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Method for preparing CAR-T cells

A technology of cells and culture medium, which is applied in the field of biotechnology and achieves the effect of excellent technical effect

Active Publication Date: 2022-03-08
深圳博雅感知药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] CN113583143B disclosed by our research team provides an excellent method for preparing CAR-T cells, however, this method still has room for improvement

Method used

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  • Method for preparing CAR-T cells
  • Method for preparing CAR-T cells
  • Method for preparing CAR-T cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: Construction of recombinant plasmids

[0068] 1. Synthesis of CAR-19-1 plasmid:

[0069] The CAR-19-1 sequence is composed of CD19 single-chain variable region, CD8a hinge region, CD8a transmembrane region, 4-1BB signal, CD3ζ patina signal, IL-7, CCL17 sequence, and the vector used is pLVX-EF1a- IRES-PGK-puro, synthesized by General Biosystems (Anhui) Co., Ltd.

[0070] The nucleotide sequence of CAR-19-1 is as follows (may be referred to as sequence 1 in the present invention):

[0071]atggccctgcccgtgaccgctctgctgctgccactggccctgctgctgcacgccgctagacctgaggtgaagctgcaggagtccggccctggcctggtggctccttcccagtccctgagcgtgacctgtacagtgtccggcgtgtccctgcctgattacggcgtgtcctggatcaggcagcctcccagaaagggcctggagtggctgggcgtgatctggggctccgagacaacctactacaattccgccctgaagtccaggctgacaatcatcaaggacaatagcaagagccaggtgtttctgaagatgaactccctgcagacagacgacaccgccatctactactgcgccaagcactactactacggcggctcctacgccatggattactggggccagggcaccagcgtgacagtgtcctccggcggcggcggaagcggaggaggaggatctggcggcggcggttccgatatccagatg...

Embodiment 2

[0086] Example 2: Viral packaging

[0087] Invitrogen Lipofectamine 3000 transfection reagent (Lip3000) was used for virus packaging, and the experimental operation was basically performed according to the instructions of the transfection reagent.

[0088] 1. 293T medium configuration

[0089] The medium was DMEM-H supplemented with 10% FBS. (DMEM-H is the DMEM medium supplemented with 4.5g / L glucose and 110mg / L sodium pyruvate).

[0090] 2. Lentivirus packaging medium configuration:

[0091] The medium is Opti-MEMI, add 1% GlutaMAX, add sodium pyruvate (1mM), 5% FBS. (The Opti-MEMI medium is a Gibco product).

[0092] 3. Lentivirus packaging:

[0093] A) 293T cells were treated with 7x10 6 Cells / well were seeded in a 10cm culture dish containing 12 mL of lentiviral packaging medium, placed at 37°C, 5% CO 2 Incubate the cells overnight.

[0094] B) Transfect when the 293T cell density reaches 95%, the transfection method is:

[0095] Solution A configuration: First, r...

Embodiment 3

[0115] Example 3: T cell preparation

[0116] 1. Preparation of T cells

[0117] A) Add antibody (20 μL, CD8 biotin antibody, biotin-labeled CD8 antibody, BD product, Cat. No. 555365) to the EP tube containing 1 mL of whole blood, and mix for 30 minutes at room temperature with a rotator. The whole blood used in the experiment was taken from 8 healthy male volunteers (26-33 years old, body weight 58-71kg), and T cells were prepared respectively. The above-mentioned biotin-labeled CD8 antibody is expensive and is an important cost item in the method for preparing T cells of the present invention.

[0118] B) Add 150 μL of microbubbles (streptavidin, streptavidin-labeled, ThermoGenesis product) to the above 1 mL of whole blood, and mix with a rotator for 20 minutes at room temperature to combine the microbubbles with the antibody-labeled cells.

[0119] C) Centrifuge cells at 400 x g for 5 minutes.

[0120] D) Use a 200µL pipette gun to gently transfer the white microbubble l...

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Abstract

The invention relates to a method for preparing CAR-T (chimeric antigen receptor T) cells, which comprises the following steps of: synthesizing plasmids which are composed of a CD19 single-chain variable region, a CD8a hinge region, a CD8a transmembrane region, a 4-1BB signal, a CD3 zeta inclusion signal, an IL-7 sequence and a CCL17 sequence; packaging the virus to obtain a lentivirus concentrated solution, and measuring the virus titer of the lentivirus concentrated solution; and preparing T cells, namely activating, transducing and amplifying the prepared T cells which are CD8 < + > T cells, and harvesting CAR-T cells. The invention also relates to a method for preparing the T cell. The invention has excellent effects as described in the specification.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a CD19-CAR-T that secretes IL-7 and CCL-17 factors, and secretes anti-PD-1 and anti-GM-CSFscFv that neutralize PD-1 and GM-CSF Cells, the present invention also relates to the preparation method and use of the CAR-T cells such as CD19-CAR-T cells. Background technique [0002] The U.S. Food and Drug Administration (FDA) approved the first gene therapy product, Kymriah, in August 2017 to treat some children and young adults with acute lymphoblastic leukemia (ALL). Kymriah is a genetically modified autologous T cell immunotherapy known as chimeric antigen receptor T (CAR-T) immunotherapy. CAR-T immunotherapy is a chimeric antigen receptor-based cellular immunotherapy program. Through in vitro gene transduction technology, the gene sequence encoding chimeric antigen receptor CAR-T is introduced into T cells to generate cells that can bind Tumor-specific T cells targeting a...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N5/0783C12N15/867C12N15/62
CPCC12N5/0636C07K14/7051C07K14/54C07K14/521C12N15/86C07K2319/02C07K2319/03C12N2740/15043
Inventor 刘庆喜许晓椿肖海蓉魏卿赵梦莲
Owner 深圳博雅感知药业有限公司
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