Tumor immunotherapy drug target and application thereof

An immunotherapeutic drug and drug action technology, applied in biochemical equipment and methods, microbial measurement/testing, instruments, etc., can solve the problems of undisclosed associations, etc., and achieve the goal of enhancing antigen-specific immune response and enhancing killing ability Effect

Active Publication Date: 2017-07-18
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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  • Tumor immunotherapy drug target and application thereof
  • Tumor immunotherapy drug target and application thereof
  • Tumor immunotherapy drug target and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0230] Example 1 Inhibition of cholesterol esterification can enhance the effector function of CD8 T cells

[0231] Test method: The cells obtained by magnetic bead sorting were resuspended in RPMI 1640 complete medium, and the corresponding concentration of inhibitors was added, and the cells were incubated at 37 degrees CO 2 Cultured in the incubator for 6 hours, then washed 3 times with RPMI 1640 complete medium, and finally resuspended in RPMI 1640 complete medium and transferred to the well plate with activating antibodies α-CD3 and α-CD28, 37 degrees CO 2 The culture was stimulated for 20 hours in the incubator, and then Brefeldin A (5 μg / ml) was added to continue the culture for 4 hours to inhibit the secretion of cytokines. After the culture was over, the cells were collected, labeled with CD8 antibody surface, and then fixed with 4% paraformaldehyde at room temperature for 10 minutes. The cell membrane was punched with 0.1% Triton-X100, and the corresponding proteins ...

Embodiment 2

[0233] Example 2 T cell-specific knockout of ACAT1 enhances CD8 T cell immune response.

[0234] The purpose of this example is to verify the function of ACAT1 in CD8 T cells. ACAT1 flox / flox mouse with CD4 Cre The mice were mated to obtain T cell-specific knockout ACAT1 mice (CD4 Cre -Acat1 flox / flox , referred to as Acat1 CKO ) and corresponding wild-type control mice (Acat1 flox / flox ).

[0235] Ex vivo test method: CD8 T cells obtained by magnetic bead sorting were resuspended in RPMI 1640 complete medium, and corresponding concentrations of inhibitors were added, and 37°C CO 2 Cultured in the incubator for 6 hours, then washed 3 times with RPMI 1640 complete medium, and finally resuspended in RPMI 1640 complete medium and transferred to the well plate with activating antibodies α-CD3 and α-CD28, 37 degrees CO 2 The culture was stimulated for 20 hours in the incubator, and then Brefeldin A (5 μg / ml) was added to continue the culture for 4 hours to inhibit the secret...

Embodiment 3

[0238] Example 3 ACAT1 gene knockout promotes the proliferation of CD8 T cells and reduces apoptosis

[0239] The purpose of this example is to detect the effect of ACAT1 on the proliferation of CD8 T cells. Through CFSE labeling experiments, CD8 cells were stimulated with α-CD3+α-CD28 antibodies, and the effect of ACAT1 on the proliferation of CD8 T cells was further detected. Compared with wild-type CD8 T cells, Acat1 CKO The proliferative ability of CD8 T cells was enhanced ( image 3 .a). At the same time, the apoptosis of CD8 T cells after activation was detected by AnnexinV and PI staining, and the results showed that Acat1 CKO CD8 T cell Annexin V - P.I. - Compared with wild-type cells, the proportion of viable cells increased significantly, while cells in apoptotic state (AnnexinV + P.I. - and AnnexinV + P.I. + ) was significantly reduced ( image 3 .b,c).

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Abstract

The invention belongs to the technical field of biology, relates to a tumor immunotherapy drug target and application thereof, discloses application of acyl-coenzyme A: cholesterol acyltransferase ACAT1 serving as a drug target on one hand, and provides a method for in-vitro screening a tumor immunotherapy drug on the other hand, According to the method, a gene of acyl-coenzyme A: cholesterol acyltransferase ACAT1 or acyl-coenzyme A: cholesterol acyltransferase ACAT1 serving as a drug object is used for selecting the inhibitor of acyl-coenzyme A: cholesterol acyltransferase ACAT1 as a tumor immunotherapy candidate primary screening medicine. A new target is provided to the tumor immunotherapy drug, and a new platform is provided to screening of tumor immunotherapy drugs.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a drug target for tumor immunotherapy and its application. Background technique [0002] Malignant tumors are currently one of the most lethal diseases. Conventional treatment methods such as surgical resection, radiotherapy and chemotherapy are widely used in tumor treatment. However, these methods have limitations in the treatment of tumors, and it is difficult to completely cure them. Tumors, especially some metastatic malignant tumors. [0003] Immunotherapy has always been a research hotspot in tumor treatment. Cytokines interleukin-2 (IL2) and interferon-α (IFNα) have been used in the treatment of renal cell carcinoma and melanoma for more than two decades, although the target specificity is weak and the toxicity is high. In recent years, with the application of anti-PD-1 and anti-CTLA-4 and other immune checkpoint blocking antibodies in tumor immunotherapy, tumor...

Claims

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Application Information

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IPC IPC(8): C12Q1/48C12Q1/02
CPCC12Q1/48G01N33/5011G01N33/505
Inventor 许琛琦李伯良杨魏白轶冰熊缨
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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