Fig.5.TR1419-28BBζ CAR-T cells showed greater
antigen-specific expansion compared with TR1419-28ζ
CAR-T cells and TR1419-BBζ CAR-T cells. (A) Expression
of CD137 on TR1419CAR-T cells was detected by flow
cytometry cultured overnight on plate-bound recombinant human TRAIL-R1
at varying protein concentrations. (B, C) Cytotoxic activity of
TR1419-28ζ CAR, TR1419-BBζ CAR or
TR1419-28BBζ CAR T cells against TRAIL-R1 positive
tumor cell lines (SW480 and HCT116). The effector cells were co-cultured
for 6 h with target cells at E:T ratio of 14:1, 7:1 and 3:1,
respectively. Cytotoxicity was determined by Calcein AM release-based
cytotoxic cell assay. The analysis was performed using Students’ t
tests. Data reflected the mean ± SEM of three triplicate wells,*p < 0.05. (D) TR1419CAR T cell
proliferation indicated by division of CFSE was assessed by flow
cytometry. TR1419-28ζ CAR,
TR1419-BBζ CAR and TR1419-28BBζ CAR
T cells were stained with CFSE, respectively. Then,
TR1419CAR T cells were cultured with SW480 for 7 d,
and analyzed by flow cytometry. (E-H) Expression of PD-1 on
TR1419CAR T cells was analyzed by FACS after 7 days of
co-culture with tumor cells. TR1419CAR T cells were
classified as PD-1 negative, PD-1low and PD-1high expressing
sub-populations. The analyses were performed using Students’ t tests.
Data reflected the mean ± SEM of three independent experiments,*p < 0.05, **p < 0.01.
Discussion
In the present work, we demonstrated that TR1419-based
CAR-T cells had potent dual anti-tumor activities in vitro. On the one
hand, TR1419CAR can mediate tumor cell apoptosis
through activation of the death receptor-dependent apoptotic pathway. On
the other hand, TR1419CAR can effectively induce
T-cell mediated cytotoxicity by inducing multiple signals with the
binding of TRAIL-R1. Meanwhile, by comparing phenotype and function
between the second- and third-generation TR1419CAR-T
cells, the third-generation TR1419-28BBζ CAR-T cells
showed higher sensitivity to target antigen, proliferated more rapidly
and expressed higher levels of PD-1 upon antigen stimulation.
Previously, we developed fully human TR1419-IgG with
tumoricidal activity, but this antibody required additional crosslinking
antibodies to induce apoptosis in the target cells
[32]. We demonstrated that the scFv from
TR1419 designed into CAR structure can directly
mediate tumor apoptosis by binding to TRAIL-R1 in the absence of
additional crosslinking antibodies. The underlying mechanism of cell
apoptosis induced by scFv from TR1419 may be related
to the spatial structure of the TR1419CAR. The
specific mechanism still requires further research and investigation.
Currently, the clinical efficacy of CAR-T cells in hematological
malignancies is rarely achieved in solid tumors, and the factors
necessary for improving its efficacy are currently being determined
[33,34]. Combinations with other
treatments will be a promising strategy to improve the efficacy of CAR-T
cells on solid tumors [35]. In our
study, TR1419-based CAR-T cells had potent dual
anti-tumor activities. The induction of apoptotic signals from TR1 on
the target cells was equivalent to that of other therapies in this case.
However, the apoptotic signals from TR1 are targeted, unlike
chemotherapy and radiotherapy, without inducing localized immune cell
damage [36]. Moreover, TR1-mediated
tumor cell apoptosis can induce immunogenic cell death of tumor cells
which enhances antigen presentation and antitumor immune responses.
Although, the role of costimulatory signals in CAR-T development is
well-established, the optimal costimulatory domains for CAR-T cells
remains to be defined, and should be evaluated case-by-case in order to
fine-tune immunotherapy approaches
[37,38]. Literature reported that the
choice of 4-1BB signaling domain in CARs conferred improved selectivity
for higher tumor antigen density, reduced T cell exhaustion phenotype
and reduced basal T-cell activation
[39,40]. In our research, there was no
significant difference in phenotypes and function between the
TR1419-28ζ and TR1419-28BBζ CAR-T
cells. However, the third-generation TR1419-28BBζ
CAR-T cells showed higher sensitivity to target antigen and proliferated
more rapidly upon antigen stimulation. The incongruent results may have
the following reasons: 1) the various preparation methods and
experimental conditions, 2) the various scFv domains, 3) the various
other components of CARs.
There are some limitations in our study that requires further
acknowledgement. Further and more comprehensive studies are required to
confirm these findings. And additional in vivo experiments are needed to
verify the results of the above in vitro. Lastly, the safety issues
related to off-tumor toxicity also needs further studies to address. In
conclusion, we designed and demonstrated that
TR1419CAR-T cells can mediate dual tumor cell killing,
and the third-generation TR141928BBCAR-T cells
exhibited a higher sensitivity at low antigen, and proliferated more
rapidly upon antigen stimulation, compared to the second-generation
TR1419CAR-T cells. This dual killing mechanism
provides a novel strategy for other CARs design.