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Polypeptide compounds for inhibiting angiogenesis and tumor growth

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20120277162 patent thumbnailZoom

Polypeptide compounds for inhibiting angiogenesis and tumor growth


In certain embodiments, this present invention provides polypeptide compositions, including compositions containing a modified polypeptide, and methods for inhibiting Ephrin B2 or EphB4 activity. In other embodiments, the present invention provides methods and compositions for treating cancer or for treating angiogenesis-associated diseases.

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Inventors: Valery Krasnoperov, Nathalie Kertesz, Ramachandra Reddy, Parkash Gill, Sergey Zozulya
USPTO Applicaton #: #20120277162 - Class: 514 193 (USPTO) - 11/01/12 - Class 514 


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The Patent Description & Claims data below is from USPTO Patent Application 20120277162, Polypeptide compounds for inhibiting angiogenesis and tumor growth.

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RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S. Provisional Application No. 60/612,488, filed Sep. 23, 2004, the specification of which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

Angiogenesis, the development of new blood vessels from the endothelium of a preexisting vasculature, is a critical process in the growth, progression, and metastasis of solid tumors within the host. During physiologically normal angiogenesis, the autocrine, paracrine, and amphicrine interactions of the vascular endothelium with its surrounding stromal components are tightly regulated both spatially and temporally. Additionally, the levels and activities of proangiogenic and angiostatic cytokines and growth factors are maintained in balance. In contrast, the pathological angiogenesis necessary for active tumor, growth is sustained and persistent, representing a dysregulation of the normal angiogenic system. Solid and hematopoetic tumor types are particularly associated with a high level of abnormal angiogenesis.

It is generally thought that the development of tumor consists of sequential, and interrelated steps that lead to the generation of autonomous clone with aggressive growth potential. These steps include sustained growth and unlimited self-renewal. Cell populations in a tumor are generally characterized by growth signal self-sufficiency, decreased sensitivity to growth suppressive signals, and resistance to apoptosis. Genetic or cytogenetic events that initiate aberrant growth sustain cells in a prolonged “ready” state by preventing apoptosis.

It is a goal of the present disclosure to provide agents and therapeutic treatments for inhibiting angiogenesis and tumor growth.

SUMMARY

OF THE INVENTION

In certain aspects, the disclosure provides polypeptide agents that inhibit EphB4 or EphrinB2 mediated functions, including monomeric ligand binding portions of the EphB4 and EphrinB2 proteins. As demonstrated herein, EphB4 and EphrinB2 participate in various disease states, including cancers and diseases related to unwanted or excessive angiogenesis. Accordingly, certain polypeptide agents disclosed herein may be used to treat such diseases.

In further aspects, the disclosure relates to the discovery that EphB4 and/or EphrinB2 are expressed, often at high levels, in a variety of tumors. Therefore, polypeptide agents that down-regulate EphB4 or EphrinB2 function may affect rumors by a direct effect on the tumor cells as well as an indirect effect on the angiogenic processes recruited by the tumor. In certain embodiments, the disclosure provides the identity of tumor types particularly suited to treatment with an agent that downregulates EphB4 or EphrinB2 function. In preferred embodiments, polypeptides disclosed herein are modified so as to have increased serum half-life in vivo.

In certain aspects, the disclosure provides soluble EphB4 polypeptides comprising an amino acid sequence of an extracellular domain of an EphB4 protein. The soluble EphB4 polypeptides bind specifically to an EphrinB2 polypeptide. The term “soluble” is used merely to indicate that these polypeptides do not contain a transmembrane domain or a portion of a transmembrane domain sufficient to compromise the solubility of the polypeptide in a physiological salt solution. Soluble polypeptides are preferably prepared as monomers that compete with EphB4 for binding to ligand such as EphrinB2 and inhibit the signaling that results from EphB4 activation. Optionally, a soluble polypeptide may be prepared in a multimeric form, by, for example, expressing as an Fc fusion protein or fusion with another multimerization domain. Such multimeric forms may have complex activities, having agonistic or antagonistic effects depending on the context. In certain embodiments the soluble EphB4 polypeptide comprises a globular domain of an EphB4 protein. A soluble EphB4 polypeptide may comprise a sequence at least 90% identical to residues 1-522 of the amino acid sequence defined by FIG. 65 (SEQ ID NO:10). A soluble EphB4 polypeptide may comprise a sequence at least 90% identical to residues 1-412 of the amino acid sequence defined by FIG. 65 (SEQ ID NO:10). A soluble EphB4 polypeptide may comprise a sequence at least 90% identical to residues 1-312 of the amino acid sequence defined by FIG. 65 (SEQ ID NO:10). A soluble EphB4 polypeptide may comprise a sequence encompassing the globular (G) domain (amino acids 29-197 of FIG. 65, SEQ ID NO:10), and optionally additional domains, such as the cysteine-rich domain (amino acids 239-321 of FIG. 65, SEQ ID NO:10), the first fibronectin type 3 domain (amino acids 324-429 of FIG. 65, SEQ ID NO:10) and the second fibronectin type 3 domain (amino acids 434-526 of FIG. 65, SEQ ID NO:10). Preferred polypeptides described herein and demonstrated as having ligand binding activity include polypeptides corresponding to 1-537, 1-427 and 1-326, respectively, of the amino acid sequence shown in FIG. 65 (SEQ ID NO:10). A soluble EphB4 polypeptide may comprise a sequence as set forth in FIG. 1 or 2 (SEQ ID Nos. 1 or 2). As is well known in the art, expression of such EphB4 polypeptides in a suitable cell, such as HEK293T cell line, will result in cleavage of a leader peptide. Although such cleavage is not always complete or perfectly consistent at a single site, it is known that EphB4 tends to be cleaved so as to remove the first 15 amino acids of the sequence shown in FIG. 65 (SEQ ID NO:10). Accordingly, as specific examples, the disclosure provides unprocessed soluble EphB4 polypeptides that bind to EphrinB2 and comprise an amino acid sequence selected from the following group (numbering is with respect to the sequence of FIG. 65, SEQ ID NO:10): 1-197, 29-197, 1-312, 29-132, 1-321, 29-321, 1-326, 29-326, 1-412, 29-412, 1-427, 29-427, 1-429, 29-429, 1-526, 29-526, 1-537 and 29-537. Additionally, heterologous leader peptides may be substituted for the endogeneous leader sequences. Polypeptides may be used in a processed form, such forms having a predicted amino acid sequence selected from the following group (numbering is with respect to the sequence of FIG. 65, SEQ ID NO:10): 16-197, 16-312, 16-321, 16-326, 16-412, 16-427, 16-429, 16-526 and 16-537. Additionally, a soluble EphB4 polypeptide may be one that comprises an amino acid sequence at least 90%, and optionally 95% or 99% identical to any of the preceding amino acid sequences while retaining EphrinB2 binding activity. Preferably, any variations in the amino acid sequence from the sequence shown in FIG. 65 (SEQ ID NO:10) are conservative changes or deletions of no more than 1, 2, 3, 4 or 5 amino acids, particularly in a surface loop region. In certain embodiments, the soluble EpbB4 polypeptide may inhibit the interaction between Ephrin B2 and EphB4. The soluble EphB4 polypeptide may inhibit clustering of or phosphorylation of Ephrin B2 or EphB4. Phosphorylation of EphrinB2 or EphB4 is generally considered to be one of the initial events in triggering intracellular signaling pathways regulated by these proteins. As noted above, the soluble EphB4 polypeptide may be prepared as a monomeric or multimeric fusion protein. The soluble polypeptide may include one or more modified amino acids. Such amino acids may contribute to desirable properties, such as increased resistance to protease digestion.

The present disclosure provides soluble EphB4 polypeptides having an additional component that confers increased serum half-life while still retaining EphrinB2 binding activity. In certain embodiments soluble EphB4 polypeptides are monomeric and are covalently linked to one or more polyoxyaklylene groups (e.g., polyethylene, polypropylene), and preferably polyethylene glycol (PEG) groups. Accordingly, one aspect of the invention provides modified EphB4 polypeptides, wherein the modification comprises a single polyethylene glycol group covalently bonded to the polypeptide. Other aspects provide modified EphB4 polypeptides covalently bonded to one, two, three, or more polyethylene glycol groups.

The one or more PEG may have a molecular weight ranging from about 1 kDa to about 100 kDa, and will preferably have a molecular weight ranging from about 10 to about 60 kDa or about 10 to about 40 kDa. The PEG group may be a linear PEG or a branched PEG. In a preferred embodiment, the soluble, monomeric EphB4 conjugate comprises an EphB4 polypeptide covalently linked to one PEG group of from about 10 to about 40 kDa (monoPEGylated EphB4), or from about 15 to 30 kDa, preferably via an ε-amino group of EphB4 lysine or the N-terminal amino group. Most preferably, EphB4 is randomly PEGylated at one amino group out of the group consisting of the ε-amino groups of EphB4 lysine and the N-terminal amino group.

In one embodiment, the pegylated polypeptides provided by the invention have a serum half-life in vivo at least 50%, 75%, 100%, 150% or 200% greater than that of an unmodified EphB4 polypeptide. In another embodiment, the pegylated EphB4 polypeptides provided by the invention inhibit EphrinB2 activity. In a specific embodiment, they inhibit EphrinB2 receptor clustering, EphrinB2 phosphorylation, and/or EphrinB2 kinase activity.

Surprisingly, it has been found that monoPEGylated EphB4 according to the invention has superior properties in regard to the therapeutic applicability of unmodified soluble EphB4 polypeptides and poly-PEGylated EphB4. Nonetheless, the disclosure also provides poly-PEGylated EphB4 having PEG at more than one position. Such polyPEGylated forms provide improved serum-half life relative to the unmodified form.

In certain embodiments, a soluble EphB4 polypeptide is stably associated with a second stabilizing polypeptide that confers improved half-life without substantially diminishing EphrinB2 binding. A stabilizing polypeptide will preferably be immunocompatible with human patients (or animal patients, where veterinary uses are contemplated) and have little or no significant biological activity.

In a preferred embodiment, the stabilizing polypeptide is a human serum albumin, or a portion thereof. A human serum albumin may be stably associated with the EphB4 polypeptide covalently or non-covalently. Covalent attachment may be achieved by expression of the EphB4 polypeptide as a co-translational fusion with human serum albumin. The albumin sequence may be fused at the N-terminus, the C-terminus or at a non-disruptive internal position in the soluble EphB4 polypeptide. Exposed loops of the EphB4 would be appropriate positions for insertion of an albumin sequence. Albumin may also be post-translationally attached to the EphB4 polypeptide by, for example, chemical cross-linking. An EphB4 polypeptide may also be stably associated with more than one albumin polypeptide. In some embodiments, the albumin is selected from the group consisting of a human serum albumin (HSA) and bovine serum albumin (BSA). In other embodiments, the albumin is a naturally occurring variant. In one preferred embodiment, the EphB4-HSA fusion inhibits the interaction between Ephrin B2 and EphB4, the clustering of Ephrin B2 or EphB4, the phosphorylation of Ephrin B2 or EphB4, or combinations thereof. In other embodiments, the EphB4-HSA fusion has enhanced in vivo stability relative to the unmodified wildtype polypeptide.

In certain aspects, the disclosure provides soluble EphrinB2 polypeptides comprising an amino acid sequence of an extracellular domain of an EphrinB2 protein. The soluble EphrinB2 polypeptides bind specifically to an EphB4 polypeptide. The term “soluble” is used merely to indicate that these polypeptides do not contain a transmembrane domain or a portion of a transmembrane domain sufficient to compromise the solubility of the polypeptide in a physiological salt solution. Soluble polypeptides are preferably prepared as monomers that compete with EphrinB2 for binding to ligand such as EphB4 and inhibit the signaling that results from EphrinB2 activation. Optionally, a soluble polypeptide may be prepared in a multimeric form, by, for example, expressing as an Fc fusion protein or fusion with another multimerization domain. Such multimeric forms may have complex activities, having agonistic or antagonistic effects depending on the context. A soluble EphrinB2 polypeptide may comprise residues 1-225 of the amino acid sequence defined by FIG. 66 (SEQ ID NO:11). A soluble EphrinB2 polypeptide may comprise a sequence defined by FIG. 3. As is well known in the art, expression of such EphrinB2 polypeptides in a suitable cell, such as HEK293T cell line, will result in cleavage of a leader peptide. Although such cleavage is not always complete or perfectly consistent at a single site, it is known that EphrinB2 tends to be cleaved so as to remove the first 26 amino acids of the sequence shown in FIG. 66 (SEQ ID NO:11). Accordingly, as specific examples, the disclosure provides unprocessed soluble EphrinB2 polypeptides that bind to EphB4 and comprise an amino acid sequence corresponding to amino acids 1-225 of FIG. 66 (SEQ ID NO:11). Such polypeptides may be used in a processed form, such forms having a predicted amino acid sequence selected from the following group (numbering is with respect to the sequence of FIG. 66, SEQ ID NO:1): 26-225. In certain embodiments, the soluble EphrinB2 polypeptide may inhibit the interaction between Ephrin B2 and EphB4. The soluble EphrinB2 polypeptide may inhibit clustering of or phosphorylation of EphrinB2 or EphB4. As noted above, the soluble EphrinB2 polypeptide may be prepared as a monomeric or multimeric fusion protein. The soluble polypeptide may include one or more modified amino acids. Such amino acids may contribute to desirable properties, such as increased resistance to protease digestion.

In certain aspects, the disclosure provides pharmaceutical formulations comprising a polypeptide reagent and a pharmaceutically acceptable carrier. The polypeptide reagent may be any disclosed herein, including, for example, soluble EphB4 or EphrinB2 polypeptides. Additional formulations include cosmetic compositions and diagnostic kits.

In certain aspects the disclosure provides methods of inhibiting signaling through Ephrin B2/EphB4 pathway in a cell. A method may comprise contacting the cell with an effective amount of a polypeptide agent, such as (a) a soluble polypeptide comprising an amino acid sequence of an extracellular domain of an EphB4 protein, wherein the EphB4 polypeptide is a monomer and binds specifically to an Ephrin B2 polypeptide; (b) a soluble polypeptide comprising an amino acid sequence of an extracellular domain of an Ephrin B2 protein, wherein the soluble Ephrin B2 polypeptide is a monomer and binds with high affinity to an EphB4 polypeptide.

In certain aspects the disclosure provides methods for reducing the growth rate of a tumor, comprising administering an amount of a polypeptide agent sufficient to reduce the growth rate of the tumor. The polypeptide agent may be selected from the group consisting of: (a) a soluble polypeptide comprising an amino acid sequence of an extracellular domain of an EphB4 protein, wherein the EphB4 polypeptide is a monomer and binds specifically to an Ephrin B2 polypeptide, and optionally comprises an additional modification to increase serum half-life, such as a PEGylation or serum albumin or both; (b) a soluble polypeptide comprising an amino acid sequence of an extracellular domain of an Ephrin B2 protein, wherein the soluble Ephrin B2 polypeptide is a monomer and binds with high affinity to an EphB4 polypeptide Optionally, the tumor comprises cells expressing a higher level of EphB4 and/or EphrinB2 than noncancerous cells of a comparable tissue.

In certain aspects, the disclosure provides methods for treating a patient suffering from a cancer. A method may comprise administering to the patient a polypeptide agent. The polypeptide agent may be selected from the group consisting of: (a) a soluble polypeptide comprising an amino acid sequence of an extracellular domain of an EphB4 protein, wherein the EphB4 polypeptide is a monomer and binds specifically to an Ephrin B2 polypeptide, and optionally comprises an additional modification to increase serum half-life, such as a PEGylation or serum albumin or both; (b) a soluble polypeptide comprising an amino acid sequence of an extracellular domain of an Ephrin B2 protein, wherein the soluble Ephrin B2 polypeptide is a monomer and binds with high affinity to an EphB4 polypeptide. Optionally, the cancer comprises cancer cells expressing EphrinB2 and/or EphB4 at a higher level than noncancerous cells of a comparable tissue. The cancer may be a metastatic cancer.

The cancer may be selected from the group consisting of colon carcinoma, breast tumor, mesothelioma, prostate tumor, squamous cell carcinoma, Kaposi sarcoma, and leukemia. Optionally, the cancer is an angiogenesis-dependent cancer or an angiogenesis independent cancer. The polypeptide agent employed may inhibit clustering or phosphorylation of Ephrin B2 or EphB4. A polypeptide agent may be co-administered with one or more additional anti-cancer chemotherapeutic agents that inhibit cancer cells in an additive or synergistic manner with the polypeptide agent.

In certain aspects, the disclosure provides methods of inhibiting angiogenesis. A method may comprise contacting a cell with an amount of a polypeptide agent sufficient to inhibit angiogenesis. The polypeptide agent may be selected from the group consisting of: (a) a soluble polypeptide comprising an amino acid sequence of an extracellular domain of an EphB4 protein, wherein the EphB4 polypeptide is a monomer and binds specifically to an Ephrin B2 polypeptide, and optionally comprises an additional modification to increase serum half-life, such as a PEGylation or serum albumin or both; (b) a soluble polypeptide comprising an amino acid sequence of an extracellular domain of an Ephrin B2 protein, wherein the soluble Ephrin B2 polypeptide is a monomer and binds with high affinity to an EphB4 polypeptide.

In certain aspects, the disclosure provides methods for treating a patient suffering from an angiogenesis-associated disease, comprising administering to the patient a polypeptide agent. The polypeptide agent may be selected from the group consisting of: (a) a soluble polypeptide comprising an amino acid sequence of an extracellular domain of an EphB4 protein, wherein the EphB4 polypeptide is a monomer and binds specifically to an Ephrin B2 polypeptide, and optionally comprises an additional modification to increase serum half-life, such as a PEGylation or serum albumin or both; (b) a soluble polypeptide comprising an amino acid sequence of an extracellular domain of an Ephrin B2 protein, wherein the soluble Ephrin B2 polypeptide is a monomer and binds with high affinity to an EphB4 polypeptide. The soluble polypeptide may be formulated with a pharmaceutically acceptable carrier. An angiogenesis related disease or unwanted angiogenesis related process may be selected from the group consisting of angiogenesis-dependent cancer, benign tumors, inflammatory disorders, chronic articular rheumatism and psoriasis, ocular angiogenic diseases, Osler-Webber Syndrome, myocardial angiogenesis, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma, telangiectasia psoriasis scleroderma, pyogenic granuloma, rubeosis, arthritis, diabetic neovascularization, vasculogenesis. A polypeptide agent may be co-administered with at least one additional anti-angiogenesis agent that inhibits angiogenesis in an additive or synergistic manner with the soluble polypeptide.

In certain aspects, the disclosure provides for the use of a polypeptide agent in the manufacture of medicament for the treatment of cancer or an angiogenesis related disorder. The polypeptide agent may be selected from the group consisting of: (a) a soluble polypeptide comprising an amino acid sequence of an extracellular domain of an EphB4 protein, wherein the EphB4 polypeptide is a monomer and binds specifically to an Ephrin B2 polypeptide, and optionally comprises an additional modification to increase serum half-life, such as a PEGylation or serum albumin or both; (b) a soluble polypeptide comprising an amino acid sequence of an extracellular domain of an Ephrin B2 protein, wherein the soluble Ephrin B2 polypeptide is a monomer and binds with high affinity to an EphB4 polypeptide.

In certain aspects, the disclosure provides methods for treating a patient suffering from a cancer, comprising: (a) identifying in the patient a tumor having a plurality of cancer cells that express EphB4 and/or EphrinB2; and (b) administering to the patient a polypeptide agent. The polypeptide agent may be selected from the group consisting of: (i) a soluble polypeptide comprising an amino acid sequence of an extracellular domain of an EphB4 protein, wherein the EphB4 polypeptide is a monomer and binds specifically to an Ephrin B2 polypeptide, and optionally comprises an additional modification to increase serum half-life, such as a PEGylation or serum albumin or both; (ii) a soluble polypeptide comprising an amino acid sequence of an extracellular domain of an Ephrin B2 protein, wherein the soluble Ephrin B2 polypeptide is a monomer and binds with high affinity to an EphB4 polypeptide.

In certain aspects, the disclosure provides methods for identifying a tumor that is suitable for treatment with an EphrinB2 or EphB4 antagonist. A method may comprise detecting in the tumor cell one or more of the following characteristics: (a) expression of EphB4 protein and/or mRNA; (b) expression of EphrinB2 protein and/or mRNA; (c) gene amplification (e.g., increased gene copy number) of the EphB4 gene; or (d) gene amplification of the EphrinB2 gene. A tumor cell having one or more of characteristics (a)-(d) may be suitable for treatment with an EphrinB2 or EphB4 antagonist, such as a polypeptide agent described herein.

Surprisingly, applicants have found that an EphB4 polypeptide lacking the globular domain can in fact inhibit tumor growth in a xenograft model, inhibit angiogenic tube formation of vascular endothelial cells and inhibit EphrinB2-activated autokinase activity of EphB4. While not wishing to be bound to any mechanism of action, it is expected that the polypeptide either prevents EphB4 aggregation or stimulates the elimination (e.g. by endocytosis) of EphB4 from the plasma membrane. Accordingly, the disclosure provides isolated soluble polypeptides comprising an amino acid sequence of a fibronectin type 3 domain of an EphB4 protein. Such polypeptides will preferably have a biological effect, such as inhibiting an activity (e.g. aggregation or kinase activity) of an EphB4 or EphrinB2 protein, and particularly the inhibition of tumor growth in a human or in a mouse xenograft model of cancer. Such polypeptides may also inhibit angiogenesis in vivo or in an cell-based assay system. Such polypeptides may not bind to EphrinB2 and may specifically exclude all of or the functional (e.g., EphrinB2 binding-) portions of the globular domain of an EphB4 protein. Such a polypeptide will preferably comprise amino acids corresponding to amino acids 324-429 and/or 434-526 of the sequence of FIG. 65 (SEQ ID NO:10), or sequences at least 90%, 95%, 98%, 99% identical thereto. An example of such a polypeptide is shown in SEQ ID NO: 15. Such a polypeptide may be modified in any of the ways described herein, and may be produced as a monomer or as a dimer or multimer comprising two or more such polypeptides, such as an Fc fusion construct. Dimers or multimers may be desirable to enhance the effectiveness of such polypeptides. All of the methods for producing and using such polypeptides are similar to those described herein with respect to other EphB4 polypeptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows amino acid sequence of the B4ECv3 protein (predicted sequence of the precursor including uncleaved Eph B4 leader peptide is shown; SEQ ID NO:1).

FIG. 2 shows amino acid sequence of the B4ECv3NT protein (predicted sequence of the precursor including uncleaved Eph B4 leader peptide is shown; SEQ ID NO:2).

FIG. 3 shows amino acid sequence of the B2EC protein (predicted sequence of the precursor including uncleaved Ephrin B2 leader peptide is shown; SEQ ID NO:3).

FIG. 4 shows amino acid sequence of the B4ECv3-FC protein (predicted sequence of the precursor including uncleaved Eph B4 leader peptide is shown; SEQ ID NO:4).

FIG. 5 shows amino acid sequence of the B2EC-FC protein (predicted sequence of the precursor including uncleaved Ephrin 82 leader peptide is shown; SEQ ID NO:5).

FIG. 6 shows B4EC-FC binding assay (Protein A-agarose based).

FIG. 7 shows B4EC-FC inhibition assay (Inhibition in solution).

FIG. 8 shows B2EC-FC binding assay (Protein-A-agarose based assay).

FIG. 9 shows chemotaxis of HUAEC in response to B4Ecv3.

FIG. 10 shows chemotaxis of HHEC in response to B2EC-FC.

FIG. 11 shows chemotaxis of HHAEC in response to B2EC.

FIG. 12 shows effect of B4Ecv3 on HUAEC tubule formation.

FIG. 13 shows effect of B2EC-FC on HUAEC tubule formation.

FIG. 14 is a schematic representation of human Ephrin B2 constructs.

FIG. 15 is a schematic representation of human EphB4 constructs.

FIG. 16 shows the domain structure of the recombinant soluble EphB4EC proteins. Designation of the domains are as follows: L—leader peptide, G—globular (ligand-binding domain), C—Cys-rich domain, F1, F2—fibronectin type III repeats, H—6×His-tag.

FIG. 17 shows purification and ligand binding properties of the EphB4EC proteins. A. SDS-PAAG gel electrophoresis of purified EphB4-derived recombinant soluble proteins (Coomassie-stained). B. Binding of Ephrin B2-AP fusion to EphB4-derived recombinant proteins immobilized on Ni-NTA-agarose beads. Results of three independent experiments are shown for each protein. Vertical axis—optical density at 420 nm.

FIG. 18 shows that EphB4v3 inhibits chemotaxis.

FIG. 19 shows that EphB4v3 inhibits tubule formation on Matrigel. A displays the strong inhibition of tubule formation by B4v3 in a representative experiment. B shows a quantitation of the reduction of tube-length obtained with B4v3 at increasing concentrations as well as a reduction in the number of junctions, in comparison to cells with no protein.

Results are displayed as mean values±S.D. obtained from three independent experiments performed with duplicate wells.

FIG. 20 shows that soluble EphB4 has no detectable cytotoxic effect as assessed by MTS assay.

FIG. 21 shows that B4v3 inhibits invasion and tubule formation by endothelial cells in the Matrigel assay. (A) to detect total invading cells, photographed at 20× magnification or with Masson\'s Trichrome Top left of A B displays section of a Matrigel plug with no GF, top right of A displays section with B4IgG containing GF and lower left section contains GF, and lower right shows GF in the presence of B4v3. Significant invasion of endothelial cells is only seen in GF containing Matrigel. Top right displays an area with a high number of invaded cells induced by B4IgG, which signifies the dimeric form of B4v3. The left upper parts of the pictures correspond to the cell layers formed around the Matrigel plug from which cells invade toward the center of the plug located in the direction of the right lower corner. Total cells in sections of the Matrigel plugs were quantitated with Scion Image software. Results obtained from two experiments with duplicate plugs are displayed as mean values±S.D.

FIG. 22 shows tyrosine phosphorylation of EphB4 receptor in PC3 cells in response to stimulation with EphrinB2-Fc fusion in presence or absence of EphB4-derived recombinant soluble proteins.

FIG. 23 shows effects of soluble EphB4ECD on viability and cell cycle. A) 3-day cell viability assay of two HNSCC cell lines. B) FACS analysis of cell cycle in HNSCC-15 cells treated as in A. Treatment of these cells resulted in accumulation in subG0/G1 and S/G2 phases as indicated by the arrows.



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stats Patent Info
Application #
US 20120277162 A1
Publish Date
11/01/2012
Document #
File Date
07/25/2014
USPTO Class
Other USPTO Classes
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