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CAR-T (chimeric antigen receptor T cell) for targeting CD19 and application of CAR-T

A chimeric antigen receptor and single-chain antibody technology, applied in the fields of immunology and molecular biology, can solve the problems of lack of tumor specificity, limited clinical application, inaccurate curative effect, etc., and achieve the effect of inhibiting proliferation

Active Publication Date: 2018-03-23
英普乐孚生物技术(上海)有限公司
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  • Summary
  • 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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  • CAR-T (chimeric antigen receptor T cell) for targeting CD19 and application of CAR-T
  • CAR-T (chimeric antigen receptor T cell) for targeting CD19 and application of CAR-T
  • CAR-T (chimeric antigen receptor T cell) for targeting CD19 and application of CAR-T

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] [Example 1] Construction of a chimeric antigen receptor lentiviral vector expressing a chimeric antigen receptor gene targeting CD19

[0028] (1) All artificially synthesized artificial CAR genes encoding chimeric antigen receptors (including the promoter of the human elongation factor 1α gene, with an EcoRV endonuclease site at the 5'-end and SalI at the 3'-end endonuclease sites).

[0029] (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

[0030] [Example 2] Using the three-vector system to package pseudolentiviral particles containing recombinant vectors of artificial genes encoding chimeric antigen receptors in 293T cells

[0031] 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).

[0032] The preparation process of the pseudolentiviral particles packaging the recombinant vector containing the artificial gene encoding the chimeric antigen receptor is as follows:

[0033] Day 0: Plank

[0034] 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

[0047] [Example 3] 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

[0048] (1) Preparation of mononuclear lymphocytes (PBMCs)

[0049] 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).

[0050] 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 ce...

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Abstract

The invention discloses a CAR-T (chimeric antigen receptor T cell) for targeting CD19 and an application of the CAR-T. A CAR for preparing the CAR-T comprises interleukin 2 signal peptides, an anti-CD19 single-chain antibody, a CD8 protein molecule hinge region, a transmembrane region, an intracellular signal structure region and a CD3 zeta protein molecule intracellular signal conduction structure region which are connected in series sequentially and has the amino acid sequence shown in SEQ ID NO:9. The CAR-T is applied to preparation of a medicine or a preparation for treating hematologicalmalignancy, wherein the hematological malignancy comprises CD19-positive B-cell acute lymphocytic leukemia, diffuse large B cell lymphoma and non-hodgkin's 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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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/02
CPCA61K35/17C07K14/7051C07K16/2803C07K2319/02C07K2319/33C12N15/86C12N2510/00C12N2740/15043
Inventor 史燕东武宁
Owner 英普乐孚生物技术(上海)有限公司
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