PEGylation enables the specific tumor accumulation of a peptide identified by phage display

Article abstract of DOI:10.1039/c3ob27475f

Peptides are excellent alternatives to small molecules and proteinaceous drugs. Their high medicinal potential for diagnostic and therapeutic applications has prompted the development of tumor targeting peptides. Despite its excellent tumor binding capacity, FROP-DOTA (H-Glu-Asn-Tyr-Glu-Leu-Met-Asp- Leu-Leu-Ala-Tyr-Leu-Lys(DOTA)-NH₂), a peptide that we had identified in phage display libraries, revealed slow binding kinetics. Consequently, biodistribution studies showed that its excretion forestalled a significant tumor accumulation. The aim of this study was to investigate whether the conjugation of PEG to FROP-DOTA resulted in a derivative with a prolonged residence time in the blood. A synthetic method for the PEGylation of the tumor specific peptide FROP-DOTA was developed. Thereafter, binding studies were done in vitro and a biodistribution was performed in tumor bearing animals. These were compared to the data obtained with FROP-DOTA. The binding kinetics of the PEGylated FROP-DOTA was even slower than that of FROP-DOTA. Biodistribution studies of the labeled conjugate in mice bearing human FRO82-2 tumors showed a time dependent increased uptake of the PEGylated peptide with a high retention (at 24 h p.i. 76% of the maximal activity concentration persisted in the tumor). The highest uptake values were determined at 120 min p.i. reaching 2.3%ID/g tumor as compared to 0.06%ID/g observed for the non-PEGylated derivative at 135 min p.i. Apparently, PEGylation provides a substantially improved stabilization in the circulation which allowed a stable tumor accumulation.

Full text of DOI:10.1039/c3ob27475f

Organic &
Biomolecular Chemistry
View Article Online
View Journal | View Issue
PAPER
PEGylation enables the specific tumor accumulation of
a peptide identified by phage display
Cite this: Org. Biomol. Chem., 2013, 11,
2706
Walter Mier,*a Susanne Krämer,a Sabine Zitzmann,a,b,c Annette Altmann,a,b
Karin Leotta,b Ursula Schierbaum,b Martina Schnölzer,d Michael Eisenhute and
Uwe Haberkorna,b
Peptides are excellent alternatives to small molecules and proteinaceous drugs. Their high medicinal
potential for diagnostic and therapeutic applications has prompted the development of tumor targeting
peptides. Despite its excellent tumor binding capacity, FROP–DOTA (H-Glu-Asn-Tyr-Glu-Leu-Met-Asp-Leu-
Leu-Ala-Tyr-Leu-Lys(DOTA)-NH2), a peptide that we had identified in phage display libraries, revealed
slow binding kinetics. Consequently, biodistribution studies showed that its excretion forestalled a signifi-
cant tumor accumulation. The aim of this study was to investigate whether the conjugation of PEG to
FROP–DOTA resulted in a derivative with a prolonged residence time in the blood. A synthetic method
for the PEGylation of the tumor specific peptide FROP–DOTA was developed. Thereafter, binding studies
were done in vitro and a biodistribution was performed in tumor bearing animals. These were compared
to the data obtained with FROP–DOTA. The binding kinetics of the PEGylated FROP–DOTA was even
slower than that of FROP–DOTA. Biodistribution studies of the labeled conjugate in mice bearing human
FRO82-2 tumors showed a time dependent increased uptake of the PEGylated peptide with a high reten-
tion (at 24 h p.i. 76% of the maximal activity concentration persisted in the tumor). The highest uptake
values were determined at 120 min p.i. reaching 2.3%ID/g tumor as compared to 0.06%ID/g observed
for the non-PEGylated derivative at 135 min p.i. Apparently, PEGylation provides a substantially improved
stabilization in the circulation which allowed a stable tumor accumulation.
Received 20th December 2012,
Accepted 19th February 2013
DOI: 10.1039/c3ob27475f
www.rsc.org/obc
DOTATOC, a peptide derivative successfully used for the diag-
nosis and therapy of neuroendocrine tumors,1,2 is the prime
Introduction
Systemic toxicity is one of the main limitations of chemo- example for this class of therapeutics. Unfortunately, the hope
therapy. While the traditional chemotherapeutic drugs affect to discover peptides that meet this performance was lost
all proliferating cells, targeted drugs preferentially pinpoint because the number of similar natural peptides is limited and
tumor cells and allow us to go beyond the possibilities of problems with unwanted accumulation in critical organs may
current drug therapy. However, also these novel agents some- occur.3 Alternatively, attempts have been made to exploit anti-
times lead to severe side effects. The success of new therapy bodies that bind epitopes specifically expressed on tumors.
modalities depends on the development of new vector systems Again, the promises for many therapeutic antibodies were dis-
to target tumors. The study of numerous naturally occurring illusioned because of their unfavorable pharmacokinetics
peptides, in particular small regulatory peptides, has demon- observed as a consequence of their high molecular weight.
strated that peptides provide excellent targeting properties.
The best results with therapeutic antibodies were obtained
with Zevalin,4 (90Y-ibritumomab tiuxetan)
radiolabeled
a
derivative of the anti-CD20 antibody rituximab – the first radio-
pharmaceutical approved for the radioimmunotherapy of
patients with B-cell non-Hodgkin lymphomas. These successes
and failures clearly define the ideal properties of tumor
binding molecules. The identification and characterization of
novel peptides that bind to cell surface receptors overexpressed
on tumors can be achieved by the screening of phage display
libraries.5 However, the high expectations for this technology
have not been met for in vivo applications. We have shown that
the coupling of DOTA to FROP-1, a peptide that enables the
aDepartment of Nuclear Medicine, University Hospital Heidelberg, INF 400,
69120 Heidelberg, Germany. E-mail: walter.mier@med.uni-heidelberg.de;
Tel: +49-6221-567720
bClinical Cooperation Unit Nuclear Medicine, German Cancer Research Center
(DKFZ), INF 280, 69120 Heidelberg, Germany. Tel: +49-6221-420
cBayer Pharma AG, Research Laboratories, Müllerstr. 178, 13353 Berlin, Germany.
Tel: +49-30 468 1111
dResearch Group Functional Proteome Analysis, German Cancer Research Center
(DKFZ), INF 280, 69120 Heidelberg, Germany. Tel: +49-6221-420
eDepartment of Radiopharmaceutical Chemistry, German Cancer Research Center
(DKFZ), INF 280, 69120 Heidelberg, Germany. Tel: +49-6221-420
2706 | Org. Biomol. Chem., 2013, 11, 2706–2711
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
selective targeting of thyroid carcinoma and a series of other
tumors,6 resulted in a dramatically improved in vitro binding
capacity. Unfortunately, the biodistribution study of this
peptide, originally identified using a phage display library,
revealed that the binding kinetics of FROP–DOTA (H-Glu-Asn-
Tyr-Glu-Leu-Met-Asp-Leu-Leu-Ala-Tyr-Leu-Lys(DOTA)-NH2) was
slow and consequently excretion impeded a significant tumor
accumulation.7 It is known that peptides can be retained in
the circulation by either albumin binding upon acylation8,9 or
by suppressing the renal filtration by increasing their mole-
cular weight.10 PEGylation, a technique first described in 1977
by Davies and Abuchowsky,11,12 is most commonly applied for
the modification of proteins.13 However, it has been shown to
be advantageous for small molecules such as MRI contrast
agents.14 More importantly several of the shortcomings of pep-
tides as pharmaceuticals15 such as short duration of action, Fig. 1 The chemical structure of the PEGylated peptide and its precursor
peptide generations.
their lack of oral bioavailability and their susceptibility to pro-
teolysis might be solved by PEGylation.
Among the different techniques of post-production modifi-
cation (i.e. amino acid manipulation, HESylation polysialyla-
tion and PEGylation) PEGylation is still the most commonly
applied technique. It allows us to increase the circulation half-
life of peptide drugs and consequently the reduction of the
doses required. Conjugation of PEG causes a significant
change in the physicochemical characteristics, as a result the
pharmacokinetic and pharmacodynamic properties are altered
– in general at a reduced immunogenic and toxicological
potential.16–18
The circulation half-life of PEG-conjugates is generally
increased by longer PEG chains. Even at extremely high con-
centrations the toxicological effects of PEG–protein conjugates
Fig. 2 Chromatographic survey of the PEGylation reaction. HPLC of PEG and
have been shown to be limited to a transient renal tubular
the maleimido-peptide versus the PEGylation reaction mixture.
vacuolization, a side effect that does not cause functional
effects.19 Therefore, PEG–protein conjugates can be considered
immunologically safe and non-toxic.
Leu-Ala-Tyr-Leu-Lys(DOTA)-Cys-NH2 (a FROP–DOTA derivative
The aim of this study was to investigate whether the conju- with a C-terminal cysteine) was obtained by solid phase syn-
gation of PEG to FROP–DOTA results in a derivative with a pro- thesis. The product was characterized by HPLC and mass spectro-
longed residence time in the blood.
metry. This cysteine derivative was reacted in solution with a
20 000 Da maleimido-modified PEG-oligomer. A first attempt
to analyze the reaction was made by using RP-HPLC as shown
in Fig. 2. Unfortunately, the chromatographic behavior of the
conjugate did not differ from the pure PEG synthesis
Results and discussion
In order to improve the retention time of FROP-1 in the circu- precursor.
lation, a PEGylated derivative of this peptide as shown in Fig. 1
was required.
Upon conjugation of the peptide the average molecular
weight of the PEG-polymer shifted from 22 401 g mol−1 to
A synthetic method for the PEGylation of the tumor specific 24 239 g mol−1 as determined by MALDI-TOF mass spectro-
peptide FROP–DOTA had to be developed. Unfortunately, all metry. This clear shift revealed that the excess of peptide
attempts to couple different PEG derivatives on the solid (5 equivalents) allowed a complete conversion of the PEG deriva-
support did not yield satisfactory results. A possible expla- tive into its peptide conjugate as judged by MALDI-TOF mass
nation is the relatively low molar concentrations achievable spectrometry as revealed by a shift of approximately 1.8 kDa
and the slow reaction kinetics to be expected with the 20 kDa corresponding to the DOTA-peptide moiety of the conjugate.
PEG building block which shows a slow diffusion rate in the
Due to the relatively slight change of the molecular weight,
polymer. The bulky PEG building block hardly reaches its reac- size exclusion chromatography did not reveal a visible change
tion partner buried at the C-terminal end of the resin bound of the retention time of the polymer as shown in Fig. 3. As the
peptide. Consequently, the PEGylation was performed in solu- mass spectrometry had revealed a complete reaction of the
tion. For this purpose H-Glu-Asn-Tyr-Glu-Leu-Met-Asp-Leu- PEG polymer, purification could be accomplished by
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 2706–2711 | 2707

View Article Online
Paper
Organic & Biomolecular Chemistry
increasing uptake of the PEGylated peptide over the first two
hours after injection, while activity in the blood and most of
the other organs decreased (Table 1). The accumulation in
the tumor showed a high retention, e.g. at 24 h p.i. 76% of the
maximal activity concentration persisted in the tumor. The
highest uptake values were determined at 120 min p.i. reach-
ing 2.3%ID/g tumor as compared to 0.06%ID/g observed for
the non-PEGylated derivative at 135 min p.i. None of the other
organs showed an increase of activity over time. In contrast to
FROP–DOTA–PEG, FROP–DOTA showed very fast elimination
with most of the activity already out of the circulation at
15 min p.i. Accumulation in the tumor is initially higher for
FROP–DOTA than for FROP–DOTA–PEG but decreases rapidly
while FROP–DOTA–PEG showed an increase in tumor activity.
In summary, the data reveal the positive effect of PEGyla-
tion including a substantially improved stabilization in the cir-
culation which allows stable tumor accumulation. Compilation
of these data as tumor to organ ratios allows a clear exposition
of this effect. The values presented in Table 2 show that the
changes of the tumor to organ ratios over time dramatically
increase in all organs in the case of the PEGylated peptide. In
the case of the tumor–blood ratio a 33-fold increase is
observed when comparing the value determined at 5 min after
injection and the value at 24 h post injection. In contrast the
relative values observed for the unconjugated peptide are
rather stable or even decreasing – apparently the result of the
lack of a specific uptake into the tumor. In order to discover
novel targeting molecules combinatorial phage display has
been extensively used for the identification of protein-
ligands.20,21 Using this technique we had identified the
thyroid carcinoma binding peptide FROP-1.6 Conjugation of
the chelator DOTA induced a secondary structure resulting in
significantly improved in vitro properties; however the peptide
retained an unfavorable pharmacokinetics. The discrepancy
between the high in vitro binding capacity and the lack of
tumor uptake was explained by the slow binding kinetics that
allowed excretion to forestall an efficient tumor uptake.7
Fig. 3 Chromatographic survey of the PEGylation reaction. Size exclusion
chromatography of the 20 kDa PEG-maleimide versus the PEGylation reaction
mixture obtained by the reaction of 5 equivalents of CH3O-PEG(20 000)C2H4-
maleimide with the peptide H-Glu-Asn-Tyr-Glu-Leu-Met-Asp-Leu-Leu-Ala-Tyr-
Leu-Lys(DOTA)-Cys-NH2.
Fig. 4 In vitro binding kinetics of the 111In-labeled peptide FROP–DOTA and its
PEGylated conjugate FROP–DOTA–PEG.
Here, we were able to show that PEGylation, an often suc-
preparative size exclusion chromatography. The final product cessfully used method to modulate the pharmacokinetics and
was desalted on a Sephadex column equilibrated and eluted immunogenicity of proteins,22,23 efficiently prolonged the resi-
with water.
dence time of the peptide in the circulation which reversed its
The in vitro binding kinetics of the PEGylated conjugate tumor accumulation profile as shown in Fig. 5. The long circu-
FROP–DOTA–PEG was compared to the kinetics of the original lation time of the PEGylated peptide was in contrast to its
peptide FROP–DOTA. Fig. 4 shows these data obtained in the moderately high molecular weight. It is not easy to extrapolate
cell line FRO82-2. Apparently, the PEGylation caused a sub- the kidney excretion limit of PEG by looking only at the kidney
stantial change in the binding kinetics of the 111In-labeled clearance threshold of macromolecules which is assumed to
compound. 111In-FROP–DOTA showed a slow accumulation to be in the range of 50 kDa.24 However, this default value cannot
reach a relatively high value of almost 400 000 cpm per 106 be assumed for PEG conjugates as the high water coordination
cells and a plateau which was reached at 2 to 3 h exposure, corres- causes strongly increased hydrodynamic volume of PEG when
ponding to 42 percent of the total activity applied. The compared to that of a globular protein having the same mole-
binding of the PEGylated conjugate 111In-FROP–DOTA–PEG cular weight.25 It is unlikely that the increased and persistent
was even slower. However, the accumulation of this compound accumulation of FROP–DOTA–PEG in the tumor is due to the
showed a continuous increase within the period of time blood unspecifically found in the highly vascularized tumor.
examined.
A comparison between tumor and blood values showed that
The biodistribution studies of the labeled conjugate in tumor accumulation increased whereas at the same time point
mice bearing human FRO82-2 showed a time dependent radioactivity in the blood decreased. Also the other organs
2708 | Org. Biomol. Chem., 2013, 11, 2706–2711
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
Table 1 %ID/g values obtained for the biodistribution in female Balb/c nu/nu mice carrying FRO82-2 tumors after i.v. injection (n = 3)
Time [min]
Blood
Heart
Lung
Spleen
Liver
Kidney
Muscle
Colon
Brain
Tumor
111In-labeled FROP–DOTA–PEG
5
23.8 8.3
23.5 3.1
11.8 3.0
11.0 1.7
8.0 1.1
1.2 0.2
6.5 1.8
6.3 0.7
3.3 0.7
3.7 1.2
2.6 0.7
0.6 0.1
13.6 3.4
14.5 3.1
7.5 2.8
5.8 1.4
5.3 1.7
1.0 0.4
4.0 1.7
4.2 1.1
2.0 0.8
1.8 0.3
1.7 0.4
1.1 0.2
8.0 3.5
7.5 2.2
3.4 0.8
3.0 0.4
2.8 0.6
1.6 0.3
23.4 10.3
32.5 6.9
27.6 10.0
27.9 5.5
31.1 1.8
23.6 6.4
1.2 0.3
1.2 0.2
0.9 0.3
0.9 0.1
0.9 0.1
0.4 0.1
3.0 1.4
3.9 0.5
3.0 1.1
2.2 0.5
1.5 0.2
0.5 0.1
0.7 0.2
0.7 0.2
0.3 0.1
0.3 0.0
0.3 0.1
0.1 0.0
1.0 0.4
1.7 0.2
2.1 0.9
2.3 0.3
2.0 0.5
1.7 0.3
15
60
120
240
1440
111In-labeled FROP–DOTA
5
3.5 1.8
1.5 0.1
0.3 0.1
0.2 0.4
1.1 0.5
0.5 0.1
0.1 0.0
0.1 0.1
2.4 1.3
1.4 0.4
0.3 0.0
0.1 0.0
0.7 0.4
0.4 0.0
0.1 0.0
0.0 0.0
4.9 2.4
1.7 0.1
0.2 0.0
0.1 0.0
11.4 3.6
5.5 0.7
3.6 1.1
3.2 1.0
0.6 0.3
0.3 0.0
0.1 0.0
0.0 0.0
nd
nd
nd
nd
0.1 0.0
0.1 0.0
0.0 0.0
0.0 0.0
1.6 0.8
1.1 0.2
0.2 0.0
0.1 0.0
15
45
135
Table 2 Tumor to organ ratios of the organ uptake values: the comparison of the biodistribution values of 111In labeled FROP–DOTA and FROP–DOTA–PEG with
respect to their tumor to organ ratios reveals the highly specific tumor accumulation of FROP–DOTA–PEG in the FRO82-2 tumors
Time [min]
tu/blood
tu/heart
tu/lung
tu/spleen
tu/liver
tu/kidney
tu/muscle
tu/brain
111In-labeled FROP–DOTA
5 min
0.5
0.3
0.6
1.4
1.0
0.7
0.7
1.1
1.7
2.4
1.4
0.6
0.3
0.6
1.7
0.1
0
0.1
2.7
2.4
0.9
15.2
4.7
0.3
135 min
Factor
111In-labeled FROP–DOTA–PEG
5 min
24 h
Factor
0.0
1.4
33.3
0.2
3.1
19.8
0.1
1.8
23.5
0.3
1.5
6
0.1
1.1
8.4
0.0
0.1
1.7
0.8
4.1
4.9
1.6
21.6
13.9
allowing for a longer exposition of the peptide to the tumor
cells is the major factor for the increased tumor accumulation.
FROP–DOTA–PEG attains a stable accumulation in the tumor
tissue, as shown in Fig. 5b. In contrast, 111In-labeled DOTA–
PEG shows only a transient uptake. Consequently, the EPR
(enhanced permeability and retention) effect may be excluded
as the cause for the tumor accumulation. As one of the motiv-
ations for the clinical application of peptides is to benefit from
their small size, the upsizing of a peptide to achieve improved
properties has to be carefully scrutinized. It might be worth-
while to compare this situation with the development process
of antibodies, where efforts were made to achieve improved
properties. The example of antibodies shows that the relation-
ship between protein size and pharmacological properties
should not be overrated. Several antibody fragments, a large
variety of miniaturized antibody forms, Fab fragments, scFv,
F(ab′)2, diabodies, minibodies, tandem scFv and nanobodies
have been developed. Interestingly, despite the numerous
efforts of antibody miniaturization the engineered smaller
formats with improved biodistribution and blood clearance
properties often do not achieve sufficient efficacy and stability
in vivo. Even formats such as ALX-0081 – a bivalent nanobody
suppressing thrombus formation,26 are far from overrating the
natural intact IgG format. Consequently, properly engineered
small protein scaffolds might represent the basis for the devel-
opment of efficient therapeutics.27 These peptides show the
Fig. 5 Kinetics of the blood (A) and the tumor accumulation (B) of 111In-
labeled FROP–DOTA–PEG, 111In-labeled FROP–DOTA and 111In-labeled DOTA–
PEG.
showed no signs of increased accumulation of FROP–DOTA– advantages of being accessible by chemical synthesis and to
PEG over time which rules out unspecific peptide–organ inter- variable modifications.28 As shown here, these modifications
action which could lead to increased tumor uptake. Therefore can be essential to achieve the pharmacokinetic profile
it seems that the slower elimination from the circulation required.
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 2706–2711 | 2709

View Article Online
Paper
Organic & Biomolecular Chemistry
mass spectrometer (Thermo Fisher Scientific), linked to an
Agilent 1200 HPLC system.
Conclusion
Phage-display is the most widely practiced display technology
for the de novo selection of peptides with selective affinity for a
specific target. Due to several limitations of the peptides ident-
ified such as their relatively low affinity, low stability and
inability to cross biological membranes several screening
methods such as CIS display,29 ribosome display30 and mRNA
display31 and maturation strategies32 and other alternatives
have been developed. While these techniques offer advantages
with respect to their screening speed, library size and the
display of unnatural amino acids the applications of the result-
ing peptides are prone to unfavorable pharmacokinetic proper-
ties which can only be ruled out by manual processing. Here
we were able to show that PEGylation can be applied to suc-
cessfully modify peptides – an eventual loss of biological
activity caused by PEGylation is compensated for by the pro-
longed body-residence time, as a result of the increased stab-
ility and higher hydrodynamic volume. Thanks to these
favorable properties, PEGylation might enhance the potential
of peptides and proteins identified by screening technologies.
PEGylation of the peptide
The PEGylation was performed in solution. For this purpose
CH3O-PEG(20 000)C2H4-maleimide (Rapp Polymere, Tübingen,
Germany) was incubated with 5 equivalents of the peptide
H-Glu-Asn-Tyr-Glu-Leu-Met-Asp-Leu-Leu-Ala-Tyr-Leu-Lys(DOTA)-
Cys-NH2. The peptide (6.7 mg = 3.2 × 10−3 mmol, 5 eq.) and
13 mg CH3O-PEG(20 000)C2H4-maleimide (Rapp Polymere,
Tübingen, Germany) (0.65 × 10−3 mmol) each dissolved in
200 μl PBS were incubated in 0.1 M phosphate buffer/0.15 M
NaCl at pH 7.2 for 20 h at room temperature. The conjugate
was purified by size exclusion chromatography on an Agilent
1100 HPLC system equipped with a Superdex 75 column
(GE Healthcare, München, Germany) using 0.05 M phosphate
buffer/0.15 M NaCl at pH 7 as the eluent. The fractions con-
taining the product were desalted on a Hi Trap Sephadex G-25
superfine desalting column (GE Healthcare, München,
Germany) equilibrated with neat H2O. A total amount of
7.61 mg, corresponding to a yield of 48%, was obtained.
The characterization of the conjugate FROP–DOTA–PEG was
performed by MALDI mass spectrometry.
Peptide labeling
Experimental procedures
The radiolabeling was performed by complexation with 111In
chloride. In brief 5 μl of a 10−3 M solution of FROP–DOTA–
PEG or FROP–DOTA was mixed with 50 μl of 0.4 M sodium
acetate buffer (pH 5) and 6 to 18 MBq 111InCl3 (Tyco Health-
care Neustadt, Germany) were added. The solution was heated
at 80 °C for 30 min. A quality control of the radiolabeled
peptide performed by HPLC on a monolithic RP HPLC 100 ×
4.6 mm column (Merck, Darmstadt, Germany) using 0.1% TFA
in water and 0.1% TFA in acetonitrile as eluents (in the case of
111In-FROP–DOTA) or thin layer chromatography on ITLC SG
showed yields >95%.
Peptide synthesis
H-Glu-Asn-Tyr-Glu-Leu-Met-Asp-Leu-Leu-Ala-Tyr-Leu-Lys(DOTA)-
Cys-NH2, (FROP-1 bearing a C-terminal DOTA residue to
allow labeling with 111In and a cysteine residue to attach a
maleimido-modified PEG oligomer) was obtained by solid
phase synthesis using Fmoc-chemistry on a Rink amide resin
(Rapp Polymere, Tübingen, Germany). Nα-Fmoc amino acids
with the following side chain protecting groups were
employed: Asp(tBu), Glu(tBu) and Tyr(tBu). The peptide chain
was constructed in an automated peptide synthesizer (ABI
433A) using reaction cycles consisting of deprotection (10 min)
with 20% piperidine in NMP and 30 min coupling with 10
equivalents of the HBTU-activated Fmoc-protected amino acid.
The Mtt protected lysine residue was deprotected with 1.5%
TFA in DCM and conjugated to 4 equivalents of tris-tBuDOTA
(CheMatech, Dijon, France) using HATU activation. The sub-
sequent cleavage from the resin was performed with
95 : 2.5 : 2.5 TFA/H2O/triisopropylsilane for 1 h at room temp-
erature and subsequent precipitation with cold diethyl ether.
The precipitate was dried and left to stand for 12 h in neat
TFA. Purification was accomplished by reversed-phase HPLC
on a Chromolith® SemiPrep 100 × 10 mm column (Merck,
Cell line
The human thyroid follicular carcinoma cell line FRO82-2
(University of California, Los Angeles, Ca, USA) was cultured in
RPMI 1640 with Glutamax containing 10% FCS, 0.15% sodium
bicarbonate, 1.5 mM sodium pyruvate, 1.5 × non-essential
amino acids (all Invitrogen Karlsruhe, Germany) and 25 mM
HEPES. The cells were cultivated at 37 °C in a 5% CO2-
incubator.
In vitro binding experiments
Darmstadt, Germany) using a linear gradient from 20% to 400 000 cells (FRO82-2) were seeded into 6-well plates and cul-
70% of acetonitrile in water (both containing 0.1% TFA) over tivated for 24 hours. The medium was replaced by 1 ml fresh
10 min, the product eluted at 4.3 min. The purified peptide medium (without FCS). 10 μl 111In-FROP–DOTA or 111In-FROP–
showed a single peak with a retention time of 2.79 min in an DOTA–PEG was added to three wells each and incubated for
analytical HPLC using a linear gradient from 0% to 100% of 10 min, 60 min, 2 h, 3 h, 4 h, 6 and 8 h. The cells were washed
acetonitrile in water (both containing 0.1% TFA) over 5 min. three times with 1 ml PBS and subsequently lysed with 0.5 ml
The mass signal at 1052.5237 m/z for the [M + 2H]2+ peak of 0.3 M NaOH. Radioactivity was determined with a gamma
(calculated: 1052.5025) confirmed the identity on an Exactive® counter and calculated as percent applied dose per 106 cells.
2710 | Org. Biomol. Chem., 2013, 11, 2706–2711
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
Animals and tumor growth
8 J. Petersen, M. Dandri, W. Mier, M. Lutgehetmann, T. Volz,
F. von Weizsacker, U. Haberkorn, L. Fischer, J. M. Pollok,
B. Erbes, S. Seitz and S. Urban, Nat. Biotechnol., 2008, 26,
335–341.
9 Q. Wang, K. Graham, T. Schauer, T. Fietz, A. Mohammed,
X. Liu, J. Hoffend, U. Haberkorn, M. Eisenhut and W. Mier,
Nucl. Med. Biol., 2004, 31, 21–30.
10 M. Behe, J. Du, W. Becker, T. Behr, C. Angerstein,
M. Marquez, J. Hiltunen, S. Nilsson and A. R. Holmberg,
Med. Oncol., 2001, 18, 59–64.
5 × 106 cells of the thyroid carcinoma cell line FRO 82-2 were
suspended in a Matrigel matrix (BD Biosciences, Heidelberg,
Germany) and subcutaneously inoculated into the anterior
region of the mouse trunk of female 7–8-week-old BALB/c
nu/nu mice (Charles River WIGA, Sulzfeld, Germany) and the
tumors were allowed to grow for about 10 to 14 days until
approximately 1 cm3 in size. All animals were cared for accord-
ing to the national animal guidelines.
11 A. Abuchowski, J. R. McCoy, N. C. Palczuk, T. van Es and
F. F. Davis, J. Biol. Chem., 1977, 252, 3582–3586.
12 A. Abuchowski, T. van Es, N. C. Palczuk and F. F. Davis,
J. Biol. Chem., 1977, 252, 3578–3581.
13 S. Jevsevar, M. Kunstelj and V. G. Porekar, Biotechnol. J.,
2010, 5, 113–128.
14 S. Duewell, R. Wuthrich, G. K. von Schulthess, H. B. Jenny,
R. N. Muller, T. Moerker and W. A. Fuchs, Invest. Radiol.,
1991, 26, 50–57.
15 C. L. Stevenson, Curr. Pharm. Biotechnol., 2009, 10,
122–137.
Organ distribution of 111In-labeled FROP–DOTA and FROP–
DOTA–PEG
The radiolabeled peptides (0.5–1 MBq per mouse) were
injected via the tail vein of the Balb/c nu/nu mice, trans-
planted with FRO82-2 thyroid tumors. The animals were sacri-
ficed at the indicated times (5, 15, 60, 120, 240 and 1440 min)
and selected organs (blood, heart, lung, spleen, liver, kidney,
muscle, colon, brain, and tumor) were removed, blotted dry
and weighed. The radioactivity was measured with a γ-counter
(Cobra II, Canberra Packard, Meriden, USA) along with a
sample of the injection solution to calculate the percentage of
injected dose per gram of the tissue (%ID/g).
16 C. S. Fishburn, J. Pharma. Sci., 2008, 97, 4167–4183.
17 J. M. Harris and R. B. Chess, Nat. Rev., 2003, 2,
214–221.
18 M. J. Roberts, M. D. Bentley and J. M. Harris, Adv. Drug
Delivery Rev., 2002, 54, 459–476.
Acknowledgements
19 A. Bendele, J. Seely, C. Richey, G. Sennello and G. Shopp,
Toxicol. Sci., 1998, 42, 152–157.
20 O. H. Aina, R. Liu, J. L. Sutcliffe, J. Marik, C. X. Pan and
K. S. Lam, Mol. Pharm., 2007, 4, 631–651.
21 E. Koivunen, W. Arap, D. Rajotte, J. Lahdenranta and
R. Pasqualini, J. Nucl. Med., 1999, 40, 883–888.
22 A. Aghemo, M. G. Rumi and M. Colombo, Nat. Rev. Gastro-
enterol. Hepatol., 2010, 7, 485–494.
This study was supported by grants from the Deutsche For-
schungsgemeinschaft (grants HA2901/5-1 and 5-2, and HA
2901/6-1) and the Bundesministerium für Bildung und For-
schung (Grant-No. 13N10269 and 01EZ0807).
Notes and references
1 C. Kratochwil, F. L. Giesel, R. Lopez-Benitez, N. Schimpfky, 23 B. B. Yang and A. Kido, Clin. Pharmacokinet, 2011, 50,
K. Kunze, M. Eisenhut, H. U. Kauczor and U. Haberkorn,
Clin. Cancer Res., 2010, 16, 2899–2905.
2 C. Kratochwil, R. Lopez-Benitez, W. Mier, S. Haufe,
295–306.
24 G. Arturson and G. Wallenius, Scand. J. Clin. Lab. Invest.,
1964, 16, 81–86.
B. Isermann, H. U. Kauczor, P. L. Choyke, U. Haberkorn 25 B. Plesner, P. Westh, S. Hvidt and A. D. Nielsen, J. Pharm.
and F. L. Giesel, Endocr. Relat. Cancer, 2011, 18, 595–602. Biomed. Anal., 2011, 55, 597–602.
3 J. C. Reubi, H. R. Macke and E. P. Krenning, J. Nucl. Med., 26 G. Sinha, Nat. Biotechnol., 2012, 30, 124.
2005, 46(Suppl 1), 67S–75S.
4 T. E. Witzig, A. Molina, L. I. Gordon, C. Emmanouilides,
27 M. Gebauer and A. Skerra, Curr. Opin. Chem. Biol., 2009,
13, 245–255.
R. J. Schilder, I. W. Flinn, M. Darif, R. Macklis, K. Vo and 28 T. Vorherr, PharmaChem, 2008, 13–16.
G. A. Wiseman, Cancer, 2007, 109, 1804–1810.
5 T. Lindner, H. Kolmar, U. Haberkorn and W. Mier, Mole-
cules, 2011, 16, 1625–1641.
29 R. Odegrip, D. Coomber, B. Eldridge, R. Hederer,
P. A. Kuhlman, C. Ullman, K. FitzGerald and
D. McGregor, Proc. Natl. Acad. Sci. U. S. A., 2004, 101,
2806–2810.
6 S. Zitzmann, W. Mier, A. Schad, R. Kinscherf,
V. Askoxylakis, S. Kramer, A. Altmann, M. Eisenhut and 30 C. Schaffitzel, J. Hanes, L. Jermutus and A. Pluckthun,
U. Haberkorn, Clin. Cancer Res., 2005, 11, 139–146. J. Immunol. Methods, 1999, 231, 119–135.
7 W. Mier, S. Zitzmann, S. Kramer, J. Reed, E. M. Knapp, 31 B. Seelig, Nat. Protoc., 2011, 6, 540–552.
A. Altmann, M. Eisenhut and U. Haberkorn, J. Nucl. Med., 32 T. Matsuura and T. Yomo, J. Biosci. Bioeng., 2006, 101,
2007, 48, 1545–1552.
449–456.
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 2706–2711 | 2711