3.2. ScFv from TR1419 CAR was proved to mediate tumor apoptosis in Jurkat cells
To explore whether scFv from TR1419 CAR could mediate tumor apoptosis through activation of the death receptor-dependent apoptotic pathway, we first constructed TR1419∆ζ lentivirus vector, which was composed of scFv from TR1419 fused to a CD8α hinge-transmembrane region, and truncated CD3ζ region (Fig. 2A). We generated TR1419-28BBζ or TR1419∆ζ-jurkat cells by lentiviral transduction, and the surface expression of scFv on the jurkat cells was measured by flow cytometry (Fig. 2B). Then, the xCELLigence RTCA DP was adopted to determine whether TR1419-28BBζ or TR1419∆ζ-jurkat cells could mediate tumor apoptosis. We have previously demonstrated with CD19-CAR that CAR-Jurkat cells have no ability to mediate the death of target antigen-positive tumor cells (Fig. S2). However, our results showed that TRAIL-R1-positive tumor cells were lysed by TR1419-28BBζ-jurkat cells and TR1419∆ζ-jurkat cells, and the specific cytotoxic effect can be blocked by the soluble target antigen TRAIL-R1 (Fig. 2C).
Next, TR1419-28BBζ-jurkat cells and TR1419∆ζ-jurkat cells were co-incubated separately with SW480 cell lines for 2 h at a 1:1 ratio, then the expression of AnnexinⅤ/7-AAD and caspase3 in SW480 cells was detected by flow cytometry. The percentages of 7-AADAnnexin V+ early (25.3%) and 7-AAD+Annexin V+ late (24.9%) apoptotic SW480 cells mediated by TR1419-28BBζ-jurkat cells and TR1419∆ζ-jurkat cells were significantly higher than the control (Fig. 2D). The percentages of Caspase3+ in SW480 cells mediated by TR1419-28BBζ-jurkat cells and TR1419∆ζ-jurkat cells were also significantly higher than the control. Meanwhile, the soluble target antigen blocked 100% of the above killing activity. Collectively, these results suggested scFv from TR1419 CAR can mediate TRAIL-R1-positive tumor apoptosis through activation of the death receptor-dependent apoptotic pathway.