Figure 6.

Enhanced Lysis of CEA⁺ Tumor Cells Depends on Simultaneous CD30, but Not CD25, Targeting

(A) Schematic representation of anti-CD30/CEA and anti-CD25/CEA CARs. (B and C) T cells express the anti-CD30/CEA and anti-CD25/CEA CAR with similar efficiency on the cell surface. T cells from the peripheral blood were grafted with anti-CD30/CEA and anti-CD25/CEA CARs, as described in the Materials and Methods, and CAR expression was recorded by flow cytometry utilizing anti-CD3 and anti-human IgG antibodies, respectively. Numbers of CD3⁺CAR⁺ T cells and mean fluorescence intensities (mfis) of CAR expression were determined. A typical dot plot (B) and mean values of CAR expression (mfis) of 5 healthy donors are shown (C). (D and E) Anti-CD30/CEA and anti-CD25/CEA CAR T cells persist similarly but expand differentially during prolonged cultivation. Anti-CEA, anti-CD30/CEA, and anti-CD25/CEA CAR T cells were cultivated in the presence of 100 U/mL IL-2 and recorded for viable CAR⁺ T cells by flow cytometry (D). The total number of viable cells was recorded by cell counting (E). Dead cells were excluded by trypan blue exclusion. A representative experiment of two is shown. (F) T cells with the anti-CD25/CEA, anti-CD30/CEA, and anti-CEA CAR, respectively, and non-modified T cells (w/o) for control (5 × 10³ CAR T cells/well), were co-cultivated for 48 h with CEA⁺ LS174T and CEA⁻ Colo320 tumor cells (each 2.5 × 10⁴ cells/well). Viability of target cells was determined by the XTT assay and cytolysis was calculated. Data represent mean values of technical replicates of a representative experiment of two ± SD. Statistical analysis was performed using the Student’s t test.