3.1. TR1419-28BBζ CAR-T cells exhibited superior killing efficacy against TRAIL-R1 positive tumor cells
In order to generate TR1419 CAR-T cells, we first constructed the lentivirus vector incorporating a CD28 and CD137 co-stimulatory domain, and CD3ζ activation domain. The single-chain variable fragment (scFv) was derived from a fully human monoclonal antibody (TR1419), which has high affinity and specificity for TRAIL-R1. We generated human TR1419-28BBζ CAR-T or mock-T cells by lentiviral transduction of PBMCs derived from healthy donors. The surface expression of CAR on the T cells was measured by flow cytometry. As shown in Fig. 1A, flow cytometric analysis confirmed that the frequency of CAR expression was 39.3%. To characterize the phenotypes of the CAR-T cells, the cells were harvested for flow cytometric detection of CD45RA, CCR7, PD-1, LAG-3 and TIM-3 by day 7 after transfection. 45%-55% T cells were naïve T cells (CCD45RA+CCR7+), and the proportions of the other three subtypes (central memory T cells, effector memory T cells, and effector T cells) were comparable (Fig.1B). No significant differences of immunoinhibitory molecule expression, PD-1, LAG-3 and TIM-3 were observed between TR1419-28BBζ CAR-T cells and mock T cells, indicating the phenotypes of T cells irrespective of gene transfection (Fig. 1C)
To determine whether TRAIL-R1 positive cancer cell lines were susceptible to TR1419-28BBζ CAR-T-cell-mediated lysis in vitro, we performed cytotoxicity assays using engineered T cells as effector cells, and different cancer cells as target cells. The TRAIL-R1 expression in different cancer cells was validated by flow cytometry (Fig. S1). Compared with mock-transduced T cells, TR1419-28BBζ CAR-T cells displayed significantly higher specific killing activity against TRAIL-R1 positive SW480, HCT116 and MDA-MB-231 cells at all E:T ratios, and accompanied by an increased IFN-γ and Granzyme B secretion (Fig. 1D-F). As for the TRAIL-R1 negative 293T cells, TR1419-28BBζ CAR-T cells had no significant killing effect and secretion of effector cytokines, compared with the mock T cells (Fig. 1D-F).