Full text of DOI:10.1039/d0md00337a

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RESEARCH ARTICLE
Synthesis and biological evaluation of a
monocyclic Fc-binding antibody-recruiting
molecule for cancer immunotherapy†
Cite this: RSC Med. Chem., 2021, 12,
406
Koichi Sasaki, ‡¶a Kyohei Muguruma,§¶b Rento Osawa,b Akane Fukuda,b
b
Atsuhiko Taniguchi, b Akihiro Kishimura,acd Yoshio Hayashi,
*
ac
acde
*
*
Takeshi Mori
and Yoshiki Katayama
Antibody-recruiting molecules (ARMs) are bispecific molecules composed of an antibody-binding motif
and a target-binding motif that redirect endogenous antibodies to target cells to elicit immune responses.
To enhance the translational potential of ARMs, it is crucial to design antibody/target-binding motifs that
have strong affinity and are easy to synthesize. Here, we synthesized a novel Fc-binding ARM (Fc-ARM) that
targets folate receptor (FR)-positive cancer cells, Reo-3, using a recently developed monocyclic peptide
15-Lys8Leu, which binds strongly to the Fc region of an antibody. Reo-3 bound to the Fc region of the
antibody with a Kd of 5.8 nM, and recruited a clinically used antibody mixture to attack FR-positive IGROV-
1 cells as efficiently as Fc-ARM2, in which a bicyclic Fc-binding peptide was used. These results indicate
that 15-Lys8Leu, which can be synthesized readily, is suitable for various applications including the
development of Fc-ARMs.
Received 2nd October 2020,
Accepted 10th January 2021
DOI: 10.1039/d0md00337a
rsc.li/medchem
infected cells,7–9 and cancer cells10–16 have been reported. We
have demonstrated previously that Fc-binding ARMs (Fc-
Introduction
Small synthetic alternatives to therapeutic antibodies can
potentially solve the intrinsic problems of antibodies such as
immunogenicity1,2 and stability.3 As one alternative, antibody-
recruiting small molecules (ARMs) are capable of bridging an
endogenous antibody and a target antigen expressed on the
surface of target cells, resulting in immune-mediated
clearance of the target cells.4 In general, ARMs are composed
of an antigen recognized by endogenous antibodies and a
targeting ligand. A variety of ARMs targeting bacteria,5,6 virus-
ARMs), which are composed of an Fc-binding peptide and a
targeting ligand, can eliminate folate receptor (FR)- or
prostate-specific membrane antigen-positive cancer cells via a
mechanism
called
antibody-dependent
cell-mediated
cytotoxicity (ADCC) (Fig. 1A).17,18 Taking advantage of the
relatively conserved structure of the Fc region of antibodies,
our approach recruits the majority of endogenous antibodies
for targeted immune responses without antigen–antibody
interactions. We also showed that the Fc affinity of Fc-ARMs
positively regulates ADCC efficacy,17 suggesting that the
development of potent Fc-binders is highly beneficial for the
advancement of Fc-ARMs as novel immunotherapeutics.
a Department of Applied Chemistry, Graduate School of Engineering, Kyushu
University, Fukuoka 819-0395, Japan. E-mail: mori.takeshi.880@m.kyushu-u.ac.jp,
ykatatcm@mail.cstm.kyushu-u.ac.jp
b Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life
Sciences, Tokyo 192-0392, Japan. E-mail: yhayashi@toyaku.ac.jp
c Graduate School of Systems Life Sciences, Kyushu University, Fukuoka 819-0395,
Japan
d International Research Center for Molecular Systems, Kyushu University, Fukuoka
819-0395, Japan
e Department of Biomedical Engineering, Chung Yuan Christian University,
Taoyuan, Taiwan
Additionally, we have conducted
a
structure–activity
relationship study and discovered a novel monocyclic peptide,
15-Lys8Leu, which has an equilibrium dissociation constant
(Kd) of 8.19 nM against the Fc region of an antibody.19 15-
Lys8Leu has great potential in various applications because of
its comparable Fc affinity and ease of synthesis when
compared with that of bicyclic Fc-binding peptides.19
Here, we used 15-Lys8Leu for the synthesis of a new Fc-
ARM that targets the FR, named Reo-3 (Fig. 1B). 15-Lys8Leu
and folic acid (FA) as the targeting ligand of the FR were
linked with an oligo-ethylene glycol linker. We tested the Fc
affinity of this new Fc-ARM, the ability to recruit antibodies
to the surface of FR positive cancer cells, and ADCC efficacy
in comparison with Fc-ARM2, a previously reported molecule
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0md00337a
‡ Present address: Department of Applied Chemistry, Graduate School of
Engineering, Osaka Prefecture University, Osaka 599-8531, Japan
§ Present address: Department of Chemical Science and Engineering, School of
Materials and Chemical Technology, Tokyo Institute of Technology, Tokyo 152-
8552, Japan
¶ These authors contributed equally to this work.
406 | RSC Med. Chem., 2021, 12, 406–409
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Fig. 1 The Fc-binding antibody-recruiting molecules used in this study. (A) Schematic representation of the mechanism of action of Fc-ARMs that
target folate receptor-positive cancer cells. (B) Molecular structures of the Fc-ARMs used.
that possesses a bicyclic Fc-III-4C peptide (Kd value is
compared with the parental 15-Lys8Leu (kon = 1.5 × 106 M−1
s−1, koff = 0.012 s−1).19 This may be caused by the bulkiness
and flexibility of the additional FA and linker moieties that
suppress fast association of the peptide and interact with the
antibody surface after the peptide binds to the Fc region.
reportedly 2.45 nM)20 as an Fc-binder.17
Results and discussion
Synthesis and evaluation of Fc-affinity
Reo-3 was synthesized by the general Fmoc-based solid-phase
peptide synthesis method,21 purified by reversed-phase high-
performance liquid chromatography (RP-HPLC, Fig. S1†) and
identified by electrospray ionization mass spectrometry (ESI-
MS). The intramolecular disulfide bond in Reo-3 was simply
formed by DMSO oxidation of the crude product, which
facilitates a single purification step that differs from the Fc-
III-4C-based peptide19 with two disulfide bonds. We first
evaluated the Fc affinity of Reo-3 by surface plasmon
resonance (SPR) binding analysis (Fig. 2). The humanized
IgG1 monoclonal antibody, trastuzumab, was immobilized
onto SPR chips, and Fc-ARMs were flowed over the chip
surface. The result showed that Reo-3 has a Kd of 5.8 nM
against the Fc region, which is very close to that of Fc-ARM2
(Kd = 6.1 nM).17 The binding affinity of Reo-3 is 1.4 times
higher because of the slower association and dissociation
steps (kon = 9.0 × 105 M−1 s−1, koff = 0.0052 s−1) when
Antibody recruitment
We next tested if Reo-3 recruits antibodies to FR-positive
IGROV-1 cancer cells. Fluorescent microscopy revealed that
Reo-3 successfully recruited human IgG antibodies labeled with
FITC (IgG-FITC) to IGROV-1 cells (Fig. 3A). Excess FA
diminished the fluorescence on the cell surface, indicating that
antibody recruitment is mediated by specific binding of Reo-3
to the FR. We also quantitatively evaluated the efficiency of
antibody recruitment by Reo-3 using flow cytometry. The
results showed that Reo-3 recruits antibodies to IGROV-1 cells
with similar efficiency when compared with that of Fc-ARM2
(Fig. 3B), in accord with the aforementioned Kd values.
Cell-mediated cytotoxicity
Finally, we evaluated the ADCC efficacy of antibodies
recruited by Reo-3. IGROV-1 cells were first treated with 10
nM of an Fc-ARM (Fc-ARM2 or Reo-3) and 100 nM of
intravenous immunoglobulin (IVIG, a clinically used mixture
of human IgG from donor sera). Then, IGROV-1 cells (target)
were co-cultured with natural killer (NK) cells (effector) at
indicated effector/target ratios for 16 h. Lactose
dehydrogenase (LDH) released from lysed target cells was
quantified (Fig. 4). “Reo-3” and “IVIG” did not induce ADCC.
In contrast, both “Fc-ARM2 + IVIG” and “Reo-3 + IVIG”
showed similar efficacy of target cell lysis in an effector/target
ratio-dependent manner. Excess FA reduced cytotoxicity of
Reo-3 + IVIG significantly. Taken together, these results
demonstrate that Reo-3 can recruit endogenous antibodies to
eliminate cancer cells in vitro, and the efficacy is comparable
with antibodies recruited by bicyclic Fc-ARM2.
Fig. 2 SPR measurement of the Fc affinity of Reo-3. Equilibrium
dissociation constant (Kd) of Reo-3 to trastuzumab was determined by
SPR analysis. kon and koff are also shown.
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Fig. 3 Antibody recruitment to FR+ IGROV-1 cells by Reo-3 and Fc-ARM2. (A) IgG-FITC (500 nM), Reo-3 (100 nM) and FA (100 μM) were used.
IgG-FITC, IgG-FITC + Reo-3, or IgG-FITC + Reo-3 + excess FA were added to the IGROV-1 cells and incubated. After a washing step and staining
with Hoechst 33342, the cells were analyzed by fluorescent microscopy. Scale bar = 20 μm. (B) The IGROV-1 cells were treated with 10 nM
of Fc-ARM (Fc-ARM2 or Reo-3) and increasing concentrations of IgG-FITC (1 to 100 nM). After washing, the cells were analyzed by flow
cytometry (n = 3, mean
SEM). Statistical analyses were carried out using two-tailed Welch's t-test. N.S. = not significant.
application studies targeting other molecules and diverse
derivatization for discovery of more potent Fc-ARMs.
Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgements
This work was in part supported by the Japanese Society for
the Promotion of Sciences (JSPS) KAKENHI Grant-in-Aid for
Challenging Research (Exploratory) (Grant number: 18K19148
to Y. K.), and Grant-in-Aid for Scientific Research (B) (Grant
number: 19H03356 to Y. H.). K. S. was supported by the
Research Fellowship for Young Scientists (JSPS, 17J05032) and
Advanced Graduate Course on Molecular Systems for Devices
(Kyushu University). We thank Dr. S. Kishimoto and Dr. Y. Ito
(Kagoshima University) for assistance with SPR experiments.
We thank the Edanz Group (https://en-author-services.
edanzgroup.com/ac) for editing a draft of this manuscript.
Fig. 4 Antibody-dependent cell-mediated cytotoxicity assay. IGROV-
1 cells (5000 cells/well) were treated with 10 nM Fc-ARM (Fc-ARM2
or Reo-3) and 100 nM IVIG. Then, 5000–40 000 cells/well of KHYG-
1/CD16a-158 V cells were added and the cells were co-cultured for
16 h. LDH released from lysed cells was quantified (n = 3, mean
SEM). FA (10 μM) was used to inhibit the binding of Fc-ARMs to the
FR. Statistically significant differences between Fc-ARM (Fc-ARM2 or
Reo-3) + IVIG and all of the other groups were observed at all
effector/target ratios. Statistical analyses were carried out using one-
way ANOVA with Tukey's multiple comparison test. **p < 0.01; N.S.
= not significant.
Notes and references
Conclusions
1 G. M. Bartelds, C. A. Wijbrandts, M. T. Nurmohamed, S.
Stapel, W. F. Lems, L. Aarden, B. A. Dijkmans, P. P. Tak and
G. J. Wolbink, Ann. Rheum. Dis., 2007, 66, 921–926.
2 T. T. Hansel, H. Kropshofer, T. Singer, J. A. Mitchell and A. J.
George, Nat. Rev. Drug Discovery, 2010, 9, 325–338.
3 M. E. Cromwell, E. Hilario and F. Jacobson, AAPS J., 2006, 8,
E572–E579.
In summary, we developed a new Fc-ARM named Reo-3, which
contains a monocyclic Fc-binding peptide 15-Lys8Leu. Reo-3
showed strong affinity for the Fc region of the human IgG1
antibody (Kd = 5.8 nM). Reo-3 recruited IVIG to induce ADCC
against FR-positive cancer cells as effective as Fc-ARM2, which
has
a bicyclic Fc-binding peptide. 15-Lys8Leu is easily
synthesized because of its relatively short amino acid sequence
and monocyclic structure, and is one of the strongest binding
peptides to the Fc region.19 Thus, 15-Lys8Leu has significant
potential for use in various applications, including the
development of Fc-ARMs and non-covalent antibody-drug
conjugates (ADCs),22 purification of antibodies and
preparations of homogenous ADCs.23 The straightforward
synthesis of the pivotal unit of the Fc-ARM should accelerate
4 P. J. McEnaney, C. G. Parker, A. X. Zhang and D. A. Spiegel,
ACS Chem. Biol., 2012, 7, 1139–1151.
5 C. R. Bertozzi and M. D. Bednarski, J. Am. Chem. Soc.,
1992, 114, 2242–2245.
6 J. Li, S. Zacharek, X. Chen, J. Wang, W. Zhang, A. Janczuk
and P. G. Wang, Bioorg. Med. Chem., 1999, 7, 1549–1558.
7 K. P. Naicker, H. Li, A. Heredia, H. Song and L.-X. Wang,
Org. Biomol. Chem., 2004, 2, 660–664.
408 | RSC Med. Chem., 2021, 12, 406–409
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RSC Medicinal Chemistry
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8 M. F. Perdomo, M. Levi, M. Sällberg and A. Vahlne, Proc.
Natl. Acad. Sci. U. S. A., 2008, 105, 12515–12520.
9 C. G. Parker, R. A. Domaoal, K. S. Anderson and D. A.
Spiegel, J. Am. Chem. Soc., 2009, 131, 16392–16394.
10 Y. Lu and P. S. Low, Cancer Immunol. Immunother., 2002, 51,
153–162.
17 K. Sasaki, M. Harada, Y. Miyashita, H. Tagawa, A.
Kishimura, T. Mori and Y. Katayama, Chem. Sci., 2020, 11,
3208–3214.
18 K. Sasaki, M. Harada, T. Yoshikawa, H. Tagawa, Y. Harada,
Y. Yonemitsu, T. Ryujin, A. Kishimura, T. Mori and Y.
Katayama, ChemBioChem, 2020, 21, 1–6.
11 R. M. Owen, C. B. Carlson, J. Xu, P. Mowery, E. Fasella and
L. L. Kiessling, ChemBioChem, 2007, 8, 68–82.
19 K. Muguruma, K. Fujita, A. Fukuda, S. Kishimoto, S.
Sakamoto, R. Arima, M. Ito, M. Kawasaki, S. Nakano, S.
Ito, K. Shimizu, A. Taguchi, K. Takayama, A. Taniguchi,
Y. Ito and Y. Hayashi, ACS Omega, 2019, 4,
14390–14397.
12 C. B. Carlson, P. Mowery, R. M. Owen, E. C. Dykhuizen and
L. L. Kiessling, ACS Chem. Biol., 2007, 2, 119–127.
13 M. K. O'Reilly, B. E. Collins, S. Han, L. Liao, C. Rillahan, P. I.
Kitov, D. R. Bundle and J. C. Paulson, J. Am. Chem. Soc.,
2008, 130, 7736–7745.
20 Y. Gong, L. Zhang, J. Li, S. Feng and H. Deng, Bioconjugate
Chem., 2016, 27, 1569–1573.
14 R. P. Murelli, A. X. Zhang, J. Michel, W. L. Jorgensen and
D. A. Spiegel, J. Am. Chem. Soc., 2009, 131, 17090–17092.
15 A. Dubrovska, C. Kim, J. Elliott, W. Shen, T.-H. Kuo, D.-I.
Koo, C. Li, T. Tuntland, J. Chang, T. Groessl, X. Wu, V.
Gorney, T. Ramirez-Montagut, D. A. Spiegel, C. Y. Cho and
P. G. Schultz, ACS Chem. Biol., 2011, 6, 1223–1231.
16 C. E. Jakobsche, P. J. McEnaney, A. X. Zhang and D. A.
Spiegel, ACS Chem. Biol., 2012, 7, 316–321.
21 C. D. Chang and J. Meienhofer, Int. J. Pept. Protein Res.,
1978, 11, 246–249.
22 K. Muguruma, F. Yakushiji, R. Kawamata, D. Akiyama, R.
Arima, T. Shirasaka, Y. Kikkawa, A. Taguchi, K. Takayama, T.
Fukuhara, T. Watabe, Y. Ito and Y. Hayashi, Bioconjugate
Chem., 2016, 27, 1606–1613.
23 S. Kishimoto, Y. Nakashimada, R. Yokota, T. Hatanaka, M.
Adachi and Y. Ito, Bioconjugate Chem., 2019, 30, 698–702.
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