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Human mesothelin chimeric antigen receptor, its T cell, its preparation method and application

A chimeric antigen receptor and cell technology, applied in the field of tumor treatment, can solve the problem of restricting the effective activation of specific T cells

Active Publication Date: 2021-03-16
英威福赛生物技术(天津)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the immune tolerance environment in the tumor tissue limits the effective activation and function of specific T cells, requiring human intervention in vitro to expand and activate mutant antigen-specific T cells in vitro

Method used

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  • Human mesothelin chimeric antigen receptor, its T cell, its preparation method and application
  • Human mesothelin chimeric antigen receptor, its T cell, its preparation method and application
  • Human mesothelin chimeric antigen receptor, its T cell, its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1. Construction of Mesothelin Recombinant Protein Expression Plasmid

[0083] The cDNA fragment of mesothelin was synthesized in vitro, the HIS tag was added at the end, and the restriction sites EcoR1 and BglII were introduced at both ends, and cloned into the expression vector pCAGGS to construct the recombinant eukaryotic expression plasmid of the full-length mesothelin protein. The above work was completed by Suzhou Synbio.

[0084] The cDNA sequence of mesothelin recombinant protein is as follows:

[0085]ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTT GCACTTGTCACGAATTCGGAAGTGGAGAAGACAGCCTGTCCTTC AGGCAAGAAGGCCCGCGAGATAGACGAGAGCCTCATCTTCTACA AGAAGTGGGAGCTGGAAGCCTGCGTGGATGCGGCCCTGCTGGCC ACCCAGATGGACCGCGTGAACGCCATCCCCTTCACCTACGAGCA GCTGGACGTCCTAAAGCATAAACTGGATGAGCTCTACCCACAAG GTTACCCCGAGTCTGTGATCCAGCACCTGGGCTACCTCTTCCTCA AGATGAGCCCTGAGGACATTCGCAAGTGGAATGTGACGTCCCTG GAGACCCTGAAGGCTTTGCTTGAAGTCAACAAAGGGCACGAAAT GAGTCCTCAGGTGGCCACCCTGATCGACCGCTTTGTGAAGGGAAGGGGCCAGCTAG...

Embodiment 2

[0086] Example 2. Expression and purification of mesothelin protein

[0087] 1) Transfection of HEK293T cells (purchased from Shanghai Cell Bank, BNCC338274): 18 hours before transfection, HEK293T cells were treated with 1.5x10 7 / ml was transferred to 30 15cm petri dishes for culture; 37.5mL DMEM (purchased from Gibco, C11995500CP) (without serum and antibiotics) was transferred to a 50mL tube, and 2970μg polyetherimide (PEI) MegaTran 1.0 (purchased from Alfa Aesar, 9002-98-6) and mix; take 37.5mL DMEM (without serum and antibiotics) to a 50mL tube, add 990μg of mesothelin plasmid DNA and mix; add PEI / DMEM solution to the prepared DNA solution , mix quickly and stand at room temperature for 15 minutes; respectively take 2.5ml PEI / DNA / DMEM mixture into each culture dish and store at 37°C, 5% CO 2 Cultivated in an incubator. After 6 hours of transfection, carefully aspirate the culture medium, and add 25ml of new culture medium DMEM+2%FBS (purchased from Gibco, 10270)+0.12% d...

Embodiment 3

[0091] Example 3. Preparation and preliminary screening of mesothelin monoclonal antibody

[0092] This part of the work was completed by Nanjing GenScript Company. The purified full-length mesothelin recombinant protein (hereinafter referred to as mesothelin antigen) obtained in Example 2 was used to immunize B6 / C57 mice according to standard methods. Fusion, screening, etc., to obtain 9 monoclonal strains.

[0093] The corresponding fusion plate cell lines are 1G7-1, 3A11-1, 3F5-1, 6H12-2, 8C7-1, 8F8-2, 8G8-2, 11E2-2, 11H3-2, and the corresponding monoclonal antibodies are labeled with this .

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Abstract

The invention provides a human mesothelin chimeric antigen receptor and a T cell, a preparation method and use thereof. The mesothelin chimeric antigen receptor comprises an anti-mesothelin scFv antibody, a CD8 hinge region, a CD28 transmembrane region, a CD3 intracellular region and a 41BB intracellular region. Nucleic acid encoding the receptor, a vector containing the nucleic acid and a host cell comprising the vector are further provided. A method for using the vector for transducing human CD8+ / CD4+ T cells to obtain the CARmesothelin-T cell is further provided. The cell is obtained by genetic modification of the T cell and can be used for treating diseases related to mesothelin expression, especially for specifically recognizing and killing mesothelin expression tumors.

Description

technical field [0001] The invention belongs to the technical field of tumor treatment, and in particular relates to a method of genetically engineering T cells to express a chimeric antigen receptor (Chimeric Antigen Receptor, CAR) for treating diseases related to the expression of mesothelin. Background technique [0002] In 2011, cancer surpassed heart disease as the leading cause of death worldwide. WHO announced in December 2013 that the number of new cancer patients worldwide has exceeded 14 million each year, which is a substantial increase compared with the 2008 statistics of 12.7 million. During the same period, the number of deaths of cancer patients also increased, from 7.6 million in the past to 8.2 million. [0003] For tumors, traditional surgical resection, chemotherapy, and radiation therapy have damage to normal tissues, have limitations, and have limited effects. The targeted therapy that has appeared in recent years is to design corresponding therapeutic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/85C12N5/10C07K19/00A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/7051C07K16/18C07K2317/565C07K2319/02C07K2319/03C12N5/0636C12N2510/00
Inventor 张卫红靳文静陈瑜谭曙光
Owner 英威福赛生物技术(天津)有限公司
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