Use of CD38 inhibitors to reduce CAR-T cell exhaustion in vitro

Abstract

The application discloses application of a CD38 inhibitor in reducing CAR-T cell exhaustion in vitro. The application provides a CD38 inhibitor-based CAR-T cell and a preparation method, and application of the CD38 inhibitor pretreated CAR-T cell in anti-tumor, the method is simple and easy to implement, the tumor is treated by adding the CD38 inhibitor in the CAR-T cell culture process, the persistence and killing function of the immune cell can be obviously increased, the refractory recurrence problem in the cell treatment process is solved to a great extent, and the survival period of the patient is prolonged.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of CD38 inhibitors in reducing CAR-T cell depletion in vitro. Background Technology

[0002] Chimeric antigen receptor T-cell therapy (CAR-T) is used to treat refractory or relapsed hematologic malignancies. However, disease relapse after treatment remains a major challenge. Existing research suggests that poor persistence of CAR-T cells due to depletion, accompanied by functional impairment, is a limiting factor in inducing sustained remission. Previous articles have reported that attenuating CAR signaling can improve depletion, enhancing memory cell subsets to improve in vivo persistence, and that targeting depletion-related genes can improve the function of tumor-infiltrating CAR-T cells (EW Weber, et al., Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling. Science 372, (2021)).

[0003] CD38 is a single-chain transmembrane glycoprotein that is highly expressed in B, T, and NK cells. CD38 is a major NAD+-consuming enzyme in mammals, and increasing research has shown that the CD38-NAD+ signaling pathway regulates the metabolic activity and anti-tumor effects of immune cells (M. Philip, et al., Chromatin states define tumor-specific T celldysfunction and reprogramming. Nature 545, 452-456 (2017).).

[0004]

[0005] The small molecule compound compound 78C (abbreviated as 78C) is a CD38 inhibitor with CAS number 1700637-55-3 and structural formula shown in Formula I. Summary of the Invention

[0006] To address the aforementioned shortcomings in the existing technology, this invention provides the application of CD38 inhibitors in reducing CAR-T cell depletion in vitro.

[0007] This invention first provides the application of a CD38 inhibitor in reducing CAR-T cell exhaustion in vitro. Preferably, the CD38 inhibitor is a small molecule compound, compound 78C. The concentration of compound 78C used is 10 μM. Preferably, the CD38 inhibitor is added to the culture medium on days 6 to 9 of CAR-T cell in vitro culture.

[0008] This invention also provides a method for in vitro CAR-T cell culture, wherein a CD38 inhibitor is added to the culture medium on days 6 to 9 of CAR-T cell in vitro culture. Preferably, the CD38 inhibitor is the small molecule compound compound 78C. Preferably, the concentration of the small molecule compound compound 78C used is 10 μM.

[0009] This invention also provides the application of CD38 inhibitors in the preparation of anticancer drugs, wherein the CD38 inhibitors are used in combination with CAR-T cells, and the CD38 inhibitors enhance the anticancer effect by reducing CAR-T cell depletion, wherein the CD38 inhibitors are small molecule compounds compound 78C.

[0010] The beneficial effects of this invention are as follows: This invention provides a CAR-T cell based on a CD38 inhibitor and a preparation method thereof, as well as the application of CAR-T cells pretreated with a CD38 inhibitor in anti-tumor therapy. This method is simple and easy to implement. By adding a CD38 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

[0011] Figure 1 Statistical graphs of CAR-T cell proliferation, apoptosis, and subset differentiation induced by CD38 inhibitors are presented. Figure A shows the average fluorescence intensity of CD38 expression on the surface of CAR-T cells after 72 h of treatment with 78C in the experimental group and DMSO in the solvent control group. Figure B shows the proliferation curves of cells after 72 h of treatment with 78C in the experimental group and DMSO in the solvent control group. Figure C shows typical flow cytometry results of differentiation indices (CD62L, CD45RO) of CAR-T cells after 72 h of treatment with 78C in the experimental group and DMSO in the solvent control group. Figure D shows the statistical graphs of differentiation indices of CAR-T cells detected by flow cytometry.

[0012] Figure 2Figure 1 shows the results of flow cytometry detection of the expression changes of PD1, LAG3, and TIM3 depletion indicators in CAR-T cells cultured with CD38 inhibitors. Figure A shows a typical flow cytometry detection of Annexin V, an apoptosis-related indicator of CAR-T cells, after 72 h of treatment with 78C in the experimental group and DMSO in the solvent control group. Figure B shows a statistical graph of the positive proportion of Annexin V in CAR-T cells after 72 h of treatment with DMSO in the experimental group and the solvent control group. Figure C shows a statistical graph of the co-expression ratio of inhibitory receptors PD-1, LAG-3, and TIM-3 in CAR-T cells after 72 h of treatment with DMSO in the experimental group and the solvent control group.

[0013] Figure 3 Figure 1 shows the results of detecting changes in cytokine release and tumor killing in CAR-T cells cultured with CD38 inhibitors. Figure A shows typical expression patterns of intracellular IL-2 and TNF-α in CAR-T cells treated with 78C (experimental group) and DMSO (solvent control group) for 72 hours by flow cytometry. Figure B shows typical expression patterns of intracellular Granzyme B and IFN-γ in CAR-T cells treated with 78C (experimental group) and DMSO (solvent control group) for 72 hours by flow cytometry. Figure C shows the statistical proportions of intracellular IL-2, TNF-α, Granzyme B, and IFN-γ in CAR-T cells treated with 78C (experimental group) and DMSO (solvent control group) for 72 hours by flow cytometry. Figure D shows the specific killing ratio of CAR-T cells treated with 78C (experimental group) and DMSO (solvent control group) in vitro after multiple rounds of stimulation, when CAR-T cells were co-cultured with tumor cells at an effector-target ratio of 1:10.

[0014] Figure 4 Biofluorescence imaging of D0-D49 mice after CAR-T cells were infused into NSG mice 3 days after treatment with different drugs.

[0015] Figure 5 A line graph showing the change in mean tumor fluorescence intensity of mice in the group with the number of days after infusion after 3 days of in vitro treatment with DMSO as the solvent control.

[0016] Figure 6 The plot shows the change in mean tumor fluorescence intensity of mice in the experimental group as a function of the number of days after infusion, after 3 days of in vitro treatment of CD19-41BB CAR-T cells with 78C.

[0017] 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

[0018] 1. Experimental Materials

[0019] HEK293T cells, ALL cell line Nalm-6, and ALL cell line B-ALL1 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.

[0020] 2. Experimental apparatus

[0021] 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); MD multi-mode microplate reader (Molecular Devices, USA).

[0022] 3. Main reagents

[0023] 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); compound 78C 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 IL-2 (APC), anti-human IFN-V (APC), anti-human TNF-α (PE), anti-human Granzyme B (PE), Annexin V (APC); PE, APC, PE-cy7, and APC-cy7 isotype controls were all purchased from Biolegend Systems, Inc., USA. EasySep TM Human T-cell negative selection kit (Stem Cell, USA, CAT#17951); FITC Annexin V Apoptosis kit (BD Bioscience, USA, CAT 556547); Membrane perforation and fixation kit (ThermoFisher, USA, CAT 88-17000-210).

[0024] 4. Solution preparation

[0025] (1) RPMI 1640 complete medium: RPMI 1640 + 10% FBS + 1% penicillin and streptomycin;

[0026] (2) DMEM (high sugar) complete medium: DMEM (high sugar) medium + 10% FBS + penicillin and streptomycin;

[0027] (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℃.

[0028] (4) Transfection reagent PEI solution: Accurately weigh 50 mg of linearized polyethyleneimine hydrochloric acid (Polyethylenimine, Linear, mw-25000), transfer it to a 50 ml centrifuge tube, add 50 mL of ddH2O, and place it in an 80℃ 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℃;

[0029] (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℃;

[0030] (6) Bright-Glo TM Preparation of Luciferase Assay reaction solution: Mix 100ml of Bright-Glo at room temperature... TM Add Luciferase Assay Buffer to 1 vial Bright-Glo™ Luciferase Assay Substrate, dissolve thoroughly, and then aliquot into 15ml centrifuge tubes and store at -80℃.

[0031] (7) 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℃.

[0032] (8) Preparation of CD38 inhibitor compound 78C solution for in vitro culture: Dissolve compound 78C powder in DMSO to prepare a stock solution with a concentration of 100 mmol / L 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.

[0033] (9) 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.

[0034] 5. Obtaining CAR-T cells

[0035] 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, T cells were infected with a lentivirus containing CAR (structure Lenti-EF1a-CD19-2nd-CAR(4-1BB), catalog number: LIC001A, purchased from ICON Biotech). Infection efficiency was assessed three days post-infection. CAR-positive cells were collected by flow cytometry and cultured in vitro.

[0036] Example 1: Effects of CD38 inhibitors on the proliferation and subpopulation differentiation of prepared CAR-T products

[0037] CAR-T cells from healthy donors, cultured to day 9 after in vitro sorting, were collected and processed at a ratio of 5 × 10⁻⁶ cells / cell. 5 Cells were seeded into 6-well plates and treated with the CD38 enzyme activity inhibitor compound 78C (10 μM) (78C group). DMSO served as a control (CTRL group). Cells were cultured at 37°C in a 5% CO2 incubator, and cell counts were performed on days 0 and 3. After drug elution, both groups were cultured again, and cell counts were performed on day 6. CAR-T cell subset markers were detected by flow cytometry 72 h post-treatment. Cell subsets were defined as follows: naive T cells (CD45RO-CD62L+), central memory T cells (CD45RO+CD62L+), effector memory T cells (CD45RO+CD62L-), and effector T cells (CD45RO-CD62L-), all expressed as percentages.

[0038] The results showed that treatment with the CD38 enzyme inhibitor compound 78C for 72 hours did not significantly affect the expression of CD38 protein on the surface of CAR-T cells compared with the control group. After drug withdrawal, CAR-T cells in the 78C-treated group exhibited stronger proliferative capacity. Simultaneously, the proportion of CD62L-positive CAR-T cells significantly increased after 72 hours of compound 78C treatment (52.6% vs 79.0%, P < 0.0001), suggesting that CD38 inhibitors can significantly increase the proportion of memory T cells. Figure 1 ).

[0039] Example 2: Effects of CD38 inhibitors on apoptosis and exhaustion in prepared CAR-T products

[0040] CAR-T cells from healthy donors, cultured in vitro for up to day 9, were collected and processed at a ratio of 5 × 10⁻⁶ cells / cell. 5 Cells were seeded into 6-well plates and treated with the CD38 inhibitor compound 78C (10 μM) (78C group). DMSO was used as a control (CTRL group). 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 activation indicators in each group. 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.

[0041] The results showed that treatment with the CD38 inhibitor compound 78C significantly reduced the proportion of Annexin V-positive CAR-T cells (35.4% vs 26.4%, P < 0.0001), indicating a significant decrease in CAR-T cell apoptosis. Furthermore, the co-expression (PD1+TIM+, TIM3+LAG3+, PD1+LAG3+) and tri-expression (PD1+TIM+LAG3+) of the inhibitory receptors PD1, TIM3, and LAG3 were significantly reduced, suggesting that CAR-T cells treated with 78C exhibited lower exhaustion and better cell function compared to the control group. Figure 2 ).

[0042] Example 3: Effects of CD38 inhibitors on cytokine release and killing in prepared CAR-T cells

[0043] CAR-T cells from healthy donors, cultured in vitro for up to day 9, were collected and processed at a ratio of 5 × 10⁻⁶ cells / cell. 5Cells were seeded into 6-well plates and treated with the CD38 inhibitor compound 78C (10 μM) (78C group), with DMSO as a control (CTRL group). Cells were cultured at 37°C in a 5% CO2 incubator. CAR-T cell cytometry was performed 72 h after treatment to detect cytokine release levels. Before collecting cell samples, cells were treated with the Golgi inhibitor Monensin (#00-4505-51, eBioscience) for 6 hours. After washing away residual culture medium with PBS, 100 μL of 4% paraformaldehyde was added to each tube for fixation at room temperature for 15 minutes. After washing away the fixative with PBS, 90% methanol was added, and the cells were permeabilized on ice for 10 minutes. Cells were washed with PBS containing 2% FBS, centrifuged, resuspended in 100 μL buffer, stained, and incubated on ice for 40 minutes in the dark before resuspending and loading onto the flow cytometer. Cells were collected 3 days after treatment and cultured at 4 × 10⁻⁶ cells / well. 3 / Inoculate into a 96-well round-bottom plate, then add 4×10 4 Luci-Nalm-6 cells (Nalm-6 cells with GFP fluorescence and luciferase) were co-cultured in 96-well plates for 24 hours. After centrifugation to remove the supernatant, the cells were resuspended in PBS, and an equal volume of chemiluminescent substrate was added. The cells were then transferred to 96-well plates and detected by chemiluminescence immunoassay using a microplate reader.

[0044] The results showed that after 72 hours of treatment with the CD38 inhibitor compound 78C, the expression ratios of IL-2 and Granzyme B in CAR-T cells were significantly reduced (IL-2 32.56% vs 16.62%, P = 0.004; Granzyme B 43.80% vs 7.85%, P < 0.0001), while the expression ratios of TNF-α and IFN-γ showed no significant difference. These data suggest that in the absence of tumor stimulation, the release levels of cytokines from CAR-T cells were significantly reduced after treatment with CD38 inhibitors, indicating a more quiescent state. Furthermore, after multiple rounds of tumor cell killing at an effector-to-target ratio of 1:10 over a 24-hour period, we observed that the specific killing rate of CAR-T cells in the 78C treatment group was significantly higher than that in the control group during all three rounds of tumor killing (round 1, 53.53% vs 83.89%, P = 0.0005; round 2, 34.02% vs 68.11%, P = 0.0002; round 3, 33.64% vs 58.69%, P = 0.0025), suggesting a stronger immune killing function. Figure 3 ).

[0045] Example 4: The tumor-killing effect of CD38 inhibitor-treated CAR-T cells on mice.

[0046] (1) Collect CAR-T cells from healthy donors on day 9 of culture, and administer them at a ratio of 1×10⁻⁶. 6 Cells were seeded into 6-well plates and treated with CD38 inhibitor compound 78C (10 μM) (78C group) and DMSO as control (CTRL group). The cells were cultured at 37°C in a 5% CO2 incubator and collected after 72 h for in vivo infusion in mice.

[0047] (2) Preparation of the ALL-NSG mouse model: 6-8 week old NCG mice were housed in an SPF-grade animal research center. Logarithmically growing luciferase(+) Nalm-6 cell lines (Nalm-6 cells, with GFP fluorescence and luciferase) were used to prepare a cell concentration of 1×10⁻⁶. 7 / ml, administered via tail vein injection at a dose of 1×10⁶ / mouse, 100μL per mouse. Tumor burden was assessed using a small animal in vivo imaging system 5 days later. Animals were randomly divided into 3 groups based on fluorescence intensity. The following day, they received 1×10⁶ / mouse. 6 Cell counts included tail vein injection of T cells, CAR-T cells pretreated with DMSO for 72 hours, and CAR-T cells pretreated with 78C for 72 hours, with 5 mice in each group. Flow cytometry was used to detect the number, differentiation, and exhaustion phenotype of CAR-T cells in bone marrow.

[0048] (3) Animal in vivo imaging was performed on days 0, 7, 14, 21, 28, 35, 42 and 49 after CAR-T cell injection to observe tumor burden and plot mouse survival curves.

[0049] The results showed that CAR-T cells exhibited strong tumor-killing function in mice, while all mice in the T group died within 28 days after infusion. Biofluorescence imaging showed that CD38 inhibitor treatment enhanced the anti-tumor effect of CAR-T cells in an ALL-NSG mouse model. The control CAR-T group gradually relapsed 21 days after infusion, and mice began to die from day 49. The CAR-T cell load in the 78C-treated group remained at a low level. Survival curve analysis indicated that CAR-T cells in the 78C-treated group significantly prolonged the survival time of ALL-NSG mice (53.5 days vs 76 days, P = 0.0014). Figure 4 , Figure 5 , Figure 6 ).

Claims

1. Application of CD38 inhibitors in reducing CAR-T cell exhaustion in vitro. The CD38 inhibitor is a small molecule compound called compound 78C.

2. The application according to claim 1, characterized in that, The concentration of compound 78C used is 10 μM.

3. The application according to claim 1, characterized in that, The CD38 inhibitor was added to the culture medium on days 6 to 9 of CAR-T cell in vitro culture.

4. A method for in vitro culture of CAR-T cells, characterized in that, CD38 inhibitors were added to the culture medium on days 6-9 of CAR-T cell in vitro culture. The CD38 inhibitor is a small molecule compound called compound 78C.

5. The method for in vitro culture of CAR-T cells according to claim 4, characterized in that, The concentration of the small molecule compound compound 78C used is 10 μM.

6. Application of CD38 inhibitors in the preparation of anticancer drugs: CD38 inhibitors enhance the anticancer effect of CAR-T cells by reducing CAR-T cell depletion in vitro. The CD38 inhibitor is a small molecule compound, compound 78C. The type of cancer is acute lymphoblastic leukemia (ALL).

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