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Method for preparing PD-1 gene-deficient type PD-1-CART cell preparation

A PD-1-CART and PD-1-CAR technology, applied in the field of molecular biology, can solve the problems of large side effects and poor targeting, and achieve the effect of reducing side effects and reducing side effects

Inactive Publication Date: 2019-12-20
王清路 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the preparations for the treatment of tumor cells have poor targeting and side effects

Method used

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  • Method for preparing PD-1 gene-deficient type PD-1-CART cell preparation
  • Method for preparing PD-1 gene-deficient type PD-1-CART cell preparation
  • Method for preparing PD-1 gene-deficient type PD-1-CART cell preparation

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Embodiment 1

[0045] (1) PD-1 gene-deficient T lymphocytes

[0046]Prepare PD-1 gene-deficient T lymphocytes according to the method described in patent ZL201710649967.6.

[0047] (2) Design of ITSM motif tyrosine mutation

[0048] The original tyrosine codon TAT in the ITSM motif was replaced with the codon TTT to mutate tyrosine into phenylalanine.

[0049] (3) PD-1+4-1BB+CD3ζ chimeric CAR design

[0050] For the chimeric 4-1BB (214-255aa) and CD3ζ (52-163aa) peptides after the PD-1 peptide, send the designed sequence to Shanghai Sangon Bioengineering Co. Add an EcoRI site, add a Spel site after the TGA stop codon at the 3' end, and then clone into the pMD18-T vector, named pMD18-T-CAR.

[0051] (4) Construction of pSin-EF2-Pur-CAR vector

[0052] a. Two plasmids, pMD18-T-CAR and pSin-EF2-Sox2-Pur, were digested with EcoR I and Spe l, and the enzyme digestion reaction system was:

[0053]

[0054] React at 37°C for half an hour, and then perform 1.0% agarose gel electrophoresis. ...

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Abstract

The present invention belongs to the technical field of molecular biology and particularly relates to a method for preparing a PD-1 gene-deficient type PD-1-CART cell preparation. The method comprisessteps of construction of PD-1-CAR chimeric sequences, construction of PD-1-CAR lentivirus and construction of PD-1 gene-deficient type PD-1-CAR cells. Firstly, after mutating ITSM motif of PD-1 protein, then T cell extracellular protein parts and transmembrane signal guide regions with PD-L1 target recognition functions are obtained, then a lentiviral plasmid with a novel CAR structure lentivirusis constructed, and the constructed lentiviral plasmid transfects with PD-1 gene-deficient type T lymphocytes to obtain the PD-1 gene-deficient type PD-1-CART cell preparation. The PD-1 gene-deficient type PD-1-CART cell preparation improves ability of the T lymphocytes to target and kill tumor cells, reduces side effects, can enhance long-term tumor immunity, and also provides a reference for development of similar new CAR gene structures.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for preparing a PD-1 gene-deficient PD-1-CART cell preparation. Background technique [0002] PD-1 (Programmed Death 1, programmed death receptor-1) is an important immunosuppressive molecule. The pathway composed of PD-1 and its ligands PD-L1 and PD-L2 plays a crucial role in maintaining peripheral immune tolerance. Tumors and pathogens that cause chronic infections can use this pathway to escape from T cell-mediated tumor-specific and pathogen-specific immunity, mainly through the production of PD-L1 on its surface, when PD-L1 is linked to a type of immune cell T The PD-1 protein on the cells inactivates the T cells, and the T cells cannot find the tumor and send a signal to the immune system to attack the tumor. [0003] PD-1 consists of a single N-terminal immunoglobulin variable (IgV)-like domain, a stem region of approximately 20 amino acids...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/90C12N9/22C12N15/66
CPCC12N9/22C12N15/66C12N15/86C12N15/907
Inventor 王清路
Owner 王清路
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