Enhancing reactivity for bioorthogonal pretargeting by unmasking antibody-conjugated trans -cyclooctenes

Article abstract of DOI:10.1021/bc500605g

The bioorthogonal cycloaddition reaction between tetrazine and trans-cyclooctene (TCO) is rapidly growing in use for molecular imaging and cell-based diagnostics. We have surprisingly uncovered that the majority of TCOs conjugated to monoclonal antibodies using standard amine-coupling procedures are nonreactive. We show that antibody-bound TCOs are not inactivated by trans-cis isomerization and that the bulky cycloaddition reaction is not sterically hindered. Instead, TCOs are likely masked by hydrophobic interactions with the antibody. We show that introducing TCO via hydrophilic poly(ethylene glycol) (PEG) linkers can fully preserve reactivity, resulting in >5-fold enhancement in functional density without affecting antibody binding. This is accomplished using a novel dual bioorthogonal approach in which heterobifunctional dibenzylcyclooctyne (DBCO)-PEG-TCO molecules are reacted with azido-antibodies. Improved imaging capabilities are demonstrated for different cancer biomarkers using tetrazine-modified fluorophore and quantum dot probes. We believe that the PEG linkers prevent TCOs from burying within the antibody during conjugation, which could be relevant to other bioorthogonal tags and biomolecules. We expect the improved TCO reactivity obtained using the reported methods will significantly advance bioorthogonal pretargeting applications.

Full text of DOI:10.1021/bc500605g

Article
pubs.acs.org/bc
Enhancing Reactivity for Bioorthogonal Pretargeting by Unmasking
Antibody-Conjugated trans-Cyclooctenes
Maha K. Rahim,† Rajesh Kota,† and Jered B. Haun*,†,‡,§
‡
§
†Department of Biomedical Engineering, Department of Chemical Engineering and Materials Science, and Chao Family
Comprehensive Cancer Center, University of California, Irvine, Irvine, California 92697, United States
S
* Supporting Information
ABSTRACT: The bioorthogonal cycloaddition reaction between tetrazine and
trans-cyclooctene (TCO) is rapidly growing in use for molecular imaging and
cell-based diagnostics. We have surprisingly uncovered that the majority of
TCOs conjugated to monoclonal antibodies using standard amine-coupling
procedures are nonreactive. We show that antibody-bound TCOs are not
inactivated by trans−cis isomerization and that the bulky cycloaddition reaction
is not sterically hindered. Instead, TCOs are likely masked by hydrophobic
interactions with the antibody. We show that introducing TCO via hydrophilic
poly(ethylene glycol) (PEG) linkers can fully preserve reactivity, resulting in
>5-fold enhancement in functional density without affecting antibody binding.
This is accomplished using a novel dual bioorthogonal approach in which
heterobifunctional dibenzylcyclooctyne (DBCO)−PEG−TCO molecules are
reacted with azido-antibodies. Improved imaging capabilities are demonstrated
for different cancer biomarkers using tetrazine-modified fluorophore and
quantum dot probes. We believe that the PEG linkers prevent TCOs from burying within the antibody during conjugation, which
could be relevant to other bioorthogonal tags and biomolecules. We expect the improved TCO reactivity obtained using the
reported methods will significantly advance bioorthogonal pretargeting applications.
residues, and TCO decorated antibodies have been shown to
retain aqueous stability and binding affinity even at high
modification levels.9,10,15,17−27,29 However, there is a large
discrepancy in the literature with respect to the number of
TCO attachments that can be obtained, and results diverge
depending on the method of analysis. Specifically, total TCO
conjugations measured by mass spectrometry have been
reported to be as high as 10−20,17,18,20 whereas functional
TCOs assessed by tetrazine reaction have been usually fewer
than 6.9,10,15,29 A potential explanation for these results is that a
significant proportion of TCO attachments have been rendered
nonreactive. Several mechanisms could be involved with TCO
inactivation, including (1) conversion of TCO to the cis
isoform that is orders of magnitude less reactive with tetrazine,
(2) steric hindrance of the bulky cycloaddition reaction near
the antibody surface, and (3) interaction of the hydrophobic
TCO with external or even internal domains of the antibody.
Trans−cis isomerizaton has been shown to occur on the order
of hours in the presence of catalysts such as free thiols or
transition metals bound to serum proteins in vivo,31,33 although
neither of these routes is expected for antibody conjugates
directly after preparation. The tetrazine/TCO cycloaddition
reaction results in a large, bulky dihydropyrazine product that
INTRODUCTION
■
There is currently strong interest in utilizing bioorthogonal
chemistries to augment the detection of diseases both inside
and outside of the body.1−5 These works utilize the concept of
pretargeting, where a chemically tagged affinity molecule such
as a monoclonal antibody is first applied to mark the target site,
followed by covalent attachment of the contrast agent.6,7 Due
to poor in vivo pharmacokinetics or other limitations of a
traditional direct conjugate, pretargeting has been shown to
improve imaging capabilities under different modalities in
animal models.8−16 Moreover, the small size of bioorthogonal
reactants enable antibodies to act as scaffolds for the
attachment of numerous nanomaterial probes to cells and
microvesicles, leading to dramatic signal amplification for in
vitro cultures and ex vivo human clinical specimens.17−27
Bioorthogonal pretargeting applications have predominantly
employed the catalyst-free inverse-electron-demand Diels−
Alder cycloaddition between 1,2,4,5-tetrazine and trans-cyclo-
octene (TCO) due to its high selectivity and unprecedented
speed.28,29 Specifically, the reaction between tetrazine/TCO is
several orders of magnitude faster than tetrazine with other
dienophiles such as norbornene and methyl-cyclopropene or
alternative catalyst-free chemistries such as the strain-promoted
azide/cyclooctyne reaction.2,30−32 This rapid kinetics is critical
to success in pretargeting scenarios, particularly within the
body. The hydrophobic TCO is typically attached to
monoclonal antibodies via standard amine-reaction to lysine
Received: December 20, 2014
Revised: January 10, 2015
Published: January 13, 2015
© 2015 American Chemical Society
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Figure 1. Direct conjugation of TCO to antibodies via standard amine-reaction results in both active (blue) and inactive (black) moieties. We report
that hydrophilic PEG linkers can fully preserve TCO reactivity. TCO−PEGs were introduced by initially modifying the antibody with azides
followed by reaction with heterobifunctional DBCO−PEGn−TCO (n = 4 or 24). The improved reactivity obtained with the PEG linker will directly
enhance binding of tetrazine-modified probes.
could be sterically hindered by the antibody surface. Steric
effects have been noted to reduce the reaction rate of TCO
with tetrazines containing bulky substituents, such as a tert-
butyl group, and these effects were not observed for smaller
cyclopropenes.34 Finally, the hydrophobic TCO may simply
bury within interior domains of the antibody to avoid the
aqueous solvent. Further investigation into these potential
inactivation mechanisms and overall TCO reactivity after
attachment to monoclonal antibodies is needed to ensure that
the remarkable speed and power of the tetrazine/TCO
cycloaddition reaction translate to applications employing
complex biomolecules. Placing the less hydrophobic tetrazine
on the antibody could potentially alleviate some of these issues,
but stability of TCO-modified secondary agents could be a
major challenge, particularly for nanoparticles. We believe the
focus should instead be on retaining TCO reactivity following
bioconjugation, thereby maximizing the power of bioorthogo-
nal pretargeting applications such as in vivo imaging and ex vivo
nanoparticle amplification for cellular diagnostics.
A simple and straightforward strategy to increase TCO
solubility and physically prevent interactions with the antibody
is to append TCO via a hydrophilic polymer linker.
Poly(ethylene glycol) (PEG) has a long history of use with
antibodies for reducing immunogenicity and improving
pharmacokinetics.35 Furthermore, it is well-known that PEG
can improve the solubility of hydrophobic molecules such as
drugs, fluorophores, and biotin. For the hydrophobic
fluorophore indocyanine green (ICG), short PEG linkers
have been shown to reduce aggregation of antibody conjugates
and prevent ICG quenching by aromatic amino acids.36,37
Finally, it was recently demonstrated that attaching TCO via a
PEG linker increased the rate of trans−cis isomerization in
serum, presumably by making the TCO more accessible to
protein-bound transition metals that can catalyze the
conversion.33 The PEG linker did not affect the reaction rate
with tetrazine; however, the number of reactive TCOs attached
to the antibody was not characterized. Thus, the activity of
bioorthogonal reactants immediately after antibody conjugation
has not been investigated to date. A potential drawback is that
PEG linkers can block antibody binding, although this appears
to be less of an issue for shorter PEGs less than 1000 Da.38−40
Herein, we characterize TCO reactivity on the surface of
different monoclonal antibodies following standard amine-
conjugation procedures. Surprisingly, we find that up to 90% of
antibody-associated TCOs are nonfunctional. We present
evidence that suggests TCOs are neither inactivated by
trans−cis isomerization nor sterically hindered from cyclo-
addition reaction near the antibody surface; thus, we believe
that inactive TCOs are masked by interactions with the
antibody. Most importantly, we successfully restore reactivity
by appending TCO using both short and long hydrophilic PEG
linkers. We attach the PEG−TCO linkers via bioorthogonal
reaction between azide and dibenzylcyclooctyne (DBCO),
which is mutually orthogonal to the tetrazine/TCO chem-
istry.41 This novel dual bioorthogonal approach involves first
decorating the antibody with varying amounts of azide followed
by cycloaddition reaction with heterobifunctional DBCO−
PEG−TCO molecules (Figure 1). Overall improvements in
functional TCO density are more than 10-fold in total and 5-
fold without significant loss of antibody binding activity.
Enhanced detection signals are also demonstrated for tetrazine-
modified fluorophores and quantum dots (QD) using flow
cytometry and confocal imaging. This work should significantly
expand the power of bioorthogonal pretargeting applications
such as in vivo molecular imaging and cell-based diagnostics
utilizing chemical amplification of nanoparticle binding.
RESULTS AND DISCUSSION
■
We first set out to explore TCO reactivity using a mouse
monoclonal antibody that is specific for the cancer biomarker
EGFR. Total TCO modification levels were measured using
matrix-assisted laser desorption/ionization time-of-flight
(MALDI-TOF) mass spectrometry (Figure S2), whereas
TCO reactivity was determined based on absorbance readings
after complete reaction with tetrazine−Oregon Green 488. We
found that TCO conjugation was efficient (Figure 2A), but,
remarkably, only 10% of the moieties were functional (Figure
2B). Reactivity was identical when measured immediately
following conjugation and after waiting up to 24 h (Figure 2C).
This suggests that TCO was not inactivated by trans−cis
isomerization, as this conversion has been shown to require
significant amounts of time even in the presence of a catalyst.
For example, 6 h or more is needed for full isomerization in the
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total modification levels and only slightly decreased reactive
sites (Figure S3). Increasing DMF concentration or adding
dimethyl sulfoxide (DMSO) during the reaction with
tetrazine−Oregon Green 488 did reveal 30% more reactive
TCOs (Figure S3). However, the majority remained inactive,
and further increases in solvent destabilized the antibody.
Further access to the masked TCOs could potentially be
obtained using a more hydrophobic tetrazine conjugate such as
a bodipy fluorophore.
We next explored whether introduction of TCO using
hydrophilic PEG linkers could maintain reactivity. We chose to
incorporate PEG−TCO onto the antibody using the mutually
orthogonal azide/DBCO reaction to improve control over
coupling density, as we have already established that azide is an
ideal reactant on the antibody at varying densities (Figure 2B).
We created heterobifunctional DBCO−PEG−TCO molecules
with 4 and 24 polymer subunits by sequentially reacting
amine−PEG−carboxylic acid with the same amine-reactive
TCO employed for direct antibody conjugations. The
carboxylic acid was then modified with amine-DBCO via N-
hydroxysuccinimide (NHS) ester activated chemistry, and the
resulting DBCO−PEG−TCO was reacted with azido-antibod-
ies (Figure 1). Total loadings of the PEG4 and PEG24 linkers
were assessed by MALDI-TOF (Figures 3A and S2) and varied
in a dose-dependent manner with the initial azide density.
Attachment levels for the PEG4−TCO were higher in all cases,
with a maximum valency of 15 that was close to the 13 azides
that were directly measured by MALDI-TOF. Conjugation of
the PEG24−TCO was less efficient, with values approximately
one-half that of the PEG4−TCO. It is unclear whether lower
conjugation levels for the PEG24 were due to physical crowding
or the presence of unreacted DBCO-amine in the preparation.
Increasing linker concentration in the reaction did not affect
results, however.
Figure 2. Conjugation of TCO and azide to an anti-EGFR antibody
using standard amine-reaction procedures. (A) Total attachment levels
determined by MALDI-TOF versus the number of reaction
equivalents, showing that modification efficiencies are similar. (B)
Reaction of the TCO−antibodies with tetrazine−fluorophore revealed
that only 10% of the TCOs were functional. In contrast, nearly all
azide modifications were reactive to DIBO−fluorophore. (C)
Functional TCO level was similar on the anti-EGFR antibody
immediately after treatment with NHS−TCO or after being
maintained 5 and 24 h at room temperature, suggesting that TCOs
were not inactivated by trans−cis isomerization. (D) Antibody binding
affinity was not affected after modification with high levels of azide or
TCO (KD ∼ 5 nM). Error bars represent the standard error of at least
three independent experiments.
We next determined whether the PEG−TCO linkers
interfered with antibody binding again using the indirect
competitive assay with A431 cells. We found that binding at 10
μg/mL concentration was unaffected for both the PEG linkers
until attachment levels exceeded 8 (Figure 3B). Further analysis
of full binding profiles confirmed that affinity was close to
control for 8 PEG24−TCOs (KD = 6 nM), but it was
dramatically reduced for 15 PEG4−TCOs (Figure 3C).
Reduced binding in the latter case can be attributed either to
decreased affinity or a population of permanently inactivated
antibody, but, in either case, binding was almost fully recovered
by increasing the antibody concentration to 30 μg/mL (Figure
S4). Importantly, TCO moieties introduced via both PEG
linkers were fully functional (Figure 3D). This was determined
under pretargeting conditions, in which A431 cells were labeled
with TCO−PEG−antibodies, reacted with tetrazine−Oregon
Green 488 at 50 μM for 30 min in the presence of BSA, and
analyzed by flow cytometry. The TCO−antibodies prepared by
direct amine reaction were also evaluated and used to convert
raw fluorescence signals (Figure S4) into the number of
functional TCOs per antibody, after adjusting for antibody
binding levels. These results were highly specific, as background
signals from a nonbinding control antibody conjugated with
TCO and PEG24−TCO were similar to autofluorescence
(Figure S4). In total, antibody reactivity enhancements were
more than 10- and 5-fold for the 4 and 24 subunit PEGs,
respectively. To illustrate these improvements, we performed
confocal imaging of A431 cells labeled with TCO- or TCO−
PEG-modified antibodies, followed by reaction with an
presence of free thiols at high concentrations and elevated
temperatures or transition metals in serum.31,33 To explore
steric hindrance, we employed the strain-promoted bioorthog-
onal reaction between azide and cyclooctyne that also yields a
bulky cycloaddition product. Azide conjugation efficiency was
similar to the TCO (Figure 2A), but nearly all moieties could
be reacted with dibenzocyclooctyne (DIBO)−Alexa Fluor 488
(Figures 2B). Discrepancies at low loadings were likely due to
the small size of azide groups, which makes mass changes
difficult to distinguish by MALDI-TOF. We expect that this
result should extend to tetrazine/TCO cycloaddition, although
the smaller azide could be less sensitive to sterics, as noted for
methyl-cyclopropene in relation to TCO.34 Interestingly,
neither TCOs nor azides interfered with antibody binding
affinity even at high modification levels (Figure 2D). This was
determined using an indirect competitive binding assay with
A431 epidermoid cancer cells. Fitting the binding data
indicated equilibrium dissociation constants (KD) were all
close to 5 nM. On the basis of the above evidence, we
speculated that hydrophobic TCOs were interacting with
external or even internal domains of the antibody, masking it
from tetrazine reaction. Internal burying could explain how an
antibody decorated with almost 20 hydrophobic TCOs can
remain stable in aqueous solution. To explore whether the
presence of a polar solvent during conjugation procedures
influenced TCO masking, we decreased the concentration of
dimethylformamide (DMF) to <1%, but this had no effect on
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Figure 3. Introduction of TCO via PEG linkers using azide/DBCO reaction. (A) Attachment of heterobifunctional DBCO−PEGn−TCO (n = 4 or
24) to azide-modified anti-EGFR antibodies. (B) Relative antibody binding at 10 μg/mL concentration showing that binding activity is retained up
to ∼8 linkers. (C) Kinetic binding curves after modification with PEG−TCOs. Binding affinity was unaffected for 8 PEG24−TCOs (KD = 6 nM), but
it was significantly reduced for 15 PEG4−TCOs (KD =100 nM). (D) TCOs introduced via PEG linkers are fully reactive, as determined for live A431
cells labeled with modified antibodies and reacted with tetrazine−Oregon Green 488 by flow cytometry. (E) Confocal images of live A431 cancer
cells labeled with (i, iii) no antibody or the anti-EGFR antibody modified with (ii) azide, (iv) TCO, (v) PEG4−TCO, and (vi) PEG24−TCO at the
highest levels. Cells were then probed with (i, ii) DIBO−Alexa Fluor 488 or (iii−vi) tetrazine−Oregon Green 488 at 50 nM for 30 min. Scale bar: 20
μm. Error bars represent the standard error of at least three independent experiments.
extremely low concentration (50 nM) of tetrazine−Oregon
Green 488 for 30 min at room temperature in the presence of 1
mg/mL BSA (Figure 3E). We similarly tested the anti-EGFR
antibody modified with 15 azides followed by reaction with
DIBO−Alexa Fluor 488. The TCO−PEG−antibodies yielded
dramatically higher fluorescent signals than the TCO and azide
cases, which were indistinguishable from autofluorescence.
Thus, the PEG linkers effectively prevented TCO inactivation
after antibody conjugation, presumably by increasing solubility
and preventing interactions with the antibody while maintain-
ing faster reaction kinetics than azide/DIBO. The PEG linkers
should also be advantageous for in vivo applications by reducing
antibody immunogenicity. A potential drawback is that higher
rates of TCO inactivation by trans−cis isomerization have been
reported in serum when using PEG linkers.33 However, this
study used a linker containing a phenyl group (benzyl ether) in
between the TCO and a 12 subunit PEG. Therefore, it is
possible that inactivation may not be as extensive for our
carbonate linker that is more hydrophilic and 4 subunit PEG
that is shorter.
the QD immunoconjugate and TCO−antibody/tetrazine−QD
cases (Figure 4A). Signals from both TCO−PEG−antibodies/
tetrazine−QD cases were greater by as much as 6-fold. It
should be noted that chemical amplification levels can increase
further over immunoconjugates at higher nanoparticle concen-
trations.17 These results were again entirely specific (Figure
S5), and PEG length did not affect QD binding levels (Figure
4B). Improved detection capacity of the PEG24−TCO was
confirmed using confocal microscopy (Figure 4C).
Finally, we modified monoclonal antibodies specific for
transferrin receptor (TfR) and EpCAM with TCO and PEG24−
TCO using the highest valency conditions. Functional and total
modification levels were similar to the anti-EGFR antibody, and
TCO reactivity was enhanced 3- to 4-fold using the PEG linker
(Figure 5A). Binding of the TCO−PEG24-modified anti-TfR
antibody was slightly diminished, however (Figure S6). Similar
5-fold signal enhancements were observed by flow cytometry
for both TCO−PEG24−antibodies after reaction with tetrazine-
modified QDs (Figure 5B). Improved detection capabilities
were also demonstrated by confocal imaging using tetrazine-
modified Oregon Green 488 (Figure S6) and QDs (Figure 5C).
These results confirm that TCO masking after conjugation to
monoclonal antibodies is not a rare phenomenon and that
reactivity can reliably be improved by introducing via PEG
linkers and our dual bioorthogonal coupling strategy.
A powerful application of tetrazine/TCO pretargeting is to
amplify nanomaterial binding to cells and microve-
sicles.17,18,20,22−27 Therefore, we labeled A431 cells with the
TCO- and PEG−TCO-modified anti-EGFR antibodies and
reacted with tetrazine-modified quantum dots (QD) at 10 nM
concentration for 30 min. A QD immunoconjugate was also
prepared and incubated under identical conditions. Fluores-
cence intensities measured by flow cytometry were similar for
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Figure 4. Amplification of QD binding. (A) QD signal determined by
flow cytometry following cycloaddition reaction to modified anti-
EGFR antibodies on A431 cancer cells. The TCO−PEG−antibodies
displayed the highest binding levels, by as much as 6-fold for the PEG4,
over the TCO−antibody and the QD immunoconjugate. (B) QD
binding levels correlated directly with the number of reactive TCOs,
showing no influence of PEG length. (C) Confocal images of A431
cells labeled with (i, iv) no antibody or the anti-EGFR antibody
conjugated with (ii) TCO or (iii) PEG24−TCO. Cells were then
probed with (i−iii) tetrazine−QD or (iv) QD immunoconjugate at 50
nM for 30 min. Scale bar: 20 μm. Error bars represent the standard
error of at least three independent experiments.
Figure 5. Translation of dual orthogonal coupling to different
antibodies. (A) Anti-TfR and EpCAM antibodies had low TCO
reactivity (12−15%) after direct amine-conjugation, but the PEG24−
TCO increased reactive TCO density 3- to 4-fold. *MALDI-TOF
analysis of the TCO−PEG24-modified anti-EpCAM antibody was
limited by difficulties with desorption. (B) Flow cytometry results for
TCO- and TCO−PEG24-modified anti-EpCAM and TfR antibodies
on A431 cells after reaction with 10 nM tetrazine−QDs for 30 min,
showing 5-fold signal enhancements for the PEG24−TCO. (C)
Confocal images of A431 cells labeled with (i, iv) anti-EpCAM, (ii,
v) anti-TfR, or (iii, vi) nonbinding control antibodies. The antibodies
were modified with (i−iii) TCO or (iv−vi) PEG24−TCO at the
highest densities and reacted with tetrazine−QD at 10 nM for 30 min.
Scale bar: 20 μm. Error bars represent the standard error of at least
three independent experiments.
CONCLUSIONS
■
In this work, we have discovered that the majority of TCOs
conjugated to antibodies using standard amine-coupling
procedures are nonreactive. To address this issue, we
introduced TCO via hydrophilic PEG linkers, which fully
preserved reactivity and improved detection signals for
tetrazine-modified fluorophores and quantum dots. Taken
together with other evidence presented, we believe that
nonfunctional TCOs are buried within the antibody. This
likely occurs during conjugation procedures, as TCO reactivity
is later maintained in the presence of cells and high
concentrations of BSA protein. We believe this work establishes
a new method to prepare TCO-modified antibodies that will
significantly advance bioorthogonal pretargeting studies such as
in vivo imaging and chemical amplification of nanoparticle
binding. Additionally, this technique could be useful for signal
amplification of activatable fluorochromes.42−45 Although we
utilized azide/DBCO cycloaddition reaction to couple PEG−
TCO to antibodies, we expect that direct amine-reaction or
alternative chemistries should provide similar results. Future
work could explore whether similar masking effects occur for
different hydrophobic reactants such as DBCO, norbornene,
and methyl-cyclopropene, alternative linkers directly distal to
the TCO such as those containing bulky phenyl groups,16 or
new bicyclic TCO derivatives that have faster reaction rates,
greater stability, and, for the dioxolane-fused TCO, potentially
greater hydrophilicity.46,47 Expanding these investigations to
different types of proteins or biological molecules, including
site-specific methods that involve genetic encoding,48−50 would
also be of interest.
EXPERIMENTAL PROCEDURES
■
Materials. All chemicals were purchased from Sigma-
Aldrich unless noted and were used as received. The
DBCO−amine was purchased from Click Chemistry Tools
(Scottsdale, AZ). Heterobifunctional carboxy−(PEG)n−amine
with n = 4 or 24 subunits and amine-reactive succinimidyl ester
(NHS)-azide were purchased from Thermo Fisher Scientific
(Rockford, IL). Primary amine-terminated quantum dots
(Qdot 605 ITK Amino), Alexa Fluor 488 DIBO Alkyne, and
amine-reactive NHS−Oregon Green 488 were purchased from
Life Technologies (Grand Island, NY). Monoclonal mouse
antibodies specific for human EpCAM (IgG2b, clone 158206)
and transferrin receptor (TfR, IgG1, clone 29806) were
purchased from R&D Systems (Minneapolis, MN). A non-
binding isotope control antibody (mouse IgG1, clone MOPC-
21) was purchased from BioLegend (San Diego, CA). All
solvents were of reagent grade or higher and were used without
further purification. HR MS (ESI LC-TOF) and MALDI-TOF
(matrix-assisted laser desorption ionization-time-of-flight) mass
spectrometry were performed for characterization of hetero-
bifunctional PEG linkers and antibodies, respectively. HR MS
was performed using a Waters LCT Premier system. For all HR
MS measurements, product was dissolved in methanol.
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MALDI-TOF was performed using a AB SCIEX TOF/TOF
5800 system with a MALDI matrix composed of 1 mg of
sinapinic acid dissolved in 100 μL of 70:30 acetonitrile/water
with 0.1% trifluororacetic acid. Absorption measurements were
recorded on a Thermo Scientific NanoDrop 2000 spectropho-
tometer. All kinetic binding analyses were performed using
GraphPad Prism. Synthesis of tetrazine−amine, NHS−
tetrazine, tetrazine−Oregon Green 488, and NHS−TCO
were as previously reported.29−31
<5% DMF for reaction with 100 or 1000 equiv of NHS−TCO,
respectively. TCO, azide, and Oregon Green 488-modified
antibodies were purified using Zeba spin desalting columns.
PEG−TCO conjugates were prepared by reacting the different
azide-modified antibodies with DBCO−PEG−TCO molecules
containing 4 or 24 subunits. Reactions were carried out using
10-fold excess relative to the number of azide moieties for 4 h at
room temperature. TCO−PEG−antibodies were purified into
PBS using Amicon Ultra-4 centrifugal filtration systems
(Millipore). Antibody concentration was determined by
absorption measurement. Oregon Green 488 density was also
measured by absorption to be 2 dyes/antibody.
Cell Culture. Human epidermoid cancer cell line A431 and
hybridoma HB-9764 producing mouse anti-human EGFR
antibody clone MAb108 were obtained from ATCC (Manassas,
VA). A431 cells were selected for antibody labeling studies due
to high-level expression of EGFR and EpCAM biomarkers as
well as moderate expression of TfR. Cells were cultured in
flasks at 37 °C with 5% CO2. A431 cells were grown in DMEM
media containing 10% fetal bovine serum, 5% penicillin/
streptomycin, and 5% L-glutamine and subcultured after
reaching confluence by treatment with trypsin−EDTA
(Corning, NY).
MALDI-TOF Analysis of Modified Antibodies. Total
TCO and azide densities were determined based on changes in
mass using MALDI-TOF mass spectrometry. The matrix was
prepared using 1 mg of sinapinic acid dissolved in 100 μL of
70:30 acetonitrile/water with 0.1% trifluororacetic acid.
Unmodified control and TCO-, TCO-PEG-, and azide-
modified antibodies were buffer-exchanged into water and
concentrated to greater than 1 mg/mL using Amicon Ultra-4
centrifugal filters (Millipore). Concentrated antibody solutions
were combined with sinapinic acid matrix in a 2:1 ratio, and 1
μL of the solution was dried on a sample plate. The sample was
loaded onto a AB SCIEX TOF/TOF 5800 MALDI-TOF
spectrometer to obtain an average molecular weight for each
antibody. Data was acquired using AB SCIEX Analyst software
and exported to MatLab (MathWorks). The number of
modifications per antibody was then calculated based on the
difference in molecular weight compared to the unmodified
antibody and assuming a specific net mass added depending on
the modification. Direct azide and TCO modifications had
expected masses of 83.0 and 152.2 g/mol, respectively. TCO−
PEG linker modifications with 4 and 24 PEG subunits had
expected masses of 758.2 and 1639.9 g/mol, respectively.
Measurement of TCO and Azide Reactivities on
Antibodies. Functional TCO loadings were measured for
direct TCO-modified antibodies using tetrazine−Oregon
Green 488. Azide-modified antibodies were similarly assessed
using DIBO−Alexa Fluor 488. Reactions were performed using
100 μg of antibody and 50 μM reactive dye in PBS containing
5% DMF at room temperature for 4 h. To study the
mechanism of TCO inactivation, reaction of direct TCO-
modified antibodies with tetrazine−Oregon Green 488 were
also performed in PBS containing 10% DMF or PBS containing
5% DMF and 5% DMSO. All samples were purified into PBS
using Zeba spin desalting columns, and the number of
fluorophores per antibody was determined by absorption
measurements.
Synthesis of DBCO−PEG−TCO Linkers. Heterobifunc-
tional DBCO−PEGn−TCO linkers were synthesized from a
carboxylic acid−PEGn−amine: 100 mg of carboxylic acid−
PEGn−amine was reacted with 2 equiv of TCO−NHS in 2 mL
of DCM containing 1.5 equiv of triethylamine for 16 h on ice.
This reaction mixture was purified using silica column
chromatography with a DCM/methanol gradient. The product
reacted with N,N′-disuccinimidyl carbonate in triethylamine
overnight to form NHS−PEGn−TCO. After evaporation,
DBCO was coupled by reacting NHS−PEGn−TCO with 1.5
equiv of amine-DBCO in 2 mL DCM containing 1.5 equiv of
triethylamine overnight. The final DBCO−PEGn−TCO was
purified using silica column chromatography with a DCM/
methanol gradient for the PEG4 linker or using Sephedex G-10
desalting media for the PEG24 linker. DBCO−PEG4−TCO was
verified by proton NMR. Both linkers where characterized by
HR MS with ESI-LC-TOF. Purified product was dissolved in
methanol and loaded into Waters LCT Premier system and
analyzed by Waters MassLynx (Version 4.0). DBCO−PEG4−
1
TCO: H NMR (400 MHz, CDCl3) δ 7.71−7.73 (d, 1H),
7.44−7.45 (m, 6H), 7.33 (d, 1H), 6.94 (s, 1H), 5.55−5.72 (m,
2H), 5.30 (d, 1H), 5.10−5.20 (m, 1H), 4.38 (s, 1H), 3.56−3.73
(m, 14H), 3.33−3.56 (m, 4H), 2.73−2.76 (m, 8H), 2.40−2.41
(m, 3H), 1.95−2.04 (m, 3H), 1.77 (m, 2H), 1.59 (m, 1H).
HRMS (TOF MS ES+) calcd for C38H49N3O8Na, 698.3520;
found, 698.3418. DBCO−PEG24−TCO: TOF MS [M + Na]
calcd for C78H129N3O28Na, 1578.87; found, 1579.03.
Anti-EGFR Antibody Production from Hybridoma.
Monoclonal mouse antibody specific for human EGFR
(IgG2a, clone Mab 108) was produced from hybridoma HB-
9764 obtained from ATCC (Manassas, VA). Hybridoma cells
were grown in RPMI media containing 10% fetal bovine serum,
5% penicillin/streptomycin, 5% L-glutamine, and 1% sodium
pyruvate and subcultured by dilution of the nonadherent cells.
For antibody production, 500 mL hybridoma cell cultures were
grown for 7 days from an initial seeding density of 5 × 105
cells/mL. The media was collected, centrifuged at 3000g for 10
min to remove cell debris, concentrated, buffer-exchanged into
PBS using a Labscale tangential flow filtration system fitted with
a Pellicon XL cassette (both from Millipore, Billerica, MA), and
purified using a HiTrap Protein G column (GE Healthcare,
Piscataway, NJ).
Antibody Modifications. Anti-EGFR, anti-EpCAM, anti-
TfR, and nonbinding control antibodies were buffer-exchanged
into PBS using Zeba spin desalting columns (Thermo
Scientific) prior to modification. Direct TCO conjugates was
prepared by reacting 200 μg of antibody with 10, 100, or 1000
equiv of NHS−TCO in 0.2 mL PBS containing 10% DMF and
0.1 M NaHCO3 (pH 8.5) for 3 h at room temperature. Azide
and Oregon Green 488A conjugates were prepared in a similar
manner using 10, 30, or 100 equiv of NHS-azide or 10 equiv of
NHS−Oregon Green 488, with DMF omitted from the solvent.
To study the effect of DMF on total TCO conjugation, direct
TCO conjugates were also prepared in PBS containing <1% or
Analysis of Antibody Binding. A431 cells (500 000 cells/
sample) were fixed using 4% formaldehyde (Sigma) in PBS and
washed using PBS. Binding experiments involved an indirect
assay since secondary antibodies may no longer recognize the
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Bioconjugate Chemistry
Article
modified antibodies, particularly for the TCO−PEG cases. This
involved labeling cells with unmodified and azide-, TCO-, and
TCO−PEG-modified antibodies at concentrations ranging
from 0.6 to 200 nM in 100 μL of PBS containing 1 mg/mL
bovine serum albumin (PBS+) for 30 min at room temperature.
After washing 3 times with ice-cold PBS+ by centrifugation,
samples were incubated with Oregon Green 488-modified
antibody at 10 μg/mL in 100 μL of PBS+ for 30 min on ice and
again washed 3 times. Finally, fluorescence was assessed using
an LSRII flow cytometer (BD Biosciences, San Jose, CA) and
analyzed using FlowJo software (Tree Star, Ashland, OR). The
ability of the first antibody to block the Oregon Green 488-
labeled antibody is representative of binding activity. Fully
blocked conditions were indicated by the signal obtained for
the unmodified antibody, and the fully unblocked condition
was represented by cells treated only with the Oregon Green
488-labeled antibody. Specifically, the mean fluorescence
intensity was subtracted from values obtained for cells
incubated only with Oregon Green 488−antibody, and all
samples were then normalized to the unmodified control
(100%).
Preparation of Quantum Dots. Amine-terminated
quantum dots (QDs) were modified with NHS−tetrazine as
described previously.18 QDs were first buffer-exchanged into
PBS using Amicon Ultra-4 centrifugal devices. The reaction was
performed using 0.8 nmoles of QD and 500 mol equiv of
NHS−tetrazine in PBS containing 5% DMF and 0.01 M
NaHCO3 for 3 h at room temperature. Tetrazine−QDs were
purified into PBS using centrifugal filters. To prepare QD
immunoconjugates, 200 μg of antibody was modified with 10
mol equiv of TCO−NHS, buffer-exchanged using a Zeba
column, and reacted with 0.15 nmoles of tetrazine−QD in 1
mL of PBS+ for 3 h at room temperature. QD immunoconju-
gates were purified with Sephacryl S-400 (GE Healthcare) in
PBS using a AKTA Pure FPLC (GE Healthcare). Final
concentrations were determined by absorption measurements
using the QD stock solution for calibration.
with tetrazine−Oregon Green 488 or DIBO−Alexa Fluor 488
at 50 nM or tetrazine−quantum dots at 10 nM for 30 min at
room temperature. Cells were then washed and imaged using
an inverted laser scanning confocal microscope (FV1000,
Olympus) with a 60× water immersion lens, and data was
acquired with Fluoview software (Olympus).
ASSOCIATED CONTENT
* Supporting Information
■
S
Characterization of the heterobifunctional DBCO−PEG−TCO
linker, MALDI-TOF results for all antibodies, investigation of
solvent concentration during antibody conjugation and TCO
reaction, binding analysis of modified antibodies, and back-
ground signals from tetrazine-modified probes. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 949-824-1243 E-mail: jered.haun@uci.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Sumi Lee for synthetic assistance and Dr.
Hongtao Chen for help with confocal imaging studies that were
performed at the Laboratory for Fluorescence Dynamics (LFD)
at the University of California Irvine. The LFD is supported
jointly by the National Institute of General Medical Sciences of
the National Institutes of Health under award no.
8P41GM103540 grant and the University of California Irvine.
This work was also supported in part by the National Cancer
Institute of the National Institutes of Health under award no.
P30CA062203 and the Interdisciplinary Innovation Initiative in
the Henry Samueli School of Engineering at the University of
California Irvine.
Quantification of Cell Labeling Using Flow Cytom-
etry. A431 cells (500 000 cells/sample) were labeled with
TCO- or TCO−PEG-modified antibodies at saturating or near-
saturating concentrations (10−30 μg/mL) in 100 μL of PBS+
for 30 min at room temperature. Cells were then washed 3
times by centrifugation with PBS+, reacted with 50 μM
tetrazine−Oregon Green 488 or 10 nM tetrazine−quantum
dots for 30 min at room temperature, and washed 3 times by
centrifugation with ice-cold PBS+. Fluorescence intensities
were assessed by flow cytometry and analyzed using FlowJo
software. Cells treated with no primary antibody, TCO-
modified nonbinding control, or TCO−PEG24-modified
nonbinding control antibody were used for controls. The
number of reactive TCOs for the TCO−PEG-modified
antibodies was determined based on fluorescence intensity of
the Oregon Green 488, relative to the signal from the TCO-
modified antibodies that were directly assessed for TCO
reactivity previously. Adjustments were made for antibody
binding level to obtain true representations of the number of
reactive TCOs per antibody.
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