Unfortunately, when imaging or treating tumors, the compounds used (e. g. , tracers, chemotherapeutic agents or radionuclides) are unspecifically distributed in the whole organism merely due to their chemical characteristics. However, e. g. , for diagnosis of a tumor and potential metastasis specific imaging only of the tumor/metastases is desirable and a prerequisite for selective medical intervention. Thus, the coupling of a tumor specific marker to a tracer is required, otherwise tumor imaging cannot be achieved. In case of tumor therapy, the unspecific distribution of the therapeutic agent, e. g., chemotherapeutic drug, in the organism requires the application of high amounts of the drug in order to achieve a sufficiently high concentration within the organ to be treated resulting in severe side effects and damages of healthy tissue. Nevertheless, often the concentration of the therapeutic drug within the desired organ is quite low, thus, not all tumor cells are killed and an effective treatment is prevented. In order to overcome this problem, for tumor imaging several alternative approaches were tried, e. g. , use of tumor specific antibodies (e. g. , directed against PSA or CEA), use of unspecific tracers like MIBI or use of tracers allowing to record tumor metabolism (e. g. , fluorodeoxyglucose) or use of peptides specific for the expression of tumor receptors (e. g. , Octreotid for recording somatostatin receptors). Unfortunately, PET using fluorodeoxyglucose gives with some kinds of tumors (e. g. , prostate carcinoma) only a very low accumulation and, thus its application for such kind of tumors is limited. Unfortunately, the approaches for tumor imaging discussed above exhibit a variety of additional disadvantages, e. g. , undesired immune responses of the patients when using humanized antibodies, slow accumulation of the antibodies due to their large size etc. Octreotid is only useful for tumors showing expression of SSTR (e. g., neuroendocrine tumors, meningiomas, small cell lung carcinomas).

Therefore, it is the object of the present invention to provide a means for the efficient, reliable and specific imaging of tumors and metastasis as well as therapy which overcomes the disadvantages of the diagnostic and therapeutic approaches presently used.

According to the present invention this is achieved by the subject matters defined in the claims.

A powerful technique utilizing phage display peptide libraries has been used that allows for the selection of peptide sequences with desired binding specifities. In this phage system, 109 different peptides motifs are expressed on the phage surface as peptides fused to phage surface proteins. The desired peptide is selected on its binding to the target cell.

By using this approach, a novel peptide, DUP-1 (aa sequence FRPNRAQDYNTN) could be isolated showing specific binding to tumor cells, i. e. , it accumulates in the body at/in tumors.

The synthesized peptide showed rapid binding kinetics, specific competition with unlabeled DUP-1 and time-dependent internalization into DU-145 cells. Biodistribution studies using radiolabeled DUP-1 in nude mice with s. c. transplanted DU-145 and PC-3 cells demonstrated a tumor accumulation of 5 and 7% ID/g in DU-145 and PC-3 tumors, respectively.

Therefore, it is generally possible to visualize tumors and metastasis through use of a peptide of the present invention when coupled to a compound that is detectable upon imaging.

Moreover, the peptide of the invention when coupled to a compound exerting a therapeutic effect, e. g. , a cytotoxin, can be used for tumor therapy. 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. The peptide of the invention also allows to evaluate the dynamics of tumor progression in a patient and to monitor the effect of therapy.

As shown in the experiments, below, this new DUP-1 lead structure shows 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.

The peptide DUP-1 contains a motif which facilitates binding to different prostate carcinoma cell lines and, presumably, further carcinoma cells but not to a benign prostate cell line or to HUVEC cells. 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/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 min p. i. , respectively. With a tumor/prostate ratio of about 3 at 15 min, DUP-1 is a promising molecule for the diagnosis of suspected prostate carcinoma. The tumor cell affinity of DUP-1 was also supported by perfusion experiments with animals bearing subcutaneously transplanted DU-145 and PC-3 tumors. Since no binding to primary cultures of endothelial cells was observed in vitro it was assumed that the peptide is able to penetrate through the basal membrane, followed by direct binding to the tumor cells. This hypothesis was sustained by the biodistribution data obtained with the perfused animals showing that most organs display reduced radioactivity levels as compared to the unperfused animals while 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 min for DU-145 and PC-3 was 5 and 7 % ID/g, respectively. This is i. e. significantly higher compared with 3.65 % ID/g delivered into tumors of the MDA-MB 435 xenograft model with 125I-RGD.

In vitro a rapid internalization of FITC-Lys-DUP-1 was observed. The experimental settings used in the pulse-chase experiment (PCE) revealed a specific internalization into cells, since unspecifically 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 min the size of the vesicles increased, indicating a fusion of the endocytotic vesicles to endosomes. This internalization is extremely useful for both imaging and potential therapeutic applications of DUP-1 derivatives.

Although the binding site is unknown, it is unlikely that DUP- 1 targets PSMA, since DU-145 as well as PC-3 cells are PSMA- negative. 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 LH-RH nor to any other peptide or protein sequence available as confirmed by a search in different protein databases such as EMBL, SwissProt and others.

In conclusion, due to its high and specific binding to prostate carcinoma cells in vitro as well as in vivo together with rapid internalization, DUP-1 represents a promising structure useful for diagnosis and treatment of cancer, in particular prostate cancer. DUP-1 or parts of it may be used for coupling with radioactive isotopes, anticancer agents or even for the modification of the envelope of virus particles such as AAV to obtain tumor specific infection.

Therapy/imaging of tumors or metastasis by use of the peptide of the invention exhibits several advantages: (a) Large amounts of peptides can be easily prepared; (b) they do not elicit undesired immune responses; (c) they can be used in combination with already approved drugs and compounds; (d) the peptide targets the tumor tissue specifically without affecting normal tissue. Thus, in therapy side effects of therapeutic compounds coupled to the peptide are minimized.

Accordingly, high concentrations of the therapeutic compound at the site of the tumor can be applied (resulting in efficient killing of the tumor) and at the same time the poisoning of the normal tissue can be reduced; and (e) as regards the diagnostic aspect, the location and size of the tumor/metastasis can be exactly determined, thus, allowing a more precise planning and carrying out of therapy.

Accordingly, the present invention relates to a peptide with selective binding to tumor cells, wherein said peptide comprises a peptide having the amino acid sequence FRPNRAQDYNTN or an amino acid sequence which differs from the amino acid sequence FRPNRAQDYNTN by one or more conservative amino acid substitutions.

The term"selective binding to tumor cells"as used herein means that, on the one hand, the peptide binds to tumor cells with a stability that is sufficient for therapeutic or diagnostic purposes and, on the other hand, does not bind to non-tumor cells or binds to the non-tumor cells to a substantially lesser extent compared to tumor cells.

The term"conservative amino acid substitutions"involves replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile ; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

Preferably, the peptide of the invention is characterized by not more than 8 conservative aa substitutions, more preferably by not more than 6 conservative aa substitutions and, even more preferably, by not more than 4 conservative aa substitutions.

The present invention also relates to a peptide with selective binding to tumor cells, wherein said peptide comprises (a) a peptide having an amino acid sequence which shows at least 60%, preferably at least 80%, more preferably 85% and even more preferably 90% identity to the amino acid sequence FRPNRAQDYNTN without losing its capability of selective binding to a tumor; or (b) a peptide which is a fragment of the peptides discussed above.

For the generation of peptides showing a particular degree of identity to the peptide DUP-1, e. g. , genetic engineering can be used to introduce amino acid changes at specific positions of a cloned DNA sequence to identify regions critical for peptide function. For example, site directed mutagenesis or alanine- scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. (Cunningham and Wells, Science 244 (1989), 1081-1085) The resulting mutant molecules can then be tested for biological activity using the assay of Example 2.

In the fragment of the peptide of the invention at least 3 contiguous aa, preferably at least 5 contiguous aa, more preferably at least contiguous 7 aa and even more preferably at least 9 contiguous aa of the amino acid sequence FRPNRAQDYNTN are left. The fragment is still capable of selective binding to a tumor cell. For evaluating whether a particular fragment (or derivative of the peptide DUP-1 characterized by substitutions of conservative amino acids) is still capable of selective binding of. tumor cells the assays of the Examples can be used.

In order to optimize the in vivo stability of the peptides of the invention by simultaneously maintaining the binding characteristics, derivatisation is expected to result in better target/non-target ratios. Among the preferred structural variations are sequence fragmentation, cyclization, generation of multimers, e. g. , dimers or trimers, D-amino acid substitution, N-or C-terminal end modifications (Hoffmann et al. , J. Nucl. Med. 44 (2003), 823-831 ; Dasgupta and Mukherjee, Br. J. Pharmacol. 129 (2000), 101-109; de Jong et al., Cancer Res. 58 (1998), 437-441), or methylation. These modifications should result in enhanced stability as well as in reduced binding to plasma proteins. D-amino acid substitutions are preferably made by substituting non-essential L-amino acids against the respective D-amino acids. This leads to a surprising enhanced stability of the whole molecule.

A further embodiment of the invention relates to peptides described above which are operatively attached to a therapeutic agent capable of exerting a therapeutic effect on a tumor. Examples of therapeutic agents useful for the present invention include an anticellular agent, chemotherapeutic agent, a radioisotope, a chelator for the binding of certain radioisotopes or a cytotoxin. Preferred cytotoxins are an A chain toxin, a ribosome inactivating protein, a-sarcin, aspergillin, restrictotin, diphtheria toxin, Pseudomonas toxin, a bacterial endotoxin or the lipid A moiety of a bacterial endotoxin. Further suitable proteins for tumor therapy are endostatin (for inhibition of tumor growth) or recombinant chimeric toxin PE37/transforming growth factor alpha (TGF-alpha) (for cytotoxicity to tumor cells).

The present invention also relates to polynucleotides encoding a peptide of the invention, preferably fused to secretory leader sequences, as well as expression vectors which are capable of expressing the peptide of the invention and which can be used for gene therapy. Preferred recombinant vectors useful for gene therapy are viral vectors, e. g. adenovirus, herpes virus, vaccinia, or, more preferably, an RNA virus such as a retrovirus. Even more preferably, the retroviral vector is a derivative of a murine or avian retrovirus. Examples of such retroviral vectors which can be used in the present invention are: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV) and Rous sarcoma virus (RSV). Most preferably, a non- human primate retroviral vector is employed, such as the gibbon ape leukemia virus (GaLV), providing a broader host range compared to murine vectors. Since recombinant retroviruses are defective, assistance is required in order to produce infectious particles. Such assistance can be provided, e. g. , by using helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. Suitable helper cell lines are well known to those skilled in the art.

Said vectors can additionally contain a gene encoding a selectable marker so that the transduced cells can be identified. Moreover, the retroviral vectors can be modified in such a way that they become target specific. This can be achieved, e. g. , by inserting a polynucleotide encoding a sugar, a glycolipid, or a protein, preferably an antibody.

Those skilled in the art know additional methods for generating target specific vectors. Further suitable vectors and methods for in vitro-or in vivo-gene theapy are described in the literature and are known to the persons skilled in the art; see, e. g. , WO 94/29469 or WO 97/00957.

In a further embodiment, the present invention relates to a pharmaceutical composition containing a peptide, polynucleotide or expression vector of the invention. For administration the compounds of the pharmaceutical composition are preferably combined with suitable pharmaceutical carriers.

Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e. g. by intravenous, intraperetoneal, subcutaneous, intramuscular, topical or intradermal administration. The route of administration, of course, depends on the nature and location of the tumor and the kind of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors. As is well known in the medical arts, dosages for any one patient depends on many factors, including the patient's size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind and stage of the tumor, general health and other drugs being administered concurrently.

Preferred pharmaceutical carriers include colloidal dispersion systems which are coupled to the peptides of the invention useful for targeting the systems to the desired sites. The colloidal dispersion systems which may be used include macromolecule complexes, nanocapsules, microspheres, beads and lipid-based systems including oil-in-water emulsions, (mixed) micelles, liposomes and lipoplexes. The preferred colloidal system is a liposome. The composition of the liposome is usually a combination of phospholipids and steroids, especially cholesterol. The skilled person is in a position to select such liposomes which are suitable for the delivery of the desired peptide. Organ-specific or cell-specific liposomes can be used in order to achieve delivery only to the desired tissue, e. g., tumor. The targeting of liposomes by the peptides of the invention can be carried out by the person skilled in the art by applying commonly known methods.

An alternative embodiment of the present invention relates to an above disclosed peptide which is linked to a diagnostic agent such as a chelator that is detectable upon imaging.

Thus, such a peptide is useful fur tumor diagnosis by imaging, e. g. , magnetic resonance imaging (MRI), PET etc.

In a preferred embodiment of the present invention, said diagnostic agent is a paramagnetic ion, radioactive ion or a fluorogenic ion. Specifically, the diagnostic agents utilized in embodiments of the invention include: chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), erbium (III), <BR> <BR> <BR> 123 9m 88<BR> ,, indium, rhenium, rhenium,<BR> <BR> <BR> <BR> copper I iodine'yttrium iodine astatine Lutetium and gallium The present invention also relates (a) to the use of a peptide, polynucleotide or expression vector of the invention for the preparation of a medicament for the treatment of a tumor and (b) to the use of a peptide of the present invention for the preparation of a diagnostic composition for tumor imaging.

Preferred tumors that can be treated/diagnosed by the compounds of the present invention are prostate tumors, thyroid carcinomas, breast cancer and neuroblastoma.

The present invention is further explained by the following figures: Figure 1 (a) Amino acid sequence of the DU-145 binding peptide DUP-1 isolated by peptide phage display (b) In vitro binding assay with DUP-1 DU-145, PC-3, HUVEC and PNT-2 were grown for 24h. 125I-DUP-1 was added to the wells and incubated for 10 min. - : no competitor was added. + : 5#10-5 M unlabeled DUP-1 as competitor was added 30 min before incubation with labeled DUP-1. Experiments were performed in triplicate, standard deviations are shown.

Figure 2: Binding of 125I-DUP-l to human tumor cell lines and primary endothelial cells JMR5-75 (neuroblastoma cells), MDA-MB435 cells (breast cancer), DU-145 and PC-3 (both prostate cancer), SW1736 and NPA-87 (both thyroid cancer), and HUVEC (primary endothelial cells) were grown for 24 hours. l25I-DUP-l was added to the wells and incubated for 10 min.- : no competitor was added. + : 5x10-5 M unlabeled DUP-1 as competitor was added 30 min before incubation with labeled DUP-1. Experiments were performed in triplicate, standard deviations are shown.

Figure 3 : In vitro competition assay with DUP-1 using various competitor concentrations DU-145 cells (a) and PC-3 (b) were grown for 24 h. Unlabeled DUP-1 in concentrations ranging from 10-4 M to 10-1l M were added to the cells 30 min before 125I-DUP-l and incubated for 10 min. Experiments were performed in triplicate, standard deviations are shown.

Figure 4 : In vitro binding kinetics of DUP-1.

DU-145 cells (a) and PC-3 (b) were grown for 24 h. 125I-DUP- was added and incubated for time points of 1 min, 5 min, 10 min, 20 min, 30 min, 60 min, 120 min and 240 min.

Figure 5: (a) Fluorescence microscopy with FITC-Lys-DUP-1 (10-5 M) (green) (I) after 10 min incubation (400x) (II) after 60 min incubation (600x) (b) PCE with 10 min preincubation of FITC-Lys-DUP-1 (5x10-5 M) (green) followed by incubation with dextran-Alexa568 (5x10-5 M) (red) for 0 min, 10 min, 30 min, 60 min and visualization by confocal microscopy White arrows show yellow vesicles.

Figure 6: Biodistribution of DUP-1 in male Balb/c nu/nu mice carrying (a) DU-145 tumors (n = 9 animals per time point) or (b) PC-3 tumors (n = 3 animals per time point) The animals were injected i. v. with I-DUP-1 and the radioactivity was measured in tumor and control organs after 5 min, 15 min, 45 min and 135 min.

(c) Male Balb/c nu/nu mice carrying DU-145 tumors (n = 6 animals per time point) injected i. v. with 13lI-DUP-l After the time-points of 5 min and 15 min the animals were perfused and the radioactivity of tumor and control organs was determined.

(d) Male COP rats (n = 3 animals per time point) bearing a Dunning R3327 subline AT1 tumor were injected i. v. with 13lI- DUP-1 After the time-points of 5 min and 15 min the animals were sacrificed and the radioactivity in various organs was measured. Standard error of the mean (S. E. M. ) is shown.

§n = 3 animals The present invention is explained by the following examples.

Example 1: Materials and Methods (A) Cell lines The human prostate cancer cell lines Du-145 and PC-3, the human thyroid carcinoma cell line SW1736, the breast cancer cell line MDA MB435, the neuroblastoma cell line IMR5-75 as well as the human embryonic kidney cell line 293 were cultivated at 37°C in a 5% C02-incubator in RPMI 1640 with Glutamax containing 10% FCS (both Invitrogen, Karlsruhe, Germany) and 25 mM HEPES. The human thyroid cancer cell line NPA-87 was cultivated in RPMI 1640 supplemented with 0. 75% sodium bicarbonate, 1.4 mM sodium pyruvate and 1.4 x non- essential amino acid solution (all Invitrogen, Karlsruhe, Germany). Dunning R3327 subline AT1 (ATCC, Manassas, VA, USA) tumors cells were cultivated in RPMI 1640 (Gibco BRL, Eggenstein, Germany) supplemented with 292 mg/1 glutamine, 100 IU/ml penicillin, 100 mg/1 streptomycin and 10% FCS. HUVEC (human umbilical vein endothelial cell) were isolated as described by Jaffe et al., J. Clin. Invest. 52: 2745-2756 (1973).

Cultivation was performed on gelatine (1%) coated cell culture flasks using medium 199 (Invitrogen, Karlsruhe, Germany) containing 20% FCS, 2 mM glutamine, 100 IU/ml penicillin, 100 IU/ml streptomycine and 2 ng bFGF/ml (Roche Diagnostics, Mannheim, Germany).

(B) 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, USA). Each selection round was conducted as follows: 1011 TU were added to 293-cells for a negative selection. After 1 h the medium was collected, centrifuged for 5 min at 1500 rpm and the supernatant was transferred to DU-145 cells grown to 90% confluence. After 1 h the cells were washed 4 x with 10 ml of HBSS (+) (Invitrogen, Karlsruhe, Germany) + 1% BSA and 4 x with 10 ml of HBSS (-) (Invitrogen, Karlsruhe, Germany) + 1% BSA.

The cells were then detached with 4 ml of (PBS + 1 mM EDTA for 5 min and centrifuged for 5 min at 1500 rpm. The cell pellet was washed 3 times in 1 ml HBSS (-) + 1% BSA and lysed with 1% Triton X-100.10 ßl of the lysate was used for titering of the phages. The remaining lysate was amplified in 50 ml ER2537 bacteria according to the manufacturer's protocol. For the next selection round 1011 TU from the previous selection round were used. Six selection rounds were performed, followed by single stranded DNA isolation from clones (QIAprep Spin M13 Kit, QIAGEN, Germany). The peptide was identified by sequencing.

(C) Animals and tumor growth Male 6-week-old BALB/c nu/nu mice and the male Copenhagen rats with a weight of 220-250 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 (5x106 cells) was injected subcutaneously into the anterior region of the mouse trunk. Tumors were grown up to a size of approximately 1.0 cm3. The rat prostate adenocarcinoma Dunning R3327 subline AT-1 (ATCC) was transplanted subcutaneously 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.

(D) 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 (Eisenhut and Mier, Radioiodination chemistry and radioionated compounds. In: Vertes, Nagy, Klencsár (ed. ), Handbook of Nuclear Chemistry, Vol. 4, pp. 257-278: Kluver Academic Publishers (2003). For fluorescence microscopy FITC was coupled via an additional lysine at the C-terminus.

(E) In vitro binding experiments 200,000 cells were seeded into 6-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 M-10-11 M) was preincubated for 30 min. 125,-labeled peptide was added to the cell culture (1-2 X 106 cpm/well) and incubated for the appropriate incubation times varying from 1 min to 4 h. The cells were washed three times with 1 ml PBS and subsequently lysed with 0.5 ml 0.3 M NaOH. Radioactivity was determined with a gamma counter and calculated as percent applied dose per 106 cells.

(F) Conventional and confocal laser scanning microscopy using FITC-labeled DUP-1 DU-145 cells were seeded subconfluently onto cover slips and cultivated for 24 hours. The medium was replaced by fresh medium (without FCS). For microscopy FITC-Lys-DUP-1 (10-5 M) was added to the medium and incubated for 10 and 60 min at 37°C. Subsequently, the cells were washed with 1 ml medium and fixed with 2% formaldehyde for 20 min on ice. For the pulse- chase experiment with confocal laser scanning microscopy 5 x 10-4 M FITC-Lys-DUP-1 was added to the medium for 10 min. The cells were washed 3 times with 1 ml PBS and incubated with 1 ml fresh medium containing 5 x 10-5 M dextran-Alexa568 (10,000 MW, fixable, Molecular Probes, Eugene, USA) for time points from 10 min to 60 min. Subsequently, the cells were washed, fixed and incubated with TO-PRO-3 (Molecular Probes, Eugene, USA, 1: 1000 dilution, 20 min) for cell nucleus staining. Then, the cells were analyzed using a Leica SP1 CLSM (Leica Microsystems Heidelberg, Mannheim, Germany).

(G) Organ distribution with radioiodinated DUP-1 3lI-DUP-l was intravenously injected into male nu/nu mice (2. 8x107 cpm/mouse), carrying the subcutaneously transplanted the human prostate tumors DU-145 or PC-3. At 5,15, 45 and 135 min post injection the mice were sacrificed. The organs were removed, weighed and the radioactivity was determined using an automated NaI (Tl) well counter (CobraII, Canberra Packard, Meriden, USA). The percentage of injected dose per gram 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 25 ml 0.9 % NaCl, tumor and control organs were removed and weighed. For the biodistribution in male COP rats bearing Dunning R3327 subline AT1 tumors, 231I-DUP-l (5 X 107 cpm/rat) was intravenously injected and the animals were sacrificed after 5 min and 15 min, the organs were removed and weighed.

Example 2: Selection of a peptide specifically binding to tumor cells For the selection of tumor specific peptides, 6 selection rounds, so called biopannings, were perfomed on DU-145 prostate carcinoma cells. The phage display library used was the Ph. D. 12 phage library from New England Biolabs. Each biopanning was conducted as follows: For the first round 5 41 of the original library (approx.

5x101°TU/ml (TU = transducing units),-109 different peptide motifs) were added to a cell culture dish with 293-cells (embryonal kidney cells) for a negative selection. After 1 h the medium was collected from the 293 cells, centrifuged for 5 min at 1500 rpm and the supernatant was transfered into a dish with DU-145 cells grown to 90% confluency. After 1 h the medium was removed from the DU-145 cells and the cells were washed 4 x with 10 ml of HBSS (+) + 1 % BSA and 4x with 10 ml of HBSS (-) + 1 % BSA. The cells were then detached with 4 ml of Versene for 5 min and resuspended in 16 ml RPMI 1640 medium and centrifuged 5 min at 1500 rpm. The cell pellet was resuspended in 1 ml HBSS (-) + 1 % BSA into an Eppendorf tube, centrifuged 5 min at 1500 rpm and the supernatend was removed.

This wash step was repeated 3 times. Then the cell pellet was lysed with 1 % Triton. 10 ul of the lysate was used for titering. The remaining lysate was added to 50 ml of a log culture of ER2537 bacterial cells and grown 5 h for amplification. The amplified phages were precipitated with PEG (see manufacturer's protocol) and the titer of the amplified phage suspension was determined. For the next biopanning 1011 TU from the previous biopanning were used. 6 biopanning were performed, then single phages clones were amplified and single stranded DNA was isolated for sequencing. From the 24 phages sequenced all phages carried the same peptide: FRPNRAQDYNTN (Figure la).

Example 3: Binding of the peptide DUP-1 on various cell types The peptide DUP-1 (FRPNRAQDYNTN) was prepared by chemical synthesis and labelled with 125I. For in vitro studies, 200,000 cells (DU-145, human prostate tumor cell line; PC-3, human prostate cell line; HUVEC, human primary endothelial cells, IMR5-75, human neuroblastoma cell line, MDA-MB-435, human breast cancer cell line, SW-1736, human thyroid cancer cell line, NPA-87, human thyroid cancer cell line) were seeded into 6-well plates and cultivated for 24 hours. Then, the medium was replaced by fresh medium (without FCS) and for the plates with competitor, unlabelled peptide was added (10-4 M) and preincubated for 30 min. Then, 125,-labelled peptide was added to the cell culture (1-2 mio cpm/well) and incubated for 10 min. The cells were collected and washed for three times with 1 ml PBS per washing. Then, the cells were lysed with 0.3 M NaOH. Radioactivity of the lysed cells was determined with a gamma counter and radioactivity as percent applied dose per 1 million cells was calculated. It could be shown that the labelled peptide specifically binds to the various tumor cells, but not the primary endothelial cells. Binding can be competitively inhibited (up to 95%). The results are presented in Figure 1b and Figure 2.

Unlabeled octreotide at the same concentration did not prevent l25I-DUP-l from binding (data not shown). The D-DUP-1 peptide, which contains the same amino acids but in their D- conformation instead of the L-conformation, did not bind to PC-3 cells (data not shown).

Example 4: Competition and binding kinetics of DKP1 to prostate carcinoma cells in vitro The peptide DUP-1 (FRPNRAQDYNTN) was prepared by chemical synthesis and labelled with 125,. 200,000 DU-145 cells (human prostate tumor cell line) were seeded into 6-well plates and cultivated for 24 hours. Then, the medium was replaced by fresh medium (without FCS).

To evaluate the inhibition and to determine the IC50-value, different competitor concentrations (unlabeled DUP-1) were added to the cells before the lzSl-labeled peptide was added.

Concentrations of 10-4 to 10-5 M inhibit greater than 95% of the binding of l25I-DUP-l to DU-145 cells (Figure 3a). At a competitor concentration of 10-8 to 10-9M the binding of DUP-1 was marginally enhanced by the presence of the competitor while at concentrations below 10-1° M the binding value reached the level of uncompeted binding again. The IC50 was calculated as 1,77 X 10-7 M. Similar results were obtained with PC-3 cells (Figure 3b). To evaluate the time course of peptide binding, 5I-labeled peptide was added to the cells and incubated for time points ranging from 1 min to 4 h. After the incubation the cells were lysed and the radioactivity as percent applied dose per 106 cells was calculated (Figure 4a and b). Binding of DUP-1 is rapid and the highest binding rate is reached after 5 min. Thereafter, the amount of bound peptide decreases and reaches a basal level of about 1.5 % applied dose/106 cells.

Example 5: Internalization of FITC-labeled DUP-1 To study the internalization process FITC-Lys-DUP-1 was added to the media and the cells were analyzed by microscopy (Figure 5a). Labeling of the cell membrane with FITC-Lys-DUP-1 was observed after 10 min incubation, whereas after 60 min incubation the peptide was intracellularly localized. To allow a more detailed analysis of the peptide localization, a pulse- chase-experiment was performed and analyzed by confocal microscopy. A 10 min pulse with 5 X 10-4 M 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 x 10-4 M dextran-Alexa568 (chase) (Figure 5b). Immediately after addition of the dextran dye, FITC-Lys-DUP-1 was found to remain bound to the cell membrane. 10 min later most of the peptide was still bound, but no internalized dextran was detected. 30 min after co-incubation of FITC-Lys-DUP-1 (green) and dextran (red), intracellularly localized yellow spots were seen indicating a co-localization of FITC-Lys-DUP-1 and dextran. At 30 and 60 min after incubation three types of spots were visible: i) red spots, showing internalized dextran-Alexa568, ii) green spots, demonstrating FITC-Lys-DUP-1 bound either intracellularly or to the cell membrane and, finally, iii) yellow spots, characterizing internalized vesicles which 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).

Example 6: Distribution of intravenously injected peptide DUP- 1 in various organs The peptide DUP-1 (FRPNRAQDYNTN) was prepared by chemical synthesis and labelled with 131I. 131I labelled peptide DUP-1 (FRPNRAQDYNTN) (28 mio cpm/mouse) was intravenously injected into male nu/nu mice, which subcutaneously carried a human prostate tumor (DU-145 or PC-3). 5 min. , 15 min. , 45 min and 135 min, respectively, postinjection the mice were sacrificed.

The organs were removed, weighed and the radioactivity was determined with a gamma counter. The results are presented in Figure 6a (DU-145 tumors) and Figure 6b (PC-3 tumors).

The biodistribution in DU-145 tumor carrying mice showed that DUP-1 accumulated after 5 min in the tumor to a level of approx. 5% ID/g (injected dose/g) which is higher than in the other organs, with the exception of kidney and blood (Figure 6a). This level was stable up to 45 min 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 min post injection (Figure 6b). The higher uptake in PC-3 tumors at 5 min. and the faster washout in PC-3 tumors at 135 min. were statistically significant in comparison with DU-145 with p < 0.05. To reduce blood background in various organs, animals carrying DU-145 tumors were perfused with NaCl (Figure 6c). Radioactivity was reduced in most organs while the tracer accumulation of 5% ID/g remained constant with or without perfusion in the tumor for 5 min and 15 min. This leads to an increase of most tumor-to-organ ratios as shown in table 1. At 5 min. lung, liver and muscle showed a statistically significant difference of unperfused to perfused organs with p < 0.05 and at 15 min. heart, lung and liver showed a statistically significant difference.

Table 1 tumor/organ ratio 5 min 15 min non-perfused perfused non-perfused perfused heart 1.75 2.12 1.78 2.79 lung 0.95 1.82 0.80 2.34 spleen 1.42 1.51 1.41 1.59 liver 1.89 2.80 1.75 3.61 kidney 0. 71 0. 83 0.88 0.84 muscle 2.70 2.06 2.15 3.06 brain 9.34 8.14 8.74 10.01 tumor 1. 00 1. 00 1. 00 1. 00 In order to demonstrate prostate binding of l3lI-DUP-l, biodistribution experiments were performed in Copenhagen rats bearing rat prostate adenocarcinoma Dunning R3327 subline AT-1 (Figure 6d). 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 to 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 min in comparison to normal prostate tissue. The tumor/muscle ratio for DU-145 tumors in mice was 3.02 at 15 min and the prostate/muscle ratio for the rats was 0.99 at 15 min p. i.