Site-specific radioiodination of HER2-targeting affibody molecules using 4-iodophenethylmaleimide decreases renal uptake of radioactivity

Article abstract of DOI:10.1002/open.201402097

Affibody molecules are small scaffold-based affinity proteins with promising properties as probes for radionuclide-based molecular imaging. However, a high reabsorption of radiolabeled Affibody molecules in kidneys is an issue. We have shown that the use of ¹²⁵I-3-iodo-((4-hydroxyphenyl)ethyl)maleimide (IHPEM) for site-specific labeling of cysteine-containing Affibody molecules provides high tumor uptake but low radioactivity retention in kidneys. We hypothesized that the use of 4-iodophenethylmaleimide (IPEM) would further reduce renal retention of radioactivity because of higher lipophilicity of radiometabolites. An anti-human epidermal growth factor receptor type 2 (HER2) Affibody molecule (Z_HER2:2395) was labeled using ¹²⁵I-IPEM with an overall yield of 45±3 %. ¹²⁵I-IPEM-Z_HER2:2395 bound specifically to HER2-expressing human ovarian carcinoma cells (SKOV-3 cell line). In NMRI mice, the renal uptake of ¹²⁵I-IPEM-Z_HER2:2395 (24±2 and 5.7±0.3 % IA g^-1at 1 and 4 h after injection, respectively) was significantly lower than uptake of ¹²⁵I-IHPEM-Z_HER2:2395 (50±8 and 12±2 % IA g^-1at 1 and 4 h after injection, respectively). In conclusion, the use of a more lipophilic linker for the radioiodination of Affibody molecules reduces renal radioactivity.

Full text of DOI:10.1002/open.201402097

DOI: 10.1002/open.201402097
Site-Specific Radioiodination of HER2-Targeting Affibody
Molecules using 4-Iodophenethylmaleimide Decreases
Renal Uptake of Radioactivity
Joanna Strand,[a] Patrik Nordeman,[b] Hadis Honarvar,[a] Mohamed Altai,[a] Anna Orlova,[b]
Mats Larhed,[b] and Vladimir Tolmachev*[a]
Affibody molecules are small scaffold-based affinity proteins
with promising properties as probes for radionuclide-based
molecular imaging. However, a high reabsorption of radiola-
beled Affibody molecules in kidneys is an issue. We have
shown that the use of 125I-3-iodo-((4-hydroxyphenyl)ethyl)ma-
leimide (IHPEM) for site-specific labeling of cysteine-containing
Affibody molecules provides high tumor uptake but low radio-
activity retention in kidneys. We hypothesized that the use of
4-iodophenethylmaleimide (IPEM) would further reduce renal
retention of radioactivity because of higher lipophilicity of radi-
ometabolites. An anti-human epidermal growth factor receptor
type 2 (HER2) Affibody molecule (ZHER2:2395) was labeled using
125I-IPEM with an overall yield of 45Æ3%. 125I-IPEM-ZHER2:2395
bound specifically to HER2-expressing human ovarian carcino-
ma cells (SKOV-3 cell line). In NMRI mice, the renal uptake of
125I-IPEM-ZHER2:2395 (24Æ2 and 5.7Æ0.3% IAgÀ1at 1 and 4 h after
injection, respectively) was significantly lower than uptake of
125I-IHPEM-ZHER2:2395 (50Æ8 and 12Æ2% IAgÀ1at 1 and 4 h after
injection, respectively). In conclusion, the use of a more lipo-
philic linker for the radioiodination of Affibody molecules
reduces renal radioactivity.
Introduction
Malignant transformation is often associated with an aberrant
expression of certain types of cell-surface proteins, for exam-
ple, receptors, cell adhesion molecules, or proteins active in
embryonic development.[1] Molecular recognition of these pro-
teins can be used for specific treatment of malignant cells, for
example, targeted therapy. Monoclonal antibodies (Mabs) are
the most used kind of targeting agents, which may act by pre-
venting mitogenic signaling[2] or by eliciting antibody-depen-
dent or complement-dependent cytotoxicity.[3] Antitumor
action of Mabs might be further enhanced by conjugation of
cytotoxic drugs or radionuclides.[4] However, there is an appre-
ciable inter- and intrapatient heterogeneity in expression of
molecular targets. Apparently, tumors that do not express par-
ticular targets would not respond to a particular targeting
therapy. Therefore, the targeted treatment should be personal-
ized, that is, adjusted to the tumor molecular abnormality pro-
file of each particular cancer case.[5] In vivo visualization of cell-
surface target proteins using radionuclide molecular imaging
can personalize anticancer treatment by the selection of pa-
tients who would most likely benefit from a particular targeted
therapy.[6]
A possible approach to the development of imaging agents
is the radiolabeling of therapeutic Mabs using nuclides emit-
ting gamma quanta that can be detected outside the patient’s
body.[7] The use of Mabs as imaging agents has, however, cer-
tain downsides. Antibodies are relatively bulky proteins
(150 kDa); this limits their rates of extravasation, tumor pene-
tration, and blood clearance of unbound tracers.[7,8] Therefore,
imaging is possible only several days after injection. In addi-
tion, antibodies have a tendency to accumulate in tumors non-
specifically due to an “enhanced permeability and retention”
(EPR) effect, which might cause false positive diagnoses.[7]
Small engineered scaffold affinity proteins, for example, Affi-
body molecules, are strong alternatives to antibodies in the
development of imaging agents.[9] Affibody molecules are
small (7 kDa) three-helical cysteine-free scaffold proteins de-
rived from the immunoglobulin-binding B domain of staphylo-
coccal receptor protein A.[10] Randomization of surface amino
acids on helices 1 and 2 of Affibody molecules creates large
combinatorial libraries enabling the selection of high-affinity
binders to different proteins, including cancer-associated
ones.[10] The small size and high affinity (in low nanomolar and
subnanomolar range) makes them good candidates for devel-
opment of imaging probes.[11] Affibody-based agents have
been generated for the imaging of several cancer-associated
molecular targets, for example human epidermal growth factor
receptor type 2 (HER2),[12] insulin-like growth factor-1 receptor
[a] J. Strand, H. Honarvar, Dr. M. Altai, Prof. Dr. V. Tolmachev
Biomedical Radiation Sciences, Faculty of Medicine
Uppsala University, 751 85 Uppsala (Sweden)
E-mail: vladimir.tolmachev@bms.uu.se
[b] Dr. P. Nordeman, Dr. A. Orlova, Prof. Dr. M. Larhed
Preclinical PET Platform, Department of Medicinal Chemistry
Faculty of Pharmacy, Uppsala University, 751 23 Uppsala (Sweden)
ORCID(s) from the author(s) for this article is/are available on the WWW
under http://dx.doi.org/10.1002/open.201402097.
ꢀ 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial License, which permits use, distribution and
reproduction in any medium, provided the original work is properly
cited and is not used for commercial purposes.
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(IGF-1R),[13] platelet derived growth factor beta (PDGFb),[14]epi-
dermal growth factor receptor (EGFR),[15] and carbonic anhy-
drase IX (CAIX).[16] Preclinical studies have demonstrated that
Affibody molecules provide a much higher contrast than radio-
labeled Mabs and enable imaging only a few hours after injec-
tion.[17] Clinical studies have demonstrated the capacity of Affi-
body molecules to image HER2 expression in breast cancer
metastases.[18,19]
Figure 1. Structures of 125I-3-iodo-((4-hydroxyphenyl)ethyl)maleimide (125I-
IHPEM) (1) and 125I-4-iodophenethylmaleimide (125I-IPEM) (2).
The major excretion route of Affibody molecules is renal due
to their small size. After glomerular filtration, Affibody mole-
cules undergo nearly quantitative reabsorption in the renal
tubuli cells followed by internalization and lysosomal degrada-
tion. The use of residualizing radiometal labels results in a long
retention of radioactivity in kidneys. This phenomenon was ob-
served for Affibody molecules specific to different targets, for
example, HER2, IGF-1R, EGFR, and PDGFRb, and labeled with
different radiometals using different chelators.[13,14,15,20,21] The
high renal retention is a major dosimetry problem in the case
of radionuclide therapy and might complicate imaging of
metastases in the lumbar region. On the other hand, radiocata-
bolites are cleared rapidly from kidneys when Affibody mole-
cules are labeled using nonresidualizing halogens, such as
18F,[22,23,24] 76Br,[25] or different iodine radioisotopes.[17,26,27] Impor-
tantly, internalization of receptor-bound Affibody molecules by
cancer cells is slow,[13,14,15,28] which makes residualizing proper-
ties of a label not critical for successful targeting. Thus, the use
and further development of nonresidualizing labels is a promis-
ing way to reduce renal retention of radioactivity of radio-
labeled Affibody molecules.
or 3-iodo-(4-hydroxyphenethyl)maleimide (IHPEM) (1, Figure 1)
as an intermediate precursor resulted in a homogenous radio-
labeled product with preserved binding specificity.[25,27] Surpris-
ingly, the use of BrHPEM and IHPEM resulted in four to seven-
fold decrease of renal retention of radioactivity at 4 h after in-
jection, in comparison with the use of para-halobenzoate-
based labels. This demonstrated that positioning of the linker
molecule influences the biodistribution profile. However, the
exact mechanism of the decrease is not clear. We speculate
that exoproteases are predominantly featured in the renal pro-
teolysis of Affibody molecules, and a terminal placement re-
sults in a more rapid formation of “leaky” lipophilic radiometa-
bolites. As lipophilicity of radiometabolites is an important
factor in their decreased intracellular retention,[29] the use of
a more lipophilic pendant group for C-terminal radioiodination
of cysteine-containing Affibody molecules should further
reduce renal retention of radioactivity. N-(4-[125I]iodophen-
ethyl)maleimide (125I-IPEM) (2), which has been earlier devel-
oped for site-specific labeling of antigen-binding fragments
(Fab fragments),[30] would be a suitable linker, as it both per-
mits site-specific labeling of cysteine-containing Affibody mole-
cules and is more lipophilic than IHPEM.
Among radiohalogens, iodine radioisotopes offer a variety of
properties as labels for targeting proteins and peptides; for ex-
123
1
ample, I (t = =13.3 h) is suitable for imaging using single-
2
124
1
photon emission computed tomography (SPECT),
I (t = =
2
131
1
4.18 d) for positron emission tomography (PET), and I (t = =
The goal of this study was to test hypothesis that the use of
125I-IPEM for indirect radioiodination of Affibody molecules pro-
vides a conjugate that is capable of specific binding to its mo-
lecular target cells and provides lower renal retention of radio-
activity than its 125I-IHPEM-labeled counterpart. The anti-HER2
Affibody molecule ZHER2:2395 with a C-terminal cysteine was
used as a model in this study. Labeling of ZHER2:2395 using 125I-
IPEM was established, and properties of 125I-IPEM-ZHER2:2395 and
125I-IHPEM-ZHER2:2395 were directly compared in vitro and in vivo.
2
125
1
8 d) for therapy. The long-lived I (t = =59.4 d) is a convenient
2
surrogate nuclide, which is commonly used in the develop-
ment of radioiodination techniques. Direct radioiodination of
Affibody molecules destroys binding, presumably due to the
presence of tyrosine in the binding site.[28] To overcome this
problem, Affibody molecules were indirectly labeled using N-
succinimidyl para-iodobenzoate (SPIB) as a precursor.[12] The re-
sulting 125I -para-iodobenzoate (125I-PIB)-ZHER2:342 has demon-
strated high-contrast imaging of HER2-expressing xenografts in
mice and low renal retention of radioactivity. A disadvantage
of that method is that it is not site-specific. There are seven Results and Discussion
amino groups in ZHER2:342 (N-terminus and six lysines), which
Synthesis of N-[4-(tri-n-butylstannylphenethylamine)phen-
ethyl]maleimide (7)
result in a mixture of proteins with a different number of la-
beled prosthetic groups per protein at different positions. A
meta-analysis of biodistribution studies suggested that the bio-
distribution profile of (125I-PIB)-ZHER2:342 was not quite reproduci-
ble, possibly because of some deviation in labeling conditions
resulting in different compositions of such a mixture.[26]
Synthesis of a precursor for iododestannylation, N-[4-(tri-n-bu-
tylstannyl)phenethyl]-maleimide (7), was performed according
to Scheme 1. 4-Bromophenethylamine (3) was protected with
phthalimde using phthalic anhydride and provided 4 in 81%.
A palladium-catalyzed metal–halogen exchange using bis(tri-
butyltin) yielded 5 in 83%. Deprotection of the phthalamide
group using methylamine (94% yield) and a subsequent reac-
tion with malemide provided 7 in 30%. Synthesis of nonra-
dioactive iodophenethylmaleimide (8) for the use as a chroma-
The introduction of a unique thiol group by engineering
a cysteine into the Affibody molecule allows a site-specific cou-
pling of a linker molecule by thiol-directed chemistry. Both in-
direct radiohalogenation of cysteine-containing Affibody mole-
cules using 3-bromo-(4-hydroxyphenethyl)maleimide (BrHPEM)
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of labeling experiments, the amount of 7 dissolved in ethanol
was varied, while the amount of oxidant was constant (40 mg,
~140 nmol) and reaction time was 10 min. The influence of the
amount of 7 on labeling yield is shown in Figure 2. Between
Scheme 1. Reagents and conditions: a) phthalic anhydride, Et2O, rt, 2 h, then
glacial HOAc, 658C, 81%; b) 4a, 2% Pd(PPh3)4, Sn2Bu6, toluene, 115 8C, 48 h,
83%; c) MeNH2 (33% in EtOH), 608C, 2 h, 94%; d) maleic anhydride, Et2O, rt,
2 h, then acetic anhydride, KOAc, 908C, 2 h, 30%.
Figure 2. Dependence of radioiodination yield on the amount of the precur-
sor 7. Note the logarithmic scale. Reagents and conditions: CAT (40 mg), reac-
tion time 10 min. Data are presented as mean Æ maximum error (n=2).
tography standard was performed according to Scheme 2.
4-Iodophenethylamine was reacted with malemide to yield N-
[4-iodophenethyl]-maleimide (9) in 38% yield.
0.01 mg (0.02 nmol) to 1 mg (2 nmol) the labeling yield in-
creased proportionally with the amount of added precursor,
from 31Æ10% up to 71.9Æ0.4%. An additional amount of pre-
cursor up to 12 mg (~24 nmol) did not further improve the la-
beling yield significantly. In a blank experiment where only sol-
vent (5% acetic acid in methanol) was used, no labeling yield
could be determined (Figure 3).
Scheme 2. Reagents and conditions: a) maleic anhydride, Et2O, rt, 2 h, then
acetic anhydride, KOAc, 908C, 2 h, 38%.
Radioiodination of 7
Precursor 7 was radioiodinated using Chloramine-T as an oxi-
dant (Scheme 3). As our goal was “one-pot” labeling, that is,
performing radioiodination of a precursor and conjugation of
radio-IPEM to prereduced Affibody molecules without inter-
mediate purification, we tried to determine a minimum
amount of 7 that provides a stable high yield. In the first series
Figure 3. Distribution of radioactivity on a TLC plate. Origin (O) and solvent
front (F) are marked. 1) Blank experiment (no precursor 7 is added). The only
peak is at Rf =0, which is the same Rf as cold iodide. 2) Analysis of a typical
reaction mixture after quenching. An additional radioactivity peak appears
with Rf =0.4, as cold 9.
Iododestannylation of 7 was apparently rapid (Figure 4).
When 3 mg (6 nmol) of 7 was used, a yield of 73Æ3% was ob-
tained already after 1 min, and further prolongation of the re-
action time did not increase the yield significantly.
For further labeling experiments, the following conditions
were selected: 2 mg (4 nmol) of 7, 40 mg (140 nmol) of Chlora-
mine-T, and a reaction time of 2 min.
Scheme 3. Reagents and conditions: a) 125IÀ, Chloramine-T, MeOH/HOAc, rt;
b) ZHER2:2395-Cys; pH 6.3.
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Figure 4. Dependence of radioiodination yield on reaction time. Reagents
and conditions: CAT (40 mg), precursor 7 (3 mg). Data are presented as mean
Æ maximum error (n=2).
Figure 5. Overall radiochemical yield of labeling of ZHER2:2395 after coupling
with 125I-IPEM. Reagents and conditions: 0.2m NH4OAc, pH 6.3, rt. Affibody:-
precursor molar ratios were 1:1, 2:1, and 3:1. Data are presented as mean Æ
maximum error (n=2).
Conjugation of IPEM (8) to ZHER2:2395
The conjugation of 125I-IPEM to a prereduced ZHER2:2395 was per-
formed using a one-pot two-step approach, that is, in the
same reaction vial as iododestannylation without purification
of 2. The coupling yield of 125I-IPEM to ZHER2:2395 was measured
by varying Affibody:IPEM molar ratios (1:1, 2:1, and 3:1). The
samples were analyzed at 20, 40, and 60 min. The overall label-
ing yield (i.e. incorporation of radioiodine into Affibody mole-
cules) after 20 min incubation was 21Æ2% and 43Æ4% for Af-
fibody:IPEM ratios of 1:1 and 2:1, respectively. The highest
overall yield, 53Æ1%, was obtained at the ratio of 3:1
(Figure 5). Since the difference between ratios 2:1 and 3:1 was
small, the ratio of 2:1 was used for preparation of conjugates
for in vivo and in vitro experiments in order to keep a higher
specific activity of the conjugate. Given that the yield remained
reasonably constant for the three time points, the incubation
time was set to 40 min. According to instant thin-layer chroma-
tography (ITLC) analysis, purification of 125I-IPEM-ZHER2:2395 using
a NAP-5 size-exclusion column provided a radiochemical purity
of 98.9Æ0.5%.
in a purity of 99.4Æ0.1%. The purity of control samples, which
were kept in phosphate-buffered saline (PBS) was 99.4Æ0.1%,
that is, the difference was within experimental accuracy. Keep-
ing samples in PBS for 24 h at 48C and 378C resulted in
a purity of 97.3Æ2.1% and 99.3Æ0.3%, respectively.
In vitro studies
To verify that the binding of the labeled ZHER2:2395 was specific
to HER2 receptors, the labeled Affibody conjugates were incu-
bated with a HER2-overexpressing human ovarian carcinoma
cell line (SKOV-3) presaturated with nonlabeled ZHER2:2395. Satu-
ration of HER2 caused significant (p<0.00005) decrease of cell-
bound activity, from 53.7Æ0.9% to 2.6Æ0.1% for 125I-IPEM-
ZHER2:2395 and from 56.8Æ0.8% to 2.8Æ0.1% for 125I-IHPEM-
ZHER2:2395(Figure 6). This demonstrates that the binding of the
conjugates to HER2-expressing cells is saturable, which sug-
gests its receptor specificity.
Data concerning binding and cellular processing of 125I-IPEM-
ZHER2:2395 and 125I-IHPEM-ZHER2:2395 by HER2-expressing SKOV-3
cells are shown in Figure 7. The two compounds showed simi-
The radioiodination of 125I-IHPEM-ZHER2:2395 resulted in an
overall labeling yield of 45Æ3% at an Affibody:HPEM molar
ratio of 2:1 and a radiochemical purity of 99% after separation
on an NAP-5 size exclusion
column.
Stability test
To ensure that radioiodine is at-
tached to the Affibody mole-
cules via a stable covalent bond,
samples of 125I-IPEM-ZHER2:2395
were incubated up to 4 h in 2m
of sodium iodide to replace non-
covalently bound 125I and in 30%
ethanol to displace a noncova-
Figure 6. Binding specificity of 125I-IPEM-ZHER2:2395 (A) and 125I-IHPEM-ZHER2:2395 (B) to HER2-expressing SKOV-3 cells.
Two groups of culture dishes containing SKOV-3 cells were incubated with radiolabeled conjugate (27 pM). One
group of culture dishes was pretreated with saturating amounts of nonlabeled Affibody molecule (blocked). Cell-
associated radioactivity was calculated as a percentage of total added radioactivity. Data are presented as mean
lently bound 2. Incubation in
sodium iodide resulted in a radio-
chemical purity of 98.5Æ0.1%,
and incubation in 30% ethanol value Æ S.D. (n=3).
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respectively) were significantly
(p<0.05) lower than the uptake
of 125I-IHPEM-ZHER2:2395 (50Æ8%
and 12Æ2% IAgÀ1 at 1 h and
4 h pi, respectively). This con-
firms the main hypothesis that
the use of a more hydrophobic
linker decreases the retention of
radioiodine in kidney after injec-
tion of Affibody molecules.
Organs expressing Na+/IÀ sym-
porter, that is, the stomach and
salivary gland, showed a signifi-
cantly (p<0.05) lower radioactiv-
ity uptake in the case of 125I-
IPEM-ZHER2:2395 in at least one
time point. Although 125I-IPEM-
ZHER2:2395 had higher uptake in
liver at 1 h pi (4.1Æ0.7% IAgÀ1
Figure 7. Binding and cellular processing of 125I-IPEM-ZHER2:2395 (A) and 125I-IHPEM-ZHER2:2395 (B) by HER2-expressing
SKOV-3 cells. Conjugates (27 pM) were incubated at 378C. Acid wash was used to determine the membrane-
bound radioactivity. Cell-bound activity is normalized to the maximum uptake. Data are presented as mean value
Æ S.D. (n=3). Error bars might not be seen because they are smaller than the point symbols.
lar binding and processing patterns. A rapid increase in cell-as-
sociated activity was followed by a slower increase phase,
a maximum, and then a decrease in cell-bound activity. The
maximum value of cellular uptake was obtained at an earlier
time point (4 h) by 125I-IPEM-ZHER2:2395 than by 125I-IHPEM-
ZHER2:2395 (8 h). The level of internalized radioactivity was low
(below 10%) during whole observation period for both conju-
gates.
compared to 2.6Æ0.1% IAgÀ1 for 125I-IHPEM-ZHER2:2395), the dif-
ference disappeared by 4 h pi (1.4% IAgÀ1 for both conju-
gates). Of note is the significantly higher radioactivity in the
gastrointestinal tract (with content) for 125I-IHPEM-ZHER2:2395
,
which indicates that a larger fraction of this conjugate or its ra-
diometabolites is excreted via bile.
Discussion
Engineered scaffold proteins such as Affibody molecules, are
an emerging alternative to Mabs in radionuclide tumor target-
ing for diagnostics and therapy. A precondition for successful
clinical application of scaffold proteins is maximizing the deliv-
ery and retention of radionuclides in tumors and minimizing
their delivery and retention in normal tissues. In the case of
imaging, this provides higher contrast and, therefore, sensitivi-
ty. In the case of radionuclide therapy, this ensures destruction
of tumors while sparing normal tissues. Hence, a decrease in
uptake and retention of radionuclides in normal tissues is as
important as an increase in tumor uptake. This study focused
on the decrease of renal radioac-
Biodistribution study in mice
Comparison of 125I-IPEM-ZHER2:2395 and 125I-IHPEM-ZHER2:2395 biodis-
tribution in NMRI mice is shown in Table 1 and Figure 8. A
common feature of both variants was rapid clearance from
blood (less than 3% injected activity (IA)gÀ1 at 1 h and less
than 1% IAgÀ1 at 4 h postinjection (pi)). It has to be noted that
blood concentration was significantly (p<0.05) lower for 125I-
IHPEM-ZHER2:2395 at all time points. Clearance from other organs
and tissues was also rapid. Importantly, the renal uptake of 125I-
IPEM-ZHER2:2395 (24Æ2% and 5.7Æ0.3% IAgÀ1 at 1 h and 4 h pi,
tivity delivered by Affibody mol-
Table 1. Comparison of the biodistribution of 125I-IPEM-ZHER2:2395 and 125I-IHPEM-ZHER2:2395 in NMRI mice.
ecules.
The decrease in renal radioac-
Uptake [% IAgÀ1
4 h
]
tivity might be achieved, in prin-
ciple, by the decrease in renal re-
absorption of targeting proteins
or by the decrease in retention
of radiocatabolites in proximal
tubuli cells. Previous studies[31]
have demonstrated that the
scavenger receptor megalin is
not involved in renal reabsorp-
tion of Affibody molecules.
Therefore, common methods for
the decrease in renal reabsorp-
tion of small proteins and pep-
tides in kidneys, e.g. saturation
1 h
24 h
IPEM
IHPEM
IPEM
IHPEM
IPEM
IHPEM
Blood
Lung
Liver
Spleen
Stomach
Kidney
Salivary gland
Muscle
GI tract[b]
Carcass
2.8Æ0.4[a]
2.8Æ0.5
4.1Æ0.7[a]
1.0Æ0.2
1.1Æ0.1[a]
24Æ2[a]
2.2Æ0.2
2.6Æ0.3
2.7Æ0.1
0.9Æ0.1
2.1Æ0.4
50Æ9
0.81Æ0.06[a]
0.57Æ0.04
1.4Æ0.1
0.56Æ0.04
0.54Æ0.06
1.4Æ0.3
0.29Æ0.05
2Æ1
0.38Æ0.03[a]
0.13Æ0.01[a]
0.18Æ0.06[a]
0.12Æ0.01[a]
0.05Æ00.01
0.90Æ0.09[a]
0.026Æ0.008
0.012Æ0.003
0.18Æ0.04
0.12Æ0.07
0.07Æ0.01
0.07Æ0.02
0.04Æ0.03
0.07Æ0.03
0.6Æ0.3
0.34Æ0.04
0.40Æ0.05[a]
5.7Æ0.3[a]
0.34Æ0.06[a]
0.11Æ0.01
9.8Æ0.9[a]
3.1Æ0.3
12Æ2
1.0Æ0.2
0.6Æ0.1
9Æ1[a]
1.2Æ0.6
1.1Æ0.9
12Æ1
1.6Æ0.3
0.12Æ0.04
18Æ3
0.040Æ0.003
0.010Æ0.002
0.32Æ0.09
1.18Æ0.66
14Æ2
14Æ1
3.8Æ1.0
0.57Æ0.09
[a] Significant (p<0.05) difference between 125I-IPEM-ZHER2:2395 and 125I-IHPEM-ZHER2:2395 at the same time point.
[b] Data from gastrointestinal (GI) tract with content and carcass are presented as % of injected radioactivity
per whole sample. Data are presented as mean Æ S.D. (n=4 mice).
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increase in relative protein amount was relatively inefficient.
The Affibody:precursor ratio of 2:1 was selected for further la-
beling to keep a reasonably high specific activity. The conjuga-
tion time of 20–30 min was sufficient to reach the maximum
overall yield (Figure 5). Importantly, the binding of 125I-IPEM-
ZHER2:2395 to HER2-expressing SKOV-3 cells was saturable (Fig-
ure 5A), which demonstrates that the binding specificity of the
Affibody molecule was preserved after labeling. Binding specif-
icity of the alternative, 125I-IHPEM-ZHER2:2395, has also been con-
firmed (Figure 5B).
The internalization rate of anti-HER2 Affibody molecules by
cancer cells is rather slow and reaches 30–40% per day in the
case of residualizing labels.[27] This internalization rate corre-
sponds to daily cell-surface renewal. In this study the internal-
ized fraction of radioactivity was below 10% at all time points
which reflects nonresidualizing properties of radiocatabolites.
After binding to a cell-surface receptor, a fraction of Affibody
molecules is internalized, transferred to the lysosomal compart-
ment, and degraded by proteolytic enzymes. Lipophilic radio-
metabolites diffuse through lysosomal and cellular membranes
and leak out from cells. A maximum cell-associated activity is
achieved when the binding rate is equal to the rate of radioca-
tabolite leakage. In the case of a higher leakage rate, the maxi-
mum is reached earlier. A comparison of binding to SKOV-3
cells (Figure 7) shows that the maximum cell-associated activity
for 125I-IPEM-ZHER2:2395 was reached earlier (4 h) than for 125I-
IHPEM-ZHER2:2395 (8 h). This suggests that 125I-IPEM provides
more “leaky” radiometabolites. It has to be noted that internal-
ization of anti-HER2 Affibody molecules by cancer cells is
rather slow,[20,21,28] and the difference is therefore relatively
small. This complicates in vitro evaluation of an internalization
rate, but is favorable for in vivo tumor targeting, as leakage of
radiocatabolites has a small effect on tumor-associated radio-
activity.[37] On the other hand, the internalization rate in kid-
neys is high, and the difference in radiocatabolite leakage rate
has to be much more pronounced.
Direct in vivo comparison of 125I-IPEM-ZHER2:2395 and 125I-
IHPEM-ZHER2:2395 biodistribution in NMRI mice confirmed the
main hypothesis of this study (Table 1). The renal radioactivity
was two times lower for 125I-IPEM-ZHER2:2395 than for 125I-IHPEM-
ZHER2:2395 at 1 h and 4 h after injection. It has to be noted that
the label nature has a clear influence in uptake in other
organs. For example, hepatic uptake of 125I-IPEM-ZHER2:2395 (4.1Æ
0.7% IAgÀ1) at 1 h pi was significantly higher than uptake of
125I-IHPEM-ZHER2:2395 (2.7Æ0.1% IAgÀ1). This might be because
of the higher overall lipophilicity of 125I-IPEM-ZHER2:2395, as higher
lipophilicity is generally associated with higher liver uptake.[38]
However, the difference disappeared at 4 h pi, a time point rel-
evant for imaging. More disturbing was the 1.5-fold higher
blood level of 125I-IPEM-ZHER2:2395, as this could negatively affect
imaging contrast. This phenomenon might be associated with
the release of radiometabolites from kidneys to blood or with
higher adhesion to blood proteins. Another interesting feature
of 125I-IPEM-ZHER2:2395 was lower radioactivity uptake in the
stomach and salivary gland. As both these organs express the
Na+/IÀsymporter, it is likely that the metabolism of 125I-IPEM re-
sults in the decreased release of free radioiodide. Overall, this
Figure 8. Comparison of biodistribution of 125I-IPEM-ZHER2:2395 and 125I-IHPEM-
ZHER2:2395 in NMRI mice. The uptake is expressed as % IAgÀ1, and presented
as mean Æ S.D. (n=4 mice).
of scavenger receptors by co- or preinjection of cationic amino
acids or Gelofusine, do not work. Therefore, minimization of re-
tention of radiocatabolites in proximal tubuli is the only viable
alternative at the moment. Our previous studies have demon-
strated that this goal might be achieved for both radiohalo-
gens[25,27] and for such radiometals as 99mTc[32,33] and radioiso-
topes of rhenium.[34,35] It has to be admitted that such decrease
was achieved as a result of structure–property relationship
studies, and molecular and biological mechanisms leading to
this are still not quite clear for us. This study should elucidate
the effect of lipophilicity of a prosthetic group, used in the at-
tachment of a radiohalogen to an Affibody molecule, on renal
retention.
Synthesis of 125I-IPEM for the labeling of Fab fragments was
reported by Hylarides and co-workers in 1991.[30] Factors influ-
encing labeling efficiency were described in the initial report.
Unfortunately, we did not find any follow-up studies concern-
ing this interesting precursor for radioiodination. 125I-IPEM at-
tracted our attention because it is suitable for thiol-directed
site-specific labeling of cysteine-containing Affibody molecules
and its hydrophobicity (logP=2.85) is higher than that of
125I-IHPEM (logP=2.46).
Iododestannylation of 7 was efficient, enabling a yield in
excess of 70% when 1–3 mg (2–6 nmol) was used for labeling
(Figure 2). The reaction was rapid, and the high yield was ach-
ieved within 1 min (Figure 4), when 3 mg of 7 was used. The ef-
ficient labeling of a small amount of precursor removed the
need for the intermediate purification of 2, i.e. we can perform
the whole conjugation “in one pot”. This approach minimizes
the handling of radioactive compounds, the associated dose
burden to personnel, the losses during transfer of labeled pre-
cursor, and the probability of human error.[36] The overall label-
ing yield of the two-step process was a modest ~20% when
an Affibody:precursor molar ratio of 1:1 was used (Figure 5). In-
creasing the ratio to 2:1 doubled the overall yield, but a further
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column chromatography was performed using silica gel 60 (0.040–
0.063 mm, Merck). Analytical liquid chromatography/mass spec-
trometry (LC-MS) was performed on a Gilson HPLC system with
a Finnigan AQA-quadropole mass spectrometer (Gilson Inc., Mid-
dleton, USA) using a C18 (5 mm, 4.6”100 mm) column, with
a 5 min gradient of 60–100% CH3CN in 0.05% aq HCOOH as
mobile phase at a flow rate of 4 mLminÀ1. All starting materials
and reagents were purchased from Sigma–Aldrich Sweden (Stock-
holm) and used without further purification. All synthesized mole-
cules are >95% pure as shown by TLC, analytical LC-MS, and NMR
spectroscopy. The radioactivity was measured using an automated
gamma counter with a 1480 Wizard three-inch NaI (Tl) detector
(Wallac Oy, Turku, Finland). The production of ZHER2:2395 was de-
scribed earlier.[20]
study demonstrated the very pronounced influence of a pros-
thetic group for radioiodination on the distribution of radioac-
tivity after injection of Affibody molecules.
This information might also be helpful in the development
of other imaging probes based on engineered scaffold pro-
teins. It has to be noted that this approach is justified when in-
ternalization of an imaging agent by cancer cells is slow but in-
ternalization by tubuli cells is rapid. In the case of rapid inter-
nalization by cancer cells, the use of a prosthetic group provid-
ing lipophilic radiometabolites would decrease tumor accumu-
lation of radioactivity. In this case, radioiodination using
prosthetic groups providing charged radiometabolites would
be recommended.[39]
Synthesis of N-(4-bromophenethyl)phthalimide (4)
To a stirred solution of 3 (1.0 g, 5 mmol in 50 mL diethyl ether) was
added phthalic anhydride (741 mg, 5 mmol). The mixture was
stirred for 2 h. Afterwards, a white precipitate (carbamoylbenzoic
acid) was collected after filtration and washed with cold diethyl
ether (3 ”30 mL). The acid was dissolved in glacial CH3COOH
(15 mL) and heated at 658C for 3 h. The mixture was cooled to rt
and was allowed to precipitate overnight. Filtration and washing
with cold CH3COOH (3”20 mL) provided 4 as a white powder
(81%, 1.33 g); 1H NMR (400 MHz, 258C, CDCl3): d=7.86–7.83 (m,
2H), 7.73–7.71 (m, 2H), 7.41–7.39 (m, 2H), 7.14–7.12 (m, 2H), 3.93–
3.89 (m, 2H), 2.98–2.94 ppm (m, 2H); 13C NMR (100 MHz, 258C,
CDCl3): d=168.1, 136.9, 134.0, 131.6, 132.0, 130.6, 123.3, 120.5,
38.9, 34.0ppm; MS (ESI): 330–332 [M+H+].
Conclusions
The use of a more lipophilic radioiodine label at the C-terminus
of Affibody molecules is associated with lower renal retention
of radioactivity. This suggests that a careful selection of the la-
beling strategy could be used in the modification and optimi-
zation of the biodistribution profile of radiohalogenated imag-
ing agents.
Experimental Section
Materials
125I was purchased from PerkinElmer (Waltham, USA). Organic sol-
vents were purchased from Merck (Darmstadt, Germany). Chlora-
mine-T (CAT) and Na2S2O5 were from Sigma (St. Louis, USA). Buffers,
including 0.1m PBS (pH 7.5) and 0.2m NH4OAc (pH 6.3), were pre-
pared using common methods from chemicals supplied by Merck
(Darmstadt, Germany). High-quality Milli-Q water (resistance higher
than 18 MWcm) was used for preparing the solutions and buffers.
Solutions of CAT, Na2S2O5, iodophenethylmaleimide, and hydroxyl-
phenethylmaleimide were prepared immediately before use. TLC
plates were developed in 1,2-dichlorethane (BDH chemicals, Poole
Dorset, UK) followed by heating. Distribution of radioactivity along
the TLC plates and ITLC strips was measured using a Cyclone Stor-
age phosphor system and analyzed with the OptiQuant image
analysis software (PerkinElmer, Waltham, USA). The radiochemical
purity of the labeled Affibody molecule construct was analyzed
using 150–771 DARK GREEN, Tec-control Chromatography Strips
(ITLC) from Biodes medical system (New York, USA). Size-exclusion
chromatography was performed on disposable NAP-5 columns
(Amersham Pharmacia Biotech AB, Uppsala, Sweden) according to
the manufacturer’s instructions.
Synthesis of N-[(tri-n-butylstannyl)phenethyl]phthalamide (5)
A pressure-resistant 50 mL glass tube was loaded with 4 (500 mg,
1.5 mmol), bis(tributyltin) (2.63 g, 4.5 mmol, 3 equiv), tetrakis(tri-
phenylphosphine)palladium(0) (35 mg, 0.03 mmol, 2 mol%), and
anhydrous toluene (15 mL). The tube was flushed with nitrogen
and, after capping, heated at 115 8C for 48 h. After cooling to rt,
the crude dark mixture was evaporated and purified by column
chromatography yielding compound 5 as a clear viscous oil (83%,
675 mg): Rf =0.2 (isohexane/EtOAc 9:1); 1H NMR (400 MHz, 258C,
CDCl3): d=7.85–7.64 (m, 4H), 7.44–7.32 (m, 2H), 7.26–7.20 (m, 2H),
3.94–3.89 (m, 2H), 2.99–2.93 (m, 2H), 1.65–1.25 (m, 12H), 1.10–0.98
(m, 6H), 0.92–0.80 ppm (m, 9H); 13C NMR (100 MHz, 258C, CDCl3):
d=168.2, 139.8, 137.6, 136.7, 133.9, 132.1, 128.5, 123.2, 39.2, 34.6,
29.0, 27.4, 13.7, 9.5 ppm; ESI (MS): 537–545 [M+H+].
Synthesis of 4-tri-n-butylstannylphenethylamine (6)
To a pressure-resistant 50 mL glass tube containing 5 (600 mg,
1.1 mmol), CH3NH2 (10 mL, 33% in absolute EtOH) was added. The
tube was capped and heated to 608C for 2 h. After cooling, the
solvent was removed leaving a white solid. NaOH (20 mL, 2.5m)
was added and then extracted with diethyl ether (3”30 mL). The
organic phases were combined, dried over MgSO4, and evaporated
leaving the desired product,[2] compound 6, as a clear low viscous
Cellular experiments were performed using human ovarian carcino-
ma cells, SKOV-3 (ATCC, purchased via LGCPromochem, Borås,
Sweden). Ketalar (ketamine, 50 mgmLÀ1, Pfizer, New York, USA),
Rompun (xylazin, 20 mgmLÀ1, Bayer, Leverkusen, Germany), and
heparin (5000 IEmLÀ1, Leo Pharma, Copenhagen, Denmark) were
obtained commercially.
1
oil (94%, 426 mg); H NMR (400 MHz, 258C, CDCl3): d=7.39 (t, J=
7.8 Hz, 2H), 7.17 (t, J=7.8 Hz, 2H), 2.97 (t, J=7.0 Hz, 2H), 2.73 (t,
J=7.0 Hz, 2H), 1.64–1.26 (m, 12H), 1.16–0.84 ppm (m, 15H);
13C NMR (100 MHz, 258C, CDCl3): d=139.5, 139.1, 136.6, 128.5, 43.5,
40.2, 29.0, 27.4, 13.7, 9.5 ppm; MS (ESI): 408–416 [M+H+].
NMR spectra were recorded on a Varian Mercury Plus spectrometer
(Santa Clara, USA) at ambient temperature. Chemical shifts are ref-
erenced via the residual solvent signals (1H: CHCl3 at 7.26 ppm;
13C: CDCl3 at 77.16 ppm). TLC was carried out on Merck precoated
60 F254 aluminum plates (0.2 mm) using UV light (254 nm). Char-
ring with 8% phosphomolybdic acid (PMA) in absolute EtOH or
0.5% ninhydrine in EtOH (95%) was used for visualization. Flash
Synthesis of N-[(4-tri-n-butylstannyl)phenethyl]maleimide (7)
Compound 6 (400 mg, 0.98 mmol) was dissolved in diethyl ether
(25 mL) where, after, maleic anhydride (115 mg, 1.2 mmol,
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1.2 equiv) was added, and the reaction was left stirring for 2 h
yielding crude maleanilinic acid. After removal of solvent in vacuo,
acetic anhydride (30 mL) was added together with KOAc (295 mg,
3 mmol). The reaction was stirred at 908C for 2 h and then cooled
to rt. To the mixture was added diethyl ether (30 mL) and then
washed with H2O (3”30 mL) and aq Na2CO3 (1”100 mL, 5%). The
organic phase was then dried with MgSO4, evaporated, and puri-
fied with column chromatography providing compound 7 as
a clear viscous oil (30%, 140 mg): Rf =0.2 (isohexane:EtOAc:Et3N
20:1:0.2); 1H NMR (400 MHz, 258C, CDCl3): d=7.38 (t, J=7.8 Hz,
2H), 7.17 (t, J=7.8 Hz, 2H), 6.66 (s, 2H), 3.78–3.72 (m, 2H), 2.90–
2.85 (m, 2H), 1.61–1.27 (m, 12H), 1.12–0.86 ppm (m, 15H); 13C NMR
(100 MHz, 258C, CDCl3): d=170.5, 139.9, 136.7, 134.0, 128.4, 39.1,
34.5, 29.0, 27.4, 13.7, 9.5 ppm; MS (ESI): 487–495 [M+H+].
used for labeling. The reduced Affibody was used for labeling ap-
proximately 10–15 min after reduction. Compound 7 was radioiodi-
nated as described above. To get the best conjugation conditions,
the Affibody:IPEM molar ratio was varied between 1:1, 2:1, and
3:1. ZHER2:2395 was added in varying amounts to the 125I-IPEM solu-
tion (2 mg of 7) and incubated at rt. Small fractions (~1.5 mL) were
analyzed on ITLC silica gel strips developed in 80% acetone in
H2O, at 20, 40, and 60 min. In this system, the Affibody molecule
remains at the origin, and IPEM and iodide have an Rf of 1.0. After
conjugation, 125I-IPEM-ZHER2:2395was purified using an NAP-5 size-ex-
clusion column pre-equilibrated with PBS. The mixture (1.6 mL) was
analyzed using radio-ITLC to determine radiochemical purity of the
final product. All experiments were done in duplicate.
Stability test
Synthesis of N-[4-iodophenethyl]-maleimide (9)
The stability of radioiodine coupling to the ZHER2:2395was assessed
by incubating the solution for at 48C for 24 h and thereafter at rt
for 4 h. Stability was further analyzed by incubating the radioiodi-
nated ZHER2:2395with 2m NaI or 30% EtOH, both for 1 h. The labeling
was done as described above and purified via NAP-5 size-exclusion
chromatography. 125I-IPEM-ZHER2:2395 was added in equal volume
(50 mL) to Eppendorf tubes containing 2m NaI in PBS, and the solu-
tion was incubated for 1 h. For the stability test in EtOH, 95%
EtOH (30 mL) was added to the sample (70 mL) in Eppendorf tubes,
and the solution was incubated for 1 h. All experiments were done
in duplicate and analyzed using ITLC strips.
To a stirred solution of 8 (124 mg, 0.5 mmol) in diethyl ether
(10 mL), maleic anhydride (57 mg, 0.6 mmol, 1.2 equiv) was added,
and the reaction was left stirring for 2 h. The precipitate was col-
lected and dissolved in glacial CH3COOH (10 mL) and heated at
658C for 3 h. The mixture was cooled to rt and was allowed to pre-
cipitate overnight. The precipitate was filtered and washed with
cold CH3COOH (3”10 mL) resulting in compound 9 as a white
1
powder (38%, 62 mg); H NMR (400 MHz, 258C, CDCl3): d=7.61 (d,
J=8.3 Hz, 1H), 7.19 (d, J=5.6 Hz, 1H), 6.99 (d, J=8.0 Hz, 1H), 6.59
(d, J=5.6 Hz, 1H), 3.87 (t, J=7.2 Hz, 1H), 2.92 ppm (t, J=7.2 Hz,
1H);13C NMR (100 MHz, 258C, CDCl3): d=166.7, 142.2, 139.1, 137.6,
134.2, 131.0, 128.8, 91.7, 50.8, 36.3 ppm; MS (ESI): 378 [M+H+].
Labeling of HPEM
Labeling of HPEM was performed as previously described.[26] In
brief, HPEM precursor solution(5 mg) in 5% HOAc in MeOH was
mixed with 125I (10 mL). To the mixture, aq CAT solution (10 mL,
4 mgmLÀ1) was added, and the solution was incubated for 5 min.
The reaction was terminated by adding aq Na2S2O5 (10 mL,
6 mguLÀ1), given in double molar excess to CAT. Conjugations of
125I-IHPEM (1) to ZHER2:2395was done as described for 125I-IPEM with
an Affibody/HPEM molar ratio of 2:1.
Radioiodination of iodophenethylmaleimide (IPEM)
The precursor was radioiodinated using CAT as an oxidant. Typically
125I (10 mL, 1 MBqmLÀ1) was added to the precursor 7 (3 mL, dis-
solved in EtOH at a predetermined concentration) and solvent
(10 mL, 5% HOAc in MeOH). To the mixture, CAT (10 mL, 4 mgmLÀ1
in Milli-Q H2O) was added. The reaction proceeded for a predeter-
mined time and then quenched by a double molar excess of
Na2S2O5 (6 mgmLÀ1 in Milli-Q H2O).
Binding specificity and cellular processing of labeled conjugates
to HER2-expressing cells
The dependence of labeling yield on the amount of precursor and
reaction time was determined by varying one parameter at a time.
The amount of 7 varied between 0.01–15 mg and the reaction time
varied between 1–10 min. When the amount of 7 varied, the reac-
tion time was set at 10 min. When the reaction time was varied,
the amount of 7 remained constant (3 mg).
The binding specificity of the 125I-IPEM- and 125I-IHPEM-ZHER2:2395
conjugates to HER2 were evaluated using HER2-expressing SKOV-3
ovarian cancer cells as previously described.[27] Briefly, a solution of
125I-IPEM-ZHER2:2395 or 125I-IHPEM-ZHER2:2395 (1.99 ng/dish, 27 pM) was
added to a set of two groups of petri dishes (1”106 cells/dish). To
block the receptors, an excess of nonlabeled ZHER2:2395was added
5 min before applying the labeled conjugate to one group of
dishes. The cells were incubated in 378C for 1 h in a humidified in-
cubator; thereafter, the cell media was collected, and the cells
were detached with trypsin-EDTA solution (0.5 mL, 0.5% trypsin,
0.02% EDTA in buffer, Biochrom AG, Berlin, Germany). Then the
complete medium (0.5 mL) was added to every dish, and cells
were resuspended and collected. The percentage of cell-bound ra-
dioactivity was calculated by measuring the radioactivity in the
media and the cells.
Silica gel 60 F254 TLC plates (20”100 mm, elution path 80 mm)
(Merck) were used for analysis. As eluent,1,2-dichlorethane was
used. In this system, iodide remains at the origin while IPEM has
an Rf of 0.4. The reaction mixture (1.8–2 mL) was applied to the
ITLC plate, which was then left to evaporate spontaneously before
being developed in the eluent. All experiments were done at least
in duplicate. Blank experiments were performed where 5% HOAc
in MeOH (3 mL) was used instead of 7.
Conjugation of IPEM (2) to prereduced ZHER2:2395
Before labeling, the Affibody molecule solution in PBS was treated
with dithiothreitol (DTT, Merck) in order to reduce spontaneously
formed disulfide bonds. Typically, the stock Affibody solution
(50 mL, 10 mgmLÀ1) in PBS was mixed with DTT (350 mL) in PBS to
get a final concentration of 50 mm DTT. The mixture was incubated
at 408C for 3 h at rt. Thereafter the mixture was applied to NAP-5
size-exclusion columns, pre-equilibrated, and eluted with well-de-
gassed 0.2m NH4OAc (pH 6.3), containing 0.04m sodium ascorbate.
The first 900 mL of the high molecular weight (HMW) fraction was
Processing of 125I-IPEM-ZHER2:2395 and 125I-IHPEM-ZHER2:2395 by SKOV-3
cells was studied according to a method previously described and
validated.[27] The labeled compounds (1.99 ng/dish, 27 pM) were
added to each dish, with three dishes per time point per conjugate
(1”106 cells/dish). The cells were incubated at 378C, 5% CO2. At
predetermined time points (1, 2, 4, 8, and 24 h after the start of in-
cubation), the media from the dishes was collected, and the cells
were washed with ice-cold serum-free medium. The cells were
then treated with 0.2m glycine buffer containing 4m urea, pH 2.5,
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[13] V. Tolmachev, J. Malmberg, C. Hofstrçm, L. AbrahmsØn, T. Bergman, A.
Sjçberg, M. Sandstrçm, T. Gräslund, A. Orlova, J. Nucl. Med. 2012, 53,
90–97.
[14] J. Strand, Z. Varasteh, O. Eriksson, L. Abrahmsen, A. Orlova, V. Tolma-
chev, Mol. Pharm. 2014, 11, 3957–3964.
[15] V. Tolmachev, D. Rosik, H. Wållberg, A. Sjçberg, M. Sandstrçm, M. Hans-
son, A. Wennborg, A. Orlova, Eur. J. Med. Mol. Imaging 2010, 37, 613–
622.
[16] H. Honarvar, J. Garousi, E. Gunneriusson, I. HçidØn-Guthenberg, M. Altai,
C. Widstrçm, V. Tolmachev, F. Y. Frejd, Int. J. Oncol. 2015, 46, 513–520.
[17] A. Orlova, H. Wållberg, S. Stone-Elander, V. Tolmachev, J. Nucl. Med.
2009, 50, 417–425.
[18] R. P. Baum, V. Prasad, D. Müller, C. Schuchardt, A. Orlova, A. Wennborg,
V. Tolmachev, J. Feldwisch, J. Nucl. Med. 2010, 51, 892–897.
[19] J. Sçrensen, D. Sandberg, M. Sandstrçm, A. Wennborg, J. Feldwisch, V.
Tolmachev, G. Šstrçm, M. Lubberink, U. Garske-Romµn, J. Carlsson, H.
Lindman, J. Nucl. Med. 2014, 55, 730–735.
[20] S. Ahlgren, A. Orlova, D. Rosik, M. Sandstrçm, A. Sjçberg, B. Baastrup, O.
Widmark, G. Fant, J. Feldwisch, V. Tolmachev, Bioconjugate Chem. 2008,
19, 235–243.
[21] J. Malmberg, A. Perols, Z. Varasteh, M. Altai, A. Braun, M. Sandstrçm, U.
Garske, V. Tolmachev, A. Orlova, A. E. Karlstrçm, Eur. J. Nucl. Med. Mol.
Imaging 2012, 39, 481–492.
[22] G. Kramer-Marek, D. O. Kiesewetter, L. Martiniova, E. Jagoda, S. B. Lee, J.
Capala, Eur. J. Nucl. Med. Mol. Imaging 2008, 35, 1008–1018.
[23] M. Namavari, O. Padilla De Jesus, Z. Cheng, A. De, E. Kovacs, J. Levi, R.
Zhang, J. K. Hoerner, H. Grade, F. A. Syud, S. S. Gambhir, Mol. Imaging
Biol. 2008, 10, 177–181.
(0.5 mL) for 5 min on ice. The solution was collected, and the cells
were washed additionally with glycine buffer (0.5 mL), the fractions
were pooled together. The radioactivity from the acid wash frac-
tions was considered to be membrane bound. The cells were then
incubated at 378C for at least 30 min with 1m NaOH (0.5 mL).The
alkaline solution was collected, the cell dishes were washed with
an additional NaOH (0.5 mL), and the alkaline fractions were
pooled. The radioactivity in the alkaline fractions was considered
internalized. The percent of internalized radioactivity was calculat-
ed for each fraction at each time point.
Biodistributions study in NMRI mice
All animal experiments were planned and performed according to
the Swedish national legislation on the protection of laboratory an-
imals, and the study plans were approved by the Uppsala commit-
tee for animal research ethics (Uppsala djurfçrsçksetiskanämd, per-
mission C224/10 given to A. Orlova). Female NMRI mice (10–12
weeks old at arrival) were acclimatized for one week at the Rud-
beck Laboratory animal facility. Groups of four animals per time
point were used for the biodistribution study. Animals were intra-
venously injected with 30 kBq of either 125I-PEM-ZHER2:2395 (4 mg) or
125I-HPEM- ZHER2:2395 (4 mg) dissolved in PBS (100 mL). At 1 h, 4 h, and
24 h after injection, a mixture of Ketala-Rompun (20 mL solution
per gram body weight, Ketalar: 10 mgmLÀ1, Rompun: 1 mgmLÀ1
)
was injected intraperitonealy. The mice were sacrificed by heart
puncture. Blood was collected, and organ samples were excised.
The samples were put in preweighed plastic vials. The samples
were weighed, and their radioactivity was measured. The uptake in
tissue and organ were calculated as percent injected activity per
gram tissue (% IAgÀ1). For the gastrointestinal tract and the car-
cass, injected activity per whole sample was calculated (% IAgÀ1).
[24] D. Rosik, A. Thibblin, G. Antoni, H. Honarvar, J. Strand, R. K. Selvaraju, M.
Altai, A. Orlova, A. Eriksson Karlstrçm, V. Tolmachev, Bioconjugate Chem.
2014, 25, 82–92.
[25] E. Mume, A. Orlova, B. Larsson, A. S. Nilsson, F. Y. Nilsson, S. Sjçberg, V.
Tolmachev, Bioconjugate Chem. 2005, 16, 1547–1555.
[26] V. Tolmachev, E. Mume, S. Sjçberg, F. Y. Frejd, A. Orlova, Eur. J. Nucl.
Med. Mol. Imaging 2009, 36, 692–701.
[27] H. Wållberg, A. Orlova, Cancer Biother. Radiopharm. 2008, 23, 435–442.
[28] A. C. Steffen, M. Wikman, V. Tolmachev, G. P. Adams, F. Y. Nilsson, S.
Ståhl, J. Carlsson, Cancer Biother. Radiopharm. 2005, 20, 239–248.
[29] V. Tolmachev, A. Orlova, H. Lundqvist, Curr. Med. Chem. 2003, 10, 2447–
2460.
[30] M. D. Hylarides, D. S. Wilbur, M. W. Reed, S. W. Hadley, J. R. Schroeder,
L. M. Grant, Bioconjugate Chem. 1991, 2, 435–440.
[31] M. Altai, Z. Varasteh, K. Andersson, A. Eek, O. Boerman, A. Orlova,
Cancer Biother. Radiopharm. 2013, 28, 187–195.
Acknowledgements
This research was financially supported by Swedish Cancer Soci-
ety (Cancerfonden) and the Swedish Research Council (Veten-
skapsradet).
[32] T. Ekblad, T. Tran, A. Orlova, C. Widstrçm, J. Feldwisch, L. AbrahmsØn, A.
Wennborg, A. E. Karlstrçm, V. Tolmachev, Eur. J. Nucl. Med. Mol. Imaging
2008, 35, 2245–2255.
Keywords: affibody
iodophenethylmaleimide · radiolabeling · radiopharmaceuticals
molecules
·
drug
design
·
[33] H. Wållberg, A. Orlova, M. Altai, S. J. Hosseinimehr, C. Widstrçm, J.
Malmberg, S. Ståhl, V. Tolmachev, J. Nucl. Med. 2011, 52, 461–469.
[34] A. Orlova, T. A. Tran, T. Ekblad, A. E. Karlstrçm, V. Tolmachev, Eur. J. Nucl.
Med. Mol. Imaging 2010, 37, 260–269.
[35] M. Altai, H. Wållberg, H. Honarvar, J. Strand, A. Orlova, Z. Varasteh, M.
Sandstrçm, J. Lçfblom, E. Larsson, S. E. Strand, M. Lubberink, S. Ståhl, V.
Tolmachev, J. Nucl. Med. 2014, 55, 1842–1848.
[36] E. Mume, A. Orlova, P. U. Malmstrçm, H. Lundqvist, S. Sjçberg, V. Tolma-
chev, Nucl. Med. Biol. 2005, 32, 613–622.
[37] J. Feldwisch, V. Tolmachev, Methods Mol. Biol. 2012, 899, 103–126.
[38] S. J. Hosseinimehr, V. Tolmachev, A. Orlova, Drug Discovery Today 2012,
17, 1224–1232.
[1] D. Hanahan, R. A. Weinberg, Cell 2011, 144, 646–674.
[2] B. Fauvel, A. Yasri, MAbs. 2014, 6, 838–851.
[3] V. Pillay, H. K. Gan, A. M. Scott, New Biotechnol. 2011, 28, 518–529.
[4] A. M. Wu, P. D. Senter, Nat. Biotechnol. 2005, 23, 1137–1146.
[5] S. E. Jackson, J. D. Chester, Int. J. Cancer. 2014, DOI: 10.1002/ijc.28940.
[6] V. Tolmachev, S. Stone-Elander, A. Orlova, Lancet Oncol. 2010, 11, 992–
1000.
[7] S. Heskamp, H. W. van Laarhoven, W. T. van der Graaf, W. J. Oyen, O. C.
Boerman, Expert Opin. Drug Delivery 2014, 11, 175–185.
[8] D. M. Goldenberg, J. Nucl. Med. 2002, 43, 693–713.
[9] Z. Miao, J. Levi, Z. Cheng, Amino Acids 2011, 41, 1037–1047.
[10] P. A. Nygren, FEBS J. 2008, 275, 2668–2676.
[39] C. A. Boswell, J. Med. Chem. 2013, 56, 9418–9426 and references there-
in.
[11] J. Lçfblom, J. Feldwisch, V. Tolmachev, J. Carlsson, S. Ståhl, F. Y. Frejd,
FEBS Lett. 2010, 584, 2670–2680.
[12] A. Orlova, M. Magnusson, T. L. Eriksson, M. Nilsson, B. Larsson, I. HçidØn-
Guthenberg, C. Widstrçm, J. Carlsson, V. Tolmachev, S. Ståhl, F. Y. Nils-
son, Cancer Res. 2006, 66, 4339–4348.
Received: October 28, 2014
Published online on January 12, 2015
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