Application of Mdivi-1 combined with CAR-T cells in the preparation of drugs for the treatment of solid tumors

Abstract

The present invention discloses the use of Mdivi-1 combined with CAR-T cells in the preparation of solid tumor therapeutic drugs. According to the present invention, compared with CD39 ‑ CAR-T and CD39 hi CAR-T cell subsets, CD39 int CAR-T cells have higher anti-tumor activity, and Mdivi-1 has the function of precisely regulating the moderate expression of CD39 in CAR-T cells. After Mdivi-1 is used to treat CAR-T cells, CD39 ‑ The proportion of CAR-T cells was significantly reduced, and CD39 int The proportion of CAR-T cells increased significantly, while CD39 hi There was no significant change in the proportion of CAR-T cells, and the cell viability of CAR-T cells was not affected. After treatment with Mdivi-1, the secretion levels of INF-γ and Ki-67 of CAR-T cells were significantly increased, and the proliferation and anti-tumor abilities of CAR-T cells were significantly enhanced, which has a high clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology. More specifically, it relates to the application of Mdivi-1 combined with CAR-T cells in the preparation of drugs for treating solid tumors. Background Art

[0002] In recent years, cell immunotherapy, led by chimeric antigen receptor T cells (CAR-T cells), has made outstanding progress in a variety of malignant tumors such as hematological malignancies (such as leukemia and lymphoma), and is an innovative therapy for the treatment of cancer. Glypican 3 (GPC3) is highly expressed in a variety of solid tumors, but its content in normal adult tissues is extremely low. CAR-T cell therapy targeting GPC3 (GPC3-CAR-T cells) is currently the most studied CAR-T cell therapy for solid tumors, especially for hepatocellular carcinoma (HCC). However, the efficacy of this therapy for HCC is not ideal at present. The main reason is that there is a very strong immunosuppressive microenvironment in HCC, which leads to functional disorders of the reinfused CAR-T cells (such as incapacity, exhaustion and apoptosis, etc.), which seriously restricts the anti-tumor effect of CAR-T cells. How to enhance the ability of CAR-T cells to antagonize the immunosuppressive microenvironment of HCC is particularly important.

[0003] CD39 is a key rate-limiting enzyme in adenosine metabolism. Its high expression on tumor cells is associated with poor prognosis in patients with various tumors. Therefore, it is considered a key molecule for regulating immunosuppression in the tumor microenvironment. However, in recent years, many studies have reported that the function of CD39 expressed on T cells is not to play an immunosuppressive role. CD39 is an important biomarker for distinguishing tumor-reactive CAR-T cells and is closely related to the anti-tumor activity of CAR-T cells. Compared with CD39 - CAR-T, CD39 + CAR-T showed stronger anti-tumor activity. Knocking down the expression of CD39 in CAR-T cells significantly inhibited the killing ability of CAR-T cells against liver cancer cells, indicating that maintaining the expression level of CD39 in CAR-T cells is particularly important for the anti-tumor efficacy of CAR-T. In theory, overexpression of CD39 can enhance the infiltration and killing ability of CAR-T cells against liver cancer, but the efficiency of constructing CAR-T cells overexpressing CD39 through a lentiviral packaging system is low and the cell utilization rate is also low, which limits the clinical application of CD39-HBVs-CAR-T cells.

[0004] Therefore, developing a method that is both simple and accurate to maintain the moderate expression of CD39 in CAR-T cells is crucial to enhancing the tumor killing ability of CAR-T cells. This will enable CAR-T cell therapy to be more effectively applied to the treatment of solid tumors such as HCC, and has important clinical application value and prospects. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the technical defects and shortcomings of the existing CAR-T cell treatment of solid tumors such as hepatocellular carcinoma, and provide a method for using Mdivi-1 to accurately regulate the moderate expression of CAR-T cell CD39, thereby improving the tumor killing ability of CAR-T cells, thereby achieving effective treatment of solid tumors including hepatocellular carcinoma.

[0006] The first objective of the present invention is to provide an application of Mdivi-1 combined with CAR-T cells in the preparation of drugs for treating solid tumors.

[0007] The second object of the present invention is to provide a drug for treating solid tumors.

[0008] The third object of the present invention is to provide Mdivi-1 in increasing CD39 in CAR-T cells int Application of CAR-T cell ratio and Mdivi-1 in the preparation of CD39 in CAR-T cells int The application of CAR-T cell ratio products, and the application of Mdivi-1 in the preparation of CAR-T cells.

[0009] The fourth object of the present invention is to provide a method for increasing CD39 int A CAR-T cell processing method with improved CAR-T cell ratio, and a method for producing CAR-T cells with improved solid tumor treatment effect.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0011] The present invention constructs CAR-T cells (GPC3-CAR-T cells) targeting HCC-related antigen Glypican 3 (GPC3). Studies have shown that GPC3-CAR-T cells (CD39) with a medium expression level of CD39 have a high expression level of CD39. int GPC3-CAR-T cells) have stronger anti-tumor activity, while CD39 high expression (CD39 hi ) cell subsets and CD39 negative (CD39 - ) cell subsets are impaired. The present invention uses Mdivi-1 to treat GPC-3-CAR-T cells, which significantly increases the CD39 intCompared with GPC-3-CAR-T cells alone, Mdivi-1 combined with GPC-3-CAR-T cells can induce a higher proportion of CD39 int GPC-3-CAR-T cells infiltrate into tumor tissues, significantly improving their ability to kill tumor cells, inducing more powerful anti-tumor immunity and achieving better therapeutic effects on solid tumors including hepatocellular carcinoma.

[0012] Therefore, the present invention first applies to protect the use of Mdivi-1 combined with CAR-T cells in the preparation of drugs for the treatment of solid tumors.

[0013] Based on this, the present invention further protects a drug for treating solid tumors, which contains Mdivi-1 and CAR-T cells.

[0014] Specifically, the CAR-T cells are GPC3-CAR-T cells.

[0015] Specifically, the solid tumor is liver cancer.

[0016] More specifically, the liver cancer is hepatocellular carcinoma.

[0017] Given that Mdivi-1 can significantly increase the CD39 int CAR-T cell ratio, therefore, the protection scope of the present invention also includes:

[0018] Mdivi-1 enhances CD39 expression in CAR-T cells int Application of CAR-T cell ratio.

[0019] Mdivi-1 is used to enhance CD39 in CAR-T cells int Application of CAR-T cell ratio products.

[0020] Application of Mdivi-1 in the preparation of CAR-T cells, wherein the prepared CAR-T cells have CD39 int The proportion of CAR-T cells was increased.

[0021] Based on this, the present invention further applies for protection of the following solutions:

[0022] The present invention applies for a method for increasing CD39 int A CAR-T cell treatment method for a CAR-T cell ratio, specifically comprising: treating the CAR-T cells with Mdivi-1.

[0023] The present invention applies to protect a method for producing CAR-T cells with improved solid tumor treatment effects, and the specific method is: treating CAR-T cells with Mdivi-1.

[0024] In the above-mentioned CAR-T cell treatment method, preferably, the concentration of Mdivi-1 is 1 to 200 μM.

[0025] More preferably, the concentration of Mdivi-1 is 10 to 100 μM.

[0026] Most preferably, the Mdivi-1 concentration is 50 μM.

[0027] In addition, the above CD39 int CAR-T cells refer to the detection of CAR-T cells by flow cytometry, which groups CAR-T cells according to the expression level of CD39: CD39 - CD39 negative group, CD39 int CD39 is at a medium level, CD39 hi CD39 is at a high expression level. int The IFN-γ expression level of CAR-T is higher than that of the other two subgroups, and the anti-tumor activity is stronger. More specifically, if the above CAR-T cells are GPC3-CAR-T cells, then according to the above cell clustering method, CD39 int GPC3-CAR-T cells.

[0028] The present invention has the following beneficial effects:

[0029] 1. This invention first proves that CD39 int CAR-T cells have higher anti-tumor activity, and high expression of CD39 (CD39 hi ) cell subsets and CD39 negative (CD39 - ) The function of the cell subset is impaired. At the same time, the present invention discovered for the first time that Mdivi-1 has the function of regulating the expression of CD39 in CAR-T cells, and is an effective regulator for regulating the expression of CD39 in CAR-T cells, which expands the clinical use of Mdivi-1.

[0030] 2. The present invention provides a method of combining Mdivi-1 with CAR-T cells, which can more accurately regulate the moderate expression of CD39 on CAR-T cells. After Mdivi-1 treats CAR-T cells, CD39 - The proportion of CAR-T cells was significantly reduced, and CD39 int The proportion of CAR-T cells increased significantly, while CD39 hiThere was no significant change in the proportion of CAR-T cells, and the cell viability of CAR-T cells was not affected. After treatment with Mdivi-1, the secretion levels of INF-γ and Ki-67 of CAR-T cells were significantly increased, and the proliferation ability and anti-tumor ability of CAR-T cells were significantly enhanced, which has a high clinical application value.

[0031] 3. The present invention achieves the regulation of CD39 expression by treating CAR-T cells with the small molecule compound Mdivi-1. Compared with the traditional genetic engineering modification method, it has the advantages of being more convenient, lower cost and better safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : Flow cytometric analysis of CD39 in tumor-infiltrating GPC3-CAR-T cells hi / int / - Proportion of cell subsets.

[0033] Figure 2 :Flowjo statistical analysis of CD39 in each group hi / int / - IFN-γ in GPC3-CAR-T cells + Cell proportion, mean±SEM, n=6, *p<0.05, **p<0.01, ***p<0.001.

[0034] Figure 3 : Cytotoxicity of three compounds, Mdivi-1, CGS-21680 and 8-Bromo-cAMP, on GPC3-CAR-T cells. The dotted lines indicate the IC50 of the three compounds on GPC3-CAR-T cells.

[0035] Figure 4 : CD39 in GPC3-CAR-T cells treated with different concentrations of Mdivi-1, CGS-21680 and 8-Bromo-cAMP int The proportion of GPC3-CAR-T cells.

[0036] Figure 5 : CD39 treated with 50 μM Mdivi-1 - / int / hi The mean fluorescence intensity of CD39 in GPC3-CAR-T cells.

[0037] Figure 6 :CD39 in GPC3-CAR-T cell group and 50μM Mdivi-1 combined with GPC3-CAR-T cell group - / int / hi The proportion of GPC3-CAR-T cells.

[0038] Figure 7: IFN-γ in GPC3-CAR-T cell group and 50μM Mdivi-1 combined with GPC3-CAR-T cell group + GPC3-CAR-T cells and Ki67 + The proportion of GPC3-CAR-T cells.

[0039] Figure 8 : The cytotoxicity of GPC3-CAR-T cell group, 50μM Mdivi-1 combined with GPC3-CAR-T cell group, Her2-CAR-T cell group, and 50μM Mdivi-1 combined with Her2-CAR-T cell group on tumor cells under different effector-target ratios.

[0040] Fig. 9 : Tumor images of GPC3-CAR-T cell group and Mdivi-1 combined with GPC3-CAR-T cell group after treatment of hepatocellular carcinoma. n=6.

[0041] Fig.10 : Tumor growth curves during the treatment of hepatocellular carcinoma in the GPC3-CAR-T cell group and the Mdivi-1 combined with GPC3-CAR-T cell group and tumor weight after treatment. n=6.

[0042] Fig.11 : Flow cytometry was used to analyze the tumor-infiltrating CAR-T cells in the GPC3-CAR-T cell group and the Mdivi-1 combined with GPC3-CAR-T cell group, and the CD39 int / hi The percentage of cells and CD39 int / hi IFN-γ in CAR-T cells + Percentage of cells. n = 6, mean ± SEM, ns: no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0044] Unless otherwise specified, the reagents and materials used in the following examples are reagents and materials available from commercial sources.

[0045] The materials and some experimental methods used in the following examples are as follows:

[0046] Experimental Materials

[0047] 1. Cells: Human hepatocellular carcinoma cell line GPC3+ Huh-7 and HEK293T cell lines were obtained from ATCC and cultured in DMEM medium containing 10% fetal bovine serum (FBS), 100 U / mL penicillin and 100 μg / mL streptomycin in a 37°C, 5% CO2 cell culture incubator.

[0048] 2. Animal: NSG (NOD-Prkdc scid IL tm1 / Bcgen, Beijing Biocytogen, Beijing, China) mice, and all mouse experiments were approved and performed in accordance with the guidelines and regulations of the Laboratory Oversight Committee of the First Affiliated Hospital of Sun Yat-sen University (SYSU-IACUC-2021-000675).

[0049] 3. Conditioned medium: The composition is: 90% RPMI 1640 (Gibco) and 10% FBS (Gibco), and contains 0.1 mM non-essential amino acids (Gibco), 2 mM GlutaMAX (Gibco) and 0.05 mM 2-mercaptoethanol.

[0050] 4. Mdivi-1: CAS number is 338967-87-6.

[0051] 5. CGS-21680: CAS number is 120225-54-9.

[0052] 6. 8-Bromo-cAMP: CAS number is 76939-46-3.

[0053] Experimental methods

[0054] (I) Preparation of GPC3-CAR-T cells

[0055] 1. Isolation and culture of primary human CD8 + T cells

[0056] (1) Healthy blood from healthy volunteers was obtained from Guangzhou Blood Center, and peripheral blood mononuclear cells (PBMCs) were extracted using the Ficoll-Hypaque gradient separation method;

[0057] (2) Using BD IMag TM Human CD8 T Lymphocyte Enrichment Set-DM, purified from PBMC by negative separation with magnetic beads + T cells were enriched to 98% purity;

[0058] (3) Separate and enrich human primary CD8 +T cells were 1×10 6 The cells were seeded at a density of 1 μg / mL in a retronectin-coated plate and cultured with conditioned medium containing 1 μg / mL CD3 antibody (Biolegend), 1 μg / mL CD28 antibody (Biolegend) and 10 ng / mL IL-2 (R&D Systems) to activate CD8 + For T cells, the culture medium containing CD3 antibody, CD28 antibody and IL-2 was replaced twice a week.

[0059] 2. Construction of lentiviral vector encoding CAR

[0060] Anti-GPC3-scFv is derived from the basic sequence of Codrituzumab antibody (patent number US16649039). The Anti-GPC3-scFv region is tandem with the intracellular domains of CD8α (nucleotides 548-623; GenBank accession number BC025715.1), CD28 (nucleotides 753-882; GenBank accession number NM_006139.3), CD137 (nucleotides 903-1026; GenBank accession number NM_001561.5) and CD3ζ (nucleotides 307-637; GenBank accession number NM_198053.2), with the GGGGS sequence inserted between each domain.

[0061] 3. Transduction of recombinant lentivirus

[0062] (1) Obtaining lentivirus: Adjust the density of HEK293T cells to 8×10 6 cells / 100 mm culture dish. After culturing for 24 h, HEK293T cells were transfected with a plasmid encoding CAR containing the pMD2.G vector (7.5 μg) of the VSV-G coding gene and the packaging vector psPAX2 (16.5 μg) using a calcium phosphate transfection kit (CAPHOS-1KT, Sigma) according to the product instructions to generate lentivirus.

[0063] (2) Collecting lentivirus: 48 hours after transfection, the supernatant was collected and filtered through a 0.45 μm membrane to remove cell debris to obtain a filtrate, which was then concentrated by ultracentrifugation at 4°C and 802,000 × g (Optima XE-100, Beckman) for 2 h to collect the lentiviral supernatant.

[0064] (3) Lentiviral transduction: After activation of CD8 +The lentiviral supernatant was added to the T cells, and then 8 μg / mL (final concentration) of polybrene transfection reagent (TR-1003-G, Sigma) was added, centrifuged at 350×g for 90 min, and incubated at 37°C. After 12 hours, the lentivirus was removed, and the conditioned medium containing 10 ng / mL IL-2 was used to continue culturing and amplifying the lentiviral-transduced CD8 + T cells, the culture medium was changed twice a week, and GPC3-CAR-T cells were obtained 14 days after lentiviral transduction.

[0065] (II) Flow cytometry

[0066] (1) Evaluation of tumor reactivity of GPC3-CAR-T cells in vitro: GPC3-CAR-T cells were washed twice in FACS buffer and stained with a combination of Anti-GGGGS (Cat No. GS-ARFT100, Hycells) (for labeling CAR-T cells), APCAnti-CD39 (Cat No. 328209, Biolegend) and Pacific Blue Anti-PD-1 (Cat No. 329916, Biolegend) antibodies at 4°C for 30 minutes. The cells were then washed twice in FACS buffer, fixed, and stained with intracellular PE Anti-IFN-γ (Cat No. 383304, Biolegend) and PE Anti-ki67 (Cat No. 350503, Biolegend) using the BD Cytofix / Cytoperm kit.

[0067] (2) Evaluation of tumor reactivity of GPC3-CAR-T cells in a mouse model of hepatocellular carcinoma: Single cell suspensions of tumor tissue were washed twice in FACS buffer and stained with a combination of Anti-GGGGS (Cat No. GS-ARFT100, Hycells) (for labeling CAR-T cells) and APC Anti-CD39 (Cat No. 328209, Biolegend) antibodies at 4°C for 30 minutes. The cells were then washed twice in FACS buffer, fixed, and stained with intracellular PE Anti-IFN-γ (Cat No. 383304, Biolegend) using the BD Cytofix / Cytoperm kit.

[0068] The stained samples were detected by flow cytometry and analyzed using FlowJo software.

[0069] Example 1 CD39 int Evaluation of the anti-tumor ability of GPC3-CAR-T

[0070] 1. Experimental methods

[0071] Using NSG (NOD-Prkdc scid IL tm1 The HCC disease model was established using mice with a 1.1% sphingomyelinase inhibitor (SGA) background. The specific method was as follows: 100 μL of 5×10 6 GPC3 + A HCC subcutaneous tumor model (n=6) was constructed by mixing Huh-7 cells with a mixed solution of Matrigel / PBS (v / v, 1:1).

[0072] Two weeks later, 200 μL of 1×10 6 The PBS solution of GPC3-CAR-T cells was added into the model mice. After 2 weeks, the tumor tissues of the mice were removed and ground into single cell suspensions, and the GPC3-CAR-T cell subpopulations and functions in the tumor tissues were detected by flow cytometry.

[0073] (II) Experimental results and analysis:

[0074] According to the results of GPC3-CAR-T cell clustering, GPC3-CAR-T cells were divided into CD39 hi PD-1 hi 、CD39 int PD-1 int 、CD39 int PD-1 - 、CD39 - PD-1 int and CD39 - PD-1 - Cell subsets (CD39 hi Indicates high CD39 expression level, CD39 int The results showed that CD39 int GPC3-CAR-T cells, especially CD39 int PD-1 int and CD39 int PD-1 - subgroups, secreting higher levels of interferon-γ (IFN-γ) (e.g. Figure 1 and Figure 2 This indicates that when CD39 expression is at a moderate level, GPC3-CAR-T cells exhibit better anti-tumor ability.

[0075] Example 2 Mdivi-1 for CD39 intEffect of GPC3-CAR-T cell proportion and function

[0076] 1. Experimental methods

[0077] The present invention screened a variety of small molecule compounds (including Mdivi-1, CGS-21680 and 8-Bromo-cAMP) for regulating CD39 in GPC3-CAR-T cells. int The proportion of GPC3-CAR-T cell subpopulations. The specific research methods are as follows:

[0078] 1. In vitro cytotoxicity test of small molecule compounds on GPC3-CAR-T cells:

[0079] GPC3-CAR-T cells were plated in 96-well plates, and small molecule compounds (Mdivi-1, CGS-21680, 8-Bromo-cAMP) with different concentration gradients were added to the GPC3-CAR-T cell culture medium. After culturing for 48 hours, the viability of GPC3-CAR-T cells was detected by CCK8 detection kit.

[0080] 2. Mdivi-1 on CD39 - / int / hi Effect of CD39 expression level in GPC3-CAR-T cells:

[0081] CD39 was isolated by flow cytometry using APC Anti-CD39 (Cat No. 328209, Biolegend) hi 、CD39 int and CD39 - After GPC3-CAR-T cells were sorted, they were plated in a 24-well plate, 50 μM Mdivi-1 was added to the culture medium, and an equal amount of PBS was used to replace Mdivi-1 as a control group. After culturing for 24 h, the mean fluorescence intensity of CD39 in CAR-T cells was detected by flow cytometry.

[0082] 3. In vitro antigen stimulation experiment to study the effect of small molecule compounds on CD39 int Effect of the proportion and function of GPC3-CAR-T cells:

[0083] GPC3-CAR-T cells were prepared by the aforementioned method for preparing GPC3-CAR-T cells, and cultured in an IL-2-free medium containing 5 μg / mL rhGPC3 (human recombinant GPC3 protein). Three small molecule compounds with different concentrations were added to the medium. After culturing for 24 h, the expression of CD39 in GPC3-CAR-T cells was detected by flow cytometry. intThe proportion of cell subpopulations, expression of INF-γ and Ki-67 molecules.

[0084] 4. Lactate dehydrogenase (LDH) release assay to detect the effect of small molecule compounds on the ability of GPC3-CAR-T cells to kill tumor cells:

[0085] (1) CAR-T cells targeting different protein molecules (GPC3 or Her2) were respectively injected into tumor cells GPC3 + Huh-7 cells were co-cultured in 96-well V-bottom plates at different cell mass ratios (8:1, 4:1, 2:1) for 24 hours. The experimental groups were specifically set as follows:

[0086] GPC3-CAR-T cells + 50 μM Mdivi-1;

[0087] GPC3-CAR-T cells + equal amount of PBS;

[0088] Her2-CAR-T cells + 50 μM Mdivi-1;

[0089] Her2-CAR-T cells + equal amount of PBS;

[0090] And set up a single GPC3-CAR-T or Her2-CAR-T cell group (effector cells), a single GPC3 + The Huh-7 cell group (target cells) served as the control group;

[0091] (2) Use CytoTox Non-radioactive cytotoxicity detection kit (G1780, Promega) was used to treat each group of samples according to the kit instructions;

[0092] (3) According to the absorbance value of each group of samples at 490nm, the cytotoxicity of CAR-T cells to tumor cells (i.e., the ability of CAR-T cells to kill tumor cells) was calculated. The cytotoxicity calculation formula is: Cytotoxicity (%) = (AB-C0) / (C1-C0) × 100%. Where A is the experimental value (CAR-T cells and GPC3 + Huh-7 cells co-cultured), B is the spontaneous release value of effector cells, C0 is the spontaneous release value of target cells, and C1 is the maximum release value of target cells (before detection, the target cells were lysed at 37°C for 30 minutes using the lysis reagent in the kit).

[0093] (II) Experimental results and analysis

[0094] 1. In vitro cytotoxicity of small molecule compounds on GPC3-CAR-T cells:

[0095] like Figure 3As shown, compared with CGS-21680 and 8-Bromo-cAMP, Mdivi-1 has a higher IC50 value and less cytotoxicity against GPC3-CAR-T cells.

[0096] 2. Effects of small molecule compounds on CD39 int Effect of GPC3-CAR-T cell ratio:

[0097] like Figure 4 As shown, at the same drug concentration, CD39 in GPC3-CAR-T treated with Mdivi-1 was significantly increased compared with that in the CGS-21680 and 8-Bromo-cAMP groups. int The proportion of GPC3-CAR-T cells is higher, and it has the ability to regulate the moderate expression of CD39 in GPC3-CAR-T cells. When the concentration of Mdivi-1 is 50 μM, CD39 in GPC3-CAR-T cells int GPC3-CAR-T cells had the highest proportion.

[0098] 3. Effects of small molecule compounds on CD39 - / int / hi Effect of CD39 expression level in GPC3-CAR-T cells:

[0099] like Figure 5 As shown, when CD39 was treated with 50 μM Mdivi-1, - 、CD39 int and CD39 hi GPC3-CAR-T cells, CD39 - GPC3-CAR-T cells and CD39 int GPC3-CAR-T cell subsets showed increased CD39 mean fluorescence intensity (MFI), while CD39 hi The CD39 fluorescence intensity of GPC3-CAR-T cells did not increase significantly. This indicates that Mdivi-1 can increase the CD39 - GPC3-CAR-T cells and CD39 int The expression level of CD39 on GPC3-CAR-T cells does not increase the expression of CD39 hi The expression level of CD39 on GPC3-CAR-T cells, that is, Mdivi-1 can accurately regulate the moderate expression of CD39 on GPC3-CAR-T cells.

[0100] 4. Effects of small molecule compounds on anti-tumor function:

[0101] like Figure 6 As shown, when GPC3-CAR-T cells were treated with 50 μM Mdivi-1, CD39 -The proportion of GPC3-CAR-T cells was significantly reduced, and CD39 int The proportion of GPC3-CAR-T cells increased significantly, and CD39 hi The proportion of GPC3-CAR-T cells did not change significantly, further indicating that Mdivi-1 could - Cell subsets transformed into CD39 int Cell subsets, increase CD39 int Moreover, the secretion levels of INF-γ and Ki-67 of GPC3-CAR-T cells were significantly increased after treatment with Mdivi-1, indicating that Mdivi-1 can promote the proliferation of GPC3-CAR-T cells and enhance their anti-tumor ability (such as Figure 7 At the same time, the present invention also proves through LDH experiments that Mdivi-1 enhances the killing ability of GPC3-CAR-T cells against tumor cells, such as Figure 8 shown.

[0102] Example 3 Study on the anti-HCC effect of Mdivi-1 combined with GPC3-CAR-T cells

[0103] 1. Experimental methods

[0104] Using NSG (NOD-Prkdc scid IL tm1 / Bcgen, Beijing Biocytogen, Beijing, China) background mice were used to construct an HCC disease model to study the in vivo anti-tumor effect of Mdivi-1 combined with GPC3-CAR-T cells. The specific method was as follows:

[0105] 6-8 week old male NSG mice were injected subcutaneously on the right side of the back with 100 μL of 5 × 10 6 GPC3 + The HCC disease model was constructed by mixing Huh-7 cells with a mixed solution of Matrigel / PBS (v / v, 1:1).

[0106] After 15 days, 200 μL of 1×10 6 The PBS solution of GPC3-CAR-T cells and 200 μL Mdivi-1 (20 mg / kg) were injected into the model mice for tumor treatment experiments. At the same time, an equal amount of PBS was used instead of Mdivi-1 as a control group.

[0107] After 2 weeks of treatment, the subcutaneous tumor volume of mice was measured (tumor volume = 0.5 × length × width 2), and the therapeutic effect was evaluated by comparing the tumor volume of mice treated with different methods in each group. After the end, the tumor tissues of mice in each group were collected, digested with 2 mg / mL type IV collagenase (Sigma) at 37°C for 30 minutes, and tumor-infiltrating T cells were separated using Percoll discontinuous density gradient, and T cell function was further detected by flow cytometry.

[0108] (II) Experimental results and analysis:

[0109] like Fig. 9 As shown in the figure, compared with the control group, the tumor volume of mice in the Mdivi-1 combined with GPC3-CAR-T cell group was significantly smaller, indicating that Mdivi-1 can significantly improve the anti-tumor activity of GPC3-CAR-T cells. The tumor growth curves of mice in each group during treatment and the tumor weight after treatment are shown in the figure. Fig.10 As shown, the tumors in the Mdivi-1 combined with GPC3-CAR-T cell group grew more slowly, the tumors were maintained at a smaller size throughout the treatment period, and the tumor weight was lighter after the treatment.

[0110] In addition, CD39 int The infiltration rate of GPC3-CAR-T cells into tumors increased significantly. int The IFN-γ secretion by GPC3-CAR-T cells also increased significantly (e.g. Fig.11 shown).

[0111] The above data show that the Mdivi-1 combined with GPC3-CAR-T cell group has excellent anti-tumor effect and tumor-infiltrating CD39 int Mdivi-1 can increase the proportion of GPC3-CAR-T cells and their IFN-γ secretion level. int The GPC3-CAR-T cell ratio is conducive to the anti-tumor activity of GPC3-CAR-T cells, thereby more effectively treating hepatocellular carcinoma (HCC).

[0112] In summary, the technical solution of the present invention significantly improves the effect of CAR-T cell therapy for HCC by regulating the expression level of CD39 on CAR-T cells, providing a new idea and new solution for cell immunotherapy for HCC.

[0113] 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. Mdivi-1 is used to increase CD39 in GPC3-CAR-T cells int Application of GPC3-CAR-T cell ratio products.

2. Application of Mdivi-1 in the preparation of GPC3-CAR-T cells, wherein the prepared GPC3-CAR-T cells have CD39 int The proportion of GPC3-CAR-T cells was increased.

3. A method to increase CD39 int The method for treating GPC3-CAR-T cells in vitro with a high GPC3-CAR-T cell ratio is characterized in that GPC3-CAR-T cells were treated with Mdivi-1.

4. An in vitro method for producing GPC3-CAR-T cells with improved treatment effects for liver cancer, characterized in that: GPC3-CAR-T cells were treated with Mdivi-1.

5. The method according to claim 3 or 4, characterized in that: The concentration of Mdivi-1 is 1-200 μM.

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