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DC (Dendritic Cell) vaccine modified by DUOX2 and applications of DC vaccine in killing and wounding pancreatic cancer initiating cells in targeted manner

A technology for initiating cells and pancreatic cancer, applied in the field of tumor cell immunotherapy, can solve problems such as failure to produce long-term therapeutic effects, failure to achieve therapeutic effects, and inability to effectively kill pancreatic cancer initiating cells.

Active Publication Date: 2017-08-08
上海尚泰生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method uses DUOX 2 Modified dendritic cells, targeted to kill the initial cells of pancreatic cancer, inhibited the recurrence and metastasis of pancreatic cancer, solved the problem that traditional surgical resection, radiotherapy and chemotherapy could not effectively kill the initial cells of pancreatic cancer, and failed to achieve the desired therapeutic effect. As well as scientific and technical issues such as the failure of DC vaccines containing only pancreatic cancer-related antigens to produce long-term therapeutic effects

Method used

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  • DC (Dendritic Cell) vaccine modified by DUOX2 and applications of DC vaccine in killing and wounding pancreatic cancer initiating cells in targeted manner
  • DC (Dendritic Cell) vaccine modified by DUOX2 and applications of DC vaccine in killing and wounding pancreatic cancer initiating cells in targeted manner
  • DC (Dendritic Cell) vaccine modified by DUOX2 and applications of DC vaccine in killing and wounding pancreatic cancer initiating cells in targeted manner

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1: Gene screening and identification of pancreatic cancer-initiating cells and pancreatic cancer-associated antigens

[0017] CD44 + CD24 + ESA + Pancreatic cancer-initiating cells and CD44 - CD24 - ESA - Gene profiles differentially expressed in non-pancreatic cancer-initiating cells and in pancreatic cancer and normal pancreatic tissue. Firstly, pancreatic cancer-initiating cells and non-pancreatic cancer-initiating cells were sorted out by flow cytometry, and the total RNA of pancreatic cancer-initiating cells and non-pancreatic cancer-initiating cells, as well as pancreatic cancer and normal pancreatic tissues were extracted respectively, and passed through Affymetrix Human Gene 1.0ST human gene expression profile chip scanning analysis, CD44 was initially screened out + CD24 + ESA + Pancreatic cancer-initiating cells and highly expressed gene DUOX in pancreatic cancer 2 , Real-time fluorescent quantitative polymerase chain reaction verified the scr...

Embodiment 2

[0018] Example 2: DUOX 2 Modified DC vaccine preparation

[0019] 1. Peripheral blood mononuclear cell isolation:

[0020] Take out the blood, slowly add it to the upper layer of Ficoll separation medium, centrifuge at 1000G for 30min, absorb the white PBMC at the boundary, and resuspend in 1640 medium; centrifuge at 350G for 8min, remove the supernatant, repeat three times, add 10ml of medium to resuspend and count. According to the counting results, DCs were cultured.

[0021] 2. Culture and transfection of DC

[0022] Adjust cell concentration to 3 x 10 6 / mL, take 6ml and add it to a T25 culture flask, at 37°C, 5% CO 2 Take it out after culturing in the incubator for 2 hours, discard the suspended cells, wash gently, add 6 mL containing GM-CSF (50ng / mL), IL-4 (50ng / mL), double antibody (100U / mL) and 10% FBS After 5 days, add 3mL containing IL-1β(10ng / mL), IL-6(100ng / mL), TNF-α(10ng / mL), PGE2(1μg / mL), CD40L with enhancer( 1 μg / mL), IFN-α (3000 μ / mL), IL-4 (800 U / mL), ...

Embodiment 3

[0023] Example 3: DUOX 2 Modified DC vaccines induce specific T cell responses

[0024] 1. Antigen-specific T cell preparation and immunogenicity detection

[0025] will load DUOX 2 Antigen mature DC cells and T cells were mixed at a ratio of 1:10, added to lymphocyte culture medium containing final concentrations of 5ng / ml IL-7 and 20IU / ml IL-2 for culture, and the medium was changed every day, and obtained on the 7th day Load DUOX 2 Antigen DC cells stimulate T cells.

[0026] 2. Immunophenotype detection of mature DC cells

[0027] Take the mature DC cells, wash them twice with PBS, and take 1×10 5 / mL were added to the corresponding flow tubes. Add 1 μl of the monoclonal antibodies to be detected, including HLA-DR, CD80, CD83 and CD86 antibodies, and incubate at 4°C in the dark for 30 minutes. Washed twice with PBS, resuspended with 400 μl of PBS, and detected by flow cytometer Cytoflex (Beckman).

[0028] 3. Detection of immunogenicity of mature DC vaccine

[002...

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Abstract

The invention relates to novel applications of DUOX2, and in particular relates to applications of the DC (Dendritic Cell) vaccine modified by DUOX2 in killing and wounding pancreatic cancer initiating cells in a targeted manner. For the first time, the invention provides the scheme that DUOX2 is taken as the relevant antigen of the pancreatic cancer initiating cell, and then the DC vaccine capable of killing and wounding pancreatic cancer initiating cells in a targeted manner is obtained through gene modification. The invention further provides a preparation method of the DC vaccine modified by DUOX2. The DC vaccine modified by DUOX2 can kill and wound the pancreatic cancer initiating cells in a targeted manner, and has the good effect of resisting pancreatic cancer. The specific immunotherapy for resisting pancreatic cancer based on the vaccine has the wide clinical application prospect.

Description

[0001] Technical field [0002] The invention belongs to the technical field of tumor cell immunotherapy, and the invention relates to double oxygenase DUOX 2 Novel uses of the enzyme DUOX specifically related to 2 Application of modified DC cells in targeting and killing pancreatic cancer-initiating cells. Background technique [0003] Pancreatic cancer is one of the malignant tumors with the highest lethality rate, with nearly 250,000 new cases and deaths worldwide each year (ShahM, Da Silva R, Gravekamp C, Libutti SK, Abraham T, Dadachova E. Targeted radionuclide therapies for pancreatic cancer. Cancer gene therapy. 2015;22(8):375-9). Because there are no obvious symptoms in the early stage of development, pancreatic cancer can usually only be diagnosed in the late stage. Clinical studies have reported that pancreatic cancer is not sensitive to chemotherapy and radiotherapy and is prone to tolerance (Bardeesy N, DePinho RA. Pancreatic cancer biology and genetics. Nature r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61P35/00
CPCA61K39/0011A61K2039/5154A61K2039/53
Inventor 李晨蔚岳荣彩王迪民
Owner 上海尚泰生物技术有限公司
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