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