Application of promoter optimized lentiviral genetically modified T cells in oncotherapy

A promoter and recombinant lentivirus technology, which is applied in the fourth-generation specific anti-tumor immunotherapy technology field, can solve the problems of low efficiency, insufficient expression, and lagging gene modification technology.

Inactive Publication Date: 2015-06-10
深圳市中美康士生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main technical support of the fourth-generation immunization technology is gene modification technology. At present, compared with western developed countries, my country's gene modification technology is seriously lagging behind, lacking service agencies and technical support that can provide genetically engineered virus vector preparation, and lack of relevant regulations to guide clinical application
At present, the more popular promoters are CMV (Human cytomegalovirus) UBC (Ubiquitin), PGK (phosphoglycerate kinase-1), EF1 (elongation factor-1alpha), etc., but their expression in T and NK cells is not durable enough and the efficiency is low , easy to be methylated and lose function

Method used

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  • Application of promoter optimized lentiviral genetically modified T cells in oncotherapy
  • Application of promoter optimized lentiviral genetically modified T cells in oncotherapy
  • Application of promoter optimized lentiviral genetically modified T cells in oncotherapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Optimization of the promoter of the lentiviral expression system

[0039] 1) Packaging and titer determination of recombinant lentivirus

[0040] Culture 293FT cells. One day before transfection, after adjusting the cell density with DMEM medium containing 10% fetal bovine serum, inoculate 25×10 cells per 15 cm cell culture dish. 6 293FT cells into a cell culture dish at 37 °C, 5% CO 2 Cultivate in an incubator, and after 16h-24h, the cell density can be used for transfection when the cell density grows to 80%-90%. On the day of transfection, the medium was replaced with a complete medium (DMEM+10% FBS) without antibiotics (P / S). The LVV-MSCV-GFP, LVV-CMV-GFP, LVV-EF1a-GFP, LVV-PGK-GFP, LVV-UBC-GFP lentiviral backbones were co-transfected with the other three packaging plasmids into 293FT cells, and Lipofectamine 2000 (Invitrogen) as the medium. After culturing for 6 hours, discard the medium, wash with PBS 3 times and replace with 20 ml of fresh complete...

Embodiment 2

[0043] Example 2: The recombinant lentiviral vectors carrying the murine TCRα and β chain genes that specifically recognize human melanoma-associated antigen gp100 (154-162) were transfected into peripheral blood autologous lymphocytes using molecular biology techniques to make the recombinant TCR is expressed in T lymphocytes to achieve the purpose of killing tumors efficiently. A lentiviral vector with an optimized promoter was used to construct a TCR expression vector containing a self-cleaving 2A peptide, Furin and a spacer sequence (see the attached table for the sequence).

[0044] 1) Optimal construction of 2A peptide, furin and spacer sequences expressing the two chains of TCR

[0045] In order to realize the co-expression of TCRα and β chain genes in the same expression vector, the present invention adopts to add 2A peptide with self-cleaving function between TCRα and β chain, and the 2A peptide adopted can be F2A (foot-and-mouth disease virus), E2A (equine Rhinitis ...

Embodiment 3

[0063] Example 3: Using MSCV lentiviral vectors packaged with chimeric antigen receptors (chimeric antigen receptors, CARs), T and NK cells can be genetically modified to express single-chain antibodies, through their specific recognition of tumor cell surface antigens, and Dual activation of T and NK cells through intracellular CD3zeta and 41-BB can not only prolong the survival time of cells, but also enhance their ability to clear tumors, and effectively kill a variety of tumors in vivo and in vitro. The present invention constructs a CAR vector through an anti-Her2 / neu single-chain antibody, genetically modifies human T and NK cells through an MSCV-optimized lentivirus system, and enables them to acquire the ability to specifically kill tumors ( Figure 7 , 8 ,9). In the in vitro cell killing test, CAR-engineered lymphocytes can significantly improve the recognition of T cells to HLA-A2+ corresponding tumor cells, and release IFN-γ, a marker factor of T cell activation. ...

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Abstract

The invention provides a promoter optimized lentiviral expression system. The promoter optimized lentiviral expression system is characterized in that an MSCV promoter is adopted; the genetically modified T cells of the promoter optimized lentiviral expression system are enabled to express an anti-tumor T cell receptor (TCR), and are characterized in that TCRalpha, furin, an interval sequence, a 2A peptide and a TCRbeta sequence are orderly inserted in multiple cloning sites; and a chimeric antigen receptor (CAR) expression vector constructed by use of a promoter optimized lentiviral vector is characterized in that a CAR sequence is inserted in the multiple cloning sites. The recombinant lentiviral vector can be applied to, for example, expressing the recombinant TCR in the T cells and expressing the CAR in T or NK cells to achieve the purpose of specifically killing tumors, and also can be applied to clinical oncotherapy.

Description

technical field [0001] The present invention belongs to a gene modification technology that uses lentivirus technology to genetically modify T and NK cells to obtain stable specific anti-tumor ability, belongs to the fourth generation of specific anti-tumor immunotherapy technology, and is a new technology for tumor immunotherapy in recent years. It is a hotspot and has made breakthroughs in cancer treatment (including leukemia) (Lee, Kochenderfer et al. 2014, Maude, Frey et al. 2014). Background technique [0002] Malignant tumors are the most serious diseases that endanger human health. Every year, about 3 million people in China are diagnosed with new tumors, and 2 million people die of cancer. Although it can be treated by surgery, radiotherapy, and chemotherapy, most patients cannot bear the side effects and treatment efficiency, and still die of tumor recurrence and metastasis. Therefore, how to remove the residual tiny tumor tissue or tumor cells is one of the proble...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10A61P35/00
Inventor 杨世成
Owner 深圳市中美康士生物科技有限公司
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