CAR-T Cell Therapy Target Proteins

Fig. 1 CAR expression were detected in CAR-T cells by FCM method using CEA protein (Cat. No. 11077-H08H). The median CAR+% is 33.7% and the range is 14.7%–43.2%.
PBMCs were activated by immobilized CD3 and CD28 antibodies. On day 2, T cells were infected with lentiviral vector in six-well plates with polybrene. After viral transduction, T cells were expanded by IL-2 for approximately 12–14 days to obtain enough cell dose. Quality control of cell products was performed by FCM. For the cell products, the CAR+ %, proportion of T cells (CD3+), NK cells (CD16+CD56+), NKT cells (CD3+CD16+CD56+), CD4/8 ratio, T memory cells, and checkpoint inhibitors (TIM-3, PD-1, LAG-3) on the T cell surface were measured on the day before CAR-T infusion. To ensure transfection, CAR+ % detection 4–5 days after viral transfection were also applied.

Fig. 2 CAR-positive rate was detected using Recombinant Human EGFR (from Sino Biological Inc.). The CAR-positive rate is relative to copy numbers of the retrovirus.
A human PBMC which had been collected after obtaining informed consent was infected two times with an undiluted solution or a 2-fold, 4-fold or 8-fold dilution of each virus solution to prepare a PBMC expressing anti-EGFR-CAR. Five days after the second virus infection, Recombinant Human EGFR in which the C-terminal end was His-tag-modified was added to the infected cells, and then a biotin-labeled anti-His-tag antibody was added. There after, the cells were stained with streptavidin-PE and a FITC labeled anti-Human CD8 antibody. Using a flow cytometer, the stained cells were subjected to measurement of a rate of PE-positive cells in FITC-positive cells, that is, a rate of cells positive for CAR that binds to EGFR in CD8-positive cells. Further, the infected cells were recovered 5 days after the second virus infection, and subjected to measurement of the number of copies of the virus incorporated into the genome.