Antibody Clicking as a Strategy to Modify Antibody Functionalities on the Surface of Targeted Cells

Article abstract of DOI:10.1021/jacs.0c05331

We established a methodology for initiating cross-linking of antibodies selectively on the cell surface through intermolecular copper-free click reactions facilitated by increased effective concentrations of antibodies binding to target antigens. Upon cross-linking of tetrazine- and bicyclononyne-modified trastuzumab on the surface of HER2-overexpressing cells, increased antibody uptake and activation of intracellular signaling were observed. Our findings demonstrate that the cross-linking reaction can significantly alter the biophysical properties of proteins, activating their unique functionalities on targeted cells to realize an increased cargo delivery and synthetic manipulation of cellular signaling.

Full text of DOI:10.1021/jacs.0c05331

pubs.acs.org/JACS
Communication
Antibody Clicking as a Strategy to Modify Antibody Functionalities
on the Surface of Targeted Cells
Toru Komatsu,* Etsu Kyo, Haruki Ishii, Kyoji Tsuchikama, Aiko Yamaguchi, Tasuku Ueno,
Kenjiro Hanaoka, and Yasuteru Urano*
Cite This: https://dx.doi.org/10.1021/jacs.0c05331
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: We established a methodology for initiating cross-linking of antibodies selectively on the cell surface through
intermolecular copper-free click reactions facilitated by increased effective concentrations of antibodies binding to target antigens.
Upon cross-linking of tetrazine- and bicyclononyne-modified trastuzumab on the surface of HER2-overexpressing cells, increased
antibody uptake and activation of intracellular signaling were observed. Our findings demonstrate that the cross-linking reaction can
significantly alter the biophysical properties of proteins, activating their unique functionalities on targeted cells to realize an increased
cargo delivery and synthetic manipulation of cellular signaling.
Copper-free click reactions, which include inverse-electron-
demand Diels−Alder reactions, are useful bioorthogonal
reactions that enable the selective and rapid formation of
covalent bonds between molecules.10−12 These reactions show
high regioselectivity, and we expected that antibody pairs
labeled with appropriate pairs of clickable linkers should form
cross-linked products when they are close to each other. We
prepared a set of antibody-clicking molecules, collectively
termed antibody clickers (AbCs), consisting of tetrazine (Tz)/
methyltetrazine (MTz) and bicyclononyne (BCN) attached
with a protein labeling site (N-hydoxysuccinimidyl ester) via
varied lengths of polyethylene glycol (PEG) linkers (Figure 1a,
Scheme S1). We hypothesized that linkers of sufficient length
are desirable for proper interactions within AbCs upon
antibody binding to cell surface antigens. In addition, the
reaction rate should be in the appropriate range such that the
desired click reaction is sufficiently slow in the diluted
extracellular medium but fast enough to trigger cross-linking
on the cell surface under increased effective concentrations.
LC-MS-based analysis of the reaction of glycine-conjugated
AbCs (Table S1, Figure S1) revealed that the second-order
reaction rates of click reactions with Tz- and BCN-linkers and
MTz- and BCN-linkers were in the ranges of k = 102−105
(M−1 s−1) and 100−101 (M−1 s−1), respectively (Figures 1b,c
and S2). Next, we studied the click reactions of antibodies
modified by the glycine-conjugated AbCs. Trastuzumab (anti-
HER2 antibody) was labeled using AbCs (4.06 0.19 AbC
molecules per antibody; Figure S3), and the reaction between
clickable pairs was monitored using SDS-PAGE. The results
ntibodies are molecules that bind to antigens with high
Aspecificity and affinity and are widely used in biological
studies and for therapeutic purposes.1 Recently, researchers
have tried to expand the functions of antibody using protein
engineering2,3 and chemical modifications.4−9 Modifications of
antibodies using chemical tools are mainly performed to add
unique functionality, such as cytotoxicity, or molecular imaging
capacity. In general, modifications are not designed for altering
the nature of the antibody as a carrier, and few studies have
reported molecular designs that aim to alter the physical
properties of antibodies to confer desired functionality.
Recent studies have reported that, in photoimmunotherapy,
the increased hydrophobicity of antibodies upon photo-
irradiation causes cytotoxicity of the conjugates, due to the
formation of protein aggregates.6,7 This indicates that the
alteration of the physical properties of antibodies (e.g.,
tendency to aggregate) can be a basis for eliciting unique
functionality (e.g., cytotoxicity). In this study, we developed a
methodology for inducing antibody aggregation using chemical
reactions that spontaneously occur on the cell surface without
photoirradiation, leading to altered behaviors and function-
alities of antibodies. We envisaged that the increased effective
concentration of antibodies binding to densely expressed target
antigens could be utilized to facilitate selective cross-linking on
the cell surface (Scheme 1).
Scheme 1. Design of Cell Surface Antibody Cross-Linking
Received: May 15, 2020
https://dx.doi.org/10.1021/jacs.0c05331
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
© XXXX American Chemical Society
A

Journal of the American Chemical Society
pubs.acs.org/JACS
Communication
antibody to reach 50% of the initial concentration) would have
been approximately 10 h. The labeling efficiency was not
significantly affected in human serum (Figure S6).
We next tested whether the cross-linking reaction of AbC-
labeled trastuzumab was facilitated after binding to the surface
of HER2-overexpressing NIH3T3 cells (3T3 HER2+).9 The
cell surface was sufficiently labeled with the antibody after 20
min under both non-click (MTzL) and click (MTzL/BCNL)
conditions (Figures 1f, S7, and S8). In the SDS-PAGE analysis
of the cell lysate, the latter condition resulted in the major
antibody population forming cross-linking products, even after
20 min (Figure 1g). The cross-linking products were observed
only at low levels in the extracellular media during the same
period (Figure S9), supporting our initial hypothesis that
antibody cross-linking was facilitated on the cell surface.
Next, we examined whether the aggregation of antibodies on
the cell surface altered their fates after target binding. We
found that the antibodies were efficiently internalized after 1−
3 h under click conditions (Figure 2a). This phenomenon was
dependent on the linker length, and shorter linkers were
relatively inefficient in forming the dot-like structures (Figure
S10), presumably because the linkers were not long enough to
connect with the antibodies when bound to the cell surface. At
this point, aggregate formation in the extracellular medium
compared with that on the cell surface contributed to the
increased antibody uptake. To test this hypothesis, previously
prepared clicked antibody products were added to the cells. As
facilitated uptake was not observed under these conditions
(Figure S11), antibodies forming aggregates after binding to
the target made a major contribution to their increased cellular
uptake.
Aggregate formation and facilitated uptake were more
robustly observed when two antibodies that targeted different
sites of HER2 were applied. The click reaction between
trastuzumab and pertuzumab, which target domain IV and
domain II of HER2, respectively, generated robust aggregates
that were also efficiently taken up by cells (Figures 2b,c and
S12). The efficiency of antibody uptake was studied by
modifying the antibody with the pH-responsive fluorescent
probe RhP-M13 to selectively detect antibodies accumulated in
the acidic intracellular environment (i.e., endosomal compart-
ments; Figure 2d). Increased fluorescence was observed under
click conditions (Figures S13−S15), confirming that an
increased number of antibodies was taken up by cells due to
the increased endocytosis.
Next, we tested whether this was a general effect using other
cells and antibodies; HER2-overexpressing tumor cells NCI-
N87 and SKBR3 were subjected to the facilitated endocytosis
of trastuzumab under click conditions (Figures S16−S18). In
addition, the facilitated endocytosis of the EGFR-targeting
antibody panitumumab was observed in the EGFR-over-
expressing tumor cells A431 (Figures S19 and S20).
The results indicated that the strategy described herein is
generally applicable for increasing the uptake and accumulation
of chemically modified antibodies for the better delivery of
cargo, and is especially useful for the development of systems
requiring efficient endosomal delivery of antibodies, such as
antibody-based therapeutics4,5 and molecular imaging.8
Besides facilitation of endocytosis, we found that cellular
shape was modified upon the cross-linking of antibodies on the
cell surface. In the system involving 3T3 HER2+ and
trastuzumab, notable rounding and clustering of the cells
were observed, seemingly by the pulling cells toward each
Figure 1. Synthesis and characterization of antibody clickers (AbCs).
(a) Structures of AbCs. Tz = tetrazine, MTz = methyltetrazine, and
BCN = bicyclononyne. (b) Monitoring of click reactions via pseudo-
first-order kinetics of tetrazine consumption. The peaks were
compared with one in the analysis without BCN (indicated with
(−) BCN). (c) Calculated second-order rate constants of the click
reactions in (b). Value
standard deviation (SD) (n = 3). (d)
Fluorescence image of the SDS-PAGE gel of trastuzumab labeled with
MTzS and TAMRA (100 nM) mixed with trastuzumab labeled with
BCNL (1000 nM) in PBS (pH 7.4). HC = heavy chain, LC = light
chain. Protein bands with large molecular weights are indicated by a
black arrow. Gel images for other click pairs are shown in Figure S4.
(e) Calculated second-order rate constant of the reactions in (d).
Value SD (n = 3). (f) Confocal fluorescence images of 3T3 HER2+
and intact NIH3T3 cells incubated with Tra-MTzL with or without
Tra-BCNL for 20 min. Antibodies were labeled with TAMRA, and
total antibody concentration was 100 nM. Scale bar = 10 μm. (g)
Analysis of cell lysate by SDS-PAGE. Cells were incubated with Tra-
MTzL with or without Tra-BCNL for 0−180 min. Antibodies were
labeled with TAMRA, and total antibody concentration was 100 nM.
confirmed that they generated larger molecular weight species
and aggregate-like structures (i.e., bands with no migration) in
a time-dependent manner (Figure 1d). The labeling rate was in
the order of k = 101−104 (M−1 s−1) in both Tz/BCN and
MTz/BCN pairs (Figures 1e, S4, and S5). The discrepancy
between small molecular and antibody click reactions might
have stemmed from the modified antibody with basal
interactions in the solution, allowing the non-ideal clickable
pairs to exhibit cross-linking. As a representative example,
trastuzumab labeled with MTzL/BCNL pairs exhibited a k =
2.7 × 102 (M−1 s−1); so, when the extracellular antibody
concentrations were 100 nM, the calculated τ1/2 (time for the
B
https://dx.doi.org/10.1021/jacs.0c05331
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
pubs.acs.org/JACS
Communication
Figure 3. Cell surface click reaction affected the cellular shape.
(Upper) Overlay of bright-field images and fluorescence images (red)
of 3T3 HER2+ cells after the addition of TAMRA-labeled Tra-TzS
(50 nM) and Tra-BCNL (50 nM). The mixture of antibodies was
added between 3 and 6 min (indicated by a black arrowhead).
(Lower) Magnified version of the upper image (white rectangle).
White arrowheads indicate the blebbing structures. Scale bar = 10 μm.
Further details are shown in Movie S1.
Figure 2. Cell surface click reactions facilitated the endocytosis of
antibodies. (a) Confocal fluorescence images of 3T3 HER2+ cells
incubated with Tra-TzS, Tra-BCNL, or both. In all conditions,
antibodies were labeled with TAMRA, and total antibody
concentration was 100 nM. The results for other click pairs are
shown in Figure S10. Scale bar = 10 μm. (b) Confocal fluorescence
images of 3T3 HER2+ cells incubated with Tra-TzS and pertuzumab
labeled with TzS (Per-TzS; left), Tra-BCNL and Per-BCNL (center),
and Tra-TzS and Per-BCNL (right). In all conditions, antibodies were
labeled with TAMRA, and total antibody concentration was 100 nM.
Scale bar = 10 μm. (c) The binding model of trastuzumab and
pertuzumab on the HER2 protein. The structure was derived from
PDB ID 6OGE.14 (d) Confocal fluorescence images of 3T3 HER2+
cells indicated mixtures of Tra-TzS, Tra-BCNL, Per-TzS, and Per-
BCNL. In all conditions, antibodies were labeled with Alexa Fluor 488
(green) and RhP-M (red). Total antibody concentration was 100 nM.
Scale bar = 10 μm.
Figure 4. Cell surface click-reaction-modified intracellular signaling
pathways of HER2-expressing tumor cells. (a) Bright-field images of
SKBR3 cells after the addition of TAMRA-labeled Tra-MTzL (50
nM) and Tra-BCNL (50 nM). Arrowheads indicate the blebbing
structures, and arrows indicate the vesicle-like structures. Scale bar =
10 μm. Further details are shown in Movie S4. (b) Result of
immunofluorescence staining of phosphor-ERK after incubation of
SKBR3 cells with Tra-MTzL with or without Tra-BCNL. Total
antibody concentration was 100 nM. For the positive control of pERK
signaling activation, cells were treated with EGF (100 ng/mL) for 1 h.
Scale bar = 10 μm. (c) Quantification of nuclear pERK signaling in
(b). Numbers in the column indicate the cell numbers included in
each analysis. (d) Viability of SKBR3 cells after treatment with Tra-
MTzL with or without Tra-BCNL for 2 d. Error bars represent SD (n
= 8). *P < 0.05 (Student’s t test). (e) Schematic view of antibody
clicking activating the intracellular growth signaling.
other by intercellular click reactions (Figures 3, S21, and S22,
Movie S1, and Movie S2). These effects were more robustly
observed in the clickable pair of trastuzumab and pertuzumab
(Figure S23, Movie S3) but were not observed for the pre-
clicked antibodies (Figure S24). Despite the risk that the
overall phenomenon could damage the cells, the cells survived
for 24 h, with no significant effects on cell viability (Figures
S22 and S25).
An interesting observation to note in the case of HER2-
expressing SKBR3 cells was that an increased growth of cells
was observed, rather than cell damage. In this, robust
membrane blebbing and the formation of vesicle-like structures
were observed (Figure 4a, Movie S4, and Movie S5), and cells
seemed to show adherent and spreading phenotypes after 3 h
(Figure S26). We speculated that these phenomena were due
to the activation of intracellular growth signaling, and we
questioned whether the phosphorylation of ERK, a major
signaling molecule downstream of HER2, involved in increased
cellular growth, would be activated; immunostaining revealed
that strong nuclear phospho-ERK signaling was observed after
C
https://dx.doi.org/10.1021/jacs.0c05331
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
pubs.acs.org/JACS
Communication
antibody clicking (Figure 4b,c). After 2 d of treatment,
trastuzumab without the click reaction reduced the cell
numbers, presumably by the blockade of growth signals, but,
under click conditions, the antibody treatment resulted in
increased cell numbers in a dose-dependent manner (Figure
4d). Thus, with and without clicking, the antibody triggered
completely different fates, growth promotion and inhibition, of
the cells. We consider that aggregation of the antibodies
induced the condensation of the targeted receptor on the cell
surface and facilitated cross-phosphorylation (Figure 4e). As
condensation of the receptors on cell surface was utilized to
activate various types of receptors,15,16 we believe that the
methodology to trigger it using the external antibodies will
serve as a unique synthetic biology tool to modify cellular fate
in clinical and biological samples as desired.
In conclusion, we developed a novel methodology for
modulating the physical properties of antibodies on cell
surfaces by facilitating intramolecular cross-linking via
increased effective concentrations. We discovered that the
cross-linking could alter antibody behaviors by increasing the
endocytosis rate and modulating the cellular signaling
pathways. The generality of the methodology will allow for
various future applications, such as increasing the effectiveness
of antibodies in cargo delivery and as synthetic biology tools to
modify intracellular signaling pathways of interest using
rational design.
orcid.org/0000-0002-1220-6327; Email: uranokun@m.u-
tokyo.ac.jp
Authors
Etsu Kyo − Graduate School of Pharmaceutical Sciences, The
University of Tokyo, Tokyo 113-0033, Japan
Haruki Ishii − Graduate School of Pharmaceutical Sciences, The
University of Tokyo, Tokyo 113-0033, Japan
Kyoji Tsuchikama − Texas Therapeutics Institute, The Brown
Foundation Institute of Molecular Medicine, The University of
Texas Health Science Center at Houston, Houston, Texas
77054, United States; orcid.org/0000-0002-2359-0408
Aiko Yamaguchi − Texas Therapeutics Institute, The Brown
Foundation Institute of Molecular Medicine, The University of
Texas Health Science Center at Houston, Houston, Texas
77054, United States
Tasuku Ueno − Graduate School of Pharmaceutical Sciences,
The University of Tokyo, Tokyo 113-0033, Japan;
orcid.org/0000-0001-6657-8209
Kenjiro Hanaoka − Graduate School of Pharmaceutical
Sciences, The University of Tokyo, Tokyo 113-0033, Japan;
orcid.org/0000-0003-0797-4038
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c05331
Notes
The authors declare no competing financial interest.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c05331.
■
sı
ACKNOWLEDGMENTS
■
Financial support for this study was provided by MEXT
(24655147, 15H05371, 15K14937, 17K19477, 18H04538,
19H02846, and 20H04694 to T.K.), JST (PRESTO, PRESTO
Network, and CREST to T.K.), AMED (CREST to Y.U.), and
the DoD (W81XWH-18-1-0004 and W81XWH-19-1-0598 to
K.T.). The project was also supported by JSPS Core-to-Core
program (JPJSCCA20170007). T.K. was supported by The
Naito Foundation, The Mochida Memorial Foundation for
Medical and Pharmaceutical Research, and The Tokyo
Biochemical Research Foundation.
Methods, detailed information on synthesis and
characterization of compounds, and supplementary
data, including Scheme S1, Table S1, and Figures S1−
S26 (PDF)
Movie S1, bright-field images of 3T3 HER2+ cells after
the addition of TAMRA-labeled Tra-TzS (50 nM) and
Tra-BCNL (50 nM) (AVI)
Movie S2, bright-field images of 3T3 HER2+ cells after
the addition of TAMRA-labeled Tra-TzS (100 nM)
(AVI)
Movie S3, bright-field images of 3T3 HER2+ cells after
the addition of TAMRA-labeled Tra-TzS (50 nM) and
Per-BCNL (50 nM) (AVI)
REFERENCES
■
(1) Rouge, L.; Chiang, N.; Steffek, M.; Kugel, C.; Croll, T. I.; Tam,
C.; Estevez, A.; Arthur, C. P.; Koth, C. M.; Ciferri, C.; Kraft, E.;
Payandeh, J.; Nakamura, G.; Koerber, J. T.; Rohou, A. Structure of
CD20 in complex with the therapeutic monoclonal antibody
rituximab. Science 2020, 367 (6483), 1224−1230.
Movie S4, bright-field images of SKBR3 cells after the
addition of TAMRA-labeled Tra-TzS (50 nM) and Tra-
BCNL (50 nM) (AVI)
Movie S5, bright-field images of SKBR3 cells after the
addition of TAMRA-labeled Tra-TzS (100 nM) (AVI)
(2) Kang, J. C.; Sun, W.; Khare, P.; Karimi, M.; Wang, X.; Shen, Y.;
Ober, R. J.; Ward, E. S. Engineering a HER2-specific antibody-drug
conjugate to increase lysosomal delivery and therapeutic efficacy. Nat.
Biotechnol. 2019, 37 (5), 523−526.
(3) Li, J. Y.; Perry, S. R.; Muniz-Medina, V.; Wang, X.; Wetzel, L. K.;
Rebelatto, M. C.; Masson Hinrichs, M. J.; Bezabeh, B. Z.; Fleming, R.
L.; Dimasi, N.; Feng, H.; Toader, D.; Yuan, A. Q.; Xu, L.; Lin, J.; Gao,
C.; Wu, H.; Dixit, R.; Osbourn, J. K.; Coats, S. R. A biparatopic
HER2-targeting antibody-drug conjugate induces tumor regression in
primary models refractory to or ineligible for HER2-targeted therapy.
Cancer Cell 2019, 35 (6), 948−949.
AUTHOR INFORMATION
Corresponding Authors
■
Toru Komatsu − Graduate School of Pharmaceutical Sciences,
The University of Tokyo, Tokyo 113-0033, Japan;
orcid.org/0000-0002-9268-6964; Email: tkomatsu@
mol.f.u-tokyo.ac.jp
Yasuteru Urano − Graduate School of Pharmaceutical Sciences
and Graduate School of Medicine, The University of Tokyo,
Tokyo 113-0033, Japan; Core Research for Evolutional Science
and Technology (CREST), Japan Agency for Medical Research
and Development (AMED), Tokyo 100-0004, Japan;
(4) Alley, S. C.; Okeley, N. M.; Senter, P. D. Antibody-drug
conjugates: targeted drug delivery for cancer. Curr. Opin. Chem. Biol.
2010, 14 (4), 529−537.
(5) Anami, Y.; Yamazaki, C. M.; Xiong, W.; Gui, X.; Zhang, N.; An,
Z.; Tsuchikama, K. Glutamic acid-valine-citrulline linkers ensure
stability and efficacy of antibody-drug conjugates in mice. Nat.
Commun. 2018, 9 (1), 2512.
D
https://dx.doi.org/10.1021/jacs.0c05331
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
pubs.acs.org/JACS
Communication
(6) Mitsunaga, M.; Ogawa, M.; Kosaka, N.; Rosenblum, L. T.;
Choyke, P. L.; Kobayashi, H. Cancer cell-selective in vivo near
infrared photoimmunotherapy targeting specific membrane mole-
cules. Nat. Med. 2011, 17 (12), 1685−1691.
(7) Sato, K.; Ando, K.; Okuyama, S.; Moriguchi, S.; Ogura, T.;
Totoki, S.; Hanaoka, H.; Nagaya, T.; Kokawa, R.; Takakura, H.;
Nishimura, M.; Hasegawa, Y.; Choyke, P. L.; Ogawa, M.; Kobayashi,
H. Photoinduced ligand release from a silicon phthalocyanine dye
conjugated with monoclonal antibodies: a mechanism of cancer cell
cytotoxicity after near-infrared photoimmunotherapy. ACS Cent. Sci.
2018, 4 (11), 1559−1569.
(8) Mestel, R. Cancer: Imaging with antibodies. Nature 2017, 543
(7647), 743−746.
(9) Urano, Y.; Asanuma, D.; Hama, Y.; Koyama, Y.; Barrett, T.;
Kamiya, M.; Nagano, T.; Watanabe, T.; Hasegawa, A.; Choyke, P. L.;
Kobayashi, H. Selective molecular imaging of viable cancer cells with
pH-activatable fluorescence probes. Nat. Med. 2009, 15 (1), 104−
109.
(10) Oliveira, B. L.; Guo, Z.; Bernardes, G. J. L. Inverse electron
demand Diels-Alder reactions in chemical biology. Chem. Soc. Rev.
2017, 46 (16), 4895−4950.
(11) Tamura, T.; Hamachi, I. Chemistry for covalent modification of
endogenous/native proteins: From test tubes to complex biological
systems. J. Am. Chem. Soc. 2019, 141 (7), 2782−2799.
(12) Sakamoto, S.; Hamachi, I. Recent Progress in Chemical
Modification of Proteins. Anal. Sci. 2019, 35 (1), 5−27.
(13) Watanabe, R.; Soga, N.; Fujita, D.; Tabata, K. V.; Yamauchi, L.;
Hyeon Kim, S.; Asanuma, D.; Kamiya, M.; Urano, Y.; Suga, H.; Noji,
H. Arrayed lipid bilayer chambers allow single-molecule analysis of
membrane transporter activity. Nat. Commun. 2014, 5, 4519.
(14) Hao, Y.; Yu, X.; Bai, Y.; McBride, H. J.; Huang, X. Cryo-EM
Structure of HER2-trastuzumab-pertuzumab complex. PLoS One
2019, 14 (5), e0216095.
(15) Fegan, A.; White, B.; Carlson, J. C.; Wagner, C. R. Chemically
controlled protein assembly: techniques and applications. Chem. Rev.
2010, 110 (6), 3315−3336.
(16) Ueki, R.; Uchida, S.; Kanda, N.; Yamada, N.; Ueki, A.;
Akiyama, M.; Toh, K.; Cabral, H.; Sando, S. A chemically unmodified
agonistic DNA with growth factor functionality for in vivo therapeutic
application. Sci. Adv. 2020, 6 (14), eaay2801.
E
https://dx.doi.org/10.1021/jacs.0c05331
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX