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Application of Caspase-1 in preparation of medicine for improving CAR-T treatment effect

A technology for therapeutic effects and drugs, applied in the fields of biomedicine and molecular biology, to relieve toxic side effects and improve therapeutic effects

Inactive Publication Date: 2020-12-11
XUZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the application of Caspase-1 in the preparation of drugs that improve the therapeutic effect of CAR-T has not been reported. This invention provides a new idea for improving the therapeutic effect of CAR-T and alleviating its side effects

Method used

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  • Application of Caspase-1 in preparation of medicine for improving CAR-T treatment effect
  • Application of Caspase-1 in preparation of medicine for improving CAR-T treatment effect
  • Application of Caspase-1 in preparation of medicine for improving CAR-T treatment effect

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Construction of cell co-culture model

[0058] 1. Experimental materials

[0059] ELISA kit was purchased from Hangzhou Lianke Biotechnology Co., Ltd., Western and IP cell lysates were purchased from Shanghai Beyond Biotechnology Co., Ltd. Raji cells (tumor cells expressing CD19) and THP-1 cells were purchased from the United States ATCC Biological Standards Resource Center, CD19 CAR-T cells were provided by the Cancer Prevention and Control Institute of Xuzhou Medical University.

[0060] 2. Model building method

[0061] (1) THP-1 cells 5×10 5 / well were induced into M2 macrophages in a 6-well plate.

[0062] (2) CD19 CAR-T cells and Raji cells were counted separately, and the ratio of CAR-T cells: Raji cells: macrophages was 1:3:1, seeded into 6-well plates, and co-cultured with macrophages, 2mL medium per well, put in 37℃, 5% CO 2 cultured in a constant temperature incubator.

[0063] (3) Drug treatment group: Before adding CAR-T cells and Raji cells...

Embodiment 2

[0064] Example 2 Inhibition of the activity of Caspase-1 on the release of IL-1β induced by CAR-T treatment

[0065] In this example, two different Caspase-1 inhibitors, namely VX-765 and Ac-YVAD-CMK, were selected to inhibit the enzymatic activity of Caspase-1 to detect the inhibition of Caspase-1 on IL-1β induced by CAR-T treatment release effects.

[0066] 1. Experimental method

[0067] CD19 CAR-T, tumor cells (Raji) and macrophages were co-cultured, and VX-765 and Ac-YVAD-CMK were added at the same time. After 24 hours, the culture supernatant and macrophages were collected for Western Blot and ELISA detection. The specific Western Blot detection steps and ELISA detection steps are as follows: Western Blot detection steps:

[0068] (1) According to the cell culture system, CD19 CAR-T, tumor cells (Raji) and macrophages were co-cultured in groups for 24 hours. After 24 hours, the cell culture supernatants of the experimental group and the control group were collected, 5×...

Embodiment 3

[0090] Example 3 The effect of inhibiting the activity of Caspase-1 on the expression of PD-L1 and IDO in macrophages induced by CAR-T therapy

[0091] This example first verified that macrophages after CAR-T treatment up-regulated the expression of their own PD-L1 and IDO through their autocrine IL-1β, and further verified that inhibiting the activity of Caspase-1 has a positive effect on CAR-T treatment-induced macrophages. Effects of PD-L1 and IDO expression on phagocytic cells.

[0092] 1. To verify whether IL-1β is involved in the expression process of PD-L1 and IDO in macrophages

[0093] (1) Experimental method

[0094] THP-1 cells were induced into M2 macrophages as a macrophage model. Recombinant human IL-1β was added to the macrophage medium, and after 24 hours, the macrophages were collected, and Western Blot was used to detect the expression of PD-L1 and IDO in the macrophages. In order to rule out the possibility of using the cell line (THP-1) as a macrophage m...

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Abstract

The invention discloses application of Caspase-1 in preparation of a medicine for improving a CART treatment effect. According to the application of the invention, cell experiments prove that IL1-betarelease caused by CAR-T treatment can be remarkably reduced by inhibiting expression and / or activity of the Caspase-1, and prove that the Caspase-1 plays an important role in macrophage IL1-beta release induced by the CART treatment. The application of the Caspase-1 provides a theoretical basis and a new thought for improving the CART treatment effect and relieving the toxic and side effects of the CART.

Description

technical field [0001] The invention belongs to the fields of biomedicine and molecular biology, and relates to the application of Caspase-1 in the preparation of drugs for improving the therapeutic effect of CAR-T. Background technique [0002] The number of deaths due to malignant tumors is 89.274 million every year in the world, becoming the second leading cause of death after cardiovascular and cerebrovascular diseases. At present, the treatment methods for malignant tumors mainly include surgery, chemotherapy, radiotherapy and other means, all of which can achieve certain therapeutic effects, but there are also shortcomings or shortcomings. Tumor immunotherapy, as an emerging therapy, has developed into the fourth tumor treatment mode after surgery, chemotherapy and radiotherapy. In recent years, tumor immunotherapy has developed rapidly. According to different mechanisms of action, tumor immunotherapy drugs are divided into six categories: T cell-targeted immunomodula...

Claims

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Application Information

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IPC IPC(8): G01N33/573C12Q1/6886A61K45/00A61P35/00
CPCG01N33/573C12Q1/6886A61K45/00A61P35/00G01N2333/96466G01N2500/04C12Q2600/158C12Q2600/136
Inventor 刘丹戴玉莲郑骏年施明徐溪悦赵璇李思瑾马文
Owner XUZHOU MEDICAL UNIV
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