Isolated culture method for tumor-specific T cells and product obtained through method
A tumor-specific, separation and culture technology, applied in the field of separation and culture methods and products obtained therefrom, achieves the effects of convenient material collection, convenient blood collection, and short time required.
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Embodiment 1
[0055] Influence of Factor Types in Culture Medium on the Proportion of Effector Memory T Lymphocytes in Cell Final Products
[0056] This example explores the effects of five different culture methods on effector memory T cells (specific phenotype: CD3) in the final cell product. + CD8 + CD45RA - CCR7 - and CD3 + CD4 + CD45RA - CCR7 - ) ratio, its specific operation includes the following steps:
[0057] (1) Take the peripheral blood of kidney cancer patients who have been treated with PD-1 antibody within 3 weeks;
[0058] (2) The peripheral blood obtained in step (1) is separated by the Ficoll density gradient method to obtain mononuclear cells;
[0059] (3) Incubate the mononuclear cells obtained in step (2) with biotinylated anti-human IgG4 antibody at 25°C for 20 minutes, then add magnetic beads coated with anti-biotin antibody and incubate at 25°C for 10 minutes, Magnetic separation of mononuclear cells positive for PD-1;
[0060] (4) The PD-1 positive mononuc...
Embodiment 2
[0063] Effects of two different PD-1 antibody pretreatment methods on the proliferation ability of PD-1 positive cells
[0064] This example explores the effects of two different PD-1 antibody pretreatment methods on the expansion speed of isolated tumor-specific T cells. In this example, two experiments were carried out on samples, one of which was peripheral blood from patients with cervical cancer. (sample 1), and the second is the peripheral blood (sample 2) of a patient with malignant melanoma. The specific operation includes the following steps:
[0065] (1) Collect peripheral blood from patients with cervical cancer and malignant melanoma before and after PD-1 antibody drug treatment (within 3 weeks). Add PD-1 antibody to the peripheral blood of patients who have not been treated with PD-1 antibody drug to make the final concentration 0.5 μg / mL, and incubate at 4°C for 0.5h, as pretreatment group 1; The patient's peripheral blood after drug treatment is directly used i...
Embodiment 3
[0070] Evaluation test for tumor specificity of isolated T cells (sample is peripheral blood of renal cancer patients)
[0071] This example evaluates the ability of isolated T cells to recognize autologous tumor cells, using IFN-γ-secreting cells to account for CD8 + and CD4 + The proportion of cells (ie, CD8 + IFN-γ + %, CD4 + IFN-γ + %) for characterization, the higher the ratio, the higher the tumor specificity of T cells.
[0072] The specific operation includes the following steps:
[0073] (1) The test is divided into three groups, which are unsorted group, PD-1 negative group and PD-1 positive group;
[0074] (2) Take peripheral blood from kidney cancer patients and add PD-1 antibody for incubation (pretreatment);
[0075] (3) The peripheral blood obtained in step (2) is separated by the Ficoll density gradient method to obtain mononuclear cells;
[0076](4) Incubate the mononuclear cells obtained in step (3) with biotinylated anti-human IgG4 antibody at 25°C f...
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