Application of carher1-nkt cells in the preparation of preparations for the treatment of advanced HER1-positive cholangiocarcinoma

A technology of NKT cells and cholangiocarcinoma, applied in gene therapy, cells modified by introducing foreign genetic material, drug combinations, etc., can solve the problems of not improving the survival rate of patients, affecting the efficacy of drugs, and drug resistance of patients, so as to avoid Effects of drug resistance, enhanced specific killing activity, enhanced ability

Active Publication Date: 2019-07-09
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the treatment of cholangiocarcinoma has made some progress recently, the survival rate of patients has not been improved, so it is urgent to explore new therapies to overcome this problem
[0003] At present, in the treatment of patients with advanced HER1-positive cholangiocarcinoma, anti-HER1 drugs (such as small molecule inhibitors targeting HER1) are already in the clinical research stage, but clinical results show that only some patients with cholangiocarcinoma are treated with HER1-targeting drugs. Small molecule inhibitors are effective in treatment, and patients will eventually develop drug resistance, which will affect the efficacy of drugs

Method used

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  • Application of carher1-nkt cells in the preparation of preparations for the treatment of advanced HER1-positive cholangiocarcinoma
  • Application of carher1-nkt cells in the preparation of preparations for the treatment of advanced HER1-positive cholangiocarcinoma
  • Application of carher1-nkt cells in the preparation of preparations for the treatment of advanced HER1-positive cholangiocarcinoma

Examples

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preparation example Construction

[0020] In the application of the present invention, preferably, the preparation method of CARHER1-NKT cells includes: packaging the lentivirus carrying pWPXL-HER1ScFv-CD8-CD137-CD3ζ to obtain a virus concentrate; using the obtained virus concentrate to infect NKT cells, so that NKT cells express the chimeric antigen receptor HER1ScFv-CD8-CD137-CD3ζ, wherein the pWPXL-HER1ScFv-CD8-CD137-CD3ζ is expressed by a lentivirus containing a gene encoding the chimeric antigen receptor HER1ScFv-CD8-CD137-CD3ζ carrier.

[0021] In the application of the present invention, the lentiviral expression vector pWPXL-HER1ScFv-CD8-CD137-CD3ζ can co-transfect packaging cells such as 293T packaging cells with an auxiliary vector to obtain virus concentrates and CARHER1-NKT cells.

[0022] The preparation method of the lentiviral expression vector pWPXL-HER1ScFv-CD8-CD137-CD3ζ is not particularly limited, and can be various methods that can be imagined by those skilled in the art. Preferably, the le...

Embodiment 1

[0074] Example 1 Preparation of NKT cells

[0075] (1) Take human venous blood in a vacuum tube containing heparin. Mononuclear cells (PBMCs) were obtained by density gradient centrifugation using lymphocyte separation medium.

[0076] (2) After PBMCs were washed three times, the final concentration of cells was adjusted to 2×10 by using NKT cell culture medium GT-T551 containing 0.6 volume % human autologous serum. 6 cells / mL; the cells were inoculated on a 75 cm 2 in cell culture flasks. Then add recombinant human interleukin-2 with a final concentration of 500U / mL, 50ng / ml CD3 monoclonal antibody and 50ng / mL recombinant human interleukin-15 to the culture medium, at 37°C and saturated humidity of 5% CO 2 cultured in an incubator.

[0077] (3) On the 4th day of culture, transfer the cells to an uncoated culture flask, add NKT cell culture medium GT-T551 every 2 days according to the number of cell growth, and control the cell concentration to 1×10 8 cells / mL, and added ...

Embodiment 2

[0078] Example 2 Construction of lentiviral expression vector pWPXL-HER1ScFv-CD8-CD137-CD3ζ

[0079] (1) Preparation of NKT cell cDNA

[0080] The NKT cells cultured in Example 1 were centrifuged to precipitate, and the total RNA of the cells was extracted with RNAiso Reagent, a total RNA extraction kit, and stored at -80°C for future use. Extracted total RNA with RevertAid Reverse Transcription Kit TM NKT cell cDNA was obtained by reverse transcription with the First StrandcDNA Synthesis Kit, and stored at -20°C for future use.

[0081] (2) Preparation of lentiviral plasmid pWPXL-CD8-CD137-CD3ζ

[0082] The following primer sequences were designed and synthesized (wherein, the underline marks are protected bases, and the square boxes are enzyme cleavage sites):

[0083]

[0084] Using the NKT cell cDNA in step (1) as a template, PCR amplification was carried out with primers P1 and P2 to obtain the hinge region and transmembrane region of CD8 with a length of 287bp. Th...

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Abstract

The invention discloses application of CARHER1-NKT cells in the preparation of preparations for the treatment of HER1 positive cholangiocarcinoma in progressive stage. CARHER1-NKT cells are NKT cells modified by a chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 zeta; the chimeric antigen receptor HER1ScFv-CD8-CD137-CD3 zeta comprises a rat growth hormone signal peptide, HER1ScFv, a hinge area and a transmembrane domain of CD8, an intracellular signal structural domain of CD137 and an intracellular signal structural domain of CD3 zeta, which are in series connection. The CARHER1-NKT cells for the treatment of HER1 positive cholangiocarcinoma in progressive stage can effectively avoid the drug resistance caused by the existing drugs targeting HER1, have certain specific killing activity on cholangiocarcinoma cells, and certain therapeutic effect on patients with HER1 positive cholangiocarcinoma in progressive stage.

Description

technical field [0001] The invention belongs to the field of tumor biological products, and specifically relates to the application of CARHER1-NKT cells in the preparation of preparations for treating advanced HER1-positive cholangiocarcinoma in adoptive immunotherapy. Background technique [0002] EGFR (epithelial growth factor receptor, epidermal growth factor receptor) or HER1 is the expression product of the proto-oncogene c-erbB1, which belongs to the human epidermal growth factor receptor (HER) family, and itself has tyrosinase kinase activity. The combination of epidermal growth factor (EGF) can activate related genes in the nucleus, thereby promoting cell division and proliferation. HER1 is highly expressed in a variety of malignant tumors. Studies have found that HER1 is overactivated in cholangiocarcinoma tissue, with an expression level as high as 80%, and its expression level is related to the progression and metastasis of cholangiocarcinoma. Although recent pro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61P35/00C12N5/10
Inventor 韩为东丰恺超郭业磊王晓慧王瑶代汉仁
Owner GENERAL HOSPITAL OF PLA
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