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CD19 targeting chimeric antigen receptor and NKT cell, and preparation method thereof and applications thereof

A technology of chimeric antigen receptors and NKT cells, applied in the field of biological products, can solve the problems of lack of specific antibodies, restrictions on targeted therapy of malignant tumors, short validity period, etc.

Active Publication Date: 2016-03-23
CELLULAR BIOMEDICINE GRP SHANGHAI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] 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

Method used

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  • CD19 targeting chimeric antigen receptor and NKT cell, and preparation method thereof and applications thereof
  • CD19 targeting chimeric antigen receptor and NKT cell, and preparation method thereof and applications thereof
  • CD19 targeting chimeric antigen receptor and NKT cell, and preparation method thereof and applications thereof

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preparation example Construction

[0133] The preparation method of the lentiviral expression vector pWPT-CD19ScFv-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-CD19ScFv-CD8-CD137 -The preparation method of CD3ζ comprises the following steps:

[0134] (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;

[0135] (2) Synthesize the nucleotide sequence encoding rat growth hormone signal peptide and CD19ScFv, and clone it into pWPT-CD8-CD137-CD3ζ, and obtain pWPT-CD19ScFv-CD8-CD137-CD3ζ with the correct sequence after sequencing verification.

[0136] In step (1), there is no particular limitation on the methods for respectively amplifying the hinge region and transmembrane region o...

Embodiment 1

[0279] The preparation of embodiment 1 NKT cell

[0280] 1.1 Preparation of NKT cells

[0281] (1) Take human venous blood in a vacuum tube containing heparin. Mononuclear cells (PBMCs) were obtained by density gradient centrifugation using lymphocyte separation medium.

[0282] (2) After the PBMCs were washed three times, the final cell concentration was adjusted to 2×10 by using NKT cell culture medium GT-T551 containing 0.6% by volume of fetal bovine serum. 6 cells / mL; the cells were inoculated on a 75 cm cell line coated with anti-CD3 monoclonal antibody at a final concentration of 5 μg / mL and retronectin at a final concentration of 10 μg / mL. 2 in cell culture flasks. Then add recombinant human protein interferon-γ with a final concentration of 1000U / mL and recombinant human interleukin-2 with a final concentration of 1000U / mL in the culture medium, and incubate at 37°C with a saturated humidity of 5% CO 2 cultured in an incubator.

[0283] (3) On the fourth day, 100 ...

Embodiment 2

[0292] Example 2 Construction of lentiviral expression vector pWPT-CD19ScFv-CD8-CD137-CD3ζ

[0293] (1) Preparation of NKT cell cDNA

[0294] 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.

[0295] (2) Preparation of lentiviral plasmid pWPT-CD8-CD137-CD3ζ

[0296] The following primer sequences were designed and synthesized (wherein, the underline marks are protected bases, and the square boxes are enzyme cleavage sites):

[0297]

[0298] Using the NKT cell cDNA in step (1) as a template, PCR amplification was performed with primers P1 and P2 to obtain the hinge region and transmembrane region of CD8 with a length of 287 bp. The nucleotide sequence is sho...

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Abstract

The present invention discloses a chimeric antigen receptor, a gene and a recombinant expression vector thereof, an engineered CD19 targeting NKT cell and applications thereof. The chimeric antigen receptor is CD19ScFv-CD8-CD137-CD3 zeta, and consists of a hinge region and a transmembrane region of the CD19ScFv and CD8, an intracellular signal structural domain of CD137, and an intracellular signal structural domain of CD3 zeta, and the the hinge region, the transmembrane region, the intracellular signal structural domain of CD137 and the intracellular signal structural domain of CD3 zeta are connected in series. When used to treat advanced stage CD19-positive B-cell acute lymphocytic leukemia, the NKT cell modified by the chimeric antigen receptor CD19ScFv-CD8-CD137-CD3 zeta provided by the present invention has good specific killing activity on leukemic cells, and has certain therapeutic effect on advanced stage CD19-positive B-cell acute lymphocytic leukemia patients who are repeatedly subjected to therapy such as radiotherapy, chemotherapy and symptomatic therapy by other drugs but cannot recover obviously.

Description

technical field [0001] The invention belongs to the field of biological products used for tumor treatment, and in particular relates to CD19-targeted chimeric antigen receptors and NKT cells and their preparation and application. More specifically, it relates to chimeric antigen receptor CD19ScFv-CD8-CD137-CD3ζ and its gene and recombinant expression vector in adoptive immunotherapy, engineered CD19-targeted NKT cells (CAR19-NKT cells) and its 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. . [0003] 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 a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K48/00A61K38/17A61K35/17A61P35/02C12N5/0783
Inventor 韩为东韩庆旺王晓慧代汉仁张文英王瑶付小兵
Owner CELLULAR BIOMEDICINE GRP SHANGHAI
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