INTEGRIN BINDING PEPTIDES AND USES THEREOF

Abstract

The invention relates to integrin binding peptides, pharmaceutical compositions comprising the peptides and to uses thereof as therapeutic, diagnostic, imaging and targeting agents.

Claims

1.-25. (canceled)

26. An isolated or recombinant integrin alpha 5 (ITGA5) binding peptide consisting of 6 to 25 amino acids and comprising an amino acid sequence TTVRYYRITYGETGGN (SEQ ID NO:3) or comprising a variant of the amino acid sequence, the variant: consisting of 6-16 consecutive amino acids of the sequence, the 6-16 consecutive amino acids comprising at least the amino acids at positions 5-10 of the sequence, and having up to three substitutions of an amino acid of the 6-16 consecutive amino acids selected from amino acids at positions 1, 2, 3, 4, 5, 7, 8, 10, 11, 12, 13, 14, 15 and 16 of the sequence by another amino acid, wherein at most 25% of the amino acids of the variant has been substituted by another amino acid.

27. The peptide of claim 26, wherein the variant of amino acid sequence TTVRYYRITYGETGGN (SEQ ID NO:3) has up to one substitution of an amino acid selected from amino acids at positions 1, 2, 4, 5, 6, 7, 8, 9, 11 and 12 of the sequence by another amino acid.

28. The peptide of claim 26, wherein the ITGA5 binding peptide consists of an amino acid sequence selected from the group consisting of RYYRITY (SEQ ID NO:8), RYYRITYC (SEQ ID NO:11), TTVRYYRITYGE (SEQ ID NO:7) and YYRITYGETGGN (SEQ ID NO:56).

29. The peptide of claim 26, wherein arginine at position 4 and/or at position 7 of the sequence is replaced by an amino acid selected from the group consisting of lysine, histidine and alanine or by a corresponding non-natural amino acid, and/or tyrosine at position 5 and/or at position 10 of the sequence is replaced by an amino acid selected from the group consisting of alanine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan or by a corresponding non-natural amino acid, and/or isoleucine at position 8 of the sequence is replaced by an amino acid selected from the group consisting of alanine, valine, leucine, methionine, phenylalanine, tyrosine and tryptophan or by a corresponding non-natural amino acid.

30. The peptide of claim 26, wherein the peptide has ?5?1 integrin and/or ITGA5 inhibiting activity.

31. A dimeric peptide comprising two peptides of claim 26.

32. The dimeric peptide of claim 31, wherein each of the two peptides of the dimeric peptide comprises a cysteine residue.

33. A compound comprising the peptide of claim 26.

34. The compound of claim 33, comprising at least one further moiety.

35. The compound of claim 34, wherein the at least one further moiety comprises a label, a linker, a N-terminal modification, a C-terminal modification and/or an internal modification.

36. The compound of claim 33, wherein the compound comprises the peptide coupled to or encapusulated into a carrier selected from the group consisting of nanoparticles, microparticles, nanocapsules, nanocomplexes, polyplexes, carbon nanotubes, quantum dots, microcapsules, liposomes, microspheres, hydrogels, polymers, micelles, dendrimers, lipid complexes, serum albumin, antibodies, antibody fragments, cyclodextrins and dextran.

37. A nucleic acid molecule comprising a nucleic acid sequence encoding the peptide of claim 26.

38. A pharmaceutical composition comprising: the peptide of claim 26, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, and/or excipient.

39. A method for the treatment of a subject that has fibrosis or a fibrosis related disorder, an inflammatory disease or cancer comprising administering to the subject a therapeutically effective amount of the peptide of claim 26,

40. A method of imaging a tissue expressing integrin alpha 5 (ITGA5), the method comprising: contacting the tissue with the peptide of claim 26, which comprises an imaging label.

41. The method according to claim 40, wherein the tissue expresses ?5?1 integrin.

42. An isolated or recombinant integrin alpha 11 (ITGA11) binding peptide consisting of 5 to 25 amino acids, and comprising an amino acid sequence SGLTEWLRWFNS (SEQ ID NO:1) or a variant of the sequence, the variant: consisting of 5-12 consecutive amino acids of the sequence, the 5-12 consecutive amino acids comprising at least the amino acids at positions 7-9 of the sequence, and having up to three substitutions of an amino acid of the 5-12 consecutive amino acids selected from amino acids at positions 1, 2, 4, 5, 6, 7, 8, 9, 11 and 12 of the sequence by another amino acid, whereby at most 25% of the amino acids of the variant has been substituted by another amino acid, or comprising an amino acid sequence SFATWTPNFERN (SEQ ID NO:2) or a variant of the sequence, the variant consisting of 5-12 consecutive amino acids of the sequence and having up to three substitutions of an amino acid by another amino acid, whereby at most 25% of the amino acids of the variant has been substituted by another amino acid, with the proviso that the peptide does not consist of the sequence MSLRWFNSGSVRPATTILFP (SEQ ID NO:4).

43. The peptide of claim 42, wherein serine at position 1 and/or at position 12 of the sequence SGLTEWLRWFNS (SEQ ID NO:1) is replaced by an amino acid selected from the group consisting of threonine, asparagine, glutamine and alanine or by a corresponding non-natural amino acid, and/or glycine at position 2 of the sequence SGLTEWLRWFNS (SEQ ID NO:1) is replaced by an amino acid selected from the group consisting of proline, alanine, cysteine, serine, threonine, asparagine and aspartic acid or by a corresponding non-natural amino acid, and/or threonine at position 4 of the sequence SGLTEWLRWFNS (SEQ ID NO:1) is replaced by an amino acid selected from the group consisting of serine, asparagine, glutamine and alanine or by a corresponding non-natural amino acid, and/or glutamic acid at position 5 of the sequence SGLTEWLRWFNS (SEQ ID NO: 1) is replaced by an amino acid selected from the group consisting of aspartic acid and alanine or by a corresponding non-natural amino acid, and/or tryptophan at position 6 and/or at position 9 of the sequence SGLTEWLRWFNS (SEQ ID NO: 1) is replaced by an amino acid selected from the group consisting of alanine, phenylalanine and tyrosine or by a corresponding non-natural amino acid, and/or leucine at position 7 of the sequence SGLTEWLRWFNS (SEQ ID NO:1) is replaced by an amino acid selected from the group consisting of alanine, valine, isoleucine and methionine or by a corresponding non-natural amino acid, and/or arginine at position 8 of the sequence SGLTEWLRWFNS (SEQ ID NO: 1) is replaced by an amino acid selected from the group consisting of lysine, histidine and alanine or by a corresponding non-natural amino acid, and/or asparagine at position 11 of the sequence SGLTEWLRWFNS (SEQ ID NO: 1) is replaced by an amino acid selected from the group consisting of threonine, serine, glutamine and alanine or by a corresponding non-natural amino acid.

44. A method for the treatment of a subject suffering from fibrosis or a fibrosis related disorder, an inflammatory disease or cancer comprising administering to the subject a therapeutically effective amount of the peptide of claim 41.

45. The peptide of claim 30, wherein the inhibiting activity comprises inhibition of binding of ?5?1 integrin and/or ITGA5 to fibroblasts, stellate cells, myofibroblasts, pericytes and/or other cells of mesenchymal origin.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0205] FIG. 1: Gene expression levels of ?-SMA and integrin ?11 in human hepatic stellate cells (A) activated with TGF-?1 and in livers isolated at different stage of liver fibrosis in CC14-induced liver fibrosis mouse models (B). *p<0.05, **p<0.01 vs. normal.

[0206] FIG. 2: A. ITGA11 in human liver cirrhosis; B. ITGA11 in pancreatic cancer C. ITGA11 in human kidney fibrosis. Images in panel A and B show immunofluorescent staining of ITGA11 (green color) and a-SMA (red color) and their merge (yellow color). Panel C shows the immunofluorescent staining of ITGA11 (red color) and a-SMA (green color) and their merge (orange-yellow color). These images show that ITGA11 is strongly expressed in fibrotic region of all pathological conditions which is co-localized with fibroblast marker a-SMA, shown with orange-yellow color in merge images.

[0207] FIG. 3: Phage ELISA assay showing the binding of phages to ?11?1 versus ?4?1.

[0208] FIG. 4: Binding of the peptide to ITGA11. A. showing the binding of AXI-I-PEG-FITC to the coated ?11?1 receptor. AXI-I-FITC bound to the receptor with increasing concentration and the binding was blocked with excess of unlabeled AXI-I. Binding of the peptide to empty well or irrelevant receptor ?5?1 led to no specific binding. B and C. showing the binding of the AXI-I-FITC (B) and AXI-II-FITC (C) peptides to LX2 hepatic stellate cells as shown with green color.

[0209] FIG. 5: Binding studies of ITGA11-binding peptide (AXI-I) labelled with FITC on the coated ?11?1 receptor (A) and on the LX2 cells (B). On the receptor, binding was competitively blocked by adding 10-fold high concentration of unlabelled peptide. The receptor binding data is an average of n=4 independent experiments, each in duplicate. Mean+sem. Knockdown of ITGA11 in LX2 led to complete inhibition of the peptide binding.

[0210] FIG. 6: AXI-I-PEG-FITC binding assay-flow cytometry in primary human pancreatic stellate cells. Different concentrations of AXI-I-PEG-FITC was incubated with suspended pancreatic stellate cells for 1 h at 4? C., washed and fixed with 0.5% formaldehyde and measured with flow cytometry. The results show that AXI-I-PEG-FITC bound to these cells concentration dependently leading to dissociation constant (kd) value of approx. 1 uM.

[0211] FIG. 7: Alanine scanning and peptide chain length scan for AXI-I peptide (SGLTEWLRWFNS).

[0212] FIG. 8. Effect of AXI-I on activation of LX2 cells in vitro (A) and in vivo in CC14-induced liver fibrosis model in mice (B,C). A) Treatment with AXI-I inhibited the expression of collagen-I at increasing concentrations in the TGF?-activated LX2 cells while scrambled peptide (SAXI) remained inactive. B) and C) In a CC14-induced liver fibrosis model, treatment with AXI-I (200 ug/kg/d) i.p. injection led to reduction of fibrogenesis, as indicated by the decrease of gene expression of ITGA5 and ITGA11 (B), markers of myofibroblasts and protein expression of collagen-I and III in livers compared to vehicle control (C). *p<0.05 mean+SEM.

[0213] FIG. 9: ITGA5 in pancreatic cancer. A. Left image shows the immunostaining of ITGA5 in normal human pancreas while right image shows the staining in pancreatic tumor. Pancreatic tumor shows a strong staining of ITGA5 while normal pancreas shows no staining. B. Co-localization of ITGA5 with a-SMA (marker for myofibroblasts) and CD31 stainings (marker for endothelial cells). Double staining shows that ITGA5 perfectly coincide with a-SMA but slightly with CD31, indicating that ITGA5 is highly expressed on myofibroblasts.

[0214] FIG. 10: ITGA5 expression in human skin fibroblasts and pancreatic stellate cells. A. Human skin fibroblasts (BJhtert) expressed an increasing levels of ITGA5 after activation with TGF-?1 for 8, 24 and 48 h. B. Activation of pancreatic stellate cells with TGF? or panc-1 tumor cell conditioned medium induced the ITGA5 expression levels after 24 h.

[0215] FIG. 11: Design of peptide against ITGA5. Image derived from Protocol Multiwell Peptide Microarrays of JPT Technologies, Berlin Germany, https://www.jpt.com/fileadmin/Multiwell-Peptide-Microarray_Protocol_Rev_1.0_V06.pdf.

[0216] FIG. 12: Binding of peptides to the coated ?5?1 receptor and LX2 cells. FITC-labeled nAV2: YYRITYGETGGN-K-PEG(6)-Fluorescein; nAV2: YYRITYGETGGN; AV3: RYYRITY. A. nAV2-FITC bound to the coated ?5?1 receptor which was blocked by excess of nAV2 peptide and much stronger with excess of AV3 peptide. B. showing the binding of nAV2-FITC to LX2 cells (stained with DAPI), as shown in green color (FITC).

[0217] FIG. 13: Alanine replacement assay of peptide RYYRUTY to find essential amino acids. Bold and underlined amino acids seem important for binding, in particular Y3 and T6.

[0218] FIG. 14: A. Anti-fibrotic effect of AV3 peptide. B. AV3-cys peptide effect on PSC activation in vitro (20 ?M concentration, 20? magnification). A. Activation of PSCs with TGF? led to activation of these cells as indicated by ?-SMA expression. Treatment with AV3 peptide significantly inhibited the ?-SMA expression at 20 ?M. B. Incubation of PSCs with TGF?1 enhanced the expression of fibrotic markers such as ?-SMA, Col-1a1 and vimentin, as shown with immunostainings. Treatment with AV3-cys peptide clearly inhibited the expression of these biomarkers. In contrast, scrambled AV3-cys did not show any inhibitory effects.

[0219] FIG. 15: Effect of peptides on migration of human fibroblasts. Human skin fibroblasts BJhtert were grown to full confluency and a scratch (wound) was made and effects of peptides were determined on the wound closure (migration of fibroblasts) after 24 h. A. showing that AV3-cys significantly inhibited the migration of fibroblasts while its scrambled version did not show any effect. B. showing the effect of different peptides on the migration of the fibroblasts. Only AV3.3 showed about 30% reduction on migration while other versions showed slight or no effects.

[0220] FIG. 16: Specificity of peptides AV3 and AXI-I. Fluorescent labeled peptides (AV3-PEG(6)-5FAM (A) and AXI-I-PEG(6)-FITC(B)) were examined for their binding affinities against ?5?1 and ?11?1 receptors, respectively.

[0221] FIG. 17: Concentration response effect of AV3 peptide on the activation of human dermal fibroblasts.

[0222] FIG. 18: Tumor imaging using AV3 peptide. Optical images showing the distribution of AV3 peptide labeled with 800CW dye (AV3-800CW). Human pancreatic tumor cells (Panc-1) combined with human pancreatic stellate cells (PSC) were co-injected into the flank of SCID mice and allowed to grow to a size of about 200 mm.sup.3. AV3-800CW (1 nmol) was injected intravenously alone (A) or with (B) 50-fold excess of unlabeled AV3 in tumor bearing mice. Images were captured using Pearl imager (LICOR).

EXAMPLES

Example 1

[0223] Materials and Methods

[0224] Materials

[0225] Peptide Phage Display Ph.D.12? Library was originally obtained by New England Biolabs. E. coli ER2738 host strain were purchased from New England Biolabs. The target protein rhIntegrin ?11?1 and control protein rhIntegrin ?4/?1 was purchased from R&D systems.

[0226] Biopanning

[0227] ER2738 host cells were cultivated overnight to use them freshly. 100 ?l of the target protein ?11?1 (50-100 g/mL) in coating buffer (0.1M NaHCO.sub.3) was incubated in 96-wells plate at 4? C. overnight. Then, each well was blocked with 300 ?L of blocking buffer 0.1M NaHCO.sub.3 (pH 8.6), 5 mg/mL BSA, 0.02% NaN3, 0.1 g/mL streptavidin at 37? C. for 2 hours. The blocked wells were then washed 6 times with 2% skimmed milk in PBST with 0.1-0.3% Tween-20. Thereafter, the pre-subtracted phages were transferred into the wells and incubated at 37? C. for 1 hour. The wells were washed 10 times with 2% Milk in PBST (0.1-0.3% Tween 20). The bound phages were eluted with acidic elution buffer and neutralize with 1M Tris-HCl (pH 9.1).

[0228] Titration of the Eluate

[0229] In brief, 1 ?L of eluate was diluted in 100 ?L of LB medium and serial dilutions were prepared. The dilutions were mixed with 200 ?L of mid-log host cells in test tubes at first and then added the mixture into 3 mL pre-warmed Agarose Top (45? C.), vortexed quickly and poured onto a pre-warmed LB/IPTG/Xgal plate immediately to spread Agarose Top evenly. After cooling at room temperature for about 5 minutes, plates were inverted and then incubated at 37? C. overnight. At last, plates were inspected and blue plaques (carrying phage vectors) on plates were counted having about 100 plaques. The pfu (plaque forming units) per mL were determined by multiplying each number by the dilution factor.

[0230] Amplification of the Eluate

[0231] The rest of the eluate of the 1.sup.st or 2.sup.nd round of screening were amplified by infecting 20 mL ER2738 cell culture and incubating at 37? C. for 4.5 hours. Amplified phages contained in the cell supernatant were precipitated by adding 1/6 volume of PEG/NaCl and incubate at 4? C. overnight. The precipitated phages were titrated on LB/IPTG/X-gal plates and preserved for the next round of screening.

[0232] Phase ELISA Assay

[0233] For each clone, 20 mL of LB medium was inoculated with ER2738 and incubated at 37? C. until slightly turbid. Single plaque of phages were inoculated to each culture and then incubated at 37? C. with vigorous aeration for 4.5 hours. The cultures were transferred to a fresh centrifuge tube and centrifuged for 10 minutes at 10,000 rpm. The upper 80% of the supernatant was transferred to a fresh tube and precipitated with PEG/NaCl twice. Then the pellet was suspended in 250 ?L TBS. The 96-well ELISA plate was coated with 100 ?L of 1 g/mL target in coating buffer and incubated at 4? C. overnight. Then, the plate was washed once with the washing buffer (0.1% Tween in TBS). All wells were blocked with 250 ?L of blocking buffer at 4? C. for 1-2 hours. Then, the plate was washed 6 times with the washing buffer and slapping on a paper towel to remove excess buffer. Thereafter, 100 ?L of phage virions was added per well. In competitive phage ELISA, competitive antigen was mixed with phages in washing buffer and added to the wells and incubated at room temperature for 1-2 hours with agitation. The plate was washed 6 times with washing buffer. HRP-conjugated anti-M13 antibody (GE healthcare, 1:5000 in blocking buffer) was added to each well and incubated at room temperature for 1 hour with agitation. Then, the plate was washed 6 times with washing buffer. The 100 ?L HRP substrate solution (22 mg OPD (Sigma) in 100 mL of 50 mM sodium citrate, pH 4.0 with freshly added 36 ?L of 30% H2O2 to 21 mL of OPD stock solution) was added to each well and incubated at room temperature for 10-60 minutes. The plates were read using a microplate reader at 490 nm.

[0234] Isolation of ssDNA for Sequencing

[0235] The single plaques were amplified by infecting 2 mL ER2738 cell culture and incubating at 37? C. for 4.5 hours. 500 ?L of the phage-containing supernatant was transferred to a fresh tube, added with 200 ?L PEG/NaCl and mixed. The tube was centrifuged at the top speed for 10 minutes and supernatant was discarded. The pellet was suspended thoroughly in 100 ?L iodide buffer, added with 250 ?L ethanol and incubated for 10 minutes at room temperature. Then the tube was centrifuged for 10 minutes and supernatant was discarded. The pellet was washed in 70% ethanol, dried briefly under vacuum. The pellet was suspended in 30 ?L TE buffer and 5 ?L of the re-suspended template was used for DNA sequencing. The sequences were translated with professional software (Vector NTI?, Version 10).

[0236] Synthesis of Peptide and Peptide-PEG-FITC

[0237] Peptides and peptide-PEG(6)-FITC were custom-synthesized by Chinapeptides, Shanghai, China at >95% purity. The successful syntheses of peptides were confirmed by mass spectrometry analyses.

[0238] Binding of Peptides to ITGA11 Receptor

[0239] Purified ITGA11 receptor (?11?1; 100 ?g/mlstock; R&D systems) was diluted to 5 ?g/ml with 1?PBS. 96-well ELISA White Maxisorb plate (Nunc) was coated with 50 ?l of 5 ?g/ml ITGA11 (or as control ?5?1) receptor for overnight at 4? C. Then, wells were blocked for 3-4 h with 200 ?l of blocking buffer (1?PBS containing 5% BSA). Wells were washed three times with 200 ?l of washing buffer (1?PBS containing 0.5% BSA and 0.05% Tween20). Peptides conjugated with FITC were diluted to different concentrations in washing buffer and then added to the plate and incubated at 37? C. for 1 h. In addition, the binding of the peptide was blocked by co-incubation with 10? higher amount of unlabeled peptide. Subsequently, wells were washed three times with washing buffer. Then, 100 ?l of 1?PBS was added and plates were read at 485 nm/520 nm with a fluorescence plate reader.

[0240] Cell Binding Experiment

[0241] Human hepatic stellate cells (LX2, 15,000 cells/well) were cultured overnight in permanox Lab-Tek 8 well chamber slides (Nunc). After overnight incubation, cells were washed 3? with 0.5% BSA containing medium and then FITC-labelled peptides (20 ?M) was added and incubated at room temperature for 2 h with intermittent shaking. The cells were then washed 3? with 0.5% BSA containing medium and 2?PBS. Thereafter, cells were fixed with 4% paraformaldehyde (prepared in 1?PBS) for 20 min followed by 3 times washing with 1?PBS. Cells were then mounted with mounting medium with DAPI (vector labs) and visualized under the fluorescence microscope (Nikon E600). For phalloidin staining, cells after fixation were incubated with TRITC-conjugated Phalloidin (1:1000 prepared in 1?PBS containing 0.1% triton X100) for 10 min. Then, cells were washed thrice with PBS and mounted with DAPI-containing mounting medium.

[0242] CC14-Induced Liver Fibrosis Mouse Model

[0243] All animal experiments were approved by ethical committee of Utrecht University. C57/BL6 mice (7-8 weeks old) were obtained from Harlan (Zeist, The Netherlands) and kept at 12/12 light dark cycle with adequate food and water supply. CC14 was intraperitoneally administered in mice (2? per week; 1.0 ml/kg prepared in olive oil) for 4 weeks (mild fibrosis) and 8 weeks (advanced fibrosis). After 24 h of the last administration, animals were sacrificed and pieces were excised out from each liver lobe and collected in Eppendorf tube and were snap frozen in liquid nitrogen for RNA isolation. Normal control mice received olive oil.

[0244] To assess the effect of AXI-I peptide on liver fibrosis, animals were injected with a single dose of CC14 (1 ml/kg) at day 0. Subsequently, mice were injected with the peptide (200 g/kg/d, i.p) or vehicle (PBS) on day 1 and 2. On day 4, mice were sacrificed and the liver samples were processed for immunohistochemistry analyses and gene expression studies.

[0245] In Vitro Gene Expression Studies

[0246] Hepatic stellate LX2 cells (80,000 cells/well) were plated in 12 wells plates and incubated overnight at 37? C./5% CO2. Then, cells were starved in 0.5% serum containing medium overnight and then activated with 5 ng/ml TGF? for 24 hrs. Subsequently, cells were washed with 1?PBS and lysed with 200 ?l of RNA lysis buffer. Total RNA was isolated using SV Total RNA Isolation System (Promega) according to manufacturer's instructions. RNA concentration was quantified using UV spectrophotometer (Nanodrop technologies). 1 ?g RNA was reverse transcribed using iScript cDNA synthesis kit (Biorad). The real time PCR reactions were performed with 20 ng cDNA using 2?SYBR green PCR master mix (Bioline) according to manufacturer's instructions and were analyzed by Biorad CFX384 Real-Time PCR detection system. Finally, the threshold cycles (Ct) were calculated and relative gene expression was normalized with GAPDH (for mouse) as housekeeping gene.

TABLE-US-00001 ITGA11humanForward: CAGCTCGCTGGAGAGATACG; Reverse: TTACAGGACGTGTTCGCCTC; GAPDHhumanForward: TCCAAAATCAAGTGGGGCGA; Reverse: TGATGACCCTTTTGGCTCCC; a-smahumanForward: GAACCCTGTGTCCTGCATCA; Reverse: TTGGAGTTCCACCTCGAAGC; ITGA11mouseForward: TTGGGCTACTACAACCGCAG; Reverse: CTTGTTGGTGCCTTCCAAGC; GAPDHmouseForward: ACAGTCCATGCCATCACTGC; Reverse: GATCCACGACGGACACATTG; ?-smamouseForward: ACTACTGCCGAGCGTGAGAT; Reverse: CCAATGAAAGATGGCTGGAA.

[0247] Binding Studies on Peptide Microarray

[0248] Peptide microarray was prepared by conjugating peptides at their N-terminal to a glass slide using a PEG linker. Each peptide was displayed three times at different positions to avoid artefacts and errors. For the binding studies, the peptide array was blocked with 3% BSA in TBST for 2 h. Then the array slide was washed 5 times in TBST and subsequently incubated with the target receptor (?11?1, 10 g/ml dissolved in PBS) for 1 h at 37? C. The slides were washed with TBST and then incubated with primary antibody (1 g/ml) against ITGA11 for 1 h at 37? C. Then slides were washed and incubated with fluorescent dye labelled secondary antibodies for 30 min. The slides were washed with TBST and water and then dried and scanned with a microarray scanner to detect binding of the peptides. The enlightened spots were analysed using ImageJ software. To determine the unspecific binding, a peptide array was incubated with only primary and secondary antibodies without the receptor incubation step. Then, the signal of unspecific binding was subtracted from the total binding to calculate specific binding.

[0249] Expression of ITGA in Human Liver Cirrhosis and Pancreatic Cancer

[0250] Immunohistochemical staining was performed as described in Example 2.

[0251] AXI-PEG FITC Binding to Human Pancreatic Stellate Cells

[0252] PSCs were trypsinized using trypsin-EDTA solution and cell numbers were diluted to 4?10.sup.4 cells/ml. Cells were incubated at 37? C. for 30 min to allow receptor recovery. Then different concentrations of AXI-I-FITC was added to the cells containing 2% FBS and incubated at 4? C. for 60 min. Thereafter, cells were centrifuged at 1500 rpm at 4? C. for 10 min. Supernatant was removed and cells were washed 3 times with PBS and then were fixed with 0.5% formaldehyde for 1 h at 4? C. and measured with flow cytometry for fluorescence.

[0253] Results

[0254] ITGA11 Expression in Hepatic Stellate Cells and Liver Fibrosis

[0255] We investigated the expression of ITGA11 in hepatic stellate cells that are the most important cell type in liver fibrosis, responsible for producing extracellular matrix. We found that activation of stellate cells with TGF?1 led to a significant increase in ITGA11 gene expression (FIG. 1A). These data corroborated with the increase in the expression level of the stellate cell activation marker ?-SMA. Furthermore, we investigated the ITGA11 expression in CC14-induced liver fibrosis model in mice. We found that the expression level of ITGA11 significantly enhanced with the progression of fibrosis after the treatment of CC14 for 4 weeks (mild fibrosis) and 8 weeks (advanced fibrosis) (FIG. 1B). This data indicates that ITGA11 is an important biomarker for liver fibrosis. Detection of ITGA11 using imaging techniques such as MRI, SPECT, PET, CT, photoacoustics or other kind of techniques with the help of a ligand against ITGA11 labelled with a radioisotope or a contrast agent can be applied for the diagnosis of liver fibrosis and to determine the progression of liver fibrosis.

[0256] We determined the expression of ITGA11 in different pathological conditions of human. We found that ITGA11 is strongly expressed in the fibrotic region of ?11 examined pathological conditions such as liver cirrhosis, pancreatic tumor stroma and kidney fibrosis (FIG. 2). The ITGA11 staining was well co-localized with the fibroblast marker a-SMA, as shown with orange-yellow color of the merge images.

[0257] Phage-Display Selected Peptides:

[0258] The phage bound to the ?11?1 receptor after subtracting from ?4?1 receptor were eluted and amplified. Randomly 40 clones were picked and examined for binding to ?11?1 and ?4?1 receptors using Phage ELISA assay (FIG. 3). Clones number clones number 11, 13, 14, 16, 19, 20, 21, 22, 24, 25, 27, 28, 29, 30, 31 and 38 showed higher binding to ?11?1 compared to ?4?1 and non-coated well. DNA sequencing data showed that clones number 11, 13, 14, 16, 19, 20, 21, 22, 24, 25, 28, 29, 30, 31, and 38 resulted into a single sequence (5-tctggtctgactgagtggttgaggtggtttaattcg-3) or amino acid sequence (AXI-I: SGLTEWLRWFNS) while clone 27 resulted into the DNA sequence (5 agttttgcgacgtggactccgaattttgagaggaat-3) or amino acid sequence (AXI-II: SFATWTPNFERN).

[0259] Peptide Binding to ITGA11

[0260] To determine whether peptides bind to ITGA11 receptor specifically, we performed binding of FITC labelled AXI-I peptide to the immobilized human ITGA11 (?11?1) receptor. We found that AXI-I-FITC bound to the ITGA11 receptor specifically, as the binding was blocked by 10-fold excess unlabeled peptide (FIG. 4A). In addition, the peptide showed very low binding to another integrin receptor (i.e. ?5?1) which was similar to the binding to the empty wells. The ?1 receptor is a common co-receptor in both ?11?1 and ?5?1 and no binding of the peptide to ?5?1 but high binding to ?11?1 indicates that the peptide is preferably bound by ?11. Furthermore, we examined the binding of AXI-I on mouse ?11?1 receptor and found that the peptide has similar binding affinity as for human, which attributes to about 80% homology between mouse and human ITGA11 receptors. After confirming the binding to the immobilized receptor, we examined the binding of the peptides on the ITGA11-expressing hepatic stellate cells. We found that both AXI-I and AXI-II bound to the stellate cells but binding of AXI-I was clearly stronger than AXI-II (FIG. 4B).

[0261] Binding studies of ITGA11-binding peptide (AXI-I) labelled with FITC on the coated ?11?1 receptor showed that the peptide bound to the receptor specifically compared to the empty well. The binding was competitively blocked by adding 10-fold high concentration of unlabelled peptide, showing specific binding of the peptide (FIG. 5A). Furthermore, we confirmed the binding of the peptide to the ITGA11 expressing LX2 cells using fluorescent microscopy. Knockdown of ITGA11 using shRNA-ITGA11 in these cells led to complete inhibition of the peptide binding, as no fluorescent signal was detected (FIG. 5B).

[0262] Alanine Scanning and Peptide Chain Length Scan

[0263] To find out the amino acids responsible for the peptide binding and the most efficient binding peptides, peptide microarrays were developed with alanine replacement and shorter peptides for AXI-I peptide (SGLTEWLRWFNS) (FIG. 7).

[0264] For AXI-I peptide (SGLTEWLRWFNS), replacement of L3 or F10 led to remarkable decrease of the peptide binding to the ?11?1 receptor, indicating that these amino acids make the epitopes. The shortening of the peptide chain showed that LTEWLRWF peptide induced the strongest binding to the receptor. As a control, the peptide array was exposed to ?5?1 to check the unspecific binding of the LTEWLRWF peptide. Table 1 shows the sequences of ITGA11 binding peptides.

TABLE-US-00002 TABLE 1 Sequence of ITGA11 binding peptides 1 2 3 4 5 6 7 8 9 10 11 12 S G L T E W L R W F N S 1 L T E W L R W F 2 G L T E W L R W F N S 3 G L T E W L R W F N 4 L T E W L R W F N S 5 S G L T E W L R W F N 6 S G L T E W A R W F N S 7 S G L T E W L R W F 8 S G L T E W L A W F N S 9 S G L T E W L R W F N S 10 S G L A E W L R W F N S 11 W L R W F N S 12 S A L T E W L R W F N S 13 S G L T E W L R W F A S 14 T E W L R W 15 S G L T E W L R W F N S 16 E W L R W F N S 17 S G L T A W L R W F N S 18 S G L T E W L R W F N A 19 S G L T E A L R W F N S 20 T E W L R W F N S 21 S G L T E W L R W 22 S G L T E W L R A F N S 23 A G L T E W L R W F N S 24 L R W F N S

[0265] AXI-PEG FITC Binding to Human Pancreatic Stellate Cells

[0266] Different concentrations of AXI-I-PEG-FITC was incubated with suspended PSCs for 1 h at 4? C., washed and fixed with 0.5% formaldehyde and measured with flow cytometry. The results demonstrate that binding of AXI-I-PEG-FITC to PSCs increased with the increasing concentration and reached to plateau at 20 ?M concentration (FIG. 6). The 50% of the total binding i.e. dissociation constant (kd) value was approx. 1 uM. Since the peptide has been modified with PEG chain and a fluorophore, the binding affinity of the peptide may be reduced due to steric hindrance.

[0267] Effect of AXI-I Peptide In Vitro on LX2 Cells and in CC14-Induced Liver Fibrosis Model in Mice

[0268] We examined the inhibitory effect of AXI-I peptide in LX2 cells. Activation of LX2 cells with TGF 1 induced the expression of collagen-1. Interestingly, treatment with increasing concentrations of AXI-I inhibited the expression of collagen-I (FIG. 8A). In contrast, the scrambled peptide showed no reduction in the collagen staining.

[0269] Furthermore, we examined the effect of AXI-I in a CC14-induced liver fibrosis model in mice. We found that treatment with AXI-I at the dose of 200 ug/kg/d i.p. led to reduction of fibrogenesis, as indicated by the decrease of gene expression of ITGA5 and ITGA11, markers of myofibroblasts and protein expression of collagen-I and III in livers compared to vehicle control (FIG. 8B). These data indicate that AXI-I possesses anti-fibrotic properties, which need to be optimized at different doses.

Example 2

[0270] Materials and Methods

[0271] ITGA5 Peptide Selection

[0272] To select the peptidomimetic binding to ITGA5, 12 amino acid overlapping peptides (8 overlap) from the sequence from human fibronectin-III domain 9-10 (Uniprot nr. P02751) were displayed on a cellular membrane as dots (FIG. 11). Peptides were attached to their c-terminal site using stable linker. The cysteine was exchanged with serine to enhance the stability and also because cysteine generally does not make an epitope. The peptide-displaying cellular membrane was soaked in methanol for 1 min and rinsed in Tris-buffered saline (TBS) and washed 3 times. Then, the membrane was blocked with 3% BSA in 0.05% tween-20 containing TBS (TBST) for 3 h at room temperature. Then, the membrane was washed for 10 min with TBST and incubated with 5 ?g/ml ?5?1 human recombinant protein (R&D systems) in 3% BSA in TBST for 1 h at room temperature and then overnight at 4? C. Subsequently, the membrane was washed 3 times with TBST. Thereafter, the bound receptor to the membrane was transferred to the PVDF membrane. To achieve this, first, the PVDF membrane was soaked in methanol for 1 min and then blocked with 5% skimmed fat milk for 2 h. Then, the membrane was incubated with primary anti-alpha5 integrin antibody (Sigma-Aldrich) for overnight in 5% skimmed milk. The membrane was washed 3 times with TBST and then incubated with anti-rabbit-HRP secondary antibody (Dako), washed and developed with chemiluminescence detection kit.

[0273] The following sequences from the fibronectin were used for developing overlapping peptides.

TABLE-US-00003 HumanFNIII-9(Uniprotnr.P02751) GLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRV PHSRNSITLT NLTPGTEYVVSIVALNGREESPLLIGQQST MouseFNIII-9(P11276) AVPPPTDLRFTNIGPDTMRVTWAPPPSIELTNLLVRYSPVKNEEDVAELS ISPSDNAVVLTNLLPGTEYLVSVSSVYEQHESIPLRGRQKT PartialHumanFNIII-10seq. TVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTG RGDSPASSKPISI

[0274] Peptide Binding Study on the Coated Receptor

[0275] The purified human recombinant ?5?1 or ?11?1 receptors (5 ?g/ml in PBS) were coated onto 96-well ELISA plates (White MaxisorbNunc) by incubating overnight at 4? C. Then, the wells were blocked with blocking buffer (5% (w/v) BSA, 150 mM Nacl, 25 mM Tris) for 2 h at room temperature. Then, the wells were washed three times with 200 ?l of washing buffer (150 mM Nacl, 25 mM Tris-base, 0.005% Tween20, 0.5% BSA). Thereafter, the peptides conjugated with PEG(6)-FITC were diluted to different concentrations in washing buffer. For the competitive studies, the wells were co-incubated with 10-fold higher amount of unlabeled peptides. The plates were incubated at 37? C. for 60 minutes. Then, the wells were washed three times with washing buffer and subsequently, the plates were read for the fluorescein signal Ex/Em 485 nm/520 nm using a fluorescent plate reader (Perkin Elmer).

[0276] Binding Studies on Peptide Microarray

[0277] Peptide microarray was prepared by conjugating peptides at their N-terminal to a glass slide using a PEG linker. Each peptide was displayed three times at different positions to avoid artefacts and errors. For the binding studies, the peptide array was blocked with 3% BSA in TBST for 2 h. Then the array slide was washed 5 times in TBST and subsequently incubated with the target receptor (?5?1, 10 g/ml dissolved in PBS) for 1 h at 37? C. The slides were washed with TBST and then incubated with primary antibody (1 g/ml) against ITGA5 for 1 h at 37? C. Then slides were washed and incubated with fluorescent dye labelled secondary antibodies for 30 min. The slides were washed with TBST and water and then dried and scanned with a microarray scanner to detect binding of the peptides. The enlightened spots were analysed using ImageJ software. To determine the unspecific binding, a peptide array was incubated with only primary and secondary antibodies without the receptor incubation step. Then, the signal of unspecific binding was subtracted from the total binding to calculate specific binding.

[0278] Effect Studies

[0279] Human primary pancreatic stellate cells (PSCs) were obtained from ScienCell (Carlsbad, Calif.) and were cultured in specified medium provided by the manufacturer, supplemented with penicillin/streptomycin. Cells were used less than the passage 9 and seeded on a Poly-L-Lysine-coated plate.

[0280] PSCs were seeded into a 12 well plate (6?10.sup.4 cells/well, for gene expression) or 24 well plate (for staining) in complete medium. After 24 h, cells were starved in serum-free medium and then after 24 h they were added with TGF-?1 (5 ng/ml) with/without the peptide.

[0281] After 24 h of incubation, cells were lysed with the lysis buffer and total RNA was isolated using the GenElute? Mammalian Total RNA Miniprep Kit. The RNA amount was measured by a NanoDrop? ND-1000 Spectrophotometer (Wilmington, Del.). Subsequently, cDNA was synthesized with iScript? cDNA Synthesis Kit (BioRad, Veenendaal, The Netherlands). 10 ng cDNA was used for each PCR reaction. The real-time qPCR primers for human ?SMA and RPS18 were purchased from Sigma (The Netherlands). Gene expression levels were normalized to the expression of the house-keeping gene RPS18s.

[0282] For immunostaining, the cells were washed and fixed with acetone-methanol and processed for immunocytochemical staining.

[0283] Immunohistochemical Staining

[0284] Human pancreatic specimens, human liver cirrhosis and kidney fibrosis specimen were obtained from the Department of Pathology, Laboratory Pathology East Netherlands (LabPON), Enschede, The Netherlands. Ethical approvals were obtained from the local Medical Ethical Committee at LabPON. Samples were cut into 2 ?m thick sections using a microtome (Leica Microsystems, Nussloch, Germany). The sections were processed for deparaffinization and then incubated at 80? C. overnight in Dako antigen retrieval buffer to expose antigens. The endogenous peroxidase activity was blocked by 3% H.sub.2O.sub.2 prepared in methanol.

[0285] Sections were then washed with PBS and incubated with the primary antibody (anti-ITGA11, anti-ITGA5 or anti-SMA or anti-CD31) for 1 hr at room temperature. Sections were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 hr at room temperature. Then incubated with Alexa488 or Alexa594-conjugated tertiary antibody for 1 hr, after which these were washed thrice with 1?PBS. Nuclei were counterstained with DAPI containing mounting medium (Sigma).

[0286] For immunocytostaining, cells were fixed with acetone-methanol (1:1) at ?20? C. for 30 min and then dried and rehydrated for 10 min. Cells were then incubated with primary antibodies for 1 h and then with HRP-labelled secondary antibodies for 30 min. Thereafter, peroxidase activity was developed using AEC (3-amino-9-ethyl carbazole) substrate kit (Life Technologies, Gaithersburg, Md.) for 20 min and nuclei were counterstained with hematoxyllin (Fluka Chemie, Buchs, Switzerland). Cells were mounted with Aquatex mounting medium (Merck, Darmstadt, Germany). The staining was visualized and the images were captured using light microscopy (Nikon eclipse E600 microscope, Nikon, Tokyo, Japan).

[0287] Results

[0288] Selection of ITGA5 Binding Peptides

[0289] ITGA5, integrin alpha5, is a known receptor for fibronectin (FN) and to select a peptide ligand against ITGA5, overlapping sequences (12 aa. long with 8 aa. overlaps) from human FN-III domains-9 and 10 were designed and displayed on a cellular membrane. The domains 9 and 10 of FN were chosen to design peptides, as these domains were reported to be responsible for binding to the ?5?1 receptor, as shown with the docking experiments (Nagae et al, 2012 J. Cell Biol. 131-140). The interaction studies were performed against human recombinant integrin ?5?1 receptor and the bound proteins were transferred to another membrane and ITGA5 was detected with antibodies. Many sequences appeared to bind to the ?5?1 receptor from human and mouse domains, the strongest binding was obtained with 2 sequences from human FN-III domain 10 as follows.

TABLE-US-00004 FromhumanFN-IIIdomain9 Seq1. ITANSFTVHWIA-weak Seq2. VALNGREESPLL-veryweak FromhumanFN-IIIdomain10 Seq3. TTVRYYRITYGE-strong Seq4. YYRITYGETGGN-verystrong Seq5. GDSPASSKPISI-moderate FrommouseFN-IIIdomain9 Seq6. SIELTNLLVRYS-moderate Seq7. TNLLVRYSPVKN-moderate

[0290] Since YYRITYGETGGN sequence provided the strongest signal, this sequence was further chemically synthesized and then PEG(6)-Fluorescein was introduced at the N-terminal side of the peptide for its detection during the peptide-binding assays to the coated ?5?1 receptor. Surprisingly, however, no binding was observed to the coated receptor. In the peptide array on the cellular membrane, the peptides were conjugated through the C-terminal, while the N-terminal of the peptides was free to bind to the receptor. However, in the synthetic peptide PEG was conjugated to the N-terminal, which might have blocked the binding of the peptide. Therefore, a new peptide (nAV2) was synthesized in which the PEG(6)-fluorescein was conjugated to the C-terminal by introducing a lysine group to allow PEG conjugation.

[0291] Interestingly, nAV2 showed a good binding to the coated receptor (FIG. 12A). Blocking of the peptide with 10 fold excess of unlabeled peptide blocked the binding of nAV2-PEG(6)-fluorescein. In addition, a new short peptide (RYYRITY, called AV3) was also designed because seq. 3 and seq. 4 (see the sequences above) had YYRITY as common amino acids and the N-terminal of the seq. 4 was crucial for the binding to the receptor. Addition of the excess of AV3 to nAV2-PEG-fluorescein strongly blocked its binding to the receptor, indicating that AV3 has higher affinity to the receptor. Therefore, AV3 was selected to move with further studies. Furthermore we performed binding of nAV2-FITC to LX2 cells and found that the peptide clearly bound to these cells compared to control cells (FIG. 12B).

[0292] Alanine Scanning and Peptide Chain Length Scan

[0293] To find out the amino acids responsible for the peptide binding and the most efficient binding peptides, peptide microarrays were developed with alanine replacement and shorter peptides. In addition a peptide having the sequence RYYRITYC (AV3-Cys) was developed. For AV3 (RYYRITY), the peptide microarray was incubated with ?5?1 receptor, which was captured by anti-?5 and then fluorescent dye labelled secondary antibody. The binding results show that replacement of Y3 led to loss of binding while replacement of R1, R4, and Y7 led to decrease in binding of AV3 peptide. Replacement of T6 induced unspecific binding of the peptide to the incubating antibodies (FIG. 13). Table 2 shows the sequences of ITGA5 binding peptides.

TABLE-US-00005 TABLE 2 Sequence of ITGA5 binding peptides. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 T T V R Y Y R I T Y G E T G G N 17 18 19 20 21 22 T T V R Y Y R I T Y G E Y Y R I T Y G E T G G N R Y Y R I T Y A Y Y R I T Y R A Y R I T Y R Y Y A I T Y R Y Y R A T Y R Y Y R I T A R Y Y R I T Y C Y Y R I T Y G E T G G N K R Y Y R I T Y G G G G L T E W L R W F

[0294] Effect Studies Primary Pancreatic Stellate Cells (PSCs)

[0295] Activation of human skin fibroblasts (BJhtert) with TGF 1 induced gene expression levels of ITGA5 at 8, 24 and 48 h (FIG. 10). In addition, activation of pancreatic stellate cells with TGF? or panc-1 tumor cell conditioned medium also induced the ITGA5 expression levels after 24 h.

[0296] Effect Studies on Skin Fibroblasts and PSC

[0297] Activation of PSCs with TGF? led to activation and differentiation of these cells to myofibroblasts, as indicated by the increased ?-SMA expression. Interestingly, treatment with AV3 peptide significantly inhibited the ?-SMA expression at 20 ?M, showing the anti-fibrotic effects of AV3 peptide (FIG. 14A). In addition, incubation of PSCs with TGF?1 enhanced the expression of fibrotic markers such as ?-SMA, Col-1a1 and vimentin, as shown with immunohistochemical stainings. Treatment with AV3-cys peptide clearly inhibited the expression of these biomarkers, indicating that AV3-cys peptide is able to inhibit the activation and differentiation of PSCs. In contrast, scrambled AV3-cys did not show any inhibitory effects (FIG. 14B).

[0298] In addition, we examined the effect of AV3 and its variants on the migration of human skin fibroblasts. We found that AV3-cys peptide significantly inhibited the migration of fibroblasts while its scrambled version did not show any effect (FIG. 15A). Furthermore, different peptide versions were tested for their effect on the migration of the fibroblasts. Only AV3.3 showed about 30% reduction on migration while other versions showed slight or no effects (FIG. 15B).

[0299] Immunohistochemical Staining of Human Pancreatic Specimens

[0300] We examined the expression of ITGA5 in normal pancreas and pancreatic tumor. We found that normal human pancreas showed no staining of ITGA5 while pancreatic tumor showed a strong staining of ITGA5 in stromal region (FIG. 9A). Co-localization of ITGA5 with ?-SMA (marker for myofibroblasts) and CD31 stainings (marker for endothelial cells) revealed that ITGA5 perfectly coincide with ?-SMA but slightly with CD31, indicating that ITGA5 is highly expressed on myofibroblasts and also on tumor vasculature (FIG. 9B).

Example 3

[0301] Materials and Methods

[0302] Microscale Thermophoresis

[0303] Fluorescent labeled peptide (AV3-PEG(6)-5FAM or AXI-I-PEG(6)-FITC) (1 ?M) was incubated with different concentrations of a human recombinant receptor (i.e. ?5?1, ?11?1 or ?v?3) for 10 min in Eppendorf tubes. The mixture of peptide and receptor was loaded in to NT.115? hydrophilic glass capillaries. In order to find the best thermophoretic setting, the binding of peptide to the target receptor was examined at low (20%), middle (40%), and high (80%) MST power and all other binding experiments were performed using the same MST-settings. Finally, the dissociation constant (Kd) value was calculated from an average of three experiments.

[0304] Tumor Imaging in Mice

[0305] All experiments were conducted under the animal ethical regulation under the Dutch law and protocols were approved by local animal ethical committee. Male SCID mice (approximately 20 g) were obtained from Charles River (Germany). Human pancreatic tumor cells (Panc-1) combined with human pancreatic stellate cells (PSC) were co-injected into the flank of the mice and tumors were allowed to grow to a size of about 200 mm3. AV3 peptide conjugated with 800CW (AV3-800CW) (1 nmol) was injected intravenously alone or with 50-fold excess of unlabeled AV3 (as a blocker) into the tumor-bearing mice. The animals were scanned under anesthesia at 3 h with Pearl optical imager (LICOR) to examine the distribution of the peptide.

[0306] Effects of AV3 Peptides on Human Dermal Fibroblasts

[0307] Human dermal fibroblasts were purchased from ScienCell (Carlsbad, Calif.) and cultured in fibroblasts medium (cat#2301, ScienCell) supplemented with penicillin and streptomycin with 2% FBS. 7?104 cells were seeded in 12-well plate and after 24 h medium was replaced with FCS-free medium and then human recombinant TGF? (5 ng/ml) was added without or with different concentration of AV3 (1, 5, 20 ?M) and scrambled AV3 (sAV3; 5 and 20 ?M) peptides. After 48 h, the cells were lysed with lysis buffer and western blot analyses was performed for analyzing ?-SMA and ?-actin expression.

[0308] Results

[0309] Specificity of Peptides AV3 and AXI-I

[0310] Fluorescent labelled peptides (AV3-PEG(6)-5FAM and AXI-I-PEG(6)-FITC) were examined for their binding affinities against ?5?1 and ?11?1 receptors, respectively using microscale thermophoresis (MST). MST allows peptides to interact with the receptors in solution phase. These peptides were also exposed to an irrelevant receptor of the integrin family ?v?3 and MST analyses were performed.

[0311] It was found that AV3 peptide has a dissociation constant (Kd) value of 97.8 nM against ?5?1 whereas the Kd value against ?v?3 is 36.1 uM (FIG. 16A). Similarly the Kd value of AXI-I peptide against ?11?1 was 149 nM (FIG. 16B) while it did not show any binding to ?v?3 (graph not shown). These data indicate that AV3 and AXI-I are highly specific for their respective integrin receptors.

[0312] Effects of AV3 Peptides on Human Dermal Fibroblasts

[0313] ?-SMA, a marker for fibroblasts activation and differentiation, was analyzed after 48 h of the activation with human recombinant TGF? (5 ng/ml) without or with different concentration of AV3 (1, 5, 20 ?M) and scrambled AV3 (sAV3, 5 and 20 ?M) peptides.

[0314] It was shown that AV3 peptide inhibited fibroblast activation concentration dependently while sAV3 did not show any inhibition (FIG. 17). These results indicate the therapeutic application of AV3 peptide in inhibiting skin scarring.

[0315] Tumor Imaging Using AV3 Peptide

[0316] The distribution of AV3 peptide labeled with 800CW dye (AV3-800CW) in human pancreatic tumor cells was analysed. Human pancreatic tumor cells (Panc-1) combined with human pancreatic stellate cells (PSC) were co-injected into the flank of SCID mice and allowed to grow to a size of about 200 mm3. AV3-800CW (1 nmol) was injected intravenously alone (FIG. 18A) or with (FIG. 18B) 50-fold excess of unlabeled AV3 in tumor bearing mice.

[0317] Images show that AV3-800CW accumulates into the tumor (arrow in FIG. 18A) while blocking with excess of AV3 blocks its accumulation in tumor specifically.