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Chimeric antigen receptor and its gene and recombinant expression vector, carmsln-nkt cell and its preparation method and application

A chimeric antigen receptor and NKT cell technology, applied in the field of tumor biological products, can solve the problems of insufficient distribution of MSLN monoclonal antibody and transient targeting effect of MSLN monoclonal antibody, achieve good industrial application prospects, and strengthen specific killing Active, empowering effects

Inactive Publication Date: 2019-08-23
伍志强
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, a large number of studies have used MSLN monoclonal antibodies or MSLN vaccines to target and kill MSLN-positive tumor cells. However, these solutions have certain effects on the basis of certain defects, such as: MSLN monoclonal antibodies target Transient effect and insufficient distribution of MSLN monoclonal antibody in the tumor site

Method used

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  • Chimeric antigen receptor and its gene and recombinant expression vector, carmsln-nkt cell and its preparation method and application
  • Chimeric antigen receptor and its gene and recombinant expression vector, carmsln-nkt cell and its preparation method and application
  • Chimeric antigen receptor and its gene and recombinant expression vector, carmsln-nkt cell and its preparation method and application

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preparation example Construction

[0027] The preparation method of the lentiviral expression vector PWPT-MSLNScFv-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-MSLNScFv-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) The nucleotide sequences encoding CD8a signal peptide and MSLNScFv were synthesized and cloned into PWPT-CD8-CD137-CD3ζ, and the correct sequence of PWPT-MSLNScFv-CD8-CD137-CD3ζ was obtained 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 CD8,...

Embodiment 1

[0082] Example 1 Preparation of NKT cells

[0083] (1) Take human venous blood in a vacuum tube containing heparin. Mononuclear cells (PBMCs) were obtained by density gradient centrifugation using lymphocyte separation medium.

[0084] (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.

[0085] (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

[0086] Example 2 Construction of lentiviral expression vector PWPT-MSLNScFv-CD8-CD137-CD3ζ

[0087] (1) Preparation of NKT cell cDNA

[0088] 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.

[0089] (2) Preparation of lentiviral plasmid PWPT-CD8-CD137-CD3ζ

[0090] The following primer sequences were designed and synthesized (wherein, the underline marks are protected bases, and the square boxes are enzyme cleavage sites):

[0091]

[0092]

[0093] 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 discloses a chimeric antigen receptor, a gene thereof and a recombinant expression vector of the gene as well as a CARMSLN-NKT cell and a preparation method and application of the CARMSLN-NKT cell. The chimeric antigen receptor is MSLNScFv-CD8-CD137-CD3 zeta including tandem a CD8a signal peptide, an MSLNScFv, shortened CD8 hinge and transmembrane regions, a CD137 intracellular signal domain and a CD3zeta intracellular signal domain; and the CARMSLN-NKT cell provided by the invention has very good specific killing activity against a mesothelioma cell when co-culture is performedwith the MSLN-positive mesothelioma cell.

Description

technical field [0001] The invention belongs to the field of tumor biological products, and in particular relates to a chimeric antigen receptor MSLNScFv-CD8-CD137-CD3ζ in adoptive immunotherapy, its gene and recombinant expression vector, and engineered MSLN-targeted NKT cells (CARMSLN -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 the corres...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K14/70517C07K14/70578C07K16/18C07K2317/622C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N5/0646C12N15/86C12N2501/2302C12N2501/2305C12N2501/515C12N2510/00C12N2740/15043
Inventor 不公告发明人
Owner 伍志强
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