Targeting T lymphocyte and preparation method and application thereof

A lymphocyte, targeting technology, applied in the field of medical biology, can solve problems such as not significantly improving the prognosis of glioma patients

Inactive Publication Date: 2019-08-23
SHENZHEN BINDEBIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, traditional therapies such as surgery and chemotherapy have not significantly improved the prognosis of patients with glioma.

Method used

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  • Targeting T lymphocyte and preparation method and application thereof
  • Targeting T lymphocyte and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Embodiment 1 A method for preparing targeted T lymphocytes, comprising:

[0105] (1) Prepare the gene sequence of chimeric antigen receptor CAR-EGFRvIII targeting EGFRvIII

[0106] The genes encoding the signal peptide, the single-chain antibody targeting EGFRvIII, the hinge region of CD8α, the transmembrane region of CD8, the signal region of 4-1BB and the signal region of CD3ζ were prepared respectively, and the genes encoding the signal peptide were shown in SEQ ID NO: 24 , the coding gene of the single-chain antibody targeting EGFRvIII is shown in SEQ ID NO: 3, the coding gene of the CD8α hinge region is shown in SEQ ID NO: 10, and the coding gene of the CD8 transmembrane region is shown in SEQ ID NO: As shown in ID NO: 13, the coding gene of the 4-1BB signal region is shown in SEQ ID NO: 16, and the coding gene of the CD3ζ signal region is shown in SEQ ID NO: 19.

[0107] The genes encoding the above signal peptide, single-chain antibody targeting EGFRvIII, CD8α h...

Embodiment 2

[0126] Embodiment 2 A method for preparing targeted T lymphocytes, comprising:

[0127] The first recombinant lentivirus with CAR-EGFRvIII and the second recombinant lentivirus with CAR-IL13Rα2 were prepared according to the same method as in Example 1, and the first recombinant lentivirus with CAR-EGFRvIII was transfected into CD3 positive T lymphocytes , on the third day of culture, count the cells and change the medium, and adjust the cell concentration to 1×10 6 cells / mL, inoculate and culture; on the 5th day of culture, observe the cell state, if the cell density increases, dilute the cell concentration to 1×10 6 cells / mL, detect cell viability, and continue to culture. After expanding and culturing to day 9-11, the cells were collected to obtain chimeric antigen receptor T cells targeting EGFRvIII. Then, the second recombinant lentivirus with CAR-IL13Rα2 was transfected into the chimeric antigen receptor T cells targeting EGFRvIII. On the third day of culture, the cell...

Embodiment 3

[0128] Embodiment 3 A method for preparing targeted T lymphocytes, comprising:

[0129] The first recombinant lentivirus with CAR-EGFRvIII and the second recombinant lentivirus with CAR-IL13Rα2 were prepared according to the same method as in Example 1. First, the second recombinant lentivirus with CAR-IL13Rα2 was transfected into CD3-positive T lymphocytes. On the third day of culture, the cells were counted and the medium was changed, and the cell concentration was adjusted to 1×10 6 cells / mL, inoculate and culture; on the 5th day of culture, observe the cell state, if the cell density increases, dilute the cell concentration to 1×10 6 cells / mL, detect cell viability, and continue to culture. After expanding and culturing to day 9-11, the cells were collected to obtain chimeric antigen receptor T cells targeting IL13Rα2; then the first recombinant lentivirus with CAR-EGFRvIII was transfected to the chimeric antigen receptor T cells targeting IL13Rα2. Somatic T cells, on th...

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PUM

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Abstract

The invention provides a targeting T lymphocyte. The targeting T lymphocyte comprises a chimeric antigen receptor CAR-EGFRvIII targeting EGFRvIII and a chimeric antigen receptor CAR-IL13Ralpha2 targeting IL13Ralpha2, the CAR-EGFRvIII can specifically target the EGFRvIII, the CAR-IL13Ralpha2 can specifically target the IL13Ralpha2, a signal area in a T cell is activated, amplification of the T cellin the body of a patient is promoted, tumor cells are efficiently and specifically killed, escape of the tumor cells can be effectively avoided, and the activity and killing capacity of the cells canbe better maintained. The invention further provides a preparation method and application of the targeting T lymphocyte.

Description

technical field [0001] The invention relates to the field of medical biology, in particular to a targeted T lymphocyte and its preparation method and application. Background technique [0002] Glioma is the most common malignant tumor of the central nervous system, originating from glial cells. Glioma accounts for about 60% of all primary nervous system tumors, and has the characteristics of "three highs and one low" of morbidity, recurrence rate, high mortality rate and low cure rate. At present, traditional therapies such as surgery and chemotherapy have not significantly improved the prognosis of glioma patients. [0003] In recent years, adoptive cellular immunotherapy has been considered as a method that may completely eliminate cancer cells in existing technologies. As one of the latest immune cell technologies, chimeric antigen receptor T cells (CAR-T) have received extensive attention and research because they can express specific receptors to target and recognize ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C07K19/00C12N15/62C12N15/867A61P35/00C12Q1/02
CPCA61K35/17A61P35/00C07K14/7051C07K16/244C07K16/2863C07K2317/622C07K2319/00C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N15/86C12N2503/02C12N2510/00C12N2740/15043C12N2800/107G01N33/5011G01N2500/10
Inventor 张宏玲钟春颖
Owner SHENZHEN BINDEBIOTECH CO LTD
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