89zr-labeled compounds for pet imaging guided personalized therapy
TRANSCRIPT
TECHNOLOGIES
DRUG DISCOVERY
TODAY
Drug Discovery Today: Technologies Vol. 8, No. 2–4 2011
Editors-in-Chief
Kelvin Lam – Blue Sky Biotech Inc., USA
Henk Timmerman – Vrije Universiteit, The Netherlands
Imaging techniques
89Zr-labeled compounds for PETimaging guided personalized therapyDanielle J. Vugts1,2,*, Guus A.M.S. van Dongen1,2
1Department of Otolaryngology/Head and Neck Surgery, VU University Medical Center, Amsterdam, The Netherlands2Department of Nuclear Medicine and PET Research, VU University Medical Center, Amsterdam, The Netherlands
89Zr-immuno-PET is an attractive option for the in vivo
evaluation of monoclonal antibodies (mAbs). For the
coupling of 89Zr to monoclonal antibodies several con-
jugation strategies are available all using desferrioxa-
mine as chelate. Here we discuss the production of89Zr, the available methods for coupling of 89Zr via
desferrioxamine to mAbs, and the evaluation of89Zr–mAb conjugates in preclinical and clinical studies.
*Corresponding author.: D.J. Vugts ([email protected])
1740-6749/$ � 2012 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddtec.2011.12.004
Guest Editors:Nicolau Beckmann – Novartis Institutes for BioMedicalResearch, Basel, Switzerland.Bert Windhorst – VU Medical Center, Amsterdam,The Netherlands.
stages of mAb development and application for example, for
Introduction
The discovery of disease-specific tissue markers has boosted
the development of molecular therapeutics like monoclonal
antibodies (mAbs). To date 30 mAbs, mostly intact immu-
noglobulins, have been approved by the U.S. Food and Drug
Administration (FDA), of which 11 are for the systemic
treatment of cancer. These approved therapeutic antibodies,
including unmodified, biologically active chimeric and/or
human antibodies, as well as radio- and drug-immunocon-
jugates represent a multibillion-dollar market. Furthermore,
hundreds of new mAbs are in early clinical development and
in preclinical evaluation and next to intact mAbs also non-
traditional scaffolds like mAb-fragments, nanobodies, affibo-
dies and minibodies are being developed. Despite the com-
mercial successes of approved mAbs, the effectiveness is
limited and only a small number of patients is benefitting
from these expensive medicines. Additionally the approval of
new mAbs for novel targets is staggering [1,2].
Immuno-positron emission tomography (immuno-PET),
the tracking and quantification of mAbs with PET at high
resolution and sensitivity can be of great value in several
(i) understanding the mechanism of action of mAbs in vivo,
(ii) anticipating on non-target toxicity, (iii) optimization of
the mAb dose and administration scheduling, (iv) facilitating
patient selection and (v) speeding up drug development by
understanding the added value and distinctive features of the
mAb during early stage clinical trials. Ultimately, this will
result in personalized therapy in which the right drug is given
at the right time to the right patient. To enable visualization
of a mAb with a PET camera, a positron emitter (b+) has to be
stably and inertly coupled. PET isotopes that would be appro-
priate for labeling mAbs and mAb-fragments are 68Ga (t1/
2 = 68 min), 18F (t1/2 = 110 min), 64Cu (t1/2 = 12.7 hours), 90Nb
(t1/2 = 14.6 hours), 86Y (t1/2 = 14.7 hours), 89Zr (t1/2 = 78.4
hours) and 124I (t1/2 = 100.2 hours). Recent reviews describe
their physical characteristics, coordination chemistry and
labeling opportunities [3–6].
In this review we will focus on the use of immuno-PET with
zirconium-89 (89Zr) in mAb development and applications.89Zr is an ideal isotope for labeling intact mAbs because its
physical half-life of 78.4 hours matches with the biological
half-life of the mAbs and the time needed to reach optimal
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Drug Discovery Today: Technologies | Imaging techniques Vol. 8, No. 2–4 2011
Physical Characteristics Production method
Purification
Zr for radiolabeling mAbs
Affinity chromatography using a
89Zr
89Y
IT
hydroxamate functionalized column
>99.99% radionuclidic and chemical
purity
Carrier free 89Zr in 1M oxalic acid with
nuclear reaction on Yttrium 78,4 h
15.7 s
EC, β+
IT= isomeric transition
Transition
EC
0.897
0.909 99.0
77.0
22.6β+
γ
Energy (MeV) Abundance (%)
EC= Electron Capture
γ
a) 89Y (p, n) 89Zr
b) 89Y (d, 2n) 89Zr
89
Drug Discovery Today: Technologies
Figure 1. Summary of the physical characteristics and production method of 89Zr.
target-to-non target ratios. 89Zr is a residualizing isotope and
is trapped inside the cell after internalization of the mAb,
while for example, 124I, another long-lived candidate radio-
isotope for immuno-PET with intact antibodies is released
from the tumor after internalization. Residualization also
occurs to some extent in organs of mAb catabolism like liver,
kidney and spleen. 89Zr emits a positron (b+ particle) with
23% efficiency and a concomitant gamma ray emission of
909 keV which decays 15.7 s later. Because PET is based on the
coincidence detection of two gamma photons of 511 keV,
which arise after combination of the b+ particle with an
electron, the delayed gamma photon of 909 keV does not
interfere with the overall image quality and accurate quanti-
fication of the PET images (Fig. 1).
General considerations regarding 89Zr-immuno-PET89Zr production89Zr can be produced through the irradiation of inexpensive
natural yttrium via the 89Y(p,n)89Zr reaction or the89Y(d,2n)89Zr reaction. The first method is mostly applied,
because a medium-to-small cyclotron can be used for this
production method. The average yield via this method is 38–
42 MBq/mAh resulting in batches of 6 GBq of 89Zr within four
to six hours, which is large taking into account that for
clinical immuno-PET studies patients receive 37 MBq. Pur-
ification of 89Zr from 89Y can be achieved via affinity chro-
matography using a hydroxamate functionalized column [7].89Zr can be eluted with 1 M oxalic acid, resulting in >99.99%
radionuclidic purity and>99.9% chemical purity. In the past,
the oxalic acid was removed by sublimation and radiolabel-
ing was performed in acetate buffer. Furthermore, H2O2 was
added to be absolutely sure that 89Zr was solely obtained in
the Zr(IV) oxidation state. Both steps, however, can be
omitted because it has been proven that without sublimation
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of oxalic acid and the addition of H2O2 transchelation is still
perfect, while potentially toxic oxalic acid can be totally
removed during subsequent purification steps [8,9]. The
method of Verel et al. [8] at the VU University Medical Center
has been further improved and 89Zr in 1 M oxalic acid is
commercially produced by BV Cyclotron VU, (http://
www.cyclotron.nl), Amsterdam, The Netherlands, and dis-
tributed by IBA Molecular Benelux (http://www.iba-molecu-
lar.com), Louvain-la-Neuve, Belgium.
89Zr chelation and labeling
Zirconium is a hard Lewis acid and thus prefers hard Lewis
bases as donor groups. Furthermore, zirconium prefers 8-
coordination. Baroncelli and Grossi and Fouche et al. showed
that zirconium(IV) can form stable complexes with hydro-
xamates [10,11]. In the past attempts have been made to
complex zirconium with diethylenetriamine pentaacetic acid
(DTPA) and porphyrins, however, the only chelate identified
up to now that forms stable complexes with zirconium is
desferrioxamine (DFO). Because DFO is clinically used for the
neutralization of iron and aluminum overload for many
years, its application as a chelate in clinical immuno-PET
studies is possible without the need of performing large
toxicity/safety studies in advance.
DFO consists of three hydroxamate groups for the com-
plexation of zirconium as well as of iron, gallium and nio-
bium and results in 6-coordination. Recently, Holland et al.
did density functional theory (DFT) calculations on the com-
plexation of DFO with zirconium [12]. The calculations pro-
vide an explanation for the observed high in vivo stability of89Zr-DFO-labeled mAbs and suggest that in aqueous solution
the coordination sphere of zirconium by DFO is expanded by
two water molecules resulting in thermodynamically stable
8-coordinated zirconium [12].
Vol. 8, No. 2–4 2011 Drug Discovery Today: Technologies | Imaging techniques
89Zr can be transchelated from oxalic acid to DFO at neutral
pH (optimum: 6.8–7.2). To this end a strong base, like sodium
carbonate, is added to the 89Zr in 1 M oxalic acid solution,
followed by 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (Hepes) buffer to obtain a final pH of 6.8–7.2. Hepes is
an excellent buffer at neutral pH and outperforms phosphate
buffer in radiolabeling efficiency: while with the latter label-
ing yields of 30% were achieved, with Hepes buffer >95%
yield was obtained. Recently Holland et al. [9] reported on the
transchelation of 89Zr-oxalate to 89Zr-chloride, with the aim
to get rid of potentially toxic oxalic acid. However, in our
hands using this 89Zr-chloride did not result in optimal
radioimmunoconjugates.
To prevent deterioration of 89Zr-labeled mAbs due to radi-
olytically formed species in the solution, gentisic acid (2,5-
dihydroxybenzoic acid) proved to be the best preservative.
Ascorbic acid is not recommended, because Zr4+ will be
reduced to Zr2+ and zirconium will dissociate from DFO.
Quality specifications
For clinical application it is important that 89Zr-labeled
mAbs are produced under current good manufacturing
practice (cGMP). After radiolabeling and purification it is
important to assess (i) appearance: a clear and colorless
solution should be observed; (ii) chelate-to-mAb ratio: in
general one to two DFO groups per mAb molecule do not
change the immunoreactivity and pharmacokinetics and
allow patient dose preparations; (iii) radiochemical purity:
this should be ideally 100%. Aggregate/dimer formation
caused by modification and radiolabeling and free 89Zr
and 89Zr-DFO should be <10%. High pressure liquid chro-
matography (HPLC) analysis can be used to evaluate both
criteria, while instant thin layer chromatography (iTLC) can
only be used to determine the percentage of free 89Zr and89Zr-DFO. It is thus important to select a HPLC column that
is able to distinguish dimers/aggregates from the native
protein and to use iTLC strips and an eluent that indeed
separates 89Zr–mAb from free 89Zr and 89Zr-DFO; (iv) immu-
noreactivity: this should not be affected by modification and
radiolabeling and can be examined by a Lindmo assay [13]
or enzyme-linked immunosorbent assay (ELISA); (v) apyr-
ogenicity: the product should be endotoxin free. This can be
assessed by, for example, an endosafe PTS reader; (vi) steri-
lity: the product should be filtered after preparation and the
filter integrity should be tested by, for example, a bubble
point test. Sterility tests have to be performed to ensure that
the product is sterile. Sterility determinations cannot be
done before injection, because of the time needed to per-
form this test. When no facilities are available for micro-
biological analysis of radioactive samples, medium can be
filtered and analyzed to monitor the filtration process.
Finally, during validation experiments it is important to
evaluate the stability of the 89Zr-labeled mAbs to ensure that
during storage the radiochemical purity and immunoreac-
tivity are preserved.
Comparison of DFO coupling strategies
There are five different DFO–mAb complexes (Scheme 1)
described in literature. Three DFO–mAb complexes use the
lysine groups of the mAb (Scheme 1, routes A–C) and two
DFO–mAb complexes use the thiol groups of specially engi-
neered ThiomAbs for coupling DFO (Scheme 1, routes D–E).
Modification of lysine groups is applicable for every mAb,
mAb fragment or peptide that contains lysine groups, while
the use of the modification via ThiomAbs is restricted to these
specially prepared mAbs.
89Zr–DFO–thioether–mAbs (A)
The synthesis of this thioether–DFO chelate and subse-
quently coupling to proteins has been described by Meijs
et al. in 1996 [14]. A S-acetylthioacetate (SATA) molecule is
coupled to DFO, a succinimidyl 4-(N-maleimidomethyl)
cyclohexane-1-carboxylate (SMCC) group to the mAb and
the resulting products are reacted with each other giving the
DFO–thioether–mAb products (Scheme 1, path A). Although
the first in vivo results looked promising for 89Zr–DFO–
thioether labeled mAbs A323/E3 and E48, showing biodis-
tribution comparable to the 99mTc and 123I-labeled analogs
[15], Verel et al. showed in 2003 [8] that 89Zr–DFO–thioether–
mAbs are not stable. Using mAb U36 they showed lower
tumor uptake, faster blood kinetics and lower uptake in all
non-target tissues of the 89Zr–DFO–thioether–U36 compared
to the hereafter described 89Zr–N-suc-DFO–U36. In vitro
experiments with 89Zr–DFO–thioether–U36 in serum showed
that the radioactivity transferred to serum albumin, while
also aggregates and 89Zr-labeled low molecular weight pro-
ducts were formed. The hypothesis for this instability is that
upon opening of the succinimide ring the chelate can break
off at either side of the sulfur atom. Once the S-containing89Zr–DFO fragment is formed, coupling with human serum
albumin (HSA) can occur, while cleavage at the other side of
the S-bond can result in the reactive S-atom at the mAb side,
making it susceptible to aggregation [8]. Therefore, this con-
jugation method is not suitable for stable coupling of DFO to
mAbs.
89Zr–N-suc-DFO–mAb (B)
In 2003 Verel et al. described the synthesis of iron-N-succinyl-
desferrioxamine-tetrafluorophenol ester (Fe–N-suc-DFO–TFP
ester) [8]. Till 2010 this was the chelate of choice for the
formation of stable 89Zr-labeled mAb conjugates (Scheme 1,
path B). In the preparation of Fe–N-suc-DFO–TFP ester, DFO is
first reacted with succinic anhydride resulting in N-succinyl-
DFO (N-suc-DFO), then the chelate DFO is temporarily
blocked with iron, and finally the acid group becomes acti-
vated to its Fe–N-suc-DFO–TFP-ester. The Fe–N-suc-DFO–TFP
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Drug Discovery Today: Technologies | Imaging techniques Vol. 8, No. 2–4 2011
O
O
O
OO
O
O
O
O
O
O
O
O
OH
OH
DFONH2
NH
HON
N
N
HN OO
O
OH S
N
NCS
NCS
SCN
H
F
F F
FS
S
H
HN
HN
H
pH 6.5
pH 8.3
N
N
DFO
DFO
DFO
DFODFODFO
HS HSSH SH
HS SH
pH 9
S S
S S
DFO-Ac-ThiomAb
DFO-Chx-Mal-ThiomAb
pH 9
pH 7.5
DFO
DFO DFO
DFO
DFODFO
DFO
Fe-DFO
H
N-suc-DFO-mAb
H2N
H2N
H2N
1. pH 9.5
DFO-Bz-NCS-MAbDFO-thioether-MAb
1. FeCl32. TFP-OH
pyridine
pH 9 A
D1D2
E
BC
pH~9
2. removal of Fe, pH 4.2-4.5, EDTA
(b)
(a) (c)
(d)
(e)
N
O
S NHN
O
O
O
O
O O
O
O
OO
O
O
OO
O
N N
O
OO
O
O
O
OO
O
O
O
N
N
SHN
SHN I
I
BrBr
Br
S
SATA
NHS
S
OO
SHN
SMCC
N
O
ONO
Drug Discovery Today: Technologies
Scheme 1. Summary of the different approaches to DFO–mAbs. Routes A–C are based on lysine modifications of mAbs and routes D1, D2 and E are
based on using specifically engineered Thiomabs having a cysteine group available for conjugation of DFO. In route A the synthesis of DFO–thioether–mAb
is depicted, in route B the synthesis of N-suc-DFO–mAb, in route C the synthesis of DFO–Bz–NCS–mAb, in route D the two synthesis possibilities of
DFO–Ac–ThiomAb and in route E the synthesis of DFO–CHx–Mal–Thiomab.
e56 www.drugdiscoverytoday.com
Vol. 8, No. 2–4 2011 Drug Discovery Today: Technologies | Imaging techniques
Table 1. Summary of preclinical data obtained with 89Zr–N-suc-DFO–mAb conjugates
mAb Target antigen Study results Refs
U36 CD44v6 Tumors as small as 19 mg containing 17 kBq 89Zr can be detected with PET. [16,17]89Zr–DFO–U36 can be used as scouting procedure for radioimmunotherapy with 90Y–DOTA–U36.
Slightly higher uptake of 89Zr in kidney, bone, liver and sternum.
cG250 G250 Comparable in vivo behavior of 89Zr–cG250 and 111In–DTPA–cG250 except for spleen, kidney
and liver, which showed a slightly higher percentage of 89Zr.
[18]
89Zr–DFO–cG250 PET images are superior to 111In–DTPA–cG250 SPECT images with respect
to tumor delineation.
Cetuximab EGFR 89Zr can predict the biodistribution of the residualizing labels 177Lu and 90Y, except for
the bone marrow uptake, which was significantly higher for 89Zr.
[19,24]
EGFR tumor expression levels do not correlate with the relative signal obtained with
PET. Possible causes: additional pharmacokinetic and pharmacodynamic mechanisms influence
tumor uptake of cetuximab.
Bevacizumab VEGF-A
binding
89Zr–DFO–bevacizumab can be used as specific VEGF tracer showing excellent
in vivo quantitative measurements.
[20,26]
89Zr–DFO–bevacizumab can be used for response monitoring upon treatment with
HSP90 inhibitor NVP-AUY922.
DN30 c-Met 89Zr–DFO–DN30 can be used as specific c-Met tracer and outperforms 124I–DN30 [21]
Zevalin CD20 89Zr–DFO–Zevalin and 88Y–DTPA–Zevalin show comparable biodistribution in tumor
bearing mice, except for the liver and bone accumulation, which was significantly higher for 89Zr.
[22]
Trastuzumab HER2 89Zr–DFO–trastuzumab can be used for imaging the HER2/neu status of the tumor. [23,25,29]89Zr–DFO–trastuzumab can be used for monitoring of HER2 downregulation by the
HSP90 inhibitor NVP–AUY922 and PU–H71
R1507 IGF-1R IGF-1R expression can be imaged with 89Zr–R1507 in triple-negative xenografts [27]
cG250–F(ab’)2 CAIX Hypoxia can be imaged with 89Zr–DFO–cG250–F(ab’)2 [28]
J591 PSMA 89Zr–DFO–J591 can be used for imaging and quantification of PSMA expression in prostate tumors [12]
Ranibizumab VEGF Alteration of VEGF expression during sunitinib treatment can be assessed
with 89Zr–DFO–ranibizumab.
[30]
ester can be stored in acetonitrile at �808C for at least a year
without hydrolysis of the TFP-ester. When the protection of
the DFO group with iron was omitted, intractable results were
obtained. In that case the TFP ester most probably reacted
with one of the hydroxamate groups and thus no conjugation
to proteins was possible anymore. When N-suc-DFO was first
radiolabeled with 89Zr, then no TFP-ester could be formed.
This prelabeling strategy is normally applied for other radio-
nuclide–chelate combinations like for 99mTc-MAG3 and this
would circumvent the protection and deprotection step with
iron. We hypothesize that zirconium is not fully saturated by
the coordination of the three hydroxamate groups of N-suc-
DFO and uses additional groups for coordination like the acid
group of N-suc-DFO, which is then not available anymore for
activation to its TFP-ester. Although the synthesis of Fe–TFP–
N-suc-DFO-ester is time consuming, the advantage of this
chelate is that the chelate-to-mAb molar ratio can easily be
determined by the analysis of the reaction mixture by size
exclusion chromatography HPLC using UV analysis at
430 nm for the assessment of the amount of Fe–N-suc-DFO
that is attached to a mAb. For obtaining optimal radioimmu-
noconjugates it is important that not too many DFO groups
are coupled per mAb molecule, because otherwise immunor-
eactivity, pharmacokinetics and pharmacodynamics of the
mAb can change. This is normally achieved when on average
less than four DFO molecules are coupled per mAb molecule,
and in practice mostly just one DFO molecule is coupled [8].
After coupling of Fe–N-suc-DFO to a mAb, iron must be
removed. It has been proven that a 100-fold molar excess of
ethylenediaminetetraacetic acid (EDTA) at pH 4.3–4.5 effi-
ciently transchelates iron to EDTA and that the integrity of
the mAb is not harmed under these conditions. Via this
method several commercial available and new therapeutic
mAbs have been labeled and their in vivo behavior assessed in
preclinical and clinical studies. An overview of the achieve-
ments with 89Zr–N-suc-DFO–mAbs in a preclinical setting is
provided in Table 1. The first studies described the compar-
able in vivo behavior of 89Zr-labeled mAbs with their 90Y, 111In
and 177Lu-labeled mAb counterparts [16–20], while later the
potential of 89Zr-immuno-PET was shown in response mon-
itoring and validation of new antibodies [12,21–30] (Table 1).
The first clinical study ever with a 89Zr-labeled mAb was
reported in 2006 by Borjesson et al. [31]. This feasibility study
with 20 patients showed that 89Zr–N-suc-DFO–U36 can be
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Drug Discovery Today: Technologies | Imaging techniques Vol. 8, No. 2–4 2011
(a)
(b)
0.28 g 0.76 g
Max
Min
A B C
Drug Discovery Today: Technologies
Figure 2. (a) PET images of FaDu xenograft bearing nude mouse obtained at 72 hours post injection of 89Zr–N-suc-DFO–cmAb U36. Coronal slices from
ventral (left) to dorsal (right). Tumor is indicated with arrows [35]; (b) PET images of a head and neck cancer patient with a tumor in the left tonsil (thick
arrow) and lymph node metastases (thin arrows) obtained at 72 hours post injection of 89Zr–N-suc-DFO–cmAb U36 (A: sagittal image; B: axial image and C:
coronal image) [31].
safely applied in patients and that it is a promising method
for imaging of primary head and neck tumors as well as
metastases in the neck. All primary tumors (n = 17) were
detected (Fig. 2). Furthermore lymph node metastases in
18 of 25 positive levels and in 11 of 15 positive sides were
detected. The missed tumor involved lymph nodes were
relatively small and contained just a small proportion of
tumor tissue and were also missed in most cases by computed
tomography (CT) and/or magnetic resonance imaging (MRI).
The sensitivity of immuno-PET for the detection of lymph
node metastases was at least as good as of CT/MRI: 72% versus
60% [31].
In aforementioned study with 89Zr-U36, also biodistribu-
tion, radiation dose and quantification potential of immuno-
PET were assessed [32]. PET quantification of blood-pool
activity in the left ventricle was in good agreement with
sampled blood activity (difference equals 0.2 � 16.9%),
except for heavy-weight patients (>100 kg). The same
accounts for the uptake in tumor tissue, where a good agree-
ment was observed between the PET-derived data and biopsy
data (mean deviation: �8.4 � 34.5%). This suggests that
patients with high and low uptake can be differentiated
and therefore it is possible to select patients who may or
may not benefit from mAb therapy. The mean radiation dose
for patients in this study receiving 74 mBq 89Zr was 40 mSv,
which is high and will limit repeated application of
e58 www.drugdiscoverytoday.com
89Zr–mAbs. However with the next generation PET/CT scan-
ners a lower 89Zr radioactivity dose could be used while the
image quality is preserved.
High quality images at lower radioactivity dose have been
shown by Dijkers et al. in 2010 [33]. In this study 37 MBq 89Zr-
trastuzumab showed excellent quality images at an effective
dose of 20 mSv. In this feasibility study with 14 patients three
different dose cohorts were evaluated: 10 or 50 mg for tras-
tuzumab-naıve patients and 10 mg for patients on trastuzu-
mab treatment. It was proven that the latter two performed
equally. Although this study was not aiming for the compar-
ison with conventional staging modalities or for assessing
specificity and sensitivity, lesions with 89Zr-trastuzumab
uptake were generally in good agreement with CT, MRI
and bone scans. PET images showed a high spatial resolution,
and a good signal-to-noise ratio, which resulted in an image
quality unapproachable by previous 111In-trastuzumab single
photon emission computed tomography (SPECT) scans.
Excellent visualization of mAb uptake in HER2-positive
lesions as well as in metastatic liver, lung, bone and even
brain HER2-positive lesions was observed. The best moment
to assess 89Zr-trastuzumab tumor uptake was four to five days
post injection. 89Zr-trastuzumab PET allowed quantification
of conjugate uptake in HER2-positive lesions, and it became
clear that for some patients with extensive tumor load no
HER2 saturation occurred during trastuzumab therapy.
Vol. 8, No. 2–4 2011 Drug Discovery Today: Technologies | Imaging techniques
Although this N-suc-DFO–TFP method for labeling of 89Zr
showed great potential in clinical immuno-PET studies, there
are also some drawbacks of this method. The lengthy proce-
dure which is relatively complicated and time consuming is
challenging with respect to good manufacturing practice
(GMP) compliancy. Furthermore the low pH (pH 4.3–4.5)
as used during the removal of iron from the chelate by EDTA
can be harmful for the mAb.
89Zr–DFO–Bz–NCS–mAb (C)
The disadvantages of the above described method of coupling
DFO to mAbs have been overcome by the introduction of a
new DFO chelate, p-isothiocyanatobenzyl-desferrioxamine
(DFO–Bz–NCS), that has been published in 2010 [34,35].
DFO–Bz–NCS has been prepared by the reaction of DFO with
1,4-phenylendiisothiocyanate and is commercially available,
also at cGMP grade, from Macrocyclics (Dallas, TX, USA). The
isothiocyanate group is frequently used as reactive group for
conjugation to mAbs or other biologicals and reacts with
lysine groups of mAb resulting in thiourea bonds (Scheme 1,
path C). Because the isothiocyanate group is not reactive
toward hydroxamate groups, DFO does not need to be tem-
porarily blocked with iron and thus making this strategy
faster, easier and more widely applicable. With this method
precautions should be taken to prevent aggregation of the
protein during coupling of the chelate to the mAb. DFO–Bz–
NCS in dimethylsulfoxide (DMSO) should be added stepwise
while shaking to prevent high local concentrations of DMSO
resulting in excessive local reaction of DFO to mAb and thus
in aggregate formation [34].
This method has been applied for the coupling of 89Zr to
mAb U36, rituximab and cetuximab by Perk et al. [35] and the
performance of this new chelate was comparable with that of
the previously discussed N-suc-DFO chelate with respect to in
vitro stability and in vivo biodistribution. In vitro stability of89Zr–DFO–Bz–NCS–mAbs, however, appeared to be depen-
dent on the storage buffer. The best storage conditions proved
to be 48C and sodium acetate buffer in the presence of the
anti-oxidant gentisic acid. The presence of Cl� should be
avoided, because this results in impaired integrity. Most
Table 2. Comparison of technologies for the conjugation of DF
Technology DFO–thioether–
mAbs (A)
N-suc-DFO–
mAbs (B)
Modificationa 3 steps 5 steps
Reaction conditions pH 6.5–9 pH 4.3–9.5
Stability Poor Good
Generally applicable Yes Yes
Clinical application No Yes, current stan
a Starting from DFO.
probably due to radiation induced formation of OCl� ions,
which react with the SH group of the enolizable thiourea
bond and thus cause detachment of the chelate-89Zr [35].
DFO–Ac–Thiomab (D) and DFO–Chx–Mal–ThiomAb (E)
In 2010 another new DFO-based conjugation method to
MAbs has been reported using site-specifically modified
mAbs. These so-called thiomAbs contain two engineered
cysteine residues in the protein, which can be used for
coupling of DFO [36]. These ThiomAbs could provide tracers
with unaltered binding affinity and scaffold stability. To this
end DFO is reacted with (i) bromoacetyl bromide resulting in
bromoacetyl–DFO (DFO–Bac), (ii) N-hydroxysuccinimidyl
iodoacetate resulting in iodoacetyl–DFO (DFO–Iac) or (iii)
SMCC resulting in maleimidocyclohexyl–DFO (DFO–Chx–
Mal). Then thio-trastuzumab is reacted with either of these
DFO-constructs resulting in DFO–Ac–thio-trastuzumab (D) or
DFO–Chx–Mal-thio-trastuzumab (E) (Scheme 1, routes D and
E). Tinianow et al. showed that these 89Zr–DFO–thio-trastu-
zumab constructs have comparable in vitro and in vivo char-
acteristics as the randomly modified lysine conjugates 89Zr–
N-suc-DFO–trastuzumab (B) and 89Zr–DFO–Bz–NCS–trastuzu-
mab (C) [36]. Therefore, side-specific conjugation of DFO to
ThiomAbs did not show an advantage over random lysine
modification of mAbs in the model employed.
Conclusion
Five different approaches to 89Zr–DFO–mAbs are reported in
literature, of which three in 2010, which exemplifies the
growing interest in 89Zr-immuno-PET. This is also supported
by the increasing number of publications in recent years:
while between 2003 and 2009 on average one or two papers
were published per year, in 2010 more than ten research
papers have been published on the application of 89Zr-labeled
compounds.
The most important characteristics of the five different
DFO–mAbs have been summarized in Table 2. Because all
chelates consist of a DFO moiety for the chelation of 89Zr the
linker determines in general the stability of the 89Zr-labeled
mAbs. Except for 89Zr–DFO–thioether–mAb (A) all other four
O to mAbs
DFO–Bz–NCS–
mAb (C)
DFO–ThiomAbs
(D/E)
2 steps 2 steps
pH 7–9 pH 7.5–9
Good Good
Yes No, specially engineered
mAbs
dard First trials will start soon –
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Drug Discovery Today: Technologies | Imaging techniques Vol. 8, No. 2–4 2011
89Zr–DFO–mAbs (B–E) show good stability and comparable in
vivo behavior. Comparison of 89Zr-labeled mAbs with other
radiometal–mAb complexes like 177Lu–mAbs in preclinical
studies revealed that bone uptake was higher for the 89Zr–
mAb constructs. This can either be explained by release of89Zr from DFO in the blood or via catabolism in tumor and for
example, liver and redistribution in the blood and subse-
quent uptake in the bone. However, up to now no conclusion
can be drawn from the available preclinical studies on what
the actual cause is of the slightly increased bone uptake seen
in some cases. Differences between the lysine modified 89Zr–
DFO–mAbs B and C and the cysteine modified 89Zr–DFO–
mAbs D and E are not the stability but the ease of synthesis
and application. While the lysine modifications are general
applicable, the cysteine modification is limited to specially
engineered antibodies, which limits the application of this
strategy. The difference between these cysteine modified
DFO–mAbs is small, the DFO–Chx–Mal–ThiomAb (E) is
slightly faster synthesized than DFO–Ac–Thiomab (D), but
their in vitro and in vivo behavior is comparable. There are
three differences between the lysine modified mAbs: (i) TFP–
N-suc-DFO needs to be synthesized from DFO, while DFO–Bz–
NCS is commercially available; (ii) preparation of 89Zr-labeled
mAbs using TFP–N-suc-DFO requires an extra de-iron step at
relatively low pH after coupling to mAbs compared to the
DFO–Bz–NCS (Table 2) and (iii) the DFO–Bz–NCS in DMSO
requires more careful handling, exemplified by the faster
aggregate formation with this DFO-chelate. In conclusion,
stable 89Zr-labeled mAbs can be prepared for clinical applica-
tion and we envision that in the near future DFO–Bz–NCS will
take over TFP–N-suc-DFO as DFO-chelate.
Acknowledgements
Funding support was provided by CTMM, the Center for
Translational Molecular Medicine (http://www.ctmm.nl,
project AIRFORCE number 030-103); and the European Com-
munity Seventh Framework Programme (FP7/2007-2012
under grant agreement no. Health-F2-2008-201342: ADA-
MANT).
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