Bio-Orthogonal T Cell Targeting Strategy for Robustly Enhancing Cytotoxicity against Tumor Cells

Article abstract of DOI:10.1002/smll.201804383

T cells can kill tumor cells by cell surface immunological recognition, but low affinity for tumor-associated antigens could lead to T cell off-target effects. Herein, a universal T cell targeting strategy based on bio-orthogonal chemistry and glycol-meta

Full text of DOI:10.1002/smll.201804383

CommuniCation
Bio-Orthogonal T Cell Targeting Strategy
www.small-journal.com
Bio-Orthogonal T Cell Targeting Strategy for Robustly
Enhancing Cytotoxicity against Tumor Cells
Wenjun Li, Hong Pan, Huamei He, Xiaoqing Meng, Qian Ren, Ping Gong, Xin Jiang,
Zhenguo Liang, Lanlan Liu, Mingbin Zheng, Ximing Shao, Yifan Ma,* and Lintao Cai*
receptor (TCR),[3b,4] which recreate T cells
T cells can kill tumor cells by cell surface immunological recognition, but low
affinity for tumor-associated antigens could lead to T cell off-target effects.
Herein, a universal T cell targeting strategy based on bio-orthogonal chemistry
and glycol-metabolic engineering is introduced to enhance recognition and
cytotoxicity of T cells in tumor immunotherapy. Three kinds of bicycle [6.1.0]
nonyne (BCN)-modified sugars are designed and synthesized, in which
Ac4ManN-BCN shows efficient incorporation into wide tumor cells with a
BCN motif on surface glycans. Meanwhile, activated T cells are treated with
Ac4GalNAz to introduce azide (N3) on the cell surface, initiating specific
tumor targeting through a bio-orthogonal click reaction between N3 and
BCN. This artificial targeting strategy remarkably enhances recognition and
migration of T cells to tumor cells, and increases the cytotoxicity 2 to 4 times
for T cells against different kinds of tumor cells. Surprisingly, based on this
strategy, the T cells even exhibit similar cytotoxicity with the chimeric antigen
receptor T-cell against Raji cells in vitro at the effector: target cell ratios (E:T) of
1:1. Such a universal bio-orthogonal T cell-targeting strategy might further
broaden applications of T cell therapy against tumors and provide a new
strategy for T cell modification.
with enhanced tumor-specific recognition
and effective cytotoxicity, but unpredict-
able on-target, off-tumor toxicity is still
the major safety concern for engineering
T cell therapy.[5] Thus it is crucial to
search novel targeting strategies for fur-
ther clinic application of T cell immune
therapeutics.[6]
Metabolic glycoengineering is an effi-
cient way to introduce various chemical
groups to cellular glycan through intrinsic
biosynthetic pathways,[7] which enable
the chemical modification of cells within
their native environment.[8] Bertozzi and
co-workers developed this technique to
wide application range in combination with
bio-orthogonal click chemistry, namely
bio-orthogonal glycometabolic labeling.[9]
The bio-orthogonal functional groups
incorporated into cell surface glycans can
be further labeled or anchored through
bio-orthogonal click reaction.[10] Based
on this technique, Kim and co-workers
In recent years, adoptive T cell therapy (ACT) has been consid-
ered a potent and feasible cancer treatment modality in cancer
immunotherapy.[1] However, due to the constant immune
selective pressure and genetic instability,[2] tumor cells would
give rise to variants displaying a multitude of evasion mecha-
nisms, including downregulate antigen presentation to render
T cells “blind” to tumor, or overexpression of decoy receptors
to inhibit T cell activity.[3] To overcome these challenges, modi-
fication of T cell has rapidly evolved by harnessing artificial
expression of chimeric antigen receptors (CARs) and T cell
and Cheng and co-workers further designed a novel tumor-
targeting strategy by introducing bio-orthogonal groups
azide (-N3) on tumor cell surface as an artificial target, which
effectively increased the tumor-targeting and accumulation
efficiency of nanoparticles through click reaction in vivo.[11]
Thus, bio-orthogonal glycometabolic labeling can provide an
artificial-targeting strategy that made tumor cells more uniform
and highly specific.[11b,12] This strategy has been widely used in
modulating the biodistribution and tumor-targeting efficacy of
probe and therapeutic nanoparticles in vitro and in vivo,[13] but
less used in improving the target recognition of immune cell
therapy, for which enhanced tumor targeting is just the urgent
issue to be solved.
Therefore, we herein report a bio-orthogonal universal-
targeting strategy in enhancing recognition and avoiding off-
target of T cell, in which complementary bio-orthogonal groups
were separately incorporated in tumor cell and T cell through
glycol-metabolic process to build implanted target reporter
(Scheme 1). Motivated by this purpose, a serial of novel unnat-
ural sugar were delicately designed and synthesized based on
bio-orthogonal molecules:bicyclo [6.1.0] nonyne (BCN)-modified
mannose, galactose, and glucose. BCN is a ring-strained alkyne
that can react with both azides (N3) and tetrazine (Tz) through
metal-free click reactions, the reaction rates (k2) of which were
Dr. W. Li, Dr. H. Pan, H. He, Dr. X. Meng, Q. Ren, Dr. P. Gong, X. Jiang,
Z. Liang, Dr. L. Liu, Dr. M. Zheng, Dr. X. Shao, Prof. Y. Ma, Prof. L. Cai
Guangdong Key Laboratory of Nanomedicine
CAS-HK Joint Lab for Biomaterials
Shenzhen Institutes of Advanced Technology
Chinese Academy of Sciences
Shenzhen 518055, P. R. China
E-mail: yf.ma@siat.ac.cn; lt.cai@siat.ac.cn
Dr. H. Pan, Dr. X. Meng, Dr. L. Liu
University of Chinese Academy of Sciences
Beijing 100049, P. R. China
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/smll.201804383.
DOI: 10.1002/smll.201804383
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Scheme 1. a) The synthesis route of Ac4ManN-BCN, one of BCN-modified nature sugars applied in subsequent experiments. b) The universal T cell
targeting strategy mediated by bio-orthogonal glycol engineering for enhancing T cell recognition and cytotoxicity to tumor cell.
−1
1.7 and 2.9 × 104
m
s−1 in phosphate buffer solution (PBS),
Before investigating the metabolic labeling performance
of these newly synthesized unnatural BCN-sugars, we eval-
uate their cytotoxicity to Raji cells in different concentra-
tion (10, 20, and 40 µm). Here we chose Raji cell because it
is a stable cultured line of lymphoblastoid cells derived from
B lymphoma, which presented sensitive response to T lympho-
cytes therapy. All of unnatural BCN-sugars showed no toxicity at
10 and 20 µm, but caused obvious lower cellular viability at 40 µm
(Figure S1, Supporting Information). Then we studied whether
these unnatural sugars could metabolically label cells with BCN
groups in vitro. Raji cells were incubated with Ac4ManN-BCN,
Ac4GalN-BCN, or Ac4GluN-BCN at 20 µm for 2 d, respectively,
and the potentially incorporated BCN groups on the cell gly-
coproteins were detected by incubation with N3-conjugated
fluorophore (N3-Cy5.5) for 30 min (Figure 1). As shown in
Figure 1, the control group treated with PBS shows negli-
gible Cy5.5 signals on the cell surface; however, cells treated
with unnatural Ac4ManN-BCN presented strong fluorescence
signals, while cells incubated with Ac4GluN-BCN or Ac4GalN-
BCN only showed slightly increased fluorescence signal. Flow
cytometry analyses of Raji cells following the same treatment
consistently showed stronger Cy5.5 fluorescence intensity in
Ac4ManN-BCN group than in Ac4GlcN-BCN and Ac4GalN-BCN
group, which might be due to that metabolic enzymes in dif-
ferent biosynthetic pathway showed different tolerance to each
BCN-sugars. In addition, cells treated by Ac4ManN-BCN were
also stained by Tz-conjugated fluorophore (Tz-cy5), another
bio-orthogonal group can react with BCN more quickly than
N3 group, and the flow cytometry results showed obvious fluo-
rescence enhancement, further confirming that BCN groups
have successfully incorporated on cell surface (Figure S2, Sup-
porting Information). Ac4ManN-BCN with different concentra-
tion (0, 10, or 20 µm) treated cells showed a dose-dependent
respectively.[11c,14] Here we chose BCN as bio-orthogonal chem-
ical reporter not only because of its fast reaction kinetics, but
also because BCN showed higher hydrophilicity than dibenzo-
cyclooctyne (DBCO) and better stability than trans-cyclooctene
(TCO), which might decrease nonspecific binding to proteins
and increase in vivo circulation.[14] These synthesized unnatural
sugars were incorporated into tumor cells via metabolic labe-
ling to introduce BCN reporters on cell surface (BCN-tumor
cell), which can be used as artificial target motif of tumor cells.
Simultaneously, commercialized Ac4GalNAz was incorporated
into activated T cells to introduce azide on cell surface (N3-T
Cell). After modification, tumor cells could fastly bond to T
cells through bio-orthogonal click reaction, which further pro-
mote natural recognition T cell toward tumor cell, subsequently
stimulate T cell and enhance its cytotoxicity. This strategy
might provide a novel insight into cell recognition and interac-
tion through bio-orthogonal glycometabolic labeling, and also
broaden the applications of T cell immunotherapy for other
tumor, even solid tumor, in vitro and in vivo.
To construct BCN-modified mannose, galactose, and glucose,
we firstly synthesized 4-nitrophenyl chloroformate activated bicycle
[6.1.0] nonyne (nPC-BCN) according to the former literatures.[15]
And then nPC-BCN was coupled with glycans (mannose, galac-
tose, and glucose) to get N-BCN-carbonyl-d-mannosamine (ManN-
BCN), N-BCN-carbonyl-d-galactosamine (GalN-BCN), and N-BCN-
carbonyl-d-glucosamine (GluN-BCN), respectively. To increase
cellular uptake, these unnatural glycans were peracetylated to form
the tetra-O-acetyl compounds including tetraacetylated-N-BCN-car-
bonyl-d-mannosamine (Ac4ManN-BCN), tetraacetylated-N-BCN-
carbonyl-d-galactosamine (Ac4GalN-BCN), and tetraacetylated-N-
BCN-carbonyl-d-glucosamine (Ac4GluN-BCN) (see Supporting
Information and Scheme S1, Supporting Information).
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Figure 1. The metabolic labeling performance of these newly synthesized unnatural BCN-sugars including Ac4ManN-BCN, Ac4GalN-BCN,
and Ac4GluN-BCN. a) Confocal imaging and b) flow cytometry analyses of Raji cell with or without BCN-sugars after staining by N3-conjugated
fluorophore (N3-Cy5.5) for 30 min (the bar in image is 10 µm). Negative group is BCN-sugars treated Raji cell but without stained by N3-Cy5.5 for
30 min. Control group is unlabeled Raji cell stained by N3-Cy5.5 for 30 min.
increase of fluorescence signal, with 82.5% N3-Cy5.5 positive
cells compared with untreated cells at 20 µm (Figure 2a). To fur-
ther verify the universal metabolic labeling ability of Ac4ManN-
BCN in different cell lines, it was incubated with human lung
carcinoma cell line (A549 cells), human rhabdomyoma cell
line (RD cells), human hepatocarcinoma cell line (HepG2
cells), human breast cancer cell line (MCF-7 cells), respec-
tively. Compared to Raji cells, these cells had higher tolerance
to Ac4ManN-BCN, not presenting any cytotoxicity even when
the treated concentration was at 50 µm (Figure S3, Supporting
Information). After modification by Ac4ManN-BCN, the tested
cells were stained by N3-Cy5.5 and tested by laser scanning con-
focal microscopy (LSCM) and flow cytometry (FCM) analysis
(Figure S4, Supporting Information). As expected, cells treated
with Ac4ManN-BCN showed significantly higher fluorescence
than control group. These results collectively demonstrated
that Ac4ManN-BCN can metabolically label cells as a proper
substrate for biosynthetic enzymes in sialic acid pathway, and
act as an optimal choice for introducing BCN on cell surface to
be an artificial target motif of tumor cells.
percentage of CD3+ positive T cells finally reached ≈99% under
this culture conditions. T cells pretreated with Ac4GlaNAz
were stained by DBCO-bearing fluorophore (DBCO-Fluor 488)
through click reaction and analyzed by confocal imaging and
flow cytometry. The data showed that T cells were efficiently
labeled by Ac4GlaNAz in a dose-dependent manner. Above
85% of DBCO-Fluor 488 positive cells were monitored in
T cells treated by 50 µm of Ac4GlaNAz, but 100 µm of
Ac4GlaNAz showed no significant increased cell fluorescent
intensity, suggesting that the dose of 50 µm was the optimum
concentration for the next experiments (Figure 2b and
Figure S5, Supporting Information).
To evaluate the effect on cell recognition for bio-orthog-
onal reaction over cell surface, the interaction between Ac4G-
laNAz treated T cells (N3-T cell) and Ac4ManN-BCN treated
Raji cells (BCN-Raji cell) was monitored by confocal micro-
scopy. As shown in Figure S6 in the Supporting Information,
the cluster of N3-T cells and BCN-Raji cells boosted gradually
from 10 to 20 min, but only less cell aggregation in control
group, indicating bio-orthogonal reaction over cell surface can
initiate the strong affinity between two types of cells, further
enhancing the recognition and activation of T cells. In addi-
tion, the dynamical process of N3-T cells binding to BCN-Raji
cells was also investigated using time lapse confocal imaging
(Figure 3a). Interestingly, when N3-T cells or BCN-Raji cells ran-
domly moved around to each other, they rapidly bound together
In order to introduce bio-orthogonal glycoengineering medi-
ated targeting strategy to immunotherapy, T cells were labeled
with different concentration of commercial Ac4GlaNAz (0, 25,
50, or 100 µm) for introducing N3 on cell surface. T cells used in
this project were extracted from human peripheral blood mono-
nuclear cells (PBMCs) isolated from healthy volunteers, and the
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Figure 2. The metabolic labeling performance of Ac4ManN-BCN and commercialized Ac4GalNAz in B lymphoma cells and active T cells, respectively.
a) Confocal imaging and b) flow cytometry analyses of Raji cell with or without treatment by Ac4ManN-BCN after incubation with N3-Cy5.5 for 30 min.
c) Confocal imaging and d) flow cytometry analyses of T cell with or without Ac4GalNAz after incubation with DBCO-Fluor 488 for 30 min. Negative
group is BCN-sugars treated Raji cell but without incubating with N3-Cy5.5 or DBCO-Fluor 488 for 30 min. Med or Control group is unlabeled Raji cell
or T cell incubated with corresponding fluorophore for 30 min. The bar in image is 10 µm.
and attracted more cells aggregating into big cell clusters
from 0 to 20 min (Figure 3b and Video S1, Supporting Infor-
mation), whereas unlabeled T cells and Raji cells exhibited a
relative random motion and less aggregation in such short time
(Figure 3b and Video S2, Supporting Information). To confirm
effect on cell migration and invasion of bio-orthogonal reaction
Figure 3. The effect of bio-orthogonal click reaction over cell surface on cell recognition and migration. a) Time lapse confocal imaging of coculture
of T cell and Raji cells with or without treatment by Ac4ManN-BCN (20 µm) or Ac4GalNAz (50 µm), respectively, at 0, 8, 16, and 24 min. The bar in
image is 15 µm. Cell with green fluorescence is T cell stained by anti-CD3. Cell without fluorescence is Raji cell. b) Count the cell cluster in the coculture
of T cell and Raji cells with or without treatment by Ac4ManN-BCN (20 µm) or Ac4GalNAz (50 µm), respectively, at 0, 8, 16, and 24 min. c) Transwell
results of the migration between T cell and Raji cells or between N3-T cell and BCN-Raji cells, respectively. Anti-CD3 (APC) and anti-CD19 (PE) separately
stained T cells and Raji cells.
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over cell surface, N3-T cells and BCN-Raji cells were examined
in transwell chambers and analyzed by flow cytometry assay
(Figure 3c). Compared to untreated T cells, the migration
rate of N3-T cells increased by 36.7% after 30 min coculture,
demonstrating that rapid bio-orthogonal reaction over cell
surface might also promote the adhesion and migration of
T cell to target cell.
T cells. Similar to LDH release assay, Calcein-AM/PI staining
assay also proved the enhanced cytotoxicity of N3-T cells against
BCN-Raji cells compared to the unlabeled T cell group at 1:1
E/T ratio (Figure 4c and Figure S7, Supporting Information),
demonstrating bio-orthogonal reaction over cell surface not
only promote intercellular recognition but also enhance T cell
cytotoxicity against tumor cell. Additional LDH assay was tested
to evaluate CAR-T cell mediated cytotoxicity as a comparison at
1:1 E/T ratio. To our surprise, N3-T cells and CAR-T cells exhib-
ited near cytotoxicity against—with or without Ac4ManN-BCN
(20 µm) treated—Raji cells, respectively (Figure 4e), indicating
that such bio-orthogonal T cell targeting strategy could be a
potentially effective method to enhance T cell treatment against
tumor.
We also investigated the universal cytotoxicity mediated by
N3-T cells against BCN-modified tumor cells (including A549
cell, RD cell, HepG2 cell, and MCF-7 cells) at 1:1 E/T ratio. As
shown in Figure S8 in the Supporting Information, N3-T cells
exhibited 2–4 times increased killing effects than untreated
T cell to all of BCN-modified tumor cells, especially for BCN-
HepG2 cells, yielding nearly 4 times higher LDH release than
control group after coculture with N3-T cells. These results
strongly proved that bio-orthogonal glycoengineering medi-
ated tumor targeting can universally enhance sensitivity and
cytotoxicity of T cell to tumor cells.
To evaluate the cytotoxicity of N3-T cells against BCN-Raji cells,
we employed Raji cell stably transfected with firefly luciferase
(Luci-Raji) as test cell line because its fluorescence intensity is
proportional to the survival rate of Raji cells, which can be visual-
ized in luminescence imaging assay. Different concentration of
Ac4ManN-BCN (10 or 20 µm) treated Luci-Raji cells (BCN-Luci-
Raji cells) were incubated with Ac4GlaNAz (50 µm) treated T cells
(N3-T cells) at 1:1 effector-to-target cell (E/T) ratio. As shown in
Figure 4a,b, luminescence intensity of BCN-Luci-Raji cell was
significantly decreased after incubated with N3-T cell, nearly
decreasing by 60% as Raji cells treated with 20 µm of Ac4ManN-
BCN. Subsequently, the N3-T cells mediated cytotoxicity against
BCN-Raji cells was further determined by lactate dehydrogenase
(LDH) assay. The results in Figure 4d showed LDH released
from BCN-Raji cells in a concentration-dependent manner
after cocultured with N3-T cells at 1:1 E/T ratio. Especially for
Raji cells treated by 20 µm of Ac4ManN-BCN, N3-T cells medi-
ated cytotoxicity increased by 2.7 times compared to untreated
Figure 4. The cytotoxicity and cytokines secretion of N3-labeled T cell (N3-T cell) against BCN-labeled Raji cell (BCN-Raji). a,b) Luminescence imaging
assay and quantitative analysis, and c) Calcein-AM/PI staining assay of cytotoxicity of Ac4GalNAz (0 and 50 µm) treated T cell against Ac4ManN-BCN
(0, 10, and 20 µm) treated Raji cell. The bar in image is 100 µm. d) LDH release for Ac4GalNAz (0 and 50 µm) treated T cell cocultured with
Ac4ManN-BCN (0, 10, 15, and 20 µm) treated Raji cell. e) LDH release for Ac4GalNAz (50 µm) treated T cell against Ac4ManN-BCN (20 µm) treated
Raji cell, and CAR-T cell against Raji cell. f–h) Cytokines secretion for Ac4GalNAz (0 and 50 µm) treated T cell cocultured with Ac4ManN-BCN (0, 10, 15,
and 20 µm) treated Raji cell.
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For further confirming apoptosis regulated by T cells, we
analyzed cytokine production of T cell or N3-T cells separately
in response to Raji cells or BCN-Raji cells (Figure 4f–h). As
shown in Figure 4f–h, N3-T cells can effectively recognize BCN-
Raji cells and produce effector cytokines including interleukin-2
(IL-2), tumor necrosis factor-α (TNF-α), and interferon-γ
(IFN-γ). With the increase of the concentration of Ac4ManN-
BCN treated Raji cells, the antitumor cytokines secretion was
significantly enhanced in N3-T cells, indicating that bio-orthog-
onal reaction over cell surface can effectively improve the recog-
nition and activation of T cells toward tumor cells, which might
be the important reason that cause the enhanced tumor toxicity
for T cells after modification.
In conclusion, the BCN modified unnatural sugars,
especially for Ac4ManN-BCN, can be efficiently and
nondestructively incorporated into wild tumor cell surface
glycans. The BCN motif on cell surface exhibited an excellent
bio-orthogonal targeting tag with rapid reaction rate, which
might enlarge the application of biodiagnostic and therapeutic
approaches. Furthermore, in conjunction with the highly effi-
cient and specific bio-orthogonal reaction, Ac4GalNAz treated
T cells presented remarkable high-efficiency targeting and
enhanced cytotoxicity against BCN-labeled tumor cells, even
showing equal cytotoxicity with CAR-T cells against BCN-Raji
cell. It suggested that T cell with robust metabolic modifica-
tion can effectively kill tumor cells and will provide a rapid and
valid therapeutic method of tumor. Such universal bio-orthog-
onal T cell targeting strategy might pave a way to improve
T cell therapies for cell targeting and tumor infiltrating, and
provide a brand-new strategy for cell immunotherapy and
solid tumor therapy.
Keywords
bio-orthogonal glycometabolic labeling, click chemistry, cytotoxicity,
T cell targeting, T cell therapy
Received: October 21, 2018
Revised: December 2, 2018
Published online:
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Acknowledgements
W.L. and H.P. contributed equally to this work. The authors gratefully
acknowledge funding support from the National Natural Science
Foundation of China (Grant No. 81601552, 31571013), Guangdong Natural
Science Foundation of Research Team (2016A030312006), Shenzhen
Science and Technology Innovation Commission (JCYJ20170818154843625,
JCYJ2017081816373945, JSGG20160331185422390, JCYJ20160429191503002,
JCYJ20150403091443298, KQCX20140521115045447, JCYJ20150521094519473),
and the SIAT Innovation Program for Excellent Young Researchers (201506).
The PBMCs were provided by the Yan Jin group. The corresponding human
experiments conform to the ethics of the Institutional Review Board of SIAT
(serial number: SIAT-IRB-170315-H0145).
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Conflict of Interest
The authors declare no conflict of interest.
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