Method for detecting CAR-T cell scFv affinity
An affinity and scfv technology, applied in the field of cell therapy, can solve the problems of inability to accurately detect the affinity of CAR-T cells, the inability to truly reflect the affinity of CAR-T cells to target antigen cells, and the neglect of steric hindrance effects
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Embodiment 1
[0081] Example 1 CD19 CAR-T cells and CD19 + Cell Affinity Assays
[0082] The preparation method of CD19 CAR-T cells used in this example is prepared by referring to Examples 1-3 in the patent literature (Invention name: Chimeric antigen receptor-modified T cells and their uses, application publication number: CN105177031A). Daudi cells (human Burkitt's lymphoma cells) and patient tumor cells were used to detect the target antigen CD19 by flow cytometry, and it was found that Daudi was a CD19 positive cell ( figure 2 Left), the proportion of CD19-positive tumor cells in the total mononuclear cells of the patient was 1.92% ( figure 2 Right), these two kinds of cells were used as the affinity detection of CD19 CAR-T cell scFv.
[0083] Affinity detection method: After fully mixing CD19 CAR-T cells with Daudi cells and patient CD19-positive tumor cells at a ratio of 3:1, they were incubated at 37°C and 5% CO 2 The cells were co-incubated in the incubator for 2 hours, and th...
Embodiment 2
[0103] Example 2 CD22 CAR-T cell scFv and CD22 + Cell Affinity Assays
[0104] The preparation method of CD22 CAR-T cells used in this example is the same as the preparation method of CD19 CAR-T cells in Example 1, the difference is only in the nucleotide and amino acid sequences of scFv. The scFv sequence refers to the patent "Mutatedanti-CD22 antibodies with increased affinity to CD22-expressing leukemia cells", patent number: US8809502B2. The Daudi cells (human Burkitt's lymphoma cells) and the patient's tumor cells were used to detect the target antigen CD22 by flow cytometry. Daudi was a CD22-positive cell, and the patient's CD22-positive tumor cells accounted for 47.82% of the total mononuclear cells ( Figure 7 ), the CD22 CAR-T cell scFv affinity detection test was carried out with these two kinds of cells.
[0105] Affinity detection method: CD22 CAR-T cells were fully mixed with Daudi cells and patient CD22 positive tumor cells at a ratio of 3:1, and incubated at 3...
Embodiment 3
[0112] Example 3 BCMA CAR-T cell scFv and different target antigen densities BCMA + Cell Affinity Assays
[0113] The preparation method of BCMA CAR-T cells used in this example is prepared by referring to Examples 1-5 in the patent document (Invention name: an anti-BCMA antibody and its application, application publication number CN112321713A). RPMI 8226 cells (human multiple myeloma cells) and NCI H929 cells (human myeloma cells) were tested for target antigen BCMA by flow cytometry, and the results showed that they were all BCMA-positive cells, but the expression density of BCMA in NCI H929 was significantly higher than that in RPMI 8226 ( Figure 12 ).
[0114] Affinity detection method: BCMA CAR-T cells were fully mixed with RPMI 8226 cells and NCI H929 cells at a ratio of 3:1, and incubated at 37°C, 5% CO 2 The cells were co-incubated in the incubator for 2 hours, and the cells were collected after 2 hours, and then the anti-CD3 flow cytometry antibody (FITC (isothioc...
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