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).