Figure 6.

Cytotoxicity of FGFR4 chimeric antigen receptor (CAR) T cells towards Rhabdomyosarcoma (RMS) cells. (A) Schematic representation of the A8FR4 CAR construct. The CAR is composed of the sdAb A8 with CD8α signal peptide sequence and C-terminal myc-tag followed by the hinge and transmembrane (TM) domains of CD8α. Intracellular signaling domains are 4-1BB and CD3ζ and are followed by a streptavidin binding peptide (SBP). (B) CD8⁺ T cell transduction efficiencies of donor B and C were determined by flow cytometry analysis of BFP signal. (C) Cytotoxicity was determined by luciferase activity of Rh4 cells co-cultured for 72 h with effector T cells of donors B and C. Relative cell death was highest for Rh4-FR4wt cells incubated with A8FR4 CAR T cells at the indicated effector:target (E:T) cell ratios in both donors. In Rh4-FR4ko cells, non-specific cell killing was observed for the co-cultivation of all CAR T cells and the non-transduced CD8+ T cells. (D) Real-time cell death analysis of Rh4 cells co-cultured with effector T cells from donor B using xCELLigence RTCA DP. A8FR4-CAR T cells showed higher killing activity at the indicated E:T cell ratios in Rh4-FR4wt compared to non-specific CD19 CAR T cells or non-transduced CD8⁺ T cells. In Rh4-FR4ko cells, no specific cytotoxicity was observed. Horizontal lines within the curves indicate the SD of the duplicate wells used during the assay. The asterisks indicate the time of addition of the effector T cells.