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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 transient targeting effect of MSLN monoclonal antibody and insufficient distribution of MSLN monoclonal antibody, achieve good industrial application prospects, and strengthen specific killing Active, empowering effects

Inactive Publication Date: 2019-08-06
GENERAL HOSPITAL OF PLA
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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] There is no particular limitation on the preparation method of the lentiviral expression vector pWPXL-MSLNScFv-CD8-CD137-CD3ζ, which can be various methods conceivable by those skilled in the art. Preferably, the lentiviral expression vector pWPXL-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 pWPXL-GFP to construct pWPXL-CD8 - CD137-CD3ζ;

[0029] (2) The nucleotide sequences encoding rat growth hormone signal peptide and MSLNScFv were synthesized and cloned into pWPXL-CD8-CD137-CD3ζ, and the correct sequence of pWPXL-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 transm...

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 pWPXL-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 pWPXL-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] P1 (SEQ ID NO.11): GATC CTGAGCAACTCCATCATGTACTTC

[0092] MluI

[0093] P2 (SEQ ID NO.12): GATC GCAGTAAAGGGTGATAACCAGTGA

[0094] BglII

[0095] P3 (SEQ ID NO.13): GATC AAACGGGGCAGAAAGAAACTC...

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Abstract

The invention discloses a chimeric antigen receptor, a gene and a recombinant expression vector thereof, a CARMSLN-NKT cell and a preparation method and application thereof. The chimeric antigen receptor is MSLNScFv-CD8-CD137-CD3 zeta, and comprises a rat growth hormone signal peptide, MSLNScFv, a hinge region and a transmembrane domain of CD8, an intracellular signaling structural domain of CD137 and an intracellular signaling structural domain of CD3 zeta, which are in series connection. The CARMSLN-NKT cells and MSLN positive mesothelioma cells are co-cultured to reach specific killing activity on mesothelioma cells.

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): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/02
Inventor 王瑶韩为东代汉仁伍志强郭业磊王晓慧
Owner GENERAL HOSPITAL OF PLA
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