Anti-tumor associated antigen WT1 specificity CTL and preparation method thereof
A tumor-associated antigen specific technology, applied in the field of anti-tumor-associated antigen WT1-specific CTL and its preparation, can solve the problem of poor anti-tumor efficacy of tumor immune cells, improve uptake efficiency and antigen presentation ability, enhance anti-tumor Tumor immunity, enhanced anti-tumor effect
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[0039] The invention provides a preparation method of anti-tumor-associated antigen WT1 specific CTL, comprising the following steps:
[0040] Step 1, preparing cationic liposome carrier targeting dendritic cell C-type lectin receptor coated with tumor-associated antigen WT1;
[0041] Step 2, using the cationic liposome carrier obtained in the step 1 to load the tumor-associated antigen WT1 on mature DC cells;
[0042] Step three, using the mature DC cells obtained in step two to induce WT1-specific T lymphocytes and central memory T lymphocytes.
[0043] The invention uses C-type lectin-type sugar-modified cationic liposome as an antigen carrier to encapsulate tumor-associated antigens for the preparation of antigen-specific T lymphocytes. The cationic liposome includes phosphatidylethanolamine bilayer, polyethylene glycol derivatized phosphatidylethanolamine and mannose; the C-type lectin type sugar is mannose or mannoside. The phospholipid bimolecular ball is composed of ...
Embodiment 1
[0045] Example 1 Preparation method of anti-tumor-associated antigen WT1-specific CTL
[0046] The preparation method of the present invention mainly includes three major steps, and this embodiment is a general preparation method of the present invention.
[0047] First, the sugar-modified cationic liposome antigen carrier of C-type lectin type is produced, and the specific operation is as follows:
[0048] Firstly, mannose or mannoside modified polyethylene glycol derivatized phosphatidylethanolamine phospholipids are prepared. 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...
Embodiment 2
[0055] Example 2 Identification of mature phenotype of DC
[0056] In this embodiment, mononuclear cells are collected and separated from peripheral blood at a rate of 3.0-5.0*10 6 Cells / ml were suspended in AIM-V serum-free medium, and cytokines IL-4 and GM-CSF were added to the medium to induce DC formation, and then placed at 37°C, 5% CO 2Culture in an incubator. On the next day, add cationic liposomes wrapped with tumor-associated WT1 antigen to the DC cell culture medium at a concentration of 1-2.5ug / ml and incubate for 6 hours, and then add DC promoting cells to the DC cells. The maturation medium is further cultured for 24-48 hours to obtain DC cells loaded with tumor-associated antigens. The mature phenotype identification results of DC are attached figure 1 . Figures A and B are the detection data of mDC maturation of two donors, CD83 is a marker of DC maturation, and CD80 and CD86 are co-stimulatory molecules. The simultaneous double-positive expression rate of m...
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