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

Pending Publication Date: 2018-08-14
GUANGZHOU BAINIFU BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Under normal circumstances, the half-life of infused NKT cells in the patient's body is about 2 weeks, and the validity period is short, requiring repeated infusions
In addition, NKT cells themselves lack specific antibodies, which are not enough to enrich around tumors or in tumor nests, which restricts the targeted treatment of malignant tumors by NKT cells
Moreover, studies have shown that NKT cells do not have a killing effect on all tumors, and the killing effect on some solid tumors is relatively weak, and the specific killing activity needs to be improved

Method used

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  • Chimeric antigen receptor, gene thereof, recombinant expression vector, CARHER2-NKT cell, and preparation method and application of CARHER2-NKT cell
  • Chimeric antigen receptor, gene thereof, recombinant expression vector, CARHER2-NKT cell, and preparation method and application of CARHER2-NKT cell
  • Chimeric antigen receptor, gene thereof, recombinant expression vector, CARHER2-NKT cell, and preparation method and application of CARHER2-NKT cell

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preparation example Construction

[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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Abstract

The invention belongs to the technical field of tumor biological products, and discloses a chimeric antigen receptor, a gene thereof, a recombinant expression vector, an engineered HER2 targeting NKTcell, and an application of the NKT cell. The chimeric antigen receptor is HER2ScFv-CD8-CD137-CD3zeta and comprises a CD8a signal peptide, HER2ScFv, a hinge region and transmembrane region for shortening CD8, an intracellular signal structural domain of CD137, and an intracellular signal structural domain of CD3zeta which are in series. The CARHER2-NKT cell provided by the invention has a certaintherapeutic effect on epithelial tumors when used for curing advanced HER2 positive epithelial tumors

Description

technical field [0001] The invention belongs to the technical field of tumor biological products, in particular, it relates to a chimeric antigen receptor HER2ScFv-CD8-CD137-CD3ζ in adoptive immunotherapy, its gene and recombinant expression vector, and engineered HER2-targeted NKT cells ( CARHER2-NKT cell) and its preparation method and application. Background technique [0002] Natural killer cells (NKT) are a special type of T lymphocyte subsets, which express both T cell surface markers and NK cell surface markers. The antigen recognition of NKT cells is different from that of traditional T cells. It cannot recognize antigenic peptides presented by classical MHC-I and class II molecules, but only glycolipids presented by non-classical MHC-class I molecules CDId Antigen activation, and rapidly produce a variety of cytokines, such as: IL-4, IFN-γ, IL-10, IL-13, etc., which can recognize and kill mutated tumor cells and virus-infected cells, while normal self-tissue cells ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/00
CPCC07K16/32A61K35/17C07K14/7051C07K14/70517C07K14/70578C07K16/2863C07K2317/622C07K2319/02C07K2319/03C12N5/0646C12N15/86C12N2501/2302C12N2501/2315C12N2501/515C12N2740/15043
Inventor 王晓慧列浦昌
Owner GUANGZHOU BAINIFU BIOTECH CO LTD
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