Fibronectin-binding peptides for use in tumor or fibrosis diagnosis and therapy

Abstract

The present invention is directed to a composition comprising at least one fibronectin binding polypeptide (FnBP) linked to at least one diagnostic or therapeutic agent, a nucleic acid encoding a fusion polypeptide comprising at least one fibronectin binding polypeptide (FnBP) linked to at least one diagnostic or therapeutic polypeptide agent as well as a corresponding recombinant vector and host cell comprising such a nucleic acid and preferably expressing said fusion polypeptide. The invention also relates to a kit of parts comprising at least one fibronectin binding polypeptide (FnBP), at least one diagnostic or therapeutic agent, and optionally one or more chemical agents for linking the fibronectin binding polypeptide (FnBP) to the diagnostic or therapeutic agent. In addition, the present invention intends said composition, nucleic acid, vector, host cell and kit for use in the therapeutic or prophylactic treatment of a disease, preferably a disease associated with abnormal fibronectin accumulation such as cancer, fibrosis or immune diseases.

Claims

1. A composition comprising at least one fibronectin binding polypeptide (FnBP) linked to at least one diagnostic or therapeutic agent, wherein the at least one FnBP is a polypeptide consisting of SEQ ID NO: 295.

2. The composition according to claim 1, wherein the FnBP binds specifically to at least one of fibronectin subunits FnI.sub.1-6, FnII.sub.1-2, FnI.sub.7-9 or FnIII.sub.7-15.

3. The composition according to claim 1, wherein the diagnostic agent is at least one of a radionuclide, magnetic resonance imaging (MRI) active compound, ultrasound contrast agent, fluorophore, positron emission tomography (PET) marker, single-photon emission computed tomography (SPECT) marker, fluorophore in the far red/near-infrared (IR) spectral region, Gd-based particle based MRI contrast agent, or Fe-oxide particle based MRI contrast agent.

4. The composition according to claim 1, wherein the therapeutic agent is at least one of a cytostatic agent, cytotoxic agent, cytokine, transcription factor inhibitor, proteasome inhibitor, protease inhibitor, apoptosis modulator, cell cycle modulator, angiogenesis inhibitor, hormone, hormone derivative, photodynamic therapy molecule, nanoparticles for thermoablation therapy, microparticles for thermoablation therapy, radionuclide, miRNA, siRNA, immunomodulatory antigen molecule, Doxorubicin, Paclitaxel, Chlorambucil, Topotecan, Vincristine, Interleukin-2, Interleukin-7, Interferon-γ, tumor necrosis factor, Curcumin, Ribavirin, Genistein, Imatinib, Erlotinib, Bryostatin, Flavopiridol, Roscovitine, Endostatin, Celexocib, ADH-1 (exherin), Sunitinib, Flutamide, Fosfestrol, Tamoxifen, Relaxin, .sup.64Cu, .sup.90Y, .sup.111In, .sup.131I, .sup.161Tb, .sup.169Er, .sup.177Lu, miRNAs specific for CD40, miRNA specific for CD80, miRNA specific for CD86, siRNAs specific for CD40, siRNA specific for CD80, siRNA specific for CD86, insulin-associated antigens, P31, whole gliadin, myelin oligodendrocyte glycoprotein, amino acids 35-55 of myelin oligodendrocyte glycoprotein (SEQ ID NO.: 297), proteolipid protein 1, amino acids 139-151 of proteolipid protein 1 (SEQ ID NO.: 298), amino acids 178-191 of proteolipid protein 1 (SEQ ID NO.: 299), Factor V, amino acids 75-89 of Factor V (SEQ ID NO.: 300), amino acids 1723-1737 of Factor V (SEQ ID NO.: 301, or amino acids 2191-2210 of Factor V (SEQ ID NO.: 302).

5. The composition according to claim 1, wherein the therapeutic agent is at least one of an antifibrotic agent, integrin inhibitor, bone morphogenic protein 7 (BMP-7), relaxin and relaxin-like peptides, lysyl oxidase (LOX) inhibitor beta-aminoproprionitrile (BAPN), or Interleukin-7 (IL-7).

6. The composition according to claim 1, wherein the therapeutic agent is at least one of an immune modulating agent, Interleukin 12, inhibitors that target the EGFR signalling cascade, myelin oligodendrocyte glycoprotein peptide sequence 35-55, a miRNA, an siRNA, PSA-TRICOM, Ipilimumab, anti-CTLA-4 antibody, anti-PD1 antibody, anti-PD-L1 antibody, or HDAC inhibitor.

7. A kit comprising the composition of claim 1.

8. The kit of claim 7, wherein the composition further comprises one or more chemical agents for linking the FnBP to the diagnostic or therapeutic agent.

9. A method for treating a subject suffering from a disease associated with pathological fibronectin accumulation, the method comprising: (a) providing the composition of claim 1, wherein the FnBP is linked to at least one therapeutic agent, and (b) administering the composition to the subject in need thereof, wherein the composition is effective for treating the disease associated with pathological fibronectin accumulation, wherein the disease is selected from the group consisting of fibrosis and cancer associated with pathological fibronectin accumulation.

10. A method for diagnosing a disease associated with pathological fibronectin accumulation in a subject, the method comprising: (a) providing the composition of claim 1, wherein the FnBP is linked to at least one diagnostic agent, (b) administering the composition to the subject in need thereof, and (c) identifying pathological fibronectin accumulation by detecting accumulation of the FnBP in said subject.

11. The method according to claim 9, wherein the disease is selected from the group consisting of pulmonary fibrosis, liver fibrosis, kidney fibrosis, breast cancer, and prostate cancer.

12. The method according to claim 10, wherein the disease is selected from the group consisting of fibrosis and cancer associated with pathological fibronectin accumulation.

13. The composition according to claim 5, wherein the relaxin peptide is selected from relaxin-1 and relaxin-2.

14. The composition according to claim 6, wherein the miRNA is selected from a miRNA specific for CD40, miRNA specific for CD80, and miRNA specific for CD86.

15. The composition according to claim 6, wherein the siRNA is selected from an siRNA specific for CD40, siRNA specific for CD80, and siRNA specific for CD86.

16. The method according to claim 12, wherein the disease is selected from the group consisting of pulmonary fibrosis, liver fibrosis, kidney fibrosis, breast cancer and prostate cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows binding of FnBPA5 peptide (solid black) to extracellular matrix protein fibronectin on different length scales. (A) Schematic representation of the modular structure of Fn consisting of three different types of modules with FnBPA5 binding to the FnI.sub.2-FnI.sub.5 domains close to the N-terminus of the Fn monomer. (B) Structure of FnBPA5 bound to FnI.sub.2-FnI.sub.5 via addition of an antiparallel beta-sheet to each FnI module. (C) Schematic representation of bacterial derived fibronectin binding peptide sequences (FnBPA5 (SEQ ID NO 295) and scrambled derivative (SEQ ID NO 296)).

(2) FIG. 2 shows in vitro binding studies of FnBPA5 and scrambled derivative to soluble and fibrillar Fn (A) Binding of FnBPA5 and scrambled derivative to manually pulled Fn fibers. Fn (left column) and peptide signal (right column) show specific peptide binding only for FnBPA5 and not for its scrambled derivative. (B) Quantification of FnBPA5 and scraFnBPA5 binding to Fn fibers showing a significantly higher binding of FnBPA5 compared to scrambled derivative. Data from 30 fibers from three independent experiments was analyzed. Shown error bars represent standard deviation, student-t-test was carried out to shown significance. (C) Fibroblast ECM was stained for fibronectin, FnBPA5 or scraFnBPA5, actin and cell nuclei. Representative images show specific binding of FnBPA5 to Fn whereas scraFnBPA5 unspecificly attached to the matrix. (D) Binding of FnBPA5 and scrambled FnBPA5 to soluble plasma Fn using anisotropy measurement, showing a K.sub.d of 75 nM for FnBPA5 and no specific binding of scrambled derivative. (E) Measurement of affinity of FnBPA5 to manually pulled relaxed Fn fibers, showing a K.sub.d of 28 nM.

(3) FIG. 3 shows SPECT/CT images of mice bearing PC-3 xenografts 96 hours post injection. Mice were injected with 12 MBq .sup.111In-FnBPA5 (A and C) respectively .sup.111In-scrambled FnBPA5 (B and D). Images were acquired post mortem 96 hours post injection. In A and B dominant kidney uptake of both FnBPA5 and scrambled control indicate unspecific clearance via the kidney. The body kidneys were removed in C and D and show uptake in tumors and liver for FnBPA5, whereas scraFnBPA5 does not show any specific uptake in other organs.

(4) FIG. 4 shows a biodistribution and blocking study of radiolabeled .sup.111In-FnBPA5 and .sup.111In-scrambled FnBPA5. (A) Biodistribution of FnBPA5 in PC-3 bearing mice was monitored at different time points in various organs. (B) Biodistribution of scrambled FnBPA5 derivative was monitored the same way. .sup.111In-FnBPA5 showed a significantly higher uptake than .sup.111In-scrambled FnBPA5 in all organs except for kidneys and pancreas, confirming the specificity of the accumulation. (C) Blocking studies were performed and uptake was analyzed 4 hour post-injection. Blocking binding sites via pre-injection of unlabeled peptide caused a significant reduction in the uptake of .sup.111In-FnBPA5 in both liver and tumor (*p<0.05) whereas it did not change uptake in kidneys. Pre-injection showed a higher influence on peptide uptake in the liver than in the tumor, demonstrating the presence of a higher amount of binding sites (fibronectin) in the tumor. (D) Higher retention time of .sup.111In-FnBPA5 in tumor compared to the other organs is reflected in increasing tumor-to-blood and tumor-to-liver ratios with increasing time.

(5) FIG. 5 shows plasma stability of FnBPA5 and its scrambled derivative measured at 37° C. at different time points in blood plasma and water. Both FnBPA5 and scraFnBPA5 show high stability at 1 hour time point.

(6) FIG. 6 shows IC.sub.50 measurement of .sup.natIn-FnBPA5. Relaxed Fn fibers were incubated with FnBPA5-Alexa488 peptide, washed and then incubated with different concentrations of FnBPA5-In to measure the concentration of competitor. At the midpoint between high and low plateau of the curve is the IC.sub.50 value at 49 nM.

(7) FIG. 7 shows a table containing biodistribution data of .sup.111In-[FnBPA5-NODAGA] in PC-3 grafted mice.

(8) FIG. 8 shows design modes for fibronectin binding peptides via combination of several Fn binding peptides into polypeptides with variable linker length. (I) Single Fn binding peptide as smallest unit. (II) Combination of several different Fn binding peptides into a Fn binding polypeptide. (III) Amino acid linker length between individual Fn binding peptides within a Fn binding polypeptide is flexible and can be adjusted for individual applications. (IV) Binding of a Fn binding peptide to a Fn module via hydrogen backbone binding. (V) Polypeptide comprised of 4 different Fn binding peptides binding to four consecutive Fn modules via hydrogen backbone binding. (VI) Different functionalization possibilities of Fn binding polypeptides at the N or C-terminus via addition of a functional molecule, such as a chelator-radionuclide complex, a fluorophore, an active component, a drug or prodrug or any kind of particle.

DETAILED DESCRIPTION OF THE INVENTION

Examples

Example 1: General Material and Methods

(9) Fn Isolation and Labelling

(10) Fn was isolated from human plasma (Zürcher Blutspendedienst SRK Switzerland) using gelatin sepharose chromatography, as previously described (E. Engvall and E. Ruoslahti, “Binding of soluble form of fibroblast surface protein, fibronectin, to collagen,” Int. J. Cancer, vol. 20, no. 1, pp. 1-5, July 1977). Plasma was thawed and passed through a PD-10 column (GE Healthcare, Little Chalfont, UK) to remove aggregates. Effluent was collected and run through a gelatin sepharose column. After washing the column Fn was eluted from the gelatin column with a 6 M urea solution. Unlabelled Fn was then rebuffered to PBS before usage. For single labelling Fn was denatured in a 4 M guanidinium hydrochloride (GdnHCI, Applichem, Darmstadt, Germany) solution to open up cryptic cysteines at FnIII7 and FnIII15. Fn was incubated with an excess of Cy5 maleimide dye (GE Healthcare, Little Chalfont, UK) and separated from the dye using a PD-10 column.

(11) Synthesis and Labelling of FnBPA5 and Derivatives

(12) Peptides were commercially synthesized (Pichem, Graz, Austria) with a spacer of three glycines and a cysteine residue at the N-terminus of the original peptide sequence from S. aureus for further labelling with a radioligand or fluorophore. FnBPA5 was labelled using a fluorophore attached to a maleimide residue or conjugated with a maleimide NODAGA complexing unit for further radiolabeling with .sup.111In. Peptides were HPLC purified after conjugation to remove remaining free binding residues. A negative control of FnBPA5 with scrambled sequence was designed to investigate whether FnBPA5 binding to Fn is sequence specific. All peptide sequences are shown in FIG. 1C (SEQ ID NOs 295 and 296). Lyophilized peptides were dissolved in water with 10% DMF and stored at −20° C. upon further usage.

(13) In Vitro Fn Fiber Assay

(14) Manually pulled Fn fibers were used as a model system for fibrillar Fn as described previously (W. C. Little, M. L. Smith, U. Ebneter, and V. Vogel, “Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage,” Matrix Biology, vol. 27, no. 5, pp. 451-461, June 2008). Fibers containing 5% of photolabeled Fn-Cy5 were deposited onto a stretchable silicone sheet, relaxed to half of their original length, corresponding to a total 7% molecular strain and after a blocking step with 4% bovine serum albumin in PBS, they were incubated with different concentrations of Alexa488 fluorescently labeled FnBPA5 to obtain a binding curve.

(15) Confocal Microscopy

(16) Manually pulled Fn fiber samples were imaged with an Olympus FV1000 confocal microscope using a 40× water immersion objective with a numerical aperture of 0.9. Alexa488-FnBPA5 and Fn-Cy5 channels were imaged with a 512×512 pixel resolution and photomultiplier tube voltage and laser powers were kept constant within an experiment.

(17) Fibroblast ECM samples (FIG. 2C) were acquired with the same microscope using an oil immersion 1.45 NA 60× objective with a pixel resolution of 1024×1024.

(18) Image Analysis

(19) Images were analyzed using Fiji-ImageJ and Matlab (MathWorks, Natick, Mass., USA). For the Fn fiber affinity study the pixelwise ratio of FnBPA5-Alexa488 signal intensity divided by Fn-Cy5 intensity was calculated for each fiber using a custom made Matlab script. Dark current values were subtracted from images and pixels with intensities below a cutoff threshold and at saturation were excluded from analysis. Approximately 10 fibers were imaged per experimental condition and each of these conditions was done in triplicate. Binding ratio of 10 μM FnBPA5-Alexa488 concentration was set to 1 and all other points were normalized to this reference point. Data points were fit using the Hill model assuming non-cooperative binding (using the below equation) and plot using Origin.

(20) $\theta =\frac{\left[L\right]}{K_d+\left[L\right]}$$\theta =\mathrm{ratio}\mathrm{of}\mathrm{occupied}\mathrm{binding}\mathrm{sites}\mathrm{divided}\mathrm{by}\mathrm{total}\mathrm{binding}\mathrm{sites};\left[L\right]=\mathrm{free}(\mathrm{unbound}\mathrm{ligand}\mathrm{concentration};K_d=\mathrm{dissociation}\mathrm{constant}.$

(21) Radiolabelling of FnBPA5-NODAGA and Scrambled FnBPA5-NODAGA

(22) The fibronectin binding peptide (FnBPA5) and its scrambled derivative (scraFnBPA5) were purchased from Peptide Specialty Laboratories GmbH (Heidelberg, Germany) conjugated with a malemide NODAGA. The compounds were dissolved in TraceSELECT® Water (Sigma Aldrich) to a final concentration of 0.25 mM. For the labelling, 14 nmol of each peptide were radiolabelled in 0.5 M ammonium acetate pH 5.5 by adding 80 MBq .sup.111InCl.sub.3 (Mallincrodt, Wollerau, Switzerland) followed by a 30 minute incubation step at 50° C. Quality control was performed by radio-HPLC (Varian Prostar, Santa Clara, USA); column Dr. Maisch Reprospher (Ammerbuch, Germany) 300 C18-TN, 4.6 cm×150 mm; 5 m with acetonitrile/water gradient starting with 15% acetonitrile up to 95% over 15 minutes with a flow rate of 1 mL/min.

(23) Tumor Model

(24) PC-3 cells (human prostate carcinoma cell line, ACC-465, DSMZ, Braunschweig, Germany) were cultured in Roswell Park Memorial Institute 1640 medium (Amimed, Bioconcept, Switzerland). Cells were cultured as monolayers at 37° C. in a humidified atmosphere containing 5% CO.sub.2.

(25) In vivo experiments were approved by the local veterinarian department and conducted in accordance with the Swiss law for animal protection. The 3-5 weeks-old female CD1 nude mice were purchased from Charles River (Germany). After 5-7 days acclimatisation period, the tumor cells were subcutaneously inoculated in both shoulders of the mice (3*106-1*107 cells in 100-150 μL PBS per side). Experiments were performed 3-4 weeks after inoculation.

(26) Statistical Analysis

(27) Statistical analysis was performed using two-tailed type 3 t-test (Microsoft Excel). Statistical significance was assumed for p-values smaller than 0.05.

Example 2: Manually Pulled Fn Fiber System to Assess Binding Specificity and Binding Affinity of FnBPA5 or Other FnBPs

(28) To assess binding constant of Alexa488-FnBPA5 and of its scrambled analogue to Fn fibers a fiber stretch assay as described above and before (Little et al., Matrix Biology, vol. 27, no. 5, pp. 451-461, June 2008) was used. Fn fibers are manually pulled from a Fn solution containing 5% fluorophore labeled Fn and deposited onto a silicone membrane. Silicone membranes can then be stretched or relaxed to desired mechanical strain state. Confocal microscopy images of manually pulled Fn fibers exposed to FnBPA5 peptide in solution are shown in FIG. 2A. FnBPA5 peptide was shown to bind to Fn much stronger than the scrambled control derivative, whose signal is within the background noise (FIG. 2A). This result is confirmed in the analysis and quantification of multiple fibers from several fields of view (FIG. 2B).

(29) To measure a quantitative binding curve Fn fibers of equal mechanical strain were incubated with different concentrations of FnBPA5-Alexa488. Peptide fluorescence intensity normalized against the Cy5-intensity from the Fn-fibers for different peptide concentrations was assessed. Intensity ratio of 10 μM FnBPA5-488 was defined as saturated and all other intensity ratios were normalized with this factor. In FIG. 2E all points of the analysis are plotted and fit leading to a binding curve with a dissociation constant K.sub.d of 28 nM. Importantly, this affinity of FnBPA5 peptide to fibrillar Fn is of the same order of magnitude as those reported for the FnBPA5 peptide binding to N-terminal Fn fragments in solution (K.sub.d=44.2 nM) (Meenan et al., J. Biol. Chem., vol. 282, no. 35, pp. 25893-25902, August 2007) and also comparable to affinities reported for several antibodies that target ECM proteins and are in clinical use (Viti et al., Cancer Research, vol. 59, no. 2, pp. 347-352, January 1999).

(30) To assess whether the presence of the chelator for the radiolabeled .sup.111In-isotope impairs FnBPA5 binding to manually pulled Fn fibers, a displacement assay was performed using .sup.natIn-FnBPA5 (cold labeled) against FnBPA5-Alexa488. Extrapolated IC.sub.50 value for .sup.natIn-FnBPA5 of 49 nM (FIG. 6) show that the radiolabeling process did not affect binding properties of FnBPA5.

Example 3: Assessment of Binding of FnBPA5 or Other FnBPs to Fibrillar Fn in Cell Culture Matrices

(31) To ensure that such tight binding can be observed also in native extracellular matrix, Fn-rich ECM assembled by fibroblasts for 2 days was incubated for 1 hour with native or scrambled FnBPA5 prior to fixation and showed specific binding of FnBPA5 to fibrillar Fn, but not of the scrambled derivative (FIG. 2C). To achieve this, normal human dermal fibroblasts (PromoCell, Heidelberg, Germany) were cultured in alpha minimum essential medium (α-MEM) with 10% fetal bovine serum (FBS) from BioWest, Nuaillé, France, and split before reaching confluence. Cells were seeded onto Fn-coated 8-well chambered coverglasses (Lab-Tek, Nalgene Nunc, Thermo Scientific, Waltham, Mass., USA) at a density of 30×10.sup.3 cells per cm.sup.2 and allowed to attach to the surface before medium exchange to medium containing 50 μg/ml unlabeled Fn. Cells were cultured for 48 hours. Fibronectin was then stained using a rabbit polyclonal anti-fibronectin antibody (ab23750, abcam, Cambridge, UK) and 5 μg/ml FnBPA5-Alexa488 respectively scr-FnBPA5-Alexa488 peptide for 1 hour before fixation with a 4% paraformaldehyde solution in PBS. After fixation cells were permeabilized for 10 minutes with PBS containing 0.01% Triton X-100. After a washing step samples were blocked in 4% BSA and 4% donkey serum for 1 hour at room temperature. Samples were then incubated for 1 hour with a donkey anti-rabbit Alexa 546 (Invitrogen) secondary antibody and Phalloidin-633 (Invitrogen, Carlsbad, Calif., USA). Before imaging cell nuclei were stained using DAPI. Fibroblast ECM samples (FIG. 2C) were acquired with an Olympus FV1000 confocal microscope using an oil immersion 1.45 NA 60× objective with a pixel resolution of 1024×1024. Specific binding can then be assessed via colocalization of peptide with Fn using a scrambled peptide derivative as negative control (FIG. 2C).

Example 4: Plasma Stability of FnBPA5

(32) To assess in vitro plasma stability 12 MBq .sup.111In-[FnBPA5-NODAGA] and .sup.111In-[scraFnBPA5-NODAGA] were incubated with 400 μL human blood plasma at 37° C. At different time points (0, 0.25, 0.5, 1, 2, 48 and 72 hours) 40 μL of plasma was taken out and precipitated by the addition of 200 μL EtOH, acetonitrile, 0.1% TFA. After filtrating the sample (MiniPrep, Qiagen, Valencia, Calif., USA) the supernatant was analysed by radio-HPLC (Varian Prostar, USA); column D-Bio Discovery C18, 25×4.6; 5 m with acetonitrile/water gradient starting with 5% acetonitrile up to 95% over 30 minutes with a flow rate of 1 mL/min. .sup.111In-FnBPA5 peptide was still intact after 72 hours (FIG. 5), thus verifying that the FnBPA5 peptide has sufficient plasma stability to be used for in vivo applications.

Example 5: Fluorescence Polarization Experiments

(33) The binding affinities of Fn to FnBPA5 were determined in three independent measurements by anisotropy titrations in a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies) equipped with automated polarizers. FnBPA5 and its scrambled derivative were synthesized with an N-terminal Alexa-488 dye. The anisotropy of 100 nM Alexa-488 labelled peptide was measured in PBS at Fn concentrations ranging from 0 to 1.4 μM. Excitation and emission were at λ.sub.ex 480 nm and λ.sub.em 520 nm respectively with both slit 10 nm, 20° C., 5 s signal acquisition and g=1.4. The K.sub.d values were determined by fitting the data to a one-site-binding model using Origin 7 (OriginLab Northampton, Mass., USA).

(34) With higher Fn concentration an increasing amount of peptide is bound to Fn leading to a shift in fluorescence anisotropy. Anisotropy values for each sample was plotted against the corresponding Fn concentration yielding to a binding curve from which a dissociation constant K.sub.d of 75 nM for Alexa 488-FnBPA5 was extrapolated. In contrast, the scrambled control did not show significant binding (FIG. 2D).

Example 6: Radiotracing of .SUP.111.In-FnBPA5 Injected into Living Mice

(35) SPECT/CT experiments were performed using a 4-head multiplexing multipinhole camera (NanoSPECT/CTplus, Bioscan Inc., Washington D.C., USA). CT scans were performed with a tube voltage of 45 kV and a tube current of 145 IA. SPECT scans at 24, 72 and 96 hours post injection were obtained with an acquisition time of 20-90 sec. per view resulting in a total scanning time of 20-45 min per mouse.

(36) The distribution of .sup.111In-radiolabeled FnBPA5 peptide injected into the tail vein of a living mouse was monitored by means of SPECT/CT for a period of 96 hours. Since Fn is upregulated in cancer stroma, PC-3-bearing CD1 nu/nu mice, a subcutaneous model for prostate carcinoma, were injected 33 days from the inoculation of the tumor cells, with 12 MBq .sup.111In-[FnBPA5-NODAGA] resp. .sup.111In-[scrambled FnBPA5-NODAGA] (2.4 nmol, 100 μL PBS) into the tail vein. The specific activity of both peptides was 6.2 MBq/nmol and the samples were scanned 96 hours post injection (p.i. and post mortem) with an acquisition time of approximately 20 seconds (.sup.111In-[FnBPA5-NODAGA]) and 200 seconds (.sup.111In-[scraFnBPA5-NODAGA]) resulting in a total scanning time of 2.5 h for .sup.111In-[scraFnBPA5]. SPECT images were reconstructed using HiSPECT software (Scivis GmbH, Goettingen, Germany). The images were reconstituted and processed with InVivoScope® software (BioscanInc., Washington D.C., USA) and zoom in videos were generated using Adobe Flash.

(37) As typically observed also for other peptides 1111n-FnBPA5 (FIG. 3A) and the negative scrambled control (FIG. 3B) mainly accumulated in the kidneys, indicating an Fn-independent uptake by this organ, presumably due to its blood filtration tasks. To visualize additional binding of the tracer to tissues throughout the body, the kidneys were subsequently removed from the sacrificed mice and the scan was repeated. Mice injected with 1111n-FnBPA5 showed activity in different organs, with a predominant uptake in tumor and liver (FIG. 3C). For mice injected with 1111n-scraFnBPA5 no uptake into other tissues was visible (FIG. 3D).

Example 7: Pharmacokinetics of .SUP.111.In-FnBPA5 Injected into Living Mice Shows Prolonged Accumulation in Mouse Prostate Tumor Xenografts

(38) The tissue-specific peptide pharmacokinetics, particularly in cancer stroma, were assessed in groups of 4 PC-3-bearing mice that were injected with approximately 150 kBq .sup.111In-FnBPA5 respectively .sup.111In-scraFnBPA5 (2.4 nmol/100 L PBS) into the tail vein and biodistribution of peptides was analyzed at different time points (1, 4, 24 and 96 hours post injection (p.i. and after sacrification)) by means of percentage of injected activity per gram tissue (% IA/g). An equal accumulation of both peptides was observed in the kidneys (FIGS. 4A, B), confirming the findings from SPECT/CT imaging (see Example 5). In both cases, a maximum at 1 hour p.i. was seen (140.58±18.10% IA/g for .sup.111In-FnBPA5 and 163.70±18.90% IA/g for .sup.111In-scra FnBPA5). .sup.111In-FnBPA5 showed an accumulation in all other examined organs (FIG. 4A), again in contrast to its scrambled derivative. Particularly, in tumor, liver and spleen, the FnBPA5 uptake is significantly higher compared to the scrambled derivative. Tumor uptake was significantly higher for all time points with a maximum at 1 h p.i. (4.74±0.77% IA/g). The retention of .sup.111In-FnBPA5 in the tumor tissue was longer compared to the other organs (FIG. 4A). In fact, the tumor-to-blood ratio increased from 3.05±1.66 at 1 h p.i. to 34.03±18.36 at 96 h p.i (FIG. 4D and FIG. 7 (ex. supplementary table 1)). Results from the biodistribution are in accordance with the SPECT/CT analysis shown in FIG. 3, and illustrate that the organ uptake of .sup.111In-FnBPA5 is, apart from the kidneys, specific and related to extracellular matrix protein fibronectin. To further confirm Fn-specific binding of .sup.111In-FnBPA5, in vivo blocking experiments were performed: an approximately 10-fold excess of unlabeled FnBPA5 (100 μg in 100 L PBS) was pre-injected directly before .sup.111In-FnBPA5 to block the binding sites (FIG. 4C). The pre-injection of unlabeled FnBPA5 causes a significant reduction (p<0.05) of .sup.111In-FnBPA5 accumulation in all examined organs with exception of the kidneys and the pancreas. The blocking effect was thereby less pronounced for the tumor tissue (uptake decrease of 35.6%) compared to the liver (58.2%). In contrast, no significant differences were seen for .sup.111In-scraFnBPA5.