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Lentiviral vector for preparation of CAR-T (chimeric antigen receptor T cells) as well as construction method and application of lentiviral vector

A lentiviral vector, recombinant lentivirus technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of low recombinant virus titer, inactivity, and insufficient recombinant virus titer, etc. Improve the effect of recombinant virus titer and rapid detection standard

Active Publication Date: 2018-01-09
CELLYAN THERAPEUTICS WUHAN CO LTD
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Problems solved by technology

For example, the titer of the recombinant virus is not high enough, except for the results reported by Naldini et al., the others are all at 10 3 TU / ml~10 4 Between TU / ml, it is difficult to meet the needs of in vivo applications
One of the main reasons for the low titer of the recombinant virus is the strong expression of the lentivirus promoter, which expresses an excessive amount of CAR gene in T cells and is often inactive.

Method used

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  • Lentiviral vector for preparation of CAR-T (chimeric antigen receptor T cells) as well as construction method and application of lentiviral vector
  • Lentiviral vector for preparation of CAR-T (chimeric antigen receptor T cells) as well as construction method and application of lentiviral vector
  • Lentiviral vector for preparation of CAR-T (chimeric antigen receptor T cells) as well as construction method and application of lentiviral vector

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Embodiment 1

[0038]1. Design and synthesis of lentiviral vector pLVCD3-MCS

[0039] (1) The pCDH-EF1-MCS lentiviral vector from SBI Company is used as the starting vector, the size of pCDH-EF1-MCS is 6kb, the vector sequence is shown in SEQ ID1, and the vector map is attached figure 1 .

[0040] (2) Synthesis of CD3-MCS sequence (Wuhan Jinkairui Bioengineering Co., Ltd.)

[0041] Using Primer5.0 software to analyze the sequence of the pCDH-EF1-MCS lentiviral vector, it was found that the restriction site ClaI and the restriction site BamHI can cut off the irrelevant part of the initial vector, and the size of the vector after restriction is 6kb.

[0042] Using the sequence of the CD3 gene promoter, plus the sequence of the multiple cloning site of the pCDH-EF1-MCS lentiviral vector, the sequence CD3-MCS is obtained, and the sequence is shown in SEQID2; the two ends of the synthetic CD3-MCS sequence are respectively added with ClaI (ATCG) Or the sequence of MluI (ACGCGT) (Wuhan Jinkairui ...

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Abstract

The invention discloses a lentiviral vector for preparation of CAR-T (chimeric antigen receptor T cells), a construction method of the lentiviral vector and an application of the lentiviral vector inanti-tumor cell product preparation. Meanwhile, the invention further provides a method for detecting virus titer with fluorescence quantitative PCR (polymerase chain reaction), and detection standardis provided for clinical application. The lentiviral vector is short, can stably and efficiently express a CAR gene in T cells and contains no genes such as GFP, Neomycin and the like irrelevant to treatment, and a high-titer lentivirus can be formed through packaging by the lentiviral vector in viral packaging cells such as HEK293T. Experiments prove that the lentiviral vector is suitable for research and clinical application of CAR-T.

Description

technical field [0001] The present invention relates to the field of cellular immunotherapy, in particular to a lentiviral vector for CAR-T preparation and its construction method and application. Background technique [0002] Immune cell therapy technology is the latest tumor treatment technology that has emerged in recent years. Compared with existing surgery, radiotherapy, chemotherapy, and targeted therapy, it has its own irreplaceable advantages and development potential. Therefore, it is called tumor therapy. the fifth largest technology. Immune cell therapy mainly includes traditional CIK (Cytokine Induced Killer cells, OCIK), DC-CIK (Dendritic Cells, DC) and the latest CAR-T technology. CAR-T technology, the full name is Chimeric Antigen Receptor T cells (CAR-T), the main structure of CAR includes a single-chain antibody domain that binds tumor-associated antigen (Tumor Associated Antigen, TAA) extracellularly And the CD28 co-stimulatory signal and CD3ζ signal of a...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10A61K35/17A61P35/00
Inventor 华权高沈鹤霄金丹李璐李国龙
Owner CELLYAN THERAPEUTICS WUHAN CO LTD
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