation, and exhaustion phenotype of CAR-T cells in the bone marrow.

[0051] (3) Animal in vivo imaging was performed on days 4, 8, 15, 22 and 28 after CAR-T cell injection to observe tumor burden and plot mouse survival curves.

[0052] The results showed that ULK1 inhibitor treatment enhanced the antitumor activity and persistence of CAR-T cells in an ALL-NSG mouse model, prolonged the survival time of tumor-bearing mice, and reduced terminal differentiation of CAR-T cells (reduction of CD62L positive population), as well as cell exhaustion and activation levels (decreased expression of PD-1 and CD25). Figures 7-10 ).

Claims

1. Application of ULK1 inhibitor in reducing CAR-T cell exhaustion and lowering CAR-T cell activation levels in vitro. The ULK1 inhibitor is SBI-0206965.

2. The application according to claim 1, characterized in that, The concentration of SBI-0206965 used is 3μM.

3. The application according to claim 1, characterized in that, The ULK1 inhibitor was added to the culture medium on days 6 to 9 of CAR-T cell culture in vitro.

4. A method for in vitro culture of CAR-T cells, characterized in that, On days 6-9 of CAR-T cell in vitro culture, a ULK1 inhibitor, SBI-0206965, was added to the culture medium.

5. The method for in vitro culture of CAR-T cells according to claim 4, characterized in that, The concentration of SBI-0206965 used is 3μM.

6. Application of ULK1 inhibitors in the preparation of anticancer drugs: ULK1 inhibitors enhance the anticancer effect of CAR-T cells by reducing CAR-T cell exhaustion and activation levels in vitro. The ULK1 inhibitor is SBI-0206965; The cancer type is B-cell acute lymphoblastic leukemia.

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