Chimeric antigen receptor and gene and recombinant expression vector thereof, engineered CD33 targeting NKT cell and application thereof

A chimeric antigen receptor and NKT cell technology, applied in the field of tumor biological products, can solve problems such as drug resistance and chemotherapy resistance, and achieve the effects of low toxic side effects, enhanced ability, and prolonged survival time

Inactive Publication Date: 2016-03-09
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, in the treatment of patients with advanced CD33-positive acute myeloid leukemia, the combination of CD33 monoclonal antibody and chemotherapy is already in the clini...

Method used

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  • Chimeric antigen receptor and gene and recombinant expression vector thereof, engineered CD33 targeting NKT cell and application thereof
  • Chimeric antigen receptor and gene and recombinant expression vector thereof, engineered CD33 targeting NKT cell and application thereof
  • Chimeric antigen receptor and gene and recombinant expression vector thereof, engineered CD33 targeting NKT cell and application thereof

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preparation example Construction

[0027] There is no particular limitation on the preparation method of the lentiviral expression vector pWPT-CD33ScFv-CD8-CD137-CD3ζ, which can be various methods conceivable by those skilled in the art. Preferably, the lentiviral expression vector pWPT-CD33ScFv-CD8-CD137 -The preparation method of CD3ζ comprises the following steps:

[0028] (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;

[0029] (2) Synthesize the nucleotide sequence encoding rat growth hormone signal peptide and CD33ScFv, and clone it into pWPT-CD8-CD137-CD3ζ, and obtain pWPT-CD33ScFv-CD8-CD137-CD3ζ with correct sequence after sequencing verification.

[0030] In step (1), there is no particular limitation on the methods for respectively amplifying the hinge region and transmembrane region of ...

Embodiment 1

[0064] The preparation of embodiment 1 NKT cell

[0065] (1) Take human venous blood in a vacuum tube containing heparin. Mononuclear cells (PBMCs) were obtained by density gradient centrifugation using lymphocyte separation medium.

[0066] (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.

[0067] (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

[0068]Example 2 Construction of lentiviral expression vector pWPT-CD33ScFv-CD8-CD137-CD3ζ

[0069] (1) Preparation of NKT cell cDNA

[0070] The NKT cells cultured in Example 1 were centrifuged to precipitate, and the total RNA of the cells was extracted with the total RNA extraction kit RNAisoReagent, and stored at -80°C for future use. Extracted total RNA with RevertAid Reverse Transcription Kit TM NKT cell cDNA was reverse transcribed by FirstStrandcDNASynthesisKit and stored at -20°C for future use.

[0071] (2) Preparation of lentiviral plasmid pWPT-CD8-CD137-CD3ζ

[0072] The following primer sequences were designed and synthesized (wherein, the underline marks are protected bases, and the square boxes are enzyme cleavage sites):

[0073]

[0074] Using the cDNA of NKT cells 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 287 bp. The nucleotide sequence i...

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Abstract

The invention discloses a chimeric antigen receptor and a gene and a recombinant expression vector thereof, an engineered CD33 targeting NKT cell and an application thereof. The chimeric antigen receptor is CD33ScFv-CD8-CD137-CD3zeta which is formed by connecting CD33ScFv, a hinge domain and a transmembrane domain of CD8, an intracellular signal structural domain of CD137 and an intracellular signal structural domain of CD3zeta in series. By adopting the chimeric antigen receptor CD33ScFv-CD8-CD137-CD3zeta modified NKT cell for treating CD33 positive acute myelogenous leukemia in a progressive stage, drug resistance and chemotherapy resistance caused by combined application of a CD33 monoclonal antibody and chemotherapy can be effectively avoided, and the cell has specific killing activity on leukemia cells.

Description

technical field [0001] The present invention belongs to the field of tumor biological products, and specifically relates to a chimeric antigen receptor CD33ScFv-CD8-CD137-CD3ζ in adoptive immunotherapy, its gene and recombinant expression vector, and engineered CD33-targeted NKT cells (CAR33 -NKT cells) and applications thereof. Background technique [0002] Acute myeloid leukemia (AML) includes all acute leukemias of non-lymphocyte origin. It is a clonal malignant proliferative disease of myeloid primitive cells in the hematopoietic system. It is a highly heterogeneous disease group. Malignant transformation of hematopoietic progenitor cells at different stages during the differentiation and development of lineage cells. [0003] CD33 is highly expressed on the surface of blast cells in more than 90% of acute myeloid leukemia patients, and CD33 is also expressed on the surface of leukemia stem cells, while only a low level of CD33 is expressed in some normal hematopoietic ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K48/00A61K38/17A61K35/17A61P35/02
Inventor 韩庆旺王晓慧韩为东王全顺王瑶付小兵
Owner GENERAL HOSPITAL OF PLA
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