Anti-tumor associated antigen WT1 specificity CTL and preparation method thereof

[0039] The present invention provides a method for preparing CTL specific for anti-tumor-associated antigen WT1, including the following steps:

[0040] Step one: preparing a cationic liposome carrier targeting the C-type lectin receptor of dendritic cells encapsulating the tumor-associated antigen WT1;

[0041] Step 2: Use the cationic liposome carrier obtained in the step 1 to load the tumor-associated antigen WT1 on mature DC cells;

[0042] In step three, the mature DC cells obtained in step two are used to induce WT1-specific T lymphocytes and central memory T lymphocytes.

[0043] The present invention adopts C-type lectin type carbohydrate-modified cationic liposomes as antigen carriers to encapsulate tumor-related 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

[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 example is a general preparation method of the present invention.

[0047] First, the preparation of C-type lectin type sugar-modified cationic liposome antigen carrier, the specific operation is as follows:

[0048] First, prepare mannose or mannoside modified polyethylene glycol derivatized phosphatidylethanolamine phospholipids. The mannose or mannoside is linked to the amino group of the polyethylene glycol derivatized phosphatidylethanolamine phospholipid by means of aldehyde-amino or hydroxy-amino condensation to obtain the polyethylene glycol derivatized phosphatidyl of mannose or mannoside Ethanolamine phospholipid. Then the cationic lipid DOTAP and the polyethylene glycol derivatized phosphatidylethanolamine phospholipid of mannose or mannoside were dissolved in chloroform-methanol (2:1), an

[0055] Example 2 Identification of mature phenotype of DC

[0056] In this example, mononuclear cells were collected and separated from peripheral blood, with a rate of 3.0-5.0*10 6 A / ml density was suspended in AIM-V serum-free medium, and then cytokine IL-4 and GM-CSF were added to the medium to induce the formation of DC, and then placed at 37℃, 5% CO 2 Culture in an incubator. On the second day, add cationic liposomes encapsulating tumor-associated WT1 antigen to the DC cell culture medium at a concentration of 1-2.5ug / ml and incubate for 6 hours. After that, add DC to the DC cells. The mature medium is cultured for 24-48 hours to obtain DC cells loaded with tumor-related antigens. The mature phenotype identification results of DC are attached figure 1 . In the figure, A and B are the mDC maturity detection data of the two donors. CD83 is the maturity marker of DC, and CD80 and CD86 are costimulatory molecules. The double positive rate of mDCCD83CD80 and CD83CD86 of two don