Chimeric antigen receptor, gene thereof and recombinant expression vector of gene as well as CAR133-NKT cell and preparation method and application of CAR133-NKT cell

A chimeric antigen receptor and NKT cell technology is applied in the field of tumor biological products to achieve the effect of enhancing specific killing activity, enhancing ability, and good industrial application prospects

Inactive Publication Date: 2018-03-13
GUANGZHOU BAINIFU BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Studies have found that the tumor-initiating cell population contains a large number of CD133-positive cells. At the same time, the high expression of CD133 is related to the poor prognosis of tum...

Method used

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  • Chimeric antigen receptor, gene thereof and recombinant expression vector of gene as well as CAR133-NKT cell and preparation method and application of CAR133-NKT cell
  • Chimeric antigen receptor, gene thereof and recombinant expression vector of gene as well as CAR133-NKT cell and preparation method and application of CAR133-NKT cell
  • Chimeric antigen receptor, gene thereof and recombinant expression vector of gene as well as CAR133-NKT cell and preparation method and application of CAR133-NKT cell

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preparation example Construction

[0039] The preparation method of the lentiviral expression vector PWPT-CD133ScFv-CD8-CD137-CD3ζ is not particularly limited, and can be various methods that can be imagined by those skilled in the art. Preferably, the lentiviral expression vector PWPT-CD133ScFv-CD8-CD137 -The preparation method of CD3ζ comprises the following steps:

[0040] (1) The hinge region and transmembrane region of CD8, the intracellular signaling domain of CD137 and the intracellular signaling domain of CD3ζ were respectively amplified from NKT cell cDNA, and cloned into the vector PWPT-GFP to construct PWPT-CD8 - CD137-CD3ζ;

[0041] (2) The nucleotide sequences encoding CD8a signal peptide and CD133ScFv were synthesized and cloned into PWPT-CD8-CD137-CD3ζ, and the correct sequence of PWPT-CD133ScFv-CD8-CD137-CD3ζ was obtained after sequencing verification.

[0042] In step (1), there is no particular limitation on the methods for respectively amplifying the hinge region and transmembrane region of ...

Embodiment 1

[0104] The preparation of embodiment 1 NKT cell

[0105] (1) Take human venous blood in a vacuum tube containing heparin. Mononuclear cells (PBMCs) were obtained by density gradient centrifugation using lymphocyte separation medium.

[0106] (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.

[0107] (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 ...

Embodiment 2

[0108]Example 2 Construction of lentiviral expression vector PWPT-CD133ScFv-CD8-CD137-CD3ζ

[0109] (1) Preparation of NKT cell cDNA

[0110] 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.

[0111] (2) Preparation of lentiviral plasmid PWPT-CD8-CD137-CD3ζ

[0112] The following primer sequences were designed and synthesized (wherein, the underline marks are protected bases, and the square boxes are enzyme cleavage sites):

[0113]

[0114]

[0115] Using 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 shortened transmembrane region of CD8 with a len...

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Abstract

The invention belongs to the field of tumor biological products, and discloses a chimeric antigen receptor and a gene thereof and a recombinant expression vector of the gene as well as a CAR133-NKT cell and a preparation method and application of the CAR133-NKT cell. The chimeric antigen receptor is CD133ScFv-CD8-CD137-CD3 zeta including tandem a CD8a signal peptide, a CD133ScFv, CD8 hinge and transmembrane regions, a CD137 intracellular signal domain and a CD3 zeta intracellular signal domain. The CAR133-NKT cell provided by the invention has very good specific killing activity against a CD133-positive epithelial tumor cell when co-culture is performed with the CD133-positive epithelial tumor cell; and the CAR133-NKT cell provided by the invention has a certain therapeutic effect on an epithelial tumor when treating the CD133-positive epithelial tumor.

Description

technical field [0001] The present invention belongs to the field of tumor biological products, and specifically relates to a chimeric antigen receptor CD133ScFv-CD8-CD137-CD3ζ in adoptive immunotherapy, its gene and recombinant expression vector, and engineered CD133-targeted NKT cells (CAR133 -NKT cell) and its preparation method and application. Background technique [0002] Natural killer cells (NKT) are a special type of T lymphocyte subsets with dual properties of T cells and NK cells. NKT cells can express TCR of T cells and NKR-P1 of NK cells. Under the mediation of TCR and NKR, NKT cells can produce a large amount of IL-4 and INFγ, which can kill tumor cells. NKT cells bind to the Fc region of specific antibodies through CD16 on their surface to exert antibody-dependent cell-mediated cytotoxicity (ADCC, antibody-dependent cell-mediated cytotoxicity). However, in the process of antibody-dependent cell-mediated killing, since the antibody can specifically bind to th...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K14/70517C07K14/70596C07K16/2896C07K2317/622C07K2319/02C07K2319/03C12N5/0646C12N15/86C12N2510/00C12N2740/15043A61P35/00C07K14/70578C07K2319/00C07K2319/33C07K2319/70
Inventor 不公告发明人
Owner GUANGZHOU BAINIFU BIOTECH CO LTD
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