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Chimeric antigen receptor T cells targeting CD19, and application of chimeric antigen receptor T cells

A technology of chimeric antigen receptors and single-chain antibodies, which is applied in the fields of immunology and molecular biology, can solve the problems of lack of tumor specificity, short duration of activity of reinfused T cells, and limitations of clinical application, and achieve the purpose of inhibiting tumor cell proliferation Effect

Inactive Publication Date: 2018-07-13
英普乐孚生物技术(上海)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Adoptive immune cell therapy (adoptive T-cell therapy, ACT) is achieved by infusing autologous or tumor-specifically induced T cells in the treatment of some metastatic malignant tumors, such as melanoma, which are almost intractable by other methods so far. Great success; however, due to many problems such as the short duration of activity of reinfused T cells in vivo, lack of tumor specificity and inaccurate curative effect, its clinical application is limited

Method used

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  • Chimeric antigen receptor T cells targeting CD19, and application of chimeric antigen receptor T cells
  • Chimeric antigen receptor T cells targeting CD19, and application of chimeric antigen receptor T cells
  • Chimeric antigen receptor T cells targeting CD19, and application of chimeric antigen receptor T cells

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Embodiment 1

[0042] Construction of chimeric antigen receptor lentiviral vectors expressing chimeric antigen receptor genes against CD19 (1) All artificially synthesized artificial CAR genes encoding chimeric antigen receptors (including the promoter of human elongation factor 1α gene, And an EcoRV endonuclease site is added at the 5'-end, and a SalI endonuclease site is added at the 3'-end).

[0043] (2) Use EcoRV and SalI (NEB Company) to cut the GFP gene and PGK promoter from the lentiviral vector pRRLSIN.cPPT.PGK-GFP.WPRE, and artificially synthesize the promoter containing the human elongation factor 1α gene The artificial CAR gene encoding the chimeric antigen receptor replaces the GFP gene and the PGK promoter to obtain a chimeric antigen receptor lentiviral vector that can express the chimeric antigen receptor gene for CD19.

Embodiment 2

[0045] Packaging of pseudolentiviral particles containing a recombinant vector containing an artificial gene encoding a chimeric antigen receptor in 293T cells using a three-vector system

[0046] 293T was cultured in DMEM medium containing 10vol% FBS, and the consumables included DMEM (Gibco, C11995500BT), FBS (Gibco, 10099-141), trypsin (Gibco, 25200-056), Dulbecco's PBS (Hyclone, cat#SH30256. 01), PEI (1 mg / ml, Life Sciences, 23966-2), Opti-MEM (Gibco, 51985-034) and 10 cm cell culture dish (Fisher Scientific, 310109011).

[0047] The preparation process of the pseudolentiviral particles packaging the recombinant vector containing the artificial gene encoding the chimeric antigen receptor is as follows:

[0048] Day 0: Plank

[0049] 24 hours before transfection, the 293FT cells in the logarithmic growth phase were digested with trypsin, the cell density was adjusted with seed medium (DMEM+10% FBS+500 μg / ml G418), and re-seeded in a 10cm cell culture dish at 37°C. 5%CO 2...

Embodiment 3

[0063] Preparation of T cells expressing chimeric antigen receptors (referred to as chimeric antigen receptor T cells) using pseudolentiviral particles containing recombinant vectors encoding artificial genes for CD19 chimeric antigen receptors

[0064] (1) Preparation of mononuclear lymphocytes (PBMCs)

[0065] Reagent consumables used include serum-free medium, serum-free freezing solution, PBS, Ficoll lymphocyte separation medium (GE Healthcare #17-5442-03), 5ml, 10ml and 25ml disposable sterile pipettes (Thermo), sterile Pipette tip (QSP), centrifuge tube (Thermo, 339652), cell freezing tube (Thermo, 375418).

[0066] Add the collected patient's autologous peripheral blood into a 50ml sterile centrifuge tube, put it in a horizontal centrifuge, centrifuge at 450g, 20°C for 8 minutes; the peripheral blood in the centrifuge tube will be divided into layers, the upper layer is plasma, and the lower layer is whole blood cells; Take the lower layer of whole blood cells and add ...

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Abstract

The invention discloses chimeric antigen receptor T cells targeting CD19, and application of the chimeric antigen receptor T cells. A chimeric antigen receptor for preparing the chimeric antigen receptor T cells comprises interleukin 2 signal peptide, an anti-CD19 single chain antibody, a CD8 protein molecular hinge region, a transmembrane region, an intracellular signal structural domain, and anintracellular signal transduction structural domain of CD3 zeta protein molecules which are sequentially connected in series. The chimeric antigen receptor T cells are used for preparing medicines orpreparations for treating hematological malignancies, wherein the hematological malignancies comprise CD19-positive acute B-lymphocytic leukemia, diffuse large B-cell lymphoma and non-Hodgkin lymphoma.

Description

technical field [0001] The invention belongs to the technical field of immunology and molecular biology, and in particular relates to a chimeric antigen receptor T cell targeting CD19 and its application. Background technique [0002] Traditional treatment methods such as radiotherapy, chemotherapy, surgery, and hematopoietic stem cell transplantation have prolonged the survival time of some patients with hematological malignancies, but recurrence, refractory and even drug resistance are still huge challenges. [0003] In recent years, due to breakthroughs in tumors, cellular immunotherapy has become an important means for the treatment of various tumors such as the blood system, and was listed by Science magazine as the first of the top ten scientific breakthroughs in 2013. Among them, the progress of chimeric antigen receptor T cell immunotherapy is particularly prominent. Chimeric antigen receptor T cell (CAR-T) immunotherapy technology is to modify the patient's autolog...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N15/867A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K16/2803C07K2317/622C07K2319/03C07K2319/33C12N5/0636C12N15/86C12N2740/15043
Inventor 韩德平
Owner 英普乐孚生物技术(上海)有限公司
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