Abstract
Tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAIL-R1) has limited expression in normal tissues but highly expression in a broad range of tumors, making it an attractive target for cancer immunotherapy. We have previously prepared a fully human monoclonal antibody targeting TRAIL-R1 (TR1419), which can specifically induce apoptosis in antigen-positive tumor cells. Here, we prepared the TR1419CAR-T cells using the single chain variable fragment (scFv) from TR1419, which were evaluated for the phenotypes and function. The TR1419CAR-T cells induced cytolysis of TRAIL-R1-positive tumor cells not only via activation of the death receptor-dependent apoptotic pathway, but also via T-cell mediated cytotoxicity. Furthermore, compared to the second-generation TR1419-28ζ and TR1419-BBζ CAR-T cells, the third-generation TR1419-28BBζ CAR-T cells had greater sensitivity to target antigen, exhibited a better proliferative ability, but showed slightly higher PD-1 expression after antigen stimulation. Altogether, TR1419CAR-T cells, especially TR1419-28BBζCAR-T cells could be a promising treatment strategy for TRAIL-R1 positive tumors.
Key words: TRAIL-R1, CAR-T, Apoptosis, Cytotoxicity, Third-generation
Introduction
Chimeric antigen receptors (CARs) are synthetic molecules composed of an antigen recognition domain of single chain variable fragment (scFv), a hinge region, a transmembrane domain, intracellular signaling region, costimulatory domains and a CD3ζ chain [1-3]. CAR-T cells have been considered as promising new modalities for adoptive cellular therapy [4]. Multiple clinical trials reported the CD19-target in the treatment of patient with malignancies [5,6]. However, there are still many encumbered challenges in solid tumor [7]. The key to the success of CAR-T cells therapy is the selection of an ideal target antigen, which should be expressed on the surface of cancer cells and shouldn’t or little be expressed on normal cells [8,9]. Therefore, finding an ideal target for CAR-T cells is one of the most important tasks for successful CAR-T therapy in solid tumors [10]. Additionally, CAR co-stimulation domains play an essential role in promoting the expansion and antitumor of CAR-T cells [11-13]. It has been reported that the phenotypic and functional differences of the second and third generation CAR-T cells are related to the selection of target antigen and the design of scFv structure [14].
TRAIL is a member of the tumor necrosis family (TNF) superfamily that interacts with its death receptors (TRAIL-R1/TR1/DR4) and induces apoptosis in a wide range of cancer cell types but not in normal cells [15]. However, some clinical trials have shown that TRAIL has little antitumor efficacy [16]. Some cancer cells are resistant to TRAIL based therapies [17]. One reason is that TRAIL appeared not to induce effective aggregation of its cognate death receptor, leading to insufficient apoptosis of some tumor cells [18]. To overcome this shortcoming, agonistic antibodies that target TRAIL-R1 have been developed for clinical application. Previously, we developed a fully human monoclonal antibody (TR1419) using the ISAAC ( immunospot array assay on a chip) technology and chimeric TransChromo (TC) mice with human immunoglobulin g and κ loci [19,20]. TR1419 could strongly induce apoptosis in TR1 positive cancer cell lines in the presence of crosslinking antibodies [21]. Moreover, TR1419can enhance TRAIL-induced apoptosis by activation of caspase-8 and upregulated expression of TR1 and increased phosphorylation of JNK [22,23].
In the present study, we first generated third-generation TR1419-28BBζ CAR-T cells, which were shown to induce cytolysis of target tumor cells not only via TR1-mediated apoptosis but also via CAR signal-induced cytolysis. Furthermore, we compared which CARs structure was the most appropriate for scFv from TR1419. The results showed that the third-generation TR1419-28BBζ CAR-T cells had higher sensitivity to target antigen, exhibited a better proliferative ability, but had a higher level of PD-1 expression after target antigen stimulation, compared to the second-generations TR1419-28ζ and TR1419-BBζ CAR-T cells. This study suggests that TR1419CAR-T cells could be a promising treatment strategy for TRAIL-R1 positive tumors, and the dual killing mechanism provides a novel optimizing strategy for other CARs design.
Materials and methods