Development of a novel antibody-tetrazine conjugate for bioorthogonal pretargeting

Article abstract of DOI:10.1039/c6ob01411a

Recently, bioorthogonal chemistry based on the Inverse Electron-Demand Diels-Alder (IEDDA) cycloaddition between 1,2,4,5-tetrazines and trans-cyclooctene (TCO) analogues added an interesting dimension to molecular imaging. Until now, antibodies (Abs) were

Full text of DOI:10.1039/c6ob01411a

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L. wyffels and K. Augustyns, Org. Biomol. Chem., 2016, DOI: 10.1039/C6OB01411A.
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Organic & Biomolecular Chemistry
ARTICLE
DEVELOPMENT OF A NOVEL ANTIBODY-TETRAZINE CONJUGATE
FOR BIOORTHOGONAL PRETARGETING
Agnese Maggi,a Eduardo Ruivo,a Jens Fissers,a Christel Vangestel,b Sneha Chatterjee,c Jurgen
Joossens,a Frank Sobott,c Steven Staelens,b Sigrid Stroobants,b,d Pieter Van der Veken,a Leonie
wyffels,b,d Koen Augustyns,a†
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Abstract. Recently, bioorthogonal chemistry based on the Inverse Electron-Demand Diels-Alder (IEDDA) cycloaddition
between 1,2,4,5-tetrazines and trans-cyclooctene (TCO) analogues added an interesting dimension to molecular imaging.
Until now, antibodies (Abs) were tagged with TCO and after pretargeting were reacted with tetrazines substituted with
reporters. However, TCO tags have the tendency to degrade under physiological conditions, and due to their hydrophobic
nature bury within the protein. This results in loss of reactivity and a low Ab functional loading. To circumvent these
problems, we report for the first time an approach in which tetrazines are used as tags for antibody (Ab) modification, and
TCO as imaging agent. We developed a new Ab-tetrazine conjugate which displays high functional loading, good stability
and reactivity. We utilized this immunoconjugate for live-cell imaging together with novel TCO probes, resulting in the
selective and rapid labeling of SKOV-3 cells. Our approach may be useful for in vivo pretargeted imaging.
between the bioorthogonal tag and an externally introduced
chemical probe (targeting step).
Introduction
A
well-established bioorthogonal reaction is the Inverse
Molecular imaging is a powerful tool which enables the non-
invasive visualization and quantification of biological processes
occurring at cellular level. Recently, the possibility to combine
molecular imaging with bioorthogonal chemistry has strongly
improved the applicability of this technique for in vitro and in vivo
imaging under different modalities.
Electron-Demand Diels-Alder (IEDDA) cycloaddition between
1,2,4,5-tetrazines and trans-cyclooctene (TCO). This reaction was
4–6
introduced in 2008
and is currently investigated for many
7
8,9
applications.
In fact, its extremely fast kinetics
enables
application for in vivo studies where reactions must occur rapidly at
micro- to nanomolar concentrations.
Generally, the term “bioorthogonal chemistry” refers to any
chemical reaction that can occur inside a living system without
interfering with native biochemical processes, and permits the
Noteworthy, tetrazine/TCO ligation has been used for
10–13
pretargeted imaging of tumors both in living cells
14–16
and in mice.
In a typical bioorthogonal pretargeting approach the target
(e.g. cell surface antigen) is first bound by a TCO-modified antibody
(Ab), and then a tetrazine probe is provided in order to achieve the
labeling through the cycloaddition reaction.
covalent attachment of a probe molecule to a biomolecule of
1–3
interest.
In order to achieve bioorthogonal ligation two
successive steps are required: (1) introduction of a bioorthogonal
tag onto the biological target (pretargeting step), and (2) reaction
Different TCO-Ab conjugates have been developed in the last
years. These conjugates can be obtained either via the reaction
aLaboratory of Medicinal Chemistry, University of Antwerp, Belgium
bMolecular Imaging Center Antwerp, University of Antwerp, Antwerp, Belgium
cBiomolecular and Analytical Mass Spectrometry, University of Antwerp, Belgium
d Antwerp University Hospital, Department of Nuclear Medicine, Edegem, Belgium
†Correspondence: koen.augustyns@uantwerpen.be
between TCO-N-hydroxysuccinimide (TCO-NHS) esters and the Ab
17,18
lysine residues,
or via a two-step procedure where the Ab is
first decorated with azide functionalities and subsequently reacted
19
with dibenzylcyclooctyne-TCO derivatives (DBCO-TCO).
Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
Moreover, the TCO moieties can be attached to the Ab either
directly or through short PEG spacers. Even though the type of
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attachment does typically not affect the Ab binding affinity, which is 3,6-di-(2-pyridyl)-1,2,4,5-tetrazines, commonly eVmiepwloArytieclde Onlianes
usually retained even at high TCO loadings, it can influence the bioorthogonal probes, exhibit fast kineticsDbOuIt: 1a0r.1e03s9u/sCc6eOptBi0b1le411tAo
effective number of reactive TCO moieties. Recently, Haun and co- degradation in biological environments. 11,29
workers have shown that when hydrophobic TCOs are directly
Here we present the synthesis and characterization of a series
attached to the antibody, up to 90% are not reactive due to their of tetrazines bearing diverse substituents. We evaluated their
tendency to bury within the protein. In contrast, the introduction of stability and reactivity for the IEDDA reaction with (E)-cyclooct-4-
TCOs through hydrophilic PEG linkers results in a fully preserved enol and validated the use of these compounds for antibody
19
reactivity.
Previous to this study, Robillard and co-workers labeling. Most importantly, we developed a novel Ab-tetrazine
described an in vivo deactivation of TCO via isomerization to the conjugate which displays a good long-term physiological stability,
unreactive cis-cyclooctene (CCO). Isomerization is probably high functional density, and fast kinetics with TCO modified-
catalyzed by copper bound to serum proteins, and its extent fluorophores. Furthermore, we demonstrate the effectiveness of
depends on the degree of exposure of the TCO tags on the Ab the Ab-tetrazine conjugate using pretargeted cancer cell studies as
surface. Particularly, it has been demonstrated that the attachment validation platform.
of TCOs through PEG linkers results in a shorter half-life of the tags
compared to direct attachment. 18
Results and discussion
Attaining a high Ab functional density is an important feature
for the application of bioorthogonal pretargeting in animal models,
where a high target-to-background ratio is desirable. The latter may
be significantly reduced by both hydrophobic burying and tag
exposure deactivation. Due to the slow clearance of intravenously
injected Abs, it can take several days to obtain an optimal
biodistribution resulting in a prolonged exposure of the TCO tags to
physiological environment with consequent deactivation.
Chemical Synthesis of 1,2,4,5-Tetrazine Derivatives
The first step towards our goal was the development of a
tetrazine derivative exhibiting an optimized compromise in terms of
reactivity and stability. To this end we investigated the effect of
different substituents on the tetrazine core and developed a total of
8
compounds (Figure 1). Furthermore, all compounds were
designed to possess a functional conjugation handle for antibody
modification, such as a 5-oxopentanoic or a propionic acid chain.
Compounds 1-5 where prepared following a modification of a
previously reported procedure which entails the reaction between
nitrile and amidine precursors with hydrazine monohydrate in
Replacement of TCO tags with more hydrophilic tetrazines
(inversion of functionalities) could offer a valid alternative to
conventional Ab-pretargeting strategies, and may address
hydrophobic burying. Moreover, with respect to future in vivo
applications, the use of more hydrophobic labeled TCOs for
targeted imaging might have an advantage to labeled tetrazines
when cell membranes or the blood-brain-barrier need to be crossed
To date, the use of tetrazines as bioorthogonal tags for
antibody-pretargeting has not been investigated. However, several
presence of a catalyst (sulfur or zinc triflate), followed by oxidation
30
of the dihydrotetrazine intermediates (Figure 1A).
After
purification, tetrazines 1-5 were obtained in yields ranging from 11
to 40%. The comparatively moderate yields apparently derive from
the use of hydrazine monohydrate, which is more convenient and
safe to use, instead of anhydrous hydrazine that was employed in
the original procedure. 30
studies demonstrated the utility of tetrazines as tags for
20–23
pretargeted protein labeling,
and construction of labeled
peptides. 24–26 Yi and co-workers reported the use of a Ab-tetrazine
The synthesis of compounds 6-8 was performed through a two-
step protocol, where tetrazines bearing thiomethyl- or 3,5-
dimethyl-1-pyrazol-1-yl (DMP) leaving groups were subjected to
nucleophilic substitution with 4-aminobenzylamine. The resulting
amino-derivatized tetrazine intermediates where subsequently
reacted with glutaric anhydride to yield the final products in good
overall yields (from 54 to 76% over two steps) (Figure 1B).
27
conjugate for protein capture. Han et al. described Ab-quantum
dot (QD) conjugates, which were assembled by reacting Ab-
28
tetrazine conjugates with norbornene-coated QDs. A challenge
for the use of tetrazine-Ab conjugates for bioorthogonal-
pretargeting is the development of tetrazines displaying long term
stability under physiological conditions and, at the same time, high
reactivity for the IEDDA reaction. Monoaryl-1,2,4,5-tetrazines and
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Figure 1. Synthesis of tetrazine derivatives and TCO fluorescent dyes
View Article Online
DOI: 10.1039/C6OB01411A
R1
R1
O
O
A
X
NH
NH2
R1
=
OH
O
i, ii
N
OH
3
,
H
+
R2
X
N
N
N
N
X = C or N
N
R2
R2 = -Me, -H,
1-5
O
O
HN
OH
3
LG
NH
N
LG = -SMe,
R1 = -Me, -H,
DMP
N
i, ii
H2N
+
B
N
N
N
N
N
N
DMP
N
N
NH2
R1
R1
6-8
SO3
N
HO
R =
O
O
H
O
O
H
H
N
O
i
H
H
C
N
O
H
N
OH
O
NH2
O
N
R
SO3
H
12
13
16
(A) Synthesis of tetrazine 1-5, reaction conditions: (i) Zn(OTf)2 or S8, NH2NH2·H2O, EtOH, r.t.; (ii) NaNO2, HCl 1M, H2O, 0 °C; (B) Synthesis of tetrazine 6-7,
reaction conditions: (i) MeOH or toluene, r.t., Ar; (ii) glutaric anhydride, THF, reflux; (C) Synthesis of TCO dyes 12 and 13, reaction conditions: (i) Dye-NHS
ester, DMF, TEA, r.t., N2.
Tetrazine Stability and Reaction Kinetics with (E)-cyclooct-4-enol
remaining intact after 4 days. On the other hand, for aryl
The stability of the newly synthesized compounds was substituted tetrazines (compounds 1-5) the stability was
investigated next. Tetrazines 1-8 were incubated in either considerably reduced by replacing a methyl group with a hydrogen
phosphate buffered-saline (PBS) or pure fetal bovine serum (FBS) at on the tetrazine core, and by the presence of electron withdrawing
37 °C, and the decrease of the tetrazine absorbance at selected groups on the aryl substituent. The least stable compounds turned
wavelengths was monitored. The proportion of intact tetrazine was out to be tetrazines 2 and 10 with measured half-lives in PBS of 2
determined after four days (see Table 1). The stability of tetrazines and 9 h respectively (Table 1).
9 and 10, which are commonly used for bioorthogonal studies and
The reactivity of tetrazines 1-9 towards the IEDDA reaction with
thus serve as reference control, was also evaluated and compared (E)-cyclooct-4-enol was also evaluated (Table 1). Since the rate of
to that of the newly synthesized compounds. As could be predicted the reaction between compounds 1-5 and the dienophile was too
tetrazines containing electron donating groups (compounds 6-8) fast for reliable determination via UV-vis experiments, the reaction
turned out to be very stable in either PBS or FBS, with up to 100% rates were measured by 1H-NMR competition experiments with 3,6-
di-2-pyridyl-1,2,4,5-tetrazine (DIPY).
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Table 1. Stability data and second order rate constants for tetrazines 1-10
View Article Online
DOI: 10.1039/C6OB01411A
O
O
O
O
O
O
O
O
O
OH
HN
OH
3
HN
OH
HN
OH
HN
OH
3
3
3
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
1
2
5
3
4
O
O
O
O
O
O
O
N
O
HN
OH
HN
OH
3
3
HN
OH
3
H
N
OH
HN
OH
3
3
O
O
NH
NH
NH
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
6
7
9
8
10
k2 (M-1 s-1)
Reaction with
Stability PBS
% Intact at 4 daysa % Intact at 4 daysa
Stability FBS
Compound
Reaction Solventc
(t½, h)b
(t½, h)b
(E)-cyclooct-4-enol
1
2
48 ± 1
0 (2)
19 ± 4 (16)
-
210 ± 42
902 ± 164
136 ± 54
875 ± 84
95 ± 15
MeOH
MeOH
MeOH
MeOH
MeOH
PBS
3
96 ± 1
41 ± 2
100 ± 2
100 ± 12
98± 2
40 ± 20 (82)
23 ± 15 (26)
84 ± 6
4
5
6
100 ± 6
95 ± 9
0.41 ± 0.02
9.3 ± 0.6
0.31 ± 0.04
807 ± 145
-
7
PBS
8
89 ± 5
34 ± 1
0 (9)
69 ± 30
19 ± 4 (23)
-
PBS
9
MeOH
-
10d
(a) Data normalized to 100% at t = 0 h, and reported as mean value of three measurament ± SD; (b) half life in brackets; (c)
temperature: 25 °C for reactions performed in MeOH, 37 °C for reactions performed in PBS; (d) the k2 of tetrazine 10 could
not be determined due to its poor solubility in MeOH.
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In fact, the second order rate constant (k2) of DIPY for the reaction the model antibody and tagged with tetrazine 3 via standard amine-
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DOI: 10.1039/C6OB01411A
with (E)-cyclooct-4-enol in MeOH is known to be 820 M-1 s-1. 9 After coupling procedure.
mixing DIPY, the dienophile and the selected tetrazine in MeOH the
The tetrazine loading was modulated using two different molar
1H NMR of the mixture was analyzed, and the conversion rates of equivalents (10 and 100) of the amino reactive tetrazine, and the
the two tetrazines determined. From the relative conversion rates number of incorporated tags was determined by quadrupole-time-
the final rate constants were calculated.31
of-flight (Q-TOF) mass spectrometry. To this end, trastuzumab was
Due to their slower kinetics, the cycloaddition rates of used as reference and its mass spectrum compared to that of the
tetrazines 6-8 could be directly measured by UV-Vis experiments. trastuzumab-tetrazine 3 conjugates. As shown in Figure 3A the
The rate constants were determined in PBS at 37°C by reacting the mass spectrum of trastuzumab reveals the presence of a 148 117
different compounds with the dienophile under pseudo first order Dalton (Da) Ab with charge states from 22+ to 27+. For comparison
conditions, and measuring the disappearance of the tetrazine we selected the most intense peak (25+ charge state), and
adsorption at selected wavelengths.
determined that treatment of trastuzumab with 10 equivalents of
From this study we determined that the reactivity of aryl the tetrazine-NHS 11 resulted in the incorporation, on average, of 2
tetrazine 1-5 is inverse to their stability. Whereas, for compounds 6- tetrazine tags per antibody. Whereas, an average of 8 tags was
8 the presence of a hydrogen substituent on the tetrazine ring incorporated after treatment with 100 equivalents (Figure 3B).
strongly affects the reactivity, with tetrazine 7 being 20 to 30 fold
more reactive than tetrazines 6 and 8.
Figure 3. Mass spectra of the trastuzumab-tetrazine 3 conjugates
Overall, the range of reactivities and stabilities covered by
compounds 1-8 make this library a valuable addition to the
tetrazine compounds currently available for bioorthogonal
pretargeting.
From these screenings we conditionally selected tetrazine 3 for
the biological studies. This compound showed the best profile with
regard to stability and reactivity with a half-life of 82 h in serum at
37 °C , and a k2 of 136 M-1 s-1 in MeOH at 25 °C (Table 1).
Noteworthy, the in vitro stability of tetrazine 3 is substantially
higher compared to that of TCO derivatives commonly used for Ab
modification, for which half-lives of circa 3 h were reported in
serum. 18 The low rate constant of compound 3 in MeOH may rise in
aqueous media. In fact, it has been previously demonstrated that
the rate of the IEDDA reaction is accelerated by increasing solvent
(A) Mass spectrum of trastuzumab (148117 Da) with charge states from 22+
to 27+; (B) 25+ charge state of trastuzumab (black) compared to
trastuzumab-tetrazine 3 conjugates after treatment with 10 (red) and 100
(blue) equivalents of tetrazine-NHS 11.
The number of reactive tetrazines per antibody was also
evaluated based on absorbance readings after reaction with an
excess of TCO-fluorescein dye (12). We observed that there are 0.4
and 5 reactive tetrazines for the conjugate bearing 2 and 8 tags,
respectively. Remarkably, 62% of tetrazine moieties resulted active
for the conjugate bearing 8 tags. This result indicates that the direct
attachment of tetrazine tags on the antibody does not influence
their reactivity, with high number of tags remaining functional
towards the cycloaddition reaction.
32
polarity and temperature. This observation has been confirmed
for tetrazine 9, which possesses a k2 of 6000 M-1 s-1 as has been
10
reported under physiological conditions.
For antibody
modification tetrazine 3 was converted to the corresponding N-
hydroxysuccinimide (NHS) ester derivative, tetrazine-NHS 11.
Development and Characterization of the Antibody-Tetrazine
Conjugate
The next step in our study was the development of an Ab-
tetrazine conjugate where tetrazine 3 is used as bioorthogonal tag.
To this end, anti-HER2/neu antibody trastuzumab was selected as
The reaction rate of the trastuzumab-tetrazine 3 conjugate with
TCO-fluorescein dye (12) was determined next. To this end, the
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immunoconjugate was reacted with different excess of 12 in PBS at
In imaging studies, a high Ab functional loadinVgiewisArdtieclseirOanblinlee
37 °C. At selected time points aliquots were taken, and the reaction therefore we selected for the binding aDssOaIy: 10th.1e039tr/aCs6tOuBzu0m14a11bA-
was quenched by addition of (E)-cyclooct-4-enol to the mixtures. tetrazine 3 conjugate with the highest tetrazine loading (8 tags per
After removal of the unreacted dienophiles the fluorescence signal Ab). In order to introduce a fluorescent label and enable the
was monitored over time and pseudo-first-order rate constants tracking of the antibody on the cells, the immunoconjugate was
(kobs) were obtained (Figure S6). From the different kobs a k2 of 3083 reacted with TCO-fluorescein dye 12.
± 352 M-1 s-1 was determined. As anticipated, the resulting rate
constant was higher than that measured for tetrazine 3 in MeOH (k2 trastuzumab-tetrazine-3-TCO-fluorescein conjugate we determined
After incubation of the cells with this “pre-clicked”
= 136 M-1 s-1).
a dissociation constant (KD) of 3.9 nM which correlates with that of
native trastuzumab (KD = 5 nM as reported by the manufacturer),
Binding affinity of the trastuzumab-tetrazine 3 conjugate for and that there is no unspecific binding to the cells (Figure 4).
HER2/neu receptors This result demonstrates that the binding affinity of the
We set out to determine whether the binding affinity of the immunoconjugate is retained even with the higher tetrazine
immunoconjugate for the receptors is retained, and performed a loading, and that this conjugate is suitable for cell imaging
saturation binding assay where triple negative human breast experiments.
adenocarcinoma (MDA-MB-231, Her-2/neu negative) and human
ovarian adenocarcinoma (SKOV-3, Her-2/neu positive) cells were Cell imaging experiments
used as negative and positive control respectively. Before
proceeding with the assay, cell lines were subjected to short pretargeted cell imaging experiments using the newly developed
tandem repeats (STR) and fluorescent in situ hybridization (FISH) trastuzumab-tetrazine conjugate in combination with TCO
To obtain a prove of concept for our approach we performed
3
analysis. The STR loci were concordant with the information given fluorescent probes (Figures 5 and 6).
by the manufacturer (ATCC Rockville, Maryland) and in all tumor
cell lines the hybridization signal was uniform. Whereas, FISH Figure 5. Pretargeting Scheme
analysis demonstrated high ratio HER-2/neu over chromosome 17
in SKOV-3 (3.16), but no detectable expression in MDA-MB-231
cells.
Figure 4. Saturation plot
Cells are pretargeted with trastuzumab-tetrazine 3 conjugates, and labeled
with TCO fluorescent dyes via the IEDDA cycloaddition.
To this end, SKOV-3 cells were incubated with 50 nM of the
immunocojugate bearing 8 tetrazine tags. After washing, Ab
labelled cells were treated with two different concentrations of TCO
dye 12 (0.5 and 10 µM), washed again, and then imaged in the
green fluorescent probe (GFP) channel. As shown in figure 6A,
considerable membrane labelling was obtained for cells treated
with trastuzumab-tetrazine 3 even at low dye concentration.
Binding of the trastuzumab-tetrazine-3-TCO-fluorescein conjugate to SKOV-
3 and MDA-MB-231 cells. Specific binding was defined as total binding
minus non-specific binding obtained by incubation of the immunoconjugate
with 1µM trastuzumab.
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Figure 6. Fluorescent microscopy images
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DOI: 10.1039/C6OB01411A
(A) Images of SKOV-3 cells treated with trastuzumab-tetrazine 3 conjugates for 45 min. (i-iii) Cells were treated with trastuzumab-tetrazine 3 0.05 µM: (i)
after treatment with dye 12 0.5 µM, (GFP channel), (ii) after treatment with dye 12 10 µM (GFP channel), (iii) example of overlay of GFP channel and
differential interference contrast (DIC). (iv-vi) Cells were treated with trastuzumab-tetrazine 3 fluorescein 0,5 µM: (iv) image in the GFP channel, (v) after
treatment with dye 13 10 µM (RFP channel), (vi) merged image of iv and v. (B) Images of SKOV-3 cells treated with trastuzumab-tetrazine 3 conjugate 0,05
µM for 48 h: (vii) after treatment with dye 12 10 µM (GFP channel), (viii) overlay of GFP and DIC. Positive and negative controls in supporting information
(FigureS7). Scale bar: 50 µm.
To track the antibody on the cells and at the same time verify after treatment with dye 12, indicating that the tetrazine moieties
the specificity of the bioorthogonal reaction, a dual labeled Ab- are still reactive (Figure 6B).
conjugate was also utilized.
These experiments clearly demonstrate the effectiveness of our
The latter was prepared by treating trastuzumab with both Ab-tetrazine conjugates as pretargeting agents for bioorthogonal
tetrazine-NHS 11 and fluorescein-NHS ester. In this case, the Ab imaging in an in vitro set-up
labeled cells where treated with Cy3 dye 13, and imaged using both
GFP and red fluorescent probe (RFP) channels. The labeling of the
Conclusions
cells was observed on both channels with good colocalization of the
Due to their high target specificity, yet long circulation times
signal (Figue 6A iv-vi).
within the body, Abs represent an excellent platform for
Negative controls were obtained by incubation of SKOV-3 cells
bioorthogonal pretargeting both in cell and animal models. In fact,
with dyes 12 and 13, showing no unspecific labeling (Figure –S7 iii-
bioorthogonal pretargeting can significantly improve the target-to-
iv). Cells were also labeled with control antibodies trastuzumab-
background ratio resulting in a better target visualization and
fluorescein and the “pre-clicked” trastuzumab-tetrazine-3-TCO-
quantification compared to conventional immunotargeting
fluorescein (Figure S7i-ii).
techniques.
With the perspective of applying our Ab-tetrazine conjugate for
Since the introduction of the tetrazine/TCO ligation, many
in vivo studies we decided to assess its stability towards prolonged
studies have reported the use of TCO modified Abs for pretargeted
exposure to biological media. Particularly, we evaluated the stability
tumor imaging. However, the inverse approach has not been
of the trastuzumab-tetrazine 3 conjugate under cell labelling
investigated yet, although inverting the TCO with the tetrazine may
conditions by extending the Ab incubation time from 45 minutes to
address some issues related to the use of Ab-TCO conjugates.
48 hours. Membranous staining of the cells could still be observed
Particularly, the higher hydrophilicity of tetrazine compounds may
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prevent hydrophobic burying of the tags within the protein residue was adsorbed onto celite and subjected to reverse phase
View Article Online
DOI: 10.1039/C6OB01411A
resulting in a higher effective functional loading.
column chromatography (H2O:MeOH gradient 95:5 to 20:80).
In this study we developed a novel Ab-tetrazine conjugate and
Method B (compounds 6-8): Compounds 6-8 were synthesized
obtain a proof of concept for its applicability using real-time by dissolving 3,6-bis-(DMP)-1,2,4,5-tetrazine (1 eq), 3-methyl-6-
33
fluorescence cell imaging as validation method. We synthesized a thiomethyl-1,2,4,5-tetrazine (1 eq)
or 6-thiomethyl-1,2,4,5-
small library of novel 1,2,4,5-tetrazines all suitable for protein tetrazine (1 eq) 33 in toluene (dry, 1 mL) or methanol (dry, 2 mL). 4-
modification, and selected compound 3 for antibody labeling. This Aminobenzylamine (1 eq) was added to the solution and the
tetrazine displays a remarkable stability towards physiological mixture was stirred at room temperature for 2 to 4 h under argon.
media and still retains a good cycloaddition reactivity with the TCO. After evaporation of the solvent the crude residue was adsorbed
Next, we developed a novel antibody-tetrazine conjugate using onto silica and subjected to column chromatography. The obtained
tetrazine 3 as bioorthogonal tag. The novel immunoconjugate was amino derivatized tetrazines were subsequently refluxed with
evaluated for pretargeted live cell imaging using two different TCO glutaric anhydride (5 eq) in THF (dry, 2 mL) for 16 h. The final
fluorescent probes as imaging agents. This study demonstrates the products were purified by reverse phase column chromatography
viability of our approach, showing that the reaction between the (H2O:MeOH, gradient 95:5 to 0:100).
Ab-tetrazine construct and TCO probes is fast even at micromolar
concentrations and, that the tetrazine tags are unaffected by General procedure for the synthesis of TCO dyes 12 and 13
prolonged exposure to physiological conditions. The high reactivity
(E)-cyclooct-4-en-1-yl-(2-aminoethyl) carbamate (16) (1 eq.) was
combined with the good functional density and stability of the Ab- dissolved in anhydrous DMF (1mL). Triethylamine (2 eq.) was
tetrazine conjugate, suggest that this strategy may be useful for in added, followed by dye-NHS derivatives (1.5 eq.). The mixture was
vivo positron emission tomography (PET) imaging. This will allow stirred overnight at room temperature under N2. After evaporation
the in vivo translation of this approach.
of the solvent, the crude residue was purified by HPLC on a C-18
column (H2O:ACN isocratic 70 : 30 + 0.1% FA) and freeze-dried. The
1H-NMR spectrum of compound 13 (Figure S3) revealed the
presence of a 1 : 1 mixture of trans and cis isomers. Due to the very
low reactivity of the cis isomer towards the iEDDA reaction the
mixture was used as such for the cell imaging experiments. For
simplicity we refer only to the trans isomer in figure 1.
Experimental
General procedures for the synthesis of 1,2,4,5-tetrazines
Method A (compounds 1-5): Compounds 1-5 were synthesized
by suspending 5-((4-cyanophenyl)amino)-5-oxopentenoic acid (14),
5-((6-cyano)-3-pyridinyl)-5-oxopentenoic acid (15) or 3-(4-
cyanophenyl)-propanoic acid (1 eq) in EtOH (2 mL). Acetamidine
hydrochloride or formamidine acetate (4 to 10 eq) was added
followed by sulfur (1 eq) or zinc triflate (Zn(OTf)2, 0.5 eq) and
hydrazine hydrate (1.8 mL). The mixture was stirred at room
temperature under argon for 16 to 48 h. Then it was diluted with
water and cooled to 0°C. NaNO2 (10 eq) was added, followed by
dropwise addition of 1M HCl. The acid was added until reaching pH
3. At this point gas evolution started and the solution turned pink or
fuchsia (Caution! This step generates a large amount of toxic
nitrogen oxide gas). The mixture was stirred at 0°C until gas
evolution ceased, then EtOAc was added and the organic phase
separated. The aqueous phase was extracted three times with
EtOAc. The combined organic phases were washed once with brine,
and dried over MgSO4. After removal of the solvent the crude
Antibody Labeling
Trastuzumab (Herceptin®, Roche) stock in PBS pH 7.4, 144 µM
(21 mg/mL) was diluted to a final concentration of 6.9 µM by
addition of a PBS solution buffered at pH 9.1. The NHS ester
derivative of tetrazine 3 (tetrazine-NHS 11) was dissolved in DMSO
to make a 60 mM stock solution. For conjugation the appropriate
excess of tetrazine was aliquoted into the antibody solution,
vortexed and reacted for 3 hours at 37 °C. The excess of tetrazine
used in this experiment were 10 and 100 equivalents with respect
to the antibody. The resulting Ab conjugates were purified via size
exclusion chromatography (PD-10 column 5K MWCO, GE
Healthcare), concentrated to 1 mg/mL and stored in PBS at 4 °C. A
dual labeled trastuzumab conjugate was obtained following a
similar procedure where trastuzumab was simultaneously
8 | J. Name., 2012, 00, 1-3
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Page 9 of 10
Organic & Biomolecular Chemistry
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Journal Name
ARTICLE
incubated with 100 equivalents of tetrazine-NHS 11, and 15 profiling. An 80-90% confluent culture was detached with trypsin-
View Article Online
equivalents of fluorescein-NHS ester.
EDTA or accutase (for SKOV-3) and washed tDhOreI:e1t0i.m10e3s9/wCi6thOB10x14P1B1AS
to remove any cell debris. Cells were counted with the Muse
System (Merck Millipore, Count and Viability Assay kit) and
Native mass spectrometry
The mass spectrometry experiments were performed on a Q- resuspended in their respective medium in a concentration of 1 x
TOF-2TM instrument (Micromass, Waters). The samples were 106 viable cells/0.5 ml.
buffer exchanged twice into 150 mM aqueous ammonium acetate
at pH 7.0 using Micro Bio-Spin P-6 columns (Bio-Rad) at Cell saturation binding assay
concentrations of 10 μM. Gold-coated nanoESI needles were
Trastuzumab-tetrazine 3 conjugate with the highest tetrazine
prepared in-house in order to transfer 5 μL aliquots of sample into loading (8 tags per antibody) was reacted with dye 12 (7.4 eq. with
the mass spectrometer. The instrument was tuned using the respect to the antibody). After purification (PD-10 column 5K
following parameters: capillary voltage 1.6 kV; nanoflow backing MWCO, GE Healthcare), the resulting fluorescent conjugate,
pressure 0.3 bar; sample cone 200 V; extractor 10 V ; collision bearing 4 fluorophore moieties, was utilized for the saturation
energy 100 V; backing pressure 4.0 mbar. The data were acquired binding assay.
and processed with Masslynx v4.1 software (Waters).
Briefly, increasing concentrations of the antibody conjugate (1
nM -80 nM, quadruplicate) were incubated with 1 x 106 SKOV-3 or
MDA-MB-231 cells in a total volume of 1 mL cell medium for 2 h at
Measurement of Tetrazine Reactive Tags on Antibody
Trastuzumab-tetrazine 3 conjugates (18 µM) were treated with 4°C. To assess non-specific binding, unmodified trastuzumab (1µM)
20 fold excess of TCO-fluorescein dye 12 with respect to the Ab. The was added (duplicates). Following incubation, the cells were
reaction was performed in PBS containing 1% of DMSO. After pelleted via centrifugation (2000 rpm for 5min at 4°C) and, after
incubation for 1h at 37 °C, the antibody conjugates were purified removal of the supernatant, washed with cold 1 x PBS + 0.2% tween
via size exclusion chromatography (PD-10 column 5K MWCO, GE 20 solution (repeated twice). After the last washing step the cells
Healthcare), and subjected to UV-vis analysis using the following were resuspended in 500 µL 1 x PBS and 200 µL aliquots from each
molar extinction coefficients: ε280
=
225.000 cm-1 M-1 for tube were transferred to a 96-well plate (Nunclon Delta Surface,
34
trastuzumab,
and ε500 = 70.000 cm-1 M-1, CF = 0.3 (Thermo Thermo scientific) and the fluorescence intensity of the samples
Scientific) for dye 12. Unmodified Trastuzumab was subjected to was measured (λex = 500 nm, λem = 550 nm). The fluorescence
the same treatment and used as reference for these experiments. intensity with Ab concentration was fitted to first-order
a
Each experiment was repeated in triplicate. Fluorescent spectra of exponential using GraphPad Prism 6, and the KD determined to be
the Ab conjugates were also recorded after treatment with dye 12 3.9 nM.
(FigureS5).
Cell microscopy
Cell culture
Her-2/neu
Trastuzumab-tetrazine 3 (0.05 µM), and trastuzumab-tetrazine-
negative
MDA-MB-231
and
Her-2/neu 3-fluorescein (0.5 µM) were incubated with 150.000 cells plated
overexpressing SKOV-3 cell lines were purchased from ATCC into 6-well plates, for 45 min in DMEM/F12 without phenol red,
(Rockville, Maryland). Cells were routinely cultured in Dulbecco’s containing 5% FBS, 2 mM glutamine, 1% sodium pyruvate and 1%
Modified Eagle Medium (DMEM) and DMEM/F12 medium penicillin/streptomycin. Cells were then washed 3x with cell
respectively, supplemented with 10% (or 5% for SKOV-3 cells) heath medium before incubating with 0.5 and 10 µM of dyes 12 and 13
inactivated FBS, 2 mM glutamine, 1% sodium pyruvate and 1% for 30 min in growth media. After washing 3 times with sterile 1 x
penicillin/streptomycin (Gibco, Life technologies). Cultures were PBS, images were taken using an EVOS fluorescence digital inverted
maintained in exponential growth at 37°C in
a humidified microscope in the GFP and RFP channels. Identical image
atmosphere containing 5% CO2. The HER-2 status was evaluated by acquisition settings were used for the experimental data. Images
FISH analysis. The identity of cell lines was confirmed with STR
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
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Page 10 of 10
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Journal Name
were analyzed using ImageJ applying identical leveling adjustments 18 R. Rossin, S. M. van den Bosch, W. Ten Hoeve, M. Carvelli, R. M.
View Article Online
DOI: 10.1039/C6OB01411A
to all experimental data across individual channels.
Versteegen, J. Lub, M. S. Robillard, Bioconjugate Chem., 2013,
24, 1210.
ACKNOWLEDGMENTS
19 M. K. Rahim, R. Kota, J. B. Haun, Bioconjugate Chem., 2015, 26,
352.
We thank Prof. Yves Guisez for his support on the mass analysis
of the immunoconjugates; and Prof. Filip Lardon for access to the
microscope. This work was supported by grants from the University
of Antwerp (GOA-BOF) and FWO-IWT.
20 R. J. Blizzard, D. R. Backus, W. Brown, C. G. Bazewicz, Y. Li, R. A.
Mehl, J. Am. Chem. Soc., 2015, 137, 10044.
21 H. E. Murrey, J. C. Judkins, C. W. Am Ende, T. E. Ballard, Y. Fang,
K. Riccardi, L. Di, E. R. Guilmette, J. W. Schwartz, J. M. Fox, D. S.
Johnson, J. Am. Chem. Soc., 2015, 137, 11461.
22 J. L. Seitchik, J. C. Peeler, M. T. Taylor, M. L. Blackman, T. W.
Rhoads, R. B. Cooley, C. Refakis, J. M. Fox, R. A. Mehl, J. Am.
Chem. Soc., 2012, 134, 2898.
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