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Preparation method and application of HLA-A0201-restrictive anti-Sox2 specific CTL

A specific and restrictive technology, applied in the field of genetic engineering, can solve the problems of low antigen uptake, poor anti-tumor treatment effect, insufficient killing activity of CTL cells, etc., and achieves simple and easy preparation method, good stability and convenience The effect of the operation

Inactive Publication Date: 2015-11-11
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

[0008] For this reason, the technical problem to be solved by the present invention is to overcome the limitations of less antigen uptake in tumor immunotherapy in the prior art, insufficient killing activity of CTL cells, and poor anti-tumor treatment effect, thereby proposing a method with strong killing ability, Preparation method and application of HLA-A0201-restricted anti-Sox2 specific CTL with strong antigenic specificity, shortened treatment time of patients and good stability, convenient operation and clinical application of tumor adoptive immune cell therapy

Method used

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  • Preparation method and application of HLA-A0201-restrictive anti-Sox2 specific CTL
  • Preparation method and application of HLA-A0201-restrictive anti-Sox2 specific CTL
  • Preparation method and application of HLA-A0201-restrictive anti-Sox2 specific CTL

Examples

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Effect test

Embodiment 1

[0048] Example 1 This example discloses the preparation of a C-type lectin-type sugar-modified cationic liposome antigen carrier. The specific operations are as follows:

[0049] (1) First prepare mannose or mannoside modified polyethylene glycol derivatized phosphatidylethanolamine phospholipids. Mannose or mannoside is connected to the amino group of polyethylene glycol derivatized phosphatidylethanolamine phospholipid through aldehyde-amino or hydroxyl-amino condensation, so as to obtain the polyethylene glycol derivatized phosphatidyl alcohol of mannose or mannoside ethanolamine phospholipids. Then the cationic lipid DOTAP and the polyethylene glycol derivatized phosphatidylethanolamine phospholipid of mannose or mannoside are respectively dissolved in chloroform-methanol (2:1), and mixed according to a certain ratio, and the mixed solution is obtained after the two are mixed. Place in a round bottom flask. Rotate the mixed solution to dryness with a stable rotary evapor...

Embodiment 2

[0050] Example 2 This example discloses the production process of 72-hour rapid maturation of dendritic cells loaded with tumor stem cell antigens, the specific operations are as follows:

[0051] (1) Collect and isolate mononuclear cells from peripheral blood, and mononuclear cells are separated at 3.0-5.0*10 6 Cells / ml suspended in AIM-V serum-free culture medium, then add cytokines IL-4 and GM-CSF DC cell induction medium to the culture medium to induce DC formation, and then culture at 37°C, 5% CO2 Cultured in a box, cultured in saturated humidity for DC cell induction.

[0052] (2) The next day, add cationic liposomes wrapped with tumor stem cell Sox2 antigen (as shown in the sequence listing) to the DC cell culture medium at a concentration of 1-2.5ug / ml and incubate for 6 hours Afterwards, DC maturation-promoting medium was added to the DC cells, and the culture was continued for 24-48 hours to obtain DC cells loaded with the tumor stem cell antigen Sox2.

[0053] Ide...

Embodiment 3

[0054] Example 3 This example discloses the process of 72-hour rapidly maturing DCs inducing the secretion of Sox2CTL cytotoxic cytokines, and the specific operations are as follows:

[0055] (1) Add CTL-inducing factors to DC cells loaded with tumor stem cell antigens, induce for 4-7 days, and add medium containing CTL-inducing factors every two and a half days in the middle.

[0056] (2) After CTL induction is completed, add BFA at a concentration of 10 ng / ml. After 4 hours, collect CTL by centrifugation, and use flow cytometry antibody IFN-r and CD8 staining to detect the secretion and expression of cytotoxic factor IFN-r, tumor stem cell antigen Sox2CTL cells Toxicity factor IFN-r test results are as follows: Figure 2A / B / C / D / E / F and Figure 3A / B / C / D / E / F shows:

[0057] Figure 2A / B / C / D / E / F and Figure 3A / B / C / D / E / F, these two sets of figures are a series of pictures of the detection data of the cytotoxic factor IFN-r of the tumor stem cell antigen SOX-2CTL induced ...

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Abstract

The invention belongs to the field of gene engineering and particularly relates to a preparation method and an application of HLA-A0201-restrictive anti-Sox2 specific CTL. In the preparation method, with a cationic liposome of a C-typed agglutinin receptor on a targeted dendritic cell as an antigen supporter, a tumor stem cell antigen Sox2 is coated. The dendritic cell is quickly matured in 72 h and meanwhile positive antigen intake efficiency and MHC-I approach antigen presentation efficiency of the dendritic cell are improved. The method, compared with a conventional method in which the matured dentritic cells are obtained in 7-8 days, is greatly reduced in best therapeutic time window of tumor patients, so that the method is beneficial to generation of the signal-inductive antigen-specific CTL by stimulating initial T cells with abundant matured dendritic cells. With combination of engineering cells and cell factors, the antigen-specific T cells and Tcm are amplified in a large scale, thereby producing the Tcm being stronger in killing capability and being higher in ratio and further improving the anti-tumor immunity effect. The method can be used for quickly and effectively preparing the tumor stem cell antigen-specific CTL and has great economic value and market prospect.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a preparation method and application of HLA-A0201-restricted anti-Sox2 specific CTL. Background technique [0002] Malignant tumors have become the number one "killer" of human beings. In the history of human anti-cancer, there have been three major weapons to fight against cancer: surgery, chemotherapy, and radiation therapy. However, although these three methods can effectively reduce the tumor burden in the short term, they still cannot effectively eliminate tumor cells. Minimal residual lesions, partial sensitivity and drug resistance to radiotherapy and chemotherapy are the root causes of tumor recurrence and metastasis, and recurrence and metastasis are the main causes of death in cancer patients. Conventional treatment methods have been unable to do what they want, and the development of new tumor treatment technologies and products is imminent. [0003] So far, immuno...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K47/46A61K47/34A61K47/26A61P35/00C12N5/0783
Inventor 陈尚刘根桃李晓祥
Owner 深圳市中美康士生物科技有限公司
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