Chimeric antigen receptor T cell preparation

A chimeric antigen receptor and preparation technology, which is applied in the fields of immunology and molecular biology, can solve the problems of restricting the application of chimeric antigen receptor T cell therapy, and achieve the effects of convenient clinical application, simplified preparation process, and simple ingredients

Active Publication Date: 2018-05-01
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

In addition, in order to preserve immune cells below -120°C, dimethyl sulfoxide (DMSO) and low-molecular-weight dextran 40 added to the cell preservation solution may cause severe allergic reactions in patients, including anaphylactic shock, so in some conditions Therapeutic applications of chimeric antigen receptor T cells are limited

Method used

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  • Chimeric antigen receptor T cell preparation
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  • Chimeric antigen receptor T cell preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] [Example 1] Construction of a chimeric antigen receptor lentiviral vector expressing a chimeric antigen receptor gene targeting CD19

[0041] (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).

[0042] (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

[0043] [Example 2] Using the three-vector system to package pseudolentiviral particles containing recombinant vectors of artificial genes encoding chimeric antigen receptors in 293T cells

[0044] 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).

[0045] The preparation process of the pseudolentiviral particles packaging the recombinant vector containing the artificial gene encoding the chimeric antigen receptor is as follows:

[0046] Day 0: Plank

[0047] 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

[0060] [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

[0061] (1) Preparation of mononuclear lymphocytes (PBMCs)

[0062] 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).

[0063] 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 an immune cell preparation. The preparation consists of immune cells and and an immune cell preservation re-infusion liquid which is free from dimethyl sulfoxide and dextran 40, wherein the immune cell preservation re-infusion liquid consists of the following components: an electrolyte solution which is isosmotic with plasma, a human serum albumin injection and a glucose injection, wherein the electrolyte solution is any one of a sodium chloride injection, a compound sodium chloride injection, a compound electrolyte injection and a lactated Ringer's solution. The immunecell preparation provided by the invention conforms to the characteristic that China is concentrated and dense in population; and the immune cell preparation is applicable to an immune cell preparation center close to a treatment agency, and the immune cell preparation only needs to be preserved at 4-15 DEG C. With the application of the immune cell preparation provided by the invention, in particular the chimeric antigen receptor T cell preparation, a preparation process can be simplified, logistics cost can be greatly reduced, the safety of the preparation can be improved and a treatment cycle can be shortened; therefore, treatment cost of the chimeric antigen receptor T cell preparation can be reduced, so as to benefit broad patients.

Description

technical field [0001] The invention belongs to the technical field of immunology and molecular biology, and relates to an immune cell preparation, in particular to a chimeric antigen receptor T cell preparation. Background technique [0002] In the field of medicine, 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/17A61K47/42A61K9/08A61P35/00A61P35/02C12N5/10
CPCA61K9/0019A61K9/08A61K35/17A61K47/42C12N5/0636C12N2510/00
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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