Model and method for detecting inhibiting effect of chimeric antigen receptor (CAR) T cells on hepatoma cells

A chimeric antigen receptor, liver cancer cell technology, applied in the field of tumor cell immunotherapy, can solve the problem of inability to effectively simulate and evaluate the in vivo efficacy of liver cancer CART cells, and achieve the effect of simulating cell effects and inhibitory effects

Inactive Publication Date: 2017-05-10
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] Aiming at the fact that the current preclinical evaluation methods targeting liver cancer CAR T cells cannot effectively simulate and evaluate the in vivo efficacy of liver cancer CAR T cells, the present invention provides a model and method for detecting the inhibitory effect of chimeric antigen receptor T cells on liver cancer c...

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  • Model and method for detecting inhibiting effect of chimeric antigen receptor (CAR) T cells on hepatoma cells
  • Model and method for detecting inhibiting effect of chimeric antigen receptor (CAR) T cells on hepatoma cells
  • Model and method for detecting inhibiting effect of chimeric antigen receptor (CAR) T cells on hepatoma cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Construction of chimeric antigen receptor T cells

[0063] (1) Synthesize human IgM signal peptide, anti-GPC3 antibody, human CD28 transmembrane region and intracellular region, human 4-1BB intracellular region and human CD3ζ intracellular region, namely CAR-GPC3-CD28-4-1BB - CD3ξ (CAR-GPC3), whose sequence is shown in SEQ ID NO.4, and then the nucleic acid sequence of CAR-CD19-CD28-4-1BB-CD3ξ (CAR CD19) was synthesized from the whole gene as a negative control, and the C-terminus of the synthesized gene contained Restriction endonuclease Pme1 digestion site and its protective base and N-terminal containing restriction endonuclease Spe1 restriction site and its protection base;

[0064] (2) Ligate the synthetic gene into the pWPXLd-EGFP lentiviral vector by restriction enzyme digestion to obtain CAR plasmid transformation vectors pWPXLd-CAR-GPC3-2A-EGFP and pWPXLd-CAR-CD19-2A-EGFP respectively;

[0065] (3) Lentiviral packaging: pWPXLd-CAR-GPC3-2A-EGFP, pWPX...

Embodiment 2

[0075] Example 2: Construction of liver cancer PDX model by subcutaneous transplantation

[0076] (1) Treatment of primary liver cancer samples: Primary liver cancer tumor tissue samples were obtained from the tumor tissue sample bank of a tertiary hospital. Liver cancer samples should be soaked in RPMI 1640 medium and stored on ice for transportation. After obtaining the tumor sample, select the liver cancer sample of the tumor tissue with light yellow / flesh color on the edge, wash it 2-3 times with PBS (1% P / S), divide the sample into three parts, one part is frozen and preserved; one part is used for tumor tissue Section and immunohistochemical analysis; one copy for tumor transplantation;

[0077] (2) Liver cancer tissue transplantation and monitoring: Subcutaneous transplantation was adopted, specifically including: using scissors to cut the primary tumor tissue sample into small pieces of tumor tissue with a diameter of 3 mm, and transplanting 2-3 small pieces into each ...

Embodiment 3

[0082] Example 3: Construction of liver cancer PDX model through liver orthotopic transplantation

[0083] (1) Treatment of primary liver cancer samples: Primary liver cancer tumor tissue samples were obtained from the tumor tissue sample bank of a tertiary hospital. Liver cancer samples should be soaked in RPMI 1640 medium and stored on ice for transportation. After obtaining the tumor sample, select the liver cancer sample of the tumor tissue with light yellow / flesh color on the edge, wash it 2-3 times with PBS (1% P / S), divide the sample into three parts, one part is frozen and preserved; one part is used for tumor tissue Section and immunohistochemical analysis; one copy for tumor transplantation;

[0084] (2) Liver cancer tissue transplantation and monitoring: The method of liver orthotopic transplantation is adopted, specifically including: using scissors to cut the primary tumor tissue sample into a volume of 2mm 3 Small pieces of tumor tissue were reserved, and each r...

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Abstract

The invention relates to the field of cellular immunotherapy of tumors, in particular to a model and a method for detecting inhibiting effect of chimeric antigen receptor (CAR) T cells on hepatoma cells. The model is obtained by transplanting primary hepatoma tissues into a model living body, and transplanting the CAR T cells into the model living body. The model provided by the invention can be used for effectively simulating the tumor malignant degree of a patient, and assessing the inhibiting effect of the CAR T cells on hepatomas of different malignant degrees; the model can be used for effectively assessing the inhibiting effect of the expression of immunosuppression check point molecules in primary hepatoma cells on the CAR T cells.

Description

technical field [0001] The invention relates to the field of tumor cell immunotherapy, in particular to a model and method for detecting the inhibitory effect of chimeric antigen receptor T cells on liver cancer cells. Background technique [0002] Liver cancer is one of the cancers with the highest mortality rate in Asia. At present, the effective treatment methods for liver cancer are partial liver resection, liver transplantation, chemotherapy, and arterial chemoembolization. Although there are many new discoveries in the diagnosis and treatment of liver cancer, the five-year survival rate of liver cancer is still only 10%. Currently, many new potential immunotherapeutic approaches are undergoing clinical trials, such as CAR T cell immunotherapy and immunosuppressive checkpoint antibodies. In view of the low survival rate, a rigorous and effective preclinical evaluation model that simulates the human body is an urgent need for the current liver cancer treatment research...

Claims

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Application Information

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IPC IPC(8): A61K35/17A61P35/00A61D1/00A61D7/00A01K67/02
CPCA01K67/02A01K2267/0337A61D1/00A61D7/00A61K35/17
Inventor 李鹏蒋治武吴迪林思妙李柏衡王素娜吴绮婷陈冬梅姚瑶
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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