Chimeric antigen receptor, gene thereof, recombinant expression vector, CARHER2-NKT cell, and preparation method and application of CARHER2-NKT cell
A chimeric antigen receptor and NKT cell technology, which is applied in the field of engineered HER2-targeted NKT cells and the preparation thereof, can solve the problems of restricting targeted therapy of malignant tumors, the specific killing activity needs to be improved, and the lack of specific antibodies, etc. , to achieve good industrial application prospects, enhance specific killing activity, and avoid the effects of drug resistance
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[0029] The method for preparing the lentiviral expression vector pWPT-HER2ScFv-CD8-CD137-CD3ζ is not particularly limited, and can be various methods conceivable by those skilled in the art. Preferably, the lentiviral expression vector pWPT-HER2ScFv-CD8-CD137 -The preparation method of CD3ζ comprises the following steps:
[0030] (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;
[0031] (2) The nucleotide sequences encoding CD8a signal peptide and HER2ScFv were synthesized and cloned into pWPT-CD8-CD137-CD3ζ, and the correct sequence of pWPT-HER2ScFv-CD8-CD137-CD3ζ was obtained after sequencing verification.
[0032]In step (1), there is no particular limitation on the methods for respectively amplifying the hinge region and transmembrane region of CD8, the intrac...
Embodiment 1
[0085] The preparation of embodiment 1 NKT cell
[0086] (1) Take human venous blood in a vacuum tube containing heparin. Mononuclear cells (PBMCs) were obtained by density gradient centrifugation using lymphocyte separation medium.
[0087] (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.
[0088] (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
[0089] Example 2 Construction of lentiviral expression vector pWPT-HER2ScFv-CD8-CD137-CD3ζ
[0090] (1) Preparation of NKT cell cDNA
[0091] 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.
[0092] (2) Preparation of lentiviral plasmid pWPT-CD8-CD137-CD3ζ
[0093] The following primer sequences were designed and synthesized (wherein, the underline marks are protected bases, and the square boxes are enzyme cleavage sites):
[0094]
[0095] Using the NKT cell cDNA in step (1) as a template, use primers P1 and P2 to carry out PCR amplification to obtain the hinge region and transmembrane region of CD8 with a length of 227bp. The nucle...
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