Characterization of CAR-transduced T cell cytotoxic potential

Characterization of CAR transduced T cell cytotoxic potential: Plate bound activation assay for cytotoxicity:

Media: RPMI-10% Human serum 1% anti-anti (RPMI-10% FBS and 1% anti-anti can also be used) 
Cells: 1 million CAR⁺ T cells from HER2-CAR transduced bulk PBMC, CD8+CD161^neg and CD8⁺CD161⁺ groups

(Calculate the cell number based on CAR transduction efficiency)

1. Coat a 96 well or a 24 well flat bottom non-tissue culture plate with HER2-Fc or CD-19Fc antibodies.
2. Dilute the antibodies 1ug/mL in sterile water and add 50-100ul to the respective wells in triplicates.
3. Wrap the plate with saran wrap and leave the antibody coated plate at 4C overnight.
4. Next day, aspirate the antibody solution, add 100-200ul media (RPMI-10% Human serum and 1% anti-anti) per well and incubate at 37°C for 30 min to an hour.
5. Aspirate the media.
6. For a 96 well plate, do not plate more than 200K cells. 50K-200K cells in 50-200ul media is ideal. For a 24 well plate, you can plate up to one million cells in one mL media.
7. Add CAR+ T cells from each group to the respective wells in triplicates against target proteins at one million per mL volume of media. No cytokines are added.
8. Leave the plate at 37°C for 48-72 hr or for an appropriate time based on the experiment.
9. Analyze the cells for activation and cytotoxicity by flow cytometry.

Repeated antigenic stimulation assay:

Media: DMEM-10% FBS 1% anti-anti

Cells: Human pancreatic cancer cell line CFPAC1

1 million CAR⁺ T cells from HER2-CAR transduced bulk PBMC, CD8+CD161^neg and CD8⁺CD161⁺ groups

(Calculate the cell number based on CAR transduction efficiency) 
Cytokines: hIL-7 (10ng/mL), hIL-15 (5ng/mL) and hIL-21 (30 ng/mL)

Day 0:

1. Grow human pancreatic cancer cell line CFPAC1 in DMEM (10% FBS 1% anti-anti).
2. When the cells are 60-70% confluent, trypsinize the cells, wash in PBS.
3. Spin down the cells (400g for 5 min), aspirate supernatant and count.
4. Resuspend cells to a concentration of 1.0x10⁶ cells/mL ofmedia
5. Place 1 mL of the cell suspension per well in a 12-well tissue culture plate (1.0x10⁶ cells/well)
6. Make sure to seed at least 1 extra well (will be used for determining CFPAC1 cell number per well on Day 1)

Day 1:

1. Trypsinize extra well(s) of CFPAC1 and count the number of cells
2. Using the number of CFPAC1 cells per well on this day, co-culture effector cells (CAR⁺T cells) at 1:4 ratio of effector:target in a final volume of 1.5-2 mL of DMEM per well. Use media that the tumors thrive on.
3. For example, add 0.25x10⁶ CAR⁺ T cells for 1.0x10⁶ CFPAC1 target cells.
4. Add IL-7 (10ng/mL), IL-15 (5ng/mL) and IL-21 (30 ng/mL) cytokines to the media.
5. After 2-3 days, move the effector CAR⁺T cells to tissue culture plate with fresh targets plated similarly as Day 0.
6. Repeat steps of Day 0 and Day 1 every 2-3 days for three-four antigenic stimulations or more.
7. Analyze the cells by flow cytometry for activation, cytotoxicity and exhaustion markers.

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