Source: http://www.google.com/patents/US8227413?ie=ISO-8859-1
Timestamp: 2014-03-17 14:30:50
Document Index: 57443446

Matched Legal Cases: ['Application No. 60', 'Application No. 60', 'Application No. 1885', 'Application No. 07827230', 'Application No. 04791852', 'Application No. 1885', 'Application No. 1885', 'Application No. 175197', 'Application No. 1885', 'Application No. 07827230', 'Application No. 1885', 'Application No. 2006']

Patent US8227413 - Compositions and methods for inducing angiogenesis - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsAn isolated peptide comprising an amino acid sequence HWRR as set forth by SEQ ID NO:5, the peptide consists of 4 or 5 amino acids, is provided. Also provided are methods of treating angiogenesis-related pathologies using the peptide of the invention or pharmaceutical compositions comprising same....http://www.google.com/patents/US8227413?utm_source=gb-gplus-sharePatent US8227413 - Compositions and methods for inducing angiogenesisAdvanced Patent SearchPublication numberUS8227413 B2Publication typeGrantApplication numberUS 12/311,866PCT numberPCT/IL2007/001256Publication dateJul 24, 2012Filing dateOct 18, 2007Priority dateOct 19, 2006Also published asEP2106404A2, US20100322856, US20120219498, WO2008047370A2, WO2008047370A3Publication number12311866, 311866, PCT/2007/1256, PCT/IL/2007/001256, PCT/IL/2007/01256, PCT/IL/7/001256, PCT/IL/7/01256, PCT/IL2007/001256, PCT/IL2007/01256, PCT/IL2007001256, PCT/IL200701256, PCT/IL7/001256, PCT/IL7/01256, PCT/IL7001256, PCT/IL701256, US 8227413 B2, US 8227413B2, US-B2-8227413, US8227413 B2, US8227413B2InventorsBritta Hardy, Alexander Battler, Annat Raiter, Chana WeissOriginal AssigneeRamot At Tel-Aviv University Ltd.Export CitationBiBTeX, EndNote, RefManPatent Citations (22), Non-Patent Citations (62), Referenced by (1), Classifications (23), Legal Events (1) External Links: USPTO, USPTO Assignment, EspacenetCompositions and methods for inducing angiogenesisUS 8227413 B2Abstract An isolated peptide comprising an amino acid sequence HWRR as set forth by SEQ ID NO:5, the peptide consists of 4 or 5 amino acids, is provided. Also provided are methods of treating angiogenesis-related pathologies using the peptide of the invention or pharmaceutical compositions comprising same.
2. An isolated peptide comprising an amino acid sequence HWRRP as set forth by SEQ ID NO:7 or HWRRA as set forth in SEQ ID NO:8, wherein the peptide consists of 12 or less amino acids.
3. The isolated peptide of claim 2, wherein said amino acid sequence is set forth by SEQ ID NO:2 or 3.
4. The isolated peptide of claim 2, wherein the peptide is a linear peptide.
5. The isolated peptide of claim 2, wherein the peptide is a cyclic peptide.
6. The isolated peptide of claim 2, wherein the peptide consists of 12 or less amino acids.
7. A composition-of-matter comprising at least one peptide of claim 2.
8. A pharmaceutical composition comprising as an active ingredient at least one peptide of claim 2 and a pharmaceutically acceptable carrier or diluent.
9. A method of inducing angiogenesis in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one peptide of claim 2, to thereby induce angiogenesis in the subject.
10. A method of treating a pathology characterized by insufficient angiogenesis in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one peptide of claim 2, to thereby treat the pathology characterized by insufficient angiogenesis in the subject.
11. The method of claim 10, wherein the pathology characterized by insufficient angiogenesis in the subject is selected from the group consisting of delayed wound-healing, delayed ulcer healing, reproduction associated disorder, arteriosclerosis, ischemic vascular disease, ischemic heart disease, myocardial ischemia, myocardial infarction, heart failure, myocardial dysfunction, myocardial remodeling, cardiomyopathies, coronary artery disease (CAD), atherosclerotic cardiovascular disease, left main coronary artery disease, arterial occlusive disease, peripheral ischemia, peripheral vascular disease, vascular disease of the kidney, peripheral arterial disease, limb ischemia, critical leg ischemia, lower extremity ischemia, cerebral ischemia, cerebro vascular disease, retinopathy, retinal repair, remodeling disorder, von Hippel-Lindau syndrome, diabetes, hereditary hemorrhagic telengiectasia, ischemic vascular disease, Buerger's disease and ischemia associated with neurodegenerative disease such as Parkinson's and Alzheimer's disease.
12. The isolated peptide of claim 2, wherein said peptide binds a glucose-regulated protein (GRP78) as set forth by SEQ ID NO:9 on endothelial cells of the tissue.
13. A composition comprising an agent attached to the peptide of claim 2, wherein the peptide targets said agent to endothelial cells.
14. The composition of claim 13, wherein the agent is selected from the group consisting of a toxin, a chemotherapeutic agent and a radioisotope.
15. A pharmaceutical composition comprising as an active ingredient the composition of claim 13 and a pharmaceutically acceptable carrier or diluent.
16. A method of identifying a putative angiogenic molecule, the method comprising:
(a) incubating the peptide of claim 2 with a glucose-regulated protein (GRP78) or cells expressing said GRP78 under conditions suitable for formation of a complex between the peptide and said GRP78 or said cells expressing GRP78, and
(b) identifying a molecule capable of displacing the peptide from said complex, to thereby identify a putative angiogenic molecule.
17. A pharmaceutical composition comprising as an active ingredient at least one peptide of claim 1 and a pharmaceutically acceptable carrier or diluent.
18. A method of inducing angiogenesis in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one peptide of claim 1 to thereby induce angiogenesis in the subject.
19. A pharmaceutical composition comprising as an active ingredient at least one peptide of claim 3 and a pharmaceutically acceptable carrier or diluent.
20. A method of inducing angiogenesis in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one peptide of claim 3 to thereby induce angiogenesis in the subject.
21. A composition comprising an agent attached to the peptide of claim 1, wherein the peptide targets said agent to endothelial cells.
22. An isolated peptide consisting of the amino acid sequence set forth in SEQ ID NO:4.
23. A pharmaceutical composition comprising as an active ingredient the peptide of claim 22 and a pharmaceutically acceptable carrier or diluent.
24. A method of inducing angiogenesis in a subject, the method comprising administering to the subject a therapeutically effective amount of the peptide of claim 22, to thereby induce angiogenesis in the subject.
RELATED APPLICATIONS This application is a National Phase Application of PCT Application No. PCT/IL2007/001256 having International Filing Date of Oct. 18, 2007, which claims benefit of U.S. Provisional Patent Application No. 60/852,645, filed on Oct. 19, 2006 and U.S. Provisional Patent Application No. 60/897,498, filed on Jan. 26, 2007. The contents of the above Applications are all incorporated herein by reference.
FIELD AND BACKGROUND OF THE INVENTION Angiogenesis is the process of generating new capillary blood vessels and involves an interplay between cells and soluble factors. Thus, activated endothelial cells migrate and proliferate to form new vessels, which are surrounded by layers of periendothelial cells; small blood vessels are surrounded by pericytes and large blood vessels are surrounded by smooth muscle cells.
The possible role of ADAM15 in neovascularization was studied in mice lacking the ADAM15 gene (i.e., ADAM15 knock out mice). The ADAM15 knock out mice exhibit a major reduction in neovascularization compared to wild-type controls (Bohm B B, Aigner T, Roy B Brodie T A, Blobel C P Burkhardt H; Arthritis Rheum. 2005 52, 4 1100-9); a strongly reduced angiogenic response in a model of hypoxia-induced proliferative retinopathy; and significantly smaller tumors which develop after implantation of melanoma cells. Specific candidate substrates for ADAM15 in the context of neovascularization include Notch1 and -4, PECAM-1, VE-cadherin, TIE-2, membrane type 1 MMP and possibly also Kit-ligand. On the other hand, although ADAM15 demonstrates strong and specific interactions with hematopoietic Src family kinases, which are known to be required for VEGF-mediated angiogenesis, nor VEGF or bFGF induce changes in ADAM15 expression in HUVECs.
SUMMARY OF THE INVENTION According to one aspect of the present invention there is provided an isolated peptide comprising an amino acid sequence HWRR as set forth by SEQ ID NO:5, wherein the peptide consists of 4 or 5 amino acids.
The term �method� refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the biotechnology art.
FIGS. 2 a-b depict the ADAM15 protein domains (FIG. 2 a) and amino acid sequence (FIG. 2 b). FIG. 2 a�the amino acid positions of the ADAM15 protein domains refer to the polypeptide set forth by SEQ ID NO:1 (GenBank Accession No. Q13444); FIG. 2 b�amino acid sequence of the ADAM15 protein. Bolded text refers to the metalloprotease domain, disintegrin like domain and EGF like domain. The underlined text corresponding to amino acids 286-297 refers to the ADOPep1 peptide being only in the metaloprotease domain but not in the disintegrin like domain or the EGF like domain.
FIG. 4 is a FACS analysis depicting the binding (in total counts) of Biotinylated ADOPep1 to endothelial cells under normoxia and hypoxia conditions. Note that the binding of ADOPep1 added at 5 micrograms per 100,000 cells to endothelial cells increased under hypoxia. X axis�Intensity of binding.
FIG. 7 is a graph depicting blood perfusion determined in a mouse ischemic hind limb model by laser Doppler blood flow analyzer and demonstrating the in vivo activity of ADOPeps on restoration of blood perfusion in ischemic mouse hind limb. A mouse hind limb model was created by excision of femoral artery of C57Bl mouse hind limb, the non-operated limb served as a control. ADOPep1 (SEQ ID NO:2; red symbols) and 3 (SEQ ID NO:4; green symbols) were injected intra-muscularly one day after surgery at 0.1 (squares) and 1 (circles) micrograms/mouse. Injection of PBS (black diamond) was used as a control. Blood flow was measured using a laser Doppler immediately (T0) or at 7 (T7), 14 (T14) and 21 (T21) days after surgery. The average perfusion of each limb was determined and the perfusion ratio [expressed as Relative Perfusion (ischemic left/control right leg)] was plotted against time. Note the significant increase in blood perfusion (p<0.05) in limb of mice treated with ADOPep1 at 0.1 microgram on day 21 after surgery.
FIGS. 9 a-b are microscopical images of histological sections subjected to von Willebrand Factor immuno-staining of mouse hind limb ischemia 21 days following injection of ADOPep1 (FIG. 9 a) or PBS (FIG. 9 b). Note the significantly higher von Willebrand Factor staining of small vessels in the ADOPep1 treated group (FIG. 9 a) as compared to the control (FIG. 9 b). Magnification �200.
FIGS. 10 a-b are images of Coomassie blue staining of polyacrylamide gel electrophoresis (PAGE) of endothelial cell lysates obtained from cells under hypoxia and analyzed before (FIG. 10 a) and after (FIG. 10 b) immunoprecipitation (IP) of the cells with biotinylated ADOPep1. FIG. 10 a�lane 1�endothelial cell lysate before IP; lane 2�molecular weight markers; FIG. 10 b�lane 3�IP endothelial cell lysate with ADOPep1. Note the major single protein band of 78 kDa following IP with biotinylated ADOPep1.
FIG. 11 is a Western Blot analysis of immune precipitation of endothelial cells lysate with biotinylated ADOPep1 under normoxia and hypoxia conditions. Staining of the nitrocellulose membrane with biotinylated ADOPep1 followed by Chemiluminescent Substrate revealed a band at 78 kDa which was further identified by Mass spectrometry as GRP78 protein (GenBank Accession No. CAB71335; SEQ ID NO:9). Lane 1�IP of endothelial cells lysate under normoxia with biotinylated ADOPep1; lane 2�IP of endothelial cells lysate under hypoxia with biotinylated ADOPep1.
FIG. 12 is Western Blot analysis of immune precipitation of endothelial cells lysate under hypoxia and normoxia with biotinylated ADOPep1. Staining of the nitrocellulose membrane with anti GRP78 antibody (Santa Cruz Biotechnologies, CA, USA) followed by Chemiluminescent Substrate confirmed the identity of the GRP78 protein in the major 78 kDa protein band. Lane 1�IP of endothelial cells lysate under hypoxia with biotinylated ADOPep1; lane 2�IP of endothelial cells lysate under normoxia with biotinylated ADOPep1.
FIGS. 13 a-b are Western blot analyses of immune precipitation of endothelial cells lysate under hypoxia with biotinylated ADOPep1. Staining of the nitrocellulose membrane was performed with Biotinylated ADOPep1 (FIG. 13 a) or anti GRP78 antibody (FIG. 13 b) followed by Chemiluminescent Substrate and confirmed the presence of GRP78 protein.
FIG. 15 is a FACS histogram depicting the binding of Biotinylated ADOPep1 to endothelial cells under normoxia and hypoxia conditions. X axis�Intensity of binding. Binding of ADOPepBiot added at 5 micrograms per 100,000 cells to endothelial cells increased under hypoxia.
FIGS. 16 a-d are FACS analyses of different tumor cell lines using the anti-GRP78 antibody. Anti-GRP78 antibody was added to MCF7 breast carcinoma (FIG. 16 a), HT-29 colon carcinoma (FIG. 16 b), SK-28 melanoma (FIG. 16 c) and K562 erytroblastoma (FIG. 16 d) and the binding to the cells was detected using FACS analysis. Note the increase in percent binding of anti-GRP78 antibody to MCF7, HT29 and SK-28 cell lines. Also note that anti-GRP78 antibody did not bind to the membrane of K562 cells.
FIGS. 18 a-e are dot plot FACS analyses depicting the inhibition of apoptosis by the ADOPep1. Endothelial cells under hypoxia were stained with both Annexin V (shown on the X axis) and Propidium Iodide (shown on the Y axis) apoptotic markers. Cells were incubated with complete medium under normoxia (FIG. 18 a), in the presence of 5% FCS and complete medium under normoxia (FIG. 18 b), in starvation medium under hypoxia for 24 hours (FIG. 18 c), in starvation medium under hypoxia for 24 hours and in the presence of ADOPep1 (FIG. 18 d) or in starvation medium under hypoxia for 24 hours and in the presence of anti-GRP78 antibody (FIG. 18 e). Note that incubation with peptide ADOPep1 induced a remarkable decrease in the stained cells (FIG. 18 d), demonstrating its feasibility to reduce hypoxia-induced apoptosis.
FIGS. 20 a-b depict binding of the anti-GRP78 antibody to endothelial cells following incubation of the cells with ADOPep1. Endothelial cells were incubated for 48 hours under either normoxia conditions, or hypoxia for 24 hours followed by normoxia for another 24 hours, in the presence or absence of the ADOPep1 (at a concentration of 10 ng/ml) and the binding to anti-GRP78 antibody was determined using FACS analysis. FIG. 20 a�a flow cytometry analysis of endothelial cells under normoxia (green plot), hypoxia (for 24 hours) followed by normoxia (for another 24 hours) in the absence (red plot) or presence (blue plot) of the ADOPep1 peptide. Note that while the binding of anti-GRP78 increased from 18.1% under normoxia to 40.1% under hypoxia, a more significant increase (up to 83.8%) was observed when the cells were incubated in the presence of the ADOPep1 under hypoxia conditions. FIG. 20 b�A histogram depicting the percent of anti-GRP78 binding to endothelial cells under normoxia, hypoxia (for 24 hours), normoxia with 10 ng/ml ADOPep1, hypoxia (24 hours) followed by normoxia (24 hours) or hypoxia (24 hours) followed by normoxia (24 hours) in the presence of 10 ng/ml ADOPep1. Results represent average�standard deviations of three independent experiments.
FIGS. 24 a-d are FACS analyses depicting hypoxia induced apoptosis. Endothelial cells were incubated for 24 hours under hypoxia conditions in the absence (FIG. 24 a) or presence of 10 ng/ml of ADOPepe1 (FIG. 24 b), peptide of Motif A (FIG. 24 c) or peptide of motif C (FIG. 24 d) and the level of apoptosis was determined using FACS analysis and the PI (shown on the Y axis)/Annexin V (shown on the X axis) markers. Note that while ADOPep1 and Motif A peptides were capable of inhibiting the hypoxia induced apoptosis from 79.3% under hypoxia to 28.6% (ADOPep1) or 35.8% (Motif A), the motif C peptide exhibited no effect of hypoxia induced apoptosis (81.1%).
FIG. 25 is a bar graph depicting the inhibition of hypoxia induced apoptosis by ADOPep1, Motif A and Motif B. Endothelial cells were incubated for 24 hours under hypoxia conditions in the absence or presence of 10 ng/ml of ADOPepe1, Motif A, Motif B or motif C and the level of apoptosis was determined using FACS analysis and quantified as the percent of Annexin V and PI positive cells. The results represent average�standard deviation of 4 independent experiments. Note that while ADOPep1 and Motif A and B peptides exhibited a significant inhibition of hypoxia induced apoptosis, the Motif C peptide exhibited no effect on apoptosis.
FIGS. 26 a-b are graphs depicting induction of endothelial cell migration under hypoxia by the ADOPEP motifs. Endothelial cells were incubated for 5 hours under hypoxia conditions in the presence of increasing concentrations of ADOPep1 (red line), peptide motif A (dark blue line), peptide motif B (green line) or peptide motif C (light blue line) and the migration of endothelial cells was detected. FIG. 26 a�experiment 1; FIG. 26 b�experiment 2. Note that while the ADOPep1, Motif A and B peptides induced endothelial cell migration at a concentration of about 10 ng/ml, motif C peptide exhibited no significant effect on endothelial cell migration.
FIG. 27 is a graph depicting induction of tube formation using AdoPep1 and Motif A (SEQ ID NO:7) but not the scrambled sROY peptide. Endothelial cells were incubated in media without supplements for 24 hours, and 50,000 cells were then transferred in 500 μl medium to 24-well plates precoated with 250 μl Cultrex Basement Membrane Extract (with reduced growth factors) (R&D Systems, Minneapolis, Minn., USA). AdoPep1, Motif A and scrambled sROY peptides were added at an optimal concentration of 10 ng/ml (based on preliminary findings), and the slides were examined by light microscopy after 18 hours incubation. The results represent average�SD of three experiments using 3 different cords. The length of the network of connected cells (tube formation) was measured in micrometers in 5 different areas of each well using Image-Pro Plus Image software (Media Cybernetics, Silver Spring, Md., USA). Note that ADOPep1 and Motif A significantly increased the length of the network of connected cells in endothelial cells under starvation and normoxic conditions while scrambled sROY (as a control peptide), did not induced tube formation.
FIGS. 28 a-d are FACS histograms (FIGS. 28 a-c) and a graph (FIG. 28 d) demonstrating that AdoPep1 and Motif A peptides compete on the binding to the same receptor on endothelial cells. Endothelial cells were cultured for 24 hours under hypoxic conditions in endothelial cell growth medium. Cells were removed by trypsin and incubated for 1 hour on ice with increasing concentrations of AdoPep1, Motif A and Motif C peptides. GRP78 polyclonal antibody (2 μg/100,000) was added to the cells for 2 hours on ice. Anti-goat FITC (Jackson ImmunoResearch) was added for 30 minutes on ice. IgG1-FITC was used as the isotype control. The samples were analyzed with a FACScan (Beckton Dickinson). Note that AdoPep1 peptide (FIG. 28 a) and Motif A peptide (FIG. 28 b) but not Motif C peptide (FIG. 28 c) inhibit the binding of GRP78 to endothelial cells. FIG. 28 d depicts the average percent binding of GRP78 to endothelial cells as a function of peptide amount as determined by 2 independent experiments.
FIGS. 30 a-b are Western blot analyses depicting induction of phosphorylation of ERK by ADoPep1 and Motif A. Endothelial cells under starvation conditions and 5 hours hypoxia were incubated for 5 (lanes 2 and 6), 20 (lanes 3 and 7), 30 (lanes 4 and 8) and 60 (lanes 5 and 9) minutes with 10 ng/ml ADoPep1 (Lanes 2-5) or Motif A (lanes 6-9) peptides or remained under hypoxia without a further incubation with a peptide (lane 1). After incubation, lysates were prepared, subject to SDS-PAGE and blotting on a nitrocellulose membrane. Western Blot analysis was performed using anti-Phospho ERK1/2 antibody. Note the induction of ERK1/2 phosphorylation after 20 minutes incubation with ADoPep1 and Motif A under hypoxia conditions. Densitometry measurements showed a maximal ERK phosphorylation after 20 minutes incubation with ADOPep1 and from 20 to 60 minutes with Motif A (data not shown). FIG. 30 a�Experiment 1; FIG. 30 b�Experiment 2. The percent adjusted volume as determined by a densitometric analysis software to compare net band densities was as follows: FIG. 30 a, lane 1�4.5, lane 2�9.3, lane 3�13.7, lane 4�13.3, lane 5�11.2, lane 6�10.7, lane 7�12.4, lane 8�12.3 and lane 9�12.4; FIG. 30 b, lane 1�4.21, lane 2�12.4, lane 3�12.5, lane 4�14.8, lane 5�10.9, lane 6�14.9, lane 7�9.8, lane 8�12.7 and lane 9�7.8.
FIG. 31 is a Western blot analysis depicting the level of phosphorylated ERK (pERK) in endothelial cells activated for 20 minutes with ADOPep1 and motif A and inhibited by pERK-inhibitor peptide. Endothelial cells (from cord 1) under starvation conditions and 5 hours hypoxia were incubated with 10 ng/ml ADOPep1 (lanes 3 and 4) or motif A (lanes 1 and 2) peptides in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of p-ERK-inhibitor peptide (Santa Cruz). For control, the endothelial cells under starvation conditions and 5 hours hypoxia (lane 5) were also incubated with the p-ERK-inhibitor peptide (lane 6). Note the inhibition of ERK phosphorylation in the presence of the p-ERK-inhibitor peptide. The percent adjusted volume as determined by a densitometric analysis software to compare net band densities was as follows: lane 1�19, lane 2�14, lane 3-14, lane 4�8.8, lane 5�11, lane 6�2.1.
FIGS. 32 a-b are Western blot analyses depicting the effect of ADOPeps on the level of phosphorylated ERK. Endothelial cells (EC) from cord 1 (FIG. 32 a) or cord 2 (FIG. 32 b) under hypoxia (5 hours) were incubated for 20 or 30 minutes with 10 ng/ml of AdoPep1, AdoPep2 and AdoPep3 or remained untreated (i.e., under hypoxia without any peptide). Western Blot analyses of samples cell lysates were performed with anti Phospho ERK antibody on nitrocellulose membranes. FIG. 32 a: Lanes 1 and 2�EC under hypoxia (untreated); lanes 3 and 4�EC under hypoxia incubated with ADOPep3 for 20 (lane 3) or 30 (lane 4) minutes; lanes 5 and 6�EC under hypoxia incubated with ADOPep2 for 20 (lane 5) or 30 (lane 6) minutes; lanes 7 and 8�EC under hypoxia incubated with ADOPep1 for 20 (lane 7) or 30 (lane 8) minutes; FIG. 32 b: Lanes 1-4�EC under hypoxia incubated for 20 minutes with ADOPep1 (lane 2), ADOPep2 (lane 3), ADOPep3 (lane 4) or remained untreated (lane 1); lanes 5-8�EC under hypoxia incubated for 30 minutes with ADOPep1 (lane 6), ADOPep2 (lane 7), ADOPep3 (lane 8) or remained untreated (lane 5); Densitometry measurements showed an increase in ERK1/2 phosphorylation after incubation of endothelial cells with ADOPep1 and 2 but not with AdoPep3. The percent adjusted volume as determined by a densitometric analysis software to compare net band densities was as follows: FIG. 32 a, lane 1�11, lane 2�13, lane 3�11, lane 4�5, lane 5�11, lane 6�13, lane 7�17, lane 8�16.7; FIG. 32 b, lane 1�17, lane 2�22, lane 3�19, lane 4�7, lane 5�8, lane 6�13, lane 7�14, lane 8�1.
FIGS. 33 a-f are representative FACS analyses depicting a specific inhibition of hypoxia-induced apoptosis of endothelial cells by ADOPep1. Endothelial cells in Petri dishes were incubated for 24 hours with 5% FCS in supplement-free endothelial cell growth medium under normoxia (FIGS. 33 a and d), exposed to 24 hours hypoxia (Figures c and f) or incubated under normoxia with 100 micromolar per 100,000 cells CoCl2 (FIGS. 33 b and e), in the absence (FIGS. 33 a-c) or presence (FIGS. 33 d-f) of 10 ng/ml Adopep1 peptide (for 24 hours). Cells were trypsinized and immediately re-suspended in PBS with 5% FCS and 0.1% Na-azide. Samples containing 100,000 cells were tested for apoptosis using the Annexin-FITC (X axis) and propidium iodide (Y axis) kit (Bender Medsystems, Vienna, Austria), according to manufacturer's instructions. Results were analyzed by FACScan (Beckton Dickinson). Note that while CoCl2 and hypoxia conditions increased the fraction of apoptotic cells (indicated by the increase of PI and Annexin-FITC positive cells in FIGS. 33 b and c as compared to FIG. 33 a), when the cells were incubated with ADOPep1 peptide, a significant fraction of hypoxia-induced apoptosis was inhibited (compare PI and Annexin-FITC positive cells in FIG. 33 f to those in FIG. 33 c), however, no effect on apoptosis induced by CoCl2 was observed (compare PI and Annexin-FITC positive cells in FIG. 33 e to those in FIG. 33 b).
FIG. 34 is a histogram depicting the FACS results of the experiments described in FIGS. 33 a-f. Data represents average�SD of 2 experiments. Note that 24 hours of hypoxia or CoCl2 treatment increased the level of apoptosis from about 27% (under normoxia) to about 70% or 67%, respectively. Also note that ADOPep1 treatment significantly inhibited the hypoxia induced apoptosis by approximately 40 percent but not the CoCl2 induced apoptosis.
DESCRIPTION OF EMBODIMENTS OF THE INVENTION Some embodiments of the invention relate to peptides which can bind to endothelial cells via the GRP78 receptor and induce angiogenesis and to uses thereof for treating pathologies characterized by insufficient angiogenesis such as ischemic diseases.
As described in the Examples section which follows, the peptides of the invention (e.g., ADOPeps-1, 2 and 3 as set forth by SEQ ID NOs: 2, 3 and 4, respectively, or the peptides set forth by SEQ ID NOs:7 and 8) bind to endothelial cells in vitro (FIGS. 3 and 4) and induce endothelial cell proliferation (FIG. 5) and migration (FIGS. 6 and 26 a-b) under hypoxia. In addition, in vivo experiments utilizing the mouse hind limb ischemia model have shown that treatment of the ischemic mice with the peptides of the invention (e.g., ADOPep1) significantly increases blood perfusion (FIG. 7) and blood vessel density (FIGS. 8, 9 a-b). Moreover, immuno precipitation (IP) and Western blot analyses revealed that the receptor of ADOPeps on endothelial cells is the glucose-regulated protein (GRP78; SEQ ID NO:9) (FIGS. 10 a-b, 11, 12) which is expressed on various tumor cells (FIGS. 16 a-d) and that the peptides of the invention (e.g., ADOPep1) bind to GRP78 on endothelial cells under hypoxia (FIGS. 13 a-b and 19). In addition, while hypoxia conditions increase the presentation of the GRP78 receptor on endothelial cells to about 40% (FIG. 15), pre-incubation of endothelial cells with the ADOPep1 further increases GRP78 presentation on endothelial cells to about 84% (FIGS. 20 a-b, 21). Furthermore, the peptides of the invention (e.g., ADOPep1, or shorter peptides such as SEQ ID NOs: 7 and 8) prevented hypoxia-induced apoptosis of endothelial cells both in vitro (FIGS. 17, 18, 24 a-d and 25) and in vivo (FIG. 22) but not Cobalt Chloride-induced apoptosis (FIGS. 33 a-f and 34). In addition, incubation of endothelial cells with the peptides of the invention (e.g., SEQ ID NO:2 or 7) result in tube formation (FIG. 27) and a significant increase in ERK1/2 phosphorylation (FIGS. 30 a-b, 31). Altogether, these results demonstrate the ability of the peptides of the invention to induce angiogenesis in a tissue under hypoxia, and suggest their use for treating ischemic diseases.
Exemplary isolated peptides of the invention
HWRRA
HWRRN
HWRRQ
HWRRE
HWRRG
HWRRH
HWRRI
HWRRL
HWRRK
HWRRF
HWRRS
HWRRV
AHWRR
RHWRR
NHWRR
QHWRR
EHWRR
GHWRR
HHWRR
IHWRR
LHWRR
KHWRR
FHWRR
PHWRR
THWRR
WHWRR
YHWRR
VHWRR
It will be appreciated that the length (�x�) of the peptide of the invention can be any integer with a value which is at least �n� and no more than �y�. Thus, n≦x≦y, wherein n<y and whereas �n� is an integer having a value between 4 to 49 and �y� is an integer having a value between 5 and 50.
The term �peptide� as used herein encompasses native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S═O, O═C�NH, CH2-O, CH2-CH2, S═C�NH, CH═CH or CF═CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein.
Further details in this respect are provided hereinunder. Peptide bonds (�CO�NH�) within the peptide may be substituted, for example, by N-methylated bonds (�N(CH3)�CO�), ester bonds (�C(R)H�C�O�O�C(R)�N�), ketomethylen bonds (�CO�CH2-), α-aza bonds (�NH�N(R)�CO�), wherein R is any alkyl, e.g., methyl, carba bonds (�CH2-NH�), hydroxyethylene bonds (�CH(OH)�CH2-), thioamide bonds (�CS�NH�), olefinic double bonds (�CH═CH�), retro amide bonds (�NH�CO�), peptide derivatives (�N(R)�CH2-CO�), wherein R is the �normal� side chain, naturally presented on the carbon atom. These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) at the same time.
As used herein in the specification and in the claims section below the term �amino acid� or �amino acids� is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other less common amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term �amino acid� includes both D- and L-amino acids.
As used herein a �retro peptide� refers to peptides that are made up of L-amino acid residues which are assembled in opposite direction to the native peptide sequence.
Retro-inverso modification of naturally occurring polypeptides involves the synthetic assembly of amino acids with α-carbon stereochemistry opposite to that of the corresponding L-amino acids, i.e., D- or D-allo-amino acids in inverse order to the native peptide sequence. A retro inverso analogue, thus, has reversed termini and reversed direction of peptide bonds, while essentially maintaining the topology of the side chains as in the native peptide sequence.
The peptides of the invention may be synthesized by any techniques that are known to those skilled in the art of peptide synthesis. For solid phase peptide synthesis, a summary of the many techniques may be found in: Stewart, J. M. and Young, J. D. (1963), �Solid Phase Peptide Synthesis,� W. H. Freeman Co. (San Francisco); and Meienhofer, J (1973). �Hormonal Proteins and Peptides,� vol. 2, p. 46, Academic Press (New York). For a review of classical solution synthesis, see Schroder, G. and Lupke, K. (1965). The Peptides, vol. 1, Academic Press (New York).
The peptides of the invention can be retrieved in �substantially pure� form. As used herein, �substantially pure� refers to a purity that allows for the effective use of the peptide in the diverse applications, described herein (e.g., for therapeutic or diagnostic purposes).
As used herein the term �subject� refers to a mammal, such as a canine, a feline, a bovine, a porcine, an equine or a human subject.
As used herein the phrase �a subject in need thereof� refers to a subject who is diagnosed with, predisposed to or suffers from an angiogenesis-dependent pathology.
As used herein the phrase �angiogenesis-dependent pathology� refers to any pathology (i.e., a condition, disease or disorder) which is characterized by and/or results from disregulated angiogenesis, i.e., insufficient angiogenesis or excess of angiogenesis.
According to an embodiment of the invention, the angiogenesis-dependent pathology according to this aspect of the invention refers to a pathology which is characterized by and/or results from insufficient angiogenesis. Examples include but are not limited to delayed wound-healing, delayed ulcer healing, reproduction associated disorders, arteriosclerosis, ischemic vascular disease, ischemic heart disease, myocardial ischemia, myocardial infarction, heart failure, myocardial dysfunction, myocardial remodeling, cardiomyopathies, coronary artery disease (CAD), atherosclerotic cardiovascular disease, left main coronary artery disease, arterial occlusive disease, peripheral ischemia, peripheral vascular disease, vascular disease of the kidney, peripheral arterial disease, limb ischemia, critical leg ischemia, lower extremity ischemia, cerebral ischemia [e.g., such as cerebral ischemia in childhood moyamoya disease (Touho H. 2007, Surg Neurol. June 20; Epub ahead of print)], cerebro vascular disease, retinopathy, retinal repair, remodeling disorder, von Hippel-Lindau syndrome, diabetes, hereditary hemorrhagic telengiectasia, ischemic vascular disease, Buerger's disease, and ischemia associated with neurodegenerative disease such as Parkinson's and Alzheimer's disease.
The term �treating� refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and/or causing the reduction, remission, or regression of a pathology. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.
As used herein a �pharmaceutical composition� refers to a preparation of one or more of the active ingredients described herein (i.e., at least one peptide of the peptides of the invention) with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
Herein the term �active ingredient� refers to the peptide of the invention accountable for the biological effect.
Fusions between the peptides of the invention and the abovedescribed agent can be generated using a variety of bifunctional protein-coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP) (e.g., essentially as described in Cumber et al. 1985, Methods of Enzymology 112: 207-224), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde; essentially as described in G. T. Hermanson, 1996, �Antibody Modification and Conjugation, in Bioconjugate Techniques, Academic Press, San Diego), bisazido compounds (such as bis-(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene) or carbodiimide conjugation procedure (as described in J. March, Advanced Organic Chemistry: Reaction's, Mechanism, and Structure, pp. 349-50 & 372-74 (3d ed.), 1985; B. Neises et al. 1978, Angew Chem., Int. Ed. Engl. 17:522; A. Hassner et al. Tetrahedron Lett. 4475; E. P. Boden et al. 1986, J. Org. Chem. 50:2394 or and L. J. Mathias 1979, Synthesis 561). For example, a ricin fusion can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the peptide. See WO94/11026; U.S. Pat. No. 6,426,400; Laske, D. W., Youle, R. J., and Oldfield, E. H. (1997) Tumor regression with regional distribution of the targeted toxin TF-CRM107 in patients with malignant brain tumors. Nature Medicine 3:1362-1368.
Additionally or alternatively, the agent of the invention can be attached to the peptide of the invention via recombinant DNA technology by constructing an expression vector which comprises the coding sequence of the agent of the invention (e.g., the PE38KDEL truncated form of pseudomonas exotoxin A) translationally fused to the coding sequence of the peptide of the invention (e.g., SEQ ID NO:2) and expressing the construct in a host cell (e.g., a prokaryotic or eukaryotic cell) for the production of a recombinant fusion peptide comprising the amino acids of the agent and the peptide of the invention. Alternatively, the expression vector can be administered to the subject in need of therapy via known gene therapy techniques (e.g., using vial vehicles).
The cancer or cancer metastases which can be treated by the composition of the invention include, but is not limited to, tumors of the gastrointestinal tract (colon carcinoma, rectal carcinoma, colorectal carcinoma, small and/or large bowel carcinoma, esophageal carcinoma, stomach carcinoma, pancreatic carcinoma), gallbladder carcinoma, Biliary tract tumors, prostate cancer, renal cancer (e.g., Wilms' tumor), liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma), bladder cancer, embryonal rhabdomyosarcoma, germ cell tumor, trophoblastic tumor, testicular germ cells tumor, immature teratoma of ovary, uterine, epithelial ovarian, sacrococcygeal tumor, choriocarcinoma, placental site trophoblastic tumor, epithelial adult tumor, ovarian carcinoma, cervical carcinoma, small-cell and non-small cell lung carcinoma, nasopharyngeal, breast carcinoma, squamous cell carcinoma (e.g., in head and neck), neurogenic tumor, astrocytoma, ganglioblastoma, neuroblastoma, lymphomas (e.g., Hodgkin's disease, non-Hodgkin's lymphoma, B cell, Burkitt, cutaneous T cell, histiocytic, lymphoblastic, T cell, thymic), gliomas, adenocarcinoma, adrenal tumor, brain malignancy (tumor), various other carcinomas (e.g., bronchogenic large cell, ductal, Ehrlich-Lettre ascites, epidermoid, large cell, Lewis lung, medullary, mucoepidermoid, oat cell, small cell, spindle cell, spinocellular, transitional cell, undifferentiated, carcinosarcoma, choriocarcinoma, cystadenocarcinoma), ependimoblastoma, epithelioma, erythroleukemia (e.g., Friend, lymphoblast), fibrosarcoma, giant cell tumor, glial tumor, glioblastoma (e.g., multiforme, astrocytoma), glioma hepatoma, heterohybridoma, heteromyeloma, histiocytoma, hybridoma (e.g., B cell), hypernephroma, insulinoma, islet tumor, keratoma, leiomyoblastoma, leiomyosarcoma, leukemia (e.g., acute lymphatic, acute lymphoblastic, acute lymphoblastic pre-B cell, acute lymphoblastic T cell leukemia, acute�megakaryoblastic, monocytic, acute myelogenous, acute myeloid, B cell, basophilic, chronic myeloid, chronic, B cell, eosinophilic, Friend, granulocytic or myelocytic, hairy cell, lymphocytic, megakaryoblastic, monocytic, monocytic-macrophage, myeloblastic, myeloid, myelomonocytic, plasma cell, pre-B cell, promyelocytic, subacute, T cell, lymphoid neoplasm), lymphosarcoma, melanoma, mammary tumor, mastocytoma, medulloblastoma, mesothelioma, metastatic tumor, monocyte tumor, multiple myeloma, myelodysplastic syndrome, myeloma, nephroblastoma, nervous tissue glial tumor, nervous tissue neuronal tumor, neurinoma, neuroblastoma, oligodendroglioma, osteochondroma, osteomyeloma, osteosarcoma (e.g., Ewing's), papilloma, transitional cell, pheochromocytoma, pituitary tumor, plasmacytoma, retinoblastoma, rhabdomyosarcoma, sarcoma (e.g., Ewing's, histiocytic cell, Jensen, osteogenic, reticulum cell), schwannoma, subcutaneous tumor, teratocarcinoma (e.g., pluripotent), teratoma, testicular tumor, thymoma and trichoepithelioma.
To facilitate complex detection, the peptides of the invention are highlighted by a tag or an antibody. It will be appreciated that highlighting can be effected prior to, concomitant with or following complex formation, depending on the highlighting method. As used herein the term �tag� refers to a molecule, which exhibits a quantifiable activity or characteristic. A tag can be a fluorescent molecule including chemical fluorescers, such as fluorescein or polypeptide fluorescers, such as the green fluorescent protein (GFP) or related proteins (www dot clontech dot corn). In such case, the tag can be quantified via its fluorescence, which is generated upon the application of a suitable excitatory light. Alternatively, a tag can be an epitope tag, a fairly unique polypeptide sequence to which a specific antibody can bind without substantially cross reacting with other cellular epitopes. Such epitope tags include a Myc tag, a Flag tag, a His tag, a leucine tag, an IgG tag, a streptavidin tag and the like.
General Materials and Methods Peptide synthesis�ADOPep1 (SEQ ID NO:2), ADOPep2 (SEQ ID NO:3) and ADOPep3 (SEQ ID NO:4) were synthesized by SynPep (Dublin, Calif., USA) according to the ADAM15 amino acid sequence (GenBank Accession No. Q13444). HPLC purity was greater than 97%. The peptides were dissolved in water in a concentration of 1 mg/ml.
Binding of ADOPep to endothelial cells�Human umbilical endothelial cells (HUVEC) were harvested by trypsin and 100,000 cells per sample were suspended in PBS+5% FCS+0.1% Na azide. Endothelial cells were either exposed for 5 hours to hypoxia or remained under normoxia conditions. Cells were stained for 2 hours on ice in dark with 0.05, 0.5, 5 μg/100,000 cells biotinylated ADOPeps. The cells were then washed twice with PBS, following which the cells were stained for 30 minutes with FITC-labeled Streptavidin (Jackson ImmunoResearch Laboratories, PA, USA). After washing, samples were analyzed using a fluorescence activated cell sorter (FACScan Beckton Dickinson, CA, USA).
Proliferation of endothelial cells incubated with ADOPeps�HUVEC from passage 3 were used for proliferation experiments. Endothelial cells (15,000 cells/well) were seeded on 24-wells in the presence of endothelial cell growth medium (Promocell, Heidelberg, Germany) with supplements and incubated for 24 hours. After then, cells were exposed to 24 hours starvation in a medium free of supplements. ADOPeps were added at concentrations of 1, 10 and 100 ng/ml for 24 hours under hypoxic conditions. 2 μCi/well of Thymidine (SIGMA, Rehovot Israel) were added or over night incubation followed by 3 washes with PBS before harvesting at 37� C. with 300 μl/well of 0.5 M NaOH. Cell lysates were transferred to scintillation vials with 2 ml scintillation liquid Ultima Gold (Packard Bioscience, Meriden, USA) and counted in a β counter. Results were obtained as counts per minute (cpm).
Migration of endothelial cells induced by ADOPeps�Endothelial cell migration was evaluated by the Chemicon QCM 96-well Migration Assay (Chemicon International, CA, USA) according to manufacturer's instructions. This kit utilizes a membrane with an 8 μm pore size. Migratory cells on the bottom of the insert membrane are dissociated from the membrane when incubated with cell detachment buffer provided by the kit. These cells are subsequently lysed and detected by a molecular probe CyQuant GR dye, a green fluorescent dye which exhibits fluorescent enhancement when bound to cellular nucleic acid. For the migration assay, HUVEC from passage 3 were incubated in endothelial cell (EC) growth medium free of supplements. After trypsinization, 25,000 EC were incubated in the migration chamber. ADOPeps were added at concentrations of 1, 10 and 100 ng/ml in the feeder tray for chemoattractant migration assay. Time of incubation was 5 hours for endothelial cells under hypoxic conditions. Results were determined in a Fluorescent ELISA reader at 480/520 nm.
Mouse hind-limb ischemia model for evaluation of the in vivo angiogenic activity of the ADOPeps�A mouse hind limb ischemia model was used. Ischemia was created in the C57Bl strain mouse by ligation and excision of the femoral artery on the left hind limb. The right hind limb served as a control. A day after the operation ADOPep1 and 3 were injected intramuscular at a site close to the operation. Each mouse was treated one day after operation, with either peptide in a total amount of 0.1 or 1 μg per mouse or PBS (50 μl) which was injected as a control. The blood perfusion was measured using a Laser Doppler Blood perfusion analyzer (Perimed, Sweden) one day post operation and 7, 14, 21 days post operation. The average perfusion of each limb was computed and percent perfusion ratio was expressed as the ischemic (left)/control (right) percent perfusion ratio.
Histological examination�Limbs from mice treated with ADOPep1, 3 or control mice injected with PBS were sacrificed at 7, 14 and 21 days post operation. Whole ischemic and non-ischemic legs were immediately fixed for 48 hours in 4% paraformaldehyde and then embedded in paraffin. Three-micrometer thick sections were prepared and cut with the muscle fibers oriented transversely. Identification of endothelial cells was performed by immunostaining for von Willebrand Factor VIII related antigen using a primary antibody of polyclonal anti-factor VIII at a 1/200 dilution (DakoCytomation, Denmark) and secondary antibody the Envision+ System-HRP (DakoCytomation, Denmark). Vessel densities were expressed as capillaries per millimeter squared. To obtain the average vessel number per cross-section area, a minimum of ten individual fields were sampled, and Image Pro-Plus (MediaCybernetics, Silver spring, MD, USA) was used to measure the counted area. The number of Factor VIII positive vessels was counted.
Preparation of HUVEC lysates�HUVEC were seeded in 90 mm Petri dishes plates for 24 hours with complete Endothelial Cell Growth Medium (PromoCell. Heildelberg, Germany). Cells were washed twice with PBS and buffer lysate (50 mM Tris Cl (pH 8, 150 mM Na Cl, 0.02% Na azide, 0.1% SDS, 100 μg/ml PMSF, 1 μg/ml protease inhibitors, 1% NP-40) was added for 20 minutes on ice. After incubation, cells were scraped with a rubber policeman and transferred to a chilled microfuge tube. After centrifugation of lysate at 12,000 g for 2 minutes at 4� C., supernatant was transferred to a fresh microfuge tube and stored at −70� C.
Immunoprecipitation (IP)�Aliquots samples of lysates (500 μl) were incubated with 10 μg of biotinylated ADOPep1 for one hour at 4� C. by gently mixing. Streptavidin Sepharose beads (Amersham Biosciences, Uppsla, Sweden) (50 μl) were added to the sample for a second incubation one hour at 4� C. by gently mixing. After then, sample was centrifuge for 20 seconds at 12,000 g and the pellet was saved and further washed 3 times. Finally the pellet was suspended in 100 μl of sample buffer for Western blot analysis. For ELISA, protein was eluted by 5 minutes incubation of the sample at room temperature in 1 ml of Tris-Glycin 0.2 M (pH 2.2), followed by titration with 1 M Tris (pH 9.1).
Polyacrylamide Gel Electrophoresis (PAGE) analysis�The protein band containing GRP78 which was immunoprecipitated with the biotinylated ADOPep1 (GRP78 immune-precipitated protein) (30 μl) was applied to minigel lane and run at standard conditions (60 mA for 2 minigels, 1.4 hours) in a 10% Tris Acrylamide gel. Gel was stained with Coomassie blue. Band was cut and sent to mass-spectroscopy for protein identification.
Western Blot analysis�Following PAGE analysis, proteins transfer was performed for 2 hours in wet conditions at 40 V in a nitrocellulose membrane, following which the gel was stained with Coomassie blue. The nitrocellulose membrane was blocked for 2 hours at room temperature by incubation with PBS containing 0.5% Tween-20 and 5% non-fat milk. Incubation of the membrane with biotinylated ADOPep1 (5 μg/ml in PBS-Tween) was performed over night at 4� C. by gently shaking. After then, membrane was washed 3 times (15 minutes each) in PBS-Tween. Incubation with the secondary antibody Peroxidase-conjugated Streptavidin (1 μg/ml) (JacksonImmunoResearch, PA, CA USA) was performed for 45 minutes at room temperature followed by 3 washes (15 minutes each) in PBS-Tween. For GRP78 immunostaining, the membrane was blocked as described hereinabove and further incubated for 24 hours at 4� C. with anti-GRP78 antibody (Santa Cruz Biotechnologies, CA, USA) at a concentration of 2 micrograms per ml, followed by 3 washes with PBS-Tween. Incubation with a secondary anti-goat FITC (Jackson ImmunoResearch Laboratories, PA, USA) at a dilution of 1:5000 was performed for 45 minutes at room temperature, followed by 3 washes in PBS-Tween. ECL was performed using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Ill., USA).
FACS analysis of anti GRP78 binding to Endothelial Cells�HUVEC were harvested by trypsin and 100,000 cells per sample were suspended in PBS+5% FCS+0.1% Na azide. Goat polyclonal anti GRP78 (Santa Cruz Biotechnologies, CA, USA) at a concentration of 1 μg/100,000 cells was added for 40 minutes on ice. Cells were washed and stained with anti-goat FITC (Jackson ImmunoResearch Laboratories, PA, USA). Samples were analyzed using a fluorescence activated cell sorter (FACScan Beckton Dickinson, CA, USA).
FACS analysis of anti GRP78 binding to different tumor cells�MCF7 breast carcinoma, SK28 Melanoma, HT 29 colon carcinoma and K562 erythroleukemia were harvested by trypsin and 300,000 cells per sample were suspended in PBS+5% FCS+0.1% Na azide. Goat polyclonal anti GRP-78 (Santa Cruz Biotechnologies, CA, USA) at a concentration of 1 μg/100,000 cells was added for 40 minutes on ice. Cells were washed and stained with anti-goat FITC (Jackson ImmunoResearch Laboratories, PA, USA). Samples were analyzed using a fluorescence activated cell sorter (FACScan Beckton Dickinson, CA, USA).
Apoptosis�EC were incubated for 24 hours with 5% FCS in EC growth media in Petri dishes plates followed by 24 hours incubation under hypoxia conditions with ADOPep1 (10 ng/ml), anti GRP78 antibody (Santa Cruz Biotechnology, CA, USA) (1 microgram/ml) or recombinant VEGF (10 ng/ml). The Annexin V FITC/PI detects the phosphatidylserin on the apoptotic cells using flow cytometry. Human Annexin V-FITC kit (Bender Medsystems, Vienna, Austria) was used for the measurement of EC apoptosis percentage following manufacturer's instructions. Samples were analyzed using a fluorescence activated cell sorter (FACScan Beckton Dickinson, CA, USA).
Competitive binding of ADOPep1, ADOPep2 and ADOPep3 peptides to GRP78�Endothelial cells (20,000 per well) were seeded in 96 well plates for 24 hours in the presence of the complete medium. Plates were washed with PBS over night and rehydrated with PBS, 0.1% Na Azide and 5% FCS. ADOPep1, ADOPep2 and ADOPep3 were added to washed plates for 2 hours at room temperature at concentrations of 0.01, 0.1 and 1 microgram per ml. Anti-GRP78 antibody (goat polyclona IgG, Santa Cruz Biotechnology, CA, USA) was added to the plates for 1 hour at room temperature at a concentration of 2 micrograms/ml per well. After washing, bound anti-GRP78 antibody was detected by incubation with anti-goat IgG Peroxidase conjugated (Jackson Immuneresearch Laboratories, PA, USA). After 5 washes with PBS-0.1% Tween 20, 100 μl/well of TMB+ Substrate-Chromogen (DAKOCytomation, CA, USA) was added for a maximum of 30 minutes. Reaction was stopped with 1 N HCl. Color developed was determined by an ELISA reader at 450 nm.
Example 1 Specific Peptides Derived from the Metalloprotease Domain of ADAM15 Bind to Endothelial Cell and Induce Proliferation and Migration Thereof Experimental Results
ADOPep1 Sequence�Several peptides were synthesized from the metalloprotease domain and preliminary experiments were conducted in order to select a peptide with the best binding ability to endothelial cells. One of these, a peptide termed ADOPep1, has the amino acid sequence set forth by SEQ ID NO:2, HWRRAHLLPRLP. Its location in the metalloprotease domain of ADAM15 molecule (SEQ ID NO:1) is presented in FIG. 2 b (underlined text corresponding to amino acids 286-297 of SEQ ID NO:1).
Binding of ADOPep1 to endothelial cells (EC) by FACS analysis�Increasing concentrations of biotinylated ADOPep1 were added to EC under normoxia conditions and the binding of ADOPep1 to EC was detected using FITC-labeled Streptavidin and FACS analysis. As can be seen in FIG. 3 an increase in percent binding of ADOPep1 to EC reached about 62% in a dose dependent manner and was maximal at 5 micrograms per ml. When EC under hypoxia conditions (for 5 hours) were incubated with biotinylated ADOPep1, the binding of the ADOPep1 at 5 μg/ml reached about 85% (FIG. 4). These results demonstrate an increase in binding of ADOPep1 to endothelial cells under hypoxia.
ADOPeps induce proliferation of EC under hypoxia�To further test the ability of ADOPeps to induce EC proliferation, increasing concentrations of ADOPeps 1, 2 and 3 were incubated with EC and the proliferation of cells under hypoxia and 24 hours starvation was determined. As shown in FIG. 5, ADOPep1 and ADOPep2 were capable of inducing proliferation of EC at a concentration of 10 ng/ml.
Novel peptide ADOPep1 induces migration of EC under hypoxia�The ability of ADOPeps to induce migration of endothelial cells under hypoxia was tested. EC were incubated in the migration chamber and ADOPeps were added at 1, 10 and 100 ng/ml in the feeder tray for 5 hours under hypoxia conditions. The migration of EC was determined in a Fluorescent ELISA reader and is expressed as Relative Fluorescent Units (RFU). As shown in FIG. 6, the most significant increase in EC migration was observed in the presence of ADOPep1 at a concentration of 10 ng/ml.
Example 2 ADOPep1 Induces Angiogenesis and Increased Perfusion of Ischemic Tissues In Vivo Experimental Results
ADOPep1 induces a significant increase in perfusion in mice with hind limb ischemia�A mouse ischemic hind limb model was used for evaluation of the in vivo potential of angiogenesis induced by ADOPeps. Ischemia was created in the mouse left hind limb by ligation and excision of the femoral artery. The right hind limb served as control. A day after the operation each of the peptides was injected into one site close to the ligation. Each mouse was treated with each of the peptides in a total amount of 0.1 or 1 μg per mouse. The blood perfusion was measured using a Laser Doppler Imager (PeriMed, Sweden) at days 7, 14 and 21 after operation. As can be seen in FIG. 7 the average perfusion of each limb was computed and expressed as the ischemic (left)/control (right) blood perfusion ratio. A statistical analysis demonstrates a significant increase in the blood perfusion ratio in mice injected with 0.1 microgram ADOPep1 at day 21 after operation in comparison to mice injected with PBS demonstrating complete recovery of the blood perfusion in the hind limb.
Histological assessment of angiogenesis in the ischemic hind limb treated with ADOPep1�After hind limb ligation and administration of 0.1 μg/per mouse of ADOPep1, the mice were sacrificed at days 7, 14 and 21 and the whole legs were embedded in paraffin. FIG. 8 shows the average of vessel number per cross section area of ten individual fields per sample expressed as mean�SE in legs of mice treated with ADOPep1 in comparison to PBS injected mice. ADOPep1 treatment resulted in a significant increase (p<0.05) in the number of blood vessels in comparison to PBS treated mice. Representative illustrations (FIGS. 9 a-b) show higher von Willebrand Factor Positive stained small vessels in the ADOPep1 treated group than in the control.
Example 3 Identification of the ADOPeps Receptor on Endothelial Cells Experimental Results
Identification of the ADOPep receptor on endothelial cells�Immune precipitation (IP) of endothelial cells lysate with biotinylated ADOPep1 was analyzed by PAGE. As can be seen in FIGS. 10 a-b, following immunoprecipitation with ADOPep1 a major single protein band is present at 78 kDa. In order to confirm that this band is the ADOPep1 peptide binding receptor, the separated proteins were transferred to a nitrocellulose membrane which was further stained with biotinylated ADOPep1 followed by Chemiluminescent Substrate. As can be seen in FIG. 11, indeed the same band was stained by the labeled peptide in two different experiments (both lanes in the PAGE shown in FIG. 11). The protein receptor band was cut from the gel and analyzed by mass-spectrometry.
IP with ADOPep1 in Normoxia
IP with ADOPep 1 SEQ
in Normoxia
AKFEELNM(+16)DLFR
KSDIDEIVLVGGSTR
VYEGERPLTK
96 88
92 92
95 86
DAGTIAGLNVM(+16)R
M(+16)KETAEAYLGK
VTHAVVTVPAYFNDAQR
DNHLLGTFDLTGIPPAPR
FEELNMDLFR
92 83
FEELNM(+16)DLFR
TWNDPSVQQDIK
96 86
SDIDEIVLVGGSTR
86 84
NTVVPTK
TFAPEEISAM(+16)VLTK
TKPYIQVDIGGGQTK
IINEPTAAAIAYGLDK
IEIESFYEGEDFSETLTR
LYGSAGPPPTGEEDTAEKD
Identification of GRP78 Receptor protein on EC under hypoxia�In order to analyze the receptor on EC that binds the ADOPep peptides under hypoxia conditions, EC were pre incubated for 5 hours under hypoxia conditions and immune precipitation was performed with Biotinylated ADOPep1, followed by Western blot analyses using biotinylated ADOPep1 (FIG. 13 a) or anti-GRP78 antibody (FIG. 13 b). Confirmation of an identical receptor on EC (glucose-regulated protein, homo sapiens, Gi 6900104) under hypoxia was done by mass spectroscopy (19 identities) as presented in Table 5, hereinbelow.
IP with ADOPep1 at Hypoxia
IP with ADOPep1
at Hypoxia
IEWLESHQDADIEDFK
LYGSAGPPPTGEEDTAEKDEL
Increased presentation of GRP78 protein on EC under hypoxia�FIGS. 14 and 15 demonstrate by FACS analysis the percentage of EC expressing GRP78 on their membrane under normoxia and hypoxia conditions. As can be seen in FIG. 14, the binding of anti-GRP78 to EC, originating from 10 different umbilical cords, was 30�13% under normoxia conditions and 52.8�8.4% after 5 hours of hypoxia. FACS histogram (FIG. 15) demonstrated presence of GRP78 receptor on ECs under normoxia or hypoxia conditions, with increased binding of anti-GRP78 antibody under hypoxia.
Receptor presence on tumor cells�To further confirm the presence of GRP78 on EC and its relationship to hypoxia, FACS analyses using the anti-GRP78 antibody performed on different lines of tumor cells including the MCF7 breast carcinoma, SK melanoma, HT colon carcinoma and K562 erythroleukemia tumor cells revealed that GRP78 is expressed on MCF7 breast carcinoma, HT-29 colon carcinoma and SK-28 melanoma cell lines but not on K562 erythroleukemia cells (FIGS. 16 a-d).
Example 4 ADOPeps Inhibit Hypoxia-Induced Apoptosis Experimental Results
Inhibition of the GRP78 receptor with an anti-GRP78 antibody or the addition of ADOPep1 result in inhibition of hypoxia-induced apoptosis of EC�The role of GRP78 in apoptosis was studied using EC under hypoxia. As shown in FIG. 17, the percentage of apoptosis of EC that were exposed for 24 hours to hypoxia was increased from 25% to 62%. Incubation of EC with ADOPep1 prevented apoptosis to levels which are similar to those seen under normoxic conditions. Incubation of EC with anti-GRP78 antibody also decreased the levels of apoptosis, however, the ADOPep was more efficient (in 29%) in decreasing apoptosis as compared to the anti-GRP78 antibody (FIG. 17). FIGS. 18 a-e depict illustration by dot plot FACS analysis of the inhibition of apoptosis by the ADOPep1 peptide. As can be seen, EC under hypoxia were stained with both Annexin V and Propidium Iodide (PI) apoptotic markers. In contrast, incubation of EC with the ADOPep1 peptide induced a remarkable decrease in the percentage of stained cells.
ADOPep1 inhibited hypoxia-induced, but not CoCl2-induced apoptosis�To further substantiate the effect of ADOPep1 on apoptosis, apoptosis was induced by hypoxia or CoCl2 (Cobalt-Chloride) treatment. As is shown in FIGS. 33 a-f and 34, 24 hours of hypoxia resulted in an increase of apoptosis to about 70%. In addition, while AdoPep1 inhibited hypoxia-induced apoptosis of endothelial cells in approximately 40 percent, AdoPep1 did not inhibit apoptosis of endothelial cells exposed to the apoptotic inducer Cobalt-Chloride. Thus, the inhibition of apoptosis by ADOPep1 is specific to the hypoxia stress conditions.
ADOPep1 induces inhibition of hypoxia-induced apoptosis in vivo�As can be seen in FIG. 22, the mean number of apoptotic cells is dramatically decreased in 7-day ischemic hind limb that was injected with ADoPep1. Thus, using the ischemic hind limb mouse model the present inventors were able to show, for the first time, that administration of ADOPep1 to ischemic hind limb results in a significant reduction of ischemia-induced apoptosis.
Example 5 ADOPeps Bind to the GRP78 Receptor on Endothelial Cells Experimental Results
Competitive binding of ADAM15 derived peptides ADOPep1, ADOPep2 and ADOPep3 to GRP78 receptor�To further test if the ADOPeps bind to the GRP78 receptor on EC, a competitive binding assay was performed on EC which were incubated with the ADOPeps prior to binding with the anti-GRP78 antibody. As demonstrated in FIG. 19 (which represents a summary of 4 experiments), all 3 ADOPeps (i.e., ADOPep1, ADOPep2 and ADOPep3) show some degree of inhibition of binding of anti-GRP78 antibody to EC while scrambled peptide (sRoY) was not effective in the competitive binding to the receptor on EC.
ADOPep1 induces upregulation of GRP78 receptor expression in vitro (under hypoxia)�FIGS. 20 a-b further depict the binding of the anti-GRP78 antibody to endothelial cells following incubation of the cells with ADOPep1 under normoxia or hypoxia conditions and demonstrate that while the presence of the GRP78 receptor on endothelial cell (as evidenced by the binding of anti-GRP78 to EC) increases under hypoxia from about 18.1% to about 40.1% (as was previously reported by others Li J, Lee A S. Stress induction of GRP/BIP and its role in cancer. Curr. Mol. Med. 2006; 6:45-54; Arap M A, Landenranta J, Mintz P J, Hajitou A, Sarkis A S, Arap W, Pasqualini R. Cell surface expression of the stress response chaperone GRP78 enables tumor targeting by circulating ligands. Cancer Cell. 2004; 6:275-84), a more significant increase in GRP78 presentation on the EC (of up to about 83.8%) is observed when the cells are incubated with the ADOPep1 under hypoxia conditions. These results demonstrate the involvement of ADOPep1 in the upregulation of GRP78 under hypoxia.
ADOPep1 induces upregulation of GRP78 receptor expression in vivo (under ischemia)�To further confirm the ADOPeps involvement in GRP78 presentation on endothelial cells, the mean number of GRP78 positive cells was determined in ischemic limb sections. As is shown in FIG. 21, 7 days following induction of ischemia the mean number of GRP78 positive cells was increased as compared to untreated hind limbs. However, injection of Adopep1 resulted in a more significant increase in mean number of GRP78 positive cells as detected 14 days after induction of ischemia. The relatively low level of GRP78 positive cells at 21 days after ischemia probably represents recovery of the ischemia in the treated animas.
Example 6 ADOPep1 Binding Causes a GRP78 Receptor Internalization Response Under Hypoxic Conditions Experimental Results
ADOPep1 binding causes a GRP78 receptor internalization response under hypoxic conditions�As is shown in FIG. 29, after 5 minutes of incubation, AdoPep1 induced GRP78 receptor internalization in endothelial cells under hypoxic conditions. The internalization response was demonstrated by inhibition of percent binding of AdoPep to membranes (less membrane GRP78 receptors) and increase in intracellular GRP78 mean fluorescence in the endothelial cells incubated for 5 minutes with AdoPep1.
Example 7 Identification of Minimal Motif Sequences from the ADOPep Peptides which Exhibit a Biological Activity Experimental Results
Identification of novel angiogenesis or tumor related motif�The present inventors have identified a 4 amino-acid sequence, HWRR, as a common motif present on ADOPep1, 2 and 3 ADAM15 derived peptides, that induces angiogenesis and binds to the GRP78 receptor on EC (Table 6, hereinbelow).
Amino acid sequences of the ADOPeptides of the invention
ADOPep1
A H L L P R L P 2
ADOPep2
E N F L A H L L 3
ADOPep3
A V T L E N F L 4
In addition, endothelial cells were incubated under hypoxia conditions in the absence or presence of ADOPep1, Motif A or C and the Q1level of apoptosis was determined using FACS analysis. As shown in FIGS. 24 a-d and 25 while ADOPep1 and Motif A and B peptides were capable of inhibiting the hypoxia induced apoptosis, the motif C peptide exhibited no effect on hypoxia induced apoptosis.
To further test the biological activity of motifs A, B, C on endothelial cells, a migration assay was performed on EC under hypoxia. As shown in FIGS. 26 a-b, while the ADOPep1, Motif A and B peptides induced endothelial cell migration at a concentration of about 10 ng/ml, motif C peptide exhibited no significant effect on endothelial cell migration.
Example 8 The ADOPeps Motifs are Capable of Tube Formation and Induction of ERK1/2 Phosphorylation Experimental Results
ADOPep1 and motif A peptides are capable of forming tubes from endothelial cells�The ability of ADOPep1 or motif A peptides to form tubes was determined in vitro. As shown in FIG. 27, while the ADOPep1 and Motif A significantly increased the length of the network of connected cells in endothelial cells under starvation and normoxic conditions, the addition of the scrambled sROY peptide had no effect on tube formation.
AdoPep1 and Motif A peptides compete on the binding to the same receptor on endothelial cells�As shown in FIGS. 28 a-d, AdoPep1 and Motif A peptides inhibited anti GRP78 binding to endothelial cells under hypoxic conditions. Ten micrograms of AdoPep1 and Motif A inhibited anti GRP78 binding in approximately 80% and 60% respectively, while motif C did not inhibit anti GRP78 binding to endothelial cells.
Incubation of endothelial cells with ADOPep1 and Motif A under hypoxia conditions increase ERK1/2 phosphorylation�As is shown in FIGS. 30 a-b, incubation of endothelial cells with ADOPep1 and Motif A under hypoxia conditions resulted in a significant increase in ERK1/2 phosphorylation as measured after 20 minutes.
Induction of ERK1/2 phosphorylation by ADOPep1 and motif A peptides is specific�In order to assess that ERK phosphorylation is specific to AdoPep1 and Motif A activation, a specific pERK peptide inhibitor was added to the endothelial cells incubated with AdoPep1 and Motif A peptides. FIG. 31 shows Western blot analysis of ERK phosphorylation inhibition by the inhibitor peptide in endothelial cells incubated with AdoPep1 and Motif A for 20 minutes under hypoxic conditions.
ADOPep1 and ADOPep2, but not ADOPep3 are capable of inducing ERK1/2 phosphorylation in endothelial cells under hypoxia�As shown in FIGS. 32 a-b, Western Blot analyses performed using anti Phospho ERK antibody (Santa Cruz Biothechnologies p-ERK(E-4),sc7383) revealed that while ADOPep1 and 2 induced ERK1/2 phosphorylation, ADOPep3 had not effect on ERK1/2 phosphorylation.
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"Arginine-Glycine-Aspartic Acid (RGD)�Peptide Binds to Both Tumor and Tumor-Endothelial Cells In Vivo", Cancer Research, 62: 5139-5143, Sep. 15, 2002.* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS20120219498 *May 10, 2012Aug 30, 2012Ramot At Tel-Aviv University Ltd.Compositions and methods for inducing angiogenesis* Cited by examinerClassifications U.S. Classification514/13.3, 530/330, 530/327, 530/329, 530/328International ClassificationA61K38/00, C07K14/515, A61K38/04, C07K17/00, C07K5/00, C07K16/00, C07K7/00, A61P35/00Cooperative ClassificationA61K38/00, G01N2333/96486, G01N33/5064, C07K7/06, C07K14/47, C07K7/08European ClassificationC07K7/06, C07K7/08, G01N33/50D2F8, C07K14/47Legal EventsDateCodeEventDescriptionFeb 22, 2010ASAssignmentFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HARDY, BRITTA;BATTLER, ALEXANDER;RAITER, ANNAT;AND OTHERS;SIGNING DATES FROM 20090325 TO 20090326;REEL/FRAME:023968/0037Owner name: RAMOT AT TEL AVIV UNIVERSITY LTD., ISRAELRotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services©2012 Google