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Method for qualitatively detecting pollution of VSVG sequence in CAR-T product

A qualitative detection and product technology, which is applied in the field of qualitative detection of VSVG sequence contamination in CAR-T products, can solve the indispensable problems of quality inspection, achieve intuitive and fast results, fill technical gaps, and have good clinical application prospects

Inactive Publication Date: 2016-11-09
IMMUNE CELL BIOTECH CO LTD
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Problems solved by technology

[0004] However, because of the need to use lentivirus to infect T cells in the preparation process to generate CAR-T, there are higher requirements for quality control
Although the progress of lentiviral packaging has gone through several generations, the safety has been greatly improved, but because the source is HIV, strict quality inspection is essential

Method used

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  • Method for qualitatively detecting pollution of VSVG sequence in CAR-T product
  • Method for qualitatively detecting pollution of VSVG sequence in CAR-T product
  • Method for qualitatively detecting pollution of VSVG sequence in CAR-T product

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Embodiment 1

[0026] Example A method for qualitative detection of VSVG sequence contamination in CAR-T products

[0027] 1. Instruments and equipment

[0028] Ultra-clean bench, Real-Time PCR instrument, desktop centrifuge, vortex suspension, Nanodrop ultra-micro spectrophotometer, etc.

[0029] 2. Reagents

[0030] Genomic DNA extraction kit (Axygen), PAGE purified PCR primers (GeneScript), positive control plasmid pMD2.G (containing VSVG gene), 2×SuperReal PreMix (Tiangen), etc.

[0031] 3. Inspection operation

[0032] (1) Preparation of the template: The final product of CAR-T was selected as the sample to be tested in a sterile environment, and the genomic DNA was extracted using the genomic DNA extraction kit produced by Axygen. After extraction, the DNA concentration and purity were analyzed using a Nanodrop instrument. It was determined that the concentration of the genomic DNA was 44.3ng / μL. The purity 260 / 280 was between 1.80-1.90.

[0033] (2) Real-Time PCR reaction:

[00...

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Abstract

The invention relates to a quality control method applied to cellular immunity CAR-T treatment, in particular to a method for qualitatively detecting pollution of a VSVG sequence in a CAR-T product. A Real-Time PCR primer of the VSVG gene sequence in an HIV is designed for overcoming the defects in the prior art of slow virus production according to the actual demands of CAR-T production. Real-Time PCR analysis is conducted on the CAR-T product through the primer, therefore, whether the VSVG gene sequence exists in the CAR-T or not can be effectively monitored, and a guarantee is provided for security of CAR-T cellular immunotherapy technique application.

Description

technical field [0001] The present invention relates to a quality control method applied in cellular immune CAR-T therapy, in particular to a method for qualitatively detecting VSVG sequence contamination in CAR-T products. Background technique [0002] CAR-T, the full name is Chimeric Antigen Receptor T-Cell Immunotherapy, chimeric antigen receptor T cell immunotherapy. This is a new type of cell therapy that has been around for many years, but has only been improved and used clinically in recent years. It has remarkable efficacy in the treatment of acute leukemia and non-Hodgkin's lymphoma, and is considered to be one of the most promising tumor treatment methods. Like all technologies, CAR-T technology has also undergone a long evolutionary process. It is during this series of evolutionary processes that CAR-T technology has gradually matured. [0003] CAR-T therapy is a specific targeted therapy, because it is based on antibody, antigen binding, or a specific binding t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/703C12Q1/686
Inventor 孙秀莲李欣徐鸿
Owner IMMUNE CELL BIOTECH CO LTD
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