Polypeptide compounds for inhibiting angiogenesis and tumor growth

Abstract

his 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.

Description

RELATED APPLICATIONS

[0001]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

[0002]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 hematopoietic tumor types are particularly associated with a high level of abnormal angiogenesis.

[0003]It is generally thought that the development of tumor consists of sequential, and interrelated steps that lead to the generation of an 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.

[0004]It is a goal of the present disclosure to provide agents and therapeutic treatments for inhibiting angiogenesis and tumor growth.

SUMMARY OF THE INVENTION

[0005]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 tumors 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.

[0006]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 EphB4 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.

[0007]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.

[0008]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.

[0009]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.

[0010]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.

[0011]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.

[0012]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.

[0013]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:11): 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.

[0014]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.

[0015]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.

[0016]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.

[0017]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.

[0018]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.

[0019]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.

[0020]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.

[0021]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.

[0022]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.

[0023]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

[0024]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).

[0025]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).

[0026]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).

[0027]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).

[0028]FIG. 5 shows amino acid sequence of the B2EC-FC protein (predicted sequence of the precursor including uncleaved Ephrin B2 leader peptide is shown; SEQ ID NO:5).

[0029]FIG. 6 shows B4EC-FC binding assay (Protein A-agarose based).

[0030]FIG. 7 shows B4EC-FC inhibition assay (Inhibition in solution).

[0031]FIG. 8 shows B2EC-FC binding assay (Protein-A-agarose based assay).

[0032]FIG. 9 shows chemotaxis of HUAEC in response to B4Ecv3.

[0033]FIG. 10 shows chemotaxis of HHEC in response to B2EC-FC.

[0034]FIG. 11 shows chemotaxis of HHAEC in response to B2EC.

[0035]FIG. 12 shows effect of B4Ecv3 on HUAEC tubule formation.

[0036]FIG. 13 shows effect of B2EC-FC on HUAEC tubule formation.

[0037]FIG. 14 is a schematic representation of human Ephrin B2 constructs.

[0038]FIG. 15 is a schematic representation of human EphB4 constructs.

[0039]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.

[0040]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.

[0041]FIG. 18 shows that EphB4v3 inhibits chemotaxis.

[0042]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.

[0043]FIG. 20 shows that soluble EphB4 has no detectable cytotoxic effect as assessed by MTS assay.

[0044]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.

[0045]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.

[0046]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.

[0047]FIG. 24 shows that B4v3 inhibits endovascular response in a murine corneal hydron micropocket assay.

[0048]FIG. 25 shows that that SCC15, B16, and MCF-7 co-injected with sB4v3 in the presence of matrigel and growth factors, inhibits the in vivo tumor growth of these cells.

[0049]FIG. 26 shows that soluble EphB4 causes apoptosis, necrosis and decreased angiogenesis in three tumor types, B16 (melanoma), SCC15 (head and neck carcinoma), and MCF-7 (breast carcinoma). Tumors were injected premixed with Matrigel plus growth factors and soluble EphB4 subcutaneously. After 10 to 14 days, the mice were injected intravenously with FITC-lectin (green) to assess blood vessel perfusion. Tumors treated with control PBS displayed abundant tumor density and a robust angiogenic response. Tumors treated with sEphB4 displayed a decrease in tumor cell density and a marked inhibition of tumor angiogenesis in regions with viable tumor cells, as well as tumor necrosis and apoptosis.

[0050]FIG. 27 shows expression of EphB4 in prostate cell lines. A) Western blot of total cell lysates of various prostate cancer cell lines, normal prostate gland derived cell line (MLC) and acute myeloblastic lymphoma cells (AML) probed with EphB4 monoclonal antibody. B) Phosphorylation of EphB4 in PC-3 cells determined by Western blot.

[0051]FIG. 28 shows expression of EphB4 in prostate cancer tissue. Representative prostate cancer frozen section stained with EphB4 monoclonal antibody (top left) or isotype specific control (bottom left). Adjacent BPH tissue stained with EphB4 monoclonal antibody (top right). Positive signal is brown color in the tumor cells. Stroma and the normal epithelia are negative. Note membrane localization of stain in the tumor tissue, consistent with trans-membrane localization of EphB4. Representative QRT-PCR of RNA extracted from cancer specimens and adjacent BPH tissues (lower right).

[0052]FIG. 29 shows downregulation of EphB4 in prostate cancer cells by tumor suppressors and RXR expression. A) PC3 cells were co-transfected with truncated CD4 and p53 or PTEN or vector only. 24 h later CD4-sorted cells were collected, lysed and analyzed sequentially by Western blot for the expression of EphB4 and β-actin, as a normalizer protein. B) Western blot as in (A) of various stable cell lines. LNCaP-FGF is a stable transfection clone of FGF-8, while CWR22R-RXR stably expresses the RXR receptor. BPH-1 was established from benign hypertrophic prostatic epithelium.

[0053]FIG. 30 shows regulation of EphB4 in prostate cancer cells by EGFR and IGFR-1. A) Western blot of PC3 cells treated with or without EGFR specific inhibitor AG1478 (1 nM) for 36 hours. Decreased EphB4 signal is observed after AG 1478 treatment. The membrane was stripped and reprobed with β-actin, which was unaffected. B) Western Blot of triplicate samples of PC3 cells treated with or without IGFR-1 specific neutralizing antibody MAB391 (2 μg/ml; overnight). The membrane was sequentially probed with EphB4, IGFR-1 and β-actin antibodies. IGFR-1 signal shows the expected repression of signal with MAB391 treatment.

[0054]FIG. 31 shows effect of specific EphB4 AS-ODNs and siRNA on expression and prostate cell functions. A) 293 cells stably expressing full-length construct of EphB4 was used to evaluate the ability of siRNA 472 to inhibit EphB4 expression. Cells were transfected with 50 nM RNAi using Lipofectamine 2000. Western blot of cell lysates 40 h post transfection with control siRNA (green fluorescence protein; GFP siRNA) or EphB4 siRNA 472, probed with EphB4 monoclonal antibody, stripped and reprobed with β-actin monoclonal antibody. B) Effect of EphB4 AS-10 on expression in 293 transiently expressing full-length EphB4. Cells were exposed to AS-10 or sense ODN for 6 hours and analyzed by Western blot as in (A). C) 48 h viability assay of PC3 cells treated with siRNA as described in the Methods section. Shown is mean±s.e.m. of triplicate samples. D) 5-day viability assay of PC3 cells treated with ODNs as described in the Methods. Shown is mean±s.e.m. of triplicate samples. E) Scrape assay of migration of PC3 cells in the presence of 50 nM siRNAs transfected as in (A). Shown are photomicrographs of representative 20× fields taken immediately after the scrape was made in the monolayer (0 h) and after 20 h continued culture. A large number of cells have filled in the scrape after 20 h with control siRNA, but not with EphB4 siRNA 472. F) Shown is a similar assay for cells treated with AS-10 or sense ODN (both 10 μM). G) Matrigel invasion assay of PC3 cells transfected with siRNA or control siRNA as described in the methods. Cells migrating to the underside of the Matrigel coated insert in response to 5 mg/ml fibronectin in the lower chamber were fixed and stained with Giemsa. Shown are representative photomicrographs of control siRNA and siRNA 472 treated cells. Cell numbers were counted in 5 individual high-powered fields and the average±s.e.m. is shown in the graph (bottom right).

[0055]FIG. 32 shows effect of EphB4 siRNA 472 on cell cycle and apoptosis. A) PC3 cells transfected with siRNAs as indicated were analyzed 24 h post transfection for cell cycle status by flow cytometry as described in the Methods. Shown are the plots of cell number vs. propidium iodide fluorescence intensity. 7.9% of the cell population is apoptotic (in the Sub G0 peak) when treated with siRNA 472 compared to 1% with control siRNA. B) Apoptosis of PC3 cells detected by Cell Death Detection ELISA^plus kit as described in the Methods. Absorbance at 405 nm increases in proportion to the amount of histone and DNA-POD in the nuclei-free cell fraction. Shown is the mean±s.e.m. of triplicate samples at the indicated concentrations of siRNA 472 and GFP siRNA (control).

[0056]FIG. 33 shows that EphB4 and EphrinB2 are expressed in mesothelioma cell lines as shown by RT-PCR (A) and Western Blot (B).

[0057]FIG. 34 shows expression of ephrin B2 and EphB4 by in situ hybridization in mesothelioma cells. NCI H28 mesothelioma cell lines cultured in chamber slides hybridized with antisense probe to ephrin B2 or EphB4 (top row). Control for each hybridization was sense (bottom row). Positive reaction is dark blue cytoplasmic stain.

[0058]FIG. 35 shows cellular expression of EphB4 and ephrin B2 in mesothelioma cultures. Immunofluorescence staining of primary cell isolate derived from pleural effusion of a patient with malignant mesothelioma and cell lines NCI H28, NCI H2373, and NCI H2052 for ephrin B2 and EphB4. Green color is positive signal for FITC labeled secondary antibody. Specificity of immunofluorescence staining was demonstrated by lack of signal with no primary antibody (first row). Cell nuclei were counterstained with DAPI (blue color) to reveal location of all cells. Shown are merged images of DAPI and FITC fluorescence. Original magnification 200×.

[0059]FIG. 36 shows expression of ephrin B2 and EphB4 in mesothelioma tumor. Immunohistochemistry of malignant mesothelioma biopsy. H&E stained section reveals tumor architecture; bottom left panel is background control with no primary antibody. EphB4 and ephrin B2 specific staining is brown color. Original magnification 200×.

[0060]FIG. 37 shows effects of EPHB4 antisense probes (A) and EPHB4 siRNAs (B) on the growth of H28 cells.

[0061]FIG. 38 shows effects of EPHB4 antisense probes (A) and EPHB4 siRNAs (B) on cell migration.

[0062]FIG. 39 shows that EphB4 is expressed in HNSCC primary tissues and metastases. A) Top: Immunohistochemistry of a representative archival section stained with EphB4 monoclonal antibody as described in the methods and visualized with DAB (brown color) localized to tumor cells. Bottom: Hematoxylin and Eosin (H&E) stain of an adjacent section. Dense purple staining indicates the presence of tumor cells. The right hand column are frozen sections of lymph node metastasis stained with EphB4 polyclonal antibody (top right) and visualized with DAB. Control (middle) was incubation with goat serum and H&E (bottom) reveals the location of the metastatic foci surrounded by stroma which does not stain. B) In situ hybridization of serial frozen sections of a HNSCC case probed with EphB4 (left column) and ephrin B2 (right column) DIG labeled antisense or sense probes generated by run-off transcription. Hybridization signal (dark blue) was detected using alkaline-phosphatase-conjugated anti-DIG antibodies and sections were counterstained with Nuclear Fast Red. A serial section stained with H&E is shown (bottom left) to illustrate tumor architecture. C) Western blot of protein extract of patient samples consisting of tumor (T), uninvolved normal tissue (N) and lymph node biopsies (LN). Samples were fractionated by polyacrylamide gel electrophoresis in 4-20% Tris-glycine gels and subsequently electroblotted onto nylon membranes. Membranes were sequentially probed with EphB4 monoclonal antibody and β-actin MoAb. Chemiluminescent signal was detected on autoradiography film. Shown is the EphB4 specific band which migrated at 120 kD and β-actin which migrated at 40 kD. The β-actin signal was used to control for loading and transfer of each sample.

[0063]FIG. 40 shows that EphB4 is expressed in HNSCC cell lines and is regulated by EGF: A) Survey of EphB4 expression in SCC cell lines. Western blot of total cell lysates sequentially probed with EphB4 monoclonal antibody, stripped and reprobed with β-actin monoclonal antibody as described for FIG. 39c. B) Effect of the specific EGFR inhibitor AG1478 on EphB4 expression: Western blot of crude cell lysates of SCC15 treated with 0-1000 nM AG 1478 for 24 h in media supplemented with 10% FCS (left) or with 1 mM AG 1478 for 4, 8, 12 or 24 h (right). Shown are membranes sequentially probed for EphB4 and β-actin. C) Effect of inhibition of EGFR signaling on EphB4 expression in SCC cell lines: Cells maintained in growth media containing 10% FCS were treated for 24 hr with 1 μM AG 1478, after which crude cell lysates were analyzed by Western blots of cell lysates sequentially probed with for EGFR, EphB4, ephrin B2 and β-actin antibodies. Specific signal for EGFR was detected at 170 kD and ephrin B2 at 37 kD in addition to EphB4 and β-actin as described in FIG. 1C. β-actin serves as loading and transfer control.

[0064]FIG. 41 shows mechanism of regulation of EphB4 by EGF: A) Schematic of the EGFR signaling pathways, showing in red the sites of action and names of specific kinase inhibitors used. B) SCC15 cells were serum-starved for 24 h prior to an additional 24 incubation as indicated with or without EGF (10 ng/ml), 3 μM U73122, or 5 μM SH-5, 5 μM SP600125, 25 nM LY294002, --μM PD098095 or 5 μM SB203580. N/A indicates cultures that received equal volume of diluent (DMSO) only. Cell lysates were subjected to Western Blot with EphB4 monoclonal antibody. β-actin signal serves as control of protein loading and transfer.

[0065]FIG. 42 shows that specific EphB4 siRNAs inhibit EphB4 expression, cell viability and cause cell cycle arrest. A) 293 cells stably expressing full length EphB4 were transfected with 50 nM RNAi using Lipofectamine®2000. 40 h post-transfection cells were harvested, lysed and processed for Western blot. Membranes were probed with EphB4 monoclonal antibody, stripped and reprobed with β-actin monoclonal antibody as control for protein loading and transfer. Negative reagent control was RNAi to scrambled green fluorescence protein (GFP) sequence and control is transfection with Lipofectamine®2000 alone. B) MTT cell viability assays of SCC cell lines treated with siRNAs for 48 h as described in the Methods section. Shown is mean±s.e.m. of triplicate samples. C) SCC15 cells transfected with siRNAs as indicated were analyzed 24 h post transfection for cell cycle status by flow cytometry as described in the Methods. Shown are the plots of cell number vs. propidium iodide fluorescence intensity. Top and middle row show plots for cells 16 h after siRNA transfection, bottom row shows plots for cells 36 h post transfection. Specific siRNA and concentration are indicated for each plot. Lipo=Lipofectamine®200 mock transfection.

[0066]FIG. 43 shows in vitro effects of specific EphB4 AS-ODNs on SCC cells. A) 293 cells transiently transfected with EphB4 full-length expression plasmid were treated 6 h post transfection with antisense ODNs as indicated. Cell lysates were collected 24 h after AS-ODN treatment and subjected to Western Blot. B) SCC25 cells were seeded on 48 well plates at equal densities and treated with EphB4 AS-ODNs at 1, 5, and 10 μM on days 2 and 4. Cell viability was measured by MTT assay on day 5. Shown is the mean±s.e.m. of triplicate samples. Note that AS-ODNs that were active in inhibiting EphB4 protein levels were also effective inhibitors of SCC15 cell viability. C) Cell cycle analysis of SCC15 cells treated for 36 h with AS-10 (bottom) compared to cells that were not treated (top). D) Confluent cultures of SCC15 cells scraped with a plastic Pasteur pipette to produce 3 mm wide breaks in the monolayer. The ability of the cells to migrate and close the wound in the presence of inhibiting EphB4 AS-ODN (AS-10) and non-inhibiting AS-ODN (AS-1) was assessed after 48 h. Scrambled ODN is included as a negative control ODN. Culture labeled no treatment was not exposed to ODN. At initiation of the experiment, all cultures showed scrapes of equal width and similar to that seen in 1 μM EphB4 AS-10 after 48 h. The red brackets indicate the width of the original scrape. E) Migration of SCC15 cells in response to 20 mg/ml EGF in two-chamber assay as described in the Methods. Shown are representative photomicrographs of non-treated (NT), AS-6 and AS-10 treated cells and 10 ng/ml Taxol as positive control of migration inhibition. F) Cell numbers were counted in 5 individual high-powered fields and the average+s.e.m. is shown in the graph.

[0067]FIG. 44 shows that EphB4 AS-ODN inhibits tumor growth in vivo. Growth curves for SCC15 subcutaneous tumor xenografts in Balb/C nude mice treated with EphB4 AS-10 or scrambled ODN at 20 mg/kg/day starting the day following implantation of 5×106 cells. Control mice received and equal volume of diluent (PBS). Shown are the mean±s.e.m. of 6 mice/group. *P=0.0001 by Student's t-test compared to scrambled ODN treated group.

[0068]FIG. 45 shows that Ephrin B2, but not EphB4 is expressed in KS biopsy tissue. (A) In situ hybridization with antisense probes for ephrin B2 and EphB4 with corresponding H&E stained section to show tumor architecture. Dark blue color in the ISH indicates positive reaction for ephrin B2. No signal for EphB4 was detected in the Kaposi's sarcoma biopsy. For contrast, ISH signal for EphB4 is strong in squamous cell carcinoma tumor cells. Ephrin B2 was also detected in KS using EphB4-AP fusion protein (bottom left). (B) Detection of ephrin B2 with EphB4/Fc fusion protein. Adjacent sections were stained with H&E (left) to show tumor architecture, black rectangle indicates the area shown in the EphB4/Fc treated section (middle) detected with FITC-labeled anti-human Fc antibody as described in the methods section. As a control an adjacent section was treated with human Fc fragment (right). Specific signal arising from EphB4/Fc binding to the section is seen only in areas of tumor cells. (C) Co-expression of ephrin B2 and the HHV8 latency protein LANA1. Double-label confocal immunofluorescence microscopy with antibodies to ephrin B2 (red) LANA1 (green), or EphB4 (red) of frozen KS biopsy material directly demonstrates co-expression of LANA1 and ephrin B2 in KS biopsy. Coexpression is seen as yellow color. Double label confocal image of biopsy with antibodies to PECAM-1 (green) in cells with nuclear propidium iodide stain (red), demonstrating the vascular nature of the tumor.

[0069]FIG. 46 shows that HHV-8 induces arterial marker expression in venous endothelial cells. (A) Immunofluorescence of cultures of HUVEC and HUVEC/BC-1 for artery/vein markers and viral proteins. Cultures were grown on chamber slides and processed for immunofluorescence detection of ephrin B2 (a, e, i), EphB4 (m, q, u), CD148 (j, v), and the HHV-8 proteins LANA1 (b, f, m) or ORF59 (r) as described in the Materials and Methods. Yellow color in the merged images of the same field demonstrate co-expression of ephrin B2 and LANA or ephrin B2 and CD148. The positions of viable cells were revealed by nuclear staining with DAPI (blue) in the third column (c, g, k, o, s, w). Photomicrographs are of representative fields. (B) RT-PCR of HUVEC and two HHV-8 infected cultures (HUVEC/BC-1 and HUVEC/BC-3) for ephrin B2 and EphB4. Ephrin B2 product (200 bp) is seen in HUVEC/BC-1, HUVEC/BC-3 and EphB4 product (400 bp) is seen in HUVEC. Shown also is β-actin RT-PCR as a control for amount and integrity of input RNA.

[0070]FIG. 47 shows that HHV-8 induces arterial marker expression in Kaposi's sarcoma cells. (A) Western blot for ephrin B2 on various cell lysates. SLK-vGPCR is a stable clone of SLK expressing the HHV-8 vGPCR, and SLK-pCEFL is control stable clone transfected with empty expression vector. SLK cells transfected with LANA or LANAΔ440 are SLK-LANA and SLK-Δ440 respectively. Quantity of protein loading and transfer was determined by reprobing the membranes with β-actin monoclonal antibody. (B) Transient transfection of KS-SLK cells with expression vector pvGPCR-CEFL resulted in the expression of ephrin B2 as shown by immunofluorescence staining with FITC (green), whereas the control vector pCEFL had no effect. KS-SLK cells (0.8×105/well) were transfected with 0.8 μg DNA using Lipofectamine 2000. 24 hr later cells were fixed and stained with ephrin B2 polyclonal antibody and FITC conjugated secondary antibody as described in the methods. (C) Transient transfection of HUVEC with vGPCR induces transcription from ephrin B2 luciferase constructs. 8×103 HUVEC in 24 well plates were transfected using Superfect with 0.8 μg/well ephrin B2 promoter constructs containing sequences from -2941 to -11 with respect to the translation start site, or two 5'-deletions as indicated, together with 80 ng/well pCEFL or pvGPCR-CEFL. Luciferase was determined 48 h post transfection and induction ratios are shown to the right of the graph. pGL3Basic is promoterless luciferase control vector. Luciferase was normalized to protein since GPCR induced expression of the cotransfected β-galactosidase. Graphed is mean±SEM of 6 replicates. Shown is one of three similar experiments.

[0071]FIG. 48 shows that VEGF and VEGF-C regulate ephrin B2 expression. A) Inhibition of ephrin B2 by neutralizing antibodies. Cells were cultured in full growth medium and exposed to antibody (100 ng/ml) for 36 hr before collection and lysis for Western blot. B) For induction of ephrin B2 expression cells were cultured in EBM growth medium containing 5% serum lacking growth factors. Individual growth factors were added as indicated and the cells harvested after 36 h. Quantity of protein loading and transfer was determined by reprobing the membranes β-actin monoclonal antibody.

[0072]FIG. 49 shows that Ephrin B2 knock-down with specific siRNA inhibits viability in KS cells and HUVEC grown in the presence of VEGF but not IGF, EGF or bFGF. A) KS-SLK cells were transfected with various siRNA to ephrin B2 and controls. After 48 hr the cells were harvested and crude cell lysates fractionated on 4-20% SDS-PAGE. Western blot was performed with monoclonal antibody to ephrin B2 generated in-house. The membrane was stripped and reprobed with β-actin monoclonal antibody (Sigma) to illustrate equivalent loading and transfer. B) 3 day cell viability assay of KS-SLK cultures in the presence of ephrin B2 and EphB4 siRNAs. 1×10⁵ cells/well in 24-well plates were treated with 0, 10 and 100 ng/ml siRNAs as indicated on the graph. Viability of cultures was determined by MTT assay as described in the methods section. Shown are the mean+standard deviation of duplicate samples. C) HUVE cells were seeded on eight wells chamber slides coated with fibronectin. The HUVE cells were grown overnight in EGM-2 media, which contains all growth supplements. On the following day, the media was replaced with media containing VEGF (10 ng/ml) or EGF, FGF and IGF as indicated. After 2 hrs of incubation at 37° C., the cells were transfected using Lipofectamine 2000 (Invitrogen) in Opti-MEM medium containing 10 nM of siRNA to ephrin B2, Eph B4 or green fluorescence protein (GFP) as control. The cells were incubated for 2 hr and then the fresh media containing growth factors or VEGF alone was added to their respective wells. After 48 hrs, the cells were stained with crystal violet and the pictures were taken immediately by digital camera at 10× magnification.

[0073]FIG. 50 shows that soluble EphB4 inhibits KS and EC cord formation and in vivo angiogenesis. Cord formation assay of HUVEC in Matrigel® (upper row). Cells in exponential growth phase were treated overnight with the indicated concentrations of EphB4 extracellular domain (ECD) prior to plating on Matrigel®. Cells were trypsinized and plated (1×10⁵ cells/well) in a 24-well plate containing 0.5 ml Matrigel®. Shown are representative 20× phase contrast fields of cord formation after 8 hr plating on Matrigel® in the continued presence of the test compounds as shown. Original magnification 200×. KS-SLK cells treated in a similar manner (middle row) in a cord formation assay on Matrigel®. Bottom row shows in vivo Matrigel® assay: Matrigel® plugs containing growth factors and EphB4 ECD or PBS were implanted subcutaneously in the mid-ventral region of mice. After 7 days the plugs were removed, sectioned and stained with H&E to visualize cells migrating into the matrix. Intact vessels with large lumens are observed in the control, whereas EphB4 ECD almost completely inhibited migration of cells into the Matrigel.

[0074]FIG. 51 shows expression of EPHB4 in bladder cancer cell lines (A), and regulation of EPHB4 expression by EGFR signaling pathway (B).

[0075]FIG. 52 shows that transfection of p53 inhibit the expression of EPHB4 in 5637 cell.

[0076]FIG. 53 shows growth inhibition of bladder cancer cell line (5637) upon treatment with EPHB4 siRNA 472.

[0077]FIG. 54 shows results on apoptosis study of 5637 cells transfected with EPHB4 siRNA 472.

[0078]FIG. 55 shows effects of EPHB4 antisense probes on cell migration. 5637 cells were treated with EPHB4AS10 (10 μM) (bottom panels). Upper panels show control cells.

[0079]FIG. 56 shows effects of EPHB4 siRNA on cell invasion. 5637 cells were transfected with siRNA 472 or control siRNA.

[0080]FIG. 57 shows comparison of EphB4 monoclonal antibodies by G250 and in pull-down assay.

[0081]FIG. 58 shows that EphB4 antibodies inhibit the growth of SCC15 xenograft tumors.

[0082]FIG. 59 shows that EphB4 antibodies cause apoptosis, necrosis and decreased angiogenesis in SCC15, head and neck carcinoma tumor type.

[0083]FIG. 60 shows that systemic administration of EphB4 antibodies leads to tumor regression.

[0084]FIG. 61 shows a genomic nucleotide sequence of human EphB4 (SEQ ID NO:6).

[0085]FIG. 62 shows a cDNA nucleotide sequence of human EphB4 (SEQ ID NO:7).

[0086]FIG. 63 shows a genomic nucleotide sequence of human Ephrin B2 (SEQ ID NO:8).

[0087]FIG. 64 shows a cDNA nucleotide sequence of human Ephrin B2 (SEQ ID NO:9).

[0088]FIG. 65 shows an amino acid sequence of human EphB4 (SEQ ID NO:10).

[0089]FIG. 66 shows an amino acid sequence of human Ephrin B2 (SEQ ID NO:11).

[0090]FIG. 67 shows a comparison of the EphrinB2 binding properties of the HSA-EphB4 fusion protein and other EphB4 polypeptides.

[0091]FIG. 68 shows a comparison between the in vivo stability of an EphB4-HSA fusion protein and an EphB4 polypeptide in mice.

[0092]FIG. 69 shows the EphrinB2 binding activity of soluble EphB4 polypeptides pegylated under specific pH conditions.

[0093]FIG. 70 shows the chromatographic separation of PEG derivatives of EphB4 protein on SP-Sepharose columns. Purity of the PEG-modified EphB4 protein was analyzed by PAGE. The EphrinB2 binding of the pegylation reaction products is also shown.

[0094]FIG. 71 shows the purity, as determined by SDS-PAGE, of chromatography-separated unpegylated, monopegylated and poly-pegylated EphB4 fractions.

[0095]FIG. 72 shows the EphrinB2-binding activity of the chromatography fractions from the EphB4 pegylation reaction.

[0096]FIG. 73 shows the retention of EphrinB2-binding activity of the chromatography fractions from the EphB4 pegylation reaction after incubation in mouse serum at 37° C. for three days.

[0097]FIG. 74 shows the in vivo stability of unpegylated, monopegylated and polypegylated EphB4 in mice over time.

DETAILED DESCRIPTION OF THE INVENTION

I. Overview

[0098]The current invention is based in part on the discovery that signaling through the ephrin/ephrin receptor (ephrin/eph) pathway contributes to tumorigenesis. Applicants detected expression of ephrin B2 and EphB4 in tumor tissues and developed anti-tumor therapeutic agents for blocking signaling through the ephrin/eph. In addition, the disclosure provides polypeptide therapeutic agents and methods for polypeptide-based inhibition of the function of EphB4 and/or Ephrin B2. Accordingly, in certain aspects, the disclosure provides numerous polypeptide compounds (agents) that may be used to treat cancer as well as angiogenesis related disorders and unwanted angiogenesis related processes. Applicants have generated modified forms of EphrinB2 and EphB4 polypeptides and have demonstrated that such modified forms have markedly improved pharmacokinetic properties. Accordingly, in certain aspects, the disclosure provides numerous polypeptide compounds (agents) that may be used to treat cancer as well as angiogenesis related disorders and unwanted angiogenesis related processes.

[0099]As used herein, the terms Ephrin and Eph are used to refer, respectively, to ligands and receptors. They can be from any of a variety of animals (e.g., mammals/non-mammals, vertebrates/non-vertebrates, including humans). The nomenclature in this area has changed rapidly and the terminology used herein is that proposed as a result of work by the Eph Nomenclature Committee, which can be accessed, along with previously-used names at web site http://www.eph-nomenclature.com.

[0100]The work described herein, particularly in the examples, refers to Ephrin B2 and EphB4. However, the present invention contemplates any ephrin ligand and/or Eph receptor within their respective family, which is expressed in a tumor. The ephrins (ligands) are of two structural types, which can be further subdivided on the basis of sequence relationships and, functionally, on the basis of the preferential binding they exhibit for two corresponding receptor subgroups. Structurally, there are two types of ephrins: those which are membrane-anchored by a glycerophosphatidylinositol (GPI) linkage and those anchored through a transmembrane domain. Conventionally, the ligands are divided into the Ephrin-A subclass, which are GPI-linked proteins which bind preferentially to EphA receptors, and the Ephrin-B subclass, which are transmembrane proteins which generally bind preferentially to EphB receptors.

[0101]The Eph family receptors are a family of receptor protein-tyrosine kinases which are related to Eph, a receptor named for its expression in an erythropoietin-producing human hepatocellular carcinoma cell line. They are divided into two subgroups on the basis of the relatedness of their extracellular domain sequences and their ability to bind preferentially to Ephrin-A proteins or Ephrin-B proteins. Receptors which interact preferentially with Ephrin-A proteins are EphA receptors and those which interact preferentially with Ephrin-B proteins are EphB receptors.

[0102]Eph receptors have an extracellular domain composed of the ligand-binding globular domain, a cysteine rich region followed by a pair of fibronectin type III repeats (e.g., see FIG. 16). The cytoplasmic domain consists of a juxtamembrane region containing two conserved tyrosine residues; a protein tyrosine kinase domain; a sterile α-motif (SAM) and a PDZ-domain binding motif. EphB4 is specific for the membrane-bound ligand Ephrin B2 (Sakano, S. et al 1996; Brambilla R. et al 1995). Ephrin B2 belongs to the class of Eph ligands that have a transmembrane domain and cytoplasmic region with five conserved tyrosine residues and PDZ domain. Eph receptors are activated by binding of clustered, membrane attached ephrins (Davis S et al, 1994), indicating that contact between cells expressing the receptors and cells expressing the ligands is required for Eph activation.

[0103]Upon ligand binding, an Eph receptor dimerizes and autophosphorylate the juxtamembrane tyrosine residues to acquire full activation (Kalo M S et al, 1999, Binns K S, 2000). In addition to forward signaling through the Eph receptor, reverse signaling can occur through the ephrin Bs. Eph engagement of ephrins results in rapid phosphorylation of the conserved intracellular tyrosines (Bruckner K, 1997) and somewhat slower recruitment of PDZ binding proteins (Palmer A 2002). Recently, several studies have shown that high expression of Eph/ephrins may be associated with increased potentials for tumor growth, tumorigenicity, and metastasis (Easty D J, 1999; Kiyokawa E, 1994; Tang X X, 1999; Vogt T, 1998; Liu W, 2002; Stephenson S A, 2001; Steube K G 1999; Berclaz G, 1996).

[0104]In certain embodiments, the present invention provides polypeptide therapeutic agents that inhibit activity of Ephrin B2, EphB4, or both. As used herein, the term "polypeptide therapeutic agent" or "polypeptide agent" is a generic term which includes any polypeptide that blocks signaling through the Ephrin B2/EphB4 pathway. A preferred polypeptide therapeutic agent of the invention is a soluble polypeptide of Ephrin B2 or EphB4. Another preferred polypeptide therapeutic agent of the invention is an antagonist antibody that binds to Ephrin B2 or EphB4. For example, such polypeptide therapeutic agent can inhibit function of Ephrin B2 or EphB4, inhibit the interaction between Ephrin B2 and EphB4, inhibit the phosphorylation of Ephrin B2 or EphB4, or inhibit any of the downstream signaling events upon binding of Ephrin B2 to EphB4. Such polypeptides may include EphB4 or EphrinB2 that are modified so as to improve serum half-life, such as by PEGylation or stable association with a serum albumin protein.

II. Soluble Polypeptides

[0105]In certain aspects, the invention relates to a soluble polypeptide comprising an extracellular domain of an Ephrin B2 protein (referred to herein as an Ephrin B2 soluble polypeptide) or comprising an extracellular domain of an EphB4 protein (referred to herein as an EphB4 soluble polypeptide). Preferably, the subject soluble polypeptide is a monomer and is capable of binding with high affinity to Ephrin B2 or EphB4. In a specific embodiment, the EphB4 soluble polypeptide of the invention comprises a globular domain of an EphB4 protein. Specific examples EphB4 soluble polypeptides are provided in FIGS. 1, 2, and 15. Specific examples of Ephrin B2 soluble polypeptides are provided in FIGS. 3 and 14.

[0106]As used herein, the subject soluble polypeptides include fragments, functional variants, and modified forms of EphB4 soluble polypeptide or an Ephrin B2 soluble polypeptide. These fragments, functional variants, and modified forms of the subject soluble polypeptides antagonize function of EphB4, Ephrin B2 or both.

[0107]In certain embodiments, isolated fragments of the subject soluble polypeptides can be obtained by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding an EphB4 or Ephrin B2 soluble polypeptides. In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. The fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments that can function to inhibit function of EphB4 or Ephrin B2, for example, by testing the ability of the fragments to inhibit angiogenesis or tumor growth.

[0108]In certain embodiments, a functional variant of an EphB4 soluble polypeptide comprises an amino acid sequence that is at least 90%, 95%, 97%, 99% or 100% identical to residues 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 of the amino acid sequence defined by FIG. 65 (SEQ ID NO: 10). Such polypeptides may be used in a processed form, and accordingly, in certain embodiments, an EphB4 soluble polypeptide comprises an amino acid sequence that is at least 90%, 95%, 97%, 99% or 100% identical to residues 16-197, 16-312, 16-321, 16-326, 16-412, 16-427, 16-429, 16-526 and 16-537 of the amino acid sequence defined by FIG. 65 (SEQ ID NO:10).

[0109]In other embodiments, a functional variant of an Ephrin B2 soluble polypeptide comprises a sequence at least 90%, 95%, 97%, 99% or 100% identical to residues 1-225 of the amino acid sequence defined by FIG. 66 (SEQ ID NO: 11) or a processed form, such as one comprising a sequence at least 90%, 95%, 97%, 99% or 100% identical to residues 26-225 of the amino acid sequence defined by FIG. 66 (SEQ ID NO: 11).

[0110]In certain embodiments, the present invention contemplates making functional variants by modifying the structure of the subject soluble polypeptide for such purposes as enhancing therapeutic or prophylactic efficacy, or stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo). Such modified soluble polypeptide are considered functional equivalents of the naturally-occurring EphB4 or Ephrin B2 soluble polypeptide. Modified soluble polypeptides can be produced, for instance, by amino acid substitution, deletion, or addition. For instance, it is reasonable to expect, for example, that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (e.g., conservative mutations) will not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains.

[0111]This invention further contemplates a method of generating sets of combinatorial mutants of the EphB4 or Ephrin B2 soluble polypeptides, as well as truncation mutants, and is especially useful for identifying functional variant sequences. The purpose of screening such combinatorial libraries may be to generate, for example, soluble polypeptide variants which can act as antagonists of EphB4, EphB2, or both. Combinatorially-derived variants can be generated which have a selective potency relative to a naturally occurring soluble polypeptide. Such variant proteins, when expressed from recombinant DNA constructs, can be used in gene therapy protocols. Likewise, mutagenesis can give rise to variants which have intracellular half-lives dramatically different than the corresponding wild-type soluble polypeptide. For example, the altered protein can be rendered either more stable or less stable to proteolytic degradation or other cellular process which result in destruction of, or otherwise inactivation of the protein of interest (e.g., a soluble polypeptide). Such variants, and the genes which encode them, can be utilized to alter the subject soluble polypeptide levels by modulating their half-life. For instance, a short half-life can give rise to more transient biological effects and, when part of an inducible expression system, can allow tighter control of recombinant soluble polypeptide levels within the cell. As above, such proteins, and particularly their recombinant nucleic acid constructs, can be used in gene therapy protocols.

[0112]There are many ways by which the library of potential homologs can be generated from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes then be ligated into an appropriate gene for expression. The purpose of a degenerate set of genes is to provide, in one mixture, all of the sequences encoding the desired set of potential soluble polypeptide sequences. The synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, S A (1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. A G Walton, Amsterdam: Elsevier pp 273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 11:477). Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al., (1990) Science 249:386-390; Roberts et al., (1992) PNAS USA 89:2429-2433; Devlin et al., (1990) Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

[0113]Alternatively, other forms of mutagenesis can be utilized to generate a combinatorial library. For example, soluble polypeptide variants (e.g., the antagonist forms) can be generated and isolated from a library by screening using, for example, alanine scanning mutagenesis and the like (Ruf et al., (1994) Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al., (1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol. Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838; and Cunningham et al., (1989) Science 244:1081-1085), by linker scanning mutagenesis (Gustin et al., (1993) Virology 193:653-660; Brown et al., (1992) Mol. Cell Biol. 12:2644-2652; McKnight et al., (1982) Science 232:316); by saturation mutagenesis (Meyers et al., (1986) Science 232:613); by PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol 1:11-19); or by random mutagenesis, including chemical mutagenesis, etc. (Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, N.Y.; and Greener et al., (1994) Strategies in Mol Biol 7:32-34). Linker scanning mutagenesis, particularly in a combinatorial setting, is an attractive method for identifying truncated (bioactive) forms of the subject soluble polypeptide.

[0114]A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations and truncations, and, for that matter, for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of the subject soluble polypeptides. The most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected. Each of the illustrative assays described below are amenable to high through-put analysis as necessary to screen large numbers of degenerate sequences created by combinatorial mutagenesis techniques.

[0115]In certain embodiments, the subject soluble polypeptides of the invention include a small molecule such as a peptide and a peptidomimetic. As used herein, the term "peptidomimetic" includes chemically modified peptides and peptide-like molecules that contain non-naturally occurring amino acids, peptoids, and the like. Peptidomimetics provide various advantages over a peptide, including enhanced stability when administered to a subject. Methods for identifying a peptidomimetic are well known in the art and include the screening of databases that contain libraries of potential peptidomimetics. For example, the Cambridge Structural Database contains a collection of greater than 300,000 compounds that have known crystal structures (Allen et al., Acta Crystallogr. Section B, 35:2331 (1979)). Where no crystal structure of a target molecule is available, a structure can be generated using, for example, the program CONCORD (Rusinko et al., J. Chem. Inf. Comput. Sci. 29:251 (1989)). Another database, the Available Chemicals Directory (Molecular Design Limited, Informations Systems; San Leandro Calif.), contains about 100,000 compounds that are commercially available and also can be searched to identify potential peptidomimetics of the EphB4 or Ephrin B2 soluble polypeptides.

[0116]In certain embodiments, the soluble polypeptides of the invention may further comprise post-translational modifications. Exemplary post-translational protein modification include phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, sumoylation, biotinylation or addition of a polypeptide side chain or of a hydrophobic group. As a result, the modified soluble polypeptides may contain non-amino acid elements, such as lipids, poly- or mono-saccharide, and phosphates. Effects of such non-amino acid elements on the functionality of a soluble polypeptide may be tested for its antagonizing role in EphB4 or Ephrin B2 function, e.g, it inhibitory effect on angiogenesis or on tumor growth.

[0117]In one specific embodiment of the present invention, modified forms of the subject soluble polypeptides comprise linking the subject soluble polypeptides to nonproteinaceous polymers. In one specific embodiment, the polymer is polyethylene glycol ("PEG"), polypropylene glycol, or polyoxyalkylenes, in the manner as set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. Examples of the modified polypeptide of the invention include PEGylated soluble Ephrin B2 and PEGylated soluble EphB4.

[0118]PEG is a well-known, water soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). The term "PEG" is used broadly to encompass any polyethylene glycol molecule, without regard to size or to modification at an end of the PEG, and can be represented by the formula:

X--O(CH₂CH₂O)_n-1CH₂CH₂OH (1), where n is 20 to 2300 and X is H or a terminal modification, e.g., a C_1-4 alkyl. In one embodiment, the PEG of the invention terminates on one end with hydroxy or methoxy, i.e., X is H or CH₃ ("methoxy PEG"). A PEG can contain further chemical groups which are necessary for binding reactions; which results from the chemical synthesis of the molecule; or which is a spacer for optimal distance of parts of the molecule. In addition, such a PEG can consist of one or more PEG side-chains which are linked together. PEGs with more than one PEG chain are called multiarmed or branched PEGs. Branched PEGs can be prepared, for example, by the addition of polyethylene oxide to various polyols, including glycerol, pentaerythriol, and sorbitol. For example, a four-armed branched PEG can be prepared from pentaerythriol and ethylene oxide. Branched PEG are described in, for example, EP-A 0 473 084 and U.S. Pat. No. 5,932,462. One form of PEGs includes two PEG side-chains (PEG2) linked via the primary amino groups of a lysine (Monfardini, C., et al., Bioconjugate Chem. 6 (1995) 62-69).

[0119]PEG conjugation to peptides or proteins generally involves the activation of PEG and coupling of the activated PEG-intermediates directly to target proteins/peptides or to a linker, which is subsequently activated and coupled to target proteins/peptides (see Abuchowski, A. et al, J. Biol. Chem., 252, 3571 (1977) and J. Biol. Chem., 252, 3582 (1977), Zalipsky, et al., and Harris et. al., in: Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications; (J. M. Harris ed.) Plenum Press: New York, 1992; Chap. 21 and 22). It is noted that an EphB4 containing a PEG molecule is also known as a conjugated protein, whereas the protein lacking an attached PEG molecule can be referred to as unconjugated.

[0120]Any molecular mass for a PEG can be used as practically desired, e.g., from about 1,000 Daltons (Da) to 100,000 Da (n is 20 to 2300), for conjugating to Eph4 or EphrinB2 soluble peptides. The number of repeating units "n" in the PEG is approximated for the molecular mass described in Daltons. It is preferred that the combined molecular mass of PEG on an activated linker is suitable for pharmaceutical use. Thus, in one embodiment, the molecular mass of the PEG molecules does not exceed 100,000 Da. For example, if three PEG molecules are attached to a linker, where each PEG molecule has the same molecular mass of 12,000 Da (each n is about 270), then the total molecular mass of PEG on the linker is about 36,000 Da (total n is about 820). The molecular masses of the PEG attached to the linker can also be different, e.g., of three molecules on a linker two PEG molecules can be 5,000 Da each (each n is about 110) and one PEG molecule can be 12,000 Da (n is about 270).

[0121]In a specific embodiment of the invention, an EphB4 polypeptide is covalently linked to one poly(ethylene glycol) group of the formula: --CO--(CH₂)_x--(OCH₂CH₂)_m--OR, with the --CO (i.e. carbonyl) of the poly(ethylene glycol) group forming an amide bond with one of the amino groups of EphB4; R being lower alkyl; x being 2 or 3; m being from about 450 to about 950; and n and m being chosen so that the molecular weight of the conjugate minus the EphB4 protein is from about 10 to 40 kDa. In one embodiment, an EphB4 ε-amino group of a lysine is the available (free) amino group.

[0122]The above conjugates may be more specifically presented by formula (II): P--NHCO--(CH₂)_x--(OCH₂CH₂)_m--OR(II), wherein P is the group of an EphB4 protein as described herein, (i.e. without the amino group or amino groups which form an amide linkage with the carbonyl shown in formula (II); and wherein R is lower alkyl; x is 2 or 3; m is from about 450 to about 950 and is chosen so that the molecular weight of the conjugate minus the EphB4 protein is from about 10 to about 40 kDa. As used herein, the given ranges of "m" have an orientational meaning. The ranges of "m" are determined in any case, and exactly, by the molecular weight of the PEG group.

[0123]One skilled in the art can select a suitable molecular mass for PEG, e.g., based on how the pegylated EphB4 will be used therapeutically, the desired dosage, circulation time, resistance to proteolysis, immunogenicity, and other considerations. For a discussion of PEG and its use to enhance the properties of proteins, see N. V. Kate, Advanced Drug Delivery Reviews 10: 91-114 (1993).

[0124]In one embodiment of the invention, PEG molecules may be activated to react with amino groups on EphB4, such as with lysines (Bencham C. O. et al., Anal. Biochem., 131, 25 (1983); Veronese, F. M. et al., Appl. Biochem., 11, 141 (1985); Zalipsky, S. et al., Polymeric Drugs and Drug Delivery Systems, adrs 9-110 ACS Symposium Series 469 (1999); Zalipsky, S. et al., Europ. Polym. J., 19, 1177-1183 (1983); Delgado, C. et al., Biotechnology and Applied Biochemistry, 12, 119-128 (1990)).

[0125]In one specific embodiment, carbonate esters of PEG are used to form the PEG-EphB4 conjugates. N,N'-disuccinimidylcarbonate (DSC) may be used in the reaction with PEG to form active mixed PEG-succinimidyl carbonate that may be subsequently reacted with a nucleophilic group of a linker or an amino group of EphB4 (see U.S. Pat. No. 5,281,698 and U.S. Pat. No. 5,932,462). In a similar type of reaction, 1,1'-(dibenzotriazolyl)carbonate and di-(2-pyridyl)carbonate may be reacted with PEG to form PEG-benzotriazolyl and PEG-pyridyl mixed carbonate (U.S. Pat. No. 5,382,657), respectively.

[0126]In one embodiment, additional sites for PEGylation are introduced by site-directed mutagenesis by introducing one or more lysine residues. For instance, one or more arginine residues may be mutated to a lysine residue. In another embodiment, additional PEGylation sites are chemically introduced by modifying amino acids on EphB4. In one specific embodiment, carboxyl groups in EphB4 are conjugated with diaminobutane, resulting in carboxyl amidation (see Li et al., Anal Biochem. 2004; 330(2):264-71). This reaction may be catalyzed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a water-soluble carbodiimide. The resulting amides can then conjugated to PEG.

[0127]PEGylation of EphB4 can be performed according to the methods of the state of the art, for example by reaction of EphB4 with electrophilically active PEGs (supplier: Shearwater Corp., USA, www.shearwatercorp.com). Preferred PEG reagents of the present invention are, e.g., N-hydroxysuccinimidyl propionates (PEG-SPA), butanoates (PEG-SBA), PEG-succinimidyl propionate or branched N-hydroxysuccinimides such as mPEG2-NHS (Monfardini, C., et al., Bioconjugate Chem. 6 (1995) 62-69). Such methods may used to PEGylated at an ε-amino group of an EphB4 lysine or the N-terminal amino group of EphB4.

[0128]In another embodiment, PEG molecules may be coupled to sulfhydryl groups on EphB4 (Sartore, L., et al., Appl. Biochem. Biotechnol., 27, 45 (1991); Morpurgo et al., Biocon. Chem., 7, 363-368 (1996); Goodson et al., Bio/Technology (1990) 8, 343; U.S. Pat. No. 5,766,897). U.S. Pat. Nos. 6,610,281 and 5,766,897 describes exemplary reactive PEG species that may be coupled to sulfhydryl groups.

[0129]In some embodiments where PEG molecules are conjugated to cysteine residues on EphB4, the cysteine residues are native to Eph4, whereas in other embodiments, one or more cysteine residues are engineered into EphB4. Mutations may be introduced into an EphB4 coding sequence to generate cysteine residues. This might be achieved, for example, by mutating one or more amino acid residues to cysteine. Preferred amino acids for mutating to a cysteine residue include serine, threonine, alanine and other hydrophilic residues. Preferably, the residue to be mutated to cysteine is a surface-exposed residue. Algorithms are well-known in the art for predicting surface accessibility of residues based on primary sequence or a protein. Alternatively, surface residues may be predicted by comparing the amino acid sequences of EphB4 an EphB2, given that the crystal structure of EphB2 has been solved (see Himanen et al., Nature. (2001) 20-27; 414(6866):933-8) and thus the surface-exposed residues identified. In one embodiment, cysteine residues are introduced into EphB4 at or near the N- and/or C-terminus, or within loop regions. Loop regions may be identified by comparing the EphB4 sequence to that of EphB2.

[0130]In some embodiments, the pegylated EphB4 comprises a PEG molecule covalently attached to the alpha amino group of the N-terminal amino acid. Site specific N-terminal reductive amination is described in Pepinsky et al., (2001) WET, 297,1059, and U.S. Pat. No. 5,824,784. The use of a PEG-aldehyde for the reductive amination of a protein utilizing other available nucleophilic amino groups is described in U.S. Pat. No. 4,002,531, in Wieder et al., (1979) J. Biol. Chem. 254, 12579, and in Chamow et al., (1994) Bioconjugate Chem. 5, 133.

[0131]In another embodiment, pegylated EphB4 comprises one or more PEG molecules covalently attached to a linker, which in turn is attached to the alpha amino group of the amino acid residue at the N-terminus of EphB4. Such an approach is disclosed in U.S. Patent Publication No. 2002/0044921 and in WO94/01451.

[0132]In one embodiment, EphB4 is pegylated at the C-terminus. In a specific embodiment, a protein is pegylated at the C-terminus by the introduction of C-terminal azido-methionine and the subsequent conjugation of a methyl-PEG-triarylphosphine compound via the Staudinger reaction. This C-terminal conjugation method is described in Cazalis et al., C-Terminal Site-Specific PEGylation of a Truncated Thrombomodulin Mutant with Retention of Full Bioactivity, Bioconjug Chem. 2004; 15(5):1005-1009.

[0133]Monopegylation of EphB4 can also be produced according to the general methods described in WO 94/01451. WO 94/01451 describes a method for preparing a recombinant polypeptide with a modified terminal amino acid alpha-carbon reactive group. The steps of the method involve forming the recombinant polypeptide and protecting it with one or more biologically added protecting groups at the N-terminal alpha-amine and C-terminal alpha-carboxyl. The polypeptide can then be reacted with chemical protecting agents to selectively protect reactive side chain groups and thereby prevent side chain groups from being modified. The polypeptide is then cleaved with a cleavage reagent specific for the biological protecting group to form an unprotected terminal amino acid alpha-carbon reactive group. The unprotected terminal amino acid alpha-carbon reactive group is modified with a chemical modifying agent. The side chain protected terminally modified single copy polypeptide is then deprotected at the side chain groups to form a terminally modified recombinant single copy polypeptide. The number and sequence of steps in the method can be varied to achieve selective modification at the N- and/or C-terminal amino acid of the polypeptide.

[0134]The ratio of EphB4 (or EphrinB2) to activated PEG in the conjugation reaction can be from about 1:0.5 to 1:50, between from about 1:1 to 1:30, or from about 1:5 to 1:15. Various aqueous buffers can be used in the present method to catalyze the covalent addition of PEG to EphB4. In one embodiment, the pH of a buffer used is from about 7.0 to 9.0. In another embodiment, the pH is in a slightly basic range, e.g., from about 7.5 to 8.5. Buffers having a pKa close to neutral pH range may be used, e.g., phosphate buffer.

[0135]In one embodiment, the temperature range for preparing a mono-PEG-EphB4 is from about 4° C. to 40° C., or from about 18° C. to 25° C. In another embodiment, the temperature is room temperature.

[0136]The pegylation reaction can proceed from 3 to 48 hours, or from 10 to 24 hours. The reaction can be monitored using SE-HPLC to distinguish EphB4, mono-PEG-EphB4 and poly-PEG-EphB4. It is noted that mono-PEG-EphB4 forms before di-PEG-EphB4. When the mono-PEG-EphB4 concentration reaches a plateau, the reaction can be terminated by adding a quenching agent to react with unreacted PEG. In some embodiments, the quenching agent is a free amino acid, such as glycine, cysteine or lysine.

[0137]Conventional separation and purification techniques known in the art can be used to purify pegylated EphB4 or EphrinB2 products, such as size exclusion (e.g. gel filtration) and ion exchange chromatography. Products may also be separated using SDS-PAGE. Products that may be separated include mono-, di-, tri- poly- and un-pegylated EphB4, as well as free PEG. The percentage of mono-PEG conjugates can be controlled by pooling broader fractions around the elution peak to increase the percentage of mono-PEG in the composition. About ninety percent mono-PEG conjugates represents a good balance of yield and activity. Compositions in which, for example, at least ninety-two percent or at least ninety-six percent of the conjugates are mono-PEG species may be desired. In an embodiment of this invention the percentage of mono-PEG conjugates is from ninety percent to ninety-six percent.

[0138]In one embodiment, pegylated EphB4 proteins of the invention contain one, two or more PEG moieties. In one embodiment, the PEG moiety(ies) are bound to an amino acid residue which is on the surface of the protein and/or away from the surface that contacts EphrinB2. In one embodiment, the combined or total molecular mass of PEG in PEG-EphB4 is from about 3,000 Da to 60,000 Da, optionally from about 10,000 Da to 36,000 Da. In a one embodiment, the PEG in pegylated EphB4 is a substantially linear, straight-chain PEG.

[0139]In one embodiment of the invention, the PEG in pegylated EphB4 or EphrinB2 is not hydrolyzed from the pegylated amino acid residue using a hydroxylamine assay, e.g., 450 mM hydroxylamine (pH 6.5) over 8 to 16 hours at room temperature, and is thus stable. In one embodiment, greater than 80% of the composition is stable mono-PEG-EphB4, more preferably at least 90%, and most preferably at least 95%.

[0140]In another embodiment, the pegylated EphB4 proteins of the invention will preferably retain at least 25%, 50%, 60%, 70% least 80%, 85%, 90%, 95% or 100% of the biological activity associated with the unmodified protein. In one embodiment, biological activity refers to its ability to bind to EphrinB2. In one specific embodiment, the pegylated EphB4 protein shows an increase in binding to EphrinB2 relative to unpegylated EphB4.

[0141]In a preferred embodiment, the PEG-EphB4 has a half-life (t₁/2) which is enhanced relative to the half-life of the unmodified protein. Preferably, the half-life of PEG-EphB4 is enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400% or 500%, or even by 1000% relative to the half-life of the unmodified EphB4 protein. In some embodiments, the protein half-life is determined in vitro, such as in a buffered saline solution or in serum. In other embodiments, the protein half-life is an in vivo half life, such as the half-life of the protein in the serum or other bodily fluid of an animal.

[0142]In certain aspects, functional variants or modified forms of the subject soluble polypeptides include fusion proteins having at least a portion of the soluble polypeptide and one or more fusion domains. Well known examples of such fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, and an immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), which are particularly useful for isolation of the fusion proteins by affinity chromatography. For the purpose of affinity purification, relevant matrices for affinity chromatography, such as glutathione-, amylase-, and nickel- or cobalt-conjugated resins are used. Another fusion domain well known in the art is green fluorescent protein (GFP). Fusion domains also include "epitope tags," which are usually short peptide sequences for which a specific antibody is available. Well known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus haemagglutinin (HA), and c-myc tags. In some cases, the fusion domains have a protease cleavage site, such as for Factor Xa or Thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby liberate the recombinant proteins therefrom. The liberated proteins can then be isolated from the fusion domain by subsequent chromatographic separation.

[0143]In certain embodiments, the soluble polypeptides of the present invention contain one pr more modifications that are capable of stabilizing the soluble polypeptides. For example, such modifications enhance the in vitro half life of the soluble polypeptides, enhance circulatory half life of the soluble polypeptides or reducing proteolytic degradation of the soluble polypeptides.

[0144]In a further embodiment, a soluble polypeptide of the present invention is fused to a cytotoxic agent. In this method, the fusion acts to target the cytotoxic agent to a specific tissue or cell (e.g., a tumor tissue or cell), resulting in a reduction in the number of afflicted cells. Such an approach can thereby reduce symptoms associated with cancer and angiogenesis-associated disorders. Cytotoxic agents include, but are not limited to, diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin and the like, as well as radiochemicals.

[0145]In certain embodiments, the soluble polypeptides of the present invention may be fused to other therapeutic proteins or to other proteins such as Fc or serum albumin for pharmacokinetic purposes. See for example U.S. Pat. Nos. 5,766,883 and 5,876,969, both of which are incorporated by reference. In some embodiments, soluble peptides of the present invention are fused to Fc variants. In a specific embodiment, the soluble polypeptide is fused to an Fc variant which does not homodimerize, such as one lacking the cysteine residues which form cysteine bonds with other Fc chains.

[0146]In some embodiments, the modified proteins of the invention comprise fusion proteins with an Fc region of an immunoglobulin. As is known, each immunoglobulin heavy chain constant region comprises four or five domains. The domains are named sequentially as follows: CH1-hinge-CH2-CH3(-CH₄). The DNA sequences of the heavy chain domains have cross-homology among the immunoglobulin classes, e.g., the CH2 domain of IgG is homologous to the CH2 domain of IgA and IgD, and to the CH3 domain of IgM and IgE. As used herein, the term, "immunoglobulin Fc region" is understood to mean the carboxyl-terminal portion of an immunoglobulin chain constant region, preferably an immunoglobulin heavy chain constant region, or a portion thereof. For example, an immunoglobulin Fc region may comprise 1) a CH1 domain, a CH2 domain, and a CH3 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, or 5) a combination of two or more domains and an immunoglobulin hinge region. In a preferred embodiment the immunoglobulin Fc region comprises at least an immunoglobulin hinge region a CH2 domain and a CH3 domain, and preferably lacks the CH1 domain.

[0147]In one embodiment, the class of immunoglobulin from which the heavy chain constant region is derived is IgG (Igγ) (γ subclasses 1, 2, 3, or 4). The nucleotide and amino acid sequences of human Fc γ-1 are set forth in SEQ ID NOS: 5 and 6. The nucleotide and amino acid sequences of murine Fcγ-2a are set forth in SEQ ID NOS: 7 and 8. Other classes of immunoglobulin, IgA (Igα), IgD (Igδ), IgE (Igε) and IgM (Igμ), may be used. The choice of appropriate immunoglobulin heavy chain constant regions is discussed in detail in U.S. Pat. Nos. 5,541,087, and 5,726,044. The choice of particular immunoglobulin heavy chain constant region sequences from certain immunoglobulin classes and subclasses to achieve a particular result is considered to be within the level of skill in the art. The portion of the DNA construct encoding the immunoglobulin Fc region preferably comprises at least a portion of a hinge domain, and preferably at least a portion of a CH₃ domain of Fc γ or the homologous domains in any of IgA, IgD, IgE, or IgM.

[0148]Furthermore, it is contemplated that substitution or deletion of amino acids within the immunoglobulin heavy chain constant regions may be useful in the practice of the invention. One example would be to introduce amino acid substitutions in the upper CH2 region to create a Fc variant with reduced affinity for Fc receptors (Cole et al. (1997) J. IMMUNOL. 159:3613). One of ordinary skill in the art can prepare such constructs using well known molecular biology techniques.

[0149]In a specific embodiment of the present invention, the modified forms of the subject soluble polypeptides are fusion proteins having at least a portion of the soluble polypeptide (e.g., an ectodomain of Ephrin B2 or EphB4) and a stabilizing domain such as albumin. As used herein, "albumin" refers collectively to albumin protein or amino acid sequence, or an albumin fragment or variant, having one or more functional activities (e.g., biological activities) of albumin. In particular, "albumin" refers to human albumin or fragments thereof (see EP 201 239, EP 322 094 WO 97/24445, WO95/23857) especially the mature form of human albumin, or albumin from other vertebrates or fragments thereof, or analogs or variants of these molecules or fragments thereof.

[0150]The present invention describes that such fusion proteins are more stable relative to the corresponding wildtype soluble protein. For example, the subject soluble polypeptide (e.g., an ectodomain of Ephrin B2 or EphB4) can be fused with human serum albumin (HSA), bovine serum albumin (BSA), or any fragment of an albumin protein which has stabilization activity. Such stabilizing domains include human serum albumin (HSA) and bovine serum albumin (BSA).

[0151]In particular, the albumin fusion proteins of the invention may include naturally occurring polymorphic variants of human albumin and fragments of human albumin (See WO95/23857), for example those fragments disclosed in EP 322 094 (namely HA (Pn), where n is 369 to 419). The albumin may be derived from any vertebrate, especially any mammal, for example human, cow, sheep, or pig. Non-mammalian albumins include, but are not limited to, hen and salmon. The albumin portion of the albumin fusion protein may be from a different animal than the EphB4.

[0152]In some embodiments, the albumin protein portion of an albumin fusion protein corresponds to a fragment of serum albumin. Fragments of serum albumin polypeptides include polypeptides having one or more residues deleted from the amino terminus or from the C-terminus. Generally speaking, an HA fragment or variant will be at least 100 amino acids long, preferably at least 150 amino acids long. The HA variant may consist of or alternatively comprise at least one whole domain of HA. Domains, with reference to SEQ ID NO:18 in U.S. Patent Publication No. 2004/0171123, are as follows: domains 1 (amino acids 1-194), 2 (amino acids 195-387), 3 (amino acids 388-585), 1+2 (1-387), 2+3 (195-585) or 1+3 (amino acids 1-194+amino acids 388-585). Each domain is itself made up of two homologous subdomains namely 1-105, 120-194, 195-291, 316-387, 388-491 and 512-585, with flexible inter-subdomain linker regions comprising residues Lys106 to Glu119, Glu292 to Val315 and Glu492 to Ala511.

[0153]In one embodiment, the EphB4-HSA fusion has one EphB4 soluble polypeptide linked to one HSA molecule, but other conformations are within the invention. For example, EphB4-HSA fusion proteins can have any of the following formula: R₁-L-R₂; R₂-L-R₁; R₁-L-R₂-L-R₁; or R₂-L-R1-L-R₂; R₁-R₂; R₂-R₁; R₁-R₂-R₁; or R₂-R₁-R₂; wherein R₁ is a soluble EphB4 sequence, R₂ is HSA, and L is a peptide linker sequence.

[0154]In a specific embodiment, the EphB4 and HSA domains are linked to each other, preferably via a linker sequence, which separates the EphB4 and HSA domains by a distance sufficient to ensure that each domain properly folds into its secondary and tertiary structures. Preferred linker sequences (1) should adopt a flexible extended conformation, (2) should not exhibit a propensity for developing an ordered secondary structure which could interact with the functional EphB4 and HSA domains, and (3) should have minimal hydrophobic or charged character, which could promote interaction with the functional protein domains. Typical surface amino acids in flexible protein regions include Gly, Asn and Ser. Permutations of amino acid sequences containing Gly, Asn and Ser would be expected to satisfy the above criteria for a linker sequence. Other near neutral amino acids, such as Thr and Ala, can also be used in the linker sequence.

[0155]In a specific embodiment, a linker sequence length of about 20 amino acids can be used to provide a suitable separation of functional protein domains, although longer or shorter linker sequences may also be used. The length of the linker sequence separating EphB4 and HSA can be from 5 to 500 amino acids in length, or more preferably from 5 to 100 amino acids in length. Preferably, the linker sequence is from about 5-30 amino acids in length. In preferred embodiments, the linker sequence is from about 5 to about 20 amino acids, and is advantageously from about 10 to about 20 amino acids. Amino acid sequences useful as linkers of EphB4 and HSA include, but are not limited to, (SerGly₄)y wherein y is greater than or equal to 8, or Gly₄SerGly₅Ser. A preferred linker sequence has the formula (SerGly₄)₄. Another preferred linker has the sequence ((Ser-Ser-Ser-Ser-Gly)3-Ser-Pro).

[0156]In one embodiment, the polypeptides of the present invention and HSA proteins are directly fused without a linker sequence. In preferred embodiments, the C-terminus of a soluble EphB4 polypeptide can be directly fused to the N-terminus of HSA or the C-terminus of HSA can be directly fused to the N-terminus of soluble EphB4.

[0157]In some embodiments, the immunogenicity of the fusion junction between HSA and EphB4 may be reduced the by identifying a candidate T-cell epitope within a junction region spanning a fusion protein and changing an amino acid within the junction region as described in U.S. Patent Publication No. 2003/0166877.

[0158]In certain embodiments, soluble polypeptides (unmodified or modified) of the invention can be produced by a variety of art-known techniques. For example, such soluble polypeptides can be synthesized using standard protein chemistry techniques such as those described in Bodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant G. A. (ed.), Synthetic Peptides: A User's Guide, W. H. Freeman and Company, New York (1992). In addition, automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Model 396; Milligen/Biosearch 9600). Alternatively, the soluble polypeptides, fragments or variants thereof may be recombinantly produced using various expression systems as is well known in the art (also see below).

III. Nucleic Acids Encoding Soluble Polypeptides

[0159]In certain aspects, the invention relates to isolated and/or recombinant nucleic acids encoding an EphB4 or Ephrin B2 soluble polypeptide. The subject nucleic acids may be single-stranded or double-stranded, DNA or RNA molecules. These nucleic acids are useful as therapeutic agents. For example, these nucleic acids are useful in making recombinant soluble polypeptides which are administered to a cell or an individual as therapeutics. Alternative, these nucleic acids can be directly administered to a cell or an individual as therapeutics such as in gene therapy.

[0160]In certain embodiments, the invention provides isolated or recombinant nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to a region of the nucleotide sequence depicted in SEQ ID Nos. 6-9. One of ordinary skill in the art will appreciate that nucleic acid sequences complementary to the subject nucleic acids, and variants of the subject nucleic acids are also within the scope of this invention. In further embodiments, the nucleic acid sequences of the invention can be isolated, recombinant, and/or fused with a heterologous nucleotide sequence, or in a DNA library.

[0161]In other embodiments, nucleic acids of the invention also include nucleotide sequences that hybridize under highly stringent conditions to the nucleotide sequence depicted in SEQ ID Nos. 6-9, or complement sequences thereof. As discussed above, one of ordinary skill in the art will understand readily that appropriate stringency conditions which promote DNA hybridization can be varied. One of ordinary skill in the art will understand readily that appropriate stringency conditions which promote DNA hybridization can be varied. For example, one could perform the hybridization at 6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C., to high stringency conditions at about 65° C. Both temperature and salt may be varied, or temperature or salt concentration may be held constant while the other variable is changed. In one embodiment, the invention provides nucleic acids which hybridize under low stringency conditions of 6×SSC at room temperature followed by a wash at 2×SSC at room temperature.

[0162]Isolated nucleic acids which differ from the subject nucleic acids due to degeneracy in the genetic code are also within the scope of the invention. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in "silent" mutations which do not affect the amino acid sequence of the protein. However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject proteins will exist among mammalian cells. One skilled in the art will appreciate that these variations in one or more nucleotides (up to about 3-5% of the nucleotides) of the nucleic acids encoding a particular protein may exist among individuals of a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention.

[0163]In certain embodiments, the recombinant nucleic acids of the invention may be operably linked to one or more regulatory nucleotide sequences in an expression construct. Regulatory nucleotide sequences will generally be appropriate for a host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Typically, said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are contemplated by the invention. The promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter. An expression construct may be present in a cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome. In a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.

[0164]In certain aspect of the invention, the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding an EphB4 or Ephrin B2 soluble polypeptide and operably linked to at least one regulatory sequence. Regulatory sequences are art-recognized and are selected to direct expression of the soluble polypeptide. Accordingly, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990). For instance, any of a wide variety of expression control sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding a soluble polypeptide. Such useful expression control sequences, include, for example, the early and late promoters of SV40, tet promoter, adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered.

[0165]This invention also pertains to a host cell transfected with a recombinant gene including a coding sequence for one or more of the subject soluble polypeptide. The host cell may be any prokaryotic or eukaryotic cell. For example, a soluble polypeptide of the invention may be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.

[0166]Accordingly, the present invention further pertains to methods of producing the subject soluble polypeptides. For example, a host cell transfected with an expression vector encoding an EphB4 soluble polypeptide can be cultured under appropriate conditions to allow expression of the EphB4 soluble polypeptide to occur. The EphB4 soluble polypeptide may be secreted and isolated from a mixture of cells and medium containing the soluble polypeptides. Alternatively, the soluble polypeptides may be retained cytoplasmically or in a membrane fraction and the cells harvested, lysed and the protein isolated. A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. The soluble polypeptides can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for particular epitopes of the soluble polypeptides. In a preferred embodiment, the soluble polypeptide is a fusion protein containing a domain which facilitates its purification.

[0167]A recombinant nucleic acid of the invention can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian), or both. Expression vehicles for production of a recombinant soluble polypeptide include plasmids and other vectors. For instance, suitable vectors include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.

[0168]The preferred mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found below in the description of gene therapy delivery systems. The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17. In some instances, it may be desirable to express the recombinant SLC5A8 polypeptide by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as the β-gal containing pBlueBac III).

[0169]Techniques for making fusion genes are well known. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).

IV. Drug Screening Assays

[0170]There are numerous approaches to screening for polypeptide therapeutic agents as antagonists of EphB4, Ephrin B2 or both. For example, high-throughput screening of compounds or molecules can be carried out to identify agents or drugs which inhibit angiogenesis or inhibit tumor growth. Test agents can be any chemical (element, molecule, compound, drug), made synthetically, made by recombinant techniques or isolated from a natural source. For example, test agents can be peptides, polypeptides, peptoids, sugars, hormones, or nucleic acid molecules. In addition, test agents can be small molecules or molecules of greater complexity made by combinatorial chemistry, for example, and compiled into libraries. These libraries can comprise, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers and other classes of organic compounds. Test agents can also be natural or genetically engineered products isolated from lysates or growth media of cells--bacterial, animal or plant--or can be the cell lysates or growth media themselves. Presentation of test compounds to the test system can be in either an isolated form or as mixtures of compounds, especially in initial screening steps.

[0171]For example, an assay can be carried out to screen for compounds that specifically inhibit binding of Ephrin B2 (ligand) to EphB4 (receptor), or vice-versa, e.g., by inhibition of binding of labeled ligand- or receptor-Fc fusion proteins to immortalized cells. Compounds identified through this screening can then be tested in animals to assess their anti-angiogenesis or anti-tumor activity in vivo.

[0172]In one embodiment of an assay to identify a substance that interferes with interaction of two cell surface molecules (e.g., Ephrin B2 and EphB4), samples of cells expressing one type of cell surface molecule (e.g., EphB4) are contacted with either labeled ligand (e.g., Ephrin B2, or a soluble portion thereof, or a fusion protein such as a fusion of the extracellular domain and the Fc domain of IgG) or labeled ligand plus a test compound (or group of test compounds). The amount of labeled ligand which has bound to the cells is determined. A lesser amount of label (where the label can be, for example, a radioactive isotope, a fluorescent or colorimetric label) in the sample contacted with the test compound(s) is an indication that the test compound(s) interferes with binding. The reciprocal assay using cells expressing a ligand (e.g., an Ephrin B2 ligand or a soluble form thereof) can be used to test for a substance that interferes with the binding of an Eph receptor or soluble portion thereof.

[0173]An assay to identify a substance which interferes with interaction between an Eph receptor and an ephrin can be performed with the component (e.g., cells, purified protein, including fusion proteins and portions having binding activity) which is not to be in competition with a test compound, linked to a solid support. The solid support can be any suitable solid phase or matrix, such as a bead, the wall of a plate or other suitable surface (e.g., a well of a microtiter plate), column pore glass (CPG) or a pin that can be submerged into a solution, such as in a well. Linkage of cells or purified protein to the solid support can be either direct or through one or more linker molecules.

[0174]In one embodiment, an isolated or purified protein (e.g., an Eph receptor or an ephrin) can be immobilized on a suitable affinity matrix by standard techniques, such as chemical cross-linking, or via an antibody raised against the isolated or purified protein, and bound to a solid support. The matrix can be packed in a column or other suitable container and is contacted with one or more compounds (e.g., a mixture) to be tested under conditions suitable for binding of the compound to the protein. For example, a solution containing compounds can be made to flow through the matrix. The matrix can be washed with a suitable wash buffer to remove unbound compounds and non-specifically bound compounds. Compounds which remain bound can be released by a suitable elution buffer. For example, a change in the ionic strength or pH of the elution buffer can lead to a release of compounds. Alternatively, the elution buffer can comprise a release component or components designed to disrupt binding of compounds (e.g., one or more ligands or receptors, as appropriate, or analogs thereof which can disrupt binding or competitively inhibit binding of test compound to the protein).

[0175]Fusion proteins comprising all, or a portion of, a protein (e.g., an Eph receptor or an ephrin) linked to a second moiety not occurring in that protein as found in nature can be prepared for use in another embodiment of the method. Suitable fusion proteins for this purpose include those in which the second moiety comprises an affinity ligand (e.g., an enzyme, antigen, epitope). The fusion proteins can be produced by inserting the protein (e.g., an Eph receptor or an ephrin) or a portion thereof into a suitable expression vector which encodes an affinity ligand. The expression vector can be introduced into a suitable host cell for expression. Host cells are disrupted and the cell material, containing fusion protein, can be bound to a suitable affinity matrix by contacting the cell material with an affinity matrix under conditions sufficient for binding of the affinity ligand portion of the fusion protein to the affinity matrix.

[0176]In one aspect of this embodiment, a fusion protein can be immobilized on a suitable affinity matrix under conditions sufficient to bind the affinity ligand portion of the fusion protein to the matrix, and is contacted with one or more compounds (e.g., a mixture) to be tested, under conditions suitable for binding of compounds to the receptor or ligand protein portion of the bound fusion protein. Next, the affinity matrix with bound fusion protein can be washed with a suitable wash buffer to remove unbound compounds and non-specifically bound compounds without significantly disrupting binding of specifically bound compounds. Compounds which remain bound can be released by contacting the affinity matrix having fusion protein bound thereto with a suitable elution buffer (a compound elution buffer). In this aspect, compound elution buffer can be formulated to permit retention of the fusion protein by the affinity matrix, but can be formulated to interfere with binding of the compound(s) tested to the receptor or ligand protein portion of the fusion protein. For example, a change in the ionic strength or pH of the elution buffer can lead to release of compounds, or the elution buffer can comprise a release component or components designed to disrupt binding of compounds to the receptor or ligand protein portion of the fusion protein (e.g., one or more ligands or receptors or analogs thereof which can disrupt binding of compounds to the receptor or ligand protein portion of the fusion protein). Immobilization can be performed prior to, simultaneous with, or after contacting the fusion protein with compound, as appropriate. Various permutations of the method are possible, depending upon factors such as the compounds tested, the affinity matrix selected, and elution buffer formulation. For example, after the wash step, fusion protein with compound bound thereto can be eluted from the affinity matrix with a suitable elution buffer (a matrix elution buffer). Where the fusion protein comprises a cleavable linker, such as a thrombin cleavage site, cleavage from the affinity ligand can release a portion of the fusion with compound bound thereto. Bound compound can then be released from the fusion protein or its cleavage product by an appropriate method, such as extraction.

V. Methods of Treatment

[0177]In certain embodiments, the present invention provides methods of inhibiting angiogenesis and methods of treating angiogenesis-associated diseases. In other embodiments, the present invention provides methods of inhibiting or reducing tumor growth and methods of treating an individual suffering from cancer. These methods involve administering to the individual a therapeutically effective amount of one or more polypeptide therapeutic agents as described above. These methods are particularly aimed at therapeutic and prophylactic treatments of animals, and more particularly, humans.

[0178]As described herein, angiogenesis-associated diseases include, but are not limited to, angiogenesis-dependent cancer, including, for example, solid tumors, blood born tumors such as leukemias, and tumor metastases; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; inflammatory disorders such as immune and non-immune inflammation; chronic articular rheumatism and psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; telangiectasia psoriasis scleroderma, pyogenic granuloma, rubeosis, arthritis, diabetic neovascularization, vasculogenesis, hematopoiesis.

[0179]It is understood that methods and compositions of the invention are also useful for treating any angiogenesis-independent cancers (tumors). As used herein, the term "angiogenesis-independent cancer" refers to a cancer (tumor) where there is no or little neovascularization in the tumor tissue.

[0180]In particular, polypeptide therapeutic agents of the present invention are useful for treating or preventing a cancer (tumor), including, but not limited to, colon carcinoma, breast cancer, mesothelioma, prostate cancer, bladder cancer, squamous cell carcinoma of the head and neck (HNSCC), Kaposi sarcoma, and leukemia.

[0181]In certain embodiments of such methods, one or more polypeptide therapeutic agents can be administered, together (simultaneously) or at different times (sequentially). In addition, polypeptide therapeutic agents can be administered with another type of compounds for treating cancer or for inhibiting angiogenesis.

[0182]In certain embodiments, the subject methods of the invention can be used alone. Alternatively, the subject methods may be used in combination with other conventional anti-cancer therapeutic approaches directed to treatment or prevention of proliferative disorders (e.g., tumor). For example, such methods can be used in prophylactic cancer prevention, prevention of cancer recurrence and metastases after surgery, and as an adjuvant of other conventional cancer therapy. The present invention recognizes that the effectiveness of conventional cancer therapies (e.g., chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery) can be enhanced through the use of a subject polypeptide therapeutic agent.

[0183]A wide array of conventional compounds have been shown to have anti-neoplastic activities. These compounds have been used as pharmaceutical agents in chemotherapy to shrink solid tumors, prevent metastases and further growth, or decrease the number of malignant cells in leukemic or bone marrow malignancies. Although chemotherapy has been effective in treating various types of malignancies, many anti-neoplastic compounds induce undesirable side effects. It has been shown that when two or more different treatments are combined, the treatments may work synergistically and allow reduction of dosage of each of the treatments, thereby reducing the detrimental side effects exerted by each compound at higher dosages. In other instances, malignancies that are refractory to a treatment may respond to a combination therapy of two or more different treatments.

[0184]When a polypeptide therapeutic agent of the present invention is administered in combination with another conventional anti-neoplastic agent, either concomitantly or sequentially, such therapeutic agent is shown to enhance the therapeutic effect of the anti-neoplastic agent or overcome cellular resistance to such anti-neoplastic agent. This allows decrease of dosage of an anti-neoplastic agent, thereby reducing the undesirable side effects, or restores the effectiveness of an anti-neoplastic agent in resistant cells.

[0185]Pharmaceutical compounds that may be used for combinatory anti-tumor therapy include, merely to illustrate: aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.

[0186]These chemotherapeutic anti-tumor compounds may be categorized by their mechanism of action into, for example, following groups: anti-metabolites/anti-cancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes--dacarbazinine (DTIC); antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (TNP-470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors.

[0187]In certain embodiments, pharmaceutical compounds that may be used for combinatory anti-angiogenesis therapy include: (1) inhibitors of release of "angiogenic molecules," such as bFGF (basic fibroblast growth factor); (2) neutralizers of angiogenic molecules, such as an anti-βbFGF antibodies; and (3) inhibitors of endothelial cell response to angiogenic stimuli, including collagenase inhibitor, basement membrane turnover inhibitors, angiostatic steroids, fungal-derived angiogenesis inhibitors, platelet factor 4, thrombospondin, arthritis drugs such as D-penicillamine and gold thiomalate, vitamin D₃ analogs, alpha-interferon, and the like. For additional proposed inhibitors of angiogenesis, see Blood et al., Bioch. Biophys. Acta., 1032:89-118 (1990), Moses et al., Science, 248:1408-1410 (1990), Ingber et al., Lab. Invest., 59:44-51 (1988), and U.S. Pat. Nos. 5,092,885, 5,112,946, 5,192,744, 5,202,352, and 6,573,256. In addition, there are a wide variety of compounds that can be used to inhibit angiogenesis, for example, peptides or agents that block the VEGF-mediated angiogenesis pathway, endostatin protein or derivatives, lysine binding fragments of angiostatin, melanin or melanin-promoting compounds, plasminogen fragments (e.g., Kringles 1-3 of plasminogen), tropoin subunits, antagonists of vitronectin α_vβ₃, peptides derived from Saposin B, antibiotics or analogs (e.g., tetracycline, or neomycin), dienogest-containing compositions, compounds comprising a MetAP-2 inhibitory core coupled to a peptide, the compound EM-138, chalcone and its analogs, and naaladase inhibitors. See, for example, U.S. Pat. Nos. 6,395,718, 6,462,075, 6,465,431, 6,475,784, 6,482,802, 6,482,810, 6,500,431, 6,500,924, 6,518,298, 6,521,439, 6,525,019, 6,538,103, 6,544,758, 6,544,947, 6,548,477, 6,559,126, and 6,569,845.

[0188]Depending on the nature of the combinatory therapy, administration of the polypeptide therapeutic agents of the invention may be continued while the other therapy is being administered and/or thereafter. Administration of the polypeptide therapeutic agents may be made in a single dose, or in multiple doses. In some instances; administration of the polypeptide therapeutic agents is commenced at least several days prior to the conventional therapy, while in other instances, administration is begun either immediately before or at the time of the administration of the conventional therapy.

VI. Methods of Administration and Pharmaceutical Compositions

[0189]In certain embodiments, the subject polypeptide therapeutic agents (e.g., soluble polypeptides or antibodies) of the present invention are formulated with a pharmaceutically acceptable carrier. Such therapeutic agents can be administered alone or as a component of a pharmaceutical formulation (composition). The compounds may be formulated for administration in any convenient way for use in human or veterinary medicine. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0190]Formulations of the subject polypeptide therapeutic agents include those suitable for oral/nasal, topical, parenteral, rectal, and/or intravaginal administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage, form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.

[0191]In certain embodiments, methods of preparing these formulations or compositions include combining another type of anti-tumor or anti-angiogenesis therapeutic agent and a carrier and, optionally, one or more accessory ingredients. In general, the formulations can be prepared with a liquid carrier, or a finely divided solid carrier, or both, and then, if necessary, shaping the product.

[0192]Formulations for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a subject polypeptide therapeutic agent as an active ingredient.

[0193]In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, and the like), one or more polypeptide therapeutic agents of the present invention may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0194]Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents.

[0195]Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0196]In particular, methods of the invention can be administered topically, either to skin or to mucosal membranes such as those on the cervix and vagina. This offers the greatest opportunity for direct delivery to tumor with the lowest chance of inducing side effects. The topical formulations may further include one or more of the wide variety of agents known to be effective as skin or stratum corneum penetration enhancers. Examples of these are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methyl or isopropyl alcohol, dimethyl sulfoxide, and azone. Additional agents may further be included to make the formulation cosmetically acceptable. Examples of these are fats, waxes, oils, dyes, fragrances, preservatives, stabilizers, and surface active agents. Keratolytic agents such as those known in the art may also be included. Examples are salicylic acid and sulfur.

[0197]Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The subject polypeptide therapeutic agents may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required. The ointments, pastes, creams and gels may contain, in addition to a subject polypeptide agent, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0198]Powders and sprays can contain, in addition to a subject polypeptide therapeutic agent, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0199]Pharmaceutical compositions suitable for parenteral administration may comprise one or more polypeptide therapeutic agents in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0200]These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

[0201]Injectable depot forms are made by forming microencapsule matrices of one or more polypeptide therapeutic agents in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.

[0202]Formulations for intravaginal or rectally administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.

[0203]In other embodiments, the polypeptide therapeutic agents of the instant invention can be expressed within cells from eukaryotic promoters. For example, a soluble polypeptide of EphB4 or Ephrin B2 can be expressed in eukaryotic cells from an appropriate vector. The vectors are preferably DNA plasmids or viral vectors. Viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Preferably, the vectors stably introduced in and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression. Such vectors can be repeatedly administered as necessary. Delivery of vectors encoding the subject polypeptide therapeutic agent can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al., 1996, TIG., 12, 510).

EXEMPLIFICATION

[0204]The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

Example 1

Soluble Derivatives of the Extracellular Domains of Human Ephrin B2 and EphB4 Proteins

[0205]Soluble derivatives of the extracellular domains of human Ephrin B2 and EphB4 proteins represent either truncated full-length predicted extracellular domains of Ephrin B2 (B4ECv3, B2EC) or translational fusions of the domains with constant region of human immunoglobulins (IgG1 Fc fragment), such as B2EC-FC, B4ECv2-FC and B4ECv3-FC. Representative human Ephrin B2 constructs and human EphB4 constructs are shown FIGS. 14 and 15.

[0206]The cDNA fragments encoding these recombinant proteins were subcloned into mammalian expression vectors, expressed in transiently or stably transfected mammalian cell lines and purified to homogeneity as described in detail in Materials and Methods section (see below). Predicted amino acid sequences of the proteins are shown in FIGS. 1-5. High purity of the isolated proteins and their recognition by the corresponding anti-Ephrin B2 and anti-EphB4 monoclonal or polyclonal antibodies were confirmed. The recombinant proteins exhibit the expected high-affinity binding, binding competition and specificity properties with their corresponding binding partners as corroborated by the biochemical assays (see e.g., FIGS. 6-8).

[0207]Such soluble derivative proteins human Ephrin B2 and EphB4 exhibit potent biological activity in several cell-based assays and in vivo assays which measure angiogenesis or anti-cancer activities, and are therefore perspective drug candidates for anti-angiogenic and anti-cancer therapy. B4ECv3 as well as B2EC and B2EC-FC proteins blocked chemotaxis of human endothelial cells (as tested with umbilical cord and hepatic AECs or VECs), with a decrease in degradation of the extracellular matrix, Matrigel, and a decrease in migration in response to growth factor stimuli (FIGS. 9-11). B4ECv3 and B2EC-FC proteins have potent anti-angiogenic effect as demonstrated by their inhibition of endothelial cell tube formation (FIGS. 12-13).

[0208]A detailed description of the materials and methods for this example may be found in U.S. Patent Publication No. 20050084873.

[0209]The sequence of the Globular domain+Cys-rich domain (B4EC-GC), precursor protein is (SEQ ID NO:12):

TABLE-US-00001 MELRVLLCWASLAAALEETLLNTKLETADLKWVTFPQVDGQWEELSGLDE EQHSVRTYEVCEVQRAPGQAHWLRTGWVPRRGAVHVYATLRFTMLECLSL PRAGRSCKETFTVFYYESDADTATALTPAWMENPYIKVDTVAAEHLTRKR PGAEATGKVNVKTLRLGPLSKAGFYLAFQDQGACMALLSLHLFYKKCAQL TVNLTRFPETVPRELVVPVAGSCVVDAVPAPGPSPSLYCREDGQWAEQPV TGCSCAPGFEAAEGNTKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSA VCQCRVGYFRARTDPRGAPCTTPPSAHHHHHH

[0210]For many uses, including therapeutic use, the leader sequence (first 15 amino acids, so that the processed form begins Leu-Glu-Glu . . . ) and the c-terminal hexahistidine tag may be removed or omitted.

[0211]Sequence of the GCF precursor protein (SEQ ID NO:13):

TABLE-US-00002 MELRVLLCWASLAAALEETLLNTKLETADLKWVTFPQVDGQWEELSGLDE EQHSVRTYEVCEVQRAPGQAHWLRTGWVPRRGAVHVYATLRFTMLECLSL PRAGRSCKETFTVFYYESDADTATALTPAWMENPYIKVDTVAAEHLTRKR PGAEATGKVNVKTLRLGPLSKAGFYLAFQDQGACMALLSLHLFYKKCAQL TVNLTRFPETVPRELVVPVAGSCVVDAVPAPGPSPSLYCREDGQWAEQPV TGCSCAPGFAEGNTKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSAVC QCRVGYFRARTDPRGAPCTTPPSAPRSVVSRLNGSSLHLEWSAPLESGGR EDLTYALRCRECRPGGSCAPCGGDLTFDPGPRDLVEPWVVVRGLRPDFTY TFEVTALNGVSSLATGPVPFEPVNVHHHHHH

[0212]For many uses, including therapeutic use, the leader sequence (first 15 amino acids, so that the processed form begins Leu-Glu-Glu . . . ) and the c-terminal hexahistidine tag may be removed or omitted.

[0213]Amino acid sequence of encoded FL-hB4EC precursor (His-tagged) (SEQ ID NO:14):

TABLE-US-00003 MELRVLLCWASLAAALEETLLNTKLETADLKWVTFPQVDGQWEELSGLDE EQHSVRTYEVCEVQRAPGQAHWLRTGWVPRRGAVHVYATLRFTMLECLSL PRAGRSCKETFTVFYYESDADTATALTPAWMENPYIKVDTVAAEHLTRKR PGAEATGKVNVKTLRLGPLSKAGFYLAFQDQGACMALLSLHLFYKKCAQL TVNLTRFPETVPRELVVPVAGSCVVDAVPAPGPSPSLYCREDGQWAEQPV TGCSCAPGFEAAEGNTKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSA VCQCRVGYFRARTDPRGAPCTTPPSAPRSVVSRLNGSSLHLEWSAPLESG GREDLTYALRCRECRPGGSCAPCGGDLIFDPGPRDLVEPWVVVRGLRPDF TYTFEVTALNGVSSLATGPVPFEPVNVTTDREVPPAVSDIRVTRSSPSSL SLAWAVPRAPSGAWLDYEVKYHEKGAEGPSSVRFLKTSENRAELRGLKRG ASYLVQVRARSEAGYGPFGQEHHSQTQLDESEGWREQGSKRAILQIEGKP IPNPLLGLDSTRTGHHHHHH

[0214]For many uses, including therapeutic use, the leader sequence (first 15 amino acids, so that the processed form begins Leu-Glu-Glu . . . ) and the c-terminal hexahistidine tag may be removed or omitted.

[0215]EphB4 CF2 protein, precursor (SEQ ID NO:15):

TABLE-US-00004 MELRVLLCWASLAAALEETLLNTKLETQLTVNLTRFPETVPRELVVPVAG SCVVDAVPAPGPSPSLYCREDGQWAEQPVTGCSCAPGFEAAEGNTKCRAC AQGTFKPLSGEGSCQPCPANSHSNTIGSAVCQCRVGYFRARTDPRGAPCT TPPSAPRSVVSRLNGSSLHLEWSAPLESGGREDLTYALRCRECRPGGSCA PCGGDLTFDPGPRDLVEPWVVVRGLRPDFTYTFEVTALNGVSSLATGPVP FEPVNVTTDREVPPAVSDIRVTRSSPSSLSLAWAVPRAPSGAWLDYEVKY HEKGAEGPSSVRFLKTSENRAELRGLKRGASYLVQVRARSEAGYGPFGQE HHSQTQLDESEGWREQGGRSSLEGPRFEGKPIPNPLLGLDSTRTGHHHHH H

[0216]The precursor sequence of the preferred GCF2 protein (also referred to herein as GCF2F) is (SEQ ID NO:16):

TABLE-US-00005 MELRVLLCWASLAAALEETLLNTKLETADLKWVTFPQVDGQWEELSGLDE EQHSVRTYEVCEVQRAPGQAHWLRTGWVPRRGAVHVYATLRFTMLECLSL PRAGRSCKETFTVFYYESDADTATALTPAWMENPYIKVDTVAAEHLTRKR PGAEATGKVNVKTLRLGPLSKAGFYLAFQDQGACMALLSLHLFYKKCAQL TVNLTRFPETVPRELVVPVAGSCVVDAVPAPGPSPSLYCREDGQWAEQPV TGCSCAPGFEAAEGNTKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSA VCQCRVGYFRARTDPRGAPCTTPPSAPRSVVSRLNGSSLHLEWSAPLESG GREDLTYALRCRECRPGGSCAPCGGDLTFDPGPRDLVEPWVVVRGLRPDF TYTFEVTALNGVSSLATGPVPFEPVNVTTDREVPPAVSDIRVTRSSPSSL SLAWAVPRAPSGAWLDYEVKYHEKGAEGPSSVRFLKTSENRAELRGLKRG ASYLVQVRARSEAGYGPFGQEHHSQTQLDESEGWREQ

[0217]The processed sequence is (SEQ ID NO:17):

TABLE-US-00006 LEETLLNTKLETADLKWVTFPQVDGQWEELSGLDEEQHSVRTYEVCEVQR APGQAHWLRTGWVPRRGAVHVYATLRFTMLECLSLPRAGRSCKETFTVFY YESDADTATALTPAWMENPYIKVDTVAAEHLTRKRPGAEATGKVNVKTLR LGPLSKAGFYLAFQDQGACMALLSLHLFYKKCAQLTVNLTRFPETVPREL VVPVAGSCVVDAVPAPGPSPSLYCREDGQWAEQPVTGCSCAPGFEAAEGN TKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSAVCQCRVGYFRARTDP RGAPCTTPPSAPRSVVSRLNGSSLHLEWSAPLESGGREDLTYALRCRECR PGGSCAPCGGDLTFDPGPRDLVEPWVVVRGLRPDFTYTFEVTALNGVSSL ATGPVPFEPVNVTYDREVPPAVSDIRVTRSSPSSLSLAWAVPRAPSGAWL DYEVKYHEKGAEGPSSVRFLKTSENRAELRGLKRGASYLVQVRARSEAGY GPFGQEHHSQTQLDESEGWREQ

Biochemical Assays

[0218]A. Binding Assay

[0219]10 μl of Ni-NTA-Agarose were incubated in microcentrifuge tubes with 50 μl of indicated amount of B4ECv3 diluted in binding buffer BB (20 mM Tris-HCl, 0.15 M NaCl, 0.1% bovine serum albumin pH 8) After incubation for 30 min on shaking platform, Ni-NTA beads were washed twice with 1.4 ml of BB, followed by application of 50 μl of B2-AP in the final concentration of 50 nM. Binding was performed for 30 min on shaking platform, and then tubes were centrifuged and washed one time with 1.4 ml of BB. Amount of precipitated AP was measured colorimetrically after application of PNPP.

[0220]B. Inhibition Assay

[0221]Inhibition in solution. Different amounts of B4ECv3 diluted in 50 μl of BB were pre-incubated with 50 μl of 5 nM B2EC-AP reagent (protein fusion of Ephrin B2 ectodomain with placental alkaline phosphatase). After incubation for 1 h, unbound B2EC-AP was precipitated with 5,000 HEK293 cells expressing membrane-associated full-length EphB4 for 20 min. Binding reaction was stopped by dilution with 1.2 ml of BB, followed by centrifugation for 10 min. Supernatants were discarded and alkaline phosphatase activities associated with collected cells were measured by adding para-nitrophenyl phosphate (PNPP) substrate.

[0222]Cell based inhibition. B4ECv3 was serially diluted in 20 mM Tris-HCl, 0.15 M NaCl, 0.1% BSA, pH 8 and mixed with 5,000 HEK293 cells expressing membrane-associated full-length Ephrin B2. After incubation for 1 h, 50 μl of 5 nM B4EC-AP reagent (protein fusion of EphB4 ectodomain with placental alkaline phosphatase were added into each tube for 30 min to detect unoccupied Ephrin B2 binding sites. Binding reactions were stopped by dilution with 1.2 ml of BB and centrifugation. Colorimetric reaction of cell-precipitated AP was developed with PNPP substrate.

[0223]C. B4EC-FC Binding Assay

[0224]Protein A-agarose based assay. 10 μl of Protein A-agarose were incubated in Eppendorf tubes with 50 μl of indicated amount of B4EC-FC diluted in binding buffer BB (20 mM Tris-HCl, 0.15 M NaCl, 0.1% BSA pH 8). After incubation for 30 min on shaking platform, Protein AAagarose beads were washed twice with 1.4 ml of BB, followed by application of 50 μl of B2ECAP reagent at the final concentration of 50 nM. Binding was performed for 30 min on shaking platform, and then tubes were centrifuged and washed once with 1.4 ml of BB. Colorimetric reaction of precipitated AP was measured after application of PNPP (FIG. 6).

[0225]Nitrocellulose based assay. B4EC-FC was serially diluted in 20 mM Tris-HCl, 0.15 M NaCl, 50 μg/ml BSA, pH 8. 2 μl of each fraction were applied onto nitrocellulose strip and spots were dried out for 3 min. Nitrocellulose strip was blocked with 5% non-fat milk for 30 min, followed by incubation with 5 nM B2EC-AP reagent. After 45 min incubation for binding, nitrocellulose was washed twice with 20 mM Tris-HCl, 0.15 M NaCl, 50 μg/ml BSA, pH 8 and color was developed by application of alkaline phosphatase substrate Sigma Fast (Sigma).

[0226]D. B4EC-FC Inhibition Assay

[0227]Inhibition in solution. See above, for B4ECv3. The results were shown in FIG. 7.

[0228]Cell based inhibition. See above, for B4ECv3.

[0229]E. B2EC-FC Binding Assay

[0230]Protein-A-agarose based assay. See above, for B4EC-FC. The results were shown in FIG. 8.

[0231]Nitrocellulose based assay. See above, for B4EC-FC.

[0232]6) Cell-Based Assays

[0233]A. Growth Inhibition Assay

[0234]Human umbilical cord vein endothelial cells (HUVEC) (1.5×103) are plated in a 96-well plate in 100 μl of EBM-2 (Clonetic # CC3162). After 24 hours (day 0), the test recombinant protein (100 μl) is added to each well at 2× the desired concentration (5-7 concentration levels) in EBM-2 medium. On day 0, one plate is stained with 0.5% crystal violet in 20% methanol for 10 minutes, rinsed with water, and air-dried. The remaining plates are incubated for 72 h at 37° C. After 72 h, plates are stained with 0.5% crystal violet in 20% methanol, rinsed with water and airdried. The stain is eluted with 1:1 solution of ethanol: 0.1 M sodium citrate (including day 0 plate), and absorbance is measured at 540 nm with an ELISA reader (Dynatech Laboratories). Day 0 absorbance is subtracted from the 72 h plates and data is plotted as percentage of control proliferation (vehicle treated cells). IC50 (drug concentration causing 50% inhibition) is calculated from the plotted data.

[0235]B. Cord Formation Assay (Endothelial Cell Tube Formation Assay)

[0236]Matrigel (60 μl of 10 mg/ml; Collaborative Lab #35423) is placed in each well of an ice-cold 96-well plate. The plate is allowed to sit at room temperature for 15 minutes then incubated at 37° C. for 30 minutes to permit the matrigel to polymerize. In the mean time, HUVECs are prepared in EGM-2 (Clonetic #CC3162) at a concentration of 2×10⁵ cells/ml. The test compound is prepared at 2× the desired concentration (5 concentration levels) in the same medium. Cells (500 μl) and 2× drug (500 μl) is mixed and 200 μl of this suspension are placed in duplicate on the polymerized matrigel. After 24 h incubation, triplicate pictures are taken for each concentration using a Bioquant Image Analysis system. Drug effect (IC50) is assessed compared to untreated controls by measuring the length of cords formed and number of junctions.

[0237]C. Cell Migration Assay

[0238]Migration is assessed using the 48-well Boyden chamber and 8 μm pore size collagen-coated (10 μg/ml rat tail collagen; Collaborative Laboratories) polycarbonate filters (Osmonics, Inc.). The bottom chamber wells receive 27-29 μl of DMEM medium alone (baseline) or medium containing chemo-attractant (bFGF, VEGF or Swiss 3T3 cell conditioned medium). The top chambers receive 45 μl of HUVEC cell suspension (1×10⁶ cells/ml) prepared in DMEM+1% BSA with or without test compound. After 5 h incubation at 37° C., the membrane is rinsed in PBS, fixed and stained in Diff-Quick solutions. The filter is placed on a glass slide with the migrated cells facing down and cells on top are removed using a Kimwipe. The testing is performed in 4-6 replicates and five fields are counted from each well. Negative unstimulated control values are subtracted from stimulated control and drug treated values and data is plotted as mean migrated cell±S.D. IC50 is calculated from the plotted data.

Example 2

Extracellular Domain Fragments of EphB4 Receptor Inhibit Angiogenesis and Tumor Growth

A. Globular Domain of EphB4 is Required for EphrinB2 Binding and for the Activity of EphB4-Derived Soluble Proteins in Endothelial Tube Formation Assay.

[0239]To identify subdomain(s) of the ectopic part of EphB4 necessary and sufficient for the anti-angiogenic activity of the soluble recombinant derivatives of the receptor, four recombinant deletion variants of EphB4EC were produced and tested (FIG. 16). Extracellular part of EphB4, similarly to the other members of EphB and EphA receptor family, contains N-terminal ligand-binding globular domain followed by cysteine-rich domain and two fibronectin type III repeats (FNIII). In addition to the recombinant B4-GCF2 protein containing the complete ectopic part of EphB4, we constructed three deletion variants of EphB4EC containing globular domain and Cys-rich domain (B4-GC); globular, Cys-rich and the first FNIII domain (GCF1) as well as the ECD version with deleted globular domain (CF2). Our attempts to produce several versions of truncated EphB4EC protein containing the globular domain alone were not successful due to the lack of secretion of proteins expressed from all these constructs and absence of ligand binding by the intracellularly expressed recombinant proteins. In addition, a non-tagged version of B4-GCF2, called GCF2-F, containing complete extracellular domain of EphB4 with no additional fused amino acids was expressed, purified and used in some of the experiments described here.

[0240]All four C-terminally 6×His tagged recombinant proteins were preparatively expressed in transiently transfected cultured mammalian cells and affinity purified to homogeneity from the conditioned growth media using chromatography on Ni²+-chelate resin (FIG. 17). Apparently due to their glycosylation, the proteins migrate on SDS-PAAG somewhat higher than suggested by their predicted molecular weights of 34.7 kDa (GC), 41.5 (CF2), 45.6 kDa (GCF1) and 57.8 kDa (GCF2). Sequence of the extracellular domain of human EphB4 contains three predicted N-glycosylation sites (NXS/T) which are located in the Cys-rich domain, within the first fibronectin type III repeat and between the first and the second fibronectin repeats.

[0241]To confirm ability of the purified recombinant proteins to bind Ephrin B2, they were tested in an in vitro binding assay. As expected, GC, GCF1 and GCF2, but not CF2 are binding the cognate ligand Ephrin B2 as confirmed by interaction between Ephrin B2-alkaline phosphatase (Ephrin B2-AP) fusion protein with the B4 proteins immobilized on Ni²+-resin or on nitrocellulose membrane (FIG. 17).

[0242]All four proteins were also tested for their ability to block ligand-dependent dimerization and activation of Eph B4 receptor kinase in PC3 cells. The PC3 human prostate cancer cell line is known to express elevated levels of human Eph B4. Stimulation of PC3 cells with Ephrin B2 IgG Fc fusion protein leads to a rapid induction of tyrosine phosphorylation of the receptor. However, preincubation of the ligand with GCF2, GCF1 or GC, but not CF2 proteins suppresses subsequent EphB4 autophosphorylation. Addition of the proteins alone to the PC3 cells or preincubation of the cells with the proteins followed by changing media and adding the ligand does not affect EphB4 phosphorylation status.

[0243]Further, we found that globular domain of EphB4 is required for the activity of EphB4-derived soluble proteins in endothelial tube formation assay.

B. Effects of Soluble EphB4 on HUV/AEC In Vitro.

[0244]Initial experiments were performed to determine whether soluble EphB4 affected the three main stages in the angiogenesis pathway. These were carried out by establishing the effects of soluble EphB4 on migration/invasion, proliferation and tubule formation by HUV/AEC in vitro. Exposure to soluble EphB4 significantly inhibited both bFGF and VEGF-induced migration in the Boyden chamber assay in a dose-dependent manner, achieving significance at nM (FIG. 18). Tubule formation by HUV/AECS on wells coated with Matrigel was significantly inhibited by soluble EphB4 in a dose-dependent manner in both the absence and presence of bFGF and VEGF (FIG. 19). We also assessed in vitro, whether nM of soluble EphB4 was cytotoxic for HUVECS. Soluble EphB4 was found to have no detectable cytotoxic effect at these doses, as assessed by MTS assay (FIG. 20).

C. Soluble EphB4 Receptor Inhibits Vascularization of Matrigel Plugs, In Vivo

[0245]To demonstrate that soluble EphB4 can directly inhibit angiogenesis in vivo, we performed a murine matrigel plug experiment. Matrigel supplemented with bFGF and VEGF with and without soluble EphB4 was injected s.c. into Balb/C nu/nu mice, forming semi-solid plugs, for six days. Plugs without growth factors had virtually no vascularization or vessel structures after 6 days (FIG. 21). In contrast, plugs supplemented with bFGF and VEGF had extensive vascularization and vessels throughout the plug. Plugs taken from mice treated with μg of soluble EphB4 had markedly reduced vascularization of plugs, comparable to plugs without growth factor (FIG. 21). Furthermore, histological examination of plugs showed decreased vessel staining (FIG. 21). Treatment at 0 μg/dose significantly inhibited the amount of infiltration in Matrigel plugs compared to control (FIG. 21).

[0246]We examined EphB4 receptor phosphorylation in HUVECs by performing Western blot analyses with lysates from soluble EphB4-treated cells and antibodies against phosphor-tyrosine. We found that soluble EphB4 treatment of serum-starved HUVECs stimulated a rapid and transient decrease in the level of phosphorylated EphB4, in the presence of EphrinB2Fc, EphB4 ligand dimer. Ephrin B2Fc without the soluble EphB4 protein induced phosphorylation of EphB4 receptor (FIG. 22).

D. Effects of Soluble EphB4 on Tumor Growth, In Vitro.

[0247]We found that soluble EphB4 inhibits the growth of SCC15 tumors grown in Balb/C Nu/Nu mice (FIG. 23).

E. Soluble EphB4 Inhibited Corneal Neovascularization

[0248]To further investigate the antiangiogenic activity of soluble EphB4 in vivo, we studied the inhibitory effect of administration of soluble EphB4 on neovascularization in the mouse cornea induced by bFGF. Hydron Pellets implanted into corneal micropocket could induce angiogenesis, in the presence of growth factors, in a typically avascular area. The angiogenesis response in mice cornea was moderate, the appearance of vascular buds was delayed and the new capillaries were sparse and grew slowly. Compared with the control group, on day 7 of implantation, the neovascularization induced by bFGF in mice cornea was markedly inhibited in soluble EphB4-treated group (FIG. 24).

F. Effects of Soluble EphB4 on Tumor Growth, In Vivo.

[0249]The same model was used to determine the effects of soluble EphB4 in vivo. SCC15 tumors implanted subcutaneously, pre-incubated with matrigel and with or w/o growth factors, as well as implanted sc alone, and mice treated sc or ip daily with 1-5 ug of soluble EphB4 were carried out.

[0250]Tumors in the control group continued to grow steadily over the treatment period, reaching a final tumor volume of mm3. However, animals injected with soluble EphB4 exhibited a significantly (p<0.0/) reduced growth rate, reaching a final tumor volume of only mm3 (FIG. 25). Similar results were obtained in two further cohorts of such tumor-bearing mice. Soluble EphB4 administration appeared to be well tolerated in vivo, with no significant effect on body weight or the general well-being of the animals (as determined by the absence of lethargy, intermittent hunching, tremors or disturbed breathing patterns).

G. Effects of Soluble EphB4 on Tumor Histology.

[0251]Histological analysis revealed the presence of a central area of necrosis in all SCC15 tumors, which was usually surrounded by a viable rim of tumor cells um in width. The central necrotic areas were frequently large and confluent and showed loss of cellular detail. Necrosis, assessed as a percentage of tumor section area, was significantly (p<0.02) more extensive in the soluble EphB4-treated group (% necrosis in treated vs. control). To determine whether the reduced volume of soluble EphB4 treated tumors was due to an effect of this protein on the tumor vascular supply, endothelial cells in blood vessels were identified in tumor sections using immunostaining with an anti-platelet cell adhesion molecule (PECAM-1; CD31) antibody (FIG. 26) and the density of microvessels was assessed. Microvessel density was similar in the outer viable rim of tumor cells (the uniform layer of cells adjacent to the tumor periphery with well defined nuclei) in control and soluble EphB4-treated tumors. Microvessel density was significantly in the inner, less viable region of tumor cells abutting the necrotic central areas in soluble EphB4-treated than control tumors. Fibrin deposition, as identified by Masson's Trichrome staining, was increased in and around blood vessels in the inner viable rim and the central necrotic core of soluble EphB4 treated than control tumors. In the outer viable rim of soluble EphB4 treated tumors, although the vessel lumen remained patent and contained red blood cells, fibrin deposition was evident around many vessels. Soluble EphB4 was found to have no such effects on the endothelium in the normal tissues examined (lungs, liver and kidneys).

H. Materials and Methods

[0252]A detailed description of the materials and methods for this example may be found in U.S. Patent Publication No. 20050084873.

[0253]Cell-Based EphB4 Tyrosine Kinase Assay

[0254]The human prostate carcinoma cell line PC3 cells were maintained in RPMI medium with 10% dialyzed fetal calf serum and 1% penicillin/streptomycin/neomycin antibiotics mix. Cells were maintained at 37° C. in a humidified atmosphere of 5% CO₂/95% air. Typically, cells were grown in 60 mm dishes until confluency and were either treated with mouse Ephrin B2-Fc fusion at 1 μg/ml in RPMI for 10 min to activate EphB4 receptor or plain medium as a control. To study the effect of different derivatives of soluble EphB4 ECD proteins on EphB4 receptor activation, three sets of cells were used. In the first set, cells were treated with various proteins (5 proteins; GC, GCF1, GCF2, GCF2-F, CF2) at 5 μg/ml for 20 min. In the second set of cells, prior to application, proteins were premixed with ephrinB2-Fc at 1:5 (EphB4 protein:B2-Fc) molar ratio, incubated for 20 min and applied on cells for 10 min. In the third set of cells, cells were first treated with the proteins for 20 min at 5 μg/ml, media was replaced with fresh media containing 1 μg/ml of EphrinB2-Fc and incubated for another 10 min.

[0255]After the stimulation, cells were immediately harvested with protein extraction buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% (v/v) Triton X100, 1 mM EDTA, 1 mM PMSF, 1 mM Sodium vanadate. Protein extracts were clarified by centrifugation at 14,000 rpm for 20 min at 4° C. Clarified protein samples were incubated overnight with protein A/G coupled agarose beads pre-coated with anti-EphB4 monoclonal antibodies. The IP complexes were washed twice with the same extraction buffer containing 0.1% Triton X100. The immunoprecipitated proteins were solubilized in 1×SDS-PAGE sample loading buffer and separated on 10% SDS-PAGE. For EphB4 receptor activation studies, electroblotted membrane was probed with anti-pTyr specific antibody 4G10 at 1:1000 dilution followed by Protein G-HRP conjugate at 1:5000 dilutions.

[0256]Endothelial Cell Tube Formation Assay

[0257]Matrigel (60 μl of 10 mg/ml; Collaborative Lab, Cat. No. 35423) was placed in each well of an ice-cold 96-well plate. The plate was allowed to sit at room temperature for 15 minutes then incubated at 37° C. for 30 minutes to permit Matrigel to polymerize. In the mean time, human umbilical vein endothelial cells were prepared in EGM-2 (Clonetic, Cat. No. CC3162) at a concentration of 2×10⁵ cells/ml. The test protein was prepared at 2× the desired concentration (5 concentration levels) in the same medium. Cells (500 μl) and 2× protein (500 μl) were mixed and 200 μl of this suspension were placed in duplicate on the polymerized Matrigel. After 24 h incubation, triplicate pictures were taken for each concentration using a Bioquant Image Analysis system. Protein addition effect (IC₅₀) was assessed compared to untreated controls by measuring the length of cords formed and number of junctions.

[0258]Cell Migration Assay

[0259]Chemotaxis of HUVECs to VEGF was assessed using a modified Boyden chamber, transwell membrane filter inserts in 24 well plates, 6.5 mm diam, 8 μm pore size, 10 μm thick matrigel coated, polycarbonate membranes (BD Biosciences). The cell suspensions of HUVECs (2×10⁵ cells/ml) in 200 μl of EBM were seeded in the upper chamber and the soluble EphB4 protein were added simultaneously with stimulant (VEGF or bFGF) to the lower compartment of the chamber and their migration across a polycarbonate filter in response to 10-20 ng/ml of VEGF with or without 100 nM-1 μM test compound was investigated. After incubation for 4-24 h at 37° C., the upper surface of the filter was scraped with swab and filters were fixed and stained with Diff Quick. Ten random fields at 200× mag were counted and the results expressed as mean # per field. Negative unstimulated control values were subtracted from stimulated control and protein treated sample values and the data was plotted as mean migrated cell±S.D. IC₅₀ was calculated from the plotted data.

[0260]Growth Inhibition Assay

[0261]HUVEC (1.5×10³ cells) were plated in a 96-well plate in 100 μl of EBM-2 (Clonetic, Cat. No. CC3162). After 24 hours (day 0), the test recombinant protein (100 μl) is added to each well at 2× the desired concentration (5-7 concentration levels) in EBM-2 medium. On day 0, one plate was stained with 0.5% crystal violet in 20% methanol for 10 minutes, rinsed with water, and air-dried. The remaining plates were incubated for 72 h at 37° C. After 72 h, plates were stained with 0.5% crystal violet in 20% methanol, rinsed with water and air-dried. The stain was eluted with 1:1 solution of ethanol: 0.1M sodium citrate (including day 0 plate), and absorbance measured at 540 nm with an ELISA reader (Dynatech Laboratories). Day 0 absorbance was subtracted from the 72 h plates and data is plotted as percentage of control proliferation (vehicle treated cells). IC₅₀ value was calculated from the plotted data.

[0262]Murine Matrigel Plug Angiogenesis Assay

[0263]In vivo angiogenesis was assayed in mice as growth of blood vessels from subcutaneous tissue into a Matrigel plug containing the test sample. Matrigel rapidly forms a solid gel at body temperature, trapping the factors to allow slow release and prolonged exposure to surrounding tissues. Matrigel (8.13 mg/ml, 0.5 ml) in liquid form at 4° C. was mixed with Endothelial Cell Growth Supplement (ECGS), test proteins plus ECGS or Matrigel plus vehicle alone (PBS containing 0.25% BSA). Matrigel (0.5 ml) was injected into the abdominal subcutaneous tissue of female nu/nu mice (6 wks old) along the peritoneal mid line. There were 3 mice in each group. The animals were cared for in accordance with institutional and NIH guidelines. At day 6, mice were sacrificed and plugs were recovered and processed for histology. Typically the overlying skin was removed, and gels were cut out by retaining the peritoneal lining for support, fixed in 10% buffered formalin in PBS and embedded in paraffin. Sections of 3 μm were cut and stained with H&E or Masson's trichrome stain and examined under light microscope

[0264]Mouse Corneal Micropocket Assay

[0265]Mouse corneal micropocket assay was performed according to that detailed by Kenyon et al., 1996. Briefly, hydron pellets (polyhydroxyethylmethacrylate [polyHEMA], Interferon Sciences, New Brunswick, N.J., U.S.A.) containing either 90 ng of bFGF (R&D) or 180 ng of VEGF (R&D Systems, Minneapolis, Minn., U.S.A.) and 40 μg of sucrose aluminium sulfate (Sigma) were prepared. Using an operating microscope, a stromal linear keratotomy was made with a surgical blade (Bard-Parker no. 15) parallel to the insertion of the lateral rectus muscle in an anesthetized animal. An intrastromal micropocket was dissected using a modified von Graefe knife (2''30 mm). A single pellet was implanted and advanced toward the temporal corneal limbus (within 0±7±1±0 mm for bFGF pellets and 0±5 mm for VEGF pellets). The difference in pellet location for each growth factor was determined to be necessary given the relatively weaker angiogenic stimulation of VEGF in this model. Antibiotic ointment (erythromycin.) was then applied to the operated eye to prevent infection and to decrease surface irregularities. The subsequent vascular response was measured extending from the limbal vasculature toward the pellet and the contiguous circumferential zone of neovascularization Data and clinical photos presented here were obtained on day 6 after pellet implantation, which was found to be the day of maximal angiogenic response.

[0266]In Vitro Invasion Assay

[0267]"Matrigel" matrix-coated 9-mm cell culture inserts (pore size, 8 μm; Becton Dickinson, Franklin Lakes, N.J.) were set in a 24-well plate. The HUVEC cells were seeded at a density of 5×10³ cells per well into the upper layer of the culture insert and cultured with serum-free EBM in the presence of EphB4 ECD for 24 h. The control group was cultured in the same media without EphB4. Then 0.5 ml of the human SCC15 cell line, conditioned medium was filled into the lower layer of the culture insert as a chemo-attractant. The cells were incubated for 24 h, then the remaining cells in the upper layer were swabbed with cotton and penetrating cells in the lower layer were fixed with 5% glutaraldehyde and stained with Diff Quick. The total number of cells passing through the Matrigel matrix and each 8 μm pore of the culture insert was counted using optical microscopy and designated as an invasion index (cell number/area).

[0268]SCC15 Tumor Growth in Mice

[0269]Subcutaneously inject logarithmically growing SCC15, head and neck squamous cell carcinoma cell line, at 5×10⁶ cell density; with or without EphB4 ECD in the presence or absence of human bFGF, into athymic Balb/c nude mice, along with Matrigel (BD Bioscience) synthetic basement membrane (1:1 v/v), and examine tumors within 2 weeks. Tumor volumes in the EphB4 ECD group, in the presence and absence of growth factor after implantation were three-fold smaller than those in the vehicle groups. There was no difference in body weight between the groups. Immunohistochemical examination of cross-sections of resected tumors and TUNEL-positive apoptosis or necrosis, CD34 immunostaining, and BrdU proliferation rate will be performed, after deparaffinized, rehydrated, and quenched for endogenous peroxidase activity, and after 10 min permeabilization with proteinase K. Quantitative assessment of vascular densities will also be performed. Local intratumoral delivery or IV delivery of EphB4 ECD will also be performed twice a week.

[0270]30 athymic nude mice, BALB/c (nu/nu), were each injected with 1×10⁶ B16 melanoma cells with 0.1 ml PBS mixed with 0.1 ml matrigel or 1.5×10⁶ SCC15 cells resuspended in 200 μl of DMEM serum-free medium and injected subcutaneously on day 0 on the right shoulder region of mice. Proteins were injected intravenously or subcutaneously, around the tumor beginning on day 1 at a loading dose of 4 μg/mg, with weekly injections of 2 ug/mg. (10 μg/g, 50 μg/kg/day), and at 2 weeks post-inoculation. Mice are sacrificed on Day 14. Control mice received PBS 50 μl each day.

[0271]Tumor Formation in Nude Mice

[0272]All animals were treated under protocols approved by the institutional animal care committees. Cancer cells (5×10⁶) were subcutaneously inoculated into the dorsal skin of nude mice. When the tumor had grown to a size of about 100 mm³ (usually it took 12 days), sEphB4 was either intraperitoneally or subcutaneously injected once/day, and tumorigenesis was monitored for 2 weeks. Tumor volume was calculated according to the formula a²×b, where a and b are the smallest and largest diameters, respectively. A Student's t test was used to compare tumor volumes, with P<0.05 being considered significant.

[0273]Quantification of Microvessel Density

[0274]Tumors were fixed in 4% formaldehyde, embedded in paraffin, sectioned by 5 μm, and stained with hematoxylineosin. Vessel density was semi-quantitated using a computer-based image analyzer (five fields per section from three mice in each group).

Example 3

EphB4 is Upregulated and Imparts Growth Advantage in Prostate Cancer

A. Expression of EphB4 in Prostate Cancer Cell Lines

[0275]We first examined the expression of EphB4 protein in a variety of prostate cancer cell lines by Western blot. We found that prostate cancer cell lines show marked variation in the abundance of the 120 kD EphB4. The levels were relatively high in PC3 and even higher in PC3M, a metastatic clone of PC3, while normal prostate gland derived cell lines (MLC) showed low or no expression of EphB4 (FIG. 27A). We next checked the activation status of EphB4 in PC3 cells by phosphorylation study. We found that even under normal culture conditions, EphB4 is phosphorylated though it can be further induced by its ligand, ephrin B2 (FIG. 27B).

B. Expression of EphB4 in Clinical Prostate Cancer Samples

[0276]To determine whether EphB4 is expressed in clinical prostate samples, tumor tissues and adjacent normal tissue from prostate cancer surgical specimens were examined. The histological distribution of EphB4 in the prostate specimens was determined by immunohistochemistry. Clearly, EphB4 expression is confined to the neoplastic epithelium (FIG. 28, top left), and is absent in stromal and normal prostate epithelium (FIG. 28, top right). In prostate tissue array, 24 of the 32 prostate cancers examined were positive. We found EphB4 mRNA is expressed both in the normal and tumor tissues of clinical samples by quantitative RT-PCR. However, tumor EphB4 mRNA levels were at least 3 times higher than in the normal in this case (FIG. 28, lower right).

C. p53 and PTEN Inhibited the Expression of EphB4 in PC3 Cells

[0277]PC3 cells are known to lack PTEN expression (Davis, et al., 1994, Science. 266:816-819) and wild-type p53 function (Gale, et al., 1997, Cell Tissue Res. 290:227-241). We investigated whether the relatively high expression of EphB4 is related to p53 and/or PTEN by re-introducing wild-type p53 and/or PTEN into PC3 cells. To compensate for the transfection efficiency and the dilution effect, transfected cells were sorted for the cotransfected truncated CD4 marker. We found that the expression of EphB4 in PC3 cells was reduced by the re-introduction of either wild-type p53 or PTEN. The co-transfection of p53 and PTEN did not further inhibit the expression of EphB4 (FIG. 29A).

D. Retinoid X Receptor (RXR α) Regulates the Expression of EphB4

[0278]We previously found that RXRα was down-regulated in prostate cancer cell lines (Thong, et al., 2003, Cancer Biol Ther. 2:179-184) and here we found EphB4 expression has the reverse expression pattern when we looked at "normal" prostate (MLC), prostate cancer (PC3), and metastatic prostate cancer (PC3M) (FIG. 27A), we considered whether RXRα regulates the expression of EphB4. To confirm the relationship, the expression of EphB4 was compared between CWR22R and CWR22R-RXRα, which constitutively expresses RXRα. We found a modest decrease in EphB4 expression in the RXRα overexpressing cell line, while FGF8 has no effect on EphB4 expression. Consistent with initial results, EphB4 was not found in "normal" benign prostate hypertrophic cell line BPH-1 (FIG. 29B).

E. Growth Factor Signaling Pathway of EGFR and IGF-1R Regulates EphB4 Expression

[0279]EGFR and IGF-1R have both been shown to have autocrine and paracrine action on PC3 cell growth. Because we found that EphB4 expression is higher in the more aggressive cell lines, we postulated that EphB4 expression might correlate with these pro-survival growth factors. We tested the relationship by independently blocking EGFR and IGF-1R signaling. EphB4 was down-regulated after blocking the EGFR signaling using EGFR kinase inhibitor AG 1478 (FIG. 30A) or upon blockade of the IGF-1R signaling pathway using IGF-1R neutralizing antibody (FIG. 30B).

F. EphB4 siRNA and Antisense ODNs Inhibit PC3 Cell Viability

[0280]To define the significance of this EphB4 overexpression in our prostate cancer model, we concentrated our study on PC3 cells, which have a relatively high expression of EphB4. The two approaches to decreasing EphB4 expression were siRNA and AS-ODNs. A number of different phosphorothioate-modified AS-ODNs complementary to different segments of the EphB4 coding region were tested for specificity and efficacy of EphB4 inhibition. Using 293 cells transiently transfected with full-length EphB4 expression vector AS-10 was found to be the most effective (FIG. 31B). A Similar approach was applied to the selection of specific siRNA. EphB4 siRNA 472 effectively knocks down EphB4 protein expression (FIG. 31A). Both siRNA 472 and antisense AS-10 ODN reduced the viability of PC3 cells in a dose dependent manner (FIGS. 31C, D). Unrelated siRNA or sense oligonucleotide had no effect on viability.

G. EphB4 siRNA and Antisense ODNs Inhibit the Mobility of PC3 Cells

[0281]PC3 cells can grow aggressively locally and can form lymph node metastases when injected orthotopically into mice. In an effort to study the role of EphB4 on migration of PC3 cells in vitro, we performed a wound-healing assay. When a wound was introduced into a monolayer of PC3 cells, over the course of the next 20 hours cells progressively migrated into the cleared area. However, when cells were transfected with siRNA 472 and the wound was introduced, this migration was significantly inhibited (FIG. 31E). Pretreatment of PC3 cells with 10 μM EphB4 AS-10 for 12 hours generated the same effect (FIG. 31F). In addition, knock-down of EphB4 expression in PC3 cells with siRNA 472 severely reduced the ability of these cells to invade Matrigel as assessed by a double-chamber invasion assay (FIG. 31G), compared to the control siRNA.

H. EphB4 siRNA Induces Cell Cycle Arrest and Apoptosis in PC3 Cells

[0282]Since knock-down of EphB4 resulted in decreased cell viability (FIG. 31C) we sought to determine whether this was due to effects on the cell cycle. In comparison to control siRNA transfected cells, siRNA 472 resulted in an accumulation of cells in the sub G0 and S phase fractions compared to cells treated with control siRNA. The sub G0 fraction increased from 1% to 7.9%, and the S phase fraction from 14.9% to 20.8% in siRNA 472 treated cells compared to control siRNA treated cells (FIG. 32A). Cell cycle arrest at sub G0 and G2 is indicative of apoptosis. Apoptosis as a result of EphB4 knock-down was confirmed by ELISA assay. A dose-dependent increase in apoptosis was observed when PC3 cells were transfected with siRNA 472, but not with control siRNA (FIG. 32B). At 100 nM there was 15 times more apoptosis in siRNA 472 transfected than control siRNA transfected PC3 cells.

I. Materials and Methods

[0283]A detailed description of the materials and methods for this example may be found in U.S. Patent Publication No. 20050084873.

Example 4

Expression of EPHB4 in Mesothelioma: a Candidate Target for Therapy

[0284]Malignant mesothelioma (MM) is a rare neoplasm that most often arises from the pleural and peritoneal cavity serous surface. The pleural cavity is by far the most frequent site affected (>90%), followed by the peritoneum (6-10%) (Carbone et al., 2002, Semin Oncol. 29:2-17). There is a strong association with asbestos exposure, about 80% of malignant mesothelioma cases occur in individuals who have ingested or inhaled asbestos. This tumor is particularly resistant to the current therapies and, up to now, the prognosis of these patients is dramatically poor (Lee et al., 2000, Curr Opin Pulm Med. 6:267-74).

[0285]Several clinical problems regarding the diagnosis and treatment of malignant mesothelioma remain unsolved. Making a diagnosis of mesothelioma from pleural or abdominal fluid is notoriously difficult and often requires a thoracoscopic or laproscopic or open biopsy and Immunohistochemical staining for certain markers such as meosthelin expressed preferentially in this tumor. Until now, no intervention has proven to be curative, despite aggressive chemotherapeutic regimens and prolonged radiotherapy. The median survival in most cases is only 12-18 months after diagnosis.

[0286]In order to identify new diagnostic markers and targets to be used for novel diagnostic and therapeutic approaches, we assessed the expression of EPHB4 and its ligand EphrinB2 in mesothelioma cell lines and clinical samples.

A. EPHB4 and EphrinB2 is Expressed in Mesothelioma Cell Lines

[0287]The expression of Ephrin B2 and EphB4 in malignant mesothelioma cell lines was determined at the RNA and protein level by a variety of methods. RT-PCR showed that all of the four cell lines express EphrinB2 and EPHB4 (FIG. 33A). Protein expression was determined by Western blot in these cell lines. Specific bands for EphB4 were seen at 120 kD. In addition, Ephrin B2 was detected in all cell lines tested as a 37 kD band on Western blot (FIG. 33B). No specific band for Ephrin B2 was observed in 293 human embryonic kidney cells, which were included as a negative control.

[0288]To confirm the presence of EphB4 transcription in mesothelioma cells, in situ hybridization was carried out on NCI H28 cell lines cultured on chamber slides. Specific signal for EphB4 was detected using antisense probe Ephrin B2 transcripts were also detected in the same cell line. Sense probes for both EphB4 and Ephrin B2 served as negative controls and did not hybridize to the cells (FIG. 34). Expression of EphB4 and Ephrin B2 proteins was confirmed in the cell lines by immunofluorescence analysis (FIG. 35). Three cell lines showed strong expression of EphB4, whereas expression of Ephrin B2 was present in H28 and H2052, and weakly detectable in H2373.

B. Evidence of Expression of EPHB4 and EphrinB2 in Clinical Samples

[0289]Tumor cells cultured from the pleural effusion of a patient diagnosed with pleural malignant mesothelioma were isolated and showed positive staining for both EphB4 and Ephrin B2 at passage 1 (FIG. 35, bottom row). These results confirm co-expression of EphB4 and Ephrin B2 in mesothelioma cell lines. To determine whether these results seen in tumor cell lines were a real reflection of expression in the disease state, tumor biopsy samples were subjected to immunohistochemical staining for EphB4 and Ephrin B2. Antibodies to both proteins revealed positive stain in the tumor cells. Representative data is shown in FIG. 36.

C. EPHB4 is Involved in the Cell Growth and Migration of Mesothelioma

[0290]The role of EphB4 in cell proliferation was tested using EPHB4 specific antisepses oligonucleotides and siRNA. The treatment of cultured H28 with EPHB4 antisense reduced cell viability. One of the most active inhibitor of EphB4 expression is EPHB4AS-10 (FIG. 37A). Transfection of EPHB4 siRNA 472 generated the same effect (FIG. 37B).

[0291]MM is a locally advancing disease with frequent extension and growth into adjacent vital structures such as the chest wall, heart, and esophagus. In an effort to study this process in vitro, we perform wound healing assay using previously described techniques (3:36). When a wound was introduced into sub confluent H28 cells, over the course of the next 28 hours cells would progressively migrate into the area of the wound. However, when cells were pretreated with EPHB4AS-10 for 24 hours, and the wound was introduced, this migration was virtually completely prevented (FIG. 38A). The migration study with Boyden Chamber assay with EPHB4 siRNA showed that cell migration was greatly inhibited with the inhibition of EPHB4 expression (FIG. 38B).

D. Materials and Methods

[0292]A detailed description of the materials and methods for this example may be found in U.S. Patent Publication No. 20050084873.

Example 5

EphB4 is Expressed in Squamous Cell Carcinoma of The Head and Neck: Regulation by Epidermal Growth Factor Signaling Pathway and Growth Advantage

[0293]Squamous cell carcinoma of the head and neck (HNSCC) is the sixth most frequent cancer worldwide, with estimated 900,000 cases diagnosed each year. It comprises almost 50% of all malignancies in some developing nations. In the United States, 50,000 new cases and 8,000 deaths are reported each year. Tobacco carcinogens are believed to be the primary etiologic agents of the disease, with alcohol consumption, age, gender, and ethnic background as contributing factors.

[0294]The differences between normal epithelium of the upper aerodigestive tract and cancer cells arising from that tissue are the result of mutations in specific genes and alteration of their expression. These genes control DNA repair, proliferation, immortalization, apoptosis, invasion, and angiogenesis. For head and neck cancer, alterations of three signaling pathways occur with sufficient frequency and produce such dramatic phenotypic changes as to be considered the critical transforming events of the disease. These changes include mutation of the p53 tumor suppressor, overexpression of epidermal growth factor receptor (EGFR), and inactivation of the cyclin dependent kinase inhibitor p16. Other changes such as Rb mutation, ras activation, cyclin D amplification, and myc overexpression are less frequent in HNSCC.

[0295]Although high expression of EphB4 has been reported in hematologic malignancies, breast carcinoma, endometrial carcinoma, and colon carcinoma, there is limited data on the protein levels of EphB4, and complete lack of data on the biological significance of this protein in tumor biology such as HNSCC.

A. HNSCC Tumors Express EphB4

[0296]We studied the expression of EphB4 in human tumor tissues by immunohistochemistry, in situ hybridization, and Western blot. Twenty prospectively collected tumor tissues following IRB approval have been evaluated with specific EphB4 monoclonal antibody that does not react with other members of the EphB and EphA family. EphB4 expression is observed in all cases, with varying intensity of staining. FIG. 39A (top left) illustrates a representative case, showing that EphB4 is expressed in the tumor regions only, as revealed by the H&E tumor architecture (FIG. 39A bottom left). Note the absence of staining for EphB4 in the stroma. Secondly, a metastatic tumor site in the lymph node shows positive staining while the remainder of the lymph node is negative (FIG. 39A, top right).

[0297]In situ hybridization was carried out to determine the presence and location of EphB4 transcripts in the tumor tissue. Strong signal for EphB4 specific antisense probe was detected indicating the presence of transcripts (FIG. 39 B, top left). Comparison with the H&E stain (FIG. 39B, bottom left) to illustrate tumor architecture reveals that the signal was localized to the tumor cells, and was absent from the stromal areas. Ephrin B2 transcripts were also detected in tumor sample, and as with EphB4, the signal was localized to the tumor cells (FIG. 39B, top right). Neither EphB4 nor ephrin B2 sense probes hybridized to the sections, proving specificity of the signals.

B. High Expression of EphB4 in Primary and Metastatic Sites of HNSCC

[0298]Western blots of tissue from primary tumor, lymph node metastases and uninvolved tissue were carried out to determine the relative levels of EphB4 expression in these sites. Tumor and normal adjacent tissues were collected on 20 cases, while lymph nodes positive for tumor were harvested in 9 of these 20 cases. Representative cases are shown in FIG. 39C. EphB4 expression is observed in each of the tumor samples. Similarly, all tumor positive lymph nodes show EphB4 expression that was equal to or greater than the primary tumor. No or minimal expression is observed in the normal adjacent tissue.

C. EphB4 Expression and Regulation by EGFR Activity in HNSCC Cell Lines

[0299]Having demonstrated the expression of EphB4 limited to tumor cells, we next sought to determine whether there was an in vitro model of EphB4 expression in HNSCC. Six HN SCC cell lines were surveyed for EphB4 protein expression by Western Blot (FIG. 40A). A majority of these showed strong EphB4 expression and thus established the basis for subsequent studies. Since EGFR is strongly implicated in HNSCC we asked whether EphB4 expression is associated with the activation of EGFR. Pilot experiments in SCC-15, which is an EGFR positive cell line, established an optimal time of 24 h and concentration of 1 mM of the specific EGFR kinase inhibitor AG 1478 (FIG. 40B) to inhibit expression of EphB4. When all the cell lines were studied, we noted robust EGFR expression in all but SCC-4, where it is detectable but not strong (FIG. 40C, top row). In response to EGFR inhibitor AG1478 marked loss in the total amount of EphB4 was observed in certain cell lines (SCC-15, and SCC-25) while no effect was observed in others (SCC-9, -12, -13 and -71). Thus SCC-15 and -25 serve as models for EphB4 being regulated by EGFR activity, while SCC-9, -12, -13 and -71 are models for regulation of EphB4 in HNSCC independent of EGFR activity, where there may be input from other factors such as p53, PTEN, IL-6 etc. We also noted expression of the ligand of EphB4, namely ephrin B2, in all of the cell lines tested. As with EphB4 in some lines ephrin B2 expression appears regulated by EGFR activity, while it is independent in other cell lines.

[0300]Clearly, inhibition of constitutive EGFR signaling repressed EphB4 levels in SCC15 cells. We next studied whether EGF could induce EphB4. We found that EphB4 levels were induced in SCC15 cells that had been serum starved for 24 h prior to 24 h treatment with 10 ng/ml EGF as shown in FIG. 41B (lanes 1 and 2). The downstream signaling pathways known for EGFR activation shown in FIG. 41A, (for review see Yarden & Slikowski 2001) were then investigated for their input into EGF mediated induction of EphB4. Blocking PLCg, AKT and JNK phosphorylation with the specific kinase inhibitors U73122, SH-5 and SP600125 respectively reduced basal levels and blocked EGF stimulated induction of EphB4 (FIG. 41B, lanes 3-8). In contrast, inhibition of ERK1/2 with PD098095 and PI3-K with LY294002 or Wortmannin had no discernible effect on EGF induction of EphB4 levels. However, basal levels of EphB4 were reduced when ERK1/2 phosphorylation was inhibited. Interestingly, inhibition of p38 MAPK activation with SB203580 increased basal, but not EGF induced EphB4 levels. Similar results were seen in the SCC25 cell line (data not shown).

D. Inhibition of EphB4 in High Expressing Cell Lines Results in Reduced Viability and Causes Cell-Cycle Arrest

[0301]We next turned to the role of EphB4 expression in HNSCC by investigating the effect of ablating expression using siRNA or AS-ODN methods. Several siRNAs to EphB4 sequence were developed (Table 1) which knocked-down EphB4 expression to varying degrees as seen in FIG. 42A. Viability was reduced in SCC-15, -25 and -71 cell lines transfected with siRNAs 50 and 472, which were most effective in blocking EphB4 expression (FIG. 42B). Little effect on viability was seen with EphB4 siRNA 1562 and 2302 or ephrin B2 siRNA 254. Note that in SCC-4, which does not express EphB4 (see FIG. 40A) there was no reduction in cell viability. The decreased cell viability seen with siRNA 50 and 472 treatment was attributable to accumulation of cells in sub G0, indicative of apoptosis. This effect was both time and dose-dependant (FIG. 42C and Table 2). In contrast, siRNA2302 that was not effective in reducing EphB4 levels and had only minor effects on viability did not produce any changes in the cell cycle when compared with the mock Lipofectamine®2000 transfection.

[0302]A detailed description of the siRNA constructs for this example may be found in U.S. Patent Publication No. 20050084873.

TABLE-US-00007 TABLE Effect of different EphB4 siRNA on Cell Cycle Treatment Sub G0 G1 S G2 36 hr Lipo alone 1.9 39.7 21.3 31.8 100 nM 2302 2.0 39.3 21.2 31.2 100 nM 50 18.1 31.7 19.7 24.4 100 nM 472 80.2 10.9 5.2 2.1 16 hr Lipo alone 7.8 55.7 15.2 18.5 100 nM 2302 8.4 57.3 14.3 17.3 10 nM 50 10.4 53.2 15.7 17.7 100 nM 50 27.7 31.3 18.1 19.6 10 nM 472 13.3 50.2 15.8 17.5 100 nM 472 30.7 31.9 16.4 18.0

[0303]In addition, over 50 phosphorothioate AS-ODNs complementary to the human EphB4 coding sequences were synthesized and tested for their ability to inhibit EphB4 expression in 293 cells transiently transfected with full length EphB4 expression plasmid. FIG. 43A shows a representative sample of the effect of some of these AS-ODNs on EphB4 expression. Note that expression is totally abrogated with AS-10, while AS-11 has only a minor effect. The effect on cell viability in SCC15 cells was most marked with AS-ODNs that are most effective in inhibiting EphB4 expression as shown in FIG. 43B. The IC₅₀ for AS-10 was approximately 1 μM, while even 10 μM AS-11 was not sufficient to attain 50% reduction of viability. When the effect that AS-10 had on the cell cycle was investigated, it was found that the sub G0 fraction increased from 1.9% to 10.5% compared to non-treated cells, indicative of apoptosis (FIG. 43C).

E. EphB4 Regulates Cell Migration

[0304]We next wished to determine if EphB4 participates in the migration of HNSCC. Involvement in migration may have implications for growth and metastasis. Migration was assessed using the wound-healing/scrape assay. Confluent SCC15 and SCC25 cultures were wounded by a single scrape with a sterile plastic Pasteur pipette, which left a 3 mm band with clearly defined borders. Migration of cells into the cleared area in the presence of test compounds was evaluated and quantitated after 24, 48 and 72 hr. Cell migration was markedly diminished in response to AS-10 that block EphB4 expression while the inactive compounds, AS-1 and scrambled ODN had little to no effect as shown in FIG. 43D. Inhibition of migration with AS-10 was also shown using the Boyden double chamber assay (FIG. 43E).

F. EphB4 AS-10 In Vivo Anti-Tumor Activity

[0305]The effect of EphB4 AS-10, which reduces cell viability and motility, was determined in SCC15 tumor xenografts in Balb/C nude mice. Daily treatment of mice with 20 mg/kg AS-10, sense ODN or equal volume of PBS by I.P. injection was started the day following tumor cell implantation. Growth of tumors in mice receiving AS-10 was significantly retarded compared to mice receiving either sense ODN or PBS diluent alone (FIG. 44). Non-specific effects attributable to ODN were not observed, as there was no difference between the sense ODN treated and PBS treated groups.

G. Materials and Methods

[0306]A detailed description of the materials and methods for this example may be found in U.S. Patent Publication No. 20050084873.

Example 6

Ephrin B2 Expression in Kaposi's Sarcoma is Induced by Human Herpesvirus Type 8: Phenotype Switch from Venous to Arterial Endothelium

[0307]Kaposi's Sarcoma (KS) manifests as a multifocal angioproliferative disease, most commonly of the skin and mucus membranes, with subsequent spread to visceral organs (1) Hallmarks of the disease are angiogenesis, edema, infiltration of lymphomononuclear cells and growth of spindle-shaped tumor cells. Pathologically, established lesions exhibit an extensive vascular network of slit-like spaces. The KS vascular network is distinct from normal vessels in the lack of basement membranes and the abnormal spindle shaped endothelial cell (tumor cell) lining these vessels. Defective vasculature results in an accumulation of the blood components including albumin, red and mononuclear cells in the lesions (1). The KS tumor is endothelial in origin; the tumor cells express many endothelial markers, including lectin binding sites for Ulex europeaus agglutinin-1 (UEA-1), CD34, EN-4, PAL-E (2) and the endothelial cell specific tyrosine kinase receptors, VEGFR-1 (Flt-1), VEGFR-2 (Flk-1/KDR), VEGFR-3 (Flt-4), Tie-1 and Tie-2 (3, RM & PSG unpublished data). KS cells co-express lymphatic endothelial cell related proteins including LYVE and podoplanin (4).

[0308]The herpesvirus HHV-8 is considered the etiologic agent for the disease. In 1994 sequences of this new herpes virus were identified in KS tumor tissue (5), and subsequent molecular-epidemiology studies have shown that nearly all KS tumors contain viral genome. Sero-epidemiology studies show that HIV infected patients with KS have the highest prevalence of HHV-8 and secondly that those with HIV infection but no KS have increased risk of developement of KS over the ensuing years if they are also seropositive for HHV-8 (6). Direct evidence for the role of HHV-8 in KS is the transformation of bone marrow endothelial cells after infection with HHV-8 (7). A number of HHV-8 encoded genes could contribute to cellular transformation (reviewed in 8). However, the most evidence has accumulated for the G-protein coupled receptor (vGPCR) in this role (9).

[0309]We investigated whether KS tumor cells are derived from arterial or venous endothelium. In addition, we investigated whether HHV-8 has an effect on expression of arterial or venous markers in a model of KS. KS tumor cells were found to express the ephrin B2 arterial marker. Further, ephrin B2 expression was induced by HHV-8 vGPCR in KS and endothelial cell lines. Ephrin B2 is a potential target for treatment of KS because inhibition of ephrin B2 expression or signaling was detrimental to KS cell viability and function.

A. KS Tumors Express Ephrin B2, but not EphB4

[0310]The highly vascular nature of KS lesions and the probable endothelial cell origin of the tumor cells prompted investigation of expression of EphB4 and ephrin B2 which are markers for venous and arterial endothelial cells, respectively. Ephrin B2, but not EphB4 transcripts were detected in tumor cells of KS biopsies by in situ hybridization (FIG. 45A). Comparison of the positive signal with ephrin B2 antisense probe and tumor cells as shown by H&E staining shows that ephrin B2 expression is limited to the areas of the biopsy that contain tumor cells. The lack of signal in KS with EphB4 antisense probe is not due to a defect in the probe, as it detected transcripts in squamous cell carcinoma, which we have shown expresses this protein (18). Additional evidence for the expression of ephrin B2 in KS tumor tissue is afforded by the localization of EphB4/Fc signal to tumor cells, detected by FITC conjugated anti human Fc antibody. Because ephrin B2 is the only ligand for EphB4 this reagent is specific for the expression of ephrin B2 (FIG. 45B, left). An adjacent section treated only with the secondary reagent shows no specific signal. Two-color confocal microscopy demonstrated the presence of the HHV-8 latency protein, LANA1 in the ephrin B2 positive cells (FIG. 45C, left), indicating that it is the tumor cells, not tumor vessels, which are expressing this arterial marker. Staining of tumor biopsy with PECAM-1 antibody revealed the highly vascular nature of this tumor (FIG. 45C, right). A pilot study of the prevalence of this pattern of ephrin B2 and EphB4 expression on KS biopsies was conducted by RT-PCR analysis. All six samples were positive for ephrin B2, while only 2 were weakly positive for EphB4 (data not shown).

B. Infection of Venous Endothelial Cells with HHV-8 Causes a Phenotype Switch to Arterial Markers

[0311]We next asked whether HHV-8, the presumed etiologic agent for KS, could itself induce expression of ephrin B2 and repress EphB4 expression in endothelial cells. Co-culture of HUVEC and BC-1 lymphoma cells, which are productively infected with HHV-8, results in effective infection of the endothelial cells (16). The attached monolayers of endothelial cells remaining after extensive washing were examined for ephrin B2 and EphB4 by RT-PCR and immunofluorescence. HUVEC express EphB4 venous marker strongly at the RNA level, but not ephrin B2 (FIG. 46B). In contrast, HHV-8 infected cultures (HUVEC/BC-1 and HUVEC/BC-3) express ephrin B2, while EphB4 transcripts are almost absent.

[0312]Immunofluorescence analysis of cultures of HUVEC and HUVEC/HHV-8 for artery/vein markers and viral proteins was undertaken to determine whether changes in protein expression mirrored that seen in the RNA. In addition, cellular localization of the proteins could be determined. Consistent with the RT-PCR data HUVEC are ephrin B2 negative and EphB4 positive (FIG. 46A(a & m)). As expected they do not express any HHV-8 latency associated nuclear antigen (LANA1) (FIG. 46A(b, n)). Co-culture of BC-1 cells, which are productively infected with HHV-8, resulted in infection of HUVEC as shown by presence of viral proteins LANA1 and ORF59 (FIG. 46A(f, r)). HHV-8 infected HUVEC now express ephrin B2 but not EphB4 (FIG. 46A(e, q, u), respectively). Expression of ephrin B2 and LANA1 co-cluster as shown by yellow signal in the merged image (FIG. 46A(h)). HHV-8 infected HUVEC positive for ephrin B2 and negative for Eph B4 also express the arterial marker CD148 (19) (FIG. 46A (j, v)). Expression of ephrin B2 and CD148 co-cluster as shown by yellow signal in the merged image (FIG. 46A(l)). Uninfected HUVEC expressing Eph B4 were negative for CD148 (not shown).

C. HHV-8 vGPCR Induces Ephrin B2 Expression

[0313]To test whether individual viral proteins could induce the expression of ephrin B2 seen with the whole virus KS-SLK cells were stably transfected with HHV-8 LANA, or LANAΔ440 or vGPCR. Western Blot of stable clones revealed a five-fold induction of ephrin B2 in KS-SLK transfected with vGPCR compared to SLK-LANA or SLK-LANAΔ440 (FIG. 47A). SLK transfected with vector alone (pCEFL) was used as a control. SLK-vGPCR and SLK-pCEFL cells were also examined for ephrin B2 and Eph B4 expression by immunofluorescence in transiently transfected KS-SLK cells. FIG. 47B shows higher expression of ephrin B2 in the SLK-vGPCR cells compared to SLK-pCEFL. No changes in Eph B4 were observed in SLK-vGPCR compared to SLK-pCEFL. This clearly demonstrates that SLK-vGPCR cells expressed high levels of ephrin B2 compared to SLK-pCEFL cells. This suggests that vGPCR of HHV-8 is directly involved in the induction of Ephrin B2 and the arterial phenotype switch in KS. Since we had shown that HHV-8 induced expression of ephrin B2 in HUVEC, we next asked if this could be mediated by a transcriptional effect. Ephrin B2 5'-flanking DNA-luciferase reporter plasmids were constructed as described in the Materials and Methods and transiently transfected into HUVECs. Ephrin B2 5'-flanking DNA sequences -2491/-11 have minimal activity in HUVEC cells (FIG. 47C). This is consistent with ephrin B2 being an arterial, not venous marker. However, we have noted that HUVEC in culture do express some ephrin B2 at the RNA level. Cotransfection of HHV-8 vGPCR induces ephrin B2 transcription approximately 10-fold compared to the control expression vector pCEFL. Roughly equal induction was seen with ephrin B2 sequences -2491/-11, -1242/-11, or -577/-11, which indicates that elements between -577 and -11 are sufficient to mediate the response to vGPCR, although maximal activity is seen with the -1242/-11 luciferase construct.

D. Expression of Ephrin B2 is Regulated by VEGF and VEGF-C

[0314]We next asked whether known KS growth factors could be involved in the vGPCR-mediated induction of ephrin B2 expression. SLK-vGPCR cells were treated with neutralizing antibodies to oncostatin-M, IL-6, IL-8, VEGF or VEGF-C for 36 hr. FIG. 48A shows that neutralization of VEGF completely blocked expression of ephrin B2 in SLK-vGPCR cells. A lesser, but significant decrease in ephrin B2 was seen neutralization of VEGF-C and IL-8. No appreciable effect was seen with neutralization of oncostatin-M or IL-6. To verify that VEGF and VEGF-C are integral to the induction of ephrin B2 expression we treated HUVEC with VEGF, VEGF-C or EGF. HUVECs were grown in EBM-2 media containing 5% FBS with two different concentration of individual growth factor (10 ng, 100 ng/ml) for 48 h. Only VEGF-A or VEGF-C induced ephrin B2 expression in a dose dependent manner (FIG. 48B). In contrast, EGF had no effect on expression of ephrin B2.

E. Ephrin B2 siRNA Inhibits the Expression of Ephrin B2 in KS

[0315]Three ephrin B2 siRNA were synthesized as described in the methods section. KS-SLK cells were transfected with siRNA and 48 h later ephrin B2 expression was determined by Western Blot. Ephrin B2 siRNAs 137 or 254 inhibited about 70% of ephrin B2 expression compared to control siRNA such as siRNA Eph B4 50 or siRNA GFP. Ephrin B2 63 siRNA was less effective than the above two siRNA Ephrin B2 (FIG. 49A).

F. Ephrin B2 is Necessary for Full KS and EC Viability, Cord Formation and In Vivo Angiogenesis Activities

[0316]The most effective ephrin B2 siRNA (254) was then used to determine whether inhibiting expression of ephrin B2 has any effect on the growth of KS-SLK or HUVEC cells. The viability of KS-SLK cells was decreased by the same siRNAs that inhibited ephrin B2 protein levels (FIG. 49B). KS-SLK express high levels of ephrin B2 and this result shows maintenance of ephrin B2 expression is integral to cell viability in this setting. HUVECs do not express ephrin B2, except when stimulated by VEGF as shown in FIG. 48B. Ephrin B2 siRNA 264 dramatically reduced growth of HUVECs cultured with VEGF as the sole growth factor. In contrast, no significant effect was seen when HUVECs were cultured with IGF, EGF and bFGF. As a control, EphB4 siRNA 50 had no detrimental effect on HUVECs in either culture condition (FIG. 49C). In addition to inhibition of viability of KS and primary endothelial cells, EphB4-ECD inhibits cord formation in HUVEC and KS-SLK and in vivo angiogenesis in the Matrigel® plug assay (FIG. 50).

G. Methods and Materials

[0317]A detailed description of the materials and methods for this example may be found in U.S. Patent Publication No. 20050084873.

Example 7

Expression of EphB4 in Bladder Cancer: a Candidate Target for Therapy

[0318]FIG. 51 shows expression of EPHB4 in bladder cancer cell lines (A), and regulation of EPHB4 expression by EGFR signaling pathway (B).

[0319]FIG. 52 shows that transfection of p53 inhibit the expression of EPHB4 in 5637 cell.

[0320]FIG. 53 shows growth inhibition of bladder cancer cell line (5637) upon treatment with EPHB4 siRNA 472.

[0321]FIG. 54 shows results on apoptosis study of 5637 cells transfected with EPHB4 siRNA 472.

[0322]FIG. 55 shows effects of EPHB4 antisense probes on cell migration. 5637 cells were treated with EPHB4AS10 (10 μM).

[0323]FIG. 56 shows effects of EPHB4 siRNA on cell invasion. 5637 cells were transfected with siRNA 472 or control siRNA.

Example 8

Inhibition of EphB4 Gene Expression by EphB4 Antisense Probes and RNAi Probes

[0324]Cell lines expressing EphB4 were treated with the synthetic phosphorothioate modified oligonucleotides and harvested after 24 hr. Cell lysates were prepared and probed by western blot analysis for relative amounts of EphB4 compared to untreated control cells.

[0325]Studies on inhibition of cell proliferation were done in HNSCC cell lines characterized to express EphB4. Loss of cell viability was shown upon knock-down of EphB4 expression. Cells were treated in vitro and cultured in 48-well plates, seeded with 10 thousand cells per well. Test compounds were added and the cell viability was tested on day 3. The results on EphB4 antisense probes were summarized below in Table 6. The results on EphB4 RNAi probes were summarized below in Table 7.

[0326]A detailed description of the antisense and siRNA constructs for this example may be found in U.S. Patent Publication No. 20050084873.

Example 9

Inhibition of Ephrin B2 Gene Expression by Ephrin B2 Antisense Probes and RNAi Probes

[0327]KS SLK, a cell line expressing endogenous high level of ephrin B2. Cell viability was tested using fixed dose of each oligonuceotide (5 uM). Gene expression downregulation was done using cell line 293 engineered to stably express full-length ephrin B2. KS SLK expressing EphrinB2 were also used to test the viability in response to RNAi probes tested at the fixed dose of 50 nM. Protein expression levels were measured using 293 cells stably expressing full-length EphrinB2, in cell lysates after 24 hr treatment with fixed 50 nM of RNAi probes.

[0328]The results on Ephrin B2 antisense probes were summarized below in Table 8. The results on Ephrin B2 RNAi probes were summarized below in Table 9.

[0329]A detailed description of the antisense and siRNA constructs for this example may be found in U.S. Patent Publication No. 20050084873.

Example 10

EphB4 Antibodies Inhibit Tumor Growth

[0330]FIG. 57 shows results on comparison of EphB4 monoclonal antibodies by G250 and in Pull-down assay.

[0331]FIG. 58 shows that EphB4 antibodies, in the presence of matrigel and growth factors, inhibit the in vivo tumor growth of SCC15 cells.

[0332]BaIbC nude mice were injected subcutaneously with 2.5×10⁶ viable tumor cells SCC15 is a head and neck squamous cell carcinoma line. Tumors were initiated in nu/nu mice by injecting 2.5-5×10⁶ cells premixed with matrigel and Growth factors, and Ab's subcutaneously to initiate tumor xenografts. Mice were opened 14 days after injections. SCC15 is a head and neck squamous cell carcinoma line, B16 is a melanoma cell line, and MCF-7 is a breast carcinoma line. The responses of tumors to these treatments were compared to control treated mice, which receive PBS injections. Animals were observed daily for tumor growth and subcutaneous tumors were measured using a caliper every 2 days. Antibodies #1 and #23 showed significant regression of SCC15 tumor size compared to control, especially with no additional growth factor added.

[0333]FIG. 59 shows that EphB4 antibodies cause apoptosis, necrosis and decreased angiogenesis in SCC15, head and neck carcinoma tumor type.

[0334]Angiogenesis was assessed by CD-31 immunohistochemistry. Tumor tissue sections from treated and untreated mice were stained for CD31. Apoptosis was assessed by immunohistochemical TUNNEL, and proliferation by BrdU assay. Following surgical removal, tumors were immediately sliced into 2 mm serial sections and embedded in paraffin using standard procedures. Paraffin embedded tissue were sectioned at 5 μm, the wax removed and the tissue rehydrated. The rehydrated tissues were microwave irradiated in antigen retreival solution. Slides were rinsed in PBS, and TUNNEL reaction mixture (Terminal deoxynucleotidyl transferase and flourescein labeled nucleotide solution), and BrdU were added in a humidity chamber completely shielded from light. The TUNNEL and BrdU reaction mixture were then removed, slides were rinsed and anti-flourescein antibody conjugated with horseradish peroxidase was added. After incubation and rinsing, 3, 3' diaminobenzidine was added. Masson's Trichrome and Hematoxylin and Eosin were also used to stain the slides to visualize morphology. Masson's Trichrome allows to visualize necrosis and fibrosis. The tumor gets blood support from tumor/skin, muscle boundary. As tumor grows, inner regions get depleted of nutrients. This leads to necrosis (cell death), preferably at the tumor center. After cells die, (tumor) tissue gets replaced with fibroblastic tissue. Slides were visualized under 20-fold magnification with digital images acquired. A different morphology was obtained on SCC tumors with each antibody administered. Ab #1 showed an increase in necrosis and fibrosis but not apoptosis. Ab #23 showed an increase in apoptosis, necrosis and fibrosis and a decrease in vessel infiltration. Ab #35 showed an increase in necrosis and fibrosis, and a small increase in apoptosis and a decrease in vessel infiltration. Ab #79 showed a large increase in apoptosis, and necrossis and fibrosis. Ab #91 showed no change in apoptosis but an increase in proliferation. And Ab #138 showed an increase in apoptosis, necrosis, fibrosis and a decrease in proliferation and vessel infiltration. Tumors treated with control PBS displayed abundant tumor density and a robust angiogenic response. Tumors treated with EphB4 antibodies displayed a decrease in tumor cell density and a marked inhibition of tumor angiogenesis in regions with viable tumor cells, as well as tumor necrosis and apoptosis.

[0335]FIG. 60 shows that systemic administration of antibodies on xenografts leads to tumor regression in SCC15 tumor xenografts.

[0336]Alternate day treatment with EphB4 monoclonal antibody or an equal volume of PBS as control were initiated on day 4, after the tumors have established, and continued for 14 days. Systemic administration was administered either IP or SC with no significant difference. All the experiments were carried out in a double-blind manner to eliminate investigator bias. Mice were sacrificed at the conclusion of the two week treatment period. Tumors were harvested immediately postmortem and fixed and processed for immunohistochemistry. EphB4 antibodies 40 mg per kg body weight were administered. Treatment with EphB4 antibody significantly inhibited human SCC tumor growth compared with control-treated mice (p<0.05). Treatment with EphB4 antibody significantly inhibited tumor weight compared with control-treated mice (p<0.05).

Example 11

HSA-EphB4 Ectodomain Fusion and PEG-Modified EphB4 Ectodomain

A. Generation of HSA-EphB4 Ectodomain Fusion

[0337]Human serum albumin fragment in XbaI-NotI form was PCR-amplified out for creating a fusion with GCF2, and TA-cloned into pEF6. In the next step, the resulting vector was cut with Xba I (partial digestion) and the HSA fragment (1.8 kb) was cloned into Xba I site of pEF6-GCF2-Xba to create fusion expression vector. The resulting vector had a point mutation C to T leading to Thr to Ile substitution in position 4 of the mature protein. It was called pEF6-GCF2-HSAmut. In the next cloning step, the mutation was removed by substituting wild type KpnI fragment from pEF6-GCF2-IF (containing piece of the vector and N-terminal part of GCF2) for the mutated one, this final vector was called pEF6-GCF2. The DNA sequence of pEF6-GCF2 was confirmed.

[0338]The predicted amino acid of the HSA-EphB4 precursor protein was as follows (SEQ ID NO:18):

TABLE-US-00008 MELRVLLCWASLAAALEETLLNTKLETADLKWVTFPQVDGQWEELSGLDE EQHSVRTYEVCDVQRAPGQAHWLRTGWVPRRGAVHVYATLRFTMLECLSL PRAGRSCKETFTVFYYESDADTATALTPAWMENPYIKVDTVAAEHLTRKR PGAEATGKVNVKTLRLGPLSKAGFYLAFQDQGACMALLSLHLFYKKCAQL TVNLTRFPETVPRELVVPVAGSCVVDAVPAPGPSPSLYCREDGQWAEQPV TGCSCAPGFEAAEGNTKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSA VCQCRVGYFRARTDPRGAPCTTPPSAPRSVVSRLNGSSLHLEWSAPLESG GREDLTYALRCRECRPGGSCAPCGGDLTFDPGPRDLVEPWVVVRGLRPDF TYTFEVTALNGVSSLATGPVPFEPVNVTTDREVPPAVSDIRVTRSSPSSL SLAWAVPRAPSGAVLDYEVKYHEKGAEGPSSVRFLKTSENRAELRGLKRG ASYLVQVRARSEAGYGPFGQEHHSQTQLDESEGWREQSRDAHKSEVAHRF KDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAEN CDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPN LPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRY KAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAF KAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLA KYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVE SKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCC AAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFKQLGEYKFQNALLVRY TKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLC VLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHA DICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKA DDKETCFAEEGKKLVAASQAALGL

[0339]The predicted amino acid sequence of the mature form of the HSA-EphB4 protein was as follows (SEQ ID NO:19):

TABLE-US-00009 LEETLLNTKLETADLKWVTFPQVDGQWEELSGLDEEQHSVRTYEVCDVQR APGQAHWLRTGWVPRRGAVHVYATLRFTMLECLSLPRAGRSCKETFTVFY YESDADTATALTPAWMENPYIKVDTVAAEHLTRKRPGAEATGKVNVKTLR LGPLSKAGFYLAFQDQGACMALLSLHLFYKKCAQLTVNLTRFPETVPREL VVPVAGSCVVDAVPAPGPSPSLYCREDGQWAEQPVTGCSCAPGFEAAEGN TKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSAVCQCRVGYFRARTDP RGAPCTTPPSAPRSVVSRLNGSSLHLEWSAPLESGGREDLTYALRCRECR PGGSCAPCGGDLTFDPGPRDLVEPWVVVRGLRPDFTYTFEVTALNGVSSL ATGPVPFEPVNVTTDREVPPAVSDIRVTRSSPSSLSLAWAVPRAPSGAVL DYEVKYHEKGAEGPSSVRFLKTSENRAELRGLKRGASYLVQVRARSEAGY GPFGQEHHSQTQLDESEGWREQSRDAHKSEVAHRFKDLGEENFKALVLIA FAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCT VATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTA FHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAA CLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKA EFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLK ECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVF LGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDE FKPLVEEPQNLIKQNCELFKQLGEYKFQNALLVRYTKKVPQVSTPTLVEV SRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKC CTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQ TALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGL

The nucleic acid sequence of the pEF6-GCF2 plasmid was as follows (SEQ ID NO: 20):

TABLE-US-00010 aatattattgaagcatttatcagggttattgtctcatgagcggatacata tttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttcc ccgaaaagtgccacctgacgtcgacggatcgggagatctcccgatcccct atggtcgactctcagtacaatctgctctgatgccgcatagttaagccagt atctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaa atttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatct gcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatata cgcgttgacattgattattgactaggcttttgcaaaaagctttgcaaaga tggataaagttttaaacagagaggaatctttgcagctaatggaccttcta ggtcttgaaaggagtgcctcgtgaggctccggtgcccgtcagtgggcaga gcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaatt gaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtc gtgtactggctccgcctttttcccgagggtgggggagaaccgtatataag tgcagtagtcgccgtgaacgttatttttcgcaacgggtttgccgccagaa cacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggt tatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgat tcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggcctt gcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctggg cgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgc tgctttcgataagtctctagccatttaaaatttttgatgacctgctgcga cgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcac actggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtc ccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaa tcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctc gcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggc accagttgcgtgagcggaaagatggccgcttcccggccctgctgcaggga gctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcaccc acacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactc cacggagtaccgggcgccgtccaggcacctcgattagttctcgagctttt ggagtacgtcgtctttaggttggggggaggggttttatgcgatggagttt ccccacactgagtgggtggagactgaagttaggccagcttggcacttgat gtaattctccttggaatttgccctttttgagtttggatcttggttcattc tcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcg tgaggaattagcttggtactaatacgactcactatagggagacccaagct ggctaggtaagcttggtaccgagctcggatccactagtccagtgtggtgg aattgcccttCAAGCTTGCCGCCACCATGGAGCTCCGGGTGCTGCTCTGC TGGGCTTCGTTGGCCGCAGCTTTGGAAGAGACCCTGCTGAACACAAAATT GGAAACTGCTGATCTGAAGTGGGTGACATTCCCTCAGGTGGACGGGCAGT GGGAGGAACTGAGCGGCCTGGATGAGGAACAGCACAGCGTGCGCACCTAC GAAGTGTGTGACGTGCAGCGTGCCCCGGGCCAGGCCCACTGGCTTCGCAC AGGTTGGGTCCCACGGCGGGGCGCCGTCCACGTGTACGCCACGCTGCGCT TCACCATGCTCGAGTGCCTGTCCCTGCCTCGGGCTGGGCGCTCCTGCAAG GAGACCTTCACCGTCTTCTACTATGAGAGCGATGCGGACACGGCCACGGC CCTCACGCCAGCCTGGATGGAGAACCCCTACATCAAGGTGGACACGGTGG CCGCGGAGCATCTCACCCGGAAGCGCCCTGGGGCCGAGGCCACCGGGAAG GTGAATGTCAAGACGCTGCGCCTGGGACCGCTCAGCAAGGCTGGCTTCTA CCTGGCCTTCCAGGACCAGGGTGCCTGCATGGCCCTGCTATCCCTGCACC TCTTCTACAAAAAGTGCGCCCAGCTGACTGTGAACCTGACTCGATTCCCG GAGACTGTGCCTCGGGAGCTGGTTGTGCCCGTGGCCGGTAGCTGCGTGGT GGATGCCGTCCCCGCCCCTGGCCCCAGCCCCAGCCTCTACTGCCGTGAGG ATGGCCAGTGGGCCGAACAGCCGGTCACGGGCTGCAGCTGTGCTCCGGGG TTCGAGGCAGCTGAGGGGAACACCAAGTGCCGAGCCTGTGCCCAGGGCAC CTTCAAGCCCCTGTCAGGAGAAGGGTCCTGCCAGCCATGCCCAGCCAATA GCCACTCTAACACCATTGGATCAGCCGTCTGCCAGTGCCGCGTCGGGTAC TTCCGGGCACGCACAGACCCCCGGGGTGCACCCTGCACCACCCCTCCTTC GGCTCCGCGGAGCGTGGTTTCCCGCCTGAACGGCTCCTCCCTGCACCTGG AATGGAGTGCCCCCCTGGAGTCTGGTGGCCGAGAGGACCTCACCTACGCC CTCCGCTGCCGGGAGTGTCGACCCGGAGGCTCCTGTGCGCCCTGCGGGGG AGACCTGACTTTTGACCCCGGCCCCCGGGACCTGGTGGAGCCCTGGGTGG TGGTTCGAGGGCTACGTCCTGACTTCACCTATACCTTTGAGGTCACTGCA TTGAACGGGGTATCCTCCTTAGCCACGGGGCCCGTCCCATTTGAGCCTGT CAATGTCACCACTGACCGAGAGGTACCTCCTGCAGTGTCTGACATCCGGG TGACGCGGTCCTCACCCAGCAGCTTGAGCCTGGCCTGGGCTGTTCCCCGG GCACCCAGTGGGGCTGTGCTGGACTACGAGGTCAAATACCATGAGAAGGG CGCCGAGGGTCCCAGCAGCGTGCGGTTCCTGAAGACGTCAGAAAACCGGG CAGAGCTGCGGGGGCTGAAGCGGGGAGCCAGCTACCTGGTGCAGGTACGG GCGCGCTCTGAGGCCGGCTACGGGCCCTTCGGCCAGGAACATCACAGCCA GACCCAACTGGATGAGAGCGAGGGCTGGCGGGAGCAGtctagaGATGCAC ACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGGAGAAGAAAATTTC AAAGCCTTGGTGTTGATTGCCTTTGCTCAGTATCTTCAGCAGTGTCCATT TGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAAAAACAT GTGTAGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTT TTTGGAGACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGA AATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCT TGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAG GTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAA AAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGG AACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGC CAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCG GGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAATGTGCCAGTC TCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTGGCTCGCCTG AGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGAC AGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAAT GTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAGGAT TCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAA ATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGC CTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTAT GCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAG AAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACAT ATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGC TATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAA TTTAATCAAACAAAACTGTGAGCTTTTTAAGCAGCTTGGAGAGTACAAAT TCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCA ACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAA ATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATC TATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTA AGTGACAGAGTCACAAAATGCTGCACAGAGTCCTTGGTGAACAGGCGACC ATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTA ATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAG GAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTTGTGAAACACAA GCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAG CTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCC GAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTATA Atagcggccgcttaagggcaattctgcagatatccagcacagtggcggcc gctcgagtctagagggcccgcggttcgaaggtaagcctatccctaaccct ctcctcggtctcgattctacgcgtaccggtcatcatcaccatcaccattg agtttaaacccgctgatcagcctcgactgtgccttctagttgccagccat ctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccact cccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgag taggtgtcattctattctggggggtggggtggggcaggacagcaaggggg aggattgggaagacaatagcaggcatgctggggatgcggtgggctctatg gcttctgaggcggaaagaaccagctggggctctagggggtatccccacgc gccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcg tgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttc ccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcg gggcatccctttagggttccgatttagtgctttacggcacctcgacccca aaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatag acggtttttcgccctttgacgttggagtccacgttctttaatagtggact cttgttccaaactggaacaacactcaaccctatctcggtctattcttttg atttataagggattttggggatttcggcctattggttaaaaaatgagctg atttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagtta gggtgtggaaagtccccaggctccccaggcaggcagaagtatgcaaagca tgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctcccca gcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagt cccgcccctaactccgcccatcccgcccctaactccgcccagttccgccc

attctccgccccatggctgactaattttttttatttatgcagaggccgag gccgcctctgcctctgagctattccagaagtagtgaggaggcttttttgg aggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcg gatctgatcagcacgtgttgacaattaatcatcggcatagtatatcggca tagtataatacgacaaggtgaggaactaaaccatggccaagcatttgtct caagaagaatccaccctcattgaaagagcaacggctacaatcaacagcat ccccatctctgaagactacagcgtcgccagcgcagctctctctagcgacg gccgcatcttcactggtgtcaatgtatatcattttactgggggaccttgt gcagaactcgtggtgctgggcactgctgctgctgcggcagctggcaacct gacttgtatcgtcgcgatcggaaatgagaacaggggcatcttgagcccct gcggacggtgtcgacaggtgcttctcgatctgcatcctgggatcaaagcg atagtgaaggacagtgatggacagccgacggcagttgggattcgtgaatt gctgccctctggttatgtgtgggagggctaagcacttcgtggccgaggag caggactgacacgtgctacgagatttcgattccaccgccgccttctatga aaggttgggcttcggaatcgttttccgggacgccggctggatgatcctcc agcgcggggatctcatgctggagttcttcgcccaccccaacttgtttatt gcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaa taaagcatttttttcactgcattctagttgtggtttgtccaaactcatca atgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcg taatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaat tccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcct aatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttc cagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgc ggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactg actcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactca aaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaa catgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgt tgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaat cgacgctcaagtcagaggtggcgaaacccgacaggactataaagatacca ggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgc cgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctt tctcaatgctcacgctgtaggtatctcagttcggtgtaggttgttcgctc caagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgcct tatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcg ccactggcagcagccactggtaacaggattagcagagcgaggtatgtagg cggtgctacagagttcttgaagtggtggcctaactacggctacactagaa ggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaa agagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtgg tttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaag aagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaac tcacgttaagggattttggtcatgagattatcaaaaaggatcttcaccta gatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatg agtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatc tcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgt gtagataactacgatacgggagggcttaccatctggccccagtgctgcaa tgataccgcgagacccacgctcaccggctccagatttatcagcaataaac cagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgc ctccatccagtctattaattgttgccgggaagctagagtaagtagttcgc cagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtg tcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatc aaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctcct tcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactc atggttatggcagcactgcataattctcttactgtcatgccatccgtaag atgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagt gtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataatacc gcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttc ggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgt aacccactcgtgcacccaactgatcttcagcatcttttactttcaccagc gtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaat aagggcgacacggaaatgttgaatactcatactcttcctttttc

B. Cell Culture and Transfections:

[0340]The human embryonic kidney cell line, 293T cells, was maintained in DMEM with 10% dialyzed fetal calf serum and 1% penicillin/streptomycin/neomycin antibiotics. Cells were maintained at 37° C. in a humidified atmosphere of 5% CO₂/95% air.Transfections of plasmids encoding EphB4 ectodomain, fragments thereof, and EphB4-HSA fusions were performed using Lipofectamine 2000 reagent (Invitrogen) according to suggested protocol. One day before transfections, 293T cells were seeded at a high density to reach 80% confluence at the time of transfection. Plasmid DNA and Lipofectamine reagent at 1:3 ratio were diluted in Opti-MEM I reduced serum medium (Invitrogen) for 5 min and mixed together to form DNA-Lipofectamine complex. For, each 10 cm culture dish, 10 μg of plasmid DNA was used. After 20 min, the above complex was added directly to cells in culture medium. After 16 hours of transfection, medium was aspirated, washed once with serum free DMEM and replaced with serum free DMEM. Secreted proteins were harvested after 48 hours by collecting conditional medium. Conditional medium was clarified by centrifugation at 10,000 g for 20 min and filtered through 0.2μ filter and used for purification.

C. Chromatographic Separation of EphB4 Ectodomain and EphB4 Ectodomain-HSA Fusion Protein

[0341]The EphB4 ectodomain fused to HSA was purified as follows: 700 ml of media was harvested from transiently transfected 293 cells grown in serum free media and concentrated up to final volume of 120 ml. Membrane: (Omega, 76 mm), 50 kDa C/O. After concentration, pH of the sample was adjusted by adding 6 ml of 1M NaAc, pH 5.5. Then sample was dialyzed against starting buffer (SB): 20 mM NaAc, 20 mM NaCl, pH 5.5 for O/N. 5 ml of SP-Sepharose was equilibrated with SB and sample was loaded. Washing: 100 ml of SB. Elution by NaCl: 12 ml/fraction and increment of 20 mM. Most of the EphrinB2 binding activity eluted in the 100 mM and 120 mM fractions.

[0342]Fractions, active in EphrinB2 binding assay (See SP chromatography, fractions #100-120 mM) were used in second step of purification on Q-column. Pulled fractions were dialyzed against starting buffer #2 (SB2): 20 mM Tris-HCl, 20 mM NaCl, pH 8 for O/N and loaded onto 2 ml of Q-Sepharose. After washing with 20 ml of SB2, absorbed protein was eluted by NaCl: 3 ml/fraction with a concentration increment of 25 mM. Obtained fractions were analyzed by PAGE and in Ephrin-B2 binding assay. The 200 mM and 225 mM fractions were found to contain the most protein and the most B2 binding activity.

[0343]Soluble EphB4 ectodomain protein was purified as follows: 300 ml of conditional medium (see: Cell culture and transfections) were concentrated up to final volume of 100 ml, using ultrafiltration membrane with 30 kDa C/O. After concentration, pH of the sample was adjusted by adding 5 ml of 1 M Na-Acetate, pH 5.5. Then sample was dialyzed against starting buffer (StB): 20 mM Na-Acetate, 20 mM NaCl, pH 5.5 for O/N. 5 ml of SP-Sepharose was equilibrated with StB and sample was loaded. After washing the column with 20 ml of StB, absorbed proteins were eluted by linear gradient of concentration of NaCl (20-250 mM and total elution volume of 20 column's volumes). Purity of the proteins was analyzed by PAGE.

D. Biotinylation of sB4 and sB4-HSA Fusion Protein.

[0344]Both soluble EphB4 ectodomain protein (sB4) and EphB4 ectodomain fused to HSA (HSA-sB4) were biotin labeled through carbohydrate chains using sodium meta-periodate as an oxidant and EZ-Link Biotin Hydrazide (PIERCE, Cat. #21339) according to manufacture's protocol. The in vitro stability of the biotinylated sB4 protein was tested by incubating 2.0×10^-9 with 40 μL of mouse serum at 37° C. for 0, 0.5, 1, 2 and 3 days. Two μL of magnetic beads and B2-AP was added for an extra hour at room temperature. After washing twice with buffer, pnPP was added for 1 hour. Biotinylated sB4 protein was found to very stable over three days, with less than 10% of the B2 binding activity being lost over that time.

E. Ephrin-B2 Binding Properties of B4-HSA

[0345]To test whether the B4-HSA fusion property retained the ability of the EphB4 extracellular domain to bind to EphrinB2, the ability of the purified B4-HSA fusion was compared to that of GCF2F, GCF2, GC, CF and B4-Fc fusion, which comprises the extracellular domain of B4 fused to hIgG1 Fc as described in Example 1. Biotinylated or His-tag protein samples were inoculated with the corresponding affinity magnetic beads and B2-AP for an hour at room temperature, before addition of PnPP. Results of binding assays are shown on FIG. 67. B4-HSA was found to retain most of its binding activity towards EphrinB2. Surprisingly, the B4-HSA protein was superior to the B4-Fc fusion in binding to EphrinB2.

[0346]An EphB4 ectodomain fusion to the C-terminus of HSA was also generated, and found to retain the ability to bind to EphrinB2 and was found to have enhanced stability in vivo over the EphB4 ectodomain.

F. Stability of B4-HSA vs. sB4 in Mice

[0347]The stability of the purified biotinylated sB4 and sB4-HSA were assayed in vivo. Each of the proteins were intravenously injected into the tail of mice in the amount of 0.5 nmoles per mouse. Blood from the eye of each mouse was taken in time frames of 15 min (0 days), 1, 2, 3 and 6 days. 10 ml of obtained serum was used in binding assay with Ephrin-B2-Alkaline Phosphatase fusion protein and Streptavidin-coated magnetic beads as a solid phase. The stability of the two proteins is shown on FIG. 68. sB4-HSA was found to have superior stability relative to sB4. For example, one day after injection, the levels of sB4-HSA in the blood of the mice were 5-fold greater than those of sB4.

G. PEGylation of Biotinylated sB4

[0348]Prior to PEGylation, biotinylated sB4 protein generated as described above was concentrated up to final concentration of 2 mg/ml using a 30 kDa MWCO ultra membrane. Sample was dialyzed O/N against coupling buffer: 30 mM phosphate, 75 mM NaCl, pH 8.00. Coupling to PEG was performed at 4° C. for 18 hours in 10 fold molar excess of reactive linear PEG unless otherwise indicated. The reactive PEG used was PEG-succinimidyl propionate, having a molecular weight of about 20 kda. Coupling to PEG may be similarly performed using branches PEGS, such as of 10 kDa, 20 kDa or 40 kDa. Other linear PEG molecules of 10 or 40 kDa may also be used.

[0349]After PEGylation, the protein sample containing EphB4 ectodomain was dialyzed against StB O/N. Three ml of SP-Sepharose was equilibrated with StB and sample was loaded. Washing and elution of absorbed proteins was performed as above (see: Purification of soluble EphB4 ectodomain and its fusion to HSA) with just one modification: total elution volume was 40 volumes of column. FIG. 69 shows chromatographic separation of PEG derivatives of EphB4 protein on SP-Sepharose columns. Purity of the PEG-modified EphB4 protein was analyzed by SDS-PAGE.

[0350]Double modified (PEGylated Biotinylated) sB4 was used on ion-exchange chromatography to separate non-PEGylated, mono-PEGylated and poly-PEGylated proteins from each other. Pegylated sample was dialyzed O/N against 20 mM Na-acetate, 20 mM NaCl, pH 5.5 and loaded onto 2 ml of SP-Sepharose. After washing with 10 ml of buffer, absorbed proteins were separated by gradual elution of NaCl: 3 ml/fraction and increment of 25 mM NaCl. Obtained fractions were analyzed by PAGE and in Ephrin-B2 binding assay.

H. Effect of PEGylation Conditions on sB4 Binding to EphrinB2

[0351]The effects of pegylating biotinylated sB4 under different pH conditions was determined. sB4 was pegylated at pH 6, 7 or 8, and the pegylated products were tested for binding to EphrinB2 as shown in FIG. 69. Ephrin2B binding activity was retained when PEGylation was performed at pH 6 and pH 7, but was partially lost at pH 8.

[0352]Additional combinations of parameters were tested, including temperature, pH and molar ratio of pegylation agent to sB4 protein, and the ability of the products of the pegylation reaction to bind to Ephrin-B2. The results of the optimization experiment are shown in FIG. 70. These results confirm the gradual decrease in B2 binding activity at basic pH.

I. Purification of Pegylated sB4 Species

[0353]Biotinylated sB4 protein was concentrated up to final concentration of 2 mg/ml using a 30 kDa MWCO ultra membrane. Sample was dialyzed O/N against coupling buffer: 30 mM phosphate, 75 mM NaCl, pH 8.00. Coupling to PEG was performed at 4° C. for 18 hours in 10 fold molar excess of reactive PEG. Double modified (PEGylated Biotinylated) sB4 was used on ion-exchange chromatography to separate non-PEGylated, mono-PEGylated and poly-PEGylated proteins from each other. Sample was dialyzed for O/N against 20 mM Na-Acetate, 20 mM NaCl, pH 5.5 and loaded onto 2 ml of SP-Sepharose. After washing with 10 ml of buffer, absorbed proteins were separated by gradual elution of NaCl: 3 refraction and increment of 25 mM NaCl. Obtained fractions were analyzed by PAGE as shown in FIG. 71. Fractions 1, 2 and 3 were found to correspond to polypegylated, monopegylated and unpegylated biotinylated sB4.

J. In Vitro Properties of PEGylated EphB4 Derivatives

[0354]Fractions 1, 2 and 3 of biotinylated and PEGylated sB4 from the SP column purification, corresponding to polypegylated, monopegylated and unpegylated biotinylated sB4, were tested for their ability to bind EphrinB2 using the standard assay. Results of this experiment are shown on FIG. 72. The order of binding activity was found to be Unpegylated>monopegylated>polypegylated.

[0355]The fractions were also tested for their stability in vitro. The fractions were tested for retention of EphrinB2 binding activity after incubation in mouse serum at 37° C. for three days. The results of this experiment are shown in FIG. 73. The order of in vitro stability was found to be monopegylated>unpegylated>polypegylated.

K. In Vivo Stability Analysis of PEGylated Derivatives of EphB4 Ectodomain in Mice

[0356]Fractions 1, 2 and 3 of biotinylated and PEGylated sB4 from the SP column purification, corresponding to polypegylated, monopegylated and unpegylated biotinylated sB4, were introduced by intravenous injection into mice in the amount of 0.5 nMoles/mouse. Blood from each mouse was taken in time frame of 10 min, 1, 2 and 3 days. 10 ml of obtained serum was used in binding assay with Ephrin-B2-Alkaline Phosphatase fusion protein and Streptavidin-coated magnetic beads as a solid phase. Signals, obtained at 10 min were taken as 100%. The two mice for each protein were of a different strain. Results are shown in FIG. 74. Pegylation was found to increase the stability of EphB4 in vivo relative to unpegylated EphB4.

INCORPORATION BY REFERENCE

[0357]All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0358]While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Sequence CWU 1

221570PRTUnknownRecombinant B4ECv3 protein 1Met Glu Leu Arg Val Leu Leu Cys Trp Ala Ser Leu Ala Ala Ala Leu 1 5 10 15Glu Glu Thr Leu Leu Asn Thr Lys Leu Glu Thr Ala Asp Leu Lys Trp 20 25 30Val Thr Phe Pro Gln Val Asp Gly Gln Trp Glu Glu Leu Ser Gly Leu 35 40 45Asp Glu Glu Gln His Ser Val Arg Thr Tyr Glu Val Cys Glu Val Gln 50 55 60Arg Ala Pro Gly Gln Ala His Trp Leu Arg Thr Gly Trp Val Pro Arg65 70 75 80Arg Gly Ala Val His Val Tyr Ala Thr Leu Arg Phe Thr Met Leu Glu 85 90 95Cys Leu Ser Leu Pro Arg Ala Gly Arg Ser Cys Lys Glu Thr Phe Thr 100 105 110Val Phe Tyr Tyr Glu Ser Asp Ala Asp Thr Ala Thr Ala Leu Thr Pro 115 120 125Ala Trp Met Glu Asn Pro Tyr Ile Lys Val Asp Thr Val Ala Ala Glu 130 135 140His Leu Thr Arg Lys Arg Pro Gly Ala Glu Ala Thr Gly Lys Val Asn145 150 155 160Val Lys Thr Leu Arg Leu Gly Pro Leu Ser Lys Ala Gly Phe Tyr Leu 165 170 175Ala Phe Gln Asp Gln Gly Ala Cys Met Ala Leu Leu Ser Leu His Leu 180 185 190Phe Tyr Lys Lys Cys Ala Gln Leu Thr Val Asn Leu Thr Arg Phe Pro 195 200 205Glu Thr Val Pro Arg Glu Leu Val Val Pro Val Ala Gly Ser Cys Val 210 215 220Val Asp Ala Val Pro Ala Pro Gly Pro Ser Pro Ser Leu Tyr Cys Arg225 230 235 240Glu Asp Gly Gln Trp Ala Glu Gln Pro Val Thr Gly Cys Ser Cys Ala 245 250 255Pro Gly Phe Glu Ala Ala Glu Gly Asn Thr Lys Cys Arg Ala Cys Ala 260 265 270Gln Gly Thr Phe Lys Pro Leu Ser Gly Glu Gly Ser Cys Gln Pro Cys 275 280 285Pro Ala Asn Ser His Ser Asn Thr Ile Gly Ser Ala Val Cys Gln Cys 290 295 300Arg Val Gly Tyr Phe Arg Ala Arg Thr Asp Pro Arg Gly Ala Pro Cys305 310 315 320Thr Thr Pro Pro Ser Ala Pro Arg Ser Val Val Ser Arg Leu Asn Gly 325 330 335Ser Ser Leu His Leu Glu Trp Ser Ala Pro Leu Glu Ser Gly Gly Arg 340 345 350Glu Asp Leu Thr Tyr Ala Leu Arg Cys Arg Glu Cys Arg Pro Gly Gly 355 360 365Ser Cys Ala Pro Cys Gly Gly Asp Leu Thr Phe Asp Pro Gly Pro Arg 370 375 380Asp Leu Val Glu Pro Trp Val Val Val Arg Gly Leu Arg Pro Asp Phe385 390 395 400Thr Tyr Thr Phe Glu Val Thr Ala Leu Asn Gly Val Ser Ser Leu Ala 405 410 415Thr Gly Pro Val Pro Phe Glu Pro Val Asn Val Thr Thr Asp Arg Glu 420 425 430Val Pro Pro Ala Val Ser Asp Ile Arg Val Thr Arg Ser Ser Pro Ser 435 440 445Ser Leu Ser Leu Ala Trp Ala Val Pro Arg Ala Pro Ser Gly Ala Trp 450 455 460Leu Asp Tyr Glu Val Lys Tyr His Glu Lys Gly Ala Glu Gly Pro Ser465 470 475 480Ser Val Arg Phe Leu Lys Thr Ser Glu Asn Arg Ala Glu Leu Arg Gly 485 490 495Leu Lys Arg Gly Ala Ser Tyr Leu Val Gln Val Arg Ala Arg Ser Glu 500 505 510Ala Gly Tyr Gly Pro Phe Gly Gln Glu His His Ser Gln Thr Gln Leu 515 520 525Asp Glu Ser Glu Gly Trp Arg Glu Gln Gly Ser Lys Arg Ala Ile Leu 530 535 540Gln Ile Glu Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser545 550 555 560Thr Arg Thr Gly His His His His His His 565 5702555PRTUnknownRecombinant B4ECv3NT protein 2Met Glu Leu Arg Val Leu Leu Cys Trp Ala Ser Leu Ala Ala Ala Leu 1 5 10 15Glu Glu Thr Leu Leu Asn Thr Lys Leu Glu Thr Ala Asp Leu Lys Trp 20 25 30Val Thr Phe Pro Gln Val Asp Gly Gln Trp Glu Glu Leu Ser Gly Leu 35 40 45Asp Glu Glu Gln His Ser Val Arg Thr Tyr Glu Val Cys Glu Val Gln 50 55 60Arg Ala Pro Gly Gln Ala His Trp Leu Arg Thr Gly Trp Val Pro Arg65 70 75 80Arg Gly Ala Val His Val Tyr Ala Thr Leu Arg Phe Thr Met Leu Glu 85 90 95Cys Leu Ser Leu Pro Arg Ala Gly Arg Ser Cys Lys Glu Thr Phe Thr 100 105 110Val Phe Tyr Tyr Glu Ser Asp Ala Asp Thr Ala Thr Ala Leu Thr Pro 115 120 125Ala Trp Met Glu Asn Pro Tyr Ile Lys Val Asp Thr Val Ala Ala Glu 130 135 140His Leu Thr Arg Lys Arg Pro Gly Ala Glu Ala Thr Gly Lys Val Asn145 150 155 160Val Lys Thr Leu Arg Leu Gly Pro Leu Ser Lys Ala Gly Phe Tyr Leu 165 170 175Ala Phe Gln Asp Gln Gly Ala Cys Met Ala Leu Leu Ser Leu His Leu 180 185 190Phe Tyr Lys Lys Cys Ala Gln Leu Thr Val Asn Leu Thr Arg Phe Pro 195 200 205Glu Thr Val Pro Arg Glu Leu Val Val Pro Val Ala Gly Ser Cys Val 210 215 220Val Asp Ala Val Pro Ala Pro Gly Pro Ser Pro Ser Leu Tyr Cys Arg225 230 235 240Glu Asp Gly Gln Trp Ala Glu Gln Pro Val Thr Gly Cys Ser Cys Ala 245 250 255Pro Gly Phe Glu Ala Ala Glu Gly Asn Thr Lys Cys Arg Ala Cys Ala 260 265 270Gln Gly Thr Phe Lys Pro Leu Ser Gly Glu Gly Ser Cys Gln Pro Cys 275 280 285Pro Ala Asn Ser His Ser Asn Thr Ile Gly Ser Ala Val Cys Gln Cys 290 295 300Arg Val Gly Tyr Phe Arg Ala Arg Thr Asp Pro Arg Gly Ala Pro Cys305 310 315 320Thr Thr Pro Pro Ser Ala Pro Arg Ser Val Val Ser Arg Leu Asn Gly 325 330 335Ser Ser Leu His Leu Glu Trp Ser Ala Pro Leu Glu Ser Gly Gly Arg 340 345 350Glu Asp Leu Thr Tyr Ala Leu Arg Cys Arg Glu Cys Arg Pro Gly Gly 355 360 365Ser Cys Ala Pro Cys Gly Gly Asp Leu Thr Phe Asp Pro Gly Pro Arg 370 375 380Asp Leu Val Glu Pro Trp Val Val Val Arg Gly Leu Arg Pro Asp Phe385 390 395 400Thr Tyr Thr Phe Glu Val Thr Ala Leu Asn Gly Val Ser Ser Leu Ala 405 410 415Thr Gly Pro Val Pro Phe Glu Pro Val Asn Val Thr Thr Asp Arg Glu 420 425 430Val Pro Pro Ala Val Ser Asp Ile Arg Val Thr Arg Ser Ser Pro Ser 435 440 445Ser Leu Ser Leu Ala Trp Ala Val Pro Arg Ala Pro Ser Gly Ala Trp 450 455 460Leu Asp Tyr Glu Val Lys Tyr His Glu Lys Gly Ala Glu Gly Pro Ser465 470 475 480Ser Val Arg Phe Leu Lys Thr Ser Glu Asn Arg Ala Glu Leu Arg Gly 485 490 495Leu Lys Arg Gly Ala Ser Tyr Leu Val Gln Val Arg Ala Arg Ser Glu 500 505 510Ala Gly Tyr Gly Pro Phe Gly Gln Glu His His Ser Gln Thr Gln Leu 515 520 525Asp Glu Ser Glu Gly Trp Arg Glu Gln Gly Ser Lys Arg Ala Ile Leu 530 535 540Gln Ile Ser Ser Thr Val Ala Ala Ala Arg Val545 550 5553233PRTUnknownRecombinant B2EC protein 3Met Ala Val Arg Arg Asp Ser Val Trp Lys Tyr Cys Trp Gly Val Leu 1 5 10 15Met Val Leu Cys Arg Thr Ala Ile Ser Lys Ser Ile Val Leu Glu Pro 20 25 30Ile Tyr Trp Asn Ser Ser Asn Ser Lys Phe Leu Pro Gly Gln Gly Leu 35 40 45Val Leu Tyr Pro Gln Ile Gly Asp Lys Leu Asp Ile Ile Cys Pro Lys 50 55 60Val Asp Ser Lys Thr Val Gly Gln Tyr Glu Tyr Tyr Lys Val Tyr Met65 70 75 80Val Asp Lys Asp Gln Ala Asp Arg Cys Thr Ile Lys Lys Glu Asn Thr 85 90 95Pro Leu Leu Asn Cys Ala Lys Pro Asp Gln Asp Ile Lys Phe Thr Ile 100 105 110Lys Phe Gln Glu Phe Ser Pro Asn Leu Trp Gly Leu Glu Phe Gln Lys 115 120 125Asn Lys Asp Tyr Tyr Ile Ile Ser Thr Ser Asn Gly Ser Leu Glu Gly 130 135 140Leu Asp Asn Gln Glu Gly Gly Val Cys Gln Thr Arg Ala Met Lys Ile145 150 155 160Leu Met Lys Val Gly Gln Asp Ala Ser Ser Ala Gly Ser Thr Arg Asn 165 170 175Lys Asp Pro Thr Arg Arg Pro Glu Leu Glu Ala Gly Thr Asn Gly Arg 180 185 190Ser Ser Thr Thr Ser Pro Phe Val Lys Pro Asn Pro Gly Ser Ser Thr 195 200 205Asp Gly Asn Ser Ala Gly His Ser Gly Asn Asn Ile Leu Gly Ser Glu 210 215 220Val Gly Ser His His His His His His225 2304771PRTUnknownRecombinant B4ECv3-FC protein 4Met Glu Leu Arg Val Leu Leu Cys Trp Ala Ser Leu Ala Ala Ala Leu 1 5 10 15Glu Glu Thr Leu Leu Asn Thr Lys Leu Glu Thr Ala Asp Leu Lys Trp 20 25 30Val Thr Phe Pro Gln Val Asp Gly Gln Trp Glu Glu Leu Ser Gly Leu 35 40 45Asp Glu Glu Gln His Ser Val Arg Thr Tyr Glu Val Cys Glu Val Gln 50 55 60Arg Ala Pro Gly Gln Ala His Trp Leu Arg Thr Gly Trp Val Pro Arg65 70 75 80Arg Gly Ala Val His Val Tyr Ala Thr Leu Arg Phe Thr Met Leu Glu 85 90 95Cys Leu Ser Leu Pro Arg Ala Gly Arg Ser Cys Lys Glu Thr Phe Thr 100 105 110Val Phe Tyr Tyr Glu Ser Asp Ala Asp Thr Ala Thr Ala Leu Thr Pro 115 120 125Ala Trp Met Glu Asn Pro Tyr Ile Lys Val Asp Thr Val Ala Ala Glu 130 135 140His Leu Thr Arg Lys Arg Pro Gly Ala Glu Ala Thr Gly Lys Val Asn145 150 155 160Val Lys Thr Leu Arg Leu Gly Pro Leu Ser Lys Ala Gly Phe Tyr Leu 165 170 175Ala Phe Gln Asp Gln Gly Ala Cys Met Ala Leu Leu Ser Leu His Leu 180 185 190Phe Tyr Lys Lys Cys Ala Gln Leu Thr Val Asn Leu Thr Arg Phe Pro 195 200 205Glu Thr Val Pro Arg Glu Leu Val Val Pro Val Ala Gly Ser Cys Val 210 215 220Val Asp Ala Val Pro Ala Pro Gly Pro Ser Pro Ser Leu Tyr Cys Arg225 230 235 240Glu Asp Gly Gln Trp Ala Glu Gln Pro Val Thr Gly Cys Ser Cys Ala 245 250 255Pro Gly Phe Glu Ala Ala Glu Gly Asn Thr Lys Cys Arg Ala Cys Ala 260 265 270Gln Gly Thr Phe Lys Pro Leu Ser Gly Glu Gly Ser Cys Gln Pro Cys 275 280 285Pro Ala Asn Ser His Ser Asn Thr Ile Gly Ser Ala Val Cys Gln Cys 290 295 300Arg Val Gly Tyr Phe Arg Ala Arg Thr Asp Pro Arg Gly Ala Pro Cys305 310 315 320Thr Thr Pro Pro Ser Ala Pro Arg Ser Val Val Ser Arg Leu Asn Gly 325 330 335Ser Ser Leu His Leu Glu Trp Ser Ala Pro Leu Glu Ser Gly Gly Arg 340 345 350Glu Asp Leu Thr Tyr Ala Leu Arg Cys Arg Glu Cys Arg Pro Gly Gly 355 360 365Ser Cys Ala Pro Cys Gly Gly Asp Leu Thr Phe Asp Pro Gly Pro Arg 370 375 380Asp Leu Val Glu Pro Trp Val Val Val Arg Gly Leu Arg Pro Asp Phe385 390 395 400Thr Tyr Thr Phe Glu Val Thr Ala Leu Asn Gly Val Ser Ser Leu Ala 405 410 415Thr Gly Pro Val Pro Phe Glu Pro Val Asn Val Thr Thr Asp Arg Glu 420 425 430Val Pro Pro Ala Val Ser Asp Ile Arg Val Thr Arg Ser Ser Pro Ser 435 440 445Ser Leu Ser Leu Ala Trp Ala Val Pro Arg Ala Pro Ser Gly Ala Trp 450 455 460Leu Asp Tyr Glu Val Lys Tyr His Glu Lys Gly Ala Glu Gly Pro Ser465 470 475 480Ser Val Arg Phe Leu Lys Thr Ser Glu Asn Arg Ala Glu Leu Arg Gly 485 490 495Leu Lys Arg Gly Ala Ser Tyr Leu Val Gln Val Arg Ala Arg Ser Glu 500 505 510Ala Gly Tyr Gly Pro Phe Gly Gln Glu His His Ser Gln Thr Gln Leu 515 520 525Asp Glu Ser Glu Gly Trp Arg Glu Gln Asp Pro Glu Pro Lys Ser Cys 530 535 540Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly545 550 555 560Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 565 570 575Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 580 585 590Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 595 600 605His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 610 615 620Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly625 630 635 640Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 645 650 655Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 660 665 670Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 675 680 685Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 690 695 700Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro705 710 715 720Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 725 730 735Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 740 745 750His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 755 760 765Pro Gly Lys 7705459PRTUnknownRecombinant B2EC-FC protein 5Met Ala Val Arg Arg Asp Ser Val Trp Lys Tyr Cys Trp Gly Val Leu 1 5 10 15Met Val Leu Cys Arg Thr Ala Ile Ser Lys Ser Ile Val Leu Glu Pro 20 25 30Ile Tyr Trp Asn Ser Ser Asn Ser Lys Phe Leu Pro Gly Gln Gly Leu 35 40 45Val Leu Tyr Pro Gln Ile Gly Asp Lys Leu Asp Ile Ile Cys Pro Lys 50 55 60Val Asp Ser Lys Thr Val Gly Gln Tyr Glu Tyr Tyr Lys Val Tyr Met65 70 75 80Val Asp Lys Asp Gln Ala Asp Arg Cys Thr Ile Lys Lys Glu Asn Thr 85 90 95Pro Leu Leu Asn Cys Ala Lys Pro Asp Gln Asp Ile Lys Phe Thr Ile 100 105 110Lys Phe Gln Glu Phe Ser Pro Asn Leu Trp Gly Leu Glu Phe Gln Lys 115 120 125Asn Lys Asp Tyr Tyr Ile Ile Ser Thr Ser Asn Gly Ser Leu Glu Gly 130 135 140Leu Asp Asn Gln Glu Gly Gly Val Cys Gln Thr Arg Ala Met Lys Ile145 150 155 160Leu Met Lys Val Gly Gln Asp Ala Ser Ser Ala Gly Ser Thr Arg Asn 165 170 175Lys Asp Pro Thr Arg Arg Pro Glu Leu Glu Ala Gly Thr Asn Gly Arg 180 185 190Ser Ser Thr Thr Ser Pro Phe Val Lys Pro Asn Pro Gly Ser Ser Thr 195 200 205Asp Gly Asn Ser Ala Gly His Ser Gly Asn Asn Ile Leu Gly Ser Glu 210 215 220Val Asp Pro Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro225 230 235 240Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 245 250 255Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 260 265 270Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 275 280 285Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 290 295 300Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val305 310 315

320Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 325 330 335Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 340 345 350Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 355 360 365Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 370 375 380Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu385 390 395 400Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 405 410 415Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 420 425 430Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 435 440 445Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455626000DNAHomo sapiens 6ggggtttcat catgttggcc aggctggtct tgaactcctg acctcaaatg atccgcctgc 60ctctgcctcc caaaatgctg ggactacagg cgtgagccac cgcgcccgcc acacccacct 120tttctttacc gttgtttcct cgatttttct ctactcccta gcgcagctta gtgcgcgcct 180cctctggaca tttttcaggg cttggttgcg cgcacagtag gtccccaaca ctgaatgttt 240atggggtgac tgtgtgaacg ttcgctgcaa ggctatccaa actgggattg ctccttgagg 300ccccctgggc ggccgtcaat tctccaaagc ttctactccc ttttccttcc ttttccccca 360aaacgcagtc cctgcgccca ctagagggtg gtgggcgcat ccaagagcgg catctagagt 420ccgcagcaag gtcagagcgg gctttgtgtg cgcggtgaac atttacgtgc acgcctgggc 480ggccctccgt gttgctgctg ggtgtgtgtt ttctctgctc cctggtgcca gccgggttcg 540ggcctgtccc gggggtccct gggccccagc cccgacatgc tcggtcctgg acagcgcgca 600ccgccacggc gcacatctgg gcggtcccgg ggttcctcac ccgccgcccc tcccccttct 660ccaaactttc tctcaacttc ccgacctgct ccactcggtg cccctctccg cttccctcat 720gaattattca gtagcgtgag ctccaatcag cgcgcccggg gctcactcgc ggagcccccg 780cgttgggaga gctgcccccg ccccccgcgc gcccctccct cccgggcccg gcgccgcccg 840gcccagttcc agcgcagctc agcccctgcc cggcccggcc cgcccggctc cgcgccgcag 900tctccctccc tcccgctccg tccccgctcg ggctcccacc atccccgccc gcgaggagag 960cactcggccc ggcggcgcga gcagagccac tccagggagg gggggagacc gcgagcggcc 1020ggctcagccc ccgccacccg gggcgggacc ccgaggcccc ggagggaccc caactccagc 1080cacgtcttgc tgcgcgcccg cccggcgcgg ccactgccag cacgctccgg gcccgccgcc 1140cgcgcgcgcg gcacagacgc ggggccacac ttggcgccgc cgcccggtgc cccgcacgct 1200cgcatgggcc cgcgctgagg gccccgacga ggagtcccgc gcggagtatc ggcgtccacc 1260cgcccaggga gagtcagacc tgggggggcg agggcccccc aaactcagtt cggatcctac 1320ccgagtgagg cggcgccatg gagctccggg tgctgctctg ctgggcttcg ttggccgcag 1380ctttggaagg tgagtttcct tgcggggggg ggcgcacccc gtcactcctg ggacctcccc 1440cccaacatct gggcctcgga gtggaggggc cggcctctga ctacccctac ccgggcactg 1500cagtcccaaa cacttcggac cgatagtgct ggaacgggag gggggcgggg aagaggcgcc 1560cgacgggtag tggagttttc ttttgtttgg gaaagagatg gagtctggct acgacccggg 1620acattcccct gcccgggctc cccgaactct cactgctgat tacatacgcc cctggctgcc 1680tttcctttcc tccctacccc actattcaaa actatctgca aagtttctgt cccagtccca 1740cctcccgccg tacatgaggg aaggtttctg gagaagcaac agcagacaag gcacaacttt 1800tcgtgctagg ccctaaaacg acccccagcg ccaattcctt agcgatcaca ccttgatcct 1860ccagttccac actcctgcaa caggatggcc tcctttgcat tcacacagca aacccccaaa 1920ccgctctccc gcccactgct cctgcccctg gtatagggtg gctccttggt ttctacaggc 1980tgcaccccat ccctttaaat gcggtctaga ccccggcccc aggtgagtcc cgggcttccc 2040ttgagaccta ggagcgggta gaaactgacc tacacagccc ccaggtagaa actgacctac 2100acagccccca catcgcccta actaacccag tctatctccc acctcctggt ctctccaagc 2160atttctttgg ccatggatcg ctgtccctcc tggtccccta aagggggagc caagagccct 2220agaaactctc ctgtgtccct aatgtccttt cagtgagctg ccaacacccc cctttctctg 2280tctggtatga aagtggttat ggggcggtag gctatgaggg actcccaaag ggaaggattc 2340agcggcgtta gaaaaaccct ctccccctgg ctgggcagga ctgccctggg ctggggatca 2400aaggctaggt gtggggttgg gagtgagggg aggcttgccc agctcagaga acggagaagg 2460gggaacaaaa accatgaacg aggggaagag gaaggccaaa ggggtggaaa aaccacgagg 2520acgaggtgtg gtgagaagga aagacgcaaa gaggaaatgg tgattgtgac acctattacc 2580tgagtgtttc caagcaccag gcctgtgctg agcgccttac aaatattaat ttcacccatc 2640cagcaacgct aagggtggtg ctattattgc ccccattttt cagatgagga ggctggggct 2700tagttaaggt taagtagttt atccaaggcc ctgtgccgcg aggaacagcg agaagtggag 2760gccgaaagcg aaggagagat agtgactgtc agaaagagaa acggaggtgg acagagagtg 2820gaggagagat aggtgagaga catgcgaact gacagatcaa agcgtggctg cagctgagct 2880gggacgcaga aagggagcct gcgcttgctc tgggctgcgg acagcccgag gcagagacag 2940tgtgtaaatt ggagacagga aaacactatc ccggctggaa caatggaggg tggagacggc 3000agcctctatc cacccccttc ccagaacccg ggcatcctgt ccccagtgag cagggctgtc 3060tcttgccacc catggggacc ttgcgcctct cacctcaggc tggctggctt cccatctgac 3120ccctagctgg aggacatcat ttggtcccca ggaagaggct gcctcaccca ccctctttct 3180cttctctcct gcagctccca tggggtggga gccaggtgtt ctggctcccc tctccaccct 3240tcccagcgcc caatgccccc cacattgccg gcccccgagg ggattcctgt accctccctc 3300ctccactctc cactgccagg ggctgtgcag tttttcctaa tcccccccct tcctccagtg 3360cctgtcccct cccccgatga tccgagccaa gccaggtgtg ttcacccctc ccattcatac 3420cgccccccag aatctcctcc cctctgcctt cccataacca aatccagatg tgaggcctcg 3480gcgggagcct gggaacccta gcatcccgac ctccagtgct tcctgatcag ggcactcgtg 3540gggagggagg tactgggatg ggggccaggg ctatgcccca ggcacggagc gctcccttca 3600aggagggaag gacggggtgt ttggtctgaa agcagagagg ggtcttggac agggaatgaa 3660attgtggggt agagaggctg attctgggac ttaggggagg aaacgtggag gctgagacaa 3720gaggttcccc tcccacacca gcagcctctg ctcgtggggg tcaggaccag ggcgcagctc 3780tcattttaac cctttctgag ctgccgcccc ttctccccgt acattttgat ctccctccct 3840cctccaggga ggcctagatc tggggtatcc caagggagcc ccatgcctac cagatgttgg 3900gggtggggtt ggcacttagc agaagaggcc agaaatcagg cgggtgcaga gggcagggct 3960tgctcccctc ttggcccccc aactcctcta gctcagagct aagaggatcc acctgcctcg 4020gttcccaggg atctggtctt cctgacctcc ctcccccacc ccaggcactg actctgtctc 4080tctgtctgtc tcagagaccc tgctgaacac aaaattggaa actgctgatc tgaagtgggt 4140gacattccct caggtggacg ggcaggtgag agctgcaccc aggagctgga gctctggagg 4200gaaactgagg gaggagaggg cgcctgtgcc gcctgctttc tgtgtgccac tcctctcccc 4260tgtcccccca gatgacagca gccccagcag tgtcgtctga gcccttctca gaggcgccct 4320cctcgcagta ccagcagccc ccctttctca gtccctctca ctttatagga ttcaccccat 4380gcagccctct ccctggcggc tccccagccc ccttgctgac ctccttctct gcacagtggg 4440aggaactgag cggcctggat gaggaacagc acagcgtgcg cacctacgaa gtgtgtgacg 4500tgcagcgtgc cccgggccag gcccactggc ttcgcacagg ttgggtccca cggcggggcg 4560ccgtccacgt gtacgccacg ctgcgcttca ccatgctcga gtgcctgtcc ctgcctcggg 4620ctgggcgctc ctgcaaggag accttcaccg tcttctacta tgagagcgat gcggacacgg 4680ccacggccct cacgccagcc tggatggaga acccctacat caaggtacct gggtgccccc 4740agggctcagc cacagccaag gtgggattcc agccagcagg cccgtggcct ggagggcagc 4800cgatgtagtt gcgaggcctc tggcccgcgc gctgggggct ggaagcagga ggcttaggtc 4860tggggaggga agggggtgat cttctgggcg gaggagcaga atatacgggg gctgcctggc 4920ccggccccca gggaggccca agggtcaggc ttctcctcca gtcacctcaa ccaccctacc 4980ccactgtgct ccagccacac tgagtttctc ccattccctg actgcacctg gctggtttcc 5040agctcaagac tttgcagcgg tgatgtctcc acctgggggc ctctctgcct ctcacacccc 5100tacttgtctt cggagttcca gctcccgaga tcttgcctgt gccaccttgg ctgactctct 5160cctccctaca atcctgcata cctctgtcca cctgcctgtc tcggcactca ttttacttta 5220tttatttttc ttttatatct atatttttaa agcggggtct tctacgttac ccaggctggt 5280ctctaactcc tgggctcaag agatttctcc cacctcggcc tcctaaagtg ctgggattat 5340aggcatgagg cactacgccc ggcctcatgg tactttataa cttccccagg attcattcat 5400cgctgtctcc ttgactctga ggtcaaggcc tggcatggcg tcagtgtcag taaatgtttg 5460tagaacgagt gaataaaaag ggggagaggt gcaggccaga ggccgggcat atcgcaggag 5520ctttgcaagg ctgaatggac agtgtggggg cctgcagaaa gtgtgccctg gggaaggtgg 5580agggaagatt ctggaacggg aaccaaggag gtccgggagg gtgagctggg aagaacacaa 5640cagtccgctg ggtcctcagg gagtggggac agcagcggtg tgcctccccc ccgccggcag 5700gtggacacgg tggccgcgga gcatctcacc cggaagcgcc ctggggccga ggccaccggg 5760aaggtgaatg tcaagacgct gcgtctggga ccgctcagca aggctggctt ctacctggcc 5820ttccaggacc agggtgcctg catggccctg ctatccctgc acctcttcta caaaaagtgc 5880gcccagctga ctgtgaacct gactcgattc ccggagactg tgcctcggga gctggttgtg 5940cccgtggccg gtagctgcgt ggtggatgcc gtccccgccc ctggccccag ccccagcctc 6000tactgccgtg aggatggcca gtgggccgaa cagccggtca cgggctgcag ctgtgctccg 6060gggttcgagg cagctgaggg gaacaccaag tgccgaggtg agagctggag cttcccctgc 6120gactgctgct catccggggg agagtcctga actccactca ggacccactt cttaagtttc 6180cattttgtat agttagatgt tgaaatggag gcttgctctg tcacccaggc tggagtgcag 6240tggcacaatc tctgctcaac tgcaaccttt gcctcccggg tccctgttca agcagttctc 6300ctgcctcagc ctcgtgagta gctgggacta caggcacacg ccaccacgcc cggctaattt 6360ttgtatttta gtagagacgg ggtttcgcca tgttggccag gctggtctcg aactcctgac 6420ctgaagtgat ttgcccgcct cggcctccca aagtgctggg attacaggcg tgcgtcacca 6480cacccagctg gaaaaaaaaa agactttatt ttcacctgaa attcattaat ttccacttga 6540aattccacct gcagttgtag caggacctga cacttgggcc ccatggaaat cacaggtatt 6600gcctgacaca gtggttcatg cccatagtgc cagcactttg agatgccaag gtgggaggat 6660cacttgagcc caggagttcg agatcagcct gggtgacaga gcaagacccc gtctctaaaa 6720aaaatttttt tttttttttc aagacagagt cttgctctgt cgcccaggct ggagtgcagt 6780ggtgcgatct cggctcactg caagctccgc ctcccaagtt aacaccattc tcctgcctca 6840gcctcccgag tagctgggac tacaggcccc gccaccacgc ccggctaatt tcttgtattt 6900ttagtagaga tggagtttca ccgtgttagc caggatggtc tcgatctcct gacctcatga 6960tctgcccgcc ttggcctccc aaagtgctgg gattacaggt gtgagccacc acacccggat 7020tacaaaaact ttttagataa ttatctgggc gacctgcctg accaacatgg agaaaccctg 7080tctctactaa aaatacaaaa ttagccggac atggtggcgc atgcctgtaa tcccagctac 7140ttgggaggct gaggcaggag aatcatttga acccaggaag cagaggttgc ggtaagccga 7200gatcatgcca ctgcactccg gtctgggagt gcactccaac aagaaggagt ttcgctcttt 7260ttgcccaggc tggagtgcag tggtgggatc tcagctcacc gcaacctcca cctcccgggt 7320tcaggcgatt ctcctgcctc agcctcccaa ggagtagctg ggattatagg tatgcatcgt 7380cacacccggc tacttttgta tttttagtag aggcaggttt ccaccatgtt ggccaggctg 7440gtcttgaact caagtgatct gccctctttg gcctccttct caggaaaaaa aaaaaatcac 7500aggtatttac aggccattcc aagtgccaaa agattgtttt tgctcatggt gacttcagta 7560tcacagatgt taggagactt gctgctatat gttaagaaag aagcacaaat gttgctgtag 7620cccaaacttt tttcctcatg tttcattgca tttcagctta attggtttcc ctggtattcc 7680tatgtatttt gtggagtgct tttaaaatca taagttggag tagaggtctt tctgtgggct 7740tcaccagact gccgagatca gggtcgaaac aggtgaggac cccttctctg gagagagtct 7800cctttctcct ctaagaggaa aggttttgag atcttttgtc cattttccca ccttagcact 7860tcatcagcct taaaagaagc tggaattttt tttttttttt ttggagatgg gatctcgata 7920tgttgcccag gctggtcttg aaccccttgg ctcaagcgat cctccagcct cagcctccca 7980aagtgctggg attcgaggca tgagccaccg agcccaccgt gcagatggat gtttttgtgc 8040atgcttttga tgaatgcttt ctctctctca gcctgtgccc agggcacctt caagcccctg 8100tcaggagaag ggtcctgcca gccatgccca gccaatagcc actctaacac cattggatca 8160gccgtctgcc agtgccgcgt cgggtacttc cgggcacgca cagacccccg gggtgcaccc 8220tgcaccagta agtgaccagc acccaggtgc agttcactgg ggaggggtca cagacctctg 8280aggtggaccc tcacatggcc cccatcctcc ctgggcttct tccctttgtc cctggcatgc 8340ttgtccctag cccggaggaa catgtggagc ccactgtctc caaggcaaga gtccagcatg 8400gctgctggtg cctccattgc cctctcccca ccaccgcaga gcaggtcggc ctctgcctga 8460ctccctggtc tcctgcagcc cctccttcgg ctccgcggag cgtggtttcc cgcctgaacg 8520gctcctccct gcacctggaa tggagtgccc ccctggagtc tggtggccga gaggacctca 8580cctacgccct ccgctgccgg gagtgccgac ccggaggctc ctgtgcgccc tgcgggggag 8640acctgacttt tgaccccggc ccccgggacc tggtggagcc ctgggtggtg gttcgagggc 8700tacgtcctga cttcacctat acctttgagg tcactgcatt gaacggggta tcctccttag 8760ccacggggcc cgtcccattt gagcctgtca atgtcaccac tgaccgagag ggtgagactt 8820gggggctggg gcggctggtg gtctggcggg agagatgtca ctgagggcct gaaggggaga 8880ggcaggggct gtgaagttgg gtaccccgga agtgtgaggg gctaaggctt tgggggcaag 8940aggcagaaag agggcaatgg ctgggcgcag tggctcacgc ctgtaatccc agcactttca 9000gaggctgaga caggcggatc acttgagccc tggagttcaa gaccagcctg ggtaacatag 9060gaagatctct ctacaaaaaa taaaaatatt agccaggcga ggtggtgcat gcctgtggtc 9120ccagctactc aagaggctga ggcaggagga ttgcttgagc ccaggagtcg gaggctgcag 9180tgagctatga tcgcaccgct gcatgccagc ctgggtgaca gagcagtgtg agatcctctc 9240tcaaaataaa tgaataagaa agagagggtg aggagctcgt aaagctgggc tggagagtta 9300agtacaggaa ggcccccagt gggactgggg ccagagagaa tcagaaggaa ttctcgaaac 9360agccaggggg aaattgagac aagtgtagcc agcagaggaa gtgttggaaa agataaggga 9420catggccagg ctgatcacaa ggtcaggagt tcaagactag cctggccaac gtggtgaaac 9480cccatgtcta ctaaaaataa aaaaattagc caggcatggt ggtgggcacc tgtaatccac 9540ttgggaagca accagaagaa ttgcttgaac ccaggaggcg gaggttgcag taagctgaga 9600ctgcgccact gcactccagc ctgggtgata gagcacgact ccgtctcgaa aaaaaaaatt 9660ttttttaagt taagggacag agctaccatg cacaagggtt ccctgtgtct ctgcctctca 9720cagtacctcc tgcagtgtct gacatccggg tgacgcggtc ctcacccagc agcttgagcc 9780tggcctgggc tgttccccgg gcacccagtg gggctgtgct ggactacgag gtcaaatacc 9840atgagaaggt aaggccatcc cccagccctg gggtgggtgg gcaatgggtt gtgctctcct 9900ggctgggaca cctgggttgc aggcacctgg caggcatttg aattccagct ctgccatgga 9960ttccctgggc agccttgggt aagccccttg gcctgtctga gcctcagact cttcatctat 10020aaaatagtta ctgtaatagt taccagcagc tggacacagt ggctgaggtt gggtgcggtg 10080gctcacgcct gtaataccaa gcactttggg aggctgaggc gggcagaatg cttgagccta 10140ggagtttgag accagcctgg gcaacatggt gaaacttcat ctctataaaa aacttaaaat 10200gggccgggcg cggtagctta cgcctgtaat cccagcactt tgggaggccg aggtgggcgg 10260atcacaaggt caggagtatc gagaccatcc tggctaacac ggtgaaaccc catctctact 10320aaaaatacaa aaaattagcc aggcgcggtg gcaggcgcct gtagtcccag ctactcggga 10380ggctgaggca ggagaatggc gtgaacccag gaggcggagc ttgcagtgag ccgagatagc 10440gccactgcag tccggcctgg gcgaaagaac aagactctgt ctccaaaaaa aaaaaaaaaa 10500aaaaaaaacg caaaaaatac ttaaaatgaa aaaaattaga ctgggcacag tggctcatgc 10560ctgtaatccc ggcactttgg gaggccgagg tgggtagaac acctggggtg aagagttcga 10620gaccagcctg gccaacaagg tgaaatcccc gtctctacta caaatagcaa aatcagctga 10680gtgtgttggc gggcccctgt aatcccagct actcaggagg ctgagacagg agaatcactg 10740gaacccaagt gattctcgac ttgaggtcga ggctgcagtg agtcgtgttt gcaccattgc 10800attccagcct gagaaagtga gaccttgtct taaaaaaaag gaatgatatt atgaatacag 10860cacatggctt gcatgcgtaa gttctcccaa aggcctcacc agttgcaagg caggctagtg 10920atgggagtgg agggcgaggg aaggaggcag gaagagcaac aggaacttgg gttcccgggt 10980gacggccacc ccactacctc tcccggacag ggcgccgagg gtcccagcag cgtgcggttc 11040ctgaagacgt cagaaaaccg ggcagagctg cgggggctga agcggggagc cagctacctg 11100gtgcaggtac gggcgcgctc tgaggccggc tacgggccct tcggccagga acatcacagc 11160cagacccaac tggatggtga gcctggggaa gggggtgagg gtgggggttg gaaagacccc 11220caaagttcct gggaagaccc caggtctcca aagtcccatc atcttttttt tttttttttt 11280tttttgagat ggagtcttgc tctgtccctc aggctggagt gcagtggcac catctccgct 11340cactgcaacc tccgcctccc ggattcaagc cattctcctg cctcagcctc ccgagtagct 11400gggattacag gcgcctgcca ccgcgcctgg ccgatttttt gtatttttag tagagacggg 11460gcttcaccgc gttggccagg ctggtctcga actcctgacc ttgtgattcg cccgcctcgg 11520cctcccgaag tgctgggatt acaggcatga gccactgcac ccggtcaaag tcctatcttc 11580atgtccttct tcctgtggat cacatggcat gccctagaga ggagagaacg taagatgtcg 11640aaaccaaaac caacagctga gttttgtgaa gtctggcctg cttcactctg tacccccagg 11700ctggagcgca gttgctcgat caaagctcac tgcacagcca ggcacagtgg ctcaccctgt 11760aaccccagca ctttgggagg ctgaagcagg aggatcactt gaggtcagga gttcgagacc 11820agtctgacca gcatggtgaa accgcgtctc tactaaaaat atagaagtta gctgagcgtg 11880gtggtgcaca cctgtaatcc cagctactcg ggaggctgag gcaggagaat cgcttgaacc 11940tgggaggtgg aggttgcagt gagctgagat tgtgccagtg cactccagcc tgggcaacag 12000agcaagactc tgtctcaaaa aaaaaaaagc tcaccgcagg cttgactttt agcaacaacc 12060tgacccctga gctccccatt ccccatccaa caaaatggga atatcatgaa gcttcctgca 12120gggctttgag gattggaggt aacaggttat ttttaatatg ctaggccagt ggctttcttt 12180tttctttcac attttttttt ttgagacgga gtctcactct gttgcccagg ctggagtgcg 12240gtggcgcgat ctcagctcac cgcaagctcc acctcctggt ctcgatctgc tgacctcctg 12300atccacccgc ctcggcttcc cgaaatgctg ggactgctgg cgtgagccac cacgcccggc 12360ctaacttttt ctttttttta agagacacgg tcttttttat cacccaggct ggagtgcggt 12420ggcaccatca tagctcattg cagcctacaa ctcccgagct caaccaatcc ttccacctta 12480gcctcccaag tagctggggc tataggcatg tgctaccgtg ctcaactaaa ttttttttta 12540tgttttgttg agacagtttc cctatgttgc ccaggctggt ctcaaattcc tgacctcgag 12600caatcctccc gcatcggcct cccaaagtgc tgggattaca ggcatgagcc gccacaccca 12660gcattggacc agtggctttc taaaccttgt aattttctgt aatagcttta ctgaaataca 12720gttcccctgc catacaattt gcctgttcaa agtgtacaat cgatgacttt tgatacattc 12780acagaattgt gcagtcacca ccacaagtaa ttttgggaca ttttcagcac cctcaaaaga 12840gaccctatag cccttagcca tcacccccca cccagatctt tctgttgcct tagtccctgg 12900caagcactaa cccactttct gtcttgaaat cttccagtgt ggtcttttgt gactgttcac 12960cgagcagaat gttttcaagg tttatgtatg ttgtagtata tatccgtggg tttttttggt 13020tgtggtttgt tttttgtttg ttttggaaac agggtctcgc tctgtcaccc aggctggagt 13080gcagtggttc aattacagct cactgcagcc tcaacctccc aggctcaagt gatcctccca 13140cctcagcctc ccaagcagct gggactgtag gcatgagcca ccatgcccag ctaatttttt 13200ttggtatttt ttgtaaagac agggtttcac catgtttccc aggctggtct cgaactcctg 13260agctcaggca atccacccac ctcagcctcc caaagtgctg tgattacagg catgagccac 13320tggacctggc ctgttttttg tttttgtttt gaacacacga ttttgctttg tcacccaggc 13380tggaatgtaa tggtctgatc atagtgcatt gcagcctcaa actcctgggc tcaagcgatc 13440ctcctacctc agcctcctga gtatctggga ccacacgtgc tcaccaccat gcttggctaa 13500ttattattat tttttgatag agacggggtc ttgctatgtt tcccaggctg gtcttgaaca 13560cctggcctca cacaatcctc ccacctcagt atctcagagt gctgggatta caggcatgag 13620ccactgctcc tggccaatat ttcatttctt tttatggaga cgtaataatc agttgtatgg 13680aaatagctga ttttgttttt tattgtatct tttggtgaac atttcaattg tatcgacttt 13740ttggataaaa acctgaaaat gtttcacctt tagaacgttt cattgaatgg agattttttt 13800gtggactctg gtatttatac tagaaccaaa tcaaaaccac tctggcggct gggcatgcct 13860aggctggttt gagactagcc tgtccaacct ggtgaaagcc catctctact aaaaatacac 13920aaattagccg agcatggtgg tacacacctg taatcccagc tactcaggag gctgaggcag 13980gagaatcgca gaacccggga ggcggagatt gcagtgagct gagattgcgc cactgcactc 14040cagcctgggc gacagagtga gactgcgtct caaaaaaaca aacaaaaaat tactctggca 14100gtaagaaaag atttcgaaac ttcctccctt gccctgaggt acttcagagg agcctgctgg 14160cccctggggg agagtttgaa acccactgtt tgttccctga

ccttgcctgc ttgtgtcctc 14220tccctccacc tgtcccctgt actggggacc tgttctcagg agatcacagt tcattgctca 14280aagccggggc tggggcctcc tacaggacca tcagtttctc ctgatcagca gcctttcctt 14340ccgcagagag cgagggctgg cgggagcagc tggccctgat tgcgggcacg gcagtcgtgg 14400gtgtggtcct ggtcctggtg gtcattgtgg tcgcagttct ctgcctcagg taagggctct 14460gacacccaga ggcccctgga agccctcagt tgatggccac ctgcctgggt gctacaggac 14520aagcctttct ggctgtcccc agcctctttt tacttgaaat cttctccaat ccctgctcct 14580tcctttggtg tgtgtgcctc ataaagatgt gtgactcagt ttaccttttg ttcctttccc 14640atcggctaca ggaagcagag caatgggaga gaagcagaat attcggacaa acacggacag 14700tatctcatcg gacatggtgg gttgccctaa tttgatggga ataggggctt ggggccgggt 14760gtggtggctc ctatctataa tcccagcact ttgggaggca gaggtgggca gatcacttga 14820ggtcaggagt tcgagaccag cctggccaac atgttgaaac tccatctcta taaaaaatac 14880atcagtcagc caggcatggt ggtgggcacc tgtaatccca gctactcagg aggctgaggc 14940agaagaatca ttttaacccg ggaggcggag attgcagtga gccaagatcg cgccactgcg 15000ctccaggcct gggtgacaga gcgagactcc atctcaggaa aaaaaaaaaa aaaaaaaaaa 15060accacggaga caggggtttg gggctaaaag ctatgagccg agcctccgag tccagtggga 15120gttaattccc agctgacggg gccctgcctg atttctcagg tactaaggtc tacatcgacc 15180ccttcactta tgaagaccct aatgaggctg tgagggaatt tgcaaaagag atcgatgtct 15240cctacgtcaa gattgaagag gtgattggtg caggtgagag ccgaaggctg cccgggcacc 15300tgggaacgaa gcgggggtgg gcagggccac actggagcgg gagagctgat gacctctgcg 15360tccttgtttg aaggtgagtt tggcgaggtg tgccgggggc ggctcaaggc cccagggaag 15420aaggagagct gtgtggcaat caagaccctg aagggtggct acacggagcg gcagcggcgt 15480gagtttctga gcgaggcctc catcatgggc cagttcgagc accccaatat catccgcctg 15540gagggcgtgg tcaccaacag catgcccgtc atgattctca cagagttcat ggagaacggc 15600gccctggact ccttcctgcg ggtgagcacc ctccctggct tctgcggcca cccggagttc 15660ccacttacac ccagaggcca cttgggttaa gaagccagga cagacagtgg gtcccaggtc 15720acctcctcca gccttttcct cttgggctaa gccctggtcc tctgcctttt ctttttttta 15780agacagagcc tcgctctgtc gcccaggctg gagtgcagtg gcgcgatctc ggctcattgc 15840tgtctccacc tccagggttc aagcgattct cctgcctcag tctcccaagt agctggtact 15900ataggcatgc accaccatgc tgactaattt ttgtattttt agtagacaca gggtttcacc 15960atgtaggcca ggctggtatc aaactcctga cctcaagtga tctccccacc tcagcctccc 16020aaagtgctgg tattacaggt gtgaggcacc acgcctggcc agccctctgc ctttaatttt 16080ccctctggga aaggctgggc tcctgggacc ttcctttccc actgccccat acagctgaag 16140gttgtcattc cttctttttt tttttaattt tgttttaatt gaattttttt tttttgagat 16200ggagtttcac tcttgttgcc caggccggag tgcaatggca agatcttggc tcaccgcaac 16260ctccgcctcc caggttcaag cgattctcct gccttagcct ccccagtagc tgggattata 16320ggcatgtgcc accacgcttg actaattttg tatttttagt agagacgggg gtttctctgt 16380gttggtcagg ctggtctcga actcccgacc tcaggtgatc cgcctgcctc ggcctcccaa 16440agtgctggga ttacagacgt gagccaccgc gcccggccaa tttttttttt ttttttttaa 16500gacagagtct cactctgtcc tctaggctgg agtgcagtgg tgcattcata gctcactgta 16560gccttgacct cctgggctca agtgatcctc ccgcctcagc ctcctgagta gctggaacta 16620cactcatgta ccaccatgct cagcaaattt ttaaaatttt ttgtagagac aggatctcga 16680taggttgccc aggctggtct gaactcctgg cctcaagcga gcctccctcc tcagcctccc 16740acagcactgg gattgcaggc atgagccact gtgcctggcc tgtcattcct tcttttgaca 16800aatatttact gagtgctttc tacgcaccgg tcatcctccc agtccccagg aataaagcta 16860tacacacggc aaactggatt tctcctcttg gggagcagag ggtctaatgg ggcaggggga 16920ctgaaaatta gcaagtaaat agacaggctt tttaaaaaag taaacaaatc atttcaaatg 16980tgaaaaaaag caaacggggt ccttcatgca gatgtggcta gagaggaaag agaactgctt 17040aatttatttg gtcactttac cagattttac tgactttttt ttttttttta actttattaa 17100gcttttcttt tttcttgaga tggagtttcc atctgtcacc caggctggag tgcagtggtg 17160cgttcttggc tcaccgcaac gtccacctcc tgggttcaag tgattctcct gcctcagcct 17220cctgagtagc ttggaattgc atggcatgca ccaccatacc cagctgatgt ttgtattttt 17280agtagagaca gggtttcatc atgttgccca ggctggtctt gaactcctgg gctcaagtga 17340tccacccatc tcggcccctc aaagtgctgg gattacaggc atgagccacc atgcctggcc 17400taggcatctt tttaaaaaaa tcaaaacatt tttctatgta gcaaaataac attgcattga 17460acagagttat agcgattccc tagcgtcatt gaatacccag ttgattttca cgtttctcta 17520gttgttctaa agatgtcctt cactgctgct ttattccaac caggatccag ttcaagaccg 17580ggctttgtac ctggttatta tatatatttt atttatttat tttagaaaca aggtcttgcc 17640ctttcgccca gtttagagtg cagtggtgca atcatagctc actgcagcct ccaaactcct 17700tggctcaggt gatcctcctg cctcagcctc ctgggtagct ggaactacag gtgcacacca 17760ccacacctgg ctaattttta aattttttac ggagatgggg gtctcgctat gttgcccagg 17820ctggtctcaa actcctggac tcaagcgatc ctccctcctt aacctctcaa agtgctggga 17880ttacaggcgt gagccaccac gcctgctgat tattatattt tcgagcctct ctaaatcttg 17940agcagttcct catgatgaca ctgacacact gaagggttag gtcccttgtc cgcctgaatg 18000tcttgatttc tggatttatg aaattcttct tatgggatca tttagcttgt ctctctgtat 18060ttcctgtaag agaagctcta tctgatgtgg ggtttttttg gttttgtttg tttgtttttt 18120gagatggagt cctgctgtcg cccaggctgg agtgcagtgg cacaatctcg gctcactgca 18180acctccgcct cctgggttca agagattctt ctgcctcagc ctcctgagta gctgggacta 18240caggcgagtg ccaccatgcc cagctaattt ttgtattttt agtagagaca gggtttcacc 18300atattggcca ggatggtctc gaacttctga cctcgtgatc tgcccaccac ctcagcctcc 18360cacagtgctg ggattacagg catgagccac tatgcccggc taatttttgt atttttagta 18420gagacagggc ttcgccatgt tggccaggct gatctgaaac ccctggcctc aagccatcca 18480ccctccttgg cctcccaaag tgctgggatt aaacgcgtga gccaccgtgc ctggtcgaag 18540agacagaaag ggtcttaaag gttcagtgac acacacctgt aatcccagca ctttgggaag 18600ctgaggctgg tggatcactc gaggccagga gttagagatc accctgggca acatggtgaa 18660accccgtctc tacacaaaat acaaaaatgg gcagagcatg atggtgcata tctgtagtcc 18720cagctactcg ggaggctgag gcgggaggat cacttaagcc tgggagatcg aggctgtagt 18780gagccatcat tgcactactg cattccagcc tgggcgatcc catctcttaa aaagagagag 18840agatgggaag accagcacag gtgaaactgg tgaacagagg agagatggta gatgctgcat 18900tgggcagtgt gacgggaacc cgctggaggg ctttggcagg agagtagttt aagaggatcc 18960cagctgggca cagtggctca cacttgtgat cccagcactt ggggaggccg gggcaggtgg 19020atcacttgag gtcaggagtt cgagaccagc ctggccaaca tggtgaaacc ctgtctgtac 19080taaaaataca aaaaccagcc aggcatggtg gtgcacccct gtaatcccag ctactcagga 19140gactaagaca ggagaatcgc ttgaactcag gaggcagagg ttgcagtgag ccaagatcac 19200gccactttac tccagcctgg gcagtagagc gagactccat ctcaaaaaaa taaataaata 19260aaaagacctc tttgctgggt gctagggagc aagagcagga gctgggagag gcctgcagca 19320gaagcctgtt gccagcatcc aggccgtggg gtgaagggaa gggtttggat ttgggacatg 19380tcttggaagc atcaccagca gaacttgctg atggattgga agtggctggt gagggagaaa 19440agggggtcaa aggaaactct gaggtctata ccctgaccat ctggcaagtg gtggtgttgc 19500cacaaactga gcggggagta gggcaggtgc aggtctggag gatggattca aaattcagtt 19560tttggagtct atgtccctgg ttctgtaggg ctgcagatgg tctgccaaat cttagcggaa 19620cccagaatac gggatttgtt tactgtctgt gacttgttgg tttccctggt gagagcaaac 19680tctttaaagg tcaaggttgg gcttcagacc ttggtttttg caccgatcat tggtcatact 19740gcagttcctc actcttctct tgcaaatcca tacacagcta gtccaagaga gctgaacagc 19800tttgtggttg gatcagcacc aatgtatctc cacctgtaga cgggttgctc aggtgactca 19860tgcctgtaat cccagcacct tgggaggcca aggtgggaag attgcttgag gccaggagtt 19920ggagacaagc ctgggaaaca cagtgagacc ccatatctac caaaaaaacc cctttgtttt 19980aattagccag gtgcagtggt gtgcacctat agtcccagct actaaggagg ctgaggcaga 20040aggatcattt gagcccagga gtttaaggct gcggtgaacc atgatcgtgc cactgcactc 20100caacctgggg gaaagaaaga gaccttgtct ctaaaaaaac taaaaaacag aaaagcattt 20160gttgagtatt tcctgggtat aaagcagtgt accaggttaa atggaaggaa aagttgaaat 20220aatttttcaa ctcataatcc gattgggaga gactgaatgc ttaccattga agcaggaacc 20280attgtaagca atgtgttgtg atactgtagc aagagctgag aaaacttggg aaaagagaaa 20340ggaggaaggc tcacctgagg gagttggggg gcttgcccta caggtgagtt gtgaggtggg 20400tctggaagtg acagatgcag tttaggaagt ggacgggagg ctgggtacgg tgactcaaca 20460tctgtaatcc cagtgctttg ggagacccag gcggaaggat cgcttcaggc caggagttaa 20520agaccagcct gggcaacata gtgggaacct atctctacta aaaattaaaa aattatccag 20580gcataatggc acatgcctat tgttccagct actcaggagg cttgcctgag cccaggaggt 20640tgaggctgca gtgagctatg atggcaccac tgcactccag cctgggcgac agaacaagac 20700cctgtctcta aaaaaaaaag atgtggatgg gagggggaac ggtgggtggg ctgtcctcac 20760caagccccca ccctatctgc tctccagcta aacgacggac agttcacagt catccagctc 20820gtgggcatgc tgcggggcat cgcctcgggc atgcggtacc ttgccgagat gagctacgtc 20880caccgagacc tggctgctcg caacatccta gtcaacagca acctcgtctg caaagtgtct 20940gactttggcc tttcccgatt cctggaggag aactcttccg atcccaccta cacgagctcc 21000ctggtaatgc tgggggtaat actgggtgtg agcttcttag ggccaggtgg gcagggcagg 21060ttggaaaggt gggaggctga gggtttggca gccctgctcc agggagagga tacaggagca 21120ggctgtgggt ggggggacag tcagctccag gaagccgact tccagatgtc taggaaaata 21180acagttggat aacctgggca acatagcaag accccatctc tacaaaaaaa ttaaaagatt 21240agccaggcgc agtggcatgc acctgtagtc ccagctactt gggaggttga ggcaggagga 21300ttgcttaagc ccaggagttg gaggctgcag tgagctatga atgtgccact gtactgcaga 21360ctgggcgaca gagcaagacc ctgtctcaaa agaacagtgg ccaggtgtgg tggctcacgc 21420ctgtaaatcc agcactttgg gaggctgagg caggaggatc gcctgaggtc aggagttcga 21480gaccagcctg gccaacatgg gaaaaccctg tcgctactaa aaatacaaaa ttagctgagg 21540gtggtggtac acgcctgtaa tccgagctac tcaggaggct gaggtaggag aaccagttga 21600acccgggagg cggagtttca gtgagccaag atcgcaccac tgcactccaa cctgggcaaa 21660cagagttgga gagtaggagg cttggggcct gagctagggg gaaaaagcag aggcaggtgg 21720gggactgggg ggcagtgtgc tgggtctggt gagtccctca gtgagtcccc cagctcacct 21780tttctccttt ttctgcaggg aggaaagatt cccatccgat ggactgcccc ggaggccatt 21840gccttccgga agttcacttc cgccagtgat gcctggagtt acgggattgt gatgtgggag 21900gtgatgtcat ttggggagag gccgtactgg gacatgagca atcaggacgt aagtgtcccg 21960tggtcctacc aagctttcct cgagtgttct ctcacctggg atttggggtg aagggtgggt 22020tcccagagag tcatcactgc tgggttcttg agaccatgga gatgacaaaa aggagaattg 22080atctttgtat caaagagttg agatacaggg ccaggcctag tggctcaagc ctgtaatccc 22140agcactttgg gaggccaagg tgggcagatc acctaaggtt aggagttcaa gaccagcctg 22200gccaacatgg tgaaaccccg tctctaaaaa aatacaaaaa attagcccag catgatgggc 22260gggtgcctgt aatcccagct actcaggagg ctgagacagg ataatcgctt gaacccagga 22320acagaggttg cagtgagctg agatcacgcc attgctttcc agcctgggca actgagcgag 22380actctgtctt aataaataaa taaaagagtt gggtacagca tatttgggtc gcagaaggat 22440gcagagatgg agggcagggt tgagaggtaa catgtctgta tcatagccca agagctgctg 22500gggccttcag ccacagagag cttcaactcc ggctaggagg attcctggat ctgttatttt 22560ttggggggct gtggctccta tcctaccatc ttccaagtca ccatttcctg ggcctgttag 22620catctttgct tttcctggac agcctcaccc agagcttctt cccctctttc caggtgatca 22680atgccattga acaggactac cggctgcccc cgcccccaga ctgtcccacc tccctccacc 22740agctcatgct ggactgttgg cagaaagacc ggaatgcccg gccccgcttc ccccaggtgg 22800tcagcgccct ggacaagatg atccggaacc ccgccagcct caaaatcgtg gcccgggaga 22860atggcgggtg aggactgcag agaatgggcc ctccttcccg ctctctgccc ccactccttg 22920cccagaagtg tccgttcatt ggtgttgggt gggagggcct ctgtccgcct ctgcaaggct 22980gggttccacc tcctcccccg gacctgggcc tggtactcag cattcctccc catccttgcc 23040ccctagggcc tcacaccctc tcctggacca gcggcagcct cactactcag cttttggctc 23100tgtgggcgag tggcttcggg ccatcaaaat gggaagatac gaagaaagtt tcgcagccgc 23160tggctttggc tccttcgagc tggtcagcca gatctctgct gagtaagcag tggcaggagc 23220tggagtgggg ctgggagagc ggggcagctg gagtcaggcc cacggggtct ccaggggctt 23280ttggggtcag cttcgggtgc caatgctgtc ttcttgcact gcgctcatgc catgcctaga 23340agggccccag aggagcagtc acagccccat ggagctgagg acccaaggac tctttggggc 23400cagcctgccc gcctcacctc ctcctgccat cacagccctg ggccatcgcg cttccgcctc 23460tcacttctag ctatctttgt gcatctatct gcattccagg cccggctctc acggtaacaa 23520tgtgtcaact cgggttctct ttttccaacc ataaaaggag aagattgggc taggttttgg 23580agatcctctt cagcttttat gtgaaatggt tttatgattc cttgcctccc aaaggctgcg 23640tatccccact tggcctttgt ctgctactcc ccctttctgc cttcccgttc ctctcccaag 23700atctcctctc accccaggtt gaataacaga aatagaagga atagaaatct gaaggccggg 23760catggtggct catgcctgta atgccagcac tttgggaggc cgaggtgggc agatcacttg 23820aggttaggag ttcgagacca ttgtggacaa cttggtgaaa ccttatgtct actaaaaata 23880caaaaattag ctgggcatgg tggtgcgtgc ctgtaatacc agctactgag gaggctgagg 23940caggagaatc gcttgaaccc gggaggtgga ggttgcagtg agccgagatc gcaccactgc 24000actccagcct ggatgacaga gtgaaattcc atctcaaaaa aaaaaaaaaa aaaaaaaaag 24060aaatgtgaag gccaggtggt ggctcacgcc tgtaatctca gcactttggg aggctcaggt 24120ggaccgattg cttgagccca ggagtttgag agcagcctgg ccaaaatagc aaaaccccat 24180ctctacaaaa caaaaacaaa aaaattagct gggcatggtg gtgcgtgcct gtggtcccag 24240ctactcagga ggctagagcc agagggtctc aggccagtct gcccctgccc cacggggcct 24300gggcacatcc ctccctaatt cttcccagcc tctctctgac ccagggggcc tcctctccct 24360tttttcccct tatctcagcc tccagccatc agcaacctcc tcttcctctc cacccagctc 24420ttcctctccc acttcggcct tttctttctc acactccatt tccctctacg gcaatctgtg 24480cagcctcttc ccccagtctc attttgcggg cttttctctc ttttctttcc ttccctggca 24540cccaagccaa aggccctgcc tctggcctcc agccctaccc ccttctgcgg ttgcacagaa 24600ggatggctgc ccagctctta aaaaaactgc ccgggaactg ttgacatctg ttctccctcc 24660cccgctggct tttctgattg gcttacaatc ctgaggctag gaccgtctca ggagccaaga 24720gaggagagcg gccacaggga acctagggtc tcaccaagct ctcctttcct tctgcaggga 24780cctgctccga atcggagtca ctctggcggg acaccagaag aaaatcttgg ccagtgtcca 24840gcacatgaag tcccaggcca agccgggaac cccgggtggg acaggaggac cggccccgca 24900gtactgacct gcaggaactc cccaccccag ggacaccgcc tccccatttt ccggggcaga 24960gtggggactc acagaggccc ccagccctgt gccccgctgg attgcacttt gagcccgtgg 25020ggtgaggagt tggcaatttg gagagacagg atttgggggt tctgccataa taggagggga 25080aaatcacccc ccagccacct cggggaactc cagaccaagg gtgagggcgc ctttccctca 25140ggactgggtg tgaccagagg aaaaggaagt gcccaacatc tcccagcctc cccaggtgcc 25200cccctcacct tgatgggtgc gttcccgcag accaaagaga gtgtgactcc cttgccagct 25260ccagagtggg ggggctgtcc cagggggcaa gaaggggtgt cagggcccag tgacaaaatc 25320attggggttt gtagtcccaa cttgctgctg tcaccaccaa actcaatcat ttttttccct 25380tgtaaatgcc cctcccccag ctgctgcctt catattgaag gtttttgagt tttgtttttg 25440gtcttaattt ttctccccgt tccctttttg tttcttcgtt ttgtttttct accgtccttg 25500tcataacttt gtgttggagg gaacctgttt cactatggcc tcctttgccc aagttgaaac 25560aggggcccat catcatgtct gtttccagaa cagtgccttg gtcatcccac atccccggac 25620cccgcctggg acccccaagc tgtgtcctat gaaggggtgt ggggtgaggt agtgaaaagg 25680gcggtagttg gtggtggaac ccagaaacgg acgccggtgc ttggaggggt tcttaaatta 25740tatttaaaaa agtaactttt tgtataaata aaagaaaatg ggacgtgtcc cagctccagg 25800ggtgatgggg gtgatggact agatttctaa ggagagtggg gctgggtagg gagggctttg 25860tggctgaccg agaggtgtca gaggtctgga ggctgcaggg ctgtaggggc tggaacttgg 25920ttatcagccc cagggtatgt ttgaggtggt ggggtggggg ccgagcgaga tgaatcattc 25980gcagctgctt ctaacgtctc 2600074235DNAHomo sapiens 7ctcggcccgg cggcgcgagc agagccactc cagggagggg gggagaccgc gagcggccgg 60ctcagccccc gccacccggg gcgggacccc gaggccccgg agggacccca actccagcca 120cgtcttgctg cgcgcccgcc cggcgcggcc actgccagca cgctccgggc ccgccgcccg 180cgcgcgcggc acagacgcgg ggccacactt ggcgccgccg cccggtgccc cgcacgctcg 240catgggcccg cgctgagggc cccgacgagg agtcccgcgc ggagtatcgg cgtccacccg 300cccagggaga gtcagacctg ggggggcgag ggccccccaa actcagttcg gatcctaccc 360gagtgaggcg gcgccatgga gctccgggtg ctgctctgct gggcttcgtt ggccgcagct 420ttggaagaga ccctgctgaa cacaaaattg gaaactgctg atctgaagtg ggtgacattc 480cctcaggtgg acgggcagtg ggaggaactg agcggcctgg atgaggaaca gcacagcgtg 540cgcacctacg aagtgtgtga cgtgcagcgt gccccgggcc aggcccactg gcttcgcaca 600ggttgggtcc cacggcgggg cgccgtccac gtgtacgcca cgctgcgctt caccatgctc 660gagtgcctgt ccctgcctcg ggctgggcgc tcctgcaagg agaccttcac cgtcttctac 720tatgagagcg atgcggacac ggccacggcc ctcacgccag cctggatgga gaacccctac 780atcaaggtgg acacggtggc cgcggagcat ctcacccgga agcgccctgg ggccgaggcc 840accgggaagg tgaatgtcaa gacgctgcgt ctgggaccgc tcagcaaggc tggcttctac 900ctggccttcc aggaccaggg tgcctgcatg gccctgctat ccctgcacct cttctacaaa 960aagtgcgccc agctgactgt gaacctgact cgattcccgg agactgtgcc tcgggagctg 1020gttgtgcccg tggccggtag ctgcgtggtg gatgccgtcc ccgcccctgg ccccagcccc 1080agcctctact gccgtgagga tggccagtgg gccgaacagc cggtcacggg ctgcagctgt 1140gctccggggt tcgaggcagc tgaggggaac accaagtgcc gagcctgtgc ccagggcacc 1200ttcaagcccc tgtcaggaga agggtcctgc cagccatgcc cagccaatag ccactctaac 1260accattggat cagccgtctg ccagtgccgc gtcgggtact tccgggcacg cacagacccc 1320cggggtgcac cctgcaccac ccctccttcg gctccgcgga gcgtggtttc ccgcctgaac 1380ggctcctccc tgcacctgga atggagtgcc cccctggagt ctggtggccg agaggacctc 1440acctacgccc tccgctgccg ggagtgccga cccggaggct cctgtgcgcc ctgcggggga 1500gacctgactt ttgaccccgg cccccgggac ctggtggagc cctgggtggt ggttcgaggg 1560ctacgtcctg acttcaccta tacctttgag gtcactgcat tgaacggggt atcctcctta 1620gccacggggc ccgtcccatt tgagcctgtc aatgtcacca ctgaccgaga ggtacctcct 1680gcagtgtctg acatccgggt gacgcggtcc tcacccagca gcttgagcct ggcctgggct 1740gttccccggg cacccagtgg ggctgtgctg gactacgagg tcaaatacca tgagaagggc 1800gccgagggtc ccagcagcgt gcggttcctg aagacgtcag aaaaccgggc agagctgcgg 1860gggctgaagc ggggagccag ctacctggtg caggtacggg cgcgctctga ggccggctac 1920gggcccttcg gccaggaaca tcacagccag acccaactgg atgagagcga gggctggcgg 1980gagcagctgg ccctgattgc gggcacggca gtcgtgggtg tggtcctggt cctggtggtc 2040attgtggtcg cagttctctg cctcaggaag cagagcaatg ggagagaagc agaatattcg 2100gacaaacacg gacagtatct catcggacat ggtactaagg tctacatcga ccccttcact 2160tatgaagacc ctaatgaggc tgtgagggaa tttgcaaaag agatcgatgt ctcctacgtc 2220aagattgaag aggtgattgg tgcaggtgag tttggcgagg tgtgccgggg gcggctcaag 2280gccccaggga agaaggagag ctgtgtggca atcaagaccc tgaagggtgg ctacacggag 2340cggcagcggc gtgagtttct gagcgaggcc tccatcatgg gccagttcga gcaccccaat 2400atcatccgcc tggagggcgt ggtcaccaac agcatgcccg tcatgattct cacagagttc 2460atggagaacg gcgccctgga ctccttcctg cggctaaacg acggacagtt cacagtcatc 2520cagctcgtgg gcatgctgcg gggcatcgcc tcgggcatgc ggtaccttgc cgagatgagc 2580tacgtccacc gagacctggc tgctcgcaac atcctagtca acagcaacct cgtctgcaaa 2640gtgtctgact ttggcctttc ccgattcctg gaggagaact cttccgatcc cacctacacg 2700agctccctgg gaggaaagat tcccatccga tggactgccc cggaggccat tgccttccgg 2760aagttcactt ccgccagtga tgcctggagt tacgggattg tgatgtggga ggtgatgtca 2820tttggggaga ggccgtactg ggacatgagc aatcaggacg tgatcaatgc cattgaacag 2880gactaccggc tgcccccgcc cccagactgt cccacctccc tccaccagct catgctggac 2940tgttggcaga aagaccggaa tgcccggccc cgcttccccc aggtggtcag cgccctggac 3000aagatgatcc ggaaccccgc cagcctcaaa atcgtggccc gggagaatgg cggggcctca 3060caccctctcc tggaccagcg gcagcctcac tactcagctt ttggctctgt gggcgagtgg 3120cttcgggcca tcaaaatggg aagatacgaa gaaagtttcg cagccgctgg ctttggctcc 3180ttcgagctgg tcagccagat

ctctgctgag gacctgctcc gaatcggagt cactctggcg 3240ggacaccaga agaaaatctt ggccagtgtc cagcacatga agtcccaggc caagccggga 3300accccgggtg ggacaggagg accggccccg cagtactgac ctgcaggaac tccccacccc 3360agggacaccg cctccccatt ttccggggca gagtggggac tcacagaggc ccccagccct 3420gtgccccgct ggattgcact ttgagcccgt ggggtgagga gttggcaatt tggagagaca 3480ggatttgggg gttctgccat aataggaggg gaaaatcacc ccccagccac ctcggggaac 3540tccagaccaa gggtgagggc gcctttccct caggactggg tgtgaccaga ggaaaaggaa 3600gtgcccaaca tctcccagcc tccccaggtg cccccctcac cttgatgggt gcgttcccgc 3660agaccaaaga gagtgtgact cccttgccag ctccagagtg ggggggctgt cccagggggc 3720aagaaggggt gtcagggccc agtgacaaaa tcattggggt ttgtagtccc aacttgctgc 3780tgtcaccacc aaactcaatc atttttttcc cttgtaaatg cccctccccc agctgctgcc 3840ttcatattga aggtttttga gttttgtttt tggtcttaat ttttctcccc gttccctttt 3900tgtttcttcg ttttgttttt ctaccgtcct tgtcataact ttgtgttgga gggaacctgt 3960ttcactatgg cctcctttgc ccaagttgaa acaggggccc atcatcatgt ctgtttccag 4020aacagtgcct tggtcatccc acatccccgg accccgcctg ggacccccaa gctgtgtcct 4080atgaaggggt gtggggtgag gtagtgaaaa gggcggtagt tggtggtgga acccagaaac 4140ggacgccggt gcttggaggg gttcttaaat tatatttaaa aaagtaactt tttgtataaa 4200taaaagaaaa tgggacgtgt cccagctcca ggggt 4235843948DNAHomo sapiens 8gcgcctcgga gctgcctgcg ggcgcacgcc gtcttccccg ccagtctgcc ccggaggatt 60gggggtccca gcctgcgtcc cgtcagtccc ttcttggccc ggagtgcgcg gagctgggag 120tggcttcgcc atggctgtga gaagggactc cgtgtggaag tactgctggg gtgttttgat 180ggttttatgc agaactgcga tttccaaatc gatagtttta gagcctatct attggaattc 240ctcgaactcc aagtaagtgg cgtccgcgat ccccctatgt ccccgccccg gggtccgccg 300cgccgtccgg gcgggaggag gggtcagtcc gcggggcctc ggagcctgtt tctggaacct 360cggttccccg tcccccaccc ccaacccccg ccccatttca ctaggtggag actcctcgct 420cggctttcca acccgagccc cgctggaacg gacggtctct ccgcctttcc tcccccgaac 480gctcccaggc gctaaaagct actatcggct cgggtgtcaa gtccgggaag gtgtccgatg 540gcgatacctg accctctcct gttttcgagg acgaaggaca tggccacaat ctaggctggc 600cggcacgcgg ggactggtgg gctctggaga gaggcggaga tgctgcattc gcggggagcg 660cgggcggcgt ggtccggggc ccgcgggcgg gcgaccgggg tggcaggacg ctggcagcga 720agcgcgttct ggagagggga gcctggagtc gctacgctgc ccgcagagcc ctggagccgg 780ggcgccttgg caccgcgccg ccagcccgag ggtgcgcggg gagctcgcct gcttcgcagg 840agaactcggg cgtcgagccc tttcctccgc gccggggaga cgggccttag gcttctccct 900gagggcccgc cgcacctcgg cctcccgctt cgttcataag ccggtagccc cggagtatgc 960ggtctcgatg gccgacctga ttgtaatgca cttcctataa aagcttaggg ccctgcccag 1020tcgacactgc tcctgaagcc ttctccctcg ggaccctggt aggaatggga tccttaggat 1080cagatttgct cttaccggac tctacagccg ggagcgagcc aggccttgtg gagagtaact 1140ttcagtttgg gccaccagag tgcattcaga atttagaaaa tcccatccat ccctaaatct 1200gtgtggtcat aactcgtagt catctgggta ttcagtactg tgtatcccct tatttcgaat 1260cacagccaaa acatatttta cagaatcttg gaattgtagt ctcgggaaac ttggagaaga 1320agtatgcaga cattagctgg tttctggaga aaacgtttga gatcagaagc aaaatcaatg 1380gcctaattga agttgagcaa gttgggcctg gttttaggag aaaagaaatg ggggattgat 1440ttagaaatca cgtcttaaag gagtgtgtcc attctcttaa aagtgtcaaa tttcaaattc 1500actaacatgt taaccaagaa tcccttcatg aaaagggcga aaacgtcggt tacaaatcgg 1560tttaaacaaa tgtttgtatg atgctagaag gcactttcaa caccgctcat acggagaagt 1620tacttagctc tgcctccttc catgtagtct gctcttgcat ggattatatt tttaatgtaa 1680attgttgtat ttgctgatga agtactggcg gcggcatctt tgcatcgatg ccggctcggg 1740aggcgccagg tggtgccgga aggagccggg ctaggacctc gcgcagcagc gggtcccgga 1800gtccgggaga ggcgggcggg cgggcgaggc ggtcgcgggg agcccgcggc gccgctgccc 1860gcccggtgcc tccagaggtc actcttccat gcggaatcgc gcagcgccag gcctcgcccc 1920tcccccaggc cgcctgctcc agccactctg cactttcact gaccggttct ctttgaggct 1980gttttttttt ttcttatgag gatttaatat ttctgtttaa atctagttga aagcaattcc 2040gttagcctct tcagcgttta gttcggtgtg tgtatcttta tctttgcgct atattaacta 2100ttagtttgtg tgtatccggt aggagaatta gaaataccta gttgggagaa aaagaaaagt 2160agaacaatag ttatttcaac ctaaggttta gacgttaata acttcttttt gtaatgtgtc 2220gagatggggg gtcctggggg gaggtgacag gtactcacca ctcccccccc ccattctgat 2280gatgaagatg agtctgtctt tccagctatg tccagacctg cgagggccct gcgtttctgg 2340aagcctgccg tttgcgcggt tgaggttgct gctgctgtct tgtcctccac agcagcattt 2400cttttaaaat tctcctgata acggcctgcc tggatgactg gataatgtgt gcctggaaaa 2460ggtctccctt gcagctgaat gctagctcca gagatcagaa agatttcttc ctgtaggagc 2520cataggaaag agtcctctct aagtttttga gaatgcatac aaccccctga tgacaggggg 2580tcgctttcct tggggaagtt ttatatttat ttccagagga aagtttgaat cggtaaatat 2640gatgtggcag gaaggtaatc aaatgcattg aagtttcaca tcagttccta tgaactgtgg 2700aacaattcat ttgtaatgaa gccgccatca gtaattagat ttgtttcatt cagaggtcag 2760cttttttagc aggtggtcga cacagggagc atgcagcagc tgtttggata cagggtccag 2820aaaacccttt gtaaattcag cgtctccgta actactttaa tcacattgtc ggctctcccg 2880tccctgactg tatgtaataa tggaaagatg tcctgcgtgc tgaaacagta gctgccctgt 2940taggttattc acattgcttt gatacgttct ggtagagttg ggtccgttgt agccattttg 3000gttgtttaaa gttttggttt tttttttgtt ttttttttaa ttcagcagag aacagtaatg 3060cctagcttcc gtttttaact taacacttca gtagaacatt ttcttccaag agggagattt 3120tggcctaagt aaagtagtgg gctctttttt aaaaaaaaat taattttact ttaatgtgag 3180caaatctgta ttggtatggt gttctgcaat gcattacact gactttgaaa atttcgagta 3240ctaatgcctt atgtctgggg ttaccattcc ctgtgcatca catactagtt agttaacata 3300gcattttgct tttcccatgt aattttttcc ctatataata ctggattcct gatactaatt 3360gacttgatac aaaagaatgg ctggatgata tccagataac gtataataca tgggcttcac 3420cacaatcagg ctctgaataa atacagacct gtcagagatt gataaaataa actacaatgg 3480atagtgctgt ttaaacagtc cattcaataa catatataag ccagcctgcc ttccattgtg 3540tctgaaattc ttatttttgt aggtaaacaa atgcacattc agcactgatt gaatagcccc 3600ttgaactatg ctccacagtt tgcgtttggg ttaatcttgt cggttttaat atagagagaa 3660aaaagctcaa agcaccaggg gtggaattgt tagtgctttc acatccacat tcctcacatt 3720ttgtcaggat gataaactgt aggtaatgga ctgtcgttgt tctgcaggac aactgagcca 3780ggcagagcac aaagactaag ctaaagcgat acctcacaac atgcttggta gccttctttt 3840cagatgagaa tttatttgag aatcatgtgt ctagggactg cacatcttaa cctcaacagt 3900tacagcttca agccccagaa acaggagctg gaggttaaga tgatttgcta agcacctggt 3960tctaaatctt ttacaaagca taagctgttg acgctggttc tgccgacgca aagacatgca 4020gatgactcca acatttccag aggcttctga cttaagctaa agtgtgtgga caggtgaatt 4080cgccatgggc ctggagacca gcttgctaaa aactatgtgt ttgaatggtt cctccagaca 4140gagtcagctg aagaacaatt ggtggattta tattaaaacc tcttgtctgt aaacttactg 4200aggtgcatcc ttcggttggt ggatcagtga gataattgcc ttcagatgga cattgcaact 4260ggagcaacta aatccttgct gtctttcctt cctctgaaat cttccaggta gctcccgaga 4320gcttcagtat gacaccaaac ttcgggcgac gttttagagt gcgttcacct aatgggaaac 4380tattcgagat cccagcgtga ctgcagtaat gcgtcatagg aatgggagtg gcaggggaaa 4440aggaaataca gattgtagac cctaataaaa aaatttttag gaaagatatt tctttaacgt 4500tttatgagaa cttcattctt aaaatactta attgcaaatt agacaaatag aagtgctctt 4560ctaaggaagg tgattaaact ggtcctccta tcagcctaat ctctgcctgc ctttgctgct 4620gacataaaga acctgttttt caggtcactt aatatacatc tacatagatt tgcttatgag 4680ctcacccttt gtgtagcgga gtagagcctt aaagaggagt gctcaactgt ttaaaatatt 4740ttgattaaaa tatgcagaac ccatagaact ataagcttct agtcaggaat tagctctttc 4800agggaacagc tccccccttc tttttaaggg gggaattaga aggaggctgg gggaggaata 4860taagaacagc aaagaaggaa ggatagcaaa tgggacatgt tccgaacagc ttggaaaaac 4920tcctgtggct tcattgtctc tataaagcca aagaatacaa agacataagc aattcagccc 4980ttctcccatg atggaagatg taaaccgttg acatgcctcc cctgtttaac ttgtttaatt 5040ctcattttaa attcagcacg atactagccg tgtgaactct gaagatttct ttagtaatcc 5100attttgtagt tccgaatcaa aaacaaagtg aaagggtctg acacaatttg cttttatttt 5160taggcaaatc aaccctggtc atagttaata aggggattac aactcagact aggtctttac 5220agatgtgatg taaatcaagg gcagagtata aagaaactga tcccttttga ttgaagtata 5280gtaaaaaggc atagagaaac tagcagcagt aatctgattg tatggcaata aaaccaccat 5340tttctgtctt tcagataaaa ataatgtggt aaatccatgc agttcataag atgtaaaggc 5400agataaaggg tgaagccatg gcaacatata gattagcttg atgttagaaa tgacacgtct 5460ctgaaaaggg cgcgggacga aggcccttgc ctccaggctg ttgggcatta tgtgagaacc 5520acacagactt ggaaactggg attaggaagt atgaaagctc tacttgtggt ctgggatggc 5580tgaggcagta aagaaaagct gctcagttct tgctcattgg tggtggataa tatggcaaag 5640gtagatttca ttgactgcct tttttataga ttgagattgg ggctgattaa aacttcagat 5700cactgcagtt gttagggcct gggagatttt cctttttaac tcctggccta acagcagcag 5760ccgttctgta ggattaactg cacttcgcgg tcgttgcctt aatctatttg ggcttcaggc 5820agggacatgc tgggaaggaa cagagaccag aggggatagg tagggctggg gttatctgaa 5880aagaaaacag agaccttttg atttcagcca tcttttcaga cccagctccc tctcccgctg 5940catgggagaa gcaaaggtaa acaggacaca ttgtccctct ccctcagcca cagagctctt 6000ctgtgagttt tgtctttccc accctggaaa aaaagataaa atacaatttt taaaagggga 6060gggaggaatt tagttttaat tcaaatgagt agtaatccaa tatgccaaaa gcagtgggct 6120ctacctagat gtaattttac tcgtaaatgt gagtcttaaa ctttgagttg aatggggcag 6180gctgttagag gtggtgtaaa ttacaggatt ataaaaatgt tagtgctgcc cagccttaaa 6240gtcaaaaaca gaaaaatctc tgtgctgttg agtcttcccg ccctctctcc tgaacaacct 6300tgtaagtaag ctagactttt gtttttgcct tccatacttt ccatttcagc cattaaacaa 6360aataagccat tgaaaccacg attgggttcc atgcagagtg acatccgcaa tcgggtcaag 6420ccagaaggaa atacttgctc gattgccccc tatttggcat tacaggaaag tctccacact 6480ttggaagagt ctgaactctc aagacattga aaatgccaaa ggctgcaaac accctgtgtc 6540tttcttgatg gagtgcatct tggtgtgttt tacaaagggg aattcagtgc tgtttttttg 6600ttgttgttgt tgtttttttt ttttaaagag cagcataggg cccttctaga ctcttggatt 6660ctgtgtctga caaaaatggt cattaaatga gcaatattat aatttagacc catttcactg 6720attttgttcc aaattctcaa ctgacttgag catctgtttg gggctgtaga tacattgccc 6780ttgttgactg tttttctcgt ttctatggga attactgtag ccattactat gtagctttca 6840tagactcaaa acatttttaa agtattgcat ataggctggc catatccagt gcctgttact 6900ttaccttctt tttctaactt aatgcagcag tctgtattaa cagatccatt tcatttgtct 6960agcttcatca gagagaggct accccctgat ttacaggctg ctcacatcca agcaccttgc 7020attctacact tgacagtgat tgctaatggc ccattcaact aaagtatttg cttgttaaca 7080gggaacagaa catgataaat gtccagcaag cttgctgcct ccttcagctt ttcaaacgca 7140gactggtgca tatttatggc aggcaaatga caaaagaaaa agctgaattg ccctggcctc 7200cagctttcta tcagaaacag ggttaaagtg attaaagcaa tcattcaaga aagccctgcc 7260gtttgtttac taaccttcat ccaacattta gctttgtagt ctacctgtga gaagatattt 7320cagaagtatt agagataagg aaggaggatc tagcaaacca gtgaaaagag taggtgacca 7380gttataaaat gctttccatg cacattgaat gccaggcgaa cctatttctg ttattccagc 7440agacaatcag cagtggctct agattattaa catattttcc tttcatgtat aaattcaaat 7500atgtaattct agtccaaagc attctgtggc tggtaagcac atacttgctg atttcaaata 7560agaaaacata gcaagggaaa gctccattaa acaagttgtt tctgccctta gtaattctct 7620aaacaagata ggaagaaaaa gtggacagta gtggagtatt aatagtgtgc tcttttcatt 7680ctctaaagca cgagtaagta agcgttcaaa ctactctgtg gtgggcatac atttagagcg 7740ctgtgaatga accactgctg ttctgccata cttaatttat ttatattatt atttttattt 7800tattgttgtt tttatgtatt attataatta tttatttata ttactaattt attttctcaa 7860tttaaatcct gttgcatcca attttaatta cagtttttgt atctgccttc ccatacttgc 7920tacccacgtc cccattgcca ctgcggcctt atccatgttt tctgtgtaca ccactctcgt 7980atcaccccag aataattatg agtgctaccc agacttttga aaccactaga gtcaacatgt 8040ttgtctttga ggaaagccaa tgatgcttta gcatttttgg caggggtgga tgtgtgttta 8100agtggggtgg gtgcagctcc ttattgtctg cctattctac tgttgttccc aatccacatt 8160ccctgcgggg cacctaacct gtgtgcatag caaagaattt ccgaccttca gagccagaag 8220tgtttctcaa ttgatctctt ccagcctagg gttatagctg atgaattata atccttgctc 8280tttccacacc tttacctggg cttaccatgg ccctaaaaca tttgcccaga atcagaattg 8340tctcatgagt gagtggggca aggcaaatcc tgttccagac cagctgagaa tgtacctagc 8400tgcagaagaa gttagaaagt gtcatctttt acttatctac cagaactata ttcgaggtac 8460attttagatt taaaaaaaaa gcaagttctc gtaggccttg aatccccccc ttgctatggg 8520aaaatggatc attattataa tggactgtcc agtaaagttc atgatttctc ctagacatgt 8580tctctctctt tatgacctag atcaagagtg atctctttaa gtcttttctt cataatccca 8640cagcactttg tacttagatg tacttagaaa gaaccatata cacggtacgt catgattgat 8700atgcaagcct tcaccactct acctgtccta aaagtcaggg acacaccttc ttcatttcat 8760cagtccctac ttctatccag cattggcatc cagtaagtat tagtggaatg gacagacaac 8820ccgaatttgt gctgatggca gtttaccctg ttttaactgt catccttctg ctactagaca 8880tggatgagac ctgagacgat gggactgctc agaggtccct ggctcttgaa ctttagggca 8940ccagaatccc ctgcagggct tgagaaaaca ggggtttctg ggccccaccc ccagagttcc 9000tgattcctga ggtctggggt ggggcttgaa gatggacatg tttaacaagc tcccaggtga 9060cgctggcaac tgctgcctca gggccatgct gagaaccctc gccctacaca aacctttctg 9120ggaaaacaac tcaacattaa agctgtttgg ggatctctga agaaatctgt agtccttgcc 9180ttgttggggg agcatcaggg atctaaccat tgatggtgga gtatttgttg ttaattcagc 9240aagcaactat taagtgttag gcctgttact cggctctaac aatacaaggc agagtgacct 9300gtaccctcga gatttaaagt ctaagtcctg tagagagaag cccaggtggg agcaagcaca 9360tttagagtta ggtgcttggt gcaaggtggg gacacagaag aagggaatgg catttgcctc 9420tggaggggtc cggaaacagc ctagggagga ggagcttgag tcttgaaata ctgtgggcat 9480ctctaagcaa agtcacagta gacagctgaa ataaagaaaa tagtaagcaa gccaaagaaa 9540cagtatttca gccaagggca gcgtgtgtct atcacgtcca cctgtgaaca cgtcccagga 9600ttctctgcat ccggccattg ctcaagacag atccctcaca ggaacagcta agccactgat 9660ttcagctacc tgttcacgtg agaattatca gtacctactg cttttcaaaa tgagtatgat 9720catggatagg tgaggcaatt cagtttcgca gagacagtag ggcaagtgcc actgtagttt 9780agttaagggc acatgcttta gagtttggct atgtgagtcc aatcccagtt tagccattta 9840ttagctgggt agctttagga gcagtagcct tagtgtctct cagttgtccc atctctataa 9900tagggacaat aacataatag tgctgaataa aagagtaaca aaattttggt caacatttaa 9960tgtatttaaa gagctaagct ccgtgattgg cacaatgaac caatcaatca aacaccagtt 10020gttattaata aaagtcagtt gaatatgtac tgtgtgcctg gccgtggttc aatttgcctt 10080tgcatacaag gaaaaaatta aaatactctg ttaataaaga ctatagcata atactttcac 10140cttaaacttc ttgatgttaa tttattttgt ttacctgcca aacttctact cattccttat 10200gactttctgc tacatgaaac accctttgta attcttttgt cctattaaat taagttctct 10260ctcctctgct ttcctgcttt tggtgctttc taataacact tttaaccctg gactttctca 10320ttcagctgtg caactgtgga ctgagaggag gctctttgaa ttcattttgt atattctagt 10380agagagtact gtgagcagtt gggttgttga atgaatacat taattcaacc tggagggatg 10440ggcagtattg cattttttac attgatatta catgatattt agaaaactgc ttaactggtg 10500gacgttgttt tattaacagc attttgtgta tagcactcac tatgtgccag ctgctattct 10560aactgcctga caaatactcc tgaaaccttc atggtaacca tatgagggaa gcacttttaa 10620tatatccata ataccaacgg ggagactgtg gccaaattgg ttaattaact tagccaaagt 10680catattgaac taataagtgg atttaaaccc agctagtctg gggccagggt ccctctttta 10740atcttctgcc tcctgcttat gctgttgcat ggagtagtct ttatcatata actaaattaa 10800gcatgcattt gcttaaagca gtgcatacat gatggatcaa aaagtttgtg gtataattgg 10860tttaattctg tcattatcca ttttgattta tagtcacttt cttatgatgg tcgtgtagtt 10920ttaaatggaa cctttgaatc tttgatataa taaggttatg tcaaatcttg ggtataataa 10980ggttataccc aatggaaaca gaataatgat cagcccattt aaaggatgac tggagagtta 11040ttacaataca taatagtcat gcatatattg agtagtattc ctttggtaac attttccttt 11100taaaaattgt aacatttgat tgttccttgt tgggagaaaa ggaggtcaga tttttgaggg 11160gagatccatt tggtgagatg ctgagtgtgt gtcaagctaa ggagatagta tgacatcttt 11220tttagagtct agtcacaatt aaatgccatt ttattttgga ttttgggatc cgtgccagct 11280tccagcttgt cagagctgag aagactcaaa tcaagtccag gcttatttct acagcaaact 11340gggattctgg cttcttgccg gtggattcat tcagtacagc ccatctggct tttgatgttc 11400tgcaagtttg gagccatttg ttgaaggaag ccaggcggtg aatattggtg gtcctggggt 11460tctcttgact ccaagtggtg ccccttggtt tgcattttca ccatgcttag catctgctta 11520cctggagacc atgcagccgc cggccagagg tctccaacaa ccaaatcttc atgcctttta 11580gaactcagag tccccagcac atcctccttc ctcctccttg tccaattact ttcatgcagt 11640tctcagtagc tgcttgtttg aatcacttat agtatttaac ttctagggtg tttttgggtt 11700ttggtcaagg taattccagg ctgaatgtgg tgactaagca ggaaataaat gggtcgtcct 11760caaagttaca gtggagcgct gtttctattt tcctaaggta cacagttgtg ggggcgatcc 11820gtatggaagt caggaaccca gtctgatttt gcttcctttt gatggtagca gtacagacct 11880ggctgttttg tagcctgctt tgtttttctt ccttttcttc cctaacttca cgggctgtgg 11940caaagccctg agacgtgcag gaaaatgtct cctgtcatac gcccacagca gacctagccc 12000tgaccctcct ctgaagccca ggaaggaggt atctgtgaag cagcctgctt gtaaagcaat 12060tgcacacagc cttgtaaact gtgttactgg gctgattata cttgattggc aaggtgaatc 12120tcttatagca aaagagaact tggagagttt tatctcatct tatgccttat taatttgttc 12180attctttaat tacacagcca cctattgagc accctattta tgcaaggtac ctggtcgggg 12240gtcagaggga gggtcccatg gtaaacgaga cagactcaat cctggaggag caggaatggc 12300agcccctcgc tgggctgttg gccccaccaa aagggaaagg tttcatttta ataatacatg 12360ggtgaatcat ttttgtcaat aggcaaaatt ctttgtagtt aaaaaaaaat atgatggtag 12420gaaggaaagg gatgggcaga gggttaaaac aaaagatatg ctctccctaa ctctagattg 12480tagtattgtt atgcttgtca ctgtagctga attccatttc tttgagtttt ttcaatgcca 12540aggcattccc tgtatgactt acgtgagcct ttcatctccg cgatttttcc cattcaggta 12600aatgagcaaa tggatttgaa cactcatatc taaaacaaga gagaaccagc tggaaatgcc 12660ctttgaattt ctttctctat gtaaaccatt tttctttctg gtgcctcacc tataaataac 12720aggagttcca ccttccttta tagactcttg ctgaaagcat ggtttggaac aagaccgtac 12780aggtgcacac aaattacagt tgggaaagaa gcctgcagtg catcttgtct ctgaaggtta 12840tgaaatcctc cttttagtaa tggagctggc gtgatcaagc cagcaggatg aaatttggca 12900tttgtgagat cacccccctt ctcacttgcc cactgtacat agcatcccag ccttactctt 12960caaatctcca cattttttct tatctagcta caaaattcat aggctgattt ttttggggtg 13020cgtgtgtggt tttttttttg tttttttggt aaataaagac ctgcattttt attttgatat 13080aggtggttga gttttgtctt taatttcatg acagagattt aactagtctc aacttttgaa 13140aagacaacaa tgatatttgg ggatcacaca cttaaagtta gatttctaga tgattaatac 13200caaagtagat gattttttag cctcagccat ttataggtat gcccttctgt gaatttttta 13260tgacagtgaa aatcatggca cagataaaaa ttaaataaat acttctgtta ttttcctgaa 13320gaaaaaaaaa aaaagcttaa actatgagaa tactgtcttt gagcacttta aaataaaatt 13380gacttcagcc agcaggattt tgagcattac atcacaaata aaaaacaaga ttaacatcaa 13440aaggagtcag ttttcattca attgtgcagc actgtgggct gtgaaattta atattatttt 13500gactcatatg ctaattgtag actgacagag gaaaatggat tgtgtttaaa taaaaggata 13560cacagcatca cacgcagctg tatcaaatac aagttgaggt ctttgggcca ggaactgggg 13620gccctctagc tctgttattg cagattcaag tttgacaaat aaaactttcc tttagactgt 13680agtttaatta ctttttttca aaggtatgcg tgatgaagag gcacaaatac acctcacctt 13740gaagagttgc taaactggtt tgtgtgccga tcagttcacc gtgtgtttga atttctgtgc 13800ttctcatctt tccttttctt gaaaagattt tgcttgtcat tggtgtgaat tgtacccccc 13860acccccaccc atctagtctt tgctctcaga tttataacac tttaatggtt ccaaattgta 13920tagcctgctc ttagacccct tttcttttcc ttgaataaat caggttcatg ttgcagacga

13980tatttgtttt aggaaagtgt gaaagaaggg gcacctgtga aaacacgcaa ttgttccaac 14040acacatatac atccaaatta aagcagaaaa tgtcaaagcc tccaatcact accttatttc 14100ttggaggttt aaagccgctg agaagatagt ggtgccctcg ctggaagttt taaggtaatt 14160actttttact ctaagcagta gtatctggta acctaattcc gtataaacct gacaccctat 14220cgctacaccc cagtatttct ctgatttcag aataagtctg cgtagaaact tgttctgatg 14280ttaaagtgca aaagggggca gtaaagtgct atccacaaaa aaggaaaaac attttccaag 14340tatttcttat tactgcctgt gtctttcgta ggccctgcct ttatttattc attttataac 14400aaaactctta tgtttggggc attcagagaa taccttatta agctgttgca gcaatctagc 14460attaaatgga agacatgcaa gactgaagat cctgcctgtt tatgaagtgt gccatcaaat 14520tcacatgctc atgatgcaga gtccttcttt gggagtattc gtattcccaa gtgcacagag 14580cacttcggaa aggagccttg gtctttggtg ttaatgctct cctagctccg tatagatgtg 14640gcaggcccaa agtacatggt ggggtgaagg gtcaagggtt tgggcttatc cagagcagcg 14700tgcatccttt gtcaggaggt gactggaaac accagccaat tacagcagaa ctgcagactg 14760ctcatctgca ttcggaattg cagatgaacc agtttgtact cgacttctct tcttcactgt 14820aggctttgac atttaattaa aaattaaagc cttttatgga aaaagtacat gttttccaaa 14880atggggtaaa ttcgaagtat acttgataca gaacactggc ttgggaataa acctgtgata 14940ttacatgact tttggtttgc aactgctagg ctgagcctct ttgtaaagct gggatttaga 15000atctttgaaa tgtttgtaca gttcaatgat taagcataaa ttgtatatat tccctttttt 15060tcacttattt gagtaaacaa gtttgttact acagcttctg tggactcaga gatttatgta 15120ttaaataggc cacaacttca actaggataa ttttatttat ctgcttgtta gggaattgca 15180tcaaaagttt aagtctgtag gcattaaata ttttaaatgc ttatttttaa agtcaattat 15240gaaagatagc acaaagtttt tctgaaacta cattaaaaaa ataatgtttt aatcttatca 15300caaaagcatt gactatttat tgcaaagaaa acacagaaag ctaaaaatca ttctaagtcc 15360accattcagt agcccaaagt ggtctcaggt aaaggcggtg tgtgtgacca tttgtttatg 15420gttgtctccg tgcagtcagc aaaataaaca gaacaacatg ccatatatta ttgatgtgta 15480tattttcaac tgaaattagc catctgctta caatgatcat atacactaat ggtataattt 15540tgaaatgaaa agaaaaataa aataattctt tgtggagagt aatgcgaatt gacttatgaa 15600tctcgccctg cttggcagtt tgctctagag gtagaagagc tttatgtgtg ggcctcctcc 15660ccccccacac atttattctg ctcacacttg caccagcatc catgtcagga ctcaccttgt 15720cctgttacat gagtaacatg gccctgattc tcaagtgcat gataactgcc ataattacac 15780ataaatatta aatatttaaa tagatcttta cgtgtgtaat attaggtaga agtggctctg 15840gatcgaatct gatgcttttt aaatagaagc tttcccacaa catttccaag cactgtcatc 15900gtgtctgtct cgatttgggg tttacctggc ctagttatct gtctgggtgt agaaactggt 15960agttcctgtt tgtatctttt ttgttctgat ctctttattc tgtgtcagct aaatattctt 16020gcagtcagtt actaacatat taactcatcc ttgtttggaa actttggcat atccttccat 16080ggtttccttc cgtggacctg tcgcgtctct caggagagcc accaggtata ttgtcacaca 16140tttcgcatgt attttcagag actacagcag catcaagtgg ccccccagcg atttgggttt 16200tcttctcggt taatctacac tctttggcca accgtgagaa aacttgtaag aaggcatcag 16260atgtttgtgc taaggtgcgt gtagtatggt cagaggaaga aagaagcagg gaaaatggag 16320tggccgtggg tgggagggga agcagggagt gcaatttcgg gttcactaca cagctctcca 16380taaacttctc cactgctggc ttcccacgga tcctcctatt acactgggca aagtgcagaa 16440atagatcagg cgaccactgc ctccgtccat ttcccaggca ccctgtgaga cccgataatg 16500caatacaggt cagcagaaaa gtccagactt gacatcccaa cgtgccatgg tctggtctgt 16560gaatgaaaat cacatgaggt gacctctgaa ctctaagtgg ctggtttatg ttttcagtgt 16620attaggcccg tgttttaaac aagcatgtgc tcgtagtgta ggttaaaact ttctgttgtc 16680ttcattaatt atgctgtgtt ctagtctatt aatattaaag aatattgtgt tgcataatga 16740ctaatttttt tattttttgg agacggagtc ttgctctgtc acccaggctg gagtgcagta 16800gtgcgatctc ggctcactgc aacctccgcc tctcggattc aagcaattct ctgtctcagc 16860ctccgagtaa ctaggactac aggcgcccgc caccatgccc agctaagtgt tgtattttta 16920atagagacgg ggttttacca tcttggccag gctggtcttg aactcctgac ctcgtgatcc 16980acccgcctca gcctcccaaa gtgctgggat tataggcgtg agccaccacg cctggcaaca 17040taaggactat tttttaaagt ttttacaatt atgactgtga agttgaaatg tctaaattat 17100tagagatcca gtttagatta ctaaatattt atgtctaatt gagatgatta gacttagcca 17160aagtatccat gtagaagtat tagagtctag attggtgaaa aacttgaaaa agcttggctt 17220aagttcaata ggtaatccaa gagtaaaaac agattccaat atcagatctt ttcaccatag 17280tcatgttaag tttggaagcc ctacttgagt gtttccagtt ttttccacat tatattgtgt 17340ctatatttga ttcaaaggca gggcatctat tgtcttgctt aggactgatt cactgggaaa 17400agccactgga gttgcctatt tccactcagt atgcctcact cttagagtag cttcccatgg 17460ttcccaggca ggccctccag tgagaatgca ccaagccaca cgccatggcc tgggaagcag 17520tcctgaacct ggagattgtc ttgatggaaa ggaagaggca gccttcccct cccaggaaga 17580tagtagagag cctgctctga cttcgctcag ggatggaact ggtctggctc agttctctct 17640cctgtgtggg acatgaatca ctcttggtgg tctttgcttt ttatttgggc ttaaaatcag 17700cagactttat taaatgacac ctctctctaa ccactctctg tctgggcgaa gtttaacaag 17760aacagcctcc ccccatgtgg tatgggttgt aactgtggcg gtttccctct gctgtttttg 17820gttacaagat gaacattatc tgaacacaca gaaagaaatc tgtatttggc atccataatg 17880gaaagtcagt ttagtaattt aaacttagcc agttatcatc atcataattc tttttaacac 17940tttcaaagtc agcataggag aagtgtattg ttgaatatta caaaatattt agggcataga 18000tagatgtgct gtgtagtttg atttgttaat gtgtctaagc aatcaaagca acagaattca 18060aatataaacc ccatcacttc caaaatagga actctgttta ctgacttgat tataacatat 18120ggaactcaat tgttttccat taaaaaatga tactattagg aaactcaccc cattttcttt 18180tcatatatat tctgctattt gcataattgt ctggagtcca tatgtaatat taaatgtaaa 18240acacaaatgc catgtagctg gtctgtttct tcctcacctt ttggttcctg gcctcctggg 18300gaagggttgc acatctgagc cgtggtctca gatgactgcc tcggaagaag cctcttccct 18360tcaggcacca ctgatgtgtg cttggtgtgg agctagactt tccctggctc tccatgtgac 18420gctcacatgt gcgtgtcttg atttccctta acttcatggc ttatctatga acagcttgat 18480ttgggggaaa aaaatgtgtt tcccaatgct ggagttataa ttgaatgtgc tgcagtcaaa 18540actgaaatgt gtgcagagaa agggggcttt tcctgtcatg ctcattgggc accagtgtgt 18600cttcacctgt tttgtgtgtt aggtccatgc gtcatgctga aatgaagaac atgggatgta 18660tggggctttg gacagtgctg agccaaaagc aagtgctcaa aagcagctgt gtttgtatta 18720ttagtggttc tggaggtggc tgattgcctt gcattttaag tagagaggga ttgtagaaga 18780ctgccaatac ttagaacttt ttccagagag gaagggtcag aaactgcatc tgcagggctc 18840cttgctctcc agaaatgcca gtgtgcctgg gagggcatct tcagaaatcc agtctctcct 18900cctcagtgtg tcctgtaccg actcagtggt tctgtcttca gaattcctat catgtctgtg 18960atctgcaaat agtggtattt aatttgactt caatttgtat aaatgttagc ttctatttgt 19020tcattcctat tttttgttca attaatacat tatttattga gcatctactc tgtgtcagcc 19080ccttgggtgt ttaatactga attagtcaca tgtgggactt gcctgccctc agggagctag 19140actataaatt cctaatgatc agtggtctcc acttttctgt cactcataat gtctggcaca 19200acataggtta cttgagttgt tacactcaca gtactgttgt ttgctgccat ggtgctttag 19260gaagtgtgag agttcccggg aggcagagtc aataatgcag actacacgta gtgaaaacat 19320ggccaggaga gctgtagttc aggctctcag ctcaactgca ctctgtccac tgagaagcca 19380taatttcttc acttaaagtg actgtgcgct atggctgttt atatatacgc ttaaaaagta 19440aaagctgcta aaccactcaa ggattggggc cttttgtatt gatttaatta aaggaacaat 19500cattgtttta atgagctcta gaaacaatta cttttgaaga gccgaggatc aaattcttgc 19560ctcacgtttt gccacagtgt gttctgaaag gtgaattaat gcttttggaa tcatcaggaa 19620tagtgagctt tgtcacgatt tactttttac aagcgtatct aatatgcata ttgaaatgtg 19680agcctcccca ccacacttcc gctttgataa gcatcccccg gattgccgtc actgaccatt 19740atagattttt aacaaagttg gacagtacac actgaatgaa aactttacat caaggaaggc 19800ctggcgtgtt tgtaaaatga attaaaaggc tcattaaatg atttatatga cttacgcctt 19860ctgaaaatat ggcctcaaac acagagatcc ccaaagccac accgacccct gcgtcccatg 19920ttctcgacct caccgcatca gcaccagcaa gacctgtcgc tgagacggtg agtgatgaga 19980gtcaagagga gtgacttgca tggcctggga ggaaacctcc tgtgaatctt tagttaagca 20040ggaaaaaaaa aatcctcatg aaggaaacag gatcttggga gcattttgaa tgaagaagga 20100gcttagtgag ccaaacttga gacatagggt gtaatgtggg agagttttaa gatttgcaga 20160gatgtacagc ttgggagggg gtgtaatgca ttttcttaaa agagctgaat gaatggttga 20220ggaaatgggt acatctggtt tggttaagga tcctaatctc tgaagcctgg gatgccccca 20280gggcttgtaa tttaggaata cttcccctaa tagtagctaa cccttatata gtgctgtctg 20340tgcaggctac aaaaggagca gattaaggat agaaaaggtt tggagtgtat gagaaaccct 20400aggcaggaat tgactcctgg tgtttgtaaa ccttaaagat gtcctaaaaa ggtcaaggaa 20460taagacagga gaaaaaggaa atgtcaggaa gatgatcaat ttaatgttta tggaatttag 20520tttgtactta ctgcccggca tcttgcctga ggtttttaac ctcagcagca catcagaatt 20580actgtgtgtg tgttggaggg gctgggggag ataaagaaat tagcctcatc ccaaacattc 20640tgattcagtc tgttacttga gaaactgaat tgtgttttgt ccataaagaa gatgaaattg 20700tctacagaga acacattgcc attcacaagg ttgaggggat accacagaga ggctcccact 20760gtgatttgca tttgtcaaaa gttctagaga attcttcaac agtacacaca tggttgtttt 20820aaatatatca ttgttataaa aattcgtttt gagttctgtt tcacagaaag tttttttgaa 20880tgaatgaatg tcatatatcc ttgctaaagg agctcagtta aaaaaaaagg gaccatcctt 20940ctcttttggg ggttgtacag taacacattc ccaagaaaga ggtaacagcc acatacattt 21000ttcttcccaa taaagagtgt gggtttttaa tatgaatcca tagtatgatt tctgttatgt 21060tttgtgctgc ttcataacca cactcatgca cttttcagaa aattaatacc attcattagc 21120ataaatcata aactattccc ttggtatggg tttgaaattg ggggtgccct atcatccttg 21180ctttatctct tagtgaatta tgaccctgta gtcatcatgg ctggtgggcg tctctggtta 21240aagaaagggt tggattggaa ggattcagag gcgattcttt gttcttaggc tttaatattt 21300taatgagcct gcaggcttgg ctgcttacga acgagctgag atttctaagt gtgttgttag 21360tgttagcact tgtagaagga tgttcattag gaagttcttg tttcagtttt tcagagaaac 21420tccccattaa gaaagatcat tcaggaacat ggctaccaag aaagaggaaa gggaggaggg 21480aggctttcag ctataagcat taaggggata ttgtatcagt agtcttagtt ctaaagattt 21540gcttctgaga attaattgga gcaaatacat ctcaagggaa gaaaaaaaaa gatttatagg 21600gcagggacag tagttgtcct tgcaagtaga ggacacttca ttttgcagct gaatcaatac 21660cacaactaat tatttctggt tatcttttac gcatttgtaa gacattgctt ttgttcagtg 21720taataaaaaa cccattgttt gatcagtgac tgactaatta tgataagtaa tttgaaacat 21780tcttgatgaa acttgtctgt taattaacat caacagcaca gggaaactaa caggacaaca 21840aagtattagt ggatccactg ttccctccaa ttgacgagct ttctctgtgg catgcccaat 21900aaactaaagc tgccaatggt taaaaaataa caaacatgtg ggagatctga ctcaccacgg 21960aggaagagtt atggtaaagt tacacaaagg agtactgaaa tattacaagc gagggggtgg 22020taaagaaatg tcagcaggta gcctgatcct acagcttaga gtaaggaaag tggtttcttt 22080ctgtctttcc tttttctttt aaagcttaat tccaaaatac attcatccca tattgatctg 22140aagtaagaga cttttgataa attaaagtgt gaatctgaaa atgtgtagtt tgggattatg 22200ggcattgcct ggctatcttg taactgtcat taatactgtt aatttttatc aactcaatgg 22260cttttttttc ttatgctttt agatttctac ctggacaagg actggtacta tacccacaga 22320taggagacaa attggatatt atttgcccca aagtggactc taaaactgtt ggccagtatg 22380aatattataa agtttatatg gttgataaag accaagcaga cagatgcact attaagaagg 22440aaaatacccc tctcctcaac tgtgccaaac cagaccaaga tatcaaattc accatcaagt 22500ttcaagaatt cagccctaac ctctggggtc tagaatttca gaagaacaaa gattattaca 22560ttatatgtaa gtataatttt attcatttat tttatagaaa ttaagataag ctatataggt 22620ttgtatcaat tttttgtttc cttaaaatta ttgtgacaaa taatttgatg aaaatctatg 22680tggaaaaatt gtcccccccc cctttttttt tttcaaagaa aacttcattg aatttgggac 22740cctgtgctac cagtattcat taagtataca tacccaaaga gaaaaaaaaa cactagaatt 22800cttaatagta ttgaaataaa tgtattatat gaatatattc agcatctcta ctgacaaaac 22860catttttaag gaccattggt ggattttgat aggtaaatct tgtgcattgc cttttctctt 22920cacccatcca tccattcatt cactcattca tttcgtattt attctgtgcc agagactgtg 22980cttaagggct agggattcag cagtgaaagg tggtaaaata gcatgttttc ctcaagaagt 23040taacagtcta gagaagatgg agctcataaa ttcgaaagat ggggatgaca ggtcacatta 23100aaaccagatt cagaagaaaa agacgaaact tggtttgctt agtacattac tcttttttgc 23160atacatatat ataatttgac acgctgtttc aagaagagat ggtacgtatc ccttgggtca 23220tatctgaggc tgacttgtga ggatgtgaag tcagctgatg agcacatttg gagcccacgc 23280ctactatgtg cagatctctc gtcagcgtca ttcccagggc cccaggtggt gttaaagtct 23340aggtgactca gacagctgtt cgcgtcattc aagcaatgaa gtcttttttc ttaatttctt 23400tggtttaaaa ttatactcat aattaattgg gttgaatttt ccagtggctt ggttaccata 23460gacttcagtt tattagggaa ctgctatctg ccactggttt attatttgcc ccaaggtgga 23520ctctaaaact ttaggtagga gactcttggt gatcaaactg aaactcttgc atctcaacct 23580atgagccgca ctttattgtt attttatttt tttagagaca gggtctagct ttgttgccga 23640ggctggcgtg cagtggcatg atcacagctc actgtagcct tgaactccag ggctcaagtg 23700atcctcccac ctcagcctcc aagtagctcg gactacaggc atgtgccact gcacccagct 23760caagagctac acttcaaagc acagaatgaa aacctatttt taaagccaac ttgatacata 23820gagtagctta ccaagaatta gtaacaacaa caacaagaaa aaaaagagag aatgtggtag 23880agtatatact tagtaaggag taattattat aaaataaaag cattctgaaa tgaaacaggt 23940agatggggtg gccaagtatg cagcatagta gggaaatctt tgaaaatgta aaatagttac 24000caggtaaaat aaatggaaac tttaagcttt tggaagccta acaatgtatt tatattagta 24060aagactttat ttttttattt tattttattt tatttttgag acggagtctc tctctttcgt 24120caggctggag tgcagtggcg tgatctcggc tcactgcaac ctccacctcc tgggttcaag 24180tgattctcct gcctcagcct cccaagtagc tgggactaca ggtgtgcgct aatttttgta 24240tttttagtca agacggggtt tcaccatgtt ggccaggatc atctggatct cttgaccttg 24300tgatccttcc gccttggcct cccaaagtac tgggattcca ggcgtgagcc accgcgcctg 24360gccttagtaa agacttttaa agtaagactt tttcagtgaa agctactgtt aggcatgaca 24420tttacaggca actgaaactg atcagatgca tttattaaga aggttaatgc ccctaggtgg 24480ggtgggagaa agaaggtcgt ggtacgggaa gaggggacac actagagatg agatgcccta 24540gggcagtgaa cgcatgtccc taatgcgtgg atgcagccca cgtccaccga taatgccgac 24600acacccagag tctctcttct tactttagct tatgacttca cgaagaatgc tttgcaaatt 24660ctaagttcgc actgggcgca agtggaattt tagtaaacat taagagttta acctttagtg 24720tgaaataata tgcaagatat gcaaataatt gtttaccaac atctctttgc ttaatgtggt 24780gagcatttaa taattgcttt ttattaatac atgagagatt tgtatttaga agcagtttaa 24840tttataatta taatattaat ctacacaata acgacatcta ttattttctt tttttggaaa 24900ctcttcatac cacactaaca ggttcattgc agttactgaa ctactctggc catcagagct 24960ctccttagag ttacgattta ccatgcaaaa gcatatggta gcctgggata aatgaatctt 25020tcttaataca gaattgaggg tctcaagttt gaaactacga gaggctattt gaatgttgct 25080ttgggggact gtcataaggg ctgggtggag gactcagggc taagaagttt gccaggaagt 25140ccagttgaga ctttcagcag agttgaaaga cttccacgat ggcgtaggca gaggaaggcg 25200tttcagatac ttgggaaaat atagaagcca atttctcacc caccctacag caaagctcat 25260tgatctacaa gtttccctag aaaggaaatg ggaaatgcag agaacaaatg ttaaaatagt 25320tttagaaatt aatattgact ttgtattgct tctgcataag ttccaagaca ccaaaacaat 25380gaatggattt taaaaagtca ctactttgca tatcagacaa atgcacacac acacacacac 25440acacacacac acacacacac acacacagtc aagctctgta ctggcttttt tgagaaggaa 25500agtgtttgaa gttagtaatt tttatatcag tacatttata aatagtgcta ggtagcatga 25560cggaaagtat taaaatttac atgtatattt ttaacacttc aaatcgttgg ttcactttga 25620gacagtaaat aatattagca tttgagttca gctttaataa attctacatg ggtttaaccc 25680caaatctgag tgtctagttg gtaagcgcct tcagaacgag cagtgttata ataaatatgt 25740tattgtgtgc tggtttcttt ccatggagag gaaaaagaga cctgatgctt tggaggagtg 25800cttgactttt ccccagtgag gagtagtcca gagggactga cttgcattgg ggagtaccct 25860acatgaacag catttcagaa gaattaaacc aggaacctag agtcctactt gctagtcctg 25920cttcctaagc ttaatgagaa agtcaatttt atttctttga actttaattt atttccctaa 25980aaaacgcttt tagtattgtc attgttctgg ctaatgatgg cggtctcctc cagtttcaag 26040ccaccttagg gctgggcata caaatgcaat ataggatcac ttgttagtgt ggtttcaaat 26100ggacatgatc ctctgtaaat tctttaaaaa catttaattt gatttgtggt gttacctgct 26160ttaaaatata gtcatcacac ttgtgagttt cagacgtgaa tatgaatttt taatttgaac 26220tgtattttta aacacactaa gtattaacta agtcccctta ggagatatgt ggcaaactga 26280tatgcatcct cattcattct tctcatagat ggttatttgt tttttaactt gtggcaaaat 26340tatatatgaa tggtcaccga cttaaaatag ttccacttaa atttttcaac tttctgatgg 26400gtttattgga gtattaaatg tattttcaat ttaatgatat tttcagctta ccttgtgctt 26460atcaagtatc aagacatagc cccacctaag tcatggagca tctgtatatg ggtttttatt 26520cttgtttaga attgactttt tcaagtgacc tatttcagta attagccctg ggcctgattt 26580gcataatgag atctcctaat cttcaagtaa tgcaaagatg gagatattat ggccatgtgg 26640tctgaagaga ccttttcttt attatgttca gatctttaat tgccttaaaa atagagtagc 26700taatttacct aacctctagt tattttatta ttgtctttaa agtttttttt aatgttcatg 26760aaataactgt tctgaaattg cctattttca agggaagctg tgtcttagac ttactaaatg 26820ctccagttga tactgggaaa gccttcttgt gttcgtagcc tttatccgta gagttttctt 26880tgcagcattt tctgtgcctg gtttagtttc ttttcagagg cgacacccag agctgaatga 26940gtcagcaggt ttggtgtgtc gaccctttgc aacagctgtc cttacgaagg ttctgtgggc 27000tggttattct accttcgcat aaaaccttgc aaaataaccc acaaagaggt tttcgtcaca 27060ctaccaaaat catgtgagtc agagatggat gaaaaatgaa tgccattgtg ttcatacttt 27120tccagtgaac agtagctaca gcagagctgt tagacaaaga aaaccgtatt aatgaagcgc 27180ctcccaattt agcttcatat ggcttttgca ttattttgct gcaaatccat agctaagaca 27240catcttgtgg catagtccgt aagtcatctt tccgaaggac tgtttgatta aaggttgttc 27300tgtgagatcc accctgtgtt gttcatggca tcctcttgga ggcctccctc actctccatg 27360ccttggcaaa gtcttcctta aggaacactg aacaagtctg gagaagctgc catttcttag 27420ggccctcatt ggttcagttg tctatagctt tttatttttt attttttttt taataaagag 27480tatgtaaaat tggaaagctt cacaaacagc tttgctattt tttagacatg tactccactt 27540ctaagcaaaa tcacaaaata aagtaaaatg cttccacaaa tataatgaaa caatattctt 27600aaagaatcaa agcagaagaa cttcagagtc tgttgcttat gttaagcata tatttgtttt 27660cttctctgct tttgatttac ttatttctgg ggtgtaggtt tggcaagtag tactgaaacg 27720tactgaatgc actgttcttt agcaagatag ttacaggagc tttcaaatgt cctcttaaca 27780tatagatttc ttttagaata tagaataatg tgtgggctgt ataaagcgat tatgtgcttt 27840atttgatgaa ttatttatgt acgataaatg tagcaaaagc cacatttcca tcattaaatg 27900taatcccatt tggtgataca gcaacatcag cctgtcattt gggtcctctg attgaggggt 27960gaggatttct gtttgatacc ttgtgcataa tggctgcgtt caagcattta aactcatttt 28020tatttctaac ctacagctgt catctttgta ataggatatt catcagaatc ttgccagaga 28080ctgtgcattt gggatcttgg gggatacagc accaccacca ccctccccct gtccaagaga 28140aacagatcaa catcttaggt tgagagtctg gggtctggaa gacccgagtt cctgagtgcc 28200ctttgacaag taacttaacc cctgtctgcc tcagtctctt catctgtaaa gtggggataa 28260tgacagcacc tgcttcacag ggttgatggg aatccagatg tggtgggata tagaaaatgc 28320ttattacttc cacctttgac accaaataca tataactaag agttaacttt ggagcagggg 28380aggaagtgtg aggctccagg ctggaggcag acctgtgttc ggctgcaagc tggagaggat 28440ggaccccaaa agcttggctg atttgaagtc catccataaa atggaactcc agagagttta 28500cacgtttcag taatgctgca taacttaatt ataagatctt ctctctttgt cttctttcag 28560tgttataaaa gctcttttgt ccttgagctt cctttaccaa gaaacatgca tttatgtatc 28620tttttgttca tggaattgcc caagcttgtt agcagatcct ttgtaagacc caaaagagac 28680agacagggga ggagtcttca gatacatata atcatttttc ccaatttcca tgttaccagc 28740cttgccagga ctttttctca gttccctgtt acacaatgaa aatagtgtct ctttattgat 28800aattttagta gcatcctaat gtggtataaa tcgtcttcca gagaagaaaa tgtgtcaggg 28860ttgcgttatc actgaggcta gctgggaaag tagatcagcc cattagtctg ataattcgaa 28920gcgttgtttc tgttatttct gaacatcatg tgaactcctt ttctgggtgt attaaaggtt 28980ttcccagtgt gtgtcagtga gactcctgat tgaatttaat atgaataaag ataaattctt

29040tacatttaag gattaaagtc tcagcttctg cttaacttga gattgcactg agaaactcct 29100ggctctcggg tatagcggag tcacgacctg gggatgtctg tcccatatgg ctctgtgtgt 29160aagaagaaaa agctgctgtg gacggagact ctgttcacat taaatgacat cacctaagcc 29220atcatgacag caagaattat ttaggaattg ctcagaataa aactgccttc attatttcat 29280aaaatgtatc ttggtatctt tagcacctta tttatggctt tttaaaggtt cactgggatt 29340tataaataat tggacaatgc tagagaccta gtacaagaat gaaagaggac aggcttcttt 29400cttaataacc tttaaacatt catcaggaag ataaaacttt aaagcaaaat aaaacacatg 29460aaaatagcca agatgcacag accagacaag caaatactac tttaacttat ttgtatagtt 29520cttaagagtc acatttgttc ctgaagtttc aaaatctcgg gctgagtgtt tgatcactta 29580gggaagtgtt gtggccttca catactcttg tctcactttg aagtctagaa acacaggtct 29640tagagcaatt tttatcactg tgagaaagct gaaacttagt gtgagtagct tagtacaatt 29700cagttggcca tcaaatgtca gaaacaaaac tcagtccagg gccgctggac ccttaggccg 29760gcgttgttag tttacaacag tgcctcctgg gtccaaacat ctaagtgcac atgtagcaat 29820agtaaagata gtatgtatgc atacataaca catatgtaga gacagcagag tatacgtaca 29880cacatgttgc atacatagca acagcagaga agctcatgaa ctataaagga tggactgtat 29940gcttgtatca gacattttgg tactgacgct ttgtcatata ttgtgtaaca tataaccagc 30000ttgcaatcat ctgcccccaa agttgaacta agaaaatcct acagggtact aggaaaggaa 30060ggccattggg aaaaggtggt tatagtggca atttgttagc tcttatgaat tttctttttc 30120tttttagaca tactcttaat tccatttttt caataaatct atactatttt gtgtttttat 30180gttagcaagt actttaagcc cctcaataga aagttgctac atcatatagt gattaaaaat 30240aaaaatctct caaacataca agtagaggtg gtatgagact tcaaattccc ttagccaagt 30300acaagtgcag cagttttgtt ggctggctgg ctgcatagaa ggactgatgg attggcagac 30360cctcaagctg gagtgtaatt gatctcatta cagaggagcc aggctgggtg acagttgtgc 30420tttgcaagtg gttttttgca ttggtgaagt agcccatttt gttgttcctg atgttaaaca 30480ggggatgaag gtattctttt attggcacaa acgcgggaaa ttgctctgga ttcttagagg 30540atagaacatg tcccctggac ggaataaggt tcatgtgtag ggcaaattta gataggggca 30600ccttattggg gttactactg gtctctagat ggtcaaagca aacaacatgt ccatctaagc 30660tgtgatgtcc atctaagctg tgtgtgtcca tgagagtgac gcattttctc ctctgcagtg 30720ttgttatatt ctaaactgtc agcagacatt aattcggtcg ctggtgaagt cccaccgcct 30780agagatgaac tctgcctccg atggatgttt tccacttcag tgccactcgt ctcgcaatta 30840ctgggtcatt aatatcattg catgcaatta gtgacagtag aaagagctag agggttgtgg 30900gatgtgcacc ctccccacca tgaacttttt actctgaccc tttcccagct agaccttttc 30960gtatcttggc aaggatattt taatgattga gactgtcaga atcttcagag caggcactgg 31020attatgtgct ggaaataatt cactcaaaca cctgcttctc catggttcag aatattttca 31080ttagatatta tcactatccc ttccctggga agtttcattt ttaaaaatct gatgcttaag 31140tacagctaat atagacaata gggaattatg ttttatcttt agaactctta cattattctt 31200ttctttaaaa atgtgagctg agtcattgct attgcagtgg tcatctggcc gcctattttt 31260aaaacacaat tcctctatct tagtagattt tggcccatat taagcatatc aagaatgact 31320tttttttttt caagacatgg ggttttattg ggggcttata tacaaggaaa gagagagtcc 31380agtggcagtg ggctggacaa gatatccaca tggccctgtg gcagtgagct gggcaggaaa 31440actgcaactg cttgcaaaca gcatgtagtt catctatagc attttcactt aacaccaccc 31500agctaatgac ttccacctgg caaccttcat ttaatccaga acttaggacc tcgagtccct 31560gtacggccca tgttccacag gatgggccga gggctcagct gttcctcata gacaaggaat 31620gactctccac attggccact cccggattcc ctagctcagg acacatattc aggtgtgtct 31680aaggctggct cttctatgtg aagttactta ttcttttacc attgactctc atgttcccac 31740tatattaagt ttttctgaat tactgtggca ataagaaacg gtcccttaaa ttatactaga 31800agaaaagctt tttttttgtt ttgtttttta ttttgaaatt atgttaaatt ttttttctta 31860actgagagat tccacctgca taaatcgtca taacttttaa cagtaagatc ttagacttag 31920aaagtgatgt ttttcctcaa cagaatttat taaaaatcaa gacaccaagc tgttccaaac 31980aatagtttga ggggaaataa aataaacaac tccataaata atcttatgtt gttaaacatg 32040tctctagcaa aacaaacaaa caaaaaagtc gggggttggg ggaggtgcag tttattgcca 32100gtactgtctg gtctttctca gaaaagcgtc agtgtacatc actgagcctg gacggtatgt 32160tttcttgatc tataccccct atgtgtacat gtgcttgcac gcacacacat gtagacacgc 32220acacatgtgc acctgccatc actttctgct cttccgtctt ttcactcttg agtgtctgta 32280gccagtagct ttccaggtct gtatagtcaa agatacctat ggccctgaat gtcttcactg 32340attgctattt gacattcata cggtttttaa tggttaaaag gctttatgcg aaagctgtga 32400tagaatttct cctgttctag atgtggtgtt tattgcttta ttttgtgact tttctctcag 32460tagattgacc ttctccctca gtgtccaagc ctcgcatagc atgatggcac ctgtaaactc 32520agttctgtat cctggtatcc tttctcttcc caagtagaag caattaagta atatatgtca 32580tcaaaacctt ttaagtgcac atacaaacaa aatcaactta ccaaactgct tcaaagttgt 32640tccatgttta acactcttct ttctgagctc tgggtagaat gtcctattat tgttcatcat 32700gaatatttga aattaaagaa ataaaactgt accattttct ttaagagcat ccatttgtac 32760ttgataacat cttcagtcat atttcaatgc tggcaaagag gaggggagtt ctaaactgtg 32820actcaatttt agaatctact ttttccaaat tattctgttt agtgcagaaa actaattaat 32880agtgttgcat agaaaagtca ctgaagctaa gccagttatt acttcttaat gcatgattta 32940ctgctttaag ttttcaaaac acaaccatag caatgtggta ttaattcaag tgattcttcc 33000tatcatattg aacgatattt tcacgggtga aaaactcaca catcctacat cactgatagt 33060ttatacagtg ttttagctgt ggctccctgc atgcaaaata agagttaatc aaatgtcagt 33120gagaaccatc tcatcaagta gagggcttgt tttgtttaaa ttaactttgc taagtataaa 33180tttcttcttg aaaataaatt ctgggccggg cgcggtggct cacgcctgta atcctagcac 33240tttgggaggc cgaggcgggc ggatcacgag gtcaggagat cgagaccaaa ctggctaaca 33300ctgtgaaacc ccgtctctac taaaaataca aaaaatgagc cgggtgtggt ggcgggctcc 33360tgtagtccca gctactcggg aggctgaggc aggagaatgg cgtgaacctg ggaggcagag 33420cttgtggtga gccaagatca caccactgca ctccagcctg ggtgacagag cgagactccg 33480tctcaaaaaa aaaaaaaagg aaaataaatt cttctgtatt tttctttctt caagtgaggc 33540catttagggg aaagtatacc ataaaacttg ctctaagata aggcaaattt ggtattatag 33600gatgaagtgc tatgtgattt gaagtaatgc tgaatttttt aaatatatta aactaaacaa 33660gaataatgag gccctcggaa agtcatgatt atatttctca tttttctcat tttaaagcca 33720cagtgaaaaa cacataaaag gaagaagtta gaaaaaaaaa tgaatgaaat tctttttttc 33780cttttggcaa attaaataga tgtttctgtt tcagaagatt ttattaatta actttaaaga 33840aacagtcatt tatttttggc attcagtgaa cactatcatt tccatgttta gaacttttct 33900tctaagttag catcttaaaa gataactgtg aaactcaagg cattcaacta cattaatttg 33960agtttcagaa attgaattct tgtttctaga gtacatagtt tgaattgatg tcagggtgtt 34020aaatagataa atcttagctt cctaggttgt atattcacac taattatttt tttatcagcc 34080ttcttatttt tcaacttacc ttattctttt tgtttttttg acactcagat ttgatagccc 34140tgtggtagaa gaaaacagta atacagtttg gtttgttgtt gtgtttgtgt ttattttaaa 34200gtcacggctt tgctttccat gttgttactg gattatgctt tttttaattc ttcagtttgc 34260caagataaca gtcttccgat cttcagaagt ctgtatcaag cttaaggaaa ctgatgtgta 34320ggaagactcg cctaagaagt ccaaattagc aaggctagca tgtgaggaca tgctggaaaa 34380gaatagttcc catagatatt gacagagaat gttcataaaa tgctacttgt tttgtggtta 34440catgagagta acttgtgtcc agtgcagctg tatgtaaggg caacgttttt attctgacga 34500ctctgtggtt ttcatgaccc tggatgctta tcatgtctct ctgttggact tcttcaacgg 34560agttgataca aatacttgct tccaagtgtc catctgccct ctcctccatc ctggccccat 34620acaaatacgc tacattttta aataatttga aataccctca atagtattta tatttcctgg 34680tgcttcattc tttccataag aactgtgata ccattattct gtaggatttt tttgtgcttc 34740cccgtttcac atctctgtgc cagtgagacc catatatcgg tgcaaatcca gaagtttgat 34800tgtccatctg attagcacac tgttagcaat gtggtggact aaacacagcc aagatgtggg 34860gctggagctt agcctcctgg gagcagagcg gtgaacatca gatgaagaca tgtgaaaatg 34920gagtactact tcctcttcct ggggatgggc taaaaagcac agccagaaat attcttgccc 34980ttccagtctg ctttacagtt actcactggt tctctttttt ttcctactca gataaccagt 35040atactcttcc cagtgactaa gaactgcaga taagtatagg tgcaaataga tggcaaaccg 35100cagatggcag ctgtgtggtt tcagatgtgc tgcagaactt ttagacgatg tgaacgcaag 35160gaactttttt gctgagcagt aatctctacc cactggaaat taggccctgg ggggaacaat 35220gtagtgactt ctatatactt actacatgca gttagacccc tgaagcaaaa gcttttaaaa 35280acaggctgta aaatgcccat gtatctttat taagcctatt ttccaactgg atagagaaat 35340tttctggtaa tttttaaatt tgtaaagtct atttttttcc tgagccaagg gaaaaaaaat 35400atctgggccc taaaagctta gttataacaa tgttattttt tctatctctg aatgattaaa 35460tgtgatttca tttatgtagc aatactatga ttgtggctgc attagatcac gctgatagaa 35520agatacaaag aaaaactaag tataatgaac taacaattta ttttcactct ttctctaagt 35580taaaaattcc cagtacattc aaatgaacaa tgaaaataat tgcagaattg tctcctgaaa 35640tggaaataga ttttttttcc caagcattag caatttcttg ttatttttca aaatcagcca 35700ctaagccttt cagagcttct tggtgactat tgcaggagaa atcagaatat taatcttgtg 35760gttttatttc agagttcgct gccaggaagg aggtataatt gggataggag actttttttt 35820tttagctgtg tcactgttca aggagggggg tttggaacct cagcataaga attacactct 35880gtgatgagga tgtagcaggg gagaagaaag gtgattttca ctatgggaag ctatacttac 35940atcaagtata aaatagactg aagtcatttt gaattacgtt atacttgtaa agtttacctc 36000ctggagtttc agttagtacc agtgtactaa ctgggttaaa acagttcatg gcaccttaga 36060tcatttctaa ctcatggcaa aaatctttcc tggtggaacg tgtaactgta ttttaaatgc 36120ccctttataa gcaaccaagt atttgggatg ttattttgat attagtagtg aatttttcag 36180tatcttccag taccctttgc aagtcacagg ttgacttaaa aggaaaagaa gcaaaatgct 36240gaatatagca gaaaaactgt ctgcattcag actgttcagc ccacttttgc tccccacgtg 36300gcaagcacac tcccccaaac aagcaatagc ctgtggcttc agaggaacct acaaaggcag 36360catctgtaga tttttccttc ttcaactcta agacttgaat gtttccctct tccccacaca 36420cttttttttt aaaccaagaa ataaaaaagt tttcactctt aaaggtgcaa agcagtttca 36480ttcttatgca acacagcctt cctcctactg tcttatagtc tgtggatgtt aaattataga 36540ttccaattga attttaatac tctagagatt ttacatttgt ggttgtcaag accccgtttt 36600ggtaaaccta gggagctccg cacaaaagca ttgatattca gaaaaggcac tgacctacaa 36660attaaaagaa aaaaaaatca aataatgtgc acctcttgtg cttccagttt gacaaagcag 36720aagtcatcag cagtttctcc ctctgcagac gcagttctca attctattta caagtaactg 36780ctctactgtg cctgtttttc tcttgctgat actcatttaa ttgtttttct tttggatctg 36840aatctttgac tgtcttttcc ccctcaagat taaaataaat acatctgtat tcctcccctt 36900tctttctgtg cactgccctt cagatctcat tttgtcattt ttcagcttag tgttgaaact 36960tttagcaaca aaaagtcagt tacttacttt gagtaagtaa ctcaaagtaa gttaactttg 37020agtttgagtg cacttttgcg tgtaggttca tttatgtgct tgtgaattta aaaacattgg 37080gattccacct gaatgaagta aaccaaacat tttaaactat cagccagata gagacatcag 37140cctttcactt ctttctatat gcagacatat cctaattttt tagaaaaatc aaataggaaa 37200attctcaaca attaattgaa gattatagct ctgctctgaa atggtccaga aataggatct 37260gctcatagaa actcatagtt tgaagcctct gggaggaaag gatactttaa aatttagtca 37320catatttgga ggagggaaaa gggaaagagc agaatgaaga actgaaaaaa atcacacacc 37380ggggcctgtc gtgaggtggg ggactggggg agggatagca ttaggagata tacctaatgt 37440aaatgacgag ttaacaggcg cagcccacca acatggcaca cgtatacata tgtaacaaac 37500ctgcacgttg tgcacatgta ccctagaact taaagtataa taaaaaaaaa ttttaatagc 37560cccattaaat aattaaaaag atttttttta gattcacaga agtgtacaaa atttttaggt 37620tttttttttt ttaagctgtc tgctgaatag tttcttaatg gtctacaatg tttgtatcta 37680caaacagata ctgtctgctt cttactaccc ttccaagaca agtattatta tggcaattat 37740tgcccagttt cccgggaaaa atttatccac agttacagaa gaatgagatg caattgtgag 37800actgtaaagt ttaagcaagc actcagagaa gcacagtgat atgtatgcac agaagaggca 37860gtctttgttt tgaggaaaac agtgaaagta aagttaattc aagaccacaa agacaagtaa 37920ataagtgcct tatttttgta gttaatataa tttcagtgga atgcatattt ctaccataaa 37980tgcatataga acttgtttgc tgacctactg tttggaaaac aaacaatccc attagaagaa 38040tgtctttggg atttattttt accagaaaat caatcctttt ttcagtccct tgcaaagtac 38100agtgttacaa gccaagactt tgataatcag gtagaaaatg gatttaaatt gcagaaatgt 38160atatgaaaca cttttgttcc ttgccccttg aactttaggg gaatgaaaat gtctagcact 38220ctccaccttc ttttctctcc tggaacttga actgtaattc aaagcctgtt tctcattaaa 38280gtacctggca gcctatctct ttacagcttg agttacaaag ctattcagag acctcgctgg 38340tctaaagaga cagaacaagg atgtgtttaa atagagcata ggctgttgaa aaaaaaaatg 38400ctgaaaatgg taaaatgatt ctgtccttcc ttccactcct cactgctgag gtggagaggg 38460aattcagttg gtgaacacca gcaagtggct ggtaaaagtc cccactttct ctccagggct 38520gccacaggac ccagaatgag tggtgggcat gtgtgtgaac cctctattca gccagagttt 38580tcccgcaaca ggtagtttgg ttgaagaggt tgactaaggt tgacattggc agtaataaca 38640cgtatgttct tctgatttac aaaacgatgg aggaaaaagg ggagattttg aagacctgat 38700ttctggtata cttcttaagc atgcataagg ctgaaaaaag aagacaaggg ttgtgggagg 38760ctcctggtct agtgtttaca gaacttggat gcttgacaaa cagagcgtca agctaattgt 38820tcttgaagca ggaaatctgc agtggaggaa gcaggtgtgg ggggatgatt accacgtttg 38880gaaatggctg cattaactat tttgctcttc tgagtttggc cccaaaagag tccatagact 38940ttttgaagga tgccatccct tttatttata gactaacatt aaatcagtca tttgtgaagg 39000aaggagaaag tgcctaaata aatttggagt cagatagcat acgtgcggca gtgtttccga 39060tatccatttc tctttatttc tttttctttt tctttttggc tttcagcatc cccatacttt 39120cagaaaactt gtgactaaga gtgaattctt atttttcaaa ttgttttcag acatttcatg 39180ttcatgtaaa cttggcttat tgatttcctg atttttcttt atttttttgt tttgtccatt 39240ttatttttaa tcagctacat caaatgggtc tttggagggc ctggataacc aggagggagg 39300ggtgtgccag acaagagcca tgaagatcct catgaaagtt ggacaaggta aagaccatct 39360gctgcttcat gacgccactg tgacctggtg tagcccccag ctagtatggt gctaatgttg 39420ccgatgccca ccttcattcg ctcttctttt tagttttcaa agcaaaccct tctgcacttt 39480gagccactga cagatttcct caagtcaatg tactaagctt ttattggaga tctaagagtt 39540aagatcagca aggtagaatg tctattgcca tagatagata gatagataga tagataatag 39600atagatagat agatagatag atatttcttt ttaaaaagca aaacactttg gttcaaaatc 39660aaaatatcca gaatgaaaac taaaagcttg tgcagttttg ctcatttctg aatcttgact 39720acagaagagt tttgttcatt gtgacttttc caatatagat aacctattgt gcagaaagaa 39780ataattattc ttctaattaa aaattggtat agtagtcaat caacttgctc agttaaattg 39840aaatgtcatc tgcaatgctt tgcctgccaa atgcaagaat ccctatagtt tccacagatg 39900gcctcacgtt ctaaacctct gaaataacta gtataaccat tttgttttaa aagaaaaatt 39960atattcttgt atttcacagt actttgcata aagactctta tgttcattgc tattcatgcc 40020tgttgaaata tatatgcagc tcctaaagct agatattgtc agatgtctgt gccgtaatta 40080atcatttgtt tttcatatag atgcaagttc tgctggatca accaggaata aagatccaac 40140aagacgtcca gaactagaag ctggtacaaa tggaagaagt tcgacaacaa gtccctttgt 40200aaaaccaaat ccaggtataa cagcatgatc tgtgtgtatg gaggtctgtg ggtaccacat 40260tcttagtagt atcttaaaag gtagggcaga gtctaaagac ttctaaccag ttaggattag 40320ctggaagtta cagtgatcag gaatctttgc tgtcagtgag tcattattaa ttacactcaa 40380taagaacaaa ataactcatt ccaatgaaag tcatatattc aaaggagtag agttcatgag 40440ctgtaagtgc cagttattag aactactctg tcaggccaaa ggtttcattg gctgacattt 40500tatcaagctg gttgtcaact ccagcttaaa gctgatgtta atgtatatgt aattaatgtg 40560ctaatccctc atctaattat atctaagcca cagagggttt aattgatcct cttctaaatt 40620ttaaatggta acatttttaa atattgcata atagtatttt ttcaggtggt tatcgttatt 40680ttgtttcaca ttttccatgt aaaagaaaat attaaacagg tccctgacaa aagtgtagaa 40740taccagataa aattgtccgt cgttgacctt cgttttctta acagtcttgg aacaaatagt 40800tctgtatttg ttaccatgct aatgaaggtt ttatagagta gctgttgagc agacatcagc 40860agttttgtat taggattgtt gtgtgcttgc ttggtcgttg tgcaaattta tcgtctgcag 40920caatattcca tccctttcca agagtcaagg agggaagttg ttatttctaa ctttcaatga 40980caagatgtgt caaattcttg tgacaaactg ataaatggat aatataatga tgccaggcag 41040ttttttagtg cttaacattt gggctggcag tctgttcggt gtgagagttt ctgctgcctt 41100ccaaatatat tttaagtgta aatcaaataa tacagacgag ttacgagctg aacattttcc 41160caggccccct cactccttcc gcgttcccga gctgttctgt tctgccagga ggcagggctc 41220ttctttagaa ggcaggccct ttgaaggttt gcatgaaact ccctttctca aaggaggcgg 41280aagagcaata ccacataaac gctcaccgct gacctggaga attggccact tccctttttc 41340ttccctgccg ctgccccagg ctggctgaca cgggttagaa gatgaagcaa gatcaagggc 41400tggctgtcac cgacagtctg tgctcttgct ggataatgat acaaaggaaa ccctgtggct 41460tgggagggta gggaagtccc tcctagagat acctctcatt tccttttgcg ttgagctctt 41520agacgaggta ttggcgaggc aaagtccagc ttctagttag taataagcct ggcttatttt 41580tcacattttt aagggtcata aaagcagtcc gtctgcactg ggacagcagt aactatctct 41640gaccttttct gtctccgcgt ctgcaggttc tagcacagac ggcaacagcg ccggacattc 41700ggggaacaac atcctcggtt ccgaagtggc cttatttgca gggattgctt caggatgcat 41760catcttcatc gtcatcatca tcacgctggt ggtcctcttg ctgaagtacc ggaggagaca 41820caggaagcac tcgccgcagc acacgaccac gctgtcgctc agcacactgg ccacacccaa 41880gcgcagcggc aacaacaacg gctcagagcc cagtgacatt atcatcccgc taaggactgc 41940ggacagcgtc ttctgccctc actacgagaa ggtcagcggc gactacgggc acccggtgta 42000catcgtccag gagatgcccc cgcagagccc ggcgaacatt tactacaagg tctgagaggg 42060accctggtgg tacctgtgct ttcccagagg acacctaatg tcccgatgcc tcccttgagg 42120gtttgagagc ccgcgtgctg gagaattgac tgaagcacag caccggggga gagggacact 42180cctcctcgga agagcccgtc gcgctggaca gcttacctag tcttgtagca ttcggccttg 42240gtgaacacac acgctccctg gaagctggaa gactgtgcag aagacgccca ttcggactgc 42300tgtgccgcgt cccacgtctc ctcctcgaag ccatgtgctg cggtcactca ggcctctgca 42360gaagccaagg gaagacagtg gtttgtggac gagagggctg tgagcatcct ggcaggtgcc 42420ccaggatgcc acgcctggaa gggccggctt ctgcctgggg tgcatttccc ccgcagtgca 42480taccggactt gtcacacgga cctcgggcta gttaaggtgt gcaaagatct ctagagttta 42540gtccttactg tctcactcgt tctgttaccc agggctctgc agcacctcac ctgagacctc 42600cactccacat ctgcatcact catggaacac tcatgtctgg agtcccctcc tccagccgct 42660ggcaacaaca gcttcagtcc atgggtaatc cgttcataga aattgtgttt gctaacaagg 42720tgccctttag ccagatgcta ggctgtctgc gaagaaggct aggagttcat agaagggagt 42780ggggctgggg aaagggctgg ctgcaattgc agctcactgc tgctgcctct gaaacagaaa 42840gttggaaagg aaaaaagaaa aaagcaatta ggtagcacag cactttggtt ttgctgagat 42900cgaagaggcc agtaggagac acgacagcac acacagtgga ttccagtgca tggggaggca 42960ctcgctgtta tcaaatagcg atgtgcagga agaaaagccc ctcttcattc cggggaacaa 43020agacgggtat tgttgggaaa ggaacaggct tggagggaag ggagaaagta ggccgctgat 43080gatatattcg ggcaggactg ttgtggtact ggcaataaga tacacagctc cgagctgtag 43140gagagtcggt ctgctttgga tgatttttta agcagactca gctgctatac ttatcacatt 43200ttattaaaca cagggaaagc atttaggaga atagcagaga gccaaatctg acctaaaagt 43260tgaaaagcca aaggtcaaac aggctgtaat tccatcatca tcgttgttat taaagaatcc 43320ttatctataa aaggtaggtc agatccccct ccccccaggt tcctccttcc cctcccgatt 43380gagccttacg acactttggt ttatgcggtg ctgtccgggt gccagggctg cagggtcggt 43440actgatggag gctgcagcgc ccggtgctct gtgtcaaggt gaagcacata cggcagacct 43500cttagagtcc ttaagacgga agtaaattat gatgtccagg gggagaagga agataggacg 43560tatttataat aggtatatag aacacaaggg atataaaatg aaagattttt actaatatat 43620attttaaggt tgcacacagt acacaccaga agatgtgaaa ttcatttgtg gcaattaagt 43680ggtcccaatg ctcagcgctt aaaaaaacaa attggacagc tacttctggg aaaaacaaca 43740tcattccaaa aagaacaata atgagagcaa atgcaaaaat aaccaagtcc tccgaaggca 43800tctcacggaa ccgtagacta ggaagtacga gccccacaga gcaggaagcc gatgtgactg 43860catcatatat ttaacaatga caagatgttc cggcgtttat ttctgcgttg ggttttccct 43920tgccttatgg gctgaagtgt tctctaga 4394894335DNAHomo sapiens 9gcgcggagct gggagtggct tcgccatggc tgtgagaagg gactccgtgt ggaagtactg 60ctggggtgtt ttgatggttt tatgcagaac tgcgatttcc

aaatcgatag ttttagagcc 120tatctattgg aattcctcga actccaaatt tctacctgga caaggactgg tactataccc 180acagatagga gacaaattgg atattatttg ccccaaagtg gactctaaaa ctgttggcca 240gtatgaatat tataaagttt atatggttga taaagaccaa gcagacagat gcactattaa 300gaaggaaaat acccctctcc tcaactgtgc caaaccagac caagatatca aattcaccat 360caagtttcaa gaattcagcc ctaacctctg gggtctagaa tttcagaaga acaaagatta 420ttacattata tctacatcaa atgggtcttt ggagggcctg gataaccagg agggaggggt 480gtgccagaca agagccatga agatcctcat gaaagttgga caagatgcaa gttctgctgg 540atcaaccagg aataaagatc caacaagacg tccagaacta gaagctggta caaatggaag 600aagttcgaca acaagtccct ttgtaaaacc aaatccaggt tctagcacag acggcaacag 660cgccggacat tcggggaaca acatcctcgg ttccgaagtg gccttatttg cagggattgc 720ttcaggatgc atcatcttca tcgtcatcat catcacgctg gtggtcctct tgctgaagta 780ccggaggaga cacaggaagc actcgccgca gcacacgacc acgctgtcgc tcagcacact 840ggccacaccc aagcgcagcg gcaacaacaa cggctcagag cccagtgaca ttatcatccc 900gctaaggact gcggacagcg tcttctgccc tcactacgag aaggtcagcg gggactacgg 960gcacccggtg tacatcgtcc aggagatgcc cccgcagagc ccggcgaaca tttactacaa 1020ggtctgagag ggaccctggt ggtacctgtg ctttcccaga ggacacctaa tgtcccgatg 1080cctcccttga gggtttgaga gcccgcgtgc tggagaattg actgaagcac agcaccgggg 1140gagagggaca ctcctcctcg gaagagcccg tcgcgctgga cagcttacct agtcttgtag 1200cattcggcct tggtgaacac acacgctccc tggaagctgg aagactgtgc agaagacgcc 1260cattcggact gctgtgccgc gtcccacgtc tcctcctcga agccatgtgc tgcggtcact 1320caggcctctg cagaagccaa gggaagacag tggtttgtgg acgagagggc tgtgagcatc 1380ctggcaggtg ccccaggatg ccacgcctgg aagggccggc ttctgcctgg ggtgcatttc 1440ccccgcagtg cataccggac ttgtcacacg gacctcgggc tagttaaggt gtgcaaagat 1500ctctagagtt tagtccttac tgtctcactc gttctgttac ccagggctct gcagcacctc 1560acctgagacc tccactccac atctgcatca ctcatggaac actcatgtct ggagtcccct 1620cctccagccg ctggcaacaa cagcttcagt ccatgggtaa tccgttcata gaaattgtgt 1680ttgctaacaa ggtgcccttt agccagatgc taggctgtct gcgaagaagg ctaggagttc 1740atagaaggga gtggggctgg ggaaagggct ggctgcaatt gcagctcact gctgctgcct 1800ctgaaacaga aagttggaaa ggaaaaaaga aaaaagcaat taggtagcac agcactttgg 1860ttttgctgag atcgaagagg ccagtaggag acacgacagc acacacagtg gattccagtg 1920catggggagg cactcgctgt tatcaaatag cgatgtgcag gaagaaaagc ccctcttcat 1980tccggggaac aaagacgggt attgttggga aaggaacagg cttggaggga agggagaaag 2040taggccgctg atgatatatt cgggcaggac tgttgtggta ctggcaataa gatacacagc 2100tccgagctgt aggagagtcg gtctgctttg gatgattttt taagcagact cagctgctat 2160acttatcaca ttttattaaa cacagggaaa gcatttagga gaatagcaga gagccaaatc 2220tgacctaaaa gttgaaaagc caaaggtcaa acaggctgta attccatcat catcgttgtt 2280attaaagaat ccttatctat aaaaggtagg tcagatcccc ctccccccag gttcctcctt 2340cccctcccga ttgagcctta cgacactttg gtttatgcgg tgctgtccgg gtgccagggc 2400tgcagggtcg gtactgatgg aggctgcagc gcccggtgct ctgtgtcaag gtgaagcaca 2460tacggcagac ctcttagagt ccttaagacg gaagtaaatt atgatgtcca gggggagaag 2520gaagatagga cgtatttata ataggtatat agaacacaag ggatataaaa tgaaagattt 2580ttactaatat atattttaag gttgcacaca gtacacacca gaagatgtga aattcatttg 2640tggcaattaa gtggtcccaa tgctcagcgc ttaaaaaaac aaattggaca gctacttctg 2700ggaaaaacaa catcattcca aaaagaacaa taatgagagc aaatgcaaaa ataaccaagt 2760cctccgaagg catctcacgg aaccgtagac taggaagtac gagccccaca gagcaggaag 2820ccgatgtgac tgcatcatat atttaacaat gacaagatgt tccggcgttt atttctgcgt 2880tgggttttcc cttgccttat gggctgaagt gttctctaga atccagcagg tcacactggg 2940ggcttcaggt gacgatttag ctgtggctcc ctcctcctgt cctcccccgc accccctccc 3000ttctgggaaa caagaagagt aaacaggaaa cctacttttt atgtgctatg caaaatagac 3060atctttaaca tagtcctgtt actatggtaa cactttgctt tctgaattgg aagggaaaaa 3120aaatgtagcg acagcatttt aaggttctca gacctccagt gagtacctgc aaaaatgagt 3180tgtcacagaa attatgatcc tctatttcct gaacctggaa atgatgttgg tccaaagtgc 3240gtgtgtgtat gtgtgagtgg gtgcgtggta tacatgtgta catatatgta taatatatat 3300ctacaatata tattatatat atctatatca tatttctgtg gagggttgcc atggtaacca 3360gccacagtac atatgtaatt ctttccatca ccccaacctc tcctttctgt gcattcatgc 3420aagagtttct tgtaagccat cagaagttac ttttaggatg ggggagaggg gcgagaaggg 3480gaaaaatggg aaatagtctg attttaatga aatcaaatgt atgtatcatc agttggctac 3540gttttggttc tatgctaaac tgtgaaaaat cagatgaatt gataaaagag ttccctgcaa 3600ccaattgaaa agtgttctgt gcgtctgttt tgtgtctggt gcagaatatg acaatctacc 3660aactgtccct ttgtttgaag ttggtttagc tttggaaagt tactgtaaat gccttgcttg 3720tatgatcgtc cctggtcacc cgactttgga atttgcacca tcatgtttca gtgaagatgc 3780tgtaaatagg ttcagatttt actgtctatg gatttggggt gttacagtag ccttattcac 3840ctttttaata aaaatacaca tgaaaacaag aaagaaatgg cttttcttac ccagattgtg 3900tacatagagc aatgttggtt ttttataaag tctaagcaag atgttttgta taaaatctga 3960attttgcaat gtatttagct acagcttgtt taacggcagt gtcattcccc tttgcactgt 4020aatgaggaaa aaatggtata aaaggttgcc aaattgctgc atatttgtgc cgtaattatg 4080taccatgaat atttatttaa aatttcgttg tccaatttgt aagtaacaca gtattatgcc 4140tgagttataa atattttttt ctttctttgt tttattttaa tagcctgtca taggttttaa 4200atctgcttta gtttcacatt gcagttagcc ccagaaaatg aaatccgtga agtcacattc 4260cacatctgtt tcaaactgaa tttgttctta aaaaaataaa atattttttt cctatggaaa 4320aaaaaaaaaa aaaaa 433510987PRTHomo sapiens 10Met Glu Leu Arg Val Leu Leu Cys Trp Ala Ser Leu Ala Ala Ala Leu 1 5 10 15Glu Glu Thr Leu Leu Asn Thr Lys Leu Glu Thr Ala Asp Leu Lys Trp 20 25 30Val Thr Phe Pro Gln Val Asp Gly Gln Trp Glu Glu Leu Ser Gly Leu 35 40 45Asp Glu Glu Gln His Ser Val Arg Thr Tyr Glu Val Cys Asp Val Gln 50 55 60Arg Ala Pro Gly Gln Ala His Trp Leu Arg Thr Gly Trp Val Pro Arg65 70 75 80Arg Gly Ala Val His Val Tyr Ala Thr Leu Arg Phe Thr Met Leu Glu 85 90 95Cys Leu Ser Leu Pro Arg Ala Gly Arg Ser Cys Lys Glu Thr Phe Thr 100 105 110Val Phe Tyr Tyr Glu Ser Asp Ala Asp Thr Ala Thr Ala Leu Thr Pro 115 120 125Ala Trp Met Glu Asn Pro Tyr Ile Lys Val Asp Thr Val Ala Ala Glu 130 135 140His Leu Thr Arg Lys Arg Pro Gly Ala Glu Ala Thr Gly Lys Val Asn145 150 155 160Val Lys Thr Leu Arg Leu Gly Pro Leu Ser Lys Ala Gly Phe Tyr Leu 165 170 175Ala Phe Gln Asp Gln Gly Ala Cys Met Ala Leu Leu Ser Leu His Leu 180 185 190Phe Tyr Lys Lys Cys Ala Gln Leu Thr Val Asn Leu Thr Arg Phe Pro 195 200 205Glu Thr Val Pro Arg Glu Leu Val Val Pro Val Ala Gly Ser Cys Val 210 215 220Val Asp Ala Val Pro Ala Pro Gly Pro Ser Pro Ser Leu Tyr Cys Arg225 230 235 240Glu Asp Gly Gln Trp Ala Glu Gln Pro Val Thr Gly Cys Ser Cys Ala 245 250 255Pro Gly Phe Glu Ala Ala Glu Gly Asn Thr Lys Cys Arg Ala Cys Ala 260 265 270Gln Gly Thr Phe Lys Pro Leu Ser Gly Glu Gly Ser Cys Gln Pro Cys 275 280 285Pro Ala Asn Ser His Ser Asn Thr Ile Gly Ser Ala Val Cys Gln Cys 290 295 300Arg Val Gly Tyr Phe Arg Ala Arg Thr Asp Pro Arg Gly Ala Pro Cys305 310 315 320Thr Thr Pro Pro Ser Ala Pro Arg Ser Val Val Ser Arg Leu Asn Gly 325 330 335Ser Ser Leu His Leu Glu Trp Ser Ala Pro Leu Glu Ser Gly Gly Arg 340 345 350Glu Asp Leu Thr Tyr Ala Leu Arg Cys Arg Glu Cys Arg Pro Gly Gly 355 360 365Ser Cys Ala Pro Cys Gly Gly Asp Leu Thr Phe Asp Pro Gly Pro Arg 370 375 380Asp Leu Val Glu Pro Trp Val Val Val Arg Gly Leu Arg Pro Asp Phe385 390 395 400Thr Tyr Thr Phe Glu Val Thr Ala Leu Asn Gly Val Ser Ser Leu Ala 405 410 415Thr Gly Pro Val Pro Phe Glu Pro Val Asn Val Thr Thr Asp Arg Glu 420 425 430Val Pro Pro Ala Val Ser Asp Ile Arg Val Thr Arg Ser Ser Pro Ser 435 440 445Ser Leu Ser Leu Ala Trp Ala Val Pro Arg Ala Pro Ser Gly Ala Val 450 455 460Leu Asp Tyr Glu Val Lys Tyr His Glu Lys Gly Ala Glu Gly Pro Ser465 470 475 480Ser Val Arg Phe Leu Lys Thr Ser Glu Asn Arg Ala Glu Leu Arg Gly 485 490 495Leu Lys Arg Gly Ala Ser Tyr Leu Val Gln Val Arg Ala Arg Ser Glu 500 505 510Ala Gly Tyr Gly Pro Phe Gly Gln Glu His His Ser Gln Thr Gln Leu 515 520 525Asp Glu Ser Glu Gly Trp Arg Glu Gln Leu Ala Leu Ile Ala Gly Thr 530 535 540Ala Val Val Gly Val Val Leu Val Leu Val Val Ile Val Val Ala Val545 550 555 560Leu Cys Leu Arg Lys Gln Ser Asn Gly Arg Glu Ala Glu Tyr Ser Asp 565 570 575Lys His Gly Gln Tyr Leu Ile Gly His Gly Thr Lys Val Tyr Ile Asp 580 585 590Pro Phe Thr Tyr Glu Asp Pro Asn Glu Ala Val Arg Glu Phe Ala Lys 595 600 605Glu Ile Asp Val Ser Tyr Val Lys Ile Glu Glu Val Ile Gly Ala Gly 610 615 620Glu Phe Gly Glu Val Cys Arg Gly Arg Leu Lys Ala Pro Gly Lys Lys625 630 635 640Glu Ser Cys Val Ala Ile Lys Thr Leu Lys Gly Gly Tyr Thr Glu Arg 645 650 655Gln Arg Arg Glu Phe Leu Ser Glu Ala Ser Ile Met Gly Gln Phe Glu 660 665 670His Pro Asn Ile Ile Arg Leu Glu Gly Val Val Thr Asn Ser Met Pro 675 680 685Val Met Ile Leu Thr Glu Phe Met Glu Asn Gly Ala Leu Asp Ser Phe 690 695 700Leu Arg Leu Asn Asp Gly Gln Phe Thr Val Ile Gln Leu Val Gly Met705 710 715 720Leu Arg Gly Ile Ala Ser Gly Met Arg Tyr Leu Ala Glu Met Ser Tyr 725 730 735Val His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Asn Ser Asn Leu 740 745 750Val Cys Lys Val Ser Asp Phe Gly Leu Ser Arg Phe Leu Glu Glu Asn 755 760 765Ser Ser Asp Pro Thr Tyr Thr Ser Ser Leu Gly Gly Lys Ile Pro Ile 770 775 780Arg Trp Thr Ala Pro Glu Ala Ile Ala Phe Arg Lys Phe Thr Ser Ala785 790 795 800Ser Asp Ala Trp Ser Tyr Gly Ile Val Met Trp Glu Val Met Ser Phe 805 810 815Gly Glu Arg Pro Tyr Trp Asp Met Ser Asn Gln Asp Val Ile Asn Ala 820 825 830Ile Glu Gln Asp Tyr Arg Leu Pro Pro Pro Pro Asp Cys Pro Thr Ser 835 840 845Leu His Gln Leu Met Leu Asp Cys Trp Gln Lys Asp Arg Asn Ala Arg 850 855 860Pro Arg Phe Pro Gln Val Val Ser Ala Leu Asp Lys Met Ile Arg Asn865 870 875 880Pro Ala Ser Leu Lys Ile Val Ala Arg Glu Asn Gly Gly Ala Ser His 885 890 895Pro Leu Leu Asp Gln Arg Gln Pro His Tyr Ser Ala Phe Gly Ser Val 900 905 910Gly Glu Trp Leu Arg Ala Ile Lys Met Gly Arg Tyr Glu Glu Ser Phe 915 920 925Ala Ala Ala Gly Phe Gly Ser Phe Glu Leu Val Ser Gln Ile Ser Ala 930 935 940Glu Asp Leu Leu Arg Ile Gly Val Thr Leu Ala Gly His Gln Lys Lys945 950 955 960Ile Leu Ala Ser Val Gln His Met Lys Ser Gln Ala Lys Pro Gly Thr 965 970 975Pro Gly Gly Thr Gly Gly Pro Ala Pro Gln Tyr 980 98511333PRTHomo sapiens 11Met Ala Val Arg Arg Asp Ser Val Trp Lys Tyr Cys Trp Gly Val Leu 1 5 10 15Met Val Leu Cys Arg Thr Ala Ile Ser Lys Ser Ile Val Leu Glu Pro 20 25 30Ile Tyr Trp Asn Ser Ser Asn Ser Lys Phe Leu Pro Gly Gln Gly Leu 35 40 45Val Leu Tyr Pro Gln Ile Gly Asp Lys Leu Asp Ile Ile Cys Pro Lys 50 55 60Val Asp Ser Lys Thr Val Gly Gln Tyr Glu Tyr Tyr Lys Val Tyr Met65 70 75 80Val Asp Lys Asp Gln Ala Asp Arg Cys Thr Ile Lys Lys Glu Asn Thr 85 90 95Pro Leu Leu Asn Cys Ala Lys Pro Asp Gln Asp Ile Lys Phe Thr Ile 100 105 110Lys Phe Gln Glu Phe Ser Pro Asn Leu Trp Gly Leu Glu Phe Gln Lys 115 120 125Asn Lys Asp Tyr Tyr Ile Ile Ser Thr Ser Asn Gly Ser Leu Glu Gly 130 135 140Leu Asp Asn Gln Glu Gly Gly Val Cys Gln Thr Arg Ala Met Lys Ile145 150 155 160Leu Met Lys Val Gly Gln Asp Ala Ser Ser Ala Gly Ser Thr Arg Asn 165 170 175Lys Asp Pro Thr Arg Arg Pro Glu Leu Glu Ala Gly Thr Asn Gly Arg 180 185 190Ser Ser Thr Thr Ser Pro Phe Val Lys Pro Asn Pro Gly Ser Ser Thr 195 200 205Asp Gly Asn Ser Ala Gly His Ser Gly Asn Asn Ile Leu Gly Ser Glu 210 215 220Val Ala Leu Phe Ala Gly Ile Ala Ser Gly Cys Ile Ile Phe Ile Val225 230 235 240Ile Ile Ile Thr Leu Val Val Leu Leu Leu Lys Tyr Arg Arg Arg His 245 250 255Arg Lys His Ser Pro Gln His Thr Thr Thr Leu Ser Leu Ser Thr Leu 260 265 270Ala Thr Pro Lys Arg Ser Gly Asn Asn Asn Gly Ser Glu Pro Ser Asp 275 280 285Ile Ile Ile Pro Leu Arg Thr Ala Asp Ser Val Phe Cys Pro His Tyr 290 295 300Glu Lys Val Ser Gly Asp Tyr Gly His Pro Val Tyr Ile Val Gln Glu305 310 315 320Met Pro Pro Gln Ser Pro Ala Asn Ile Tyr Tyr Lys Val 325 33012332PRTUnknownRecombinant B4EC-GC 12Met Glu Leu Arg Val Leu Leu Cys Trp Ala Ser Leu Ala Ala Ala Leu 1 5 10 15Glu Glu Thr Leu Leu Asn Thr Lys Leu Glu Thr Ala Asp Leu Lys Trp 20 25 30Val Thr Phe Pro Gln Val Asp Gly Gln Trp Glu Glu Leu Ser Gly Leu 35 40 45Asp Glu Glu Gln His Ser Val Arg Thr Tyr Glu Val Cys Glu Val Gln 50 55 60Arg Ala Pro Gly Gln Ala His Trp Leu Arg Thr Gly Trp Val Pro Arg65 70 75 80Arg Gly Ala Val His Val Tyr Ala Thr Leu Arg Phe Thr Met Leu Glu 85 90 95Cys Leu Ser Leu Pro Arg Ala Gly Arg Ser Cys Lys Glu Thr Phe Thr 100 105 110Val Phe Tyr Tyr Glu Ser Asp Ala Asp Thr Ala Thr Ala Leu Thr Pro 115 120 125Ala Trp Met Glu Asn Pro Tyr Ile Lys Val Asp Thr Val Ala Ala Glu 130 135 140His Leu Thr Arg Lys Arg Pro Gly Ala Glu Ala Thr Gly Lys Val Asn145 150 155 160Val Lys Thr Leu Arg Leu Gly Pro Leu Ser Lys Ala Gly Phe Tyr Leu 165 170 175Ala Phe Gln Asp Gln Gly Ala Cys Met Ala Leu Leu Ser Leu His Leu 180 185 190Phe Tyr Lys Lys Cys Ala Gln Leu Thr Val Asn Leu Thr Arg Phe Pro 195 200 205Glu Thr Val Pro Arg Glu Leu Val Val Pro Val Ala Gly Ser Cys Val 210 215 220Val Asp Ala Val Pro Ala Pro Gly Pro Ser Pro Ser Leu Tyr Cys Arg225 230 235 240Glu Asp Gly Gln Trp Ala Glu Gln Pro Val Thr Gly Cys Ser Cys Ala 245 250 255Pro Gly Phe Glu Ala Ala Glu Gly Asn Thr Lys Cys Arg Ala Cys Ala 260 265 270Gln Gly Thr Phe Lys Pro Leu Ser Gly Glu Gly Ser Cys Gln Pro Cys 275 280 285Pro Ala Asn Ser His Ser Asn Thr Ile Gly Ser Ala Val Cys Gln Cys 290 295 300Arg Val Gly Tyr Phe Arg Ala Arg Thr Asp Pro Arg Gly Ala Pro Cys305 310 315 320Thr Thr Pro Pro Ser Ala His His His His His His 325 33013431PRTUnknownRecombinant GCF 13Met Glu Leu Arg Val Leu Leu Cys Trp Ala Ser Leu Ala Ala Ala Leu 1 5 10 15Glu Glu Thr Leu Leu Asn Thr Lys Leu Glu Thr Ala Asp Leu Lys Trp 20 25 30Val Thr Phe Pro Gln Val Asp Gly Gln Trp Glu Glu Leu Ser Gly Leu 35 40 45Asp Glu Glu Gln His Ser Val Arg Thr Tyr Glu Val Cys Glu Val Gln 50 55 60Arg Ala Pro Gly Gln Ala His Trp Leu Arg Thr Gly Trp Val Pro Arg65 70 75 80Arg Gly Ala Val His Val Tyr Ala Thr Leu Arg Phe Thr Met Leu Glu 85 90 95Cys Leu Ser Leu Pro Arg Ala Gly Arg Ser Cys Lys Glu Thr

Phe Thr 100 105 110Val Phe Tyr Tyr Glu Ser Asp Ala Asp Thr Ala Thr Ala Leu Thr Pro 115 120 125Ala Trp Met Glu Asn Pro Tyr Ile Lys Val Asp Thr Val Ala Ala Glu 130 135 140His Leu Thr Arg Lys Arg Pro Gly Ala Glu Ala Thr Gly Lys Val Asn145 150 155 160Val Lys Thr Leu Arg Leu Gly Pro Leu Ser Lys Ala Gly Phe Tyr Leu 165 170 175Ala Phe Gln Asp Gln Gly Ala Cys Met Ala Leu Leu Ser Leu His Leu 180 185 190Phe Tyr Lys Lys Cys Ala Gln Leu Thr Val Asn Leu Thr Arg Phe Pro 195 200 205Glu Thr Val Pro Arg Glu Leu Val Val Pro Val Ala Gly Ser Cys Val 210 215 220Val Asp Ala Val Pro Ala Pro Gly Pro Ser Pro Ser Leu Tyr Cys Arg225 230 235 240Glu Asp Gly Gln Trp Ala Glu Gln Pro Val Thr Gly Cys Ser Cys Ala 245 250 255Pro Gly Phe Ala Glu Gly Asn Thr Lys Cys Arg Ala Cys Ala Gln Gly 260 265 270Thr Phe Lys Pro Leu Ser Gly Glu Gly Ser Cys Gln Pro Cys Pro Ala 275 280 285Asn Ser His Ser Asn Thr Ile Gly Ser Ala Val Cys Gln Cys Arg Val 290 295 300Gly Tyr Phe Arg Ala Arg Thr Asp Pro Arg Gly Ala Pro Cys Thr Thr305 310 315 320Pro Pro Ser Ala Pro Arg Ser Val Val Ser Arg Leu Asn Gly Ser Ser 325 330 335Leu His Leu Glu Trp Ser Ala Pro Leu Glu Ser Gly Gly Arg Glu Asp 340 345 350Leu Thr Tyr Ala Leu Arg Cys Arg Glu Cys Arg Pro Gly Gly Ser Cys 355 360 365Ala Pro Cys Gly Gly Asp Leu Thr Phe Asp Pro Gly Pro Arg Asp Leu 370 375 380Val Glu Pro Trp Val Val Val Arg Gly Leu Arg Pro Asp Phe Thr Tyr385 390 395 400Thr Phe Glu Val Thr Ala Leu Asn Gly Val Ser Ser Leu Ala Thr Gly 405 410 415Pro Val Pro Phe Glu Pro Val Asn Val His His His His His His 420 425 43014570PRTUnknownRecombinant FL-hB4EC 14Met Glu Leu Arg Val Leu Leu Cys Trp Ala Ser Leu Ala Ala Ala Leu 1 5 10 15Glu Glu Thr Leu Leu Asn Thr Lys Leu Glu Thr Ala Asp Leu Lys Trp 20 25 30Val Thr Phe Pro Gln Val Asp Gly Gln Trp Glu Glu Leu Ser Gly Leu 35 40 45Asp Glu Glu Gln His Ser Val Arg Thr Tyr Glu Val Cys Glu Val Gln 50 55 60Arg Ala Pro Gly Gln Ala His Trp Leu Arg Thr Gly Trp Val Pro Arg65 70 75 80Arg Gly Ala Val His Val Tyr Ala Thr Leu Arg Phe Thr Met Leu Glu 85 90 95Cys Leu Ser Leu Pro Arg Ala Gly Arg Ser Cys Lys Glu Thr Phe Thr 100 105 110Val Phe Tyr Tyr Glu Ser Asp Ala Asp Thr Ala Thr Ala Leu Thr Pro 115 120 125Ala Trp Met Glu Asn Pro Tyr Ile Lys Val Asp Thr Val Ala Ala Glu 130 135 140His Leu Thr Arg Lys Arg Pro Gly Ala Glu Ala Thr Gly Lys Val Asn145 150 155 160Val Lys Thr Leu Arg Leu Gly Pro Leu Ser Lys Ala Gly Phe Tyr Leu 165 170 175Ala Phe Gln Asp Gln Gly Ala Cys Met Ala Leu Leu Ser Leu His Leu 180 185 190Phe Tyr Lys Lys Cys Ala Gln Leu Thr Val Asn Leu Thr Arg Phe Pro 195 200 205Glu Thr Val Pro Arg Glu Leu Val Val Pro Val Ala Gly Ser Cys Val 210 215 220Val Asp Ala Val Pro Ala Pro Gly Pro Ser Pro Ser Leu Tyr Cys Arg225 230 235 240Glu Asp Gly Gln Trp Ala Glu Gln Pro Val Thr Gly Cys Ser Cys Ala 245 250 255Pro Gly Phe Glu Ala Ala Glu Gly Asn Thr Lys Cys Arg Ala Cys Ala 260 265 270Gln Gly Thr Phe Lys Pro Leu Ser Gly Glu Gly Ser Cys Gln Pro Cys 275 280 285Pro Ala Asn Ser His Ser Asn Thr Ile Gly Ser Ala Val Cys Gln Cys 290 295 300Arg Val Gly Tyr Phe Arg Ala Arg Thr Asp Pro Arg Gly Ala Pro Cys305 310 315 320Thr Thr Pro Pro Ser Ala Pro Arg Ser Val Val Ser Arg Leu Asn Gly 325 330 335Ser Ser Leu His Leu Glu Trp Ser Ala Pro Leu Glu Ser Gly Gly Arg 340 345 350Glu Asp Leu Thr Tyr Ala Leu Arg Cys Arg Glu Cys Arg Pro Gly Gly 355 360 365Ser Cys Ala Pro Cys Gly Gly Asp Leu Thr Phe Asp Pro Gly Pro Arg 370 375 380Asp Leu Val Glu Pro Trp Val Val Val Arg Gly Leu Arg Pro Asp Phe385 390 395 400Thr Tyr Thr Phe Glu Val Thr Ala Leu Asn Gly Val Ser Ser Leu Ala 405 410 415Thr Gly Pro Val Pro Phe Glu Pro Val Asn Val Thr Thr Asp Arg Glu 420 425 430Val Pro Pro Ala Val Ser Asp Ile Arg Val Thr Arg Ser Ser Pro Ser 435 440 445Ser Leu Ser Leu Ala Trp Ala Val Pro Arg Ala Pro Ser Gly Ala Trp 450 455 460Leu Asp Tyr Glu Val Lys Tyr His Glu Lys Gly Ala Glu Gly Pro Ser465 470 475 480Ser Val Arg Phe Leu Lys Thr Ser Glu Asn Arg Ala Glu Leu Arg Gly 485 490 495Leu Lys Arg Gly Ala Ser Tyr Leu Val Gln Val Arg Ala Arg Ser Glu 500 505 510Ala Gly Tyr Gly Pro Phe Gly Gln Glu His His Ser Gln Thr Gln Leu 515 520 525Asp Glu Ser Glu Gly Trp Arg Glu Gln Gly Ser Lys Arg Ala Ile Leu 530 535 540Gln Ile Glu Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser545 550 555 560Thr Arg Thr Gly His His His His His His 565 57015401PRTUnknownRecombinant B4-CF2 15Met Glu Leu Arg Val Leu Leu Cys Trp Ala Ser Leu Ala Ala Ala Leu 1 5 10 15Glu Glu Thr Leu Leu Asn Thr Lys Leu Glu Thr Gln Leu Thr Val Asn 20 25 30Leu Thr Arg Phe Pro Glu Thr Val Pro Arg Glu Leu Val Val Pro Val 35 40 45Ala Gly Ser Cys Val Val Asp Ala Val Pro Ala Pro Gly Pro Ser Pro 50 55 60Ser Leu Tyr Cys Arg Glu Asp Gly Gln Trp Ala Glu Gln Pro Val Thr65 70 75 80Gly Cys Ser Cys Ala Pro Gly Phe Glu Ala Ala Glu Gly Asn Thr Lys 85 90 95Cys Arg Ala Cys Ala Gln Gly Thr Phe Lys Pro Leu Ser Gly Glu Gly 100 105 110Ser Cys Gln Pro Cys Pro Ala Asn Ser His Ser Asn Thr Ile Gly Ser 115 120 125Ala Val Cys Gln Cys Arg Val Gly Tyr Phe Arg Ala Arg Thr Asp Pro 130 135 140Arg Gly Ala Pro Cys Thr Thr Pro Pro Ser Ala Pro Arg Ser Val Val145 150 155 160Ser Arg Leu Asn Gly Ser Ser Leu His Leu Glu Trp Ser Ala Pro Leu 165 170 175Glu Ser Gly Gly Arg Glu Asp Leu Thr Tyr Ala Leu Arg Cys Arg Glu 180 185 190Cys Arg Pro Gly Gly Ser Cys Ala Pro Cys Gly Gly Asp Leu Thr Phe 195 200 205Asp Pro Gly Pro Arg Asp Leu Val Glu Pro Trp Val Val Val Arg Gly 210 215 220Leu Arg Pro Asp Phe Thr Tyr Thr Phe Glu Val Thr Ala Leu Asn Gly225 230 235 240Val Ser Ser Leu Ala Thr Gly Pro Val Pro Phe Glu Pro Val Asn Val 245 250 255Thr Thr Asp Arg Glu Val Pro Pro Ala Val Ser Asp Ile Arg Val Thr 260 265 270Arg Ser Ser Pro Ser Ser Leu Ser Leu Ala Trp Ala Val Pro Arg Ala 275 280 285Pro Ser Gly Ala Trp Leu Asp Tyr Glu Val Lys Tyr His Glu Lys Gly 290 295 300Ala Glu Gly Pro Ser Ser Val Arg Phe Leu Lys Thr Ser Glu Asn Arg305 310 315 320Ala Glu Leu Arg Gly Leu Lys Arg Gly Ala Ser Tyr Leu Val Gln Val 325 330 335Arg Ala Arg Ser Glu Ala Gly Tyr Gly Pro Phe Gly Gln Glu His His 340 345 350Ser Gln Thr Gln Leu Asp Glu Ser Glu Gly Trp Arg Glu Gln Gly Gly 355 360 365Arg Ser Ser Leu Glu Gly Pro Arg Phe Glu Gly Lys Pro Ile Pro Asn 370 375 380Pro Leu Leu Gly Leu Asp Ser Thr Arg Thr Gly His His His His His385 390 395 400His16537PRTUnknownRecombinant B4-GCF2F 16Met Glu Leu Arg Val Leu Leu Cys Trp Ala Ser Leu Ala Ala Ala Leu 1 5 10 15Glu Glu Thr Leu Leu Asn Thr Lys Leu Glu Thr Ala Asp Leu Lys Trp 20 25 30Val Thr Phe Pro Gln Val Asp Gly Gln Trp Glu Glu Leu Ser Gly Leu 35 40 45Asp Glu Glu Gln His Ser Val Arg Thr Tyr Glu Val Cys Glu Val Gln 50 55 60Arg Ala Pro Gly Gln Ala His Trp Leu Arg Thr Gly Trp Val Pro Arg65 70 75 80Arg Gly Ala Val His Val Tyr Ala Thr Leu Arg Phe Thr Met Leu Glu 85 90 95Cys Leu Ser Leu Pro Arg Ala Gly Arg Ser Cys Lys Glu Thr Phe Thr 100 105 110Val Phe Tyr Tyr Glu Ser Asp Ala Asp Thr Ala Thr Ala Leu Thr Pro 115 120 125Ala Trp Met Glu Asn Pro Tyr Ile Lys Val Asp Thr Val Ala Ala Glu 130 135 140His Leu Thr Arg Lys Arg Pro Gly Ala Glu Ala Thr Gly Lys Val Asn145 150 155 160Val Lys Thr Leu Arg Leu Gly Pro Leu Ser Lys Ala Gly Phe Tyr Leu 165 170 175Ala Phe Gln Asp Gln Gly Ala Cys Met Ala Leu Leu Ser Leu His Leu 180 185 190Phe Tyr Lys Lys Cys Ala Gln Leu Thr Val Asn Leu Thr Arg Phe Pro 195 200 205Glu Thr Val Pro Arg Glu Leu Val Val Pro Val Ala Gly Ser Cys Val 210 215 220Val Asp Ala Val Pro Ala Pro Gly Pro Ser Pro Ser Leu Tyr Cys Arg225 230 235 240Glu Asp Gly Gln Trp Ala Glu Gln Pro Val Thr Gly Cys Ser Cys Ala 245 250 255Pro Gly Phe Glu Ala Ala Glu Gly Asn Thr Lys Cys Arg Ala Cys Ala 260 265 270Gln Gly Thr Phe Lys Pro Leu Ser Gly Glu Gly Ser Cys Gln Pro Cys 275 280 285Pro Ala Asn Ser His Ser Asn Thr Ile Gly Ser Ala Val Cys Gln Cys 290 295 300Arg Val Gly Tyr Phe Arg Ala Arg Thr Asp Pro Arg Gly Ala Pro Cys305 310 315 320Thr Thr Pro Pro Ser Ala Pro Arg Ser Val Val Ser Arg Leu Asn Gly 325 330 335Ser Ser Leu His Leu Glu Trp Ser Ala Pro Leu Glu Ser Gly Gly Arg 340 345 350Glu Asp Leu Thr Tyr Ala Leu Arg Cys Arg Glu Cys Arg Pro Gly Gly 355 360 365Ser Cys Ala Pro Cys Gly Gly Asp Leu Thr Phe Asp Pro Gly Pro Arg 370 375 380Asp Leu Val Glu Pro Trp Val Val Val Arg Gly Leu Arg Pro Asp Phe385 390 395 400Thr Tyr Thr Phe Glu Val Thr Ala Leu Asn Gly Val Ser Ser Leu Ala 405 410 415Thr Gly Pro Val Pro Phe Glu Pro Val Asn Val Thr Thr Asp Arg Glu 420 425 430Val Pro Pro Ala Val Ser Asp Ile Arg Val Thr Arg Ser Ser Pro Ser 435 440 445Ser Leu Ser Leu Ala Trp Ala Val Pro Arg Ala Pro Ser Gly Ala Trp 450 455 460Leu Asp Tyr Glu Val Lys Tyr His Glu Lys Gly Ala Glu Gly Pro Ser465 470 475 480Ser Val Arg Phe Leu Lys Thr Ser Glu Asn Arg Ala Glu Leu Arg Gly 485 490 495Leu Lys Arg Gly Ala Ser Tyr Leu Val Gln Val Arg Ala Arg Ser Glu 500 505 510Ala Gly Tyr Gly Pro Phe Gly Gln Glu His His Ser Gln Thr Gln Leu 515 520 525Asp Glu Ser Glu Gly Trp Arg Glu Gln 530 53517522PRTUnknownRecombinant processed B4-GCF2F 17Leu Glu Glu Thr Leu Leu Asn Thr Lys Leu Glu Thr Ala Asp Leu Lys 1 5 10 15Trp Val Thr Phe Pro Gln Val Asp Gly Gln Trp Glu Glu Leu Ser Gly 20 25 30Leu Asp Glu Glu Gln His Ser Val Arg Thr Tyr Glu Val Cys Glu Val 35 40 45Gln Arg Ala Pro Gly Gln Ala His Trp Leu Arg Thr Gly Trp Val Pro 50 55 60Arg Arg Gly Ala Val His Val Tyr Ala Thr Leu Arg Phe Thr Met Leu65 70 75 80Glu Cys Leu Ser Leu Pro Arg Ala Gly Arg Ser Cys Lys Glu Thr Phe 85 90 95Thr Val Phe Tyr Tyr Glu Ser Asp Ala Asp Thr Ala Thr Ala Leu Thr 100 105 110Pro Ala Trp Met Glu Asn Pro Tyr Ile Lys Val Asp Thr Val Ala Ala 115 120 125Glu His Leu Thr Arg Lys Arg Pro Gly Ala Glu Ala Thr Gly Lys Val 130 135 140Asn Val Lys Thr Leu Arg Leu Gly Pro Leu Ser Lys Ala Gly Phe Tyr145 150 155 160Leu Ala Phe Gln Asp Gln Gly Ala Cys Met Ala Leu Leu Ser Leu His 165 170 175Leu Phe Tyr Lys Lys Cys Ala Gln Leu Thr Val Asn Leu Thr Arg Phe 180 185 190Pro Glu Thr Val Pro Arg Glu Leu Val Val Pro Val Ala Gly Ser Cys 195 200 205Val Val Asp Ala Val Pro Ala Pro Gly Pro Ser Pro Ser Leu Tyr Cys 210 215 220Arg Glu Asp Gly Gln Trp Ala Glu Gln Pro Val Thr Gly Cys Ser Cys225 230 235 240Ala Pro Gly Phe Glu Ala Ala Glu Gly Asn Thr Lys Cys Arg Ala Cys 245 250 255Ala Gln Gly Thr Phe Lys Pro Leu Ser Gly Glu Gly Ser Cys Gln Pro 260 265 270Cys Pro Ala Asn Ser His Ser Asn Thr Ile Gly Ser Ala Val Cys Gln 275 280 285Cys Arg Val Gly Tyr Phe Arg Ala Arg Thr Asp Pro Arg Gly Ala Pro 290 295 300Cys Thr Thr Pro Pro Ser Ala Pro Arg Ser Val Val Ser Arg Leu Asn305 310 315 320Gly Ser Ser Leu His Leu Glu Trp Ser Ala Pro Leu Glu Ser Gly Gly 325 330 335Arg Glu Asp Leu Thr Tyr Ala Leu Arg Cys Arg Glu Cys Arg Pro Gly 340 345 350Gly Ser Cys Ala Pro Cys Gly Gly Asp Leu Thr Phe Asp Pro Gly Pro 355 360 365Arg Asp Leu Val Glu Pro Trp Val Val Val Arg Gly Leu Arg Pro Asp 370 375 380Phe Thr Tyr Thr Phe Glu Val Thr Ala Leu Asn Gly Val Ser Ser Leu385 390 395 400Ala Thr Gly Pro Val Pro Phe Glu Pro Val Asn Val Thr Thr Asp Arg 405 410 415Glu Val Pro Pro Ala Val Ser Asp Ile Arg Val Thr Arg Ser Ser Pro 420 425 430Ser Ser Leu Ser Leu Ala Trp Ala Val Pro Arg Ala Pro Ser Gly Ala 435 440 445Trp Leu Asp Tyr Glu Val Lys Tyr His Glu Lys Gly Ala Glu Gly Pro 450 455 460Ser Ser Val Arg Phe Leu Lys Thr Ser Glu Asn Arg Ala Glu Leu Arg465 470 475 480Gly Leu Lys Arg Gly Ala Ser Tyr Leu Val Gln Val Arg Ala Arg Ser 485 490 495Glu Ala Gly Tyr Gly Pro Phe Gly Gln Glu His His Ser Gln Thr Gln 500 505 510Leu Asp Glu Ser Glu Gly Trp Arg Glu Gln 515 520181124PRTUnknownRecombinant HSA-EphB4 precursor protein 18Met Glu Leu Arg Val Leu Leu Cys Trp Ala Ser Leu Ala Ala Ala Leu 1 5 10 15Glu Glu Thr Leu Leu Asn Thr Lys Leu Glu Thr Ala Asp Leu Lys Trp 20 25 30Val Thr Phe Pro Gln Val Asp Gly Gln Trp Glu Glu Leu Ser Gly Leu 35 40 45Asp Glu Glu Gln His Ser Val Arg Thr Tyr Glu Val Cys Asp Val Gln 50 55 60Arg Ala Pro Gly Gln Ala His Trp Leu Arg Thr Gly Trp Val Pro Arg65 70 75 80Arg Gly Ala Val His Val Tyr Ala Thr Leu Arg Phe Thr Met Leu Glu 85

90 95Cys Leu Ser Leu Pro Arg Ala Gly Arg Ser Cys Lys Glu Thr Phe Thr 100 105 110Val Phe Tyr Tyr Glu Ser Asp Ala Asp Thr Ala Thr Ala Leu Thr Pro 115 120 125Ala Trp Met Glu Asn Pro Tyr Ile Lys Val Asp Thr Val Ala Ala Glu 130 135 140His Leu Thr Arg Lys Arg Pro Gly Ala Glu Ala Thr Gly Lys Val Asn145 150 155 160Val Lys Thr Leu Arg Leu Gly Pro Leu Ser Lys Ala Gly Phe Tyr Leu 165 170 175Ala Phe Gln Asp Gln Gly Ala Cys Met Ala Leu Leu Ser Leu His Leu 180 185 190Phe Tyr Lys Lys Cys Ala Gln Leu Thr Val Asn Leu Thr Arg Phe Pro 195 200 205Glu Thr Val Pro Arg Glu Leu Val Val Pro Val Ala Gly Ser Cys Val 210 215 220Val Asp Ala Val Pro Ala Pro Gly Pro Ser Pro Ser Leu Tyr Cys Arg225 230 235 240Glu Asp Gly Gln Trp Ala Glu Gln Pro Val Thr Gly Cys Ser Cys Ala 245 250 255Pro Gly Phe Glu Ala Ala Glu Gly Asn Thr Lys Cys Arg Ala Cys Ala 260 265 270Gln Gly Thr Phe Lys Pro Leu Ser Gly Glu Gly Ser Cys Gln Pro Cys 275 280 285Pro Ala Asn Ser His Ser Asn Thr Ile Gly Ser Ala Val Cys Gln Cys 290 295 300Arg Val Gly Tyr Phe Arg Ala Arg Thr Asp Pro Arg Gly Ala Pro Cys305 310 315 320Thr Thr Pro Pro Ser Ala Pro Arg Ser Val Val Ser Arg Leu Asn Gly 325 330 335Ser Ser Leu His Leu Glu Trp Ser Ala Pro Leu Glu Ser Gly Gly Arg 340 345 350Glu Asp Leu Thr Tyr Ala Leu Arg Cys Arg Glu Cys Arg Pro Gly Gly 355 360 365Ser Cys Ala Pro Cys Gly Gly Asp Leu Thr Phe Asp Pro Gly Pro Arg 370 375 380Asp Leu Val Glu Pro Trp Val Val Val Arg Gly Leu Arg Pro Asp Phe385 390 395 400Thr Tyr Thr Phe Glu Val Thr Ala Leu Asn Gly Val Ser Ser Leu Ala 405 410 415Thr Gly Pro Val Pro Phe Glu Pro Val Asn Val Thr Thr Asp Arg Glu 420 425 430Val Pro Pro Ala Val Ser Asp Ile Arg Val Thr Arg Ser Ser Pro Ser 435 440 445Ser Leu Ser Leu Ala Trp Ala Val Pro Arg Ala Pro Ser Gly Ala Val 450 455 460Leu Asp Tyr Glu Val Lys Tyr His Glu Lys Gly Ala Glu Gly Pro Ser465 470 475 480Ser Val Arg Phe Leu Lys Thr Ser Glu Asn Arg Ala Glu Leu Arg Gly 485 490 495Leu Lys Arg Gly Ala Ser Tyr Leu Val Gln Val Arg Ala Arg Ser Glu 500 505 510Ala Gly Tyr Gly Pro Phe Gly Gln Glu His His Ser Gln Thr Gln Leu 515 520 525Asp Glu Ser Glu Gly Trp Arg Glu Gln Ser Arg Asp Ala His Lys Ser 530 535 540Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala545 550 555 560Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu 565 570 575Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys 580 585 590Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu 595 600 605Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly 610 615 620Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys625 630 635 640Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg 645 650 655Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr 660 665 670Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe 675 680 685Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe 690 695 700Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys705 710 715 720Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg 725 730 735Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala 740 745 750Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala 755 760 765Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys 770 775 780Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala785 790 795 800Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu 805 810 815Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val 820 825 830Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe 835 840 845Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val 850 855 860Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr865 870 875 880Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu 885 890 895Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val 900 905 910Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys 915 920 925Gln Asn Cys Glu Leu Phe Lys Gln Leu Gly Glu Tyr Lys Phe Gln Asn 930 935 940Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro945 950 955 960Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys 965 970 975Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu 980 985 990Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val 995 1000 1005Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg 1010 1015 1020Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu1025 1030 1035 1040Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser 1045 1050 1055Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val 1060 1065 1070Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp 1075 1080 1085Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu 1090 1095 1100Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala1105 1110 1115 1120Ala Leu Gly Leu191109PRTUnknownRecombinant HSA-EphB4 mature protein 19Leu Glu Glu Thr Leu Leu Asn Thr Lys Leu Glu Thr Ala Asp Leu Lys 1 5 10 15Trp Val Thr Phe Pro Gln Val Asp Gly Gln Trp Glu Glu Leu Ser Gly 20 25 30Leu Asp Glu Glu Gln His Ser Val Arg Thr Tyr Glu Val Cys Asp Val 35 40 45Gln Arg Ala Pro Gly Gln Ala His Trp Leu Arg Thr Gly Trp Val Pro 50 55 60Arg Arg Gly Ala Val His Val Tyr Ala Thr Leu Arg Phe Thr Met Leu65 70 75 80Glu Cys Leu Ser Leu Pro Arg Ala Gly Arg Ser Cys Lys Glu Thr Phe 85 90 95Thr Val Phe Tyr Tyr Glu Ser Asp Ala Asp Thr Ala Thr Ala Leu Thr 100 105 110Pro Ala Trp Met Glu Asn Pro Tyr Ile Lys Val Asp Thr Val Ala Ala 115 120 125Glu His Leu Thr Arg Lys Arg Pro Gly Ala Glu Ala Thr Gly Lys Val 130 135 140Asn Val Lys Thr Leu Arg Leu Gly Pro Leu Ser Lys Ala Gly Phe Tyr145 150 155 160Leu Ala Phe Gln Asp Gln Gly Ala Cys Met Ala Leu Leu Ser Leu His 165 170 175Leu Phe Tyr Lys Lys Cys Ala Gln Leu Thr Val Asn Leu Thr Arg Phe 180 185 190Pro Glu Thr Val Pro Arg Glu Leu Val Val Pro Val Ala Gly Ser Cys 195 200 205Val Val Asp Ala Val Pro Ala Pro Gly Pro Ser Pro Ser Leu Tyr Cys 210 215 220Arg Glu Asp Gly Gln Trp Ala Glu Gln Pro Val Thr Gly Cys Ser Cys225 230 235 240Ala Pro Gly Phe Glu Ala Ala Glu Gly Asn Thr Lys Cys Arg Ala Cys 245 250 255Ala Gln Gly Thr Phe Lys Pro Leu Ser Gly Glu Gly Ser Cys Gln Pro 260 265 270Cys Pro Ala Asn Ser His Ser Asn Thr Ile Gly Ser Ala Val Cys Gln 275 280 285Cys Arg Val Gly Tyr Phe Arg Ala Arg Thr Asp Pro Arg Gly Ala Pro 290 295 300Cys Thr Thr Pro Pro Ser Ala Pro Arg Ser Val Val Ser Arg Leu Asn305 310 315 320Gly Ser Ser Leu His Leu Glu Trp Ser Ala Pro Leu Glu Ser Gly Gly 325 330 335Arg Glu Asp Leu Thr Tyr Ala Leu Arg Cys Arg Glu Cys Arg Pro Gly 340 345 350Gly Ser Cys Ala Pro Cys Gly Gly Asp Leu Thr Phe Asp Pro Gly Pro 355 360 365Arg Asp Leu Val Glu Pro Trp Val Val Val Arg Gly Leu Arg Pro Asp 370 375 380Phe Thr Tyr Thr Phe Glu Val Thr Ala Leu Asn Gly Val Ser Ser Leu385 390 395 400Ala Thr Gly Pro Val Pro Phe Glu Pro Val Asn Val Thr Thr Asp Arg 405 410 415Glu Val Pro Pro Ala Val Ser Asp Ile Arg Val Thr Arg Ser Ser Pro 420 425 430Ser Ser Leu Ser Leu Ala Trp Ala Val Pro Arg Ala Pro Ser Gly Ala 435 440 445Val Leu Asp Tyr Glu Val Lys Tyr His Glu Lys Gly Ala Glu Gly Pro 450 455 460Ser Ser Val Arg Phe Leu Lys Thr Ser Glu Asn Arg Ala Glu Leu Arg465 470 475 480Gly Leu Lys Arg Gly Ala Ser Tyr Leu Val Gln Val Arg Ala Arg Ser 485 490 495Glu Ala Gly Tyr Gly Pro Phe Gly Gln Glu His His Ser Gln Thr Gln 500 505 510Leu Asp Glu Ser Glu Gly Trp Arg Glu Gln Ser Arg Asp Ala His Lys 515 520 525Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys 530 535 540Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe545 550 555 560Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr 565 570 575Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr 580 585 590Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr 595 600 605Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu 610 615 620Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val625 630 635 640Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu 645 650 655Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr 660 665 670Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala 675 680 685Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro 690 695 700Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln705 710 715 720Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys 725 730 735Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe 740 745 750Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu 755 760 765Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu 770 775 780Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys785 790 795 800Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu 805 810 815Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp 820 825 830Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp 835 840 845Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp 850 855 860Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr865 870 875 880Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys 885 890 895Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile 900 905 910Lys Gln Asn Cys Glu Leu Phe Lys Gln Leu Gly Glu Tyr Lys Phe Gln 915 920 925Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr 930 935 940Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys945 950 955 960Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr 965 970 975Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro 980 985 990Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg 995 1000 1005Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys 1010 1015 1020Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu1025 1030 1035 1040Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu 1045 1050 1055Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met 1060 1065 1070Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys 1075 1080 1085Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln 1090 1095 1100Ala Ala Leu Gly Leu1105209244DNAArtificial SequencePEF6-GCF2 plasmid sequence 20aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 60tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 120tcgacggatc gggagatctc ccgatcccct atggtcgact ctcagtacaa tctgctctga 180tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg 240cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct 300gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca 360ttgattattg actaggcttt tgcaaaaagc tttgcaaaga tggataaagt tttaaacaga 420gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa ggagtgcctc gtgaggctcc 480ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg 540gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc 600gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc 660gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt 720ggttcccgcg ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc 780acctggctgc agtacgtgat tcttgatccc gagcttcggg ttggaagtgg gtgggagagt 840tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt gagttgaggc ctggcctggg 900cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat 960aagtctctag ccatttaaaa tttttgatga cctgctgcga cgcttttttt ctggcaagat 1020agtcttgtaa atgcgggcca agatctgcac actggtattt cggtttttgg ggccgcgggc 1080ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg 1140ccaccgagaa tcggacgggg gtagtctcaa gctggccggc ctgctctggt gcctggcctc 1200gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg 1260tgagcggaaa gatggccgct tcccggccct gctgcaggga gctcaaaatg gaggacgcgg 1320cgctcgggag agcgggcggg tgagtcaccc acacaaagga aaagggcctt tccgtcctca 1380gccgtcgctt catgtgactc cacggagtac cgggcgccgt ccaggcacct cgattagttc 1440tcgagctttt ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt 1500ccccacactg agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc 1560ttggaatttg ccctttttga gtttggatct tggttcattc tcaagcctca gacagtggtt 1620caaagttttt ttcttccatt tcaggtgtcg tgaggaatta gcttggtact aatacgactc 1680actataggga gacccaagct ggctaggtaa gcttggtacc gagctcggat ccactagtcc 1740agtgtggtgg aattgccctt caagcttgcc gccaccatgg agctccgggt gctgctctgc 1800tgggcttcgt tggccgcagc tttggaagag accctgctga acacaaaatt ggaaactgct 1860gatctgaagt gggtgacatt ccctcaggtg gacgggcagt gggaggaact gagcggcctg

1920gatgaggaac agcacagcgt gcgcacctac gaagtgtgtg acgtgcagcg tgccccgggc 1980caggcccact ggcttcgcac aggttgggtc ccacggcggg gcgccgtcca cgtgtacgcc 2040acgctgcgct tcaccatgct cgagtgcctg tccctgcctc gggctgggcg ctcctgcaag 2100gagaccttca ccgtcttcta ctatgagagc gatgcggaca cggccacggc cctcacgcca 2160gcctggatgg agaaccccta catcaaggtg gacacggtgg ccgcggagca tctcacccgg 2220aagcgccctg gggccgaggc caccgggaag gtgaatgtca agacgctgcg cctgggaccg 2280ctcagcaagg ctggcttcta cctggccttc caggaccagg gtgcctgcat ggccctgcta 2340tccctgcacc tcttctacaa aaagtgcgcc cagctgactg tgaacctgac tcgattcccg 2400gagactgtgc ctcgggagct ggttgtgccc gtggccggta gctgcgtggt ggatgccgtc 2460cccgcccctg gccccagccc cagcctctac tgccgtgagg atggccagtg ggccgaacag 2520ccggtcacgg gctgcagctg tgctccgggg ttcgaggcag ctgaggggaa caccaagtgc 2580cgagcctgtg cccagggcac cttcaagccc ctgtcaggag aagggtcctg ccagccatgc 2640ccagccaata gccactctaa caccattgga tcagccgtct gccagtgccg cgtcgggtac 2700ttccgggcac gcacagaccc ccggggtgca ccctgcacca cccctccttc ggctccgcgg 2760agcgtggttt cccgcctgaa cggctcctcc ctgcacctgg aatggagtgc ccccctggag 2820tctggtggcc gagaggacct cacctacgcc ctccgctgcc gggagtgtcg acccggaggc 2880tcctgtgcgc cctgcggggg agacctgact tttgaccccg gcccccggga cctggtggag 2940ccctgggtgg tggttcgagg gctacgtcct gacttcacct atacctttga ggtcactgca 3000ttgaacgggg tatcctcctt agccacgggg cccgtcccat ttgagcctgt caatgtcacc 3060actgaccgag aggtacctcc tgcagtgtct gacatccggg tgacgcggtc ctcacccagc 3120agcttgagcc tggcctgggc tgttccccgg gcacccagtg gggctgtgct ggactacgag 3180gtcaaatacc atgagaaggg cgccgagggt cccagcagcg tgcggttcct gaagacgtca 3240gaaaaccggg cagagctgcg ggggctgaag cggggagcca gctacctggt gcaggtacgg 3300gcgcgctctg aggccggcta cgggcccttc ggccaggaac atcacagcca gacccaactg 3360gatgagagcg agggctggcg ggagcagtct agagatgcac acaagagtga ggttgctcat 3420cggtttaaag atttgggaga agaaaatttc aaagccttgg tgttgattgc ctttgctcag 3480tatcttcagc agtgtccatt tgaagatcat gtaaaattag tgaatgaagt aactgaattt 3540gcaaaaacat gtgtagctga tgagtcagct gaaaattgtg acaaatcact tcataccctt 3600tttggagaca aattatgcac agttgcaact cttcgtgaaa cctatggtga aatggctgac 3660tgctgtgcaa aacaagaacc tgagagaaat gaatgcttct tgcaacacaa agatgacaac 3720ccaaacctcc cccgattggt gagaccagag gttgatgtga tgtgcactgc ttttcatgac 3780aatgaagaga catttttgaa aaaatactta tatgaaattg ccagaagaca tccttacttt 3840tatgccccgg aactcctttt ctttgctaaa aggtataaag ctgcttttac agaatgttgc 3900caagctgctg ataaagctgc ctgcctgttg ccaaagctcg atgaacttcg ggatgaaggg 3960aaggcttcgt ctgccaaaca gagactcaaa tgtgccagtc tccaaaaatt tggagaaaga 4020gctttcaaag catgggcagt ggctcgcctg agccagagat ttcccaaagc tgagtttgca 4080gaagtttcca agttagtgac agatcttacc aaagtccaca cggaatgctg ccatggagat 4140ctgcttgaat gtgctgatga cagggcggac cttgccaagt atatctgtga aaatcaggat 4200tcgatctcca gtaaactgaa ggaatgctgt gaaaaacctc tgttggaaaa atcccactgc 4260attgccgaag tggaaaatga tgagatgcct gctgacttgc cttcattagc tgctgatttt 4320gttgaaagta aggatgtttg caaaaactat gctgaggcaa aggatgtctt cctgggcatg 4380tttttgtatg aatatgcaag aaggcatcct gattactctg tcgtgctgct gctgagactt 4440gccaagacat atgaaaccac tctagagaag tgctgtgccg ctgcagatcc tcatgaatgc 4500tatgccaaag tgttcgatga atttaaacct cttgtggaag agcctcagaa tttaatcaaa 4560caaaactgtg agctttttaa gcagcttgga gagtacaaat tccagaatgc gctattagtt 4620cgttacacca agaaagtacc ccaagtgtca actccaactc ttgtagaggt ctcaagaaac 4680ctaggaaaag tgggcagcaa atgttgtaaa catcctgaag caaaaagaat gccctgtgca 4740gaagactatc tatccgtggt cctgaaccag ttatgtgtgt tgcatgagaa aacgccagta 4800agtgacagag tcacaaaatg ctgcacagag tccttggtga acaggcgacc atgcttttca 4860gctctggaag tcgatgaaac atacgttccc aaagagttta atgctgaaac attcaccttc 4920catgcagata tatgcacact ttctgagaag gagagacaaa tcaagaaaca aactgcactt 4980gttgagcttg tgaaacacaa gcccaaggca acaaaagagc aactgaaagc tgttatggat 5040gatttcgcag cttttgtaga gaagtgctgc aaggctgacg ataaggagac ctgctttgcc 5100gaggagggta aaaaacttgt tgctgcaagt caagctgcct taggcttata atagcggccg 5160cttaagggca attctgcaga tatccagcac agtggcggcc gctcgagtct agagggcccg 5220cggttcgaag gtaagcctat ccctaaccct ctcctcggtc tcgattctac gcgtaccggt 5280catcatcacc atcaccattg agtttaaacc cgctgatcag cctcgactgt gccttctagt 5340tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact 5400cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat 5460tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc 5520aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc 5580tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 5640acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc 5700ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg gggcatccct 5760ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat 5820ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc 5880acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc 5940tattcttttg atttataagg gattttgggg atttcggcct attggttaaa aaatgagctg 6000atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa 6060agtccccagg ctccccaggc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca 6120accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc 6180aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc 6240agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag 6300gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc 6360ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca gcacgtgttg 6420acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg aggaactaaa 6480ccatggccaa gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa 6540tcaacagcat ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg 6600gccgcatctt cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg 6660tggtgctggg cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg 6720gaaatgagaa caggggcatc ttgagcccct gcggacggtg tcgacaggtg cttctcgatc 6780tgcatcctgg gatcaaagcg atagtgaagg acagtgatgg acagccgacg gcagttggga 6840ttcgtgaatt gctgccctct ggttatgtgt gggagggcta agcacttcgt ggccgaggag 6900caggactgac acgtgctacg agatttcgat tccaccgccg ccttctatga aaggttgggc 6960ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg 7020gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat 7080agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc 7140aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg 7200taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 7260atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 7320ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 7380taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 7440tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 7500aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 7560aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 7620ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 7680acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 7740ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 7800tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 7860tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 7920gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 7980agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 8040tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 8100agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 8160tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 8220acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 8280tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 8340agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 8400tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 8460acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 8520tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 8580ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 8640agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 8700tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 8760acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 8820agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 8880actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 8940tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 9000gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 9060ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 9120tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 9180aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 9240tttc 92442111PRTUnknownlinker peptide sequence 21Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser 1 5 102217PRTUnknownlinker peptide sequence 22Ser Ser Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser Ser Ser Gly Ser 1 5 10 15Pro

Claims

1. An isolated soluble polypeptide comprising an amino acid sequence of an extracellular domain of an EphB4 protein, wherein the polypeptide is a monomer and binds specifically to an Ephrin B2 polypeptide.

2. The polypeptide of claim 1, comprising a globular domain of an EphB4 protein or a sequence that is at least 90% identical to a globular domain of EphB4.

3. The polypeptide of claim 1, comprising a sequence at least 90% identical to residues 29-197 of the amino acid sequence defined by FIG. 65 (SEQ ID NO:10).

4. The polypeptide of claim 1, further comprising a modification that increases serum half-life.

5. The polypeptide of claim 4, wherein said modification comprises a polyethylene glycol group.

6. The polypeptide of claim 5, wherein said modification is a single polyethylene glycol group covalently bonded to the polypeptide.

7. The polypeptide of claim 5, wherein said polypeptide is covalently bonded to two polyethylene glycol groups.

8. The polypeptide of claim 5, wherein said polypeptide is covalently bonded to multiple polyethylene glycol groups.

9. The polypeptide of claim 5, wherein said polyethylene glycol group has a molecular weight of from about 10 to about 40 kDa.

10. The polypeptide of claim 5, wherein the polyethylene glycol group has a molecular weight of from about 30 to about 40 kDa.

11. The polypeptide of claim 5, wherein said polyethylene glycol group is selected from linear PEG chains and branched PEG chains.

12. The polypeptide of claim 5, wherein said polyethylene glycol group is attached to a group selected from the lysine side chains and the N-terminal amino group of the EphB4 polypeptide.

13. The polypeptide of claim 4, wherein said polypeptide has a serum half-life in vivo at least 50% greater than that of an unmodified EphB4 polypeptide.

14. The polypeptide of claim 4, wherein said polypeptide has a serum half-life in vivo at least 100% greater than that of an unmodified EphB4 polypeptide.

15. The polypeptide of claim 4, wherein the polypeptide is a fusion protein.

16. The polypeptide of claim 15, wherein the polypeptide comprises an albumin protein or fragments thereof.

17. The polypeptide of claim 16, wherein said albumin protein is selected from a human serum albumin (HSA) and bovine serum albumin (BSA).

18. The polypeptide of claim 16, wherein the albumin is a naturally occurring variant.

19. The polypeptide of claim 1, wherein the polypeptide has one or more activities selected from:(a) inhibition of EphrinB2 activity;(b) inhibition of EphrinB2 kinase activity;(c) inhibition of the interaction between EphB4 and EphrinB2;(d) inhibition of EphB4 kinase activity;(e) inhibition of clustering of Ephrin B2; and(f) inhibition of clustering of EphB4.

20. The polypeptide of claim 4, wherein the polypeptide has enhanced in vivo stability relative to the unmodified wildtype polypeptide.

21. A pharmaceutical composition comprising a polypeptide of claim 1, and a pharmaceutically acceptable carrier.

22. A method of inhibiting signaling through Ephrin B2/EphB4 pathway in a cell, comprising contacting the cell with an effective amount of a polypeptide of claim 1.

23. A method of reducing the growth rate of a tumor, comprising administering an amount of a polypeptide of claim 1, sufficient to reduce the growth rate of the tumor.

24. A method for treating a patient suffering from a cancer, comprising administering to the patient a polypeptide of claim 1.

25. A method of inhibiting angiogenesis, comprising contacting a cell with a polypeptide of claim 1.

26. A method for treating a patient suffering from an angiogenesis-associated disease, comprising administering to the patient a polypeptide of claim 1.

27. The polypeptide of claim 1, wherein the polypeptide comprises one or more modified amino acid residues.

28. A cosmetic composition comprising the polypeptide of claim 1, and a pharmaceutically acceptable carrier.

29. A method of reducing the growth rate of a tumor, comprising administering an amount of a polypeptide agent sufficient to reduce the growth rate of the tumor, wherein the polypeptide agent is selected from:(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(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.

30. The method of claim 29, wherein the tumor comprises cells expressing a higher level of EphB4 and/or EphrinB2 than noncancerous cells of a comparable tissue.

31. A method for treating a patient suffering from a cancer, comprising administering to the patient a polypeptide agent selected from:(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(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.

32. The method of claim 31, wherein the cancer comprises cancer cells expressing EphrinB2 and/or EphB4 at a higher level than noncancerous cells of a comparable tissue.

33. The method of claim 31, wherein the cancer is metastatic cancer.

34. The method of claim 31, wherein the tumor is selected from colon carcinoma, breast tumor, mesothelioma, prostate tumor, squamous cell carcinoma, Kaposi sarcoma, and leukemia.

35. The method of claim 31, wherein the cancer is an angiogenesis-dependent cancer.

36. The method of claim 31, wherein the cancer is an angiogenesis-independent cancer.

37. The method of claim 31, wherein the polypeptide agent is 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 and further comprises a modification that increases serum half-life.

38. The method of claim 31, further including administering at least one additional anti-cancer chemotherapeutic agent that inhibits cancer cells in an additive or synergistic manner with the polypeptide agent.

39. A method of inhibiting angiogenesis, comprising contacting a cell with an amount of a polypeptide agent sufficient to inhibit angiogenesis, wherein the polypeptide agent is selected from:(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(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.

40. A method for treating a patient suffering from an angiogenesis-associated disease, comprising administering to the patient a polypeptide agent selected from:(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(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.

41-47. (canceled)

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