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\begin{document}

\title{TRAIL-R1-targeted chimeric antigen receptor T cells exhibit dual
antitumor effects}



\author[1]{AiShun Jin}%
\author[1]{Yaru Nai}%
\author[1]{Li Du}%
\author[2]{Meiying Shen}%
\author[1]{Tingting Li}%
\author[1]{Jingjing Huang}%
\author[1]{xiaojian  Han}%
\author[1]{Feiyang Luo}%
\author[2]{Da Pang}%
\affil[1]{Chongqing Medical University}%
\affil[2]{Harbin Medical University Cancer Hospital}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{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 TR1419-28\selectlanguage{greek}ζ \selectlanguage{english}and TR1419-BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells, the
third-generation TR1419-28BB\selectlanguage{greek}ζ \selectlanguage{english}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 TR1419-28BB\selectlanguage{greek}ζ\selectlanguage{english}CAR-T cells could be a
promising treatment strategy for TRAIL-R1 positive tumors.%
\end{abstract}%



\sloppy


\textbf{TRAIL-R1-targeted chimeric antigen receptor T cells exhibit dual
antitumor effects}

Yaru Nai \textsuperscript{a, 1}, Li Du\textsuperscript{a, 1}, Meiying
Shen\textsuperscript{b}, Tingting Li\textsuperscript{a}, Jingjing
Huang\textsuperscript{a}, Xiaojian Han\textsuperscript{a}, Feiyang
Luo\textsuperscript{a}, Da Pang\textsuperscript{b}, Aishun
Jin\textsuperscript{a, *}

\textsuperscript{a} \emph{Chongqing Key Laboratory of Basic and
Translational Research of Tumor Immunology, Chongqing Medical
University, Chongqing 400016, China}

\textsuperscript{b} \emph{Department of Breast Surgery, Harbin Medical
University Cancer Hospital, Harbin 150081, China}

* Corresponding authors at: Chongqing Key Laboratory of Basic and
Translational Research of Tumor Immunology, Chongqing Medical
University, No.1 Medical College Road, Yuzhong District, Chongqing
400016, People's Republic of China.

\emph{E-mail addresses} : aishunjin@cqmu.edu.cn.

\textsuperscript{1} These authors contributed equally to this research.

\textbf{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 (TR1\textsuperscript{419}), which
can specifically induce apoptosis in antigen-positive tumor cells. Here,
we prepared the TR1\textsuperscript{419}CAR-T cells using the single
chain variable fragment (scFv) from TR1\textsuperscript{419}, which were
evaluated for the phenotypes and function. The
TR1\textsuperscript{419}CAR-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
TR1\textsuperscript{419}-28\selectlanguage{greek}ζ \selectlanguage{english}and TR1\textsuperscript{419}-BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T
cells, the third-generation TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}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, TR1\textsuperscript{419}CAR-T cells,
especially TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ\selectlanguage{english}CAR-T cells could be a
promising treatment strategy for TRAIL-R1 positive tumors.

Key words: TRAIL-R1, CAR-T, Apoptosis, Cytotoxicity, Third-generation

\textbf{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 CD3\selectlanguage{greek}ζ \selectlanguage{english}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 (TR1\textsuperscript{419}) using the ISAAC (
immunospot array assay on a chip) technology and chimeric TransChromo
(TC) mice with human immunoglobulin g and \selectlanguage{greek}κ \selectlanguage{english}loci {[}19,20{]}.
TR1\textsuperscript{419} could strongly induce apoptosis in TR1 positive
cancer cell lines in the presence of crosslinking antibodies {[}21{]}.
Moreover, TR1\textsuperscript{419}can 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
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}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
TR1\textsuperscript{419}. The results showed that the third-generation
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}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 TR1\textsuperscript{419}-28\selectlanguage{greek}ζ \selectlanguage{english}and
TR1\textsuperscript{419}-BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells. This study suggests that
TR1\textsuperscript{419}CAR-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.

\textbf{Materials and methods}

\emph{2.1. Plasmid construction and lentivirus preparation}

The target TR1\textsuperscript{419}CAR consisted of
TR1\textsuperscript{419} scFv, CD28 or 4-1BB costimulatory domains, and
CD3-\selectlanguage{greek}ζ \selectlanguage{english}signaling domain {[}24,25{]}. They were obtained by overlap PCR
amplification, PCR products were clone into lentivirus vector pWPXL and
verified by enzymatic digestion and sequencing. To produce the
lentivirus supernatant, 8\selectlanguage{ngerman}×10\textsuperscript{5} HEK-293T cells were
plated in 6-well. After 24h, 293T cells were transfected with pMD.2G
encoding VSV-G envelope, pSPAX2 lentivirus plasmid, and the CAR-pWPXL
plasmids by using xfect Transfection Reagent (Takara), according to the
manufacturer's instructions. The lentivirus supernatants were collected
and filtered with 0.45 \selectlanguage{greek}μ\selectlanguage{english}M filter at 48h after transfection.

\emph{2.2. Cell lines and cell culture conditions}

SW480, HCT116, K562 and huh7 were obtained from ATCC. Human Jurkat cell
line was maintained in our laboratory. These cells were cultured in RPMI
1640 medium (Gibco, Invitrogen, Carlsbad, CA) supplemented with 10\%
fetal bovine serum (FBS) (Biological Industries, ISR). Human breast
cancer cell lines MDA-MB-231 and HS578T were obtained from ATCC and
cultured in Dulbecco's modified Eagle medium (Gibco, Invitrogen,
Carlsbad, CA) supplemented with 10\% fetal bovine serum (FBS)
(Biological Industries, ISR). The HEK-293T cell lines were purchased
from ATCC and maintained in complete growth medium (Gibco invitrogen),
containing with 10\% fetal bovine serum (FBS) (Biological Industries,
ISR), 2mM L-glutamine (Gibco, Invitrogen) and 1mM sodium pyruvate
solution (Gibco, Invitrogen). All above cells were cultured in a
humidified atmosphere containing 5\% CO\textsubscript{2} at 37.

\emph{2.3. Human CAR-T cells generation}

Human peripheral blood mononuclear cells (PBMCs) samples were obtained
from healthy donors according to the manufacturer's protocol. Written
informed consents were obtained from all donors. PBMC were stimulated
with anti-CD3/CD28 for 48 h. Then, the activated T cells were infected
with the CAR-encoding lentiviral supernatant. 24h after lentiviral
infection, the lentiviral supernatant was replaced with fresh medium
containing RPMI-1640 and supplemented with 10\% FBS (Gibco), 200U/ml
IL-2, 25 mM HEPPES, 55 \selectlanguage{greek}μ\selectlanguage{english}M 2-M, 100 U/ml penicillin and 100 \selectlanguage{greek}μ\selectlanguage{english}g/ml
streptomycin. The infected T cells were cultured for 7 days.

\emph{2.4. Flow cytometry}

For cell-surface staining, the lentivirus-infected T cells were
harvested and washed with phosphate buffered saline (PBS) followed by
incubation with 1ug/ml recombinant protein TRIL-R1-Fc (R\&D Systems,
Minneapolis, MN) at room temperature for 20 min. After being washed, the
cells were incubation with 1ug/ml APC conjugated anti-human IgG
secondary antibodies and other cocktail antibodies (Biolegend) at room
temperature for 15 min in the dark. After being washed with PBS, the
cells were analyzed with a BD celesta (BD Biosciences) software by flow
cytometry. The following monoclonal antibodies were used with the
indicated subtypes: APC-lableled human IgG-Fc (clone HP6017),
Percy5.5-lableled anti-CD4 (clone RPA-T4), Alex700-lableled anti-CD8
(clone HIT8a), BV421-lableled anti-CCR7 (clone G043H7), PE-lableled
anti-CD45RO (clone UCHL1), AF488-lableled anti PD-1(clone EH12.2H7),
BV605-lableled anti-LAG-3 (clone 11C3C65), BV785-lableled anti-TIM-3
(clone F38-2E2). All sample data analyzed was done on[?]10,000 events
using the FlowJo V 10 data analysis software.

\emph{2.5. Killing activity assay}

The ability of CAR-T cells to kill tumor cells was determined via
Calcein AM (CAM, Dojindo) release-based cytotoxic cell assay {[}26{]}.
Briefly, target cells were stained with 10\selectlanguage{greek}μ\selectlanguage{english}M CAM for 30 minutes at 37,
followed by terminating the reaction with addition of Durbe phosphate
buffered saline without calcium (DPBS) buffer. The target tumor cells
were washed five times with DPBS. Non-transduced T cells were used to
normalize the percentage of CAR-positive cells. Then, CAR-T cells and
target cells were plated in a 96-well microplate at various
effector/target (E:T) ratios. After incubation for 6h at 37 \selectlanguage{ngerman}°C, the 50ul
supernatant were transferred to 96-black plates to measure fluorescence
intensity (FI) at 485\selectlanguage{english} nm excitation and 520\selectlanguage{english} nm emission wavelengths. The
percentage of cytotxicity was calculated according to the following
formula: lysis\% = (test release--spontaneous release)/(maximal
release--spontaneous release)x100\%.

The ability of CAR-T cells to kill 293T cells was detected via Real Time
Cell Analysis (RTCA, ACEA). One prior day to seeded approximately
1*10\textsuperscript{4} 293T cells on 16 E-plate, the cells were grown
to mid-logarithmic growth phase. Non-transduced T cells were used to
normalize the percentage of CAR-positive cells, and CAR-T cells were
added to the cultures at indicated effector-to-target ratio. RTCA DP
analyzer was used to monitor real-time target cell growth, and the
results were analyzed by RTCA software {[}27{]}. The percentage of
cytotxicity was calculated according to the following formula: lysis\% =
{[}1 -- (experiments/empty culture){]} x100\%.

\emph{2.6. Enayme-linked immunosorbent assay}

The CAR-T cells and target tumor cells were co-cultured at various
effector/target (E:T) ratios in a 96-well plate for 24hr at 37\selectlanguage{english}. The
culture supernatants were harvested and subjected to ELISA to detect
INF-\selectlanguage{greek}γ \selectlanguage{english}and Granzyme B, according to the manufacturer's instructions.
Every value represented the mean of triplicate wells.

\emph{2.7. Tumor cells apoptosis assay}

CAR-T cells and target cells were incubated for 2h at 37\selectlanguage{ngerman}°C, and target
cell apoptosis was assayed using an Annexin V/7-AAD Apoptosis Detection
Kit (Biolegend), according to the manufacturer's instructions. Target
cell caspase-3 activity was detected used a caspase-3 activity detection
assay kit (BD). In brief, the cells were washed twice with cold PBS,
followed by fixing and permeabilizing in Cytofix/CytoPerm for 20 min on
ice (BD Biosciences). Subsequent staining was performed with 1×PermWash
as the staining and wash buffer and incubated for 15 min at room
temperature. Washed cells were analyzed with a BD celesta (BD
Biosciences) software by flow cytometry.

\emph{2.8. Statistical analysis}

Statistical analyses in this study were performed with Graph Pad Prism
software 8.0.2 version. Data are presented as the mean ± SEM. When there
were two experiment groups, Student's t-test was used or ANOVA analysis
for multiple comparisons. \emph{P\textless{}0.05} were considered
statistically significance.

\textbf{Results}

\selectlanguage{greek}\emph{3.1. TR1\textsuperscript{419}-28BBζ CAR-T cells exhibited superior
killing efficacy against TRAIL-R1 positive tumor cells}\selectlanguage{english}

In order to generate TR1\textsuperscript{419} CAR-T cells, we first
constructed the lentivirus vector incorporating a CD28 and CD137
co-stimulatory domain, and CD3\selectlanguage{greek}ζ \selectlanguage{english}activation domain. The single-chain
variable fragment (scFv) was derived from a fully human monoclonal
antibody (TR1\textsuperscript{419}), which has high affinity and
specificity for TRAIL-R1. We generated human
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}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 na\selectlanguage{ngerman}ïve T cells
(CCD45RA\textsuperscript{+}CCR7\textsuperscript{+}), 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 TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}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 TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}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,
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}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-\selectlanguage{greek}γ \selectlanguage{english}and Granzyme B secretion (Fig. 1D-F). As for the TRAIL-R1 negative
293T cells, TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells had no
significant killing effect and secretion of effector cytokines, compared
with the mock T cells (Fig. 1D-F).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Fig.1.}TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells had specific
killing to tumor cells. (A) CAR construct with humanized anti-TRAIL-R1
scFv, CD8a hinge, CD28 transmembrane domain and intracellular
co-stimulatory domain, CD137 intracellular co-stimulatory domain, and
intracellular CD3\selectlanguage{greek}ζ \selectlanguage{english}activation domain. (B) Expression rate of the CAR on
T cells was confirmed by flow cytometry on day 7 following lentiviral
transfection. Mock T cells were served as a negative control. (C-D)
Expression of inhibitory receptors and subtypes on CAR-T cells was
examined by flow cytometry,
CD45RA\textsuperscript{+}CCR7\textsuperscript{+}: na\selectlanguage{ngerman}ïve T cells,
CD45RA\textsuperscript{-}CCR7\textsuperscript{+}: central memory T
cells, CD45RA\textsuperscript{-}CCR7\textsuperscript{-}: effector memory
T cells, CD45RA\textsuperscript{+}CCR7\textsuperscript{-}: effector T
cells. (E) Cytotoxic activity of TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR- or
mock transduced T cells against TRAIL-R1 positive tumor cell lines. The
effector cells were co-cultured for 6 h with target cells at E:T ratio
of 10:1, 5:1 and 2.5:1, respectively. Cytotoxicity was determined by
Calcein AM release-based cytotoxic cell assay. (F-G) IFN-\selectlanguage{greek}γ \selectlanguage{english}and Granzyme
B production by TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells or
mock-transduced T cells was detected by ELISA when co-cultured with the
indicated cells for 24 h. The analyses were performed using Students' t
tests. Data reflected the mean \selectlanguage{ngerman}± SEM of three separate
experiments.\emph{*p} \textless{} 0.05, \emph{**p} \textless{} 0.01 and
\emph{***p}\textless{} 0.001.

\emph{3.2. ScFv from TR1\textsuperscript{419} CAR was proved to mediate
tumor apoptosis in Jurkat cells}

To explore whether scFv from TR1\textsuperscript{419} CAR could mediate
tumor apoptosis through activation of the death receptor-dependent
apoptotic pathway, we first constructed TR1\textsuperscript{419}\selectlanguage{english}[?]\selectlanguage{greek}ζ
\selectlanguage{english}lentivirus vector, which was composed of scFv from
TR1\textsuperscript{419} fused to a CD8\selectlanguage{greek}α \selectlanguage{english}hinge-transmembrane region, and
truncated CD3\selectlanguage{greek}ζ \selectlanguage{english}region (Fig. 2A). We generated
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}or TR1\textsuperscript{419}[?]\selectlanguage{greek}ζ-\selectlanguage{english}jurkat
cells by lentiviral transduction, and the surface expression of scFv on
the jurkat cells was measured by flow cytometry (Fig. 2B). Then, the
xCELLigence RTCA DP was adopted to determine whether
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}or TR1\textsuperscript{419}[?]\selectlanguage{greek}ζ-\selectlanguage{english}jurkat
cells could mediate tumor apoptosis. We have previously demonstrated
with CD19-CAR that CAR-Jurkat cells have no ability to mediate the death
of target antigen-positive tumor cells (Fig. S2). However, our results
showed that TRAIL-R1-positive tumor cells were lysed by
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ-\selectlanguage{english}jurkat cells and
TR1\textsuperscript{419}[?]\selectlanguage{greek}ζ-\selectlanguage{english}jurkat cells, and the specific cytotoxic
effect can be blocked by the soluble target antigen TRAIL-R1 (Fig. 2C).

Next, TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ-\selectlanguage{english}jurkat cells and
TR1\textsuperscript{419}[?]\selectlanguage{greek}ζ-\selectlanguage{english}jurkat cells were co-incubated separately
with SW480 cell lines for 2 h at a 1:1 ratio, then the expression of
AnnexinV/7-AAD and caspase3 in SW480 cells was detected by flow
cytometry. The percentages of 7-AAD\textsuperscript{-}Annexin
V\textsuperscript{+} early (25.3\%) and 7-AAD\textsuperscript{+}Annexin
V\textsuperscript{+} late (24.9\%) apoptotic SW480 cells mediated by
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ-\selectlanguage{english}jurkat cells and
TR1\textsuperscript{419}[?]\selectlanguage{greek}ζ-\selectlanguage{english}jurkat cells were significantly higher than
the control (Fig. 2D). The percentages of Caspase3\textsuperscript{+} in
SW480 cells mediated by TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ-\selectlanguage{english}jurkat cells and
TR1\textsuperscript{419}[?]\selectlanguage{greek}ζ-\selectlanguage{english}jurkat cells were also significantly higher
than the control. Meanwhile, the soluble target antigen blocked 100\% of
the above killing activity. Collectively, these results suggested scFv
from TR1\textsuperscript{419} CAR can mediate TRAIL-R1-positive tumor
apoptosis through activation of the death receptor-dependent apoptotic
pathway.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Fig.2.} scFv from TR1\textsuperscript{419} CAR mediated tumor
cell apoptosis by binding with TRAIL-R1. (A) Schematic representation of
TR1\textsuperscript{419}[?]\selectlanguage{greek}ζ \selectlanguage{english}and the lentiviral vector. The construct
consisted of the extracellular portion of anti-TRAIL-R1 scFv, linking
via a CD8\selectlanguage{greek}α \selectlanguage{english}hinge-transmembrane domains to the truncated CD3\selectlanguage{greek}ζ \selectlanguage{english}molecule.
(B) The expression of TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}and
TR1\textsuperscript{419}[?]\selectlanguage{greek}ζ \selectlanguage{english}in Jurkat cells was detected by flow
cytometry. (C) Cytotoxicity of TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ- \selectlanguage{english}and
TR1\textsuperscript{419}[?]\selectlanguage{greek}ζ -\selectlanguage{english}Jurkat cells against SW480 in the absence or
in the presence of soluble TRAIL-R1 protein was determined by RTCA. The
analysis was performed using Students' t tests. Data reflected the mean
\selectlanguage{ngerman}± SEM of triplicate wells, \emph{***p} \textless{} 0.01. (D) SW480 were
fixed and labeled with annexin V and 7-AAD and then analyzed by flow
cytometry to identify apoptotic cells (annexin
V\textsuperscript{+}/7-AAD\selectlanguage{english}\textsuperscript{-} and annexin
V\textsuperscript{+}/7-AAD\textsuperscript{+}), after incubation with
effector cells for 2h. (E) The expression of caspase3 protein in SW480
analyzed by flow cytometry, after incubation with effector cells for 2h.

\selectlanguage{greek}\emph{3.3. TR1\textsuperscript{419}-28BBζ CAR-T cells exhibited dual
antitumor effects}\selectlanguage{english}

To further investigate whether TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T
cells can mediate tumor apoptosis through binding to death receptor
TRAIL-R1, we first generated TRAIL-R1 (full length) and TRAIL-R1
(truncation) lentivirus vectors. The TRAIL-R1 (full length) was composed
of full TRAIL-R1, and the death domain of TRAIL-R1 was truncated in the
TRAIL-R1 (truncation) {[}28{]} (Fig. 3A). We produced 293T (TRAIL-R1
full length) and 293T (TRAIL-R1 trunc) cells by lentiviral transfecting
the associated plasmids (Fig. 3B).

Next, we generated TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells and
TR1\textsuperscript{419}[?]\selectlanguage{greek}ζ-\selectlanguage{english}T cells by lentiviral transduction, which
transfection rates were detected by flow cytometry (Fig. 3C). Then, we
performed cytotoxicity assays using TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T
cells and TR1\textsuperscript{419}[?]\selectlanguage{greek}ζ-\selectlanguage{english}T cells as effector cells, and
different 293T cells as target cells. As for the TRAIL-R1 negative 293T
cells, TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells and
TR1\textsuperscript{419}[?]\selectlanguage{greek}ζ-\selectlanguage{english}T cells had no significant killing effect and
secretion of effector cytokines, compared with the mock T cells (Fig.
3D-F). The TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells and
TR1\textsuperscript{419}[?]\selectlanguage{greek}ζ-\selectlanguage{english}T cells effectively killed the 293T (TRAIL-R1
full length) cells, whereas TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells
displayed significantly higher specific killing compared to
TR1\textsuperscript{419}[?]\selectlanguage{greek}ζ-\selectlanguage{english}T cells (Fig. 3D). Meanwhile, the data showed
that much higher levels of IFN-\selectlanguage{greek}γ \selectlanguage{english}and Granzyme B were produced by
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells compared to
TR1\textsuperscript{419}[?]\selectlanguage{greek}ζ-\selectlanguage{english}T cells when co-culturing with 293T (TRAIL-R1
full length) cells. However, no significant difference was found in
cytokine secretion between TR1\textsuperscript{419}[?]\selectlanguage{greek}ζ-\selectlanguage{english}T cells and mock T
cells (Fig. 3E-F). As for the 293T (TRAIL-R1 trunc) cells,
TR1\textsuperscript{419}[?]\selectlanguage{greek}ζ-\selectlanguage{english}T cells had no significant killing effect and
secretion of effector cytokines compared with the mock T cells, but
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells displayed significantly
killing effect and IFN-\selectlanguage{greek}γ \selectlanguage{english}and Granzyme B production (Fig. 3D-F). The
results above suggested that TR1\textsuperscript{419}-CAR-T cells can
not only effectively induce T-cell mediated cytotoxicity toward tumor
cells, but also mediate tumor cell apoptosis through activation of the
death receptor-dependent apoptotic pathway, indicating dual anti-tumor
activity.

.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Fig.3.}TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells mediated dual
tumour cell killing. (A) Schematic representation of TRAIL-R1 (full
length) and TRAIL-R1 (truncation). TRAIL-R1 (full length) comprised the
extracellular region, the transmembrane domain, and the intracellular
region with death domain. TRAIL-R1 (truncation) comprised the
extracellular region, the transmembrane domain, and the intracellular
region without death domain, SP: signal peptide, TM: transmembrane
domain, DD: death domain. (B) 293T cells were transfected with the
lentiviral expression TRAIL-R1 trunc and TRAIL-R1 full length plasmids,
respectively. Then, the expression of TRAIL-R1 in 293T cells was
determined by flow cytometry. (C) Mock (untransduced),
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells or
TR1\textsuperscript{419}[?]\selectlanguage{greek}ζ-\selectlanguage{english}T cells were evaluated by flow cytometry for
scFv expression. (D) Cytotoxic activity of
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR- and TR1\textsuperscript{419}[?]\selectlanguage{greek}ζ-\selectlanguage{english}T
cells against 293T, 293T (TRAIL-R1 trunc) and 293T (TRAIL-R1 full
length) was determined by RTCA assay. (E, F) IFN-\selectlanguage{greek}γ \selectlanguage{english}and Granzyme B
production by TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells or
TR1\textsuperscript{419}[?]\selectlanguage{greek}ζ-\selectlanguage{english}T cells was detected by ELISA when
co-cultured with the indicated cells for 24 h. The analyses were
performed using Students' t tests. Data reflected the mean \selectlanguage{ngerman}± SEM of
three triplicate wells. \emph{*p} \textless{} 0.05,
\emph{**p}\textless{} 0.01 and \emph{***p} \textless{} 0.001.

\subsection*{\texorpdfstring{\emph{3.4. The costimulatory domains did not
influence the phenotypes of TR1\textsuperscript{419}-CAR-T
cells}}{3.4. The costimulatory domains did not influence the phenotypes of TR1419-CAR-T cells}}

{\label{the-costimulatory-domains-did-not-influence-the-phenotypes-of-tr1419-car-t-cells}}

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 {[}29,30{]}, which
suggested that we should select the most appropriate CARs based on our
own scFv structure. To explore which CARs structure is the most
appropriate for scFv from TR1\textsuperscript{419}, we first constructed
CD28-containing (TR1\textsuperscript{419}-28\selectlanguage{greek}ζ\selectlanguage{english}) and 4-1BB-containing
(TR1\textsuperscript{419}-BB\selectlanguage{greek}ζ\selectlanguage{english}) TR1\textsuperscript{419}-CARs (Fig. 4A).
For the following studies, we generated TR1\textsuperscript{419}-28\selectlanguage{greek}ζ,
\selectlanguage{english}TR1\textsuperscript{419}-BB\selectlanguage{greek}ζ \selectlanguage{english}and TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T
cells by lentiviral transduction. The surface expression of CAR on the T
cells was measured by flow cytometry. Three
TR1\textsuperscript{419}-CARs showed comparable transfection rates and
mean fluorescence intensity (Fig. 4B). Meanwhile,
TR1\textsuperscript{419}-28\selectlanguage{greek}ζ, \selectlanguage{english}TR1\textsuperscript{419}-BB\selectlanguage{greek}ζ \selectlanguage{english}and
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells exhibited similar cell
surface T cell phenotypes, of which approximately 60\% were Tn (Fig.
4C). CD8\textsuperscript{+}TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells
showed higher levels of PD-1 and LAG-3 expression than
CD8\textsuperscript{+}TR1\textsuperscript{419}-28\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells. However,
CD4\textsuperscript{+} TR1\textsuperscript{419}-28\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells showed
higher levels of TIM-3 expression than
CD4\textsuperscript{+}TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells (Figure
4D). (Fig. 4D).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

\textbf{Fig.4.} The costimulatory domains did not impact
TR1\textsuperscript{419}CAR-T phenotypes. (A) Diagram of the lentiviral
expression cassette with the second generation
TR1\textsuperscript{419}-CARs containing the humanized scFv
(TR1\textsuperscript{419}) targeting TRAIL-R1, with a CD8\selectlanguage{greek}α \selectlanguage{english}hinge domain,
a CD28 transmembrane domain, a cytoplasmic CD28 or 4-1BB costimulatory
domain, and a cytolytic CD3\selectlanguage{greek}ζ \selectlanguage{english}domain. (B) Mock (untransduced),
TR1\textsuperscript{419}-28\selectlanguage{greek}ζ, \selectlanguage{english}TR1\textsuperscript{419}-BB\selectlanguage{greek}ζ \selectlanguage{english}or
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR T cells were evaluated by flow
cytometry for scFv expression to detect lentiviral transduction of CARs.
(C) Representative CAR-T cell phenotyping plot based on CD45RA and CCR7.
CD45RA\textsuperscript{+}CCR7\textsuperscript{+}: na\selectlanguage{ngerman}ïve T cells,
CD45RA\textsuperscript{-}CCR7\textsuperscript{+}: central memory T
cells, CD45RA\textsuperscript{-}CCR7\textsuperscript{-}: effector memory
T cells, CD45RA\textsuperscript{+}CCR7\textsuperscript{-}: effector T
cells. (D) Quantification of inhibitory molecules (PD-1, LAG-3 and
TIM-3) expression on CD4\textsuperscript{+}CAR and
CD8\textsuperscript{+}CAR T cells on day 7 following lentiviral
transfection. The analyses were performed using Students' t tests. Data
reflected the mean ± SEM of three independent experiments,
\emph{*p}\textless{} 0.05, \emph{**p} \textless{} 0.01.

\selectlanguage{greek}\emph{3.5. TR1\textsuperscript{419}-28BBζ CAR-T cells proliferated more
rapidly than TR1\textsuperscript{419}-28ζ CAR-T cells and
TR1\textsuperscript{419}-BBζ CAR-T cells upon antigen stimulation}\selectlanguage{english}

To explore the functional differences among
TR1\textsuperscript{419}-28\selectlanguage{greek}ζ, \selectlanguage{english}TR1\textsuperscript{419}-BB\selectlanguage{greek}ζ \selectlanguage{english}and
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells, we first examined the
expression of CD137 in TR1\textsuperscript{419}CAR-T cells after 24
hours of stimulation with different concentrations of plate-bound
recombinant human TRAIL-R1 protein. While TR1\textsuperscript{419}-28\selectlanguage{greek}ζ,
\selectlanguage{english}TR1\textsuperscript{419}-BB\selectlanguage{greek}ζ \selectlanguage{english}and TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T
cells exhibited similar CD137 expression against 0.5ug/ml, 0.01ug/ml and
0.005ug/ml antigen protein, TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells
induced higher CD137 expression against 0.1ug/ml and 0.05ug/ml antigen
protein (Fig. 5A). Then, we performed in vitro tumor killing assays.
TR1\textsuperscript{419}-28\selectlanguage{greek}ζ, \selectlanguage{english}TR1\textsuperscript{419}-BB\selectlanguage{greek}ζ \selectlanguage{english}and
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells were co-cultured with various
tumor targets, and Calcein AM release-based cytotoxic cell assay was
used to quantify tumor cell killing. The results showed that
TR1\textsuperscript{419}-28\selectlanguage{greek}ζ, \selectlanguage{english}TR1\textsuperscript{419}-BB\selectlanguage{greek}ζ \selectlanguage{english}and
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells killed TRAIL-R1-expressing
tumor cells with similar efficiency (Fig. 5B).

The proliferation of TR1\textsuperscript{419}-CAR-T cells upon antigen
stimulation was assessed using CFSE staining, and the CFSE fluorescence
declined following each cell division. The expression CFSE of
TR1\textsuperscript{419}-CAR-T cells was detected after co-cultured with
TRAIL-R1 positive SW480 cells for 7 days. TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ
\selectlanguage{english}CAR-T cells showed superior expansion compared with
TR1\textsuperscript{419}-28\selectlanguage{greek}ζ \selectlanguage{english}and TR1\textsuperscript{419}-BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T
cells against TRAIL-R1 positive tumors, and no significant difference
was found in cell proliferation between TR1\textsuperscript{419}-28\selectlanguage{greek}ζ \selectlanguage{english}and
TR1\textsuperscript{419}-BB\selectlanguage{greek}ζ\selectlanguage{english}CAR-T cells (Fig. 5C). Meanwhile, we also
detected the expression of PD-1 in TR1\textsuperscript{419}CAR-T cells
after co-incubation with SW480. The results indicated that PD-1 was more
frequently expressed by TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ\selectlanguage{english}CAR-T cells than
TR1\textsuperscript{419}-28\selectlanguage{greek}ζ \selectlanguage{english}and TR1\textsuperscript{419}-BB\selectlanguage{greek}ζ\selectlanguage{english}CAR-T
cells. However, the frequencies of PD-1\textsuperscript{+} CAR-T cells
between TR1\textsuperscript{419}-28\selectlanguage{greek}ζ \selectlanguage{english}and TR1\textsuperscript{419}-BB\selectlanguage{greek}ζ
\selectlanguage{english}groups were similar (Fig. 5D). There has shown that the highest level of
PD-1 expression reflects T-cell exhaustion status {[}31{]}. Thus, based
on positive PD-1 expression,
PD-1\textsuperscript{+}TR1\textsuperscript{419}CAR-T cells were divided
into two subgroups: high PD-1 expressing cells
(PD-1\textsuperscript{high}) and low PD-1 positive cells
(PD-1\textsuperscript{low}). There was no significant difference in the
expression of PD-1\textsuperscript{low} among the
TR1\textsuperscript{419}-28\selectlanguage{greek}ζ, \selectlanguage{english}TR1\textsuperscript{419}-BB\selectlanguage{greek}ζ \selectlanguage{english}and
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells. However,
PD-1\textsuperscript{high} was more frequently expressed by
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ\selectlanguage{english}CAR-T cells than
TR1\textsuperscript{419}-BB\selectlanguage{greek}ζ\selectlanguage{english}CAR-T cells (Fig. 5E). In summary,
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells proliferated more rapidly
than TR1\textsuperscript{419}-28\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells and
TR1\textsuperscript{419}-BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells upon antigen stimulation, but
expressed higher levels of PD-1.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image10/image10}

\end{center}
\end{figure}

\textbf{Fig.5.}TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells showed greater
antigen-specific expansion compared with TR1\textsuperscript{419}-28\selectlanguage{greek}ζ
\selectlanguage{english}CAR-T cells and TR1\textsuperscript{419}-BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells. (A) Expression
of CD137 on TR1\textsuperscript{419}CAR-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
TR1\textsuperscript{419}-28\selectlanguage{greek}ζ \selectlanguage{english}CAR, TR1\textsuperscript{419}-BB\selectlanguage{greek}ζ \selectlanguage{english}CAR or
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}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 \selectlanguage{ngerman}± SEM of three triplicate wells,\emph{*p}
\textless{} 0.05. (D) TR1\textsuperscript{419}CAR T cell proliferation
indicated by division of CFSE was assessed by flow cytometry.
TR1\textsuperscript{419}-28\selectlanguage{greek}ζ \selectlanguage{english}CAR, TR1\textsuperscript{419}-BB\selectlanguage{greek}ζ \selectlanguage{english}CAR and
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR T cells were stained with CFSE,
respectively. Then, TR1\textsuperscript{419}CAR T cells were cultured
with SW480 for 7 d, and analyzed by flow cytometry. (E-H) Expression of
PD-1 on TR1\textsuperscript{419}CAR T cells was analyzed by FACS after 7
days of co-culture with tumor cells. TR1\textsuperscript{419}CAR 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 \selectlanguage{ngerman}± SEM of three independent experiments,\emph{*p}
\textless{} 0.05, \emph{**p} \textless{} 0.01.

\textbf{Discussion}

In the present work, we demonstrated that TR1\textsuperscript{419}-based
CAR-T cells had potent dual anti-tumor activities in vitro. On the one
hand, TR1\textsuperscript{419}CAR can mediate tumor cell apoptosis
through activation of the death receptor-dependent apoptotic pathway. On
the other hand, TR1\textsuperscript{419}CAR 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 TR1\textsuperscript{419}CAR-T
cells, the third-generation TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}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 TR1\textsuperscript{419}-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 TR1\textsuperscript{419} 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
TR1\textsuperscript{419} may be related to the spatial structure of the
TR1\textsuperscript{419}CAR. 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, TR1\textsuperscript{419}-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 TR1\textsuperscript{419}-28\selectlanguage{greek}ζ \selectlanguage{english}and
TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}CAR-T cells. However, the
third-generation TR1\textsuperscript{419}-28BB\selectlanguage{greek}ζ \selectlanguage{english}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
TR1\textsuperscript{419}CAR-T cells can mediate dual tumor cell killing,
and the third-generation TR1\textsuperscript{419}28BBCAR-T cells
exhibited a higher sensitivity at low antigen, and proliferated more
rapidly upon antigen stimulation, compared to the second-generation
TR1\textsuperscript{419}CAR-T cells. This dual killing mechanism
provides a novel strategy for other CARs design.

\textbf{Author Contributions:}

\textbf{Aishun Jin:} Conceptualization, Supervision, Project
administration. \textbf{YN} and \textbf{LD:} Investigation, Methodology,
Writing---original draft preparation\textbf{:} \textbf{MS}
and\textbf{JH:} Methodology, Project administration \textbf{TL:}
Software, Methodology. \textbf{FL} : Software, Data
curation\textbf{,}Bioinformatics analysis. \textbf{XH:} Writing-review
and Editing.\textbf{LD:} Formal analysis, Investigation, Resources.
\textbf{MS:}Investigation, Formal analysis. \textbf{DP:} Supervision.

\textbf{Funding:}

This work was supported by National Natural Science Foundation of China,
grant number 81572807 and 81872329; and by Chongqing Municipal Education
Commission of Science and Technology Research Project of China, grant
number: KJZD-K201800403

\textbf{Acknowledgements:}

We would like to thank all team members involved in tumor immunotherapy
project at Chongqing Key Laboratory of Basic and Translational Research
of Tumor Immunology, Chongqing Medical University for their
technological assistance and help in developing this article. We thank
Professor Tatsuhiko Ozawac, Eiji Kobayashic and Hiroyuki Kishi for help
with experiments. We also thank all healthy individuals participated in
this study. We appreciate supporter Mr. Yuling Feng providing Chongqing
Medical University fund (X4457).

\textbf{Conflict of Interests}

The authors declare no competing interests.

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