Figure 7.

Enhanced Lysis of CEA⁺ Tumor Cells Depends on the CD30-Blocking HRS3-scFv, but Not the CD30-Activating SH31355B scFv, Domain within the anti-CD30/CEA CAR

(A) Schematic representation of anti-CD30/CEA CARs. (B and C) T cells express the anti-CD30(HRS3)/CEA and anti-CD30(SH313B5)/CEA CAR with similar efficiency on the cell surface. T cells from the peripheral blood were grafted with anti-CD30/CEA CARs, 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) (C) of 5 healthy donors are shown. (D) Anti-CD30(HRS3)/CEA and anti-CD30(SH313B5)/CEA CAR T cells suppress CD30 expression with different efficiencies. Peripheral blood T cells were grafted with CARs as described above, and they were analyzed 24 h after transduction for the expression of CD30 by flow cytometry, utilizing anti-CD3 and anti-CD30 mAbs, respectively. Data represent mean values ± SD of 5 healthy blood donors. (E) Anti-CD30(SH313B)/CEA CAR and anti-CD30(HRS3)/CAR T cells mediated an anti-CEA CAR T cell attack with different efficiencies. Anti-CD30/CEA CAR T cells (7.5 × 10³ CAR T cells/well) were co-cultivated for 48 h with CEA⁺ LS174T or CEA⁻ Colo320 tumor cells (each 2.5 × 10⁴ cells/well). T cells with the anti-CEA CAR and cells without CAR (PBLs), respectively, served as controls. Viability of target cells was determined by the XTT assay and cytolysis was calculated. Data represent the mean values of technical replicates of a representative experiment of three ± SD. Significant differences were determined by the Student’s t test.