New fluorine-18 pretargeting PET imaging by bioorthogonal chlorosydnone-cycloalkyne click reaction

Article abstract of DOI:10.1039/c9cc05486c

We report the first pretargeting in vivo study using the Strain-Promoted Sydnone-Alkyne Cycloaadition (SPSAC) reaction. The injection of a fluorine-18 labeled cyclooctyne three days after cetuximab bearing chlorosydnone moieties allowed a significant detection of the tumor by PET imaging suggesting an efficient click reaction inside the tumoral site. With a kinetic constant superior to 300 M^-1 s^-1, the SPSAC reaction might be an interesting tool, in addition to tetrazine-cyclooctene ligation, for in vivo chemistry.

Full text of DOI:10.1039/c9cc05486c

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New fluorine-18 pretargeting PET imaging by bioorthogonal
chlorosydnone-cycloalkyne click reaction
Mylène Richard,a Charles Truillet,a Vu Long Tran,a Hui Liu,b Karine Porte,b Davide Audisio, b Mélanie
Roche,a Benoit Jego,a Sophie Cholet,c François Fenaille,c Bertrand Kuhnast,a Frédéric Taranb* and
Simon Specklina*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
We report the first pretargeting in vivo study using the Strain-Promoted Sydnone-Alkyne Cycloaadition (SPSAC) reaction.
The injection of a fluorine-18 labeled cyclooctyne three days after Cetuximab bearing chlorosydnones moieties allowed a
significant detection of the tumor by PET imaging suggesting efficient click reaction inside the tumoral site. With a kinetic
constant superior to 300 M-1.s-1, the SPSAC reaction might be an interesting tool, in addition to the tetrazine-cyclooctene
ligation, for in vivo chemistry.
www.rsc.org/
two partners, the ligation reaction itself is critical for the success
of this approach and has to be as fast and selective as possible.
Introduction
Since the first reports involving the concept of bioorthogonal
chemistry by the group of Bertozzi,1 the challenge posed by the
When considering bioorthogonal reactions, the biggest player
in this field is undoubtedly the Inverse Electron Demand Diels
Alder (IEDDA, Scheme 1b) cycloaddition with tetrazines and
trans-cyclooctenes that has been particularly studied for
pretargeting since its first in vivo demonstration by the group of
Robillard.5 Characterized by very high reaction rates, the IEDDA
cycloaddition has been successfully applied in animal models
for both imaging6 and radioimmunotherapy7 of several
oncological targets with an associated dosimetry that bears no
comparison with the conventional approach.
compatibility of an organic reaction with
a biological
environment has tickled the creativity of researchers, resulting
in the development of fast and highly specific bioorthogonal
reactions enabling new applications in chemical biology.2
However, out of this arsenal of bioorthogonal transformations,
only a few have been successfully applied to living animals.3 To
cross this ultimate barrier, the considered ligation must gather
various requirements such as high reaction rate, total
chemoselectivity, metabolic stability and low toxicity.
One of the most prominent application of in vivo bioorthogonal
reactions is the pretargeting approach used in nuclear medicine
for antibody-based diagnosis and therapy.4 Antibodies are
characterized by a very slow in vivo pharmacokinetics and often
reach their target after one to several days after injection. For
routine nuclear imaging, injection of radiolabeled antibodies is
restricted to isotopes with decay half-lives matching their
distribution time, thus inducing unnecessary radiation exposure
to the patient. The pretargeting strategy involves the in vivo
ligation of a radiolabeled molecule to the antibody injected
several days before, thus allowing to strongly decrease the
isotope biological residence time and therefore the associated
radiation exposure. In addition to biodistribution issues of the
a) Strain promoted cycloaddition of 4-chloro-sydnones with cyclooctynes
O
O
Cl
Cl
CO2
O
CO2H
4
CO2H
4
N
25 °C
N
HO2C
N
+
N
HO2C
O
N
N
Blood plasma
k = 343 ± 41 M-1. s-1
b) Bioothogonal reaction in mAb pretargeting
103-104
101-102
102-103
10-1-100
10-3
second order
reaction rate
(M-1 s-1
)
N
N
N
N
N
N
N
Cl
N
N
N
N
N
N
N
N
N
O
O
RO2
C
PPh2
R
R
Staudinger
ligation
SPAAC
IEDDA
SPSAC
IEDDA
validated in
mAb
pretargeting
this work
a. UMR 1023 IMIV, Service Hospitalier Frédéric Joliot (SHFJ), CEA, Inserm,
Université Paris Sud, CNRS, Université Paris-Saclay, Orsay, France.
Email: simon.specklin@cea.fr
b. Service de Chimie Bio-organique et Marquage DRF-JOLIOT-SCBM, CEA,
Université Paris-Saclay, 91191 Gif-sur-Yvette (France)
Scheme 1 Strategies for antibody pretargeting. SPAAC: Strain-Promoted Azide-
Alkyne Cycloaddition; IEDDA : inverse-Electron-Demand Diels–Alder tetrazine-
cyclooctene
cycloaddition;
SPSAC:
Strain-Promoted
Sydnone-Alkyne
Cycloaddition.
Email: frederic.taran@cea.fr
c. Service de Pharmacologie et d’Immunoanalyse (SPI), CEA/DRF/JOLIOT,
Université Paris Saclay, F-91191 Gif-sur-Yvette, France.
†Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
The requirement of such high kinetics for pretargeting
applications is still an open question as the much slower IEDDA
cycloaddition of tetrazines and the Reppe anhydride also
This journal is © The Royal Society of Chemistry 20xx
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provided an efficient pretargeted PET imaging of epidermal lipophilicity was thus designed and prepared fromV1iebwyAratilcilpe hOanltinice
growth factor receptor (EGFR) receptors.8 Nonetheless, slower substitution with fluorine-18 (Scheme 2). Both the precursor 1
DOI: 10.1039/C9CC05486C
reactions such as the Strain-Promoted Azide-Alkyne and the non-radioactive reference 2 were obtained in a
Cycloaddition (SPAAC) and the Staudinger ligation were found straightforward synthesis of 4 and 5 steps respectively (see SI).
unsuccessful for in vivo pretargeting,9 delineating the kinetic We studied the radiofluorination of 1 with K[18F]F/K222 complex
cut-off for this application.
and identified the best conditions to reach satisfactory
With the aim of finding reactions with a potential for conversion without pronounced degradation. These conditions
bioorthogonal chemistry, we identified sydnones as a promising were implemented in a fully automated synthesis including
dipole scaffold for copper-catalyzed cycloaddition with radiofluoration of 1, purification of [18F]-2 by semi-preparative
alkynes.10 Based on this work, we11 and others12 highlighted the HPLC and formulation in ethanol. This method allows the
reactivity of these mesoionics with strained alkynes for a production of the cyclooctyne [18F]-2 at high activity levels with
copper-free Strain-Promoted Sydnone Alkyne Cycloaddition good radiochemical yield and excellent radiochemical purity (≥
(SPSAC) reaction. Sydnones can easily be tuned up to 95%).
outperform the reaction rate of the SPAAC reaction. Notably,
the presence of an halogen atom in position 4 of the mesoionic
ring has a spectacular beneficial effect on the reaction rate.11,13
O
N
OTs
Particularly, 4-chloro-sydnones and dibenzocyclooctynes
N
O
O
H
2
(DBCO) appears as a promising combination of reactants to
perform very fast ligation reactions. In preliminary experiments
we indeed observed fast SPSAC reaction in blood plasma, with
second order rate constant up to 343 M-1.s-1 (Scheme 1a) which
is more than 100-times faster than the SPAAC reaction with
azides. Furthermore, despite lower kinetics than IEDDA, SPSAC
involve stable and readily accessible reactive partners and represents
thus an interesting alternative to the use of strained alkene. In
particular, 4-chloro-sydnones can be easily obtained in a few steps
and are stable for days in biological media.
1
2
A
17% RCY
UV
K[18F]F-K222
MeCN
(decay-corrected)
4,13 GBq after 80 min
SA : 124 ± 9 GBq/µmol
(typical synthesis)
15 min, 90 °C
[
[
18F]-2
18F]-2
B
C
UV
O
N
O
18F
N
O
2
H
RAD
[
18F]-2
Scheme 2 Synthesis of [18F]-2. (A) UV chromatogram of non-radioactive reference
2, (B) UV chromatogram of [18F]-2, (C) radioactive chromatogram of [18F]-2.
In the present work, we report the first evaluation of in vivo
SPSAC with 4-chloro-sydnones and cyclooctynes for
pretargeting imaging. For this study, we selected Cetuximab
pretargeting in nude mice xenograft model based on fluorine-
18 PET imaging. This clinical monoclonal antibody targets the
extracellular domain of EGFR overexpressed in several cancers.
Despite being rather challenging due to its internalization over
time, Cetuximab stands as a suitable benchmark for our study
as biodistribution data in murine models by 89Zr PET imaging 14
and pretargeting imaging results using different strategies are
available on this antibody.15
We then focused on the preparation of a 4-chloro-sydnone-
modified Cetuximab and designed compound 4 as a suitable
coupling reagent to be conjugated with the antibody. A short
PEG linker separating the 4-chloro-sydnone core from the
attachment point was considered to bring flexibility in order to
maximize the accessibility of the sydnone for SPSAC reaction.
Conjugation of 4 to lysine residues of Cetuximab was performed
according to standard conditions. Different amounts of the
activated ester of 4 were considered and an optimum of 100
equivalents of activated ester relative to Cetuximab provided
satisfactory results. Purification by filtration on steric-exclusion
column furnished the pure Cetuximab conjugate CTX-Syd,
which was analyzed by MALDI-TOF mass spectrometry (Fig. SI.
3). An average number of eight 4-chloro-sydnones per antibody
was found, which represents the modification of about 10% of
the total number of lysine residues of Cetuximab.
Results and discussion
The first step was to select the reactive partner to be
conjugated to the antibody and the one to be radiolabeled.
When looking at the reported results on TCO/tetrazine-based
pretargeting strategies, both combinations were deployed with
little rationale beside their related pharmacokinetics and
metabolic stability. However, labeling
a
compound
functionalized with a 4-chloro-sydnone could require a complex
multistep radiosynthesis as sydnones were described as
valuable leaving groups toward radiofluorination.12c Regarding
the many existing prosthetic groups derived from radiolabeled
cyclooctynes,16 we then decided to involve a cyclooctyne
labeled with fluorine-18 as the radioactive effector in
conjunction with Cetuximab conjugated to 4-chloro-sydnones.
[18F]-2 bearing a short PEG linker to compensate the DBCO
We next considered the SPSAC reaction between CTX-Syd and
[18F]-2 in vitro. Several parameters such as antibody
concentration, reaction time and temperature have been
modulated to determine the optimum conjugation conditions.
In our hands, reaction at 37 °C for 1 h of [18F]-2 (6.5 µM, 1 equiv.,
24.1 MBq) with CTX-Syd (8.9 µM, 1.4 equiv.) gave the best
conjugation yields. Purification by size-exclusion provided
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radiofluorinated Cetuximab [18F]-CTX-Pyr with a 48 % labeling
yield. SDS-PAGE analysis indicated that both light and heavy
chains of the antibody were radiolabeled, with a ratio of 1.8 in
favour of the heavy chain (Scheme 3). Control experiments with
unmodified Cetuximab showed no incorporation of [18F]-2, thus
validating the selectivity of the SPSAC reaction.
intestine, suggesting
a
hepato-biliary eliminationViewoAfrtic[le18OFn]l-in2e,
independently of the presence of theDOI: 10.1039/C9CC05486C
antibody. A blocking
experiment was also conducted to confirm the specific labeling of the
antibody at the tumor site. The mice were injected with a mixture of
CTX-Syd (4 µg/g) and an 20 fold excess of unmodified Cetuximab (80
µg/g), followed by injection of [18F]-2 three days later. No significant
activity was observed in the tumor at 4 h post-injection with a T/M
ratio of 1.2 (±0.4), matching the tumoral uptake of the
biodistribution of [18F]-2 alone. The in vivo blocking thus confirmed
the EGFR specific tumoral uptake and afforded the proof of concept
for the pretargeting using SPSAC bioorthogonal reaction.
Scheme 3 Preparation and characterization of Cetuximab conjugates.
We then evaluated whether the modifications introduced to the
antibody would affect its affinity for the targeted EGFR receptors
prior to in vivo pretargeting experiments. This control was performed
by a standard Lindmo excess antigen method involving the in-vitro
labeled [18F]-CTX-Pyr and EGFR overexpressing cells A431.17 An
immunoreactive fraction > 90% was observed, indicating a retention
of the affinity of the modified antibody CTX-Syd toward its target
(Fig. SI. 4).
We then performed the pretargeting experiment consisting in
injecting first CTX-Syd to the tumor-bearing mice, followed by
injection of [18F]-2 in a second phase. A period of 3 days between the
two injections was set to reach the maximal uptake of the antibody
in the tumor, as determined by the pharmacokinetics of 89Zr labeled
Cetuximab (see SI, Fig SI. 5). After injection of CTX-Syd (4 µg/g), [18F]-
2 was administered 3 days later and the mice were imaged by a
dynamic scan during 1 h followed by a static scan 4 h post-injection.
A progressive accumulation of activity was observed in the tumor
with Tumor-to-Muscle ratio (T/M) significantly increasing from 1.1
(±0.1) after 30 min to 2.3 (±0.3) at 4 h post-injection.
Several controls were achieved to link this accumulation of activity
with the pretargeting by SPSAC. First, the biodistribution of [18F]-2 in
mice without injection of antibody showed a T/M ratio of 1.4 (±0.3)
at 4 h post-injection, indicating low non-specific tumoral uptake of
the labeled cyclooctyne. Similarly to the pretargeting experiment,
most of the residual activity was found in the spleen and the
Figure 1 PET imaging data of the in vivo targeting of CTX-Syd with [18F]-2 by
SPSAC reaction in nude mice. A. PET images of [18F]-2 alone, with CTX-Syd and
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with a large excess of Cetuximab (blocking) at 4 h post injection. B. Biodistribution
in % of injected dose per cm3 (%ID/cc) 4 h post injections. C. Ratio tumor over
muscle at 4 h. *P<0.05.
7
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Conclusions
In conclusion, we have reported the first use of SPSAC reaction
in a pretargeting proof of concept study for PET imaging. We
demonstrated that the therapeutic antibody Cetuximab
modified with chlorosydnones is able to react in vivo with a
fluorine-18 labeled cyclooctyne leading to significant tumoral
radiolabeling. Further work to optimize the pharmacokinetics of
the labeled cyclooctyne should provide improved image
contrast, leading to a new practical tool for pretargeting
imaging.
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Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by CEA (program Bottom-up) and the
French Research National Agency (ANR-14-CE06-0004).
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DOI: 10.1039/C9CC05486C
New fluorine-18 pretargeting PET imaging by bioorthogonal
chlorosydnone-cycloalkyne click reaction
Mylène Richard,a Charles Truillet,a Vu Long Tran,a Hui Liu,b Karine Porte,b Davide Audisio, b Mélanie
Roche,a Benoit Jego,a Sophie Cholet,c François Fenaille,c Bertrand Kuhnast,a Frédéric Taranb* and
Simon Specklina*
A PET pretargeting approach using the Strain-Promoted Sydnone-Alkyne Cycloaddition.
Tumor