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
View PDF1 Cites 14 Cited by
  • 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 intracta

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0041] Example 1

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

[0043] (2) The GFP gene and the PGK promoter were excised from the lentiviral vector pRRLSIN.cPPT.PGK-GFP.WPRE with EcoRV and SalI (NEB) double enzyme digestion, and the promoter containing the human elongation factor 1α gene was artificially synthesized 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.

Example Embodiment

[0044] Example 2

[0045] Using a three-vector system to package pseudo-lentiviral particles containing recombinant vectors containing artificial genes encoding chimeric antigen receptors in 293T cells

[0046] 293T is cultured in DMEM medium containing 10vol% FBS. Consumables include DMEM (Gibco, C11995500BT), FBS (Gibco, 10099-141), pancreatin (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 dishes (Fisher Scientific, 310109011).

[0047] The preparation process of pseudo-lentiviral particles packaging recombinant vectors containing artificial genes encoding chimeric antigen receptors is as follows:

[0048] Day 0: Planking

[0049] Twenty-four hours before transfection, digest 293FT cells in logarithmic growth phase with trypsin, adjust the cell density with seed medium (DMEM+10% FBS+500μg / ml G418), and re-inoculate them in a 10cm cell culture dish at 37℃. 5% CO 2 Cultivate in a ...

Example Embodiment

[0062] Example 3

[0063] Use pseudo-lentiviral particles containing a recombinant vector encoding an artificial gene targeting CD19 chimeric antigen receptor to prepare T cells expressing chimeric antigen receptors (referred to as chimeric antigen receptor T cells)

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

[0065] The reagents and consumables used include serum-free medium, serum-free cryopreservation medium, PBS, Ficoll lymphocyte separation solution (GEHealthcare#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 peripheral blood of the patient to a 50ml sterile centrifuge tube, place it in a horizontal centrifuge, and centrifuge at 450g at 20°C for 8 minutes; the peripheral blood in the centrifuge tube will be separated into layers, with plasma in the upper layer and whole blood cells in the lower layer; Take the lower l...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap