Thiol-Reactive Bifunctional Chelators for the Creation of Site-Selectively Modified Radioimmunoconjugates with Improved Stability

Article abstract of DOI:10.1021/acs.bioconjchem.8b00081

Maleimide-bearing bifunctional chelators have been used extensively for the site-selective bioconjugation and radiolabeling of peptides and proteins. However, bioconjugates obtained using these constructs inevitably suffer from limited stability in vivo,

Full text of DOI:10.1021/acs.bioconjchem.8b00081

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Thiol-reactive bifunctional chelators for the creation of site-
selectively modified radioimmunoconjugates with improved stability
Pierre Adumeau, Maria Davydova, and Brian M. Zeglis
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00081 • Publication Date (Web): 06 Mar 2018
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Thiol-reactive bifunctional chelators for the creation of
site-selectively modified radioimmunoconjugates with
improved stability
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Pierre Adumeau1,2, Maria Davydova1, Brian M. Zeglis1,2,3,4
1. Department of Chemistry, Hunter College of the City University of New York, New York,
NY, 10028
2. Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY, 10065
3. Ph.D. Program in Chemistry, the Graduate Center of the City University of New York, New
York, NY, 10016
4. Department of Radiology, Weill Cornell Medical College, New York, NY, 10065
Author e-mail address: bz102@hunter.cuny.edu
Received: January 29th, 2018
Running Title: Improved Thiol-Reactive Bifunctional Chelators
Corresponding Author: *Phone: 1-212-896-0443. Fax: 1-212-896-0484. E-mail:
bz102@hunter.cuny.edu
Keywords: site-specific bioconjugation; site-selective bioconjugation; maleimide; thiol;
sulfhydryl; radioimmunoconjugate; positron emission tomography; PET; immunoPET.
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ABSTRACT
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Maleimide-bearing bifunctional chelators have been used extensively for the site-selective
bioconjugation and radiolabeling of peptides and proteins. However, bioconjugates obtained
using these constructs inevitably suffer from limited stability in vivo, a trait which translates into
suboptimal activity concentrations in target tissue and higher uptake levels in healthy, non-target
tissues. To circumvent this issue, phenyloxadiazolyl methylsulfones have previously been
reported as alternatives to maleimides for thiol-based ligations, but these constructs have
scarcely been used in the field of radiochemistry. In this report, we describe the synthesis of two
thiol-reactive bifunctional chelators for 89Zr and 177Lu based on a new, easy-to-make
phenyloxadiazolyl methylsulfone reagent: PODS. Radioimmunoconjugates created using these
novel bifunctional chelators displayed higher in vitro stability than their maleimide-derived
cousins. More importantly, PET imaging in murine models of cancer revealed that a 89Zr-labeled
radioimmunoconjugate created using a PODS-bearing bifunctional chelator produced
significantly lower uptake in non-target tissues than its analogous maleimide-based counterpart.
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INTRODUCTION
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The ability of many biomolecules to selectively and specifically target cellular biomarkers
has long been harnessed for both nuclear imaging and radiotherapy. This exploitation requires
the attachment of a radioactive payload to the biomolecule. In the case of peptides and proteins,
this is typically performed by reacting bifunctional probes with the amino acids within the
biomolecule, most often lysines. While controlling the site of this conjugation is fairly easy with
small peptides ⎯ which rarely possess more than one or two copies of each amino acid ⎯ this
becomes a much bigger problem with larger biomolecules. For example, most antibodies contain
dozens of lysines distributed throughout their macromolecule structure. The indiscriminate
attachment of a bifunctional chelator to these lysines can lead to the formation of thousands of
different regioisomers. Not surprisingly, this so-called “random” approach to bioconjugation has
low reproducibility and produces highly heterogeneous conjugates. In addition, this strategy can
result in the inadvertent grafting of payloads to bioactive sites in the biomolecule, rendering a
portion of the immunoconjugates inoperative.
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To circumvent these issues, researchers have turned their attention to strategies that allow for
better control over the site of the ligation reaction, known as “site-specific” bioconjugations.1–5
Conjugations to cysteine ⎯ a thiol-bearing amino acid present in small numbers in proteins ⎯
with maleimide-bearing probes have become a staple of this field and have been used extensively
over the last three decades.6 The prevalence of this strategy is rooted mainly in its simplicity and
efficiency. Many proteins contain disulfide bridges that can be easily reduced to form reactive
thiols, and the Michael addition between a maleimide and a sulfhydryl group can be performed at
physiological pH and room temperature, reliably leading to the formation of a succinimidyl
thioether linkage within an hour (Figure 1).
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Figure 1. Michael addition of a thiol-bearing biomolecule (green) and a radionuclide-bearing
maleimide (yellow) to form a radiolabeled bioconjugate as well as the additional reactions that
the radiolabeled construct can undergo in the presence of endogenous thiol-bearing molecules
(pink).
While the ligation between thiols and maleimides represents an undeniable improvement
over random conjugation methods, the reaction suffers from drawbacks as well. Indeed,
maleimide-based conjugates display limited stability in physiological media because the
succinimidyl thioether linkage can undergo a retro-Michael reaction that leads to the release of
the payload or its exchange with other molecules containing free thiols (most often serum
albumin, cysteine, and glutathione).7–11 Several studies focused on the development antibody-
drug conjugates have reported on this phenomenon as well as the off-target uptake of payload
that it creates.7,9–11 In response to this issue, these studies and others have explored the possibility
of modifying the chemical environment of the succinimidyl moiety in order to accelerate its
hydrolysis to a more stable thioether form that is immune to thiol exchange reactions. In the
context of nuclear imaging and radiotherapy, this retro-Michael reaction can lead to the release
of the radioactive payload and the subsequent in vivo radiolabeling of endogeneous biomolecules
through thiol exchange reactions (Figure 1; top right). This “leakage” causes higher uptake in
non-target tissues and lower uptake in target tissues, ultimately resulting in higher radiation
doses to healthy tissues, reduced imaging contrast, and lower therapeutic ratios. Several
alternative reagents which create more-stable linkages with thiols have been used for the
bioconjugation and radiolabeling of antibodies, most notably tosylates, bromo- and iodo-acetyls,
and vinyl sulfones.7,12–17 However, each of these options has drawbacks as well, including lower
reaction rates with thiols as well as reactivity with other amino acids.18
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An article published in 2013 holds particular promise in this area.19 In this work, the Barbas
Laboratory described the creation of an oxadiazolyl methyl sulfone-based reagent that could
selectively react with thiols at a rate comparable to maleimides and form more stable conjugates
than those obtained with the latter (Scheme 1). This reagent was used to label different
THIOMABs ⎯ antibodies engineered to bear free cysteine residues ⎯ with fluorescein, and
these phenyloxadiazole-based conjugates displayed higher stability over time in serum than their
maleimide-based cousins.20 Yet despite the benefits of this ligation over maleimide-thiol
conjugations, this construct has scarcely been used since its publication. This is especially
obvious in radiochemistry, in which only two reports describe the use of the reagent or its
derivatives. In the first, Zhang et al. used this reagent along with other methylsulfone-oxadiazole
derivatives to incorporate an azide moiety into an RGD peptide.21 In the second, a team in
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18
Switzerland designed an F-labeled prosthetic group based on this reagent which seemed to
offer substantial advantages over traditional maleimide-based prosthetic groups for 18F-
radiofluorinations.22
Scheme 1. Structure of the reagent reported by Barbas, et al. and scheme of its reactivity with
thiols.19
The infrequent use of this reagent was somewhat surprising at first, especially since it is
commercially available from Sigma-Aldrich. However, our experience in ordering it three
separate times provides some hint as to this scarcity. In each instance, we received a complex
mixture of degradation products containing less than 15% of the desired compound
(Supplementary Figure S1). In light of these setbacks, we considered synthesizing the reagent
according to the published procedure, but it appeared that this synthesis was far from trivial for
radiochemical and molecular imaging laboratories that lack sophisticated organic chemistry
equipment.
With this in mind, we have aimed to create an easily accessible phenyloxadiazole-based
reagent for thiol conjugations that can be obtained through a robust route that could be carried
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out with basic organic chemistry equipment. Using this reagent ⎯ dubbed PODS ⎯ we have
synthesized two bifunctional chelators for the bioconjugation and radiolabeling of thiol-bearing
biomolecules with 89Zr and 177Lu. It is our hope that the efficacy of this PODS reagent as well as
the simple and straightforward chemistry associated with its synthesis will encourage researchers
from all fields to move from maleimide-based probes to a more reliable and stable alternative.
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RESULTS AND DISCUSSION
Synthesis
The synthesis originally published by Toda, et al. involves extensive chromatographic
purifications and overnight refluxes over its five steps.19 This approach requires specific
equipment and skills, and while both are undoubtedly available in any organic chemistry
laboratory, they are less common in laboratories focused on the synthesis of bioconjugates.
Therefore, the first step of our investigation was designing an easily synthesized
phenyloxadiazole-based reagent. Along these lines, our goal was to devise a synthetic route with
minimal equipment, simple procedures, and no chromatographic purifications.
In the original synthesis, the formation of the oxadiazole ring is particularly tricky.
Fortunately, several 5-phenyl-1,3,4-oxadiazole-2-thiol derivatives bearing substitutions at the
phenyl para-position are commercially available, allowing us to circumvent this cumbersome
synthesis. As amide linkages can be formed under milder conditions than the ether linkage
created by Toda, et al., 5-(4-aminophenyl)-1,3,4-oxadiazole-2-thiol seemed like an ideal
precursor for an easy-to-make reagent. Two other essential elements were included in the reagent
as well: (i) a short PEG chain to make the construct more water-soluble and provide a spacer
between the biomolecule and the payload and (ii) a terminal amine that can be readily coupled
with amine-reactive bifunctional chelators and fluorophores.
Our phenyloxadiazolyl methyl sulfone derivative ⎯ dubbed ‘PODS’ ⎯ was obtained in four
steps with a global yield of 48% (Scheme 2). All intermediates were obtained in >95% purity
using minimal equipment and simple procedures without chromatographic purifications. The
methyl thioether 1 was obtained quantitatively via the methylation of the thiol with methyl
iodide; unfortunately, we were not able to replace the use of the toxic methyl iodide with any
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safer reagent. The coupling between 1 and the carboxylic acid-bearing PEG chain was performed
using EDCI as a coupling agent. Overnight reaction at room temperature led to the complete
consumption of the starting material but also the formation of degradation products. It was
possible to separate 2 from the two unidentified impurities by precipitating it with a mixture of
ethyl acetate and cyclohexane. The oxidation of 2 was performed using the peracid mCPBA.
This lead to the formation of the methylsulfone 3 with a yield of 90%. Finally, PODS was
obtained from 3 after the quantitative deprotection of the terminal amine using a solution of
trifluoroacetic acid in dichloromethane. Solutions of PODS in DMSO were stable at room
temperature and have been kept at room temperature for 8 months without any noticeable signs
of degradation (Supplementary Figure S2).
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Scheme 2. Synthesis of PODS
Reactivity with model thiols
In order to study the reactivity of PODS with thiols, Boc-L-cysteine was used as a model
thiol in conjunction with the mild reducing agent tris(2-carboxyethyl)phosphine (TCEP), whose
role was the reduction of any latent cystine species in solution (Scheme 3). The reaction between
PODS and Boc-L-cysteine was quantitative in less than 2 minutes at pH 7.5 (Supplementary
Figure S3). In a move toward a model closer to proteins and peptides, a tripeptide containing a
central cysteine ⎯ glutathione ⎯ was reacted with PODS as well. Again, the conjugation
reaction reached completion before the first measurement could be acquired (<3 min at pH 7.5).
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Scheme 3. Coupling of PODS to thiol-bearing biomolecules.
Conjugation to a model antibody
Next, the utility of PODS for the site-selective modification of proteins was investigated. To
this end, the well-characterized HER2-targeting humanized immunoglobulin trastuzumab was
chosen as the model protein, and a fluorescent derivative of PODS (PODS-FL) was prepared via
the reaction of PODS with fluorescein isothiocyanate. PODS-FL was conjugated with
trastuzumab in the presence of TCEP, a mild reducing agent used to cleave the four inter-chain
disulfide bonds within the immunoglobulin (Figure 2). The presence of several thiol moieties
capable of reacting with PODS means that this approach to bioconjugation can best be
characterized as “site-selective” rather than “site-specific”, as the latter suggests the formation of
completely homogeneous immunoconjugates. Indeed, the grafting of cargoes onto any of the 8
available cysteine residues can lead to the formation of several regioisomers. Of course, “site-
specificity” represents the ideal scenario. Yet still, site-selective conjugations easily outclass
random modification methods due to the controlled location of the conjugation sites — i.e. in the
hinge region, far from the CDR domains — as well as their limited number. The number of
fluorescein moieties present on the final conjugate was assessed using UV-Vis
spectrophotometry. As expected, the degree of labeling (DOL) increased with the number of
equivalents of PODS-FL in the reaction mixture, reaching 2.04 ± 0.03 after 2 hours of incubation
with 10 equivalents of PODS-FL. Interestingly, the DOL was only minimally affected by the
temperature of the reaction: similar results were obtained at 25 °C and 37 °C. Even more
importantly, the DOL of the immunoconjugates created via the incubation of 10 equivalents of
PODS-FL with the antibody in the absence of TCEP or with the antibody following re-
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oxidization remained close to zero: 0.03 ± 0.02 and 0.1 ± 0.1, respectively. This last piece of data
demonstrates the specificity of the reagent especially well.
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Figure 2. (A) The site-selective modification of trastuzumab with PODS-FL. The structure of
trastuzumab-PODS-FL depicts only one regioisomer resulting from the conjugation reaction,
though several are possible. (B) Plot of the degree of labeling (DOL) of the conjugates obtained
after 2 h of reaction using different numbers of equivalent of PODS-FL under various conditions
(10 equivalents of TCEP, unless specified otherwise).
In order to investigate the influence of antibody type on the efficacy of the conjugation
reaction, several other antibodies ⎯ including human, humanized, chimeric, and murine
constructs ⎯ were incubated with 10 equivalents of PODS-FL under the conditions described
above (Table 1). Intriguingly, while the DOL of the fully human, humanized, and chimeric
immunoconjugates all remained slightly above 2, the number of fluorophores per antibody
dropped to ∼1.5 for the murine immunoglobulins. This difference may be due to dissimilarities in
the regions surrounding the disulfide linkages in murine antibodies, but this hypothesis will
require further investigation.
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Table 1: Degree of labeling of different antibodies following conjugation with PODS-FL
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Antibody
Human plasma IgG
Trastuzumab
huA33
Cetuximab
AR 9.6
Mouse plasma IgG
Type
Human
Humanized
Humanized
Chimeric
Murine
Constant region
Human IgG
Ratio FL:Ab
2.1±0.1
2.0±0.1
2.1±0.1
2.2±0.1
1.4±0.1
1.5±0.1
Human IgG1
Human IgG1
Human IgG1
Murine IgG1
Murine IgG
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Murine
Synthesis of bifunctional chelators
In light of these results, we moved forward with the synthesis of thiol-reactive bifunctional
chelators based on PODS. Zirconium-89 and lutetium-177 were chosen as the proof-of-concept
radionuclides for this study. Over the last decade, 89Zr has become an essential nuclide for
immunoPET, and 177Lu is a β-emitting nuclide of growing interest for radioimmunotherapy.23–25
Accordingly, we chose to modify PODS with desferrioxamine (DFO) and CHX-A′′-DTPA, the
‘gold standard’ chelators for 89Zr and 177Lu, respectively.26,27
PODS-CHX-A′′-DTPA was obtained in 69% yield via the reaction of PODS with an
isothiocyanate-bearing derivative of CHX-A′′-DTPA chelator in the presence of base (Scheme
4). The synthesis PODS-DFO through a similarly straightforward approach turned out to be
much trickier, as degradation products were commonly seen upon the analysis of the crude
reaction mixture. Upon investigation, it became apparent that the hydroxylamines of the chelator
react with PODS under basic conditions by substituting at the methylsulfonyl position, causing
the formation of undesired side products (Supplemental Figure S5). To circumvent this, we
‘protected’ the hydroxylamine groups via the coordination of iron. The resulting p-SCN-Bn-
DFO-Fe was conjugated with PODS in presence of base, leading to the formation of PODS-
DFO-Fe in good yield (72%) (Scheme 4). We were able to obtain pure PODS-DFO by removing
the iron with an aqueous solution of oxalic acid 1M. However ⎯ as could perhaps have been
expected ⎯ free PODS-DFO underwent degradation in aqueous solution at pH > 4.5, almost
certainly due to the hydroxylamine-mediated reactions discussed above. Given the low reaction
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rate between PODS and thiols at this pH (Supplemental Figure S3), unprotected PODS-DFO was
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deemed unsuitable for the modification of peptides and proteins.
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Scheme 4. Synthesis of the PODS-based bifunctional chelators for 89Zr and 177Lu.
Bioconjugation
The bifunctional chelators were then conjugated to a model antibody ⎯ trastuzumab ⎯ to
produce DFO-PODS-trast and CHX-A′′-DTPA-PODS-trast. A strategy similar to that developed
by Verel et al. was used to obtain the former: PODS-DFO-Fe was conjugated to the antibody,
and the iron was removed thereafter.28 The ideal conditions for this decomplexation procedure
were investigated and optimized for PODS-DFO-Fe using UV-Vis spectrophotometry, and 30
min of incubation at pH 4.5 and 25 °C in presence of EDTA ― 2.2 g/L ― was found to lead to
the removal of >95% of the iron from DFO (Supplementary Table S1). Informed by the
experiments with PODS-FL, 10 equivalents of PODS-DFO-Fe and PODS-CHX-A′′-DTPA were
used with a goal of a final DOL of 2 chelators/mAb. Mass spectrometry confirmed that the
immunoconjugates were indeed modified with PODS-DFO-Fe and PODS-CHX-A′′-DTPA.
However, the cleavage of the disulfide bonds resulted in the fragmentation of the antibody
during MALDI-TOF which, in turn, rendered the accurate determination of the exact DOLs
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impossible. To facilitate in vitro comparisons, the maleimide-based counterparts of these two
immunoconjugates ⎯ DFO-mal-trast and CHX-A′′-DTPA-mal-trast ⎯ were also synthesized
using identical reaction conditions.
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Radiolabeling and in vitro stability
89Zr-DFO-PODS-trast and 89Zr-DFO-mal-trast were each obtained in good yield, high purity,
and similar specific activity ⎯ 2.5 ± 0.2 and 2.4 ± 0.4 Ci/g, respectively ⎯ using standard 89Zr-
radiolabeling protocols (Supplemental Table S2). Likewise, typical 177Lu-labeling protocols were
used to produce 177Lu-CHX-A′′-DTPA-PODS-trast and 177Lu-CHX-A′′-DTPA-mal-trast in high
yield, high purity, and identical specific activities: 1.3 ± 0.1 Ci/g.
All four radioimmunoconjugates were incubated in human serum at 37 °C for one week,
during which the integrity of the constructs was assessed using instant thin layer chromatography
(ITLC) (Supplemental Figure S6). This trial clearly revealed that the PODS-based conjugates
were more stable than their maleimide-based cousins. 89Zr-DFO-PODS-trast was 81 ± 5% intact
after seven days of incubation, whereas the integrity of 89Zr-DFO-mal-trast fell well below 70%
by the same point: 63 ± 12%. A similar difference ⎯ though admittedly of lesser magnitude ⎯
was obtained with the 177Lu-labeled immunoconjugates: 177Lu-CHX-A′′-DTPA-PODS-trast and
177Lu-CHX-A′′-DTPA-mal-trast were 82 ± 1% and 76 ± 2 % intact, respectively, after 7 days.
These difference in the stability of the maleimide- and PODS-based immunoconjugates are
consistent with previously obtained data for antibody-fluorophore conjugates.20
In vivo behavior
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We chose a different model system for a comparison of the in vivo behavior of Zr-DFO-
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PODS- and Zr-DFO-mal-based radioimmunoconjugates: huA33, a humanized antibody that
targets the A33 antigen, a transmembrane glycoprotein expressed in more than 95% of colorectal
cancers.29–31 There are two reasons for this switch. First, we wanted to interrogate the modularity
of our PODS-based approach by validating our bioconjugation, stability, and in vitro results with
a different antibody. Second, many HER2-expressing cell lines (e.g. BT474, SKOV3, etc.)
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exhibit irregular growth patterns in mice, resulting in heterogeneous tumors. In contrast, the two
most often used A33 antigen-expressing cell lines ⎯ SW1222 and LS174T ⎯ grow extremely
predictably in athymic nude mice, reliably forming xenografts with similar size and shape. Given
that the goal of this phase of this investigation was to make comparisons between the in vivo
behavior of PODS- and maleimide-based radioimmunoconjugates, we decided to make the
homogeneity of our xenografts a top priority.
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Using the procedures we have described above, DFO-PODS-huA33 and DFO-mal-huA33
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were successfully synthesized and radiolabeled with Zr to produce Zr-DFO-PODS-huA33
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and Zr-DFO-mal-huA33 in high yield, purity, and specific activity (2.8 ± 0.4 and 2.8 ± 0.2
Ci/g, respectively). The incubation of these radioimmunoconjugates in human serum for seven
days at 37 °C yielded stability profiles similar to those obtained for the trastuzumab-based
radioimmunoconjugates: after a week, 89Zr-DFO-PODS-huA33 remained 86 ± 1 % intact, while
its maleimide-based cousin was only 61 ± 5 % intact (Supplementary Figure S6). The in vitro
immunoreactivity of the two radioimmunoconjugates was determined via saturation binding
assay with A33 antigen-expressing SW1222 human colorectal cancer cells. The immunoreactive
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fractions were similar for both constructs: 0.95 ± 0.04 and 0.92 ± 0.06 for Zr-DFO-PODS-
huA33 and 89Zr-DFO-mal-huA33, respectively.
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89Zr-DFO-PODS-huA33 and Zr-DFO-mal-huA33 were injected into athymic mice bearing
subcutaneous A33 antigen-expressing SW1222 human colorectal cancer xenografts. PET
imaging revealed striking differences between the in vivo behavior of the two constructs (Figure
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3). These disparities are particularly evident in the maximum intensity projections. Zr-DFO-
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PODS-huA33 displayed better tumor-to-background activity concentration ratios than the Zr-
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DFO-mal-huA33 at 24, 48, and 120 h post-injection. For Zr-DFO-mal-huA33, uptake in the
bone was clearly visible as early as 24 h after administration and increased over time, and
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significant uptake in the kidneys and liver could also be observed. Zr-DFO-PODS-huA33, in
contrast, produced much lower activity concentrations in each of these three tissues. The two
constructs produced similar activity concentrations in the tumor: around 50-60 %ID/g for each.
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Figure 3. Planar (left) and maximum intensity projection (right) PET images of athymic nude
mice bearing A33 antigen-expressing SW1222 colorectal cancer xenografts (white arrow)
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following the injection of Zr-DFO-PODS-huA33 and Zr-DFO-mal-huA33 (140 µCi, 60-65
µg). The coronal slices intersect the center of the tumors.
The biodistribution data tells a similar story (Figure 4 and Supplemental Table S3). Most
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notably, Zr-DFO-mal-huA33 produces higher activity concentrations in the kidneys and bone
than 89Zr-DFO-PODS-huA33. Similar tumoral activity concentrations were observed for the two
radioimmunoconjugates; the decreases observed for both constructs between 48 h and 120 h
post-injection were likely due to the growth of the tumors between the two time points
(Supplemental Figure S7). The efficient clearance of 89Zr-DFO-PODS-huA33 from healthy
organs over the course of the study led to it producing lower uptake values in all non-target
tissues ⎯ except the large intestine ⎯ at 120 h post-injection compared to 89Zr-DFO-mal-
huA33. In contrast, 89Zr-DFO-mal-huA33 produced increasing activity concentrations in the
bone over the course of the study and did not display any appreciable clearance from the liver
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and the spleen over 5 days. The tumor-to-organ activity concentration ratios obtained with Zr-
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DFO-PODS-huA33 are generally superior to those created by Zr-DFO-mal-huA33 at 120 h
post-injection (Supplemental Table S4). More specifically, the tumor-to-organ activity
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concentration ratios for the four organs with the highest uptake ⎯ liver, spleen, kidney and bone
⎯ are nearly double for 89Zr-DFO-PODS-huA33 compared to 89Zr-DFO-mal-huA33: 24.4 ± 8.5
to 13.6 ± 2.4 (tumor-to-liver), 23.2 ± 13.4 and 14.9 ± 1.6 (tumor-to-spleen), 19.4 ± 5.2 and 12.0
± 2.3 (tumor-to-kidney), and 6.5 ± 1.9 and 3.0 ± 0.7 (tumor-to-bone). Indeed, the only organ for
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which the activity concentration ratio is lower for Zr-DFO-PODS-huA33 compared to Zr-
DFO-mal-huA33 is the large intestine: 67 ± 18 and 132 ± 30, respectively.
Figure 4. Biodistribution data after the administration of 89Zr-DFO-PODS-huA33 and 89Zr-
DFO-mal-huA33 (30 µCi, 15-18 µg) to athymic nude mice bearing A33 antigen-expressing
subcutaneous SW1222 human colorectal cancer xenografts. The values for the stomach, small
intestine, and large intestine include contents.
The superior stability of the PODS-thiol linkage can explain these results. Indeed, the in vivo
radiolabeling of endogenous thiol-bearing molecules and proteins caused by the retro-Michael
reaction leads to off-target uptake of the radiometal and slower clearance from the non-target
organs. Further, the metabolism of these radiolabeled endogeneous biomolecules likely causes
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the release of 89Zr into the blood and translates into higher activity concentrations in the bone.32–
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CONCLUSION
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In this investigation, we have described the synthesis of a new thiol-reactive
phenyloxadiazolyl methylsulfone reagent ⎯ dubbed PODS ⎯ for the site-selective modification
of peptides and proteins. This reagent was obtained in four steps with a global yield of 48%
using a simple and straightforward synthetic route. PODS displayed rapid and selective reactivity
with thiols and was successfully used to site-selectively modify a model antibody (trastuzumab)
with a fluorophore. We then synthesized two bifunctional chelators based on PODS: PODS-DFO
(for 89Zr) and PODS- CHX-A′′-DTPA (for 177Lu). Both of these constructs were used
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successfully for the site-selective modification and radiolabeling of trastuzumab, and Zr- and
177Lu-labeled radioimmunoconjugates based on PODS proved more stable in vitro than their
maleimide-based analogs. Finally, the in vivo behavior of 89Zr-DFO-PODS-huA33 was
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compared to that of Zr-DFO-mal-huA33 in a murine model of human colorectal cancer. The
performance of the former was clearly superior, especially at later time points (>48 h p.i.). In
light of the clinical preference for imaging time points ranging from 72 – 144 h, the use of
PODS-based radioimmunoconjugates in lieu of their maleimide-derived cousins could
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substantially improve the quality of clinical Zr-immunoPET scans. Moreover, the improved
stability of 177Lu-DOTA-PODS-labeled antibodies compared to maleimide-based constructs
could be beneficial in radioimmunotherapy as well, providing radioimmunoconjugates with
lower uptake in healthy tissues and thus higher therapeutic indices.
While this study has focused on the modification of IgGs, PODS-based reagents hold equal
promise for the site-selective modification of cysteine-bearing antibody fragments, peptides, and
proteins as well. In addition, bifunctional chelators similar to the ones described in this report
could be created to accommodate virtually any radiometal, as amine-reactive derivatives of a
wide range of chelators are currently commercially available. Ultimately, we sincerely hope that
this work ⎯ and especially the simple and straightforward chemistry that we have used here ⎯
will help promote the use of phenyloxadiazolyl reagents for thiol-based conjugations across a
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wide variety of disciplines and encourage researchers to move from maleimides to more reliable
and more stable alternatives.
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EXPERIMENTAL PROCEDURES
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Synthesis of 4-(5-(methylthio)-1,3,4-oxadiazol-2-yl)aniline (1): In a glass vessel protected
from light with aluminium foil, 5-(4-aminophenyl)-1,3,4-oxadiazole-2-thiol (100 mg; 0.517
mmol; 1 eq.) was dissolved in 3 mL of methanol, and diisopropylethylamine (DIPEA; 360 µL;
2.07 mmol; 4 eq.) was added to the solution. The mixture was stirred at room temperature for 10
minutes before the slow addition of iodomethane (32 µL; 0.517 mmol; 1 eq.). After 45 minutes,
the solvent was removed under reduced pressure. The white solid was dissolved in 3 mL of ethyl
acetate and washed with a 0.1 M solution of sodium carbonate and then washed with water until
reaching pH 7. The organic phase was dried over MgSO4 before the evaporation of the volatiles
under reduced pressure, ultimately affording white needles (107 mg; yield: 100%). Because of its
slight light-sensitiveness, this compound was kept in foil-covered glass vials. TLC (Ethyl
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acetate:triethylamine, 9:1): Rf 0.65. H NMR (500 MHz, CDCl3) 7.79 (2H, d, J = 8.5 Hz), 6.72
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(2H, d, J = 8.5 Hz), 4.04 (2H, br s), 2.75 (3H, s). C NMR (125 MHz, CDCl3)166.3, 163.7,
149.7, 128.5, 114.8, 113.5, 14.8. HRMS-ESI m/z Calcd for [C9H9N3OS+H]+: 208.0539; found:
208.0539; Δ: -0.80 ppm.
Synthesis of tert-Butyl (18-((4-(5-(methylthio)-1,3,4-oxadiazol-2-yl)phenyl)amino)-15,18-
dioxo-4,7,10-trioxa-14-azaoctadecyl)carbamate (2): To a solution of N-Boc-N’-succinyl-4,7,10-
trioxa-1,13-tridecanediamine (386.5 mg; 0.919 mmol; 1 eq.) in dichloromethane (3 mL) in a
glass vessel protected from light with aluminum foil was added diisopropylethylamine (480 µL;
3 eq.) and N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI; MW = 191.7
g/mol; 1.379 mmol; 1.5 eq.). To this solution was added 200 mg of 1 (0.965 mmol; 1.05 eq.).
The reaction mixture was stirred at room temperature overnight. The mixture was then washed
three times with 5 mL of an aqueous solution of 1 M hydrochloric acid. The organic phase was
washed twice with 5 mL of an aqueous solution of 1 M Na2CO3 and then with water until pH
neutral. The organic phase was dried on MgSO4 and evaporated. The off-white solid residue was
dissolved in 10 mL of ethyl acetate, and the target compound was precipitated by slow addition
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of 30 mL of cyclohexane. After filtration, the final product was obtained as a white powder (310
mg; yield: 55%). Because of its slight light-sensitiveness, this compound was kept in foil-
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covered glass vials. TLC (acetonitrile:water, 3:1): Rf 0.73. H NMR (500 MHz, CDCl3) 9.68
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(1H, s), 7.91 (2H, d, J= 9.0 Hz), 7.71 (2H, d, J= 8.5 Hz), 6.82 (1H, s), 4.99 (1H, s), 3.70-3.45
(12H, m), 3.41 (2H, q, J= 6.0 Hz), 3.20 (2H, q, J= 6.5 Hz), 2.76 (3H, s), 2.71 (2H, m), 2.63 (2H,
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m), 1.80-1.70 (4H, m), 1.42 (9H, s). C NMR (125 MHz, CDCl3) 172.6, 171.3, 165.8, 164.6,
156.2, 141.8, 127.7, 119.6, 118.6, 79.2, 70.6, 70.5, 70.3, 70.1, 69.6, 38.8, 38.5, 33.5, 31.6, 29.9,
28.6, 14.8. HRMS-ESI m/z Calcd for [C28H43N5O8S+Na]+: 632.2725; found: 632.2722; Δ: 0.35
ppm.
Synthesis of tert-Butyl (18-((4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)amino)-15,18-
dioxo-4,7,10-trioxa-14-azaoctadecyl)carbamate (3): In a glass vessel protected from light with
aluminium foil, 30 mg of 2 (0.049 mmol, 1 eq.) was dissolved in 4 mL of dichloromethane
before the slow addition of 48.5 mg of m-chloroperbenzoic acid (70%, 0.197 mmol, 4 eq.). The
reaction mixture was stirred at room temperature overnight. The resulting yellow mixture was
washed three times with 8 mL of an aqueous 0.1 M solution of NaOH and then with water until
the pH of the solution reached 7. The organic phase was dried over MgSO4 and evaporated under
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reduced pressure to give a pale solid (28.5 mg, yield: 90%). TLC (acetonitrile): Rf 0.51. H
NMR (500 MHz, CDCl3) 9.99 (1H, s), 7.98 (2H, d, J= 9.0 Hz), 7.75 (2H, d, J= 8.5 Hz), 6.88
(1H, s), 4.99 (1H, s), 3.66-3.50 (15H, m), 3.41 (2H, q, J= 6.0 Hz), 3.20 (2H, q, J= 6.5 Hz), 2.71
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(2H, m), 2.65 (2H, m), 1.80-1.70 (4H, m), 1.43 (9H, s). C NMR (125 MHz, CDCl3) 172.6,
171.5, 166.5, 161.6, 156.1, 143.4, 128.7, 119.6, 116.4, 79.1, 70.5, 70.4, 70.2, 70.0, 69.4, 43.0,
38.8, 38.4, 33.2, 31.3, 29.7, 28.4. HRMS-ESI m/z Calcd for [C28H43N5O10S+H]+: 642.2803;
found: 642.2797; Δ: 1.06 ppm.
Synthesis of N1-(3-(2-(2-(3-Aminopropoxy)ethoxy)ethoxy)propyl)-N4-(4-(5-(methylsulfonyl)-
1,3,4 oxadiazol-2-yl)phenyl)succinamide (PODS): To a solution of 3 in dichloromethane (30.0
mg, 46.8 µmol; in 1.6 mL) was added trifluoroacetic acid (400 µL). The reaction mixture was
stirred at room temperature for 3 hours, and the volatiles were then removed by evaporation
under reduced pressure. The oily residue was dissolved in 7 mL of water and 4 mL of ethyl
acetate. The aqueous phase was then washed twice with 4 mL of ethyl acetate. The aqueous layer
was lyophilized to afford the final product as a white powder (25.0 mg, yield: 98%). TLC
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(acetonitrile:water, 3:1): Rf 0.38.1H NMR (500 MHz, D2O) 7.85 (2H, d, J = 9.0 Hz), 7.55 (2H, d,
J = 8.5 Hz), 3.60-3.45 (15H, m), 3.45 (2H, t, J= 6.5 Hz), 3.20 (2H, t, J= 6.5 Hz), 3.04 (2H, t, J=
7.0 Hz), 2.67 (2H, t, J= 6.5 Hz), 2.54 (2H, t, J= 6.5 Hz), 1.87 (2H, qt, J= 6.5 Hz), 1.70 (2H, qt, J=
6.5 Hz).13C NMR (125 MHz, D2O) 174.5, 173.2, 166.8, 161.4, 142.2, 128.6, 120.3, 116.6, 69.4,
69.4, 69.3, 69.2, 68.2, 68.2, 42.5, 37.6, 36.2, 31.9, 30.7, 28.2, 26.4. HRMS-ESI m/z Calcd. for
[C23H35N5O8S+H]+: 542.2279; found: 542.2281; Δ: -0.36 ppm.
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Synthesis of PODS-FL: To a solution of 6.0 mg of PODS in 600 uL of DMF (11.1 µmol, 1
eq.) were added 3.9 µL of DIPEA (22.2 µmol, 2 eq.) and 122 µL of a 0.1 M solution of
fluorescein-isothiocyanate in DMSO (12.2 µmol, 1.1 eq.). The mixture was protected from light
and let to react overnight at room temperature. The product was then purified by HPLC (gradient
MeCN/H2O + 0.1% TFA, 0% MeCN to 100% in 30 min, Rt = 22.5 min) to afford 3.8 mg of an
orange powder (yield: 37%). HRMS-ESI m/z Calcd. for [C44H46N6O13S2+H]+: 931.2637; found:
931.2634; Δ: 0.27 ppm.
Synthesis of PODS-CHX-A″-DTPA: To a solution of PODS (2.1 mg, 3.84 µmol, 1 eq.) in
200 µL of DMSO was added DIPEA (6.7 µL, 10 eq.). To this solution was added 3.0 mg of p-
SCN-Bn-CHX-A″-DTPA•3HCl (4.25 µmol, 1.1 eq.; Macrocyclics, Inc.), and the mixture was
stirred at room temperature overnight. The product was then purified by HPLC (gradient
MeCN/H2O + 0.1% TFA, 0% MeCN to 100% in 25 min, Rt = 19.5 min) to afford 3.0 mg of a
white powder (yield: 69%).HRMS-ESI m/z Calcd. for [C49H69N9O18S2+H]+: 1136.4275; found:
1136.4273; Δ: 0.18 ppm.
Synthesis of PODS-DFO-Fe: To a solution of p-SCN-Bn-DFO (5.0 mg, 6.65 µmol, 1.2 eq.;
Macrocyclics, Inc.) in 100 µL of DMSO was added 1.8 mg of FeCl3 hexahydrate (6.65 µmol, 1.2
eq.). The resulting dark red solution was added to a solution of PODS (3.0 mg, 5.54 µmol, 1 eq.)
in 100 µL of DMSO with DIPEA (4.83 µL, 5 eq.). The mixture was stirred at room temperature
overnight. The product was then purified by HPLC (gradient MeCN/H2O +0.1% TFA, 30%
MeCN to 100% in 30 min, Rt = 16.5 min) to afford 5.2 mg of a dark red solid (yield:
72%).HRMS-ESI m/z Calcd. for [C56H84FeN13O16S3+H]+: 1347.4745; found: 1347.4735; Δ: 0.74
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Preparation of mAb-PODS-FL conjugates: To a suspension of 200 µg of antibody in PBS pH
7.4 (1 mg/mL) was added 1.33 µL of a fresh TCEP solution (10 mM in water, 10 eq.) and the
appropriate volume of a solution of PODS-FL (1 mM in DMSO). The reaction mixture was
stirred on a thermomixer (25°C or 37°C) for 30 min, 2 h, or 24 h. The conjugate was then
purified on a size exclusion column (Sephadex G-25 M, PD-10 column, GE Healthcare; dead
volume = 2.5 mL, eluted with 2 mL of PBS, pH 7.4) and concentrated using centrifugal filtration
units with a 50,000 Da molecular weight cut off (AmiconTM Ultra 4 Centrifugal Filtration Units,
Millipore Corp. Billerica, MA).
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Preparation of mAb-PODS-DFO conjugates: To a suspension of 1 mg of antibody in PBS
pH 7.4 (1 mg/mL) was added 6.7 µL of a fresh TCEP solution (10 mM in water, 10 eq.) and 33
µL of a solution of PODS-DFO-Fe (2 mM in DMSO). The reaction mixture was stirred on a
thermomixer at 2 5°C for 2 hours. To this yellow solution was then added 100 µL of EDTA
(tetrasodic salt, 25 g/L in water), and the pH was adjusted to 4.5 with 0.25 M H2SO4. The
mixture was stirred at 25 °C for 30 min to yield a colorless solution, indicating the transchelation
of the Fe(III) from the DFO. The conjugate was then purified on a size exclusion column
(Sephadex G-25 M, PD-10 column, GE Healthcare; dead volume = 2.5 mL, eluted with 2 mL of
PBS, pH 7.4) and concentrated using centrifugal filtration units with a 50,000 Da molecular
weight cut off (AmiconTM Ultra 4 Centrifugal Filtration Units, Millipore Corp. Billerica, MA).
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ACKNOWLEDGEMENTS
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The authors are grateful for the generous financial support of the National Institutes of Health
(4R00CA178205-02 and R01CA204167), the TeamConnor Childhood Cancer Foundation, and
the National Institute on Minority Health and Health Disparities (G12MD007599). Services
provided by the MSKCC Small-Animal Imaging Core Facility were supported in part by NIH
grants R24 CA83084 and P30 CA08748.
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SUPPORTING INFORMATION DESCRIPTION
General information; synthetic protocols; chemical characterization data; PODS-thiol
reactivity assay; preparation of mAb-PODS-FL conjugates; preparation of mAb-PODS-DFO and
mAb-PODS-CHX-A′′-DTPA conjugates; radiolabeling protocols; in vitro stability assays; PET
imaging; biodistribution assay; supplementary data.
ABBREVIATIONS
DOL = Degree of labeling; FL = fluorescein; IgG = immunoglobulin G; DFO =
desferrioxamine; CHX-A′′-DTPA = 2,2'-((2-(((1S,2S)-2-(bis(carboxymethyl)amino)cyclohexyl)
(carboxymethyl)amino)ethyl)azanediyl)diacetic acid; PET = positron emission tomography.
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REFERENCES
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