A New Prostate Carcinoma Binding Peptide (DUP-1) for Tumor ... · A New Prostate Carcinoma Binding Peptide (DUP-1) for Tumor Imaging and Therapy Sabine Zitzmann,1,3 Walter Mier,3

A New Prostate Carcinoma Binding Peptide (DUP-1) for Tumor

Imaging and Therapy

Sabine Zitzmann,1,3 Walter Mier,3 Arno Schad,4

Ralf Kinscherf,5 Vasileios Askoxylakis,1,3

Susanne Kramer,3 Annette Altmann,1,3

Michael Eisenhut,2 and Uwe Haberkorn1,3

1Clinical Cooperation Unit Nuclear Medicine and 2Department ofRadiopharmaceutical Chemistry, German Cancer Research Center;Departments of 3Nuclear Medicine, 4Anatomy and Cell Biology II,and 5Anatomy and Cell Biology III, University of Heidelberg,Heidelberg, Germany

ABSTRACT

Purpose: Prostate carcinomas belong to the most

widespread tumors, and their number is increasing. Imaging

modalities used for diagnosis, such as ultrasound, computed

tomography, and positron emission tomography, often

produce poor results. Radiolabeled peptides with high

sensitivity and specificity for prostate cancer would be a

desirable tool for tumor diagnosis and treatment.

Experimental Design: We used phage display and the

prostate-specific membrane antigen–negative cell line DU-

145 to identify a peptide. The isolated DUP-1 was tested

in vitro for its binding specificity, kinetics, and affinity.

Internalization of the peptide was evaluated with confocal

microscopy. The tumor accumulation in a nude mouse model

was analyzed with 131I-labeled DUP-1 in PC-3 and DU-145

prostate tumors as well as in the rat prostate tumor

model AT-1.

Results: The synthesized peptide showed rapid binding

kinetics peaking at 10 minutes. It shows specific binding to

prostate carcinoma cells but low binding affinity to non-

tumor cells. Peptide binding is competed with unlabeled

DUP-1, and a time-dependent internalization into DU-145

cells was shown. Biodistribution studies of DUP-1 in nude

mice with s.c. transplanted DU-145 and PC-3 tumors showed

a tumor accumulation of 5% and 7% injected dose per gram,

and bound peptide could not be removed by perfusion. The

rat prostate tumor model showed an increase of radioactivity

in the prostate tumor up to 300% in comparison with normal

prostate tissue.

Conclusions: DUP-1 holds promise as a lead peptide

structure applicable in the development of new diagnostic

tracers or anticancer agents that specifically target prostate

carcinoma.

INTRODUCTION

More than 30% of newly diagnosed cancers in males are

prostate cancer, making it a leading cause of death from cancer

worldwide (1, 2). Despite the initial effectiveness of hormone

therapy, many patients with metastatic disease eventually

progress to an androgen-resistant state (3). Various new

treatment modalities are currently being developed, but none

has yet shown a survival benefit in patients with hormone-

refractory prostate cancer (4).

New imaging procedures for diagnosis, treatment planning,

and therapy of prostate cancer are necessary for accurate staging

because current imaging methods are not satisfying i.e., positron

emission tomography with 2-[18F]fluoro-2-deoxyglucose does

not allow metabolic labeling in the majority of untreated primary

prostate cancers (5). Preliminary studies using 11C-choline show

potential for the primary staging of prostate cancer, but these

findings have to be confirmed in larger clinical studies (6).

Capromab pendetide (ProstaScint) is an 111In-labeled monoclo-

nal antibody against the prostate-specific membrane antigen

(PSMA) and used for imaging lymph node metastases, but the

interpretation of scintigraphic data obtained with ProstaScint is

demanding (7).

Molecular therapeutic approaches use PSMA as target

molecule (8). PSMA is a transmembrane folate hydrolase with

enhanced expression in prostate cancer tissue in comparison

with benign and neoplastic epithelial prostate cells (9, 10).

A weak extraprostatic expression of the protein has been noted

in small intestine mucosa, brain, salivary glands, and a subset of

renal proximal tubules (11, 12). Therefore, the monoclonal

antibody HuJ591, which recognizes the extracellular domain of

PSMA, has been used for treatment (13, 14). However,

heterogeneous expression of the target structure may lead to

treatment failure (15).

Peptides are promising molecules to deliver radionuclides

or therapeutic drugs into tumors. The application of a tumor-

selective peptide requires enough binding sites, such as overex-

pressed receptors, high affinity of the ligand, and sufficient

in vivo stability. Peptides that have by now been examined in

detail are somatostatin (16), gastrin (17), luteinizing hormone-

releasing hormone (18, 19), and bombesin (20, 21). The most

prominent example for a tumor-specific peptide is octreotide

(Sandostatin; ref. 22), which recognizes mainly the somatostatin

receptor subtype 2, and is used for diagnosis (23) as well as for

radiopeptide therapy (24, 25). Peptides also facilitate selective

transport of cytotoxic compounds into tumor tissue. For

example, the conjugation of a somatostatin analogue to the

topoisomerase inhibitors doxorubicin or 2-pyrrolinodoxorubicin

resulted in an effective growth inhibition of somatostatin

receptor–expressing tumors in vivo (26, 27). The coupling of

doxorubicin to the luteinizing hormone-releasing hormone, a

peptide with 10 amino acids, was evaluated in human epithelial

Received 8/4/04; revised 9/21/04; accepted 10/7/04.Grant support: Deutsche Forschungsgemeinschaft grants HA2901/2-1and HA2901/2-2.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.Requests for reprints: Sabine Zitzmann, Clinical Cooperation UnitNuclear Medicine, German Cancer Research Center, Im NeuenheimerFeld 280, D-69120 Heidelberg, Germany. Phone: 49-6221-567571;Fax: 49-6221-567585; E-mail: [email protected].

D2005 American Association for Cancer Research

Vol. 11, 139–146, January 1, 2005 Clinical Cancer Research 139

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ovarian cancers (28). In a nude mouse model with luteinizing

hormone-releasing hormone–expressing prostate tumors, the

cytotoxic luteinizing hormone-releasing hormone analogue

reduced tumor growth by 62% compared with castrated animals

(29). Even large peptide nucleic acid sequences conjugated with

octreotate are selectively taken up by somatostatin receptor–

expressing tumors leading to the suppression of oncogen

expression (30, 31).

In this study, a new peptide with specificity for PSMA-

negative prostate tumor cell lines, such as DU-145 and PC-3,

was identified by phage display techniques. Affinity and

kinetics of this peptide were determined in cell binding assays.

Confocal microscopy showed the internalization of the peptide

in a time-dependent manner. Biodistribution experiments in

DU-145 and PC-3 tumor carrying nude mice and rats were

done showing high uptake of the peptide in tumor tissue,

recommending this peptide as a promising lead structure for

improved targeting of prostate carcinomas.

MATERIAL AND METHODS

Cell Lines. The human prostate carcinoma cell lines DU-

145 and PC-3 (both American Type Culture Collection,

Manassas, VA), the human prostate cell line (normal,

immortalized with SV40) PNT-2 (European Collection of

Animal Cell Cultures, Salisbury, United Kingdom), and the

human embryonic kidney cell line 293 (American Type Culture

Collection) were cultivated at 37jC in a 5% CO2 incubator in

RPMI 1640 with Glutamax containing 10% FCS (both

Invitrogen, Karlsruhe, Germany) and 25 mmol/L HEPES.

Dunning R3327 subline AT-1 (American Type Culture

Collection) tumors cells were cultivated in RPMI 1640 (Life

Technologies, Eggenstein, Germany) supplemented with 292

mg/L glutamine, 100 IU/mL penicillin, 100 mg/L streptomycin,

and 10% FCS. Human umbilical vein endothelial cells were

isolated as described in the literature (32). Cultivation was done

on gelatin (1%)–coated cell culture flasks using medium 199

(Invitrogen) containing 20% FCS, 2 mmol/L glutamine, 100 IU/

mL penicillin, 100 IU/mL streptomycin, and 2 ng/mL basic

fibroblast growth factor (Roche Diagnostics, Mannheim,

Germany).

Selection of Tumor Cell Binding Peptides. The phage

display library was a linear 12–amino acid peptide library

(Ph.D.12, New England Biolabs, Beverly, MA). Each selection

round was conducted as follows: 1011 transducing units were

added to 293 cells for a negative selection. After 1 hour, the

medium was collected and centrifuged for 5 minutes at 1,500

rpm, and the supernatant was transferred to DU-145 cells grown

to 90% confluency. After 1 hour, the cells were washed four

times with 10 mL HBSS(+) (Invitrogen) + 1% bovine serum

albumin (BSA) and four times with 10 mL HBSS(�) + 1% BSA.

The cells were then detached with 4 mL PBS + 1 mmol/L EDTA

for 5 minutes and centrifuged for 5 minutes at 1,500 rpm. The

cell pellet was washed thrice in 1 mL HBSS(�) + 1% BSA and

lysed with 1% Triton X-100. Lysate (10 AL) was used for titeringof the phages. The remaining lysate was amplified in 50 mL

ER2537 bacteria according to the manufacturer’s protocol. For

the next selection round, 1011 transducing units from the

previous selection round were used. Six selection rounds were

done followed by ssDNA isolation from clones (QIAprep Spin

M13 Kit, Qiagen, Hilden, Germany). The peptide was identified

by sequencing.

Animals and Tumor Growth. Male 6-week-old BALB/c

nu/nu mice and the male Copenhagen rats weighing 220 to 250 g

were obtained from Charles River WIGA (Sulzfeld, Germany)

and housed in VentiRacks. For inoculation of the tumors in nude

mice, a Matrigel matrix/cell suspension (5 � 106 cells) was

injected s.c. into the anterior region of the mouse trunk. Tumors

were grown up to a size of f1.0 cm3. The rat prostate

adenocarcinoma Dunning R3327 subline AT-1 (American Type

Culture Collection) was transplanted s.c. into the leg of the

Copenhagen rats by using a tumor piece (4 mm2) from a rat host.

All animals were cared for according to the German animal

guidelines.

Peptide. The DUP-1 peptide (FRPNRAQDYNTN) was

obtained by solid-phase peptide synthesis using Fmoc chemistry.

The radiolabeling was achieved by iodination using the

chloramine-T method (33). The labeled peptide was purified

by high-pressure liquid chromatography on a LiChrosorb RP-

select B 5 Am, 250 � 4 mm column (Merck, Darmstadt,

Germany) using Tris-phosphate and methanol as eluents. The

specific activities obtained were 90 GBq/Amol for the 125I-

labeled peptide and 110 GBq/Amol for the 131I-labeled peptide.

For fluorescence microscopy, FITC was coupled via an

additional lysine at the COOH terminus.

In vitro Binding Experiments. Cells (n = 200,000) were

seeded into six-well plates and cultivated for 24 hours. The

medium was replaced by 1 mL fresh medium (without FCS).

When using the competitor, unlabeled peptide (10�4–10�11 mol/

L) was preincubated for 30 minutes. 125I-labeled peptide was

added to the cell culture (1–2 � 106 cpm per well) and

incubated for the appropriate incubation times varying from 1

minute to 4 hours. The cells were washed thrice with 1 mL PBS

and subsequently lysed with 0.3 mol/L NaOH (0.5 mL).

Radioactivity was determined with a g-counter and calculated

as percentage applied dose per 106 cells. If BSA or dry milk

powder were used as blocking agents, it was added to a final

concentration of 1% in medium without FCS.Stability Experiments. Serum stability measurements

were done with unlabeled and 131I-labeled DUP-1. Aliquots of

the peptide were incubated in human serum for several time

points at room temperature or 37jC. After incubation, 1

volume of acetonitrile was added to the sample to precipitate

serum proteins, which were pelleted by centrifugation. The

supernatant was then analyzed by reverse-phase high-pressure

liquid chromatography. Samples of were taken and analyzed

by matrix-assisted laser desorption ionization-time of flight

mass spectrometry.Conventional and Confocal Laser Scanning Microscopy

Using FITC-Labeled DUP-1. DU-145 cells were seeded

subconfluently onto coverslips and cultivated for 24 hours.

The medium was replaced by fresh medium (without FCS).

For microscopy, FITC-Lys-DUP-1 (10�5 mol/L) was added to

the medium and incubated for 10 and 60 minutes at 37jC.Subsequently, the cells were washed with 1 mL medium and

fixed with 2% formaldehyde for 20 minutes on ice. For the

pulse-chase experiment with confocal laser scanning

microscopy, 5 � 10�4 mol/L FITC-Lys-DUP-1 were added

New Prostate Carcinoma Binding Peptide140



to the medium for 10 minutes. The cells were washed thrice

with 1 mL PBS and incubated with 1 mL fresh medium

containing 5 � 10�5 mol/L dextran-Alexa568 (10,000

molecular weight, fixable, Molecular Probes, Eugene, OR)

for time points from 10 to 60 minutes. Subsequently, the cells

were washed, fixed, and incubated with TO-PRO-3 (Molecular

Probes, 1:1,000 dilution, 20 minutes) for cell nucleus staining.

Then, the cells were analyzed using a Leica SP1 CLSM (Leica

Microsystems Heidelberg, Mannheim, Germany).

Organ Distribution with Radioiodinated DUP-1. 131I-

DUP-1 was injected i.v. into male nu/nu mice (2.8 � 107 cpm

per mouse), carrying the s.c. transplanted human prostate

tumors DU-145 or PC-3. At 5, 15, 45, and 135 minutes

postinjection, the mice were sacrificed. The organs were

removed and weighed and the radioactivity was determined

using an automated NaI(Tl) well counter (CobraII, Canberra

Packard, Meriden, CT). The percentage of injected dose per

gram (ID/g) of tissue was calculated. For the perfusion

experiments, the mice were anesthesized with 5 mg Ketanest

(Parke-Davis, Berlin, Germany) and 400 AL of 0.2% Rompun

(BayerVital, Leverkusen, Germany) both injected i.p. Under

full anesthesia, the mice were perfused through the heart with

0.9% NaCl (25 mL) and tumor and control organs were

removed and weighed. For the biodistribution in male COP rats

bearing Dunning R3327 subline AT-1 tumors, 131I-DUP-1 (5 �107 cpm per rat) was injected i.v., the animals were sacrificed

after 5 and 15 minutes, and the organs were removed and

weighed.

RESULTS

Selection of a Peptide Binding to the Prostate Carci-

noma Cell Line DU-145. For the selection of tumor specific

peptides, in vitro selection rounds were done on DU-145 cells.

For each round, f1011 transducing units of the phages were

added to a cell culture dish with 293 cells for a negative selection

for 60 minutes followed by positive selection with DU-145

cells for 60 minutes. The unbound phages were washed off and

bound phages were recovered by lysing the DU-145 cells. After

six rounds, single phage clones were selected and amplified

and ssDNA was isolated for sequencing. Among 24 clones

sequenced, all peptides showed the same sequence. For in vitro

evaluation of the peptide, we used the identified peptide as

well as its inverse form. Because the inverse peptide showed

up to 40% better binding, we used the FRPNRAQDYNTN

(DUP-1) peptide for further evaluation (Fig. 1A).

Binding, Competition, Binding Kinetics, and Stability

of DUP-1 to Prostate Tumor Cell Lines. The peptide DUP-1

was prepared by Fmoc solid-phase synthesis and labeled with125I. Cells were incubated for 10 minutes with the 125I-labeled

peptide with or without unlabeled peptide as competitor (10�4

mol/L). Radioactivity of the lysed cells was calculated as

percentage applied dose per 106 cells. A high binding capacity

with up to 12% of the applied dose per 106 cells was found

for the prostate tumor cells DU-145 and PC-3 but not for the

immortalized benign prostate cells PNT-2 or for the primary

cultures of human umbilical vein endothelial cells (Fig. 1B).

The binding capacity of PC-3 cells was not significantly

higher in comparison with DU-145 cells. To show the

specificity of this binding, cells were preincubated with

10�4 mol/L DUP-1 followed by the radiolabeled DUP-1.

The recovered lysates showed that the unlabeled DUP-1 could

competitively inhibit (up to 95%) 125I-DUP-1 binding.

Unlabeled octreotide at the same concentration did not

prevent 125I-DUP-1 from binding (data not shown). The D-

DUP-1 peptide, which contains of the same amino acids but

in their D-conformation instead of the L-conformation, did not

bind to PC-3 cells (data not shown). To evaluate the inhibition

and to determine the IC50 value, different competitor

concentrations (unlabeled DUP-1) were added to the cells

before the 125I-labeled peptide was added. Concentrations of

10�4 to 10�5 mol/L inhibit >95% of the binding of 125I-DUP-

1 to DU-145 cells (Fig. 2A). At a competitor concentration of

10�8 to 10�9 mol/L, the binding of DUP-1 was marginally

enhanced by the presence of the competitor, whereas at

concentrations below 10�10 mol/L the binding value reached

the level of uncompeted binding again. The IC50 was

calculated as 1.77 � 10�7 mol/L. Similar results were

obtained with PC-3 cells (Fig. 2B).To evaluate the time

course of peptide binding, 125I-labeled peptide was added to

the cells and incubated for time points ranging from 1 minute

to 4 hours. After the incubation, the cells were lysed and the

radioactivity as percentage applied dose per 106 cells was

calculated (Fig. 2C and D). Binding of DUP-1 is rapid and

the highest binding rate is reached after 5 minutes. Thereafter,

the amount of bound peptide decreases and reaches a basal

level of f1.5% applied dose per 106 cells. The stability of

the peptide was evaluated in heparinized human serum at

25jC and 37jC. High-pressure liquid chromatography analysis

showed the peptide is rapidly degraded with a half-life of f2

minutes (Fig. 2E).

Fig. 1 A, amino acid sequence of the DU-145 binding peptide DUP-1isolated by peptide phage display. B, in vitro binding assay with DUP-1.DU-145, PC-3, human umbilical vein endothelial cells (HUVEC), andPNT-2 were grown for 24 hours. 125I-DUP-1 was added to the wells andincubated for 10 minutes. -, no competitor was added; +, 5 � 10�5 mol/Lunlabeled DUP-1 as competitor was added 30 minutes before incubationwith labeled DUP-1. Experiments were done in triplicate. Bars, SD.

Clinical Cancer Research 141



Internalization of FITC-Labeled DUP-1. To study the

internalization process, FITC-Lys-DUP-1 was added to the

medium and the cells were analyzed by microscopy (Fig. 3A).

Labeling of the cell membrane with FITC-Lys-DUP-1 was

observed after 10-minute incubation, whereas after 60-minute

incubation the peptide was intracellularly localized. To allow a

more detailed analysis of the peptide localization, a pulse-chase

experiment was done and analyzed by confocal microscopy. A

10-minute pulse with 5 � 10�4 mol/L FITC-Lys-DUP-1 was

applied to DU-145 cells followed by the removal of unbound

peptide and the incubation in the presence of 5 � 10�4 mol/L

dextran-Alexa568 (chase; Fig. 3B). Immediately after addition of

the dextran dye, FITC-Lys-DUP-1 was found to remain bound to

the cell membrane. Ten minutes later, most of the peptide was

still bound, but no internalized dextran was detected. Thirty

minutes after coincubation of FITC-Lys-DUP-1 (green) and

dextran (red), intracellularly localized yellow spots were seen,

indicating a colocalization of FITC-Lys-DUP-1 and dextran. At

30 and 60 minutes after incubation, three types of spots were

visible: (a) red spots, showing internalized dextran-Alexa568; (b)

green spots, demonstrating FITC-Lys-DUP-1 bound either

intracellularly or to the cell membrane; and (c) yellow spots,

characterizing internalized vesicles that contain FITC-Lys-DUP-

1 as well as dextran-Alexa568. A series of images obtained from

different layers of a DU-145 cell showed the vesicle-like

structure of the fluorescent spots (data not shown).

Biodistribution of Radiolabeled DUP-1. To investigate

the distribution of DUP-1 in vivo , the peptide was labeled with131I and injected i.v. into male nu/nu mice, carrying human

prostate tumors (DU-145 or PC-3). The biodistribution in

DU-145 tumor carrying mice showed that DUP-1 accumulated

after 5 minutes in the tumor to a level of f5% ID/g (injected

dose/g), which is higher than in the other organs, with the

exception of kidney and blood (Fig. 4A). This level was stable

up to 45 minutes before a distinct decrease was noted. PC-3

tumors showed a higher tumor uptake, amounting up to 7%

ID/g in the tumor, but with a faster washout resulting in values

comparable values of the DU-145 tumor at 45 minutes

postinjection (Fig. 4B). The higher uptake in PC-3 tumors at

5 minutes and the faster washout in PC-3 tumors at 135

minutes were statistically significant in comparison with DU-

145 tumors (P < 0.05). To reduce blood background in various

organs, animals carrying DU-145 tumors were perfused with

NaCl (Fig. 4C). Radioactivity was reduced in most organs,

whereas the tracer accumulation of 5% ID/g remained constant

Fig. 2 In vitro competition assay with DUP-1 using various competitorconcentrations. DU-145 cells (A) and PC-3 (B) were grown for 24 hours.Unlabeled DUP-1 in concentrations ranging from 10�4 to 10�11 mol/Lwere added to the cells 30 minutes before 125I-DUP-1 and incubated for10 minutes. Experiments were done in triplicate. Bars, SD. In vitrobinding kinetics of DUP-1. DU-145 cells (C) and PC-3 (D) were grownfor 24 hours. 125I-DUP-1 was added and incubated for time points of 1,5, 10, 20, 30, 60, 120, and 240 minutes. E, high-pressure liquidchromatography chromatographic analysis of 131I-labeled DUP-1incubated in human serum at 25jC. Samples were taken at the timepoints indicated and the serum proteins were precipitated withacetonitrile before chromatography.




with or without perfusion in the tumor for 5 and 15 minutes.

This leads to an increase of most tumor-to-organ ratios as

shown in Table 1. At 5 minutes, lung, liver, and muscle

showed a statistically significant difference of unperfused to

perfused organ (P < 0.05), and at 15 minutes, heart, lung, and

liver showed a statistically significant difference of unperfused

to perfused organ (P < 0.001). To show prostate binding of131I-DUP-1, biodistribution experiments were done in

Copenhagen rats bearing rat prostate adenocarcinoma

Dunning R3327 subline AT-1 (Fig. 4D). The prostates in

young mice used in our experiments were too small for reliable

measurements. For this reason, we used the AT-1 rat prostate

tumor model. The biodistribution in the rats was comparable

with the data obtained in mice, with all organs showing a

decrease of radioactivity over time, except for the tumor. The

radioactivity in the prostate tumor increased up to 300% after

15 minutes in comparison with normal prostate tissue. The

tumor-to-muscle ratio for DU-145 tumors in mice was 3.02 at

15 minutes and the prostate-to-muscle ratio for the rats was

0.99 at 15 minutes postinjection.

DISCUSSION

In this study, we introduce a new lead structure with

specific binding to prostate carcinoma cell lines in vitro and

selective accumulation in prostate carcinomas in vivo . This 12–

amino acid lead structure can be the launching platform for the

development of an improved mode of delivery for radionuclides

or pharmaceutical drugs to prostate tumors. Phage display is a

successful tool for identifying novel peptides with high

specificity (34, 35) and has been employed to isolate a NG2

proteoglycan-binding peptide to target tumor neovasculature

(36) or to identify specifically binding peptides for human lung

tumor cells (37).

The peptide DUP-1 contains a motif that facilitates

binding to different prostate carcinoma cell lines but not to a

benign prostate cell line or to human umbilical vein endothelial

cell. This specificity for prostate carcinoma cells is reproduced

in animal experiments. DUP-1 shows enhanced uptake even in

undifferentiated rat prostate adenocarcinomas (AT-1) versus

normal prostate tissue. The rat model showed comparable

tumor-to-muscle ratio for the AT-1 tumors in rats as for the

DU-145 tumors in mice with 2.54 and 3.02 at 15 minutes

postinjection, respectively. With a tumor-to-prostate ratio of f3

at 15 minutes, DUP-1 is a promising molecule for the diagnosis

of suspected prostate carcinoma. However, data in humans are

needed to assess its potential for the differential diagnosis

between tumor and benign hyperplasia. The tumor cell affinity

of DUP-1 was also supported by perfusion experiments with

animals bearing s.c. transplanted DU-145 and PC-3 tumors.

Because no binding to primary cultures of endothelial cells was

observed in vitro , we assumed that the peptide is able to

penetrate through the basal membrane followed by direct

binding to the tumor cells (38). This hypothesis was sustained

by the biodistribution data obtained with the perfused animals

showing that most organs display reduced radioactivity levels

compared with the unperfused animals, whereas the tracer

accumulation in the tumor remains unaffected. This indicates

that the high activity value observed in the tumor is due to

specific binding. The total amount of radioactivity obtained

with DUP-1 in the tumors at 15 minutes for DU-145 and PC-3

was 5% and 7% ID/g, respectively. This is significantly higher

compared with 3.65% ID/g delivered into tumors of the MDA-

MB-435 xenograft model with 125I-RGD (39).

In vitro , we observed a rapid internalization of FITC-

Lys-DUP-1. The experimental settings used in the pulse-chase

experiment revealed internalization into cells, because unspe-

cifically bound peptide was removed to ensure that only

bound peptide can be internalized during the following

incubation period. Dextran-Alexa586 does not bind to the

cells (data not shown) and high concentrations of this dye

allow visualization of internalized molecules only. Confocal

laser scanning microscopy showed intracellularly localized

vesicle-like structures. In addition, confocal micrograph slices

through the cells showed small areas of localized fluorescence

(data not shown), which was attributed to endocytotic vesicles.

After 60 minutes, the size of the vesicles increased, indicating

a fusion of the endocytotic vesicles to endosomes. This

internalization is useful for both imaging and potential

therapeutic applications of DUP-1 derivatives.

Fig. 3 A, fluorescence microscopy with FITC-Lys-DUP-1 (10�5 mol/L; green ; I) after 10-minute incubation (�400; II) and after 60-minuteincubation (�600). B, pulse-chase experiment with 10-minute preincu-bation of FITC-Lys-DUP-1 (5 � 10�5 mol/L; green) followed byincubation with dextran-Alexa568 (5 � 10�5 mol/L; red) for 0, 10, 30,and 60 minutes and visualization by confocal microscopy. White arrows,yellow vesicles.




Although the binding site is unknown, it is unlikely that

DUP-1 targets PSMA, because DU-145 and PC-3 cells are

PSMA negative (40). The competitive binding with unlabeled

DUP-1 points to saturable cell surface site, which after binding

leads to an internalization process. DUP-1 has no sequence

similarity to bombesin or luteinizing hormone-releasing hor-

mone or to any other peptide or protein sequence available as

confirmed by a search in different protein databases such as

European Molecular Biology Laboratory, SwissProt, etc. The

target structure for DUP-1 will be investigated in further

experiments using display cloning procedures (41).

The elevated blood values can be due to various reasons.

One possible explanation is the interaction of DUP-1 with serum

albumin. The use of 1% BSA as blocking solution strongly

inhibited the binding of DUP-1, suggesting that BSA bound the

peptide and prevented binding (data not shown). Similar results

were obtained with human albumin. A second reason is the

relative low stability of the peptide, which might lead to labeled

peptide fragments circulating in the bloodstream before they are

secreted via the kidneys. Analyses of serum stability of DUP-1

in vitro with high-pressure liquid chromatography has proven

degradation of DUP-1 within 10 minutes (data not shown).

Similar results were obtained with the blood of mice and rats

(data not shown).

This in vivo instability of peptides resulting from phage

display libraries was expected. Peptides displayed on the phage

surface are protected from proteolysis and may be displayed in a

defined conformation. This may result in reduced stability and

different binding properties of the corresponding peptides (42).

The focus of this work therefore was to find lead peptide

structures, which are able to bind selectively PSMA-negative

Fig. 4 Biodistribution of DUP-1 in male BALB/c nu/nu mice carryingDU-145 tumors (A ; n = 9 animals per time point) or PC-3 tumors (B ; n =3 animals per time point). Animals were injected i.v. with 131I-DUP-1and the radioactivity was measured in tumor and control organs after 5,15, 45, and 135 minutes. C, male BALB/c nu/nu mice carrying DU-145tumors (n = 6 animals per time point) injected i.v. with 131I-DUP-1. After5 and 15 minutes, the animals were perfused and the radioactivity oftumor and control organs was determined. D, male COP rats (n = 3animals per time point) bearing a Dunning R3327 subline AT-1 tumorwere injected i.v. with 131I-DUP-1. After 5 and 15 minutes, the animalswere sacrificed and the radioactivity in various organs was measured.Bars, SE.

Table 1 Tumor-to-tissue ratios from %ID/g for unperfused andperfused animals carrying DU-145 tumors

Tumor-to-organ ratio 5 min 15 min

Unperfused Perfused Unperfused Perfused

Heart 1.75 2.12 1.78 2.79Lung 0.95 1.82 0.80 2.34Spleen 1.42 1.51 0.80 2.34Liver 1.89 2.80 1.75 3.61Kidney 0.71 0.83 0.88 0.84Muscle 2.70 2.06 2.15 3.06Brain 9.34 8.14 8.74 10.01Tumor 1.00 1.00 1.00 1.00




prostate cancer cells. In a next step, the lead sequence DUP-1 is

now used to derivatize and optimize the in vivo stability by

simultaneously maintaining the binding characteristics. This is

expected to result in better target/nontarget ratios. Among the

structure evolution steps, we consider sequence fragmentation,

cyclization, D-amino acid substitution, and NH2- or COOH-

terminal end modifications (43–45). These modifications should

result in enhanced stability as well as in reduced binding to

plasma proteins.

In conclusion, due to its high and specific binding to

prostate carcinoma cells in vitro and in vivo together with rapid

internalization, DUP-1 represents a promising structure useful

for diagnosis and treatment of prostate cancer. DUP-1 or parts of

it may be used for coupling with radioactive isotopes and

anticancer agents or even for the modification of the envelope of

virus particles such as adeno-associated virus to obtain tumor

specific infection.

ACKNOWLEDGMENTS

We thank S. Peschke, M. Mahmut, and F. Birkle for contributing to

the in vitro experiments; H. Eskerski, U. Schierbaum, and K. Leotta for

contributing to the animal experiments; K. Schmidt for sharing her

human umbilical vein endothelial cell; and A. Tietz and R. Kuhnlein for

transplanting the rat prostate adenocarcinoma Dunning R3327 subline

AT-1 tumors.

REFERENCES

1. Crawford ED. Epidemiology of prostate cancer. Urology 2003;62:3–12.

2. Parkin DM, Bray FI, Devesa SS. Cancer burden in the year 2000. Theglobal picture. Eur J Cancer 2001;37 Suppl 8:S4–66.

3. Sandblom G, Varenhorst E. Incidence rate and management ofprostate carcinoma. Biomed Pharmacother 2001;55:135–43.

4. Sternberg CN. What’s new in the treatment of advanced prostatecancer? Eur J Cancer 2003;39:136–46.

5. Effert PJ, Bares R, Handt S, Wolff JM, Bull U, Jakse G. Metabolicimaging of untreated prostate cancer by positron emission tomographywith 18fluorine-labeled deoxyglucose. J Urol 1996;155:994–8.

6. Kotzerke J, Prang J, Neumaier B, et al. Experience with carbon-11choline positron emission tomography in prostate carcinoma. Eur J NuclMed 2000;27:1415–9.

7. Rosenthal SA, Haseman MK, Polascik TJ. Utility of capromabpendetide (ProstaScint) imaging in the management of prostate cancer.Tech Urol 2001;7:27–37.

8. Horoszewicz JS, Kawinski E, Murphy GP. Monoclonal antibodies to anew antigenic marker in epithelial prostatic cells and serum of prostaticcancer patients. Anticancer Res 1987;7:927–35.

9. Bostwick DG, Pacelli A, Blute M, Roche P, Murphy GP. Prostatespecific membrane antigen expression in prostatic intraepithelialneoplasia and adenocarcinoma: a study of 184 cases. Cancer 1998;82:2256–61.

10. Ross JS, Sheehan CE, Fisher HA, et al. Correlation of primary tumorprostate-specific membrane antigen expression with disease recurrence inprostate cancer. Clin Cancer Res 2003;9:6357–62.

11. Renneberg H, Friedetzky A, Konrad L, et al. Prostate specificmembrane antigen (PSM) is expressed in various human tissues:implication for the use of PSM reverse transcription polymerase chainreaction to detect hematogenous prostate cancer spread. Urol Res 1999;27:23–7.

12. Silver DA, Pellicer I, Fair WR, Heston WD, Cordon-Cardo C.Prostate-specific membrane antigen expression in normal and malignanthuman tissues. Clin Cancer Res 1997;3:81–5.

13. Liu H, Moy P, Kim S, et al. Monoclonal antibodies to theextracellular domain of prostate-specific membrane antigen also reactwith tumor vascular endothelium. Cancer Res 1997;57:3629–34.

14. Nanus DM, Milowsky MI, Kostakoglu L, et al. Clinical use ofmonoclonal antibody HuJ591 therapy: targeting prostate specificmembrane antigen. J Urol 2003;170:S84–8; discussion S8–9.

15. Smith-Jones PM, Vallabhajosula S, Navarro V, Bastidas D,Goldsmith SJ, Bander NH. Radiolabeled monoclonal antibodies specificto the extracellular domain of prostate-specific membrane antigen:preclinical studies in nude mice bearing LNCaP human prostate tumor.J Nucl Med 2003;44:610–7.

16. Pawlikowski M, Melen-Mucha G. Perspectives of new potentialtherapeutic applications of somatostatin analogs. Neuroendocrinol Lett2003;24:21–7.

17. Smith CJ, Volkert WA, Hoffman TJ. Gastrin releasing peptide (GRP)receptor targeted radiopharmaceuticals: a concise update. Nucl Med Biol2003;30:861–8.

18. Halmos G, Arencibia JM, Schally AV, Davis R, Bostwick DG. Highincidence of receptors for luteinizing hormone-releasing hormone(LHRH) and LHRH receptor gene expression in human prostate cancers.J Urol 2000;163:623–9.

19. Schally AV, Nagy A. Cancer chemotherapy based on targeting ofcytotoxic peptide conjugates to their receptors on tumors. Eur JEndocrinol 1999;141:1–14.

20. Anastasi A, Erspamer V, Bucci M. Isolation and structure ofbombesin and alytesin, 2 analogous active peptides from the skin of theEuropean amphibians Bombina and Alytes . EXS 1971;27:166–7.

21. Scopinaro F, De Vincentis G, Varvarigou AD, et al. 99mTc-bombesin detects prostate cancer and invasion of pelvic lymph nodes.Eur J Nucl Med Mol Imaging 2003;30:1378–82.

22. Bauer W, Briner U, Doepfner W, et al. SMS 201-995: a very potentand selective octapeptide analogue of somatostatin with prolongedaction. Life Sci 1982;31:1133–40.

23. Henze M, Schuhmacher J, Dimitrakopoulou-Strauss A, et al.Exceptional increase in somatostatin receptor expression in pancreaticneuroendocrine tumour, visualised with (68)Ga-DOTATOC PET. EurJ Nucl Med Mol Imaging 2004;31:466.

24. Bodei L, Cremonesi M, Zoboli S, et al. Receptor-mediatedradionuclide therapy with 90Y-DOTATOC in association with aminoacid infusion: a phase I study. Eur J Nucl Med Mol Imaging 2003;30:207–16.

25. Waldherr C, Pless M, Maecke HR, et al. Tumor response and clinicalbenefit in neuroendocrine tumors after 7.4 GBq (90)Y-DOTATOC. J NuclMed 2002;43:610–6.

26. Benali N, Cordelier P, Calise D, et al. Inhibition of growth andmetastatic progression of pancreatic carcinoma in hamster after somato-statin receptor subtype 2 (sst2) gene expression and administration ofcytotoxic somatostatin analog AN-238. Proc Natl Acad Sci U S A2000;97:9180–5.

27. Nagy A, Schally AV, Halmos G, et al. Synthesis and biologicalevaluation of cytotoxic analogs of somatostatin containing doxorubicinor its intensely potent derivative, 2-pyrrolinodoxorubicin. Proc Natl AcadSci U S A 1998;95:1794–9.

28. Miyazaki M, Nagy A, Schally AV, et al. Growth inhibition of humanovarian cancers by cytotoxic analogues of luteinizing hormone-releasinghormone. J Natl Cancer Inst 1997;89:1803–9.

29. Letsch M, Schally AV, Szepeshazi K, Halmos G, Nagy A. Preclinicalevaluation of targeted cytotoxic luteinizing hormone-releasing hormoneanalogue AN-152 in androgen-sensitive and insensitive prostate cancers.Clin Cancer Res 2003;9:4505–13.

30. Mier W, Eritja R, Mohammed A, Haberkorn U, Eisenhut M. Peptide-PNA conjugates: targeted transport of antisense therapeutics into tumors.Angew Chem Int Ed Engl 2003;42:1968–71.

31. Sun L, Fuselier JA, Murphy WA, Coy DH. Antisense peptide nucleicacids conjugated to somatostatin analogs and targeted at the n-myconcogene display enhanced cytotoxity to human neuroblastoma IMR32cells expressing somatostatin receptors. Peptides 2002;23:1557–65.




32. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture ofhuman endothelial cells derived from umbilical veins. Identificationby morphologic and immunologic criteria. J Clin Invest 1973;52:2745–56.

33. Eisenhut M, Mier W. Radioiodination chemistry and radioiodinatedcompounds. In: Vertes A, Nagy S, Klencsar Z, editors. Handbook ofnuclear chemistry. Vol. 4. Amsterdam: Kluwer Academic Publishers;2003. p. 257–78.

34. Koivunen E, Arap W, Valtanen H, et al. Tumor targeting with aselective gelatinase inhibitor. Nat Biotechnol 1999;17:768–74.

35. Pasqualini R. Vascular targeting with phage peptide libraries. Q JNucl Med 1999;43:159–62.

36. Burg MA, Pasqualini R, Arap W, Ruoslahti E, Stallcup WB. NG2proteoglycan-binding peptides target tumor neovasculature. Cancer Res1999;59:2869–74.

37. Oyama T, Sykes KF, Samli KN, Minna JD, Johnston SA, BrownKC. Isolation of lung tumor specific peptides from a random peptidelibrary: generation of diagnostic and cell-targeting reagents. Cancer Lett2003;202:219–30.

38. Jain RK. Delivery of molecular and cellular medicine to solidtumors. J Control Release 1998;53:49–67.

39. Chen X, Park R, Tohme M, Shahinian AH, Bading JR, Conti PS.

MicroPET and autoradiographic imaging of breast cancer av-integrinexpression using 18F- and 64Cu-labeled RGD peptide. Bioconjug Chem2004;15:41–9.

40. Israeli RS, Powell CT, Corr JG, Fair WR, Heston WD. Expres-sion of the prostate-specific membrane antigen. Cancer Res 1994;54:1807–11.

41. Rhyner C, Kodzius R, Crameri R. Direct selection of cDNAs fromfilamentous phage surface display libraries: potential and limitations.Curr Pharm Biotechnol 2002;3:13–21.

42. Nilsson F, Tarli L, Viti F, Neri D. The use of phage display for thedevelopment of tumour targeting agents. Adv Drug Deliv Rev 2000;43:165–96.

43. Hoffman TJ, Gali H, Smith CJ, et al. Novel series of 111In-labeledbombesin analogs as potential radiopharmaceuticals for specific targetingof gastrin-releasing peptide receptors expressed on human prostatecancer cells. J Nucl Med 2003;44:823–31.

44. Dasgupta P, Mukherjee R. Lipophilization of somatostatin analogRC-160 with long chain fatty acid improves its antiproliferative andantiangiogenic activity in vitro . Br J Pharmacol 2000;129:101–9.

45. de Jong M, Breeman WA, Bakker WH, et al. Comparison of (111)In-labeled somatostatin analogues for tumor scintigraphy and radionuclidetherapy. Cancer Res 1998;58:437–41.




2005;11:139-146. Clin Cancer Res Sabine Zitzmann, Walter Mier, Arno Schad, et al. Tumor Imaging and TherapyA New Prostate Carcinoma Binding Peptide (DUP-1) for

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A New Prostate Carcinoma Binding Peptide (DUP-1) for Tumor ... · A New Prostate Carcinoma Binding Peptide (DUP-1) for Tumor Imaging and Therapy Sabine Zitzmann,1,3 Walter Mier,3