CN118178625A - Application of Mdivi-1 combined with CAR-T cells in the preparation of drugs for the treatment of solid tumors - Google Patents

Abstract

The present invention discloses the application of Mdivi-1 combined with CAR-T cells in the preparation of drugs for treating solid tumors. According to the research of 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 accurately regulating the moderate expression of CD39 in CAR-T cells. After Mdivi-1 treated CAR-T cells, the proportion of CD39 ^- CAR-T cells was significantly reduced, the proportion of CD39 ^-int- CAR-T cells was significantly increased, and the proportion of CD39 ^-hi- CAR-T cells did not change significantly, and did not affect the cell viability of CAR-T cells. After being treated 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.

Description

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

Technical Field

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 technique

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.

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, and is therefore 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 exhibits stronger anti-tumor activity. Knocking down the expression of CD39 in CAR-T cells significantly inhibits 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.

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

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.

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.

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

The third object of the present invention is to provide an application of Mdivi-1 in increasing the proportion of CD39 ^int CAR-T cells in CAR-T cells, an application of Mdivi-1 in preparing a product for increasing the proportion of CD39 ^int CAR-T cells in CAR-T cells, and an application of Mdivi-1 in preparing CAR-T cells.

The fourth object of the present invention is to provide a CAR-T cell treatment method for increasing the proportion of CD39 ^int CAR-T cells, and a method for producing CAR-T cells with improved solid tumor treatment effects.

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

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 ^int GPC3-CAR-T cells) with a medium expression level of CD39 have stronger anti-tumor activity, while CD39 high expression (CD39 ^hi ) cell subpopulations and CD39 negative (CD39 ^- ) cell subpopulations have impaired functions. The present invention uses Mdivi-1 to treat GPC-3-CAR-T cells, significantly increasing the proportion of CD39 ^int GPC-3-CAR-T cells. Compared 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 to infiltrate into tumor tissues, significantly improve the ability to kill tumor cells, induce more powerful anti-tumor immunity, and achieve better therapeutic effects on solid tumors including hepatocellular carcinoma.

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.

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

Specifically, the CAR-T cells are GPC3-CAR-T cells.

Specifically, the solid tumor is liver cancer.

More specifically, the liver cancer is hepatocellular carcinoma.

In view of the fact that Mdivi-1 can significantly increase the proportion of CD39 ^int CAR-T cells in CAR-T cells, the protection scope of the present invention also includes:

Application of Mdivi-1 in increasing the proportion of CD39 ^int CAR-T cells in CAR-T cells.

Application of Mdivi-1 in the preparation of products for increasing the proportion of CD39 ^int CAR-T cells in CAR-T cells.

Use of Mdivi-1 in the preparation of CAR-T cells, wherein the proportion of CD39 ^int CAR-T cells in the prepared CAR-T cells is increased.

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

The present invention applies to protect a CAR-T cell treatment method for increasing the proportion of CD39 ^int CAR-T cells, and the specific method is: treating CAR-T cells with Mdivi-1.

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.

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

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

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

In addition, the above-mentioned CD39 ^int CAR-T cells refer to the detection of CAR-T cells by flow cytometry, and the CAR-T cells are grouped according to the expression level of CD39: CD39 ^- is a CD39 expression negative group, CD39 ^int is CD39 at a medium level, and CD39 ^hi is CD39 at a high expression level. Among them, the IFN-γ expression level of CD39 ^int CAR-T is higher than that of the other two subgroups, and the anti-tumor activity is stronger. More specifically, if the above-mentioned CAR-T cells are GPC3-CAR-T cells, CD39 ^int GPC3-CAR-T cells can be obtained according to the above-mentioned cell grouping method.

The present invention has the following beneficial effects:

1. The present invention proves for the first time that CD39 ^int CAR-T cells have higher anti-tumor activity, while the functions of CD39 high expression (CD39 ^hi ) cell subsets and CD39 negative (CD39 ^- ) cell subsets are impaired. At the same time, the present invention discovers 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.

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 treated CAR-T cells, the proportion of CD39 ^- CAR-T cells was significantly reduced, the proportion of CD39 ^int CAR-T cells was significantly increased, and the proportion of CD39 ^hi CAR-T cells did not change significantly, and did not affect the cell viability of CAR-T cells. 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 high clinical application value.

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

Figure 1: Flow cytometry analysis of the proportion of CD39 ^hi/int/- cell subsets in tumor-infiltrating GPC3-CAR-T cells.

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

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

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

Figure 5: Mean fluorescence intensity of CD39 in CD39 ^-/int/hi GPC3-CAR-T cells treated with 50 μM Mdivi-1.

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

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

Figure 8: 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.

Figure 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.

Figure 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.

Figure 11: Flow cytometry analysis of tumor-infiltrating CAR-T cells in the GPC3-CAR-T cell group and the Mdivi-1 combined with GPC3-CAR-T cell group, comparing the percentage of CD39 ^int/hi cells in tumor-infiltrating CAR-T cells and the percentage of IFN-γ ⁺ cells in CD39 ^int/hi CAR-T cells in each group. n=6, mean±SEM, ns: no significant difference, *p<0.05, **p<0.01, ***p<0.001.

Detailed ways

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.

Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

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

Experimental Materials

1. Cells: Human hepatoma cell lines 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, and cultured in a 37°C, 5% _CO2 cell culture incubator.

2. Animals: NSG (NOD-Prkdc ^scid IL2rg ^tm1 /Bcgen, Beijing Biocytogen, Beijing, China) mice. All experiments with mice 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).

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.

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

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

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

experimental method

(I) Preparation of GPC3-CAR-T cells

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

(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;

(2) using BD IMag ^TM Human CD8 T Lymphocyte Enrichment Set-DM, primary human CD8 ⁺ T cells were purified from PBMCs using magnetic beads to a purity of 98% and enriched;

(3) The isolated and enriched human primary CD8 ⁺ T cells were seeded at a density of 1×10 ⁶ cells/mL in retronectin-coated well plates 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 ⁺ T cells. The medium containing the above CD3 antibody, CD28 antibody and IL-2 was replaced twice a week.

2. Construction of lentiviral vector encoding CAR

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.

3. Transduction of recombinant lentivirus

(1) Obtaining lentivirus: The density of HEK293T cells was adjusted to 8 × 10 ⁶ cells/100 mm culture dish. After culturing for 24 h, HEK293T cells were transfected with a plasmid encoding CAR, the pMD2.G vector (7.5 μg) containing 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.

(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.

(3) Lentiviral transduction: Add lentiviral supernatant to the activated CD8 ⁺ T cells obtained in method 1, then add 8 μg/mL (final concentration) polybrene transfection reagent (TR-1003-G, Sigma), centrifuge at 350×g for 90 min, and incubate at 37°C. After 12 hours, remove the lentivirus, and continue to culture and expand the lentiviral-transduced CD8 ⁺ T cells using conditioned medium containing 10 ng/mL IL-2. Change the medium twice a week. GPC3-CAR-T cells are obtained 14 days after lentiviral transduction.

(II) Flow cytometry

(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.

(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.

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

Example 1 Evaluation of the anti-tumor ability of CD39 ^int GPC3-CAR-T

1. Experimental methods

The HCC disease model was constructed using mice with NSG (NOD-Prkdc ^scid IL2rg ^tm1 /Bcgen, Beijing Biocytogen, Beijing, China) background. The specific method was as follows: 100 μL of a mixed solution of matrix gel/PBS (v/v, 1:1) containing 5×10 ⁶ GPC3 ⁺ Huh-7 cells was subcutaneously injected on the right side of the back of 6-8 week old male NSG mice to construct a subcutaneous HCC tumor model (n=6).

Two weeks later, 200 μL of PBS solution containing 1×10 ⁶ GPC3-CAR-T cells was injected into the model mice through the tail vein. Two weeks later, the mouse tumor tissue was removed and ground into a single cell suspension, and the GPC3-CAR-T cell subpopulations and functions in the tumor tissue were detected by flow cytometry.

(II) Experimental results and analysis:

By flow cytometry, according to the clustering results of GPC3-CAR-T cells, 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 indicates medium CD39 expression level). The results showed that CD39 ^int GPC3-CAR-T cells, especially CD39 ^int PD-1 ^int and CD39 ^int PD-1 ^- subsets, secreted higher levels of interferon-γ (IFN-γ) (as shown in Figures 1 and 2), indicating that when CD39 expression was at a medium level, GPC3-CAR-T cells exhibited better anti-tumor ability.

Example 2 Effect of Mdivi-1 on the proportion and function of CD39 ^int GPC3-CAR-T cells

1. Experimental methods

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

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

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.

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

CD39 ^hi , CD39 ^int and CD39 ^- GPC3-CAR-T cells were sorted out by flow cytometry using CD39 flow cytometry antibody (APC Anti-CD39 (Cat No.328209, Biolegend)) and plated in 24-well plates. 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 hours, the average fluorescence intensity of CD39 in CAR-T cells was detected by flow cytometry.

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

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 hours, the proportion of CD39 ^int cell subsets, and the expression of INF-γ and Ki-67 molecules in GPC3-CAR-T cells were detected by flow cytometry.

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:

(1) CAR-T cells targeting different protein molecules (GPC3 or Her2) were co-cultured with tumor cells GPC3 ⁺ Huh-7 at different cell ratios (8:1, 4:1, 2:1) in a 96-well V-bottom plate for 24 hours. The specific experimental groups were set as follows:

GPC3-CAR-T cells + 50 μM Mdivi-1;

GPC3-CAR-T cells + equal amount of PBS;

Her2-CAR-T cells + 50 μM Mdivi-1;

Her2-CAR-T cells + equal amount of PBS;

A single GPC3-CAR-T or Her2-CAR-T cell group (effector cells) and a single GPC3 ⁺ Huh-7 cell group (target cells) were set up as control groups;

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

(3) According to the absorbance value of each group of samples at 490 nm, 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 was: Cytotoxicity (%) = (ABC ₀ )/(C ₁ -C ₀ ) × 100%. Wherein, A is the experimental value (CAR-T cells co-cultured with GPC3 ⁺ Huh-7 cells), B is the spontaneous release value of effector cells, C ₀ is the spontaneous release value of target cells, and C ₁ is the maximum release value of target cells (before detection, the target cells were lysed at 37°C for 30 min using the lysis reagent in the kit).

(II) Experimental results and analysis

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

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

2. Effect of small molecule compounds on the proportion of CD39 ^int GPC3-CAR-T cells:

As shown in Figure 4, at the same drug concentration, the proportion of CD39 ^int GPC3-CAR-T cells in GPC3-CAR-T treated with Mdivi-1 was higher than that in the CGS-21680 and 8-Bromo-cAMP groups, which has the ability to regulate the moderate expression of CD39 in GPC3-CAR-T cells. When the concentration of Mdivi-1 was 50 μM, the proportion of CD39 ^int GPC3-CAR-T cells in GPC3-CAR-T cells was the highest.

3. Effects of small molecule compounds on CD39 expression levels in CD39 ^-/int/hi GPC3-CAR-T cells:

As shown in Figure 5, when CD39 ^- , CD39 ^int and CD39 ^hi GPC3-CAR-T cells were treated with 50 μM Mdivi-1, the CD39 ^- GPC3-CAR-T cells and CD39 ^int GPC3-CAR-T cell subsets showed an increase in CD39 mean fluorescence intensity (MFI), while the CD39 fluorescence intensity of CD39 ^hi GPC3-CAR-T cells did not increase significantly. This indicates that Mdivi-1 can increase the expression level of CD39 on CD39 ^- GPC3-CAR-T cells and CD39 ^int GPC3-CAR-T cells, but will not increase the expression level of CD39 on CD39 ^hi GPC3-CAR-T cells, that is, Mdivi-1 can accurately regulate the moderate expression of CD39 on GPC3-CAR-T cells.

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

As shown in Figure 6, when GPC3-CAR-T cells were treated with 50μM Mdivi-1, the proportion of CD39 ^- GPC3-CAR-T cells was significantly reduced, the proportion of ^CD39int GPC3-CAR-T cells was significantly increased, and the proportion of ^CD39hi GPC3-CAR-T cells did not change significantly, further illustrating that Mdivi-1 can convert CD39 ^- cell subsets into ^CD39int cell subsets and increase the proportion of ^CD39int cells. In addition, the INF-γ and Ki-67 secretion levels of GPC3-CAR-T 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 (as shown in 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 to tumor cells, as shown in Figure 8.

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

1. Experimental methods

The HCC disease model was established using mice with NSG (NOD-Prkdc ^scid IL2rg ^tm1 /Bcgen, Beijing Biocytogen, Beijing, China) background to study the in vivo anti-tumor effect of Mdivi-1 combined with GPC3-CAR-T cells. The specific method was as follows:

100 μL of a mixed solution of Matrigel/PBS (v/v, 1:1) containing 5×10 ⁶ GPC3 ⁺ Huh-7 cells was subcutaneously injected on the right side of the back of 6-8 week-old male NSG mice to establish an HCC disease model.

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

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.

(II) Experimental results and analysis:

As shown in Figure 9, 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 Figure 10. The tumor in the Mdivi-1 combined with GPC3-CAR-T cell group grew more slowly, the tumor was maintained at a smaller volume throughout the treatment period, and the tumor weight was lighter after treatment.

In addition, the infiltration rate of CD39 ^int GPC3-CAR-T cells into the tumor increased significantly in the Mdivi-1 combined with GPC3-CAR-T cell group. At the same time, the IFN-γ secreted by CD39 ^int GPC3-CAR-T cells also increased significantly in the Mdivi-1 combined with GPC3-CAR-T cell group (as shown in Figure 11).

The above data show that the excellent anti-tumor effect of Mdivi-1 combined with GPC3-CAR-T cells is related to the increase in the proportion of tumor-infiltrating CD39 ^int GPC3-CAR-T cells and the increase in the level of IFN-γ secretion. Mdivi-1 can increase the proportion of CD39 ^int GPC3-CAR-T cells, which is beneficial for GPC3-CAR-T cells to exert anti-tumor activity, thereby more effectively treating hepatocellular carcinoma (HCC).

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.

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 (10)

1. Application of Mdivi-1 combined with CAR-T cells in the preparation of drugs for the treatment of solid tumors. 2. A drug for treating solid tumors, characterized in that it contains Mdivi-1 and CAR-T cells. 3. The use according to claim 1 or the drug according to claim 2, characterized in that the CAR-T cells are GPC3-CAR-T cells. 4. The use according to claim 1 or the medicine according to claim 2, characterized in that the solid tumor is liver cancer. 5. Application of Mdivi-1 in increasing the proportion of CD39 ^int CAR-T cells. 6. Application of Mdivi-1 in the preparation of products for increasing the proportion of CD39 ^int CAR-T cells. 7. Use of Mdivi-1 in the preparation of CAR-T cells, wherein the proportion of CD39 ^int CAR-T cells in the prepared CAR-T cells is increased. 8. A CAR-T cell treatment method for increasing the proportion of CD39 ^int CAR-T cells, characterized in that the CAR-T cells are treated with Mdivi-1. 9. A method for producing CAR-T cells with improved solid tumor treatment effects, characterized in that the CAR-T cells are treated with Mdivi-1. 10 . The method according to claim 8 or 9 , wherein the concentration of Mdivi-1 is 1 to 200 μM.