TROP2 chimeric antigen receptor, T cell, and preparing method and application of thereof

A technology of chimeric antigen receptors and cells, which is applied in the field of tumor treatment, can solve the problems of restricting the effective activation of specific T cells, and achieve the effects of high secretion level, killing ability, good vitality, and large number of cell proliferation

Active Publication Date: 2019-10-11
盈凯赛威(北京)生物科技有限公司
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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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  • TROP2 chimeric antigen receptor, T cell, and preparing method and application of thereof
  • TROP2 chimeric antigen receptor, T cell, and preparing method and application of thereof
  • TROP2 chimeric antigen receptor, T cell, and preparing method and application of thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1. Construction of trophoblast cell surface antigen 2 recombinant protein expression plasmid

[0081] The cDNA fragment of trophoblast surface antigen 2 was synthesized in vitro, and the HIS tag was added to the end, and the restriction sites EcoR1 and BglII were introduced into the two ends respectively, and cloned into the expression vector pCAGGS to construct the recombinant eukaryotic expression plasmid of the full-length protein of trophoblast surface antigen 2 . The above work was completed by Suzhou Synbio.

[0082] The cDNA sequence of the trophoblast surface antigen 2 recombinant protein is as follows:

[0083] ATGGCTCGGGGCCCCGGCCTCGCGCCGCCACCGCTGCGGCTGCCGCTGCTGCTGCTGGTGCTGGCGGCGGTGACCGGCCACACGGCCGCGCAGGACAACTGCACGTGTCCCACCAACAAGATGACCGTGTGCAGCCCCGACGGCCCCGGCGGCCGCTGCCAGTGCCGCGCGCTGGGCTCGGGCATGGCGGTCGACTGCTCCACGCTGACCTCCAAGTGTCTGCTGCTCAAGGCGCGCATGAGCGCCCCCAAGAACGCCCGCACGCTGGTGCGGCCGAGTGAGCACGCGCTCGTGGACAACGATGGCCTCTACGACCCCGACTGCGACCCCGAGGGCCGCTTCAAGGC...

Embodiment 2

[0084] Example 2. Expression and purification of trophoblast cell surface antigen 2 protein

[0085] 1) Transfection of HEK293T cells (purchased from Shanghai Cell Bank, BNCC338274): 18 hours before transfection, HEK293T cells were treated with 1.5x10 7 / ml spread to 30 15cm culture dishes; take 37.5mL DMEM (purchased from Gibco, C11995500CP) (without serum and antibiotics) to a 50mL tube, add 2970μg polyetherimide (PEI) MegaTran 1.0 (purchased from AlfaAesar , 9002-98-6) and mix; take 37.5mL DMEM (without serum and antibiotics) to a 50mL tube, add 990μg of trophoblast cell surface antigen 2 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 cultured 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,...

Embodiment 3

[0089] Example 3. Preparation and preliminary screening of trophoblast surface antigen 2 monoclonal antibody This part of the work was completed by Nanjing GenScript, and the purified full-length trophoblast surface antigen 2 recombinant protein obtained in Example 2 was used according to the standard method (hereinafter referred to as trophoblast surface antigen 2 antigen) was used for immunization, cell fusion, screening, etc. of B6 / C57 mice to obtain 7 monoclonal strains.

[0090] The corresponding fusion plate cell lines are 5H3, 6H5, 6A7, 6E12, 8F5, 8A11, 8H12, and the corresponding monoclonal antibodies are labeled with this.

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Abstract

The invention provides an anti-trophoblast cell-surface antigen-2 chimeric antigen receptor, a T cell, preparing method and application thereof. The anti-trophoblast cell-surface antigen-2 chimeric antigen receptor comprises an anti-trophoblast cell-surface antigen-2 scFv antibody, an IgG4 hinge region, a CD28 transmembrane region, a CD3 intracellular region and a 41BB intracellular region. The invention further provides nucleic acid coding the receptor, a carrier comprising the nucleic acid, and a host cell comprising the carrier. The invention further provides a method for utilizing the carrier for transducing the human CD8+/CD4+T cell to obtain a CARTROP2-T cell. The cell is obtained through gene modification and transformation on the T cell. The receptor can be used for treating diseases related to expression of the trophoblast cell-surface antigen-2, and particularly used for specifically recognizing and killing tumors for expressing the trophoblast cell-surface antigen-2.

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 treatment with trophoblast cell-surface antigen-2 (Trophoblast cell-surface antigen-2). , TROP2) expression-related diseases. Background technique [0002] In 2011, cancer surpassed heart disease as the leading cause of death worldwide. The 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. According to the 2015 Chinese cancer statistics published in CA: A Cancer Journal for Clinjicians (impact factor 144.8): 2015 my country New cancer cases were 4.292 million. New cancer ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N5/10C12N15/867A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/7051C07K2317/565C07K2319/02C07K2319/03C12N5/0636C12N15/86C12N2510/00C12N2740/15043
Inventor 张卫红靳文静陈瑜谭曙光
Owner 盈凯赛威(北京)生物科技有限公司
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