Application of pantoprazole in preparation of enhancer for MSLN-car-t cell therapy against brain metastases

Abstract

An application of pantoprazole in the preparation of an enhancer for an MSLN-CAR-T cell therapy against brain metastases. Treating with pantoprazole prior to MSLN-CAR-T therapy increases MSLN protein expression, particularly on cell surfaces, reduces MSLN target antigen expression heterogeneity, and improves the homing ability and killing ability of MSLN-CAR-T cells.

Description

Use of pantoprazole in preparation of enhancer for MSLN-CAR-T cell treatment of brain metastasis TECHNICAL FIELD

[0001] The present application relates to the field of tumor treatment, in particular to the use of pantoprazole in the preparation of an enhancer for MSLN-CAR-T cell treatment of brain metastasis. BACKGROUND

[0002] With the improvement of diagnosis and treatment technology, early detection and early treatment of cancer have prolonged the life of patients, but the incidence of brain metastasis has increased year by year. Brain metastasis usually occurs in patients with lung cancer, breast cancer and melanoma, with an incidence of 40%-50%, 20% and 10%-20%, respectively. At present, the treatment options for brain metastasis include surgery, whole brain radiotherapy, stereotactic radiosurgery and systemic drug therapy, but the efficacy is limited, and new treatment options are urgently needed.

[0003] Chimeric antigen receptor (CAR)-T cell immunotherapy has shown encouraging results in different types of cancer and has been approved for the treatment of hematologic malignancies. A preclinical study in a mouse model of breast cancer brain metastasis showed that intracerebroventricular administration of HER2-CAR-T cells resulted in the regression of multiple brain metastases and meningeal metastases and prolonged overall survival. Based on this preclinical study, a phase I clinical trial of HER2-CAR-T cell treatment of recurrent brain / meningeal metastases is underway (NCT03696030). This brings new hope for the treatment of brain metastasis.

[0004] MSLN is a glycosylated phosphatidylinositol-anchored protein that is usually present in small amounts on the surface of mesothelial cells in the pleura, pericardium, peritoneum and tunica vaginalis (male). Currently, MSLN has been found to be overexpressed in a variety of cancers, including malignant mesothelioma, ovarian cancer, breast cancer, pancreatic cancer, lung cancer, gastric cancer, cervical cancer, uterine serous carcinoma, and bile duct cancer. Unfortunately, analysis of published studies on MSLN-CAR-T cell treatment of solid tumors shows that MSLN-CAR-T cell monotherapy is safe, but has not shown significant clinical efficacy beyond disease stabilization. Therefore, it is important to explore the mechanisms behind the poor clinical efficacy of MSLN-CAR-T cells and to propose targeted measures.

[0005] In clinic, proton pump inhibitors are widely used for treating peptic ulcer, gastroesophageal reflux disease and other digestive system diseases. Many clinical research results show that pretreatment with proton pump inhibitors combined with metronomic chemotherapy can inhibit autophagy, reverse drug resistance of refractory tumors and induce chemotherapy sensitization of various solid tumors such as osteosarcoma, breast cancer and digestive tract tumors. Proton pump inhibitors include pantoprazole, omeprazole, lansoprazole, rabeprazole and esomeprazole, which can pass through the blood-brain barrier. Among them, pantoprazole is the third generation of proton pump inhibitors, which is more stable in weak acid environment than similar drugs. After being activated, it only binds to two sites of the activated site of proton pump (while omeprazole and lansoprazole show more site binding to the activated site), which reflects the high selectivity of the combination of proton pump at the molecular level. Pantoprazole has weak inhibitory effect on cytochrome P450 enzyme and can be metabolized by the second enzyme system, so it is not easy to cause drug interaction and is safer when combined with other drugs. Whether pantoprazole can improve the efficacy of MSLN-CAR-T cell treatment of brain metastasis needs to be further explored. SUMMARY

[0006] Therefore, one of the purposes of the present application is to provide a pharmaceutical preparation for treating brain metastasis; and the second purpose of the present application is to provide a method for treating brain metastasis.

[0007] In order to achieve the above purposes, the present application provides the following technical solutions:

[0008] 1. A pharmaceutical preparation for treating brain metastasis, the pharmaceutical preparation comprising pantoprazole and MSLN-CAR-T cells.

[0009] Pantoprazole as an enhancer for MSLN-CAR-T cell treatment of brain metastasis improves the homing ability and killing ability of MSLN-CAR-T cells

[0010] Preferably, in the present application, the preparation is used for intraperitoneal administration of pantoprazole and intratumoral administration of MSLN-CAR-T cells.

[0011] 2. A method for treating brain metastasis, wherein an effective amount of pantoprazole is used every day one week before MSLN-CAR-T cell treatment.

[0012] Preferably, in the present application, the brain metastasis is malignant mesothelioma, ovarian cancer, breast cancer, pancreatic cancer, lung cancer, gastric cancer, cervical cancer, uterine serous carcinoma or cholangiocarcinoma.

[0013] Preferably, in the present application, the effective amount is 6 mg / kg.

[0014] The application has the beneficial effects that the application provides the use of pantoprazole in the preparation of an enhancer for MSLN-CAR-T cell treatment of brain metastases, provides a new use of pantoprazole, and improves the homing ability and killing ability of MSLN-CAR-T cells by improving the expression of MSLN protein, especially on the cell membrane, and reducing the heterogeneity of MSLN target antigen expression. It provides a new idea for CAR-T cell treatment of solid tumors and lays a preliminary foundation for related clinical experiments. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to make the purpose, technical scheme and beneficial effects of the application clearer, the application provides the following drawings for illustration:

[0016] Fig. 1 is a fluorescence intensity diagram of pantoprazole combined with MSLN-CAR-T cell treatment of triple negative breast cancer brain metastases.

[0017] Fig. 2 is a fluorescence intensity quantitative analysis diagram of pantoprazole combined with MSLN-CAR-T cell treatment of triple negative breast cancer brain metastases.

[0018] Fig. 3 is a survival analysis diagram of pantoprazole combined with MSLN-CAR-T cell treatment of triple negative breast cancer brain metastases.

[0019] Fig. 4 is a representative picture of immunohistochemistry of pantoprazole increasing MSLN protein expression of triple negative breast cancer brain metastases cells.

[0020] Fig. 5 is a fluorescence intensity diagram of pantoprazole combined with MSLN-CAR-T cell treatment of non-small cell lung cancer brain metastases.

[0021] Fig. 6 is a fluorescence intensity quantitative analysis diagram of pantoprazole combined with MSLN-CAR-T cell treatment of non-small cell lung cancer brain metastases.

[0022] Fig. 7 is a survival analysis diagram of pantoprazole combined with MSLN-CAR-T cell treatment of non-small cell lung cancer brain metastases.

[0023] Fig. 8 is a representative picture of immunohistochemistry of pantoprazole increasing MSLN protein expression of non-small cell lung cancer brain metastases cells. DETAILED DESCRIPTION

[0024] The application will be further described below in combination with the drawings and specific examples, so that those skilled in the art can better understand the application and implement it, but the examples are not limiting the application.

[0025] Pantoprazole in the present application is purchased from the United States selleck company (item number: S5608); MSLN-CAR plasmid is purchased from ICARTAB company in China (item number: LIC012A); T cells are taken from healthy donors in the blood transfusion department of the first affiliated hospital of the army medical university; MSLN antibody is purchased from the United States abcam company (activation: ab133489); immunohistochemical kit is purchased from Denmark Dako company (item number: K5007); NOG mice are purchased from Vantian Lihua company and are raised in the animal experiment center of the first affiliated hospital of the army medical university.

[0026] Example 1

[0027] Pantoprazole combined with MSLN-CAR-T cells inhibits triple negative breast cancer brain metastasis, and the specific steps are as follows:

[0028] A, adopt craniostereotaxic apparatus to construct triple negative breast cancer brain transplantation tumor model:

[0029] (1) Collect tumor cells in good growth state, adjust the cell density to 2×10 4 μL with PBS, put the cells on ice and take them to the animal experiment center.

[0030] (2) According to the weight of mice (female NOG mice, 18-22g), anesthetize the mice with appropriate amount of chloral hydrate by intraperitoneal injection.

[0031] (3) After successful anesthesia, fix the mice with craniostereotaxic apparatus (prone position), disinfect the surgical area with alcohol cotton ball, cut the skin of the mouse head, drill a hole on the skull (position: 1.5mm to the right of bregma, 1.5mm forward), inject 10μL cell suspension into the mouse intracranially through microsyringe, and the needle depth is 2.5mm.

[0032] (4) When injecting cells, keep uniform speed and slow speed. After injection, leave for 2min and then slowly pull out the syringe. After disinfection with alcohol cotton ball, suture the wound.

[0033] (5) Send the mice back to the cage, observe closely, and select tumor-bearing mice as experimental mice.

[0034] B, divide the mice into control group, pantoprazole group, MSLN-CAR-T cell group and pantoprazole combined with MSLN-CAR-T cell group;

[0035] C, start intraperitoneal injection of pantoprazole 6mg / kg on the fourth day of tumor bearing, once a day, for 1 week;

[0036] D, give MSLN-CAR-T cells intratumorally on the seventh day of tumor bearing;

[0037] E. The change in tumor fluorescence intensity (representing tumor size) was observed using a small animal live imaging instrument (Figures 1 and 2), and it was found that the tumor fluorescence intensity of the pantoprazole combined with MSLN-CAR-T cell group was reduced compared to the other three groups; survival analysis results showed that the survival time of the pantoprazole combined with MSLN-CAR-T cell group was increased by 1 times compared to the MSLN-CAR-T cell group (Figure 3);

[0038] F. MSLN immunohistochemical staining was performed on the tumor tissues of the MSLN-CAR-T cell group and the pantoprazole combined with MSLN-CAR-T cell group, and the results showed that the MSLN protein expression of the pantoprazole combined with MSLN-CAR-T cell group was significantly increased (Figure 4).

[0039] The above results show that the use of pantoprazole reduces the heterogeneity of MSLN target antigen expression by increasing the expression of MSLN protein, especially on the cell membrane, and improves the homing ability and killing ability of MSLN-CAR-T cells.

[0040] Example 2

[0041] Pantoprazole combined with MSLN-CAR-T cells inhibits non-small cell lung cancer brain metastasis, and the specific steps are as follows:

[0042] A. A stereotactic instrument was used to construct a non-small cell lung cancer brain metastasis model (method same as above); the mice were divided into a control group, a pantoprazole group, a MSLN-CAR-T cell group and a pantoprazole combined with MSLN-CAR-T cell group;

[0043] B. Abdominal injection of pantoprazole 6 mg / kg was given on the fourth day of tumor bearing, once a day, for 1 week;

[0044] C. MSLN-CAR-T cells were administered intratumorally on the seventh day of tumor bearing;

[0045] D. The change in tumor fluorescence intensity (representing tumor size) was observed using a small animal live imaging instrument (Figures 5 and 6), and survival analysis was performed (Figure 7); the results showed that the tumor fluorescence intensity of the pantoprazole combined with MSLN-CAR-T cell group was reduced; survival analysis results showed that the survival time of the pantoprazole combined with MSLN-CAR-T cell group was increased compared to the MSLN-CAR-T cell group.

[0046] E. MSLN immunohistochemical staining was performed on the tumor tissues of the MSLN-CAR-T cell group and the pantoprazole combined with MSLN-CAR-T cell group (Figure 8), and the results showed that the MSLN protein expression of the pantoprazole combined with MSLN-CAR-T cell group was significantly increased.

[0047] The above-described embodiments are merely preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Any equivalent substitutions or transformations made by those skilled in the art based on the present application are within the protection scope of the present application. The protection scope of the present application is subject to the claims.

Claims

1. A pharmaceutical preparation for the treatment of brain metastases, characterized in that: The pharmaceutical preparation comprises pantoprazole and MSLN-CAR-T cells.

2. The pharmaceutical preparation according to claim 1, characterized in that: The preparation is for intraperitoneal administration of pantoprazole and intratumoral administration of MSLN-CAR-T cells.

3. A method of treating brain metastases, characterized by: An effective amount of pantoprazole is used every day in the week before MSLN-CAR-T cell treatment.

4. Use according to claim 1, characterized in that: The brain metastases are malignant mesothelioma, ovarian cancer, breast cancer, pancreatic cancer, lung cancer, gastric cancer, cervical cancer, uterine serous carcinoma or cholangiocarcinoma.

5. The use according to claim 1, characterized in that: The effective amount is 6 mg / kg.

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