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Clinical Cancer Research Vol. 11, 6705-6712, September 15, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

Preclinical Evaluation of the Breast Cancer Cell-Binding Peptide, p160

Vasileios Askoxylakis1,4, Sabine Zitzmann1,4, Walter Mier4, Keith Graham4, Susanne Krämer4, Frederic von Wegner5, Rainer H.A. Fink5, Manfred Schwab2, Michael Eisenhut3 and Uwe Haberkorn1,4

Authors' Affiliations: 1 Clinical Cooperation Unit Nuclear Medicine and Departments of 2 Cytogenetics and 3 Radiopharmaceutical Chemistry, German Cancer Research Center; 4 Department of Nuclear Medicine and 5 Medical Biophysics, Institute for Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany

Requests for reprints: Uwe Haberkorn, Department of Nuclear Medicine, University of Heidelberg, Im Neuenheimer Feld 400, D-69120 Heidelberg, Germany. Phone: 49-6221-567732; Fax: 49-6221-565463; E-mail: uwe_haberkorn{at}med.uni-heidelberg.de.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Selective delivery of drugs into the target tissue is expected to result in high drug concentrations in the tissue of interest and therefore enhanced drug efficacy. To develop a peptide-based radiopharmaceutical, we investigated the properties of a peptide with affinity for human breast cancer, which has been selected through phage display.

Experimental Design: The bioactivity of the p160 peptide (VPWMEPAYQRFL) was evaluated in vitro and in vivo. The specific binding to human breast cancer MDA-MB-435 cells was confirmed in competition experiments. Internalization of the peptide was investigated with confocal microscopy. Furthermore, the biodistribution of 131I-labeled p160 was studied in tumor-bearing mice. In vivo stability was evaluated at different periods after tracer administration using high-performance liquid chromatography analysis.

Results: The binding of 125I-labeled p160 was inhibited up to 95% by the unlabeled peptide with an IC50 value of 0.6 µmol/L. In addition, 40% of the total bound activity was found to be internalized into the human breast cancer cells. Although a rapid degradation was seen, biodistribution studies in nude mice showed a higher uptake in tumor than in most of the organs. Perfusion of the animals caused a reduction of the radioligand accumulation in the healthy tissues, whereas the tumor uptake remained constant. A comparison of [131I]p160 with a 131I-labeled Arg-Gly-Asp peptide revealed a higher tumor-to-organ ratio for [131I]p160.

Conclusions: p160 has properties that make it an attractive carrier for tumor imaging and the intracellular delivery of isotopes or chemotherapeutic drugs.

Breast cancer is the most common cancer in women representing >30% of malignancies in women. Because breast cancer may exist for a long period as noninvasive or invasive but nonmetastatic disease, there is an urgent need for early diagnosis and therapy in these patients. This is addressed by different diagnostic techniques, such as mammography, magnetic resonance imaging, and spectroscopy, scintigraphy, or positron emission tomography. Treatment of breast cancer is stage dependent and includes surgical management, radiation therapy, chemotherapy, and hormonal therapy, whereas different experimental approaches, such as gene therapy and antiangiogenic therapies, are under investigation (1).

Major drawbacks of current cancer therapies are side effects and low specificity for tumor cells. To improve the specific uptake of therapeutic agents in the tumor, different strategies have been pursued. One of those is the use of molecular addresses for tumor targeting. In this direction, "engineered antibodies" or antibody fragments are the most common vehicles used. A prominent example of an antibody developed to target specific proteins involved in the development of breast cancer is the monoclonal antibody trastuzumab (2). The corresponding antigen is a member of the epidermal growth factor receptor family, called erbB2 (HER-2/neu) and its overexpression, caused by amplification of the gene, can result in a malignant transformation of human breast epithelium (3). However, the size of intact antibody molecules results in a limited tumor penetration and a slow clearance from the circulation, which decreases the tumor-to-blood ratio (4).

As an alternative to antibodies, recent efforts to identify new targeting molecules on carcinomas and metastases have focused on the use of peptides. Peptides possess pharmacokinetic properties that make them ideal shuttles for tumor targeting, as they are smaller and usually characterized by rapid clearance from blood and nontarget tissues and high uptake into target tissues. Such a homing peptide with the potential to bind specific and preferentially to the target structure is the three–amino acid motif Arg-Gly-Asp (RGD). RGD is present in many extracellular matrix components, like fibronectin. The RGD peptide is capable to target molecules both to angiogenic endothelial cells and to tumor cells (5). RGD analogues are used in tumor imaging (6, 7), antiangiogenesis approaches (8), and tumor targeting with chemotherapeutic drugs or radionuclides (9).

The identification of new efficient peptides with specific targeting abilities and reduced background binding is a major challenge in cancer-related peptide research. Peptides with tumor affinity can be identified via selections using complex random peptide libraries, containing a high number of peptides that are displayed on bacteriophages (10). Phage display libraries have been used to select phages expressing peptides on their surface with organ- or tumor-binding specificity (11–13).

A potential candidate peptide with promising targeting properties is the p160 peptide (14). The peptide p160 (VPWMEPAYQRFL), identified through random peptide phage display, is a linear dodecapeptide with specificity for the breast cancer cell line MDA-MB-435 and the neuroblastoma cell line WAC 2. The bacteriophage t160, displaying the peptide p160, was isolated by selection on the neuroblastoma cell line WAC 2. Inhibition of phage binding through the chemically synthesized p160 peptide determined that the phage binding to WAC 2 cells is mediated through the displayed peptide. In addition, confocal light microscopy studies revealed an internalization of t160 in WAC 2 neuroblastoma cells.

For in vivo application, the peptide has to be chemically synthesized and used without the phage backbone. In this study, we investigated the properties of the peptide p160 on the human breast cancer cell line MDA-MB-435 as the phages were found to have high affinity for this cell line. For a systematic investigation of the pharmacologic properties of p160, binding, internalization, and organ biodistribution studies were carried out. Affinity and binding kinetics of p160 were determined to study its cellular handling and to optimize its properties for drug targeting purposes.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Peptides. The peptides p160 (VPWMEPAYQRFL) and D-p160 (all amino acids in D-isoform) were obtained by solid-phase peptide synthesis using Fmoc coupling protocols (15). All standard reagents and solvents for the peptide synthesis were purchased from Merck (Darmstadt, Germany) or Novabiochem (Laeufelfingen, Switzerland). The FITC-RGD-4C (CDCRGDCFC) peptide was obtained from Synthem (Nimes, France).

Radiolabeling of p160 and FITC-RGD-4C with 125I or 131I was done using the chloramine-T method (16). The radioisotopes Na125I and Na131I were obtained from Amersham Pharmacia Biotech (Freiburg, Germany). For conventional and confocal laser scanning microscopy studies, FITC was coupled via an additional lysine at the COOH terminus of p160.

Cell lines. All cell lines were cultivated at 37°C in a 5% CO2 incubator. The human breast cancer cell line MDA-MB-435 (National Cancer Institute, Frederick, MD) was cultured in RPMI 1640 with Glutamax containing 10% FCS (Invitrogen, Karlsruhe, Germany) and 25 mmol/L HEPES. The human breast cancer cell line MCF-7 (American Type Culture Collection, Manassas, VA) was cultured in DMEM with Glutamax containing 10% FCS (Invitrogen) and 25 mmol/L HEPES. Human umbilical vein endothelial cells (HUVEC) were isolated as described (17) and cultivated on 1% gelatin-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).

In vitro binding experiments and competition experiments. Binding assays were done using breast cancer MDA-MB-435, MCF-7, or HUVEC cells as target. For the in vitro experiments, cells were plated in six-well plates in 3 mL volumes of medium, supplemented with 10% FCS and 25 mmol/L HEPES, at a density of 400,000 cells per well. After 24 hours of cultivation, the medium was replaced by 1 mL fresh medium (without FCS) containing 1 x 106 to 2 x 106 cpm 125I-labeled p160 (3.6 x 10–10-7.1 x 10–10 mol/L) and incubation was done at 37°C. After 1 hour, incubation was stopped by removing the medium and washing the cells thrice with PBS. Subsequently, cells were lysed with 0.5 mL of 0.3 mol/L NaOH and the radioactivity was measured with a gamma counter and calculated as percent applied dose per 106 cells. Competition experiments were carried out using the unlabeled p160 peptide as inhibitor for radioligand binding at different concentrations (10–4-10–10 mol/L). Same binding experiments were carried out using the peptides D-p160 and octreotide as competitors for binding of the radioligand [125I]p160. For kinetic analysis, 125I-labeled p160 was incubated with MDA-MB-435 breast cancer cells for different incubation periods varying from 10 minutes to 3 hours.

Internalization experiments. Internalization experiments were done as described (18, 19). Subconfluent cell cultures of MDA-MB-435 breast cancer cells were incubated with [125I]p160 for 60 minutes at 37°C and 4°C. Cells were incubated without or with an excess of unlabeled peptide (10–4 mol/L). Cellular uptake was stopped by removing medium from the cells and washing thrice with 1 mL PBS. Subsequently, cells were washed twice with 1 mL of 20 mmol/L sodium acetate (pH 5.0) in PBS for 10 minutes at room temperature to remove the surface-bound radioactivity. The cells were washed again thrice with 1 mL ice-cold PBS and lysed with 0.5 mL NaOH (0.3 mol/L). Surface-bound and internalized radioactivity was measured with a gamma counter and calculated as percent applied dose per 106 cells.

Confocal laser scanning microscopy using FITC-labeled p160. For the confocal microscopy experiments, 50,000 MDA-MB-435 cells were seeded onto coverslips. After 24 hours of cultivation, the medium was replaced by fresh medium (without FCS) and FITC-Lys-p160 (10–6 mol/L) was added to the cells. The FITC-labeled peptide was incubated with the cells for 30 minutes at 37°C. After incubation, the medium was removed and the cells were washed thrice with 1 mL medium. Subsequently, the cells were fixed with 2% formaldehyde for 20 minutes on ice. The cells were washed again thrice with medium and incubated with TO-PRO-3 (Molecular Probes, Eugene, OR, 1:1,000 dilution, 30 minutes) to stain the cell nuclei. Finally, the cells were washed thrice with 1 mL PBS and the coverslips were put on slides using fluorescent mounting medium (DAKO, Carpinteria, CA). The experiments were also carried out using the unlabeled p160 peptide (10–4 mol/L) as competitor for the binding of FITC-Lys-p160. Samples without FITC-Lys-p160 were analyzed to determine autofluorescence of the MDA-MB-435 cells. After treatment of the cells, confocal imaging was done on an inverted microscope (Leica DM IRBE) with a confocal laser scanning unit (Leica SP2 MP).

In vivo studies. Biodistribution was done on 9-week-old female BALB/c nu/nu mice obtained from Charles River WIGA (Sulzfeld, Germany) and housed in VentiRacks. For the in vivo studies, breast cancer MDA-MB-435 tumors were transplanted in Matrigel-Matrix (Falcon) s.c. into the anterior region of the mouse trunk. The tumors were allowed to grow for ~2 weeks to a volume of 1.0 cm3, and 0.1 mL 131I-labeled p160 (~1 MBq) was injected via the tail vein. At 1 hour postinjection, the animals were sacrificed and dissected. Samples of tumor, blood, and selected tissues were removed, drained of blood, and weighed and the radioactivity in each organ was determined with a gamma counter (LB 951G, Berthold, Germany) and calculated as percent injected dose per gram tissue (%ID/g). To determine the uptake in blood-free organs, perfusion experiments were carried out. For the perfusion studies, [131I]p160 was injected in tumor carrying mice as described. At 1 hour postinjection, the animals were anesthetized by an i.p. injection of 5 mg Ketanest (Parke-Davis, Berlin, Germany) and 400 µL of 0.2% Rompun (BayerVital, Leverkusen, Germany). A catheter was put in the ascending aorta through a small cut in the left ventricle of the heart of the mouse, and perfusion was done with 25 mL of 0.9% NaCl through a cut in the liver. After perfusion, samples of tumor and organs of interest were removed, weighed, and counted for radioactivity. Radioactivity concentration was expressed as %ID/g. Organ distribution of [131I]FITC-RGD-4C was done without perfusion in BALB/c nu/nu mice carrying MDA-MB-435 breast cancer tumors. All animal experiments were carried out in conformity with the German law for protection of animals and are in compliance with European laws.

Metabolism. The serum stability of p160 was investigated by high-performance liquid chromatography. 131I-labeled p160 was injected in the tail vein of a BALB/c nu/nu mouse. At different time points varying from 1 to 60 minutes, blood samples were taken and centrifuged to collect serum. Serum was deproteinized by protein precipitation with equal volume of acetonitrile. After centrifugation for 15 minutes at 12,000 x g, serum proteins were pelleted and the supernatant was injected into an analytic LiChrosorb RP-select B 5 µm, 250 x 4 mm (Merck) high-performance liquid chromatography column (20). Tris/phosphate and methanol were used as eluents to separate the peptide and its fragments according to their hydrophilicity.

Data analysis and statistics. Statistical comparisons between groups were done by the unpaired Student's t test using the Sigmastat program (Jandel Scientific, Erkrath, Germany). P ≤ 0.05 was considered statistically significant.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
In vitro binding experiments and competition experiments. To determine the affinity of p160, competition binding assays with the human breast cancer cell line MDA-MB-435 were done. For the characterization of the binding of p160, the peptide was synthesized by Fmoc solid-phase peptide synthesis and labeled with 125I. The radioligand [125I]p160 was incubated with MDA-MB-435 cells for 1 hour at 37°C with or without unlabeled peptide as competitor (10–4 mol/L). No FCS was present in the medium during the incubation to avoid degradation by serum proteins and to allow analysis of the peptide characteristics without the influence of additional variables. After incubation, the cells were washed and lysed and the bound radioactivity was calculated as percent applied dose per 106 cells. The 125I-labeled p160 peptide showed a binding capacity of 1.5% to 2% of the applied dose per 106 cells. The unlabeled peptide p160 at a concentration of 10–4 mol/L caused an up to 95% decrease of the binding of the radioligand [125I]p160. Using the peptides D-p160 and octreotide as competitor at the same concentration, the binding of the radioligand could not be competitively abolished (Fig. 1A). The same binding experiments with the breast cancer cell line MCF-7 revealed a binding capacity of ~7% of the applied dose per 106 cells, which could be inhibited by unlabeled p160 but not by the peptides D-p160 and octreotide (Fig. 1C). Performing the binding experiments on HUVECs, the binding capacity was found to be lower. Only 0.4% of the applied dose per 106 cells was measured on the HUVEC cells. Furthermore, the binding of [125I]p160 on HUVEC cells was not competitively abolished by the unlabeled p160 peptide at a concentration of 10–4 mol/L (Fig. 1B).



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  		Fig. 1. Binding and displacement of the 125I-labeled p160 peptide. A and C, specific binding of [125I]p160 to human breast cancer cell lines MDA-MB-435 and MCF-7. Nonspecific binding was determined in the presence of 10–4 mol/L unlabeled p160. The peptides octreotide and D-p160 were used at the same concentration (10–4 mol/L) as negative control competitors. B, binding of [125I]p160 with and without an excess of the unlabeled peptide (10–4 mol/L) in MDA-MB-435 and HUVEC cells. Incubation was done for 1 hour at 37°C. D, binding of [125I]p160 to MDA-MB-435 cells as a function of time. Incubation was done from 5 to 180 minutes. All experiments were done in triplicate. Bars, SD.

 
Kinetic studies of [125I]p160 in MDA-MB-435 cells, with incubation periods varying from 5 minutes to 3 hours, revealed a time-dependent increase of the radioligand uptake for incubation periods up to 10 to 20 minutes. Thereafter, a time-dependent decrease of [125I]p160 uptake was noticed, with the bound activity reduced to the background level after 3 hours of incubation (Fig. 1D).

The affinity of p160 in MDA-MB-435 cells was evaluated through competition studies using the unlabeled p160 peptide as competitor for radioligand binding at different concentrations varying from 10–4 to 10–10 mol/L. At a competitor concentration of 10–4 mol/L, up to 95% of the binding of [125I]p160 were inhibited. At concentrations <10–9 to 10–10 mol/L, the bound activity reached the level of uncompeted binding. Evaluation of the competition binding data gave an IC50 value of 0.6 ± 0.9 µmol/L with a Kd of 0.86 µmol/L (Fig. 2). The Scatchard analysis of the data led to a maximum number of binding sites of 1.03 x 10–11 µmol/cell.



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  		Fig. 2. Displacement of bound [125I]p160 by the unlabeled p160 peptide. MDA-MB-435 cells were incubated with 1 x 106 to 2 x 106 cpm radioligand and increasing concentrations of the unlabeled p160 peptide. Inset, Scatchard plot derived from the same data.

 
Internalization and confocal laser scanning microscopy. In vitro internalization studies were done to determine the rate of internalization of [125I]p160 in MDA-MB-435 human breast cancer cells. After 1-hour incubation at 37°C, the cells were subjected to an acid wash procedure with sodium acetate (pH 5.0) to remove membrane-bound ligand molecules. Internalization was investigated both under conditions where internalization is active (37°C) and under conditions where it is suppressed (4°C). To determine nonspecific uptake, incubation with an excess of the unlabeled p160 peptide (10–4 mol/L) was done. The results of the internalization experiments at 37°C showed that 40% of the total bound activity were found to be internalized into the MDA-MB-435 cells (Fig. 3). After incubation at 37°C with an excess of the unlabeled peptide, the internalized activity was measured to be decreased to the level of 2.5% of the internalized activity without the presence of competitor. After 1-hour incubation at 4°C, the internalization of [125I]p160 was strongly suppressed to the value of 5% to 10% of the internalized activity at 37°C, whereas the presence of the unlabeled peptide at a concentration of 10–4 mol/L resulted in a total suppression of the radioligand uptake (Fig. 3).



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  		Fig. 3. Binding and internalization of [125I]p160 in MDA-MB-435 cells. Cells were grown for 24 hours and incubated with 1 x 106 to 2 x 106 cpm radioligand for 1 hour at 37°C or at 4°C. After washing the cells with an acid buffer (surface-bound activity), the cells were lysed and the internalized radioactivity was measured (internalized activity). Surface-bound and internalized radioactivity was also measured after treatment in the presence of the unlabeled p160 peptide at a concentration of 10–4 mol/L.

 
The results of the binding and internalization studies of the radioactive p160 were confirmed by the results of the confocal laser microscopy studies. After 30-minute incubation of the FITC-Lys-p160 with MDA-MB-435 cells, an intensive fluorescence signal in the cells was found. Further investigation revealed a concentration of the fluorescence in irregular clusters at the periphery of the cells (Fig. 4A). After incubation with excess of the unlabeled p160 peptide as competitor, no FITC fluorescence was detected (Fig. 4B). To exclude autofluorescence of the MDA-MB-435 cells, confocal laser microscopy studies were done without treatment of the cells with FITC-labeled p160, revealing no fluorescence signal at all (data not shown).



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  		Fig. 4. Confocal laser scanning microscopy with FITC-Lys-p160. A, MDA-MB-435 cells were incubated with FITC-Lys-p160 (10–6 mol/L) for 30 minutes (green). B, cells were incubated with FITC-Lys-p160 (10–6 mol/L) for 30 minutes in the presence of unlabeled p160 at a concentration of 10–4 mol/L. Cell nuclei were stained with TO-PRO-3 (blue).

 
Metabolism. In vivo investigation of the stability of p160 in serum was done through injection of 131I-labeled p160 in mice and high-performance liquid chromatography analysis of serum samples taken from the animal at different time periods. The stability studies revealed a fast degradation of p160 by serum proteases. Immediately after injection, only the full-length peptide eluted at 17.7 minutes (Fig. 5). After 2-minute circulation of [131I]p160 in blood, a first degradation product of p160 was detected yielding a fragment that eluted at 13.2 minutes. After 5-minute circulation in the blood, a second fragment appeared at 4.0 minutes, and the amount of this fragment increased with time. After 30 minutes, only the 4-minute fragment remained and was still present after 1 hour.



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  		Fig. 5. Metabolites analysis by high-performance liquid chromatography in serum collected at time points from 2 to 60 minutes after i.v. injection of 131I-labeled p160. Before chromatography, the serum proteins were precipitated with acetonitrile and centrifugation at 12,000 x g for 15 minutes. t0 = 0 minute.

 
In vivo studies. Biodistribution experiments of p160 labeled with 131I were done in female BALB/c nu/nu mice carrying human breast cancer MDA-MB-435 tumors s.c. into the trunk. The organ biodistribution in MDA-MB-435 tumor carrying mice at 1 hour after i.v. injection of [131I]p160 showed a tumor accumulation of 6%ID/g (Fig. 6). The uptake in the tumor was higher than in heart, spleen, liver, and brain and almost the same compared with the kidneys. Only the blood value (8%) was higher than the accumulation in the tumor (data not shown). The lung tissue showed almost the same radioactivity concentration as the tumor tissue, but the perfusion experiments showed a >60% decrease in lung radioactivity. To reduce blood background in the tumor and the other organs, biodistribution studies were done followed by perfusion of the mice with 0.9% NaCl. The perfusion experiments showed a reduction of the uptake in all organs, whereas the uptake in the tumor remained almost constant (Fig. 6). Heart, lung, and liver showed a statistically significant decrease of unperfused to perfused organ with P < 0.005. The reduction of the uptake in the healthy tissues but not in the tumor after perfusion results in an increase of the tumor-to-organ ratios. The tumor-to-liver ratio was 1.5 before perfusion and 4 after perfusion (data not shown).



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  		Fig. 6. Organ distribution of p160 in female BALB/c nu/nu mice carrying MDA-MB-435 tumors. Black columns, activity concentration (%ID/g) in tumor and control organs after 1-hour circulation of 131I-labeled p160 in the mice. Gray columns, radioactivity concentration (%ID/g) in tumor and control organs after perfusion of the animals (n = 3 animals per experiment).

 
Organ distribution of [131I]FITC-RGD-4C in BALB/c nu/nu mice, carrying MDA-MB-435 breast cancer tumors, revealed a similar RGD-4C uptake in the tumor and most of the organs (blood, heart, spleen, and brain) like [131I]p160. RGD-4C showed a higher accumulation in kidney and liver (data not shown), resulting in lower tumor-to-organ ratios. The tumor-to-kidney ratio of [131I]FITC-RGD-4C was calculated to be 10 times lower than the tumor-to-kidney ratio of [131I]p160. The tumor-to-liver ratio of [131I]FITC-RGD-4C was also only half the tumor-to-liver ratio of [131I]p160 (Table 1).


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  		Table 1. Tumor-to-organ ratios calculated from the organ distribution of [131I]p160 in unperfused female BALB/c nu/nu mice carrying MDA-MB-435 tumors (n = 7 animals) and the organ distribution of [131I]FITC-RGD-4C in unperfused BALB/c nu/nu mice carrying MDA-MB-435 tumors (n = 3 animals)

 

   Discussion
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 Materials and Methods
 Results
 Discussion
References
 
Although chemotherapy and hormonal therapy play an important role in the treatment of breast cancer, the results of prospective studies show that a significant number of patients does not respond to these therapeutic modalities (21). Drug resistance, in addition to side effects of chemotherapy and hormonal therapy, necessitates the search for specific tumor targeting agents. A novel target for the treatment of breast cancer is the HER-2 protein, which has been reported to be overexpressed in breast malignancies and is associated with aggressive tumor growth (22). In this respect, a tumor-specific liposome system has been developed for the delivery of anti-HER-2 antisense oligonucleotides, which are known to sensitize breast cancer cells to chemotherapy (23). The specific targeting of drug-carrying liposomes into breast cancer cells required an addition of folate receptor ligands on the surface of the liposomes, as it is known that the human {alpha}-isoform folate receptor is overexpressed in tumors (24). This example of how tumor specificity can promote tumor therapy shows the importance of the identification of molecules specifically binding to breast cancer cells. These can offer new specific ligands that can be conjugated on liposomes and play the role of lead structure for efficient tumor targeting.

P160 is a peptide with specificity for the breast cancer cell line MDA-MB-435 identified through phage display by Zhang et al. (14). The peptide p160 was chosen for further investigation among the other peptides identified by Zhang et al. because of its high specificity. Binding studies with phages expressing the p160 motif revealed that from various tumor and normal cell lines only the breast cancer cell line MDA-MB-435 and a subset of neuroblastoma cell lines were targeted. Further evaluation of the properties of the chemically synthesized p160 showed that this specificity was mediated by the peptide moiety of the phages expressing p160. Our data show that p160 binds to the MDA-MB-435 breast cancer cells but does not bind to primary endothelial HUVEC cells.

The hypothesis that the cellular binding of p160 could be mediated through a specific receptor was strongly supported by two results. First, the uptake in MDA-MB-435 breast cancer cells was reduced with increasing concentration of the unlabeled p160 peptide as competitor, whereas studies with unspecific competitors like octreotide and D-p160 showed no effect. Second, we found evidence for an internalization of the radiolabeled and the FITC-labeled p160, which was inhibited in the presence of the unlabeled peptide. This result indicates that a receptor-mediated process might be involved in the internalization of p160. This hypothesis was sustained by internalization studies at 4°C, revealing a suppression of the internalization as expected for a receptor-mediated endocytotic process. Confocal laser scanning microscopy showed a concentration of FITC-Lys-p160 in irregular clusters at the periphery of the cells, which might be caused by the accumulation of peptide-receptor complexes in endocytosis-specialized areas of the cell membrane.

A prerequisite for the use of an agent as targeting vehicle is a selective binding to the tissue of interest and limited uptake by healthy tissues. After i.v. administration in tumor-bearing mice, [131I]p160 showed a higher uptake in tumors than in most normal organs. In addition, perfusion experiments showed a specific binding to tumor tissue. The perfusion decreased the uptake selectively in the normal tissues, resulting in higher tumor-to-organ ratios. This indicates the contribution of the blood pool to the high radioactivity values especially in the highly perfused organs lung and kidney. Consequently, these tissues showed the greatest decrease after perfusion, although the radioactivity level in the tumor remained almost constant. The perfusion experiment also suggests specific uptake into tumor tissue and only unspecific accumulation in the other organs. Although the perfusion has no therapeutic relevance, it reveals peptide characteristics necessary to generate a stable and more active compound. The high uptake in the kidneys can be explained by renal excretion of the peptide. The elevated blood values might be explained by various mechanisms. One possibility is the interaction of p160 with serum proteins such as albumin. Another possibility could be the rapid degradation of 131I-labeled p160, which might lead to labeled peptide fragments that are unable to bind to the tumor but circulate in the bloodstream. The instability of p160 is not uncommon. Peptides, identified through phage display technology, are presented at the surface of filamentous bacteriophages and are therefore protected against proteolysis. The chemically synthesized linear peptides are not shielded by the macromolecular phage, which might result in reduced serum stability (25). Therefore, one major issue of further investigation is the stabilization of p160. In this respect, different methods can be used. One of those is the cyclization of the peptide through a disulfide bridge between two conjugated cysteine molecules at the NH2- and COOH-terminal of p160, whereas a second method is the exchange of amino acids with unnatural amino acids that cannot be recognized by serum proteases, like D-amino acids or N-methylated amino acids. PEGylation is another modification that could be exploited. PEGylation has been used successfully to prolong peptide half-life in vivo (26). The use of peptides coupled to liposomes or other carriers might prove to be favorable in two ways: the large liposome particle might protect the peptide against degradation, as in the case of the phage-bound peptide, and the peptide could facilitate specific accumulation of the particle in the tumor (27).

The biodistribution of p160 was compared with the biodistribution of the RGD-4C peptide. The three–amino acid motif RGD is known to bind integrins (28, 29). The RGD-4C peptide was compared with p160 in this study for two reasons. First, the MDA-MB-435 cells are known to express {alpha}vß3 integrins on their surface and are potential targets for RGD peptides (30). Second, the RGD-4C peptide was found to accumulate to integrin-expressing tumors using in vivo phage display (31). The comparison revealed a similar organ distribution of p160 and the RGD-4C peptide, except for a higher liver and kidney accumulation of RGD-4C. It has been shown that the pharmacokinetics of RGD peptides can be improved in several ways. Particularly, glycosylation of a cyclic RGD peptide resulted in significantly reduced liver uptake and increased tumor accumulation (32). Coupling of the chelator diethylenetriaminepentaacetic acid to RGD caused a shift from predominantly liver clearance to renal clearance (33). These and other modifications led to the development of attractive RGD peptides for tumor therapy and can be used as model for further development of p160. However, it should be noted that despite its rapid degradation the unmodified p160 reached similar uptake values as RGD-4C and may therefore have great potential in further development. To allow use of p160 as an tumor imaging agent, certainly, the radioactivity level in the blood needs to be reduced. A first step will be the addition of a chelator to the peptide to allow labeling with radiometals. Chelated radiometal complexes are usually very firmly attached; therefore, the noise caused by deiodination events will be eliminated. In addition, the interaction of p160 with serum proteins has to be analyzed to assess a possible depot effect. An improvement of peptide stability will allow to take advantage of the rapid blood clearance typical for small peptides.

In conclusion, the peptide p160 seems to be a promising candidate for tumor imaging and cancer therapy. The uptake experiments in vitro show that the binding of the radiolabeled peptide to the breast cancer cells might be mediated through a specific receptor, whereas the organ distribution in tumor-bearing mice showed a higher binding to the tumor than to the other organs, which is favorable for cancer treatment.


   Acknowledgments
 
We thank Q. Wang for her contribution to the in vitro experiments, H. Eskerski, U. Schierbaum, and K. Leotta for their help with the biodistribution experiments, and S. Peschke for her contribution to the cell culture.


   Footnotes
 
Grant support: Deutsche Forschungsgemeinschaft grants HA2901/2-1 and 2-2.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: K. Graham is currently at Schering AG, Berlin, Germany.

Received 2/25/05; revised 5/31/05; accepted 6/16/05.


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  1. Rakhmilevich AL, Hooper AT, Hicklin DJ, Sondel PM. Treatment of experimental breast cancer using interleukin-12 gene therapy combined with anti-vascular endothelial growth factor receptor-2 antibody. Mol Cancer Ther 2004;3:969–76.[Abstract/Free Full Text]
  2. Toi M, Takada M, Bando H, et al. Current status of antibody therapy for breast cancer. Breast Cancer 2004;11:10–4.[Medline]
  3. Takahashi M, Inoue K, Goto R, et al. Metastatic breast cancer of HER2 scored 2+ by IHC and HER2 gene amplification assayed by FISH has a good response to single agent therapy with trastuzumab: a case report. Breast Cancer 2003;10:170–4.[Medline]
  4. Colcher D, Goel A, Pavlinkova G, Beresford G, Booth B, Batra SK. Effects of genetic engineering on the pharmacokinetics of antibodies. Q J Nucl Med 1999;43:132–9.[Medline]
  5. Zitzmann S, Ehemann V, Schwab M. Arginine-glycine-aspartic acid (RGD)-peptide binds to both tumor and tumor-endothelial cells in vivo. Cancer Res 2002;62:5139–43.[Abstract/Free Full Text]
  6. Chen X, Hou Y, Tohme M, et al. Pegylated Arg-Gly-Asp peptide: 64Cu labeling and PET imaging of brain tumor {alpha}vß3-integrin expression. J Nucl Med 2004;45:1776–83.[Abstract/Free Full Text]
  7. Haubner R, Wester HJ, Weber WA, et al. Noninvasive imaging of {alpha}(v)ß3 integrin expression using 18F-labeled RGD-containing glycopeptide and positron emission tomography. Cancer Res 2001;61:1781–5.[Abstract/Free Full Text]
  8. Pasqualini R, Koivunen E, Kain R, et al. Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis. Cancer Res 2000;60:722–7.[Abstract/Free Full Text]
  9. Janssen ML, Oyen WJ, Dijkgraaf I, et al. Tumor targeting with radiolabeled {alpha}(v)ß(3) integrin binding peptides in a nude mouse model. Cancer Res 2002;62:6146–51.[Abstract/Free Full Text]
  10. Landon LA, Deutscher SL. Combinatorial discovery of tumor targeting peptides using phage display. J Cell Biochem 2003;90:509–17.[CrossRef][Medline]
  11. Pasqualini R, Ruoslahti E. Organ targeting in vivo using phage display peptide libraries. Nature 1996;380:364–6.[CrossRef][Medline]
  12. Poul MA, Becerril B, Nielsen UB, Morisson P, Marks JD. Selection of tumor-specific internalizing human antibodies from phage libraries. J Mol Biol 2000;301:1149–61.[CrossRef][Medline]
  13. Barry MA, Dower WJ, Johnston SA. Toward cell-targeting gene therapy vectors: selection of cell-binding peptides from random peptide-presenting phage libraries. Nat Med 1996;2:299–305.[CrossRef][Medline]
  14. Zhang J, Spring H, Schwab M. Neuroblastoma tumor cell-binding peptides identified through random peptide phage display. Cancer Lett 2001;171:153–64.[CrossRef][Medline]
  15. Wellings DA, Atherton E. Standard Fmoc protocols. Methods Enzymol 1997;289:44–67.[CrossRef][Medline]
  16. Crim JW, Garczynski SF, Brown MR. Approaches to radioiodination of insect neuropeptides. Peptides 2002;23:2045–51.[CrossRef][Medline]
  17. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 1973;52:2745–56.
  18. Okarvi SM. Synthesis, radiolabeling and in vitro and in vivo characterization of a technetium-99m-labeled {alpha}-M2 peptide as a tumor imaging agent. J Pept Res 2004;63:460–8.[CrossRef][Medline]
  19. Garcia-Garayoa E, Allemann-Tannahill L, Blauenstein P, et al. In vitro and in vivo evaluation of new radiolabeled neurotensin(8-13) analogues with high affinity for NT1 receptors. Nucl Med Biol 2001;28:75–84.[CrossRef][Medline]
  20. Kuhnast B, Bodenstein C, Haubner R, et al. Targeting of gelatinase activity with a radiolabeled cyclic HWGF peptide. Nucl Med Biol 2004;31:337–44.[CrossRef][Medline]
  21. Buzdar AU, Singletary SE, Theriault RL, et al. Prospective evaluation of paclitaxel versus combination chemotherapy with fluorouracil, doxorubicin, and cyclophosphamide as neoadjuvant therapy in patients with operable breast cancer. J Clin Oncol 1999;17:3412–7.[Abstract/Free Full Text]
  22. Slamon DJ, Godolphin W, Jones LA, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 1989;244:707–12.[Abstract/Free Full Text]
  23. Rait AS, Pirollo KF, Xiang L, Ulick D, Chang EH. Tumor-targeting, systemically delivered antisense HER-2 chemosensitizes human breast cancer xenografts irrespective of HER-2 levels. Mol Med 2002;8:475–86.[Medline]
  24. Jhaveri MS, Rait AS, Chung KN, Trepel JB, Chang EH. Antisense oligonucleotides targeted to the human {alpha} folate receptor inhibit breast cancer cell growth and sensitize the cells to doxorubicin treatment. Mol Cancer Ther 2004;3:1505–12.[Abstract/Free Full Text]
  25. Jain RK. Delivery of molecular and cellular medicine to solid tumors. J Control Release 1998;53:49–67.[CrossRef][Medline]
  26. Lee SH, Lee S, Youn YS, et al. Synthesis, characterization, and pharmacokinetic studies of PEGylated glucagon-like peptide-1. Bioconjug Chem 2005;16:377–82.[CrossRef][Medline]
  27. Ichikawa K, Hikita T, Maeda N, et al. Antiangiogenic photodynamic therapy (PDT) by using long-circulating liposomes modified with peptide specific to angiogenic vessels. Biochim Biophys Acta 2005;1669:69–74.[Medline]
  28. Pfaff M, Tangemann K, Muller B, et al. Selective recognition of cyclic RGD peptides of NMR defined conformation by {alpha}IIbß3, {alpha}Vß3, and {alpha}5ß1 integrins. J Biol Chem 1994;269:20233–8.[Abstract/Free Full Text]
  29. Ruoslahti E. RGD and other recognition sequences for integrins. Annu Rev Cell Dev Biol 1996;12:697–715.[CrossRef][Medline]
  30. Wong NC, Mueller BM, Barbas CF, et al. {alpha}v Integrins mediate adhesion and migration of breast carcinoma cell lines. Clin Exp Metastasis 1998;16:50–61.[CrossRef][Medline]
  31. Arap W, Pasqualini R, Ruoslahti E. Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science 1998;279:377–80.[Abstract/Free Full Text]
  32. Haubner R, Wester HJ, Burkhart F, et al. Glycosylated RGD-containing peptides: tracer for tumor targeting and angiogenesis imaging with improved biokinetics. J Nucl Med 2001;42:326–36.[Abstract/Free Full Text]
  33. van Hagen PM, Breeman WA, Bernard HF, et al. Evaluation of a radiolabelled cyclic DTPA-RGD analogue for tumour imaging and radionuclide therapy. Int J Cancer 2000;90:186–98.[CrossRef][Medline]



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