METHODS AND COMPOSITIONS FOR INHIBITING ANGIOGENESIS AND

TUMOR GROWTH

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 61/415,788, filed on November 19, 2010, the entire disclosure of which is incorporated herein by this reference.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH OR

DEVELOPMENT

The invention described herein was supported, in whole or in part, by the U.S. National Cancer Institute (ROl CA 079218-07). The United States government has certain rights in the invention.

BACKGROUND OF THE FNVENTION

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.

It is generally thought that the development of tumors 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. Additional agents and therapeutic treatments for inhibiting angiogenesis and tumor growth are needed.

SUMMARY OF THE INVENTION

In certain aspects, the disclosure provides a method for inhibiting angiogenesis in a subject in need thereof by conjointly administering a D114/Notch pathway antagonist and an Ephrin/Eph pathway antagonist. In certain aspects, the disclosure provides a method for treating cancer in a subject in need thereof by conjointly administering a D114/Notch pathway antagonist and an Ephrin/Eph pathway antagonist. In certain aspects, the disclosure provides a method for treating or preventing D114/Notch pathway inhibition-related toxicity in a subject in need thereof comprising administering an Ephrin/Eph pathway antagonist.

In certain embodiments, the D114/Notch pathway antagonist is a polypeptide comprising an extracellular region of D114 that binds to D114. In certain embodiments, the polypeptide comprises the DSL domain of SEQ ID NO: 1 , or a variant thereof. In certain embodiments, the polypeptide comprises amino acids 27-524 of SEQ ID NO: 1 , or a variant thereof. In certain embodiments, the polypeptide comprises amino acids 1-486 of SEQ ID NO: 1, or a variant thereof. In certain embodiments, the polypeptide comprises amino acids 27-486 of SEQ ID NO: 1, or variant thereof. In certain embodiments, the polypeptide comprises amino acids 1-442 of SEQ ID NO: 1, or a variant thereof. In certain embodiments, the polypeptide comprises amino acids 27-442 of SEQ ID NO: 1, or variant thereof. In certain embodiments as set forth above, the polypeptide is covalently linked to a moiety selected from an Fc domain, His tag, or a polyoxyalkylene moiety. In certain embodiments as set forth above, the polypeptide is covalently linked to a moiety that confers enhanced pharmacokinetic properties, such as an Fc domain, His tag, or a polyoxyalkylene moiety.

In certain embodiments, the D114/Notch pathway antagonist is an antibody, or a fragment thereof, that binds to D114. In certain embodiments, the antibody is a monoclonal antibody. In certain embodiments, the antibody is a human or humanized antibody. In certain embodiments, the antibody comprises SEQ ID NO: 74 or SEQ ID NO: 75. In certain embodiments, the antibody comprises SEQ ID NO: 76 or SEQ ID NO: 77. In certain embodiments as set forth above, the antibody is covalently linked to a moiety selected from an Fc domain, His tag, or a polyoxyalkylene moiety. In certain embodiments as set forth above, the antibody is covalently linked to a moiety that confers enhanced pharmacokinetic properties, such as an Fc domain, His tag, or a polyoxyalkylene moiety.

In certain embodiments, D114/Notch pathway inhibition comprises chronic D114 blockade. In certain embodiments, D114/Notch pathway inhibition-related toxicity results from soluble D114 treatment. In certain embodiments, D114/Notch pathway inhibition-related toxicity comprises vascular proliferative lesions, e.g., in the liver.

In certain embodiments, the Ephrin/Eph pathway is the Ephrin B2/EphB4 pathway. In certain such embodiments, the Ephrin/Eph pathway antagonist is selected from a soluble antagonist comprising the extracellular domain of Ephrin B2; a soluble antagonist comprising the extracellular domain of EphB4; an antibody, or a fragment thereof, that binds to EphB4; an antibody, or a fragment thereof, that binds to Ephrin B2; a nucleic acid compound that hybridizes to an EphB4 transcript under physiological conditions and decreases the expression of EphB4 in a cell; and a nucleic acid compound that hybridizes to an EphrinB2 transcript under physiological conditions and decreases the expression of EphrinB2 in a cell. In certain embodiments, the Ephrin/Eph pathway antagonist inhibits the interaction between Ephrin B2 and EphB4. In certain embodiments, the Ephrin/Eph pathway antagonist inhibits clustering of Ephrin B2 or EphB4. In certain embodiments, the Ephrin/Eph pathway antagonist inhibits phosphorylation of Ephrin B2 or EphB4.

In certain embodiments, the soluble Ephrin/Eph pathway antagonist is a monomeric polypeptide. In certain embodiments, the soluble Ephrin/Eph pathway antagonist is a fusion protein. In certain embodiments, the fusion protein comprises a first portion comprising an EphB4 extracellular domain, wherein the EphB4 extracellular domain has a sequence selected from residues 16-522, residues 16-412, residues 16-312, residues 1-522, residues 1- 412, and residues 1-312 of SEQ ID NO: 12, and a second portion comprising a heterologous sequence. In certain embodiments, the fusion protein comprises albumin or a fragment thereof or a polyethylene glycol (PEG) moiety. In certain embodiments, the fusion protein comprises an Fc domain of an antibody or a fragment thereof. In certain such embodiments, the antibody is a monoclonal antibody. In certain embodiments, the antibody is selected from antibody 47 and 131 as described herein. In certain embodiments, the antibody is a human or humanized antibody. In certain embodiments, the antibody is a deimmunized antibody. In certain embodiments, the subject is a human. In certain embodiments, the subject has an angiogenesis-associated disease. In certain embodiments, the angiogenesis-associated disease is selected from angiogenesis-dependent cancer, benign tumors, inflammatory disorders, chronic articular rheumatism, ocular angiogenic diseases, Osier- Webber

Syndrome, myocardial angiogenesis, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma, wound granulation, wound healing, psoriasis, scleroderma, pyogenic granuloma, coronary collaterals, ischemic limb angiogenesis, rubeosis, arthritis, and diabetic neovascularization. In certain embodiments, the patient is diagnosed with a cancer selected from colon carcinoma, breast tumor, mesothelioma, prostate tumor, squamous cell carcinoma, Kaposi sarcoma, and leukemia. In certain embodiments, the Ephrin/Eph pathway antagonist is administered systemically. In certain embodiments, the Ephrin/Eph pathway antagonist is administered locally to a site of angiogenesis.

In certain aspects, the disclosure provides a composition comprising a D114/Notch pathway antagonist and an Ephrin/Eph pathway antagonist, such as any of the antagonists described above or elsewhere herein. In certain embodiments, the Ephrin/Eph pathway antagonist is selected from a soluble antagonist comprising the extracellular domain of Ephrin B2; a soluble antagonist comprising the extracellular domain of EphB4; an antibody, or a fragment thereof, that binds to EphB4; an antibody, or a fragment thereof, that binds to Ephrin B2; a nucleic acid compound that hybridizes to an EphB4 transcript under

physiological conditions and decreases the expression of EphB4 in a cell; and a nucleic acid compound that hybridizes to an EphrinB2 transcript under physiological conditions and decreases the expression of EphrinB2 in a cell. In certain embodiments, the D114/Notch pathway antagonist is selected from a soluble D114 or an antibody that binds to D114. In certain embodiments, the composition is a pharmaceutical composition and/or comprises a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1D show DU4 allelic deletion reduced tumor burden in RT2 mice due to increased nonproductive tumor angiogenesis. A, Number of tumors per animal, average tumor volume and overall tumor burden, are calculated as the sum of tumor volumes per mouse, in 13.5 week-old RT2 DU4 ^+/+ (n=8) and RT2 DU4 ^+/~ (n=8) mice. B, Vascular response examined in RT2 DU4 ^+/+ (n=5) and RT2 DU4 ^+/~ (n=5) insulinomas by PECAM immunostaining. Compared to RT2 DU4 ^+/+ controls, the RT2 mice with deleted DU4 allele showed reduced vessel calibers, increased sprouting, branching irregularities, and network disorganization {left). Dotted lines mark tumor border. / indicates insulinoma while P indicates normal pancreatic tissue surrounding the tumor. Vascular density was estimated as percentage of PECAM-positive area per tumor section surface and presented for two experimental groups {right). C, Mural cell coverage on newly formed vessels was examined by double staining of PECAM and a-SMA. While PECAM and a-SMA were co-localized in RT2 DU4 ^+/+ tumors, SMA-positive cells lining PECAM-positive endothelium were profoundly reduced in RT2 DU4 ^+/~ insulinomas {left). The percentage of PECAM-positive structures covered by SMA-positive area was measured as an indicator of vessel maturation {right). D, Tumor vessel competence evaluated by lectin perfusion. Simultaneous staining of endothelial cells with PECAM and visualization of perfused vessels with streptavidin-Alexa 488 demonstrates a reduction in the fraction of perfused vessels in RT2 D114+/- vs. RT2 D114+/+ mice {left). The percentage of PECAM-positive area co-localized with lectin (Alexa 488 signal) was measured to estimate the proportion of functional vessels within insulinomas {right). The two group values for each parameter were statistically analyzed using Mann- Whitney- Wilcoxon test. Error bars represent SEM. * P < 0.05 was considered significant.

Figure 2 shows differential gene expression in RT2 DU4 ^+/+ vs. RT2 DU4 ^+/~ insulinomas. Total RNA was isolated from harvested insulinomas (2-3 tumors/mouse, n=5 for each genotype) and gene expression analysis of tumor tissues was performed by quantitative RT-PCR for indicated genes involved in angiogenesis. Gene expression levels were normalized to β-actin levels. Error bars represent SD.

Figures 3A-3D show sEphB4-Alb inhibited insulinoma growth and extended longevity in RT2 DU4 ^+/+ and DU4 ^+/~ mice. A, Average tumor volumes developed by 13.5- week old RT2 {DU4 ^+/+ or DU4 ^+/~ ) male mice previously treated for 3.5 weeks with drug vehicle (PBS) or sEphB4-Alb (10 mg/kg). All the groups had 6 animals and were treated i.p. 3 times a week. B, Survival rates in RT2 DU4 ^+/+ and RT2 DU4 ^+/~ treated with vehicle (PBS) or sEphB4-Alb (5 mg/kg). All the groups had 10 animals and were treated i.p. 3 times a week. Survival rate was calculated as the percentage of live mice at the end of each week relative to the initial number of the animals per experimental group. C, Sections of tumors harvested at the end of the experiment mentioned in A were co-stained with anti-PECAM {red) and anti-a-SMA {green) as described in Figure 1C. D, Sections of tumors harvested at the end of the experiment mentioned in A were co-stained with anti-PECAM {red) and anti- NG2 (green). The percentage of PECAM-positive structures covered by NG2 -positive area was measured as a parameter of pericyte recruitment (right). Kaplan-Meier product-limit estimation with Breslow generalized Wilcoxon test was used for survival analyses. Other data were analyzed using Mann-Whitney-Wilcoxon test. Error bars represent SEM. * P < 0.05 and ** P < 0.01 were considered significant and highly significant, respectively.

Figures 4A-4D show combination therapy of sD114-Fc and sEphB4Alb inhibits RT2 tumor growth with greater efficacy than either molecule alone. A, Average tumor volumes developed by 13.5 -week old RT2 male mice previously treated for 3.5 weeks with drug vehicle (PBS, i.p. 3x/wk, control group), sEphB4-Alb (lOmg/kg i.p., 3x/wk), sD114-Fc (lOmg/kg i.p. 3x/wk), or combination of sEphB4-Alb and sD114-Fc (lOmg/kg, i.p. 3x/wk for both). The results were obtained from two independent trials involving 6 animals per treatment group and per trial. B, Sections of tumors harvested at the end of the experiment mentioned in A were immunostained with PECAM (red) and DAPI (blue) as described in Figure IB. C. Sections of tumors harvested at the end of the experiment mentioned in A were stained as described in Figure 1C. D, Tumor vessel functionality evaluated by lectin perfusion as described in Figure ID. Perfusion in normal pancreas tissue was also analyzed and shown on the bottom left. The data were analyzed using Mann-Whitney-Wilcoxon test. Error bars represent SEM. * P < 0.05 and ** P < 0.01 were considered significant and highly significant, respectively.

Figures 5A-5F show hepatic vascular lesions observed in endothelial-specific DU4 knock-out mice are prevented by sEphB4-Alb administration. Representative pictures of hematoxylin and eosin staining of liver sections from induced, tamoxifen-treated (A and B) and non- induced, PBS-treated (C and D) DU4 ^lox/lox Cre+ mice, and from induced, tamoxifen- treated DU4 ^lox/lox Cre+ mice administered with PBS vehicle (E) or sEphB4-Alb (F) for 12 weeks to assess the effect of Ephrin-B2/EphB4 blockade combined with total inhibition of D114/Notch endothelial signaling. A, Ten weeks after the induction of endothelium-specific D114 loss-of- function, all studied 14-week-old DU4 ^lox/lox Cre+ presented excessive sub- capsular vascular proliferation. The image shows vessels (arrows), some of which extremely dilated, occupying sub-capsular regions throughout the liver. B, Higher magnification of subcapsular blood vessels (inset in A); C, Age-matched non- induced DU4 ^lox/lox Cre+ presented normal liver histology. D, Higher magnification of the inset in C showing a single hepatic vessel that cannot be considered sub-capsular. E and F, Excessive vascular proliferation forming an hemangioma-like sub-capsular structure in a tamoxifen-induced DU4 ^{ox ox} Cre+ mouse injected with PBS and normal hepatic structure in a sEpfiB4-treated tamoxifen- induced DU4 ^lox/lox Cre+ mouse, respectively. V, blood vessel; nEC, endothelial cell nucleus, RBC, red blood cells; H, hepatocytes.

Figures 6A-6B show upregulation of Rgs5 and PSENEN by sEphB4-Alb. A. Total RNA used for global gene analysis was used to validate upregulation of Rgs5 and PSENEN. Gene expression level was normalized to β-actin. Another housekeeping gene GAPDH was also

included in this analysis. B. Human umbilical artery endothelial cells and smooth muscle cells (both from Lonza) were co-cultured (1 : 1) for overnight, followed by treatment with sEphB4-Alb (10 ug/ml) for 6 hours. Cells were harvested and total RNA was isolated for quantitative RT-PCR. Gene expression level was normalized to β-actin. Another

housekeeping gene GAPDH was also included in this analysis.

DETAILED DESCRIPTION OF THE INVENTION /. Overview

The current disclosure is based in part on the discovery that signaling through both the D114/Notch pathway and the ephrin/ephrin receptor (ephrin/eph) pathway contribute to tumorigenesis. Applicants have shown that inhibition of the ephrin/eph pathway relieves toxicity induced by D114/Notch pathway inhibition. In addition, the disclosure provides therapeutic agents and methods for inhibiting the function of the ephrin/eph pathway in combination with the D114/Notch pathway. Accordingly, in certain aspects, the disclosure provides numerous compositions (agents) that may be used to treat cancer as well as angiogenesis-related disorders and unwanted angiogenesis-related processes.

The Notch pathway, particularly Notchl and Notch4, participates in angiogenic processes. Notch signalling is generally involved in the regulation of processes as diverse as cellular proliferation, differentiation, specification and survival (Artavanis-Tsakonas et al. , 1999). Its complexity in vertebrates is illustrated by the existence of multiple Notch receptor and ligands, each with distinct patterns of expression. In mammals there are four Notch receptors (notchl-4) and five ligands (jaggedl, 2 and Dill, 3 and 4). Mutations of Notch receptors and ligands in mice lead to abnormalities in various organs, from all three germ lines, including the vascular system (Iso et al, 2003). The Notch pathway functions through local cell interactions, the extracellular domain of the ligand, present on the surface of one cell, interacts with the extracellular domain of the receptor on an adjacent cell. This interaction allows the action of two ADAM proteases on the extracellular domain of Notch followed by the action of a γ-secretase on the transmembrane domain releasing the intracellular domain from the cell membrane and allowing it to be directed to the nucleus, where it functions with CSL to activate the expression of transcriptional repressors of the enhancer-of-split family (Mumm & Kopan, 2000).

Arterial versus venous differentiation has long been thought to be mainly dependent on physical factors such as blood pressure and oxygen concentration. Recently, however, the identification of a number of genes that are specifically expressed in arterial or venous endothelial cells well before the onset of circulation, seems to indicate an important role for genetic determination of endothelial cells in the primary differentiation events between arteries and veins. Among these genes are eph-B4, specifically expressed in venous endothelial cells (Adams et al, 1999) and ephrin-B2 (Adams et al, 1999; Gale et al, 2001), notchl (Krebs et al, 2000), notch4 (Uyttendaele et al, 1996) and dll4 (Shutter et al, 2000), among others, which are specifically expressed in arterial endothelial cells.

Studies with mutations in zebrafish Notch homologues demonstrate the importance of this pathway in regulating the arterial versus venous endothelial differentiation, downstream of vascular endothelial growth factor and sonic-hedgehog and upstream of the ephrin pathway (Lawson et al, 2002), being the earliest genes expressed in an endothelial arterial specific fashion. There is mounting evidence, in both zebrafish and mouse, that Notch function is essential in the establishment of the arterial endothelial cell fate (Lawson et al, 2002; Fischer et al, 2004; Duarte et al, 2004). 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 on the worldwide web at eph-nomenclature.com. The work described herein, refers to Ephrin B2 and EphB4. However, the present invention contemplates any ephrin ligand and/or Eph receptor within their respective family that 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.

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.

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. The cytoplasmic domain consists of a juxtamembrane region containing two conserved tyrosine residues; a protein tyrosine kinase domain; a sterile a-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.

Upon ligand binding, an Eph receptor dimerizes and autophosphorylate the juxtamembrane tyrosine residues to acquire full activation (Kalo MS et al, 1999, Binns KS, 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 DJ, 1999; Kiyokawa E, 1994; Tang XX, 1999; Vogt T, 1998; Liu W, 2002; Stephenson SA, 2001; Steube KG 1999; Berclaz G, 1996). Targeting tumor angiogenesis, in particular through blocking vascular endothelial growth factor (VEGF) activity, has been successful, with several drugs now approved for use in many different cancer types [1]. Rapid development of resistance to these [2], however, highlights the need for other vascular targeted therapies.

D114/Notch pathway, which plays prominent role in angiogenesis, has become such a target. D114 is critical for embryonic vascular development and arterial specification and is markedly induced in murine and human tumor vessels [3-8]. Notchl and Notch4 are also expressed in tumor vessels [6]. Notch ligand expression and Notch activation is induced by VEGF [9, 10]. D114/Notch signaling in turn attenuates VEGF signaling, thus arresting endothelial cell proliferation, followed by recruitment of mural cells and vessel maturation [7-9, 11, 12].

Not surprisingly, targeted DU4 allele deletion results in increased vascular

proliferation but, unexpectedly, impaired vessel structure and function [8]. Overall, inhibition of D114/Notch causes reduced tumor growth. Careful evaluation of the tumor vessels reveals increased vessel proliferation, reduced lumen size, reduced mural cell recruitment, increased leakiness and reduced perfusion. Furthermore, tumors resistant to VEGF targeted therapy remain responsive to D114/Notch inhibitors [7, 12].

Another ligand-receptor pair downstream from the VEGF and Notch pathways that plays a critical role in artery- vein endothelium specification is Ephrin-B2 and EphB4. Ephrin- B2 is specifically expressed in arterial angioblasts, endothelial cells, and perivascular mesenchymal cells, whereas EphB4 is expressed in endothelial cells belonging to the venous lineage only. Targeted disruption of either EphB4 or EfnB2 results in early lethality in the developing embryo as a result of arrested angiogenesis but not vasculogenesis [13-15].

On binding, the receptor and ligand on adjacent cells undergo dimerization and clusterization, activating forward and reverse signaling in receptor-expressing and ligand- expressing cells respectively to achieve vascular maturation. The monomeric form of the extracellular domain of EphB4 functions as an antagonist of EphB4-Ephrin-B2 signaling, thus blocking endothelial cell migration and tube formation and retarding angiogenesis in tumor models [16, 17]. Fusion of this protein with albumin at the C-terminus (sEphB4-Alb) results in favorable pharmokinetics for clinical development.

The work described herein makes use of the RIPl-Tag2 transgenic mouse model [18], wherein pancreatic islet carcinogenesis occurs secondary to the expression of the SV-40 large T-antigen (Tag) expression under the Rat Insulin Promoter (RIP). In this model, angiogenic islets become hyperplastic and dysplastic by week 5, and acquire angiogenic switch by week 10, progressing to adenomas (insulinomas) and invasive carcinomas [19]. Predictable stepwise progression and angiogenic switch permits investigation of tumor angiogenesis and their inhibitors.

The term "antibody" as used herein is intended to include fragments thereof which are also specifically reactive with antigen. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab)2 fragments can be generated by treating antibody with pepsin. The resulting F(ab)2 fragment can be treated to reduce disulfide bridges to produce Fab fragments. The antibody of the present invention is further intended to include bispecific, single-chain, and chimeric and humanized molecules having affinity for antigen conferred by at least one CDR region of the antibody. Techniques for the production of single chain antibodies (US Patent No. 4,946,778) can also be adapted to produce single chain antibodies. Also, transgenic mice or other organisms including other mammals, may be used to express humanized antibodies. In certain embodiments, the antibodies further comprise a label attached thereto and able to be detected (e.g., the label can be a radioisotope, fluorescent compound, enzyme or enzyme co-factor).

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 polypeptides of the disclosure.

II. DlWNotch pathway antagonists

D114 is a Notch ligand and contains a signal sequence, a DSL domain, eight epidermal growth factor-like repeats, a transmembrane domain, and an intracellular region, all of which are characteristics of members of the Delta protein family. The tissue distribution of Delta-4 mRNA resembles that previously described for Notch-4 (Int-3) transcripts. Soluble forms of the extracellular portion of Delta-4 inhibit the apparent proliferation of human aortic endothelial cells, but not human pulmonary arterial endothelial cells. Yoneya et al. J.

Biochem. Vol. 129, pp. 27-34 (2001).

Members of the Notch family of proteins are transmembrane receptors that contain characteristic multiple epidermal growth factor (EGF)-like repeats as well as conserved domains such as RAM, ankyrin-like repeat, and PEST sequences. Ligands for Notch proteins include Delta and Serrate in Drosophila melanogaster, LAG -2 and APX-1 in Caenorhabditis elegans, and Delta and Serrate (or Jagged) in vertebrates. These ligands are also

transmembrane proteins and contain a highly conserved DSL (Delta-Serrate-LAG-2) motif upstream of a variable number of EGF-like repeats. The DSL domain is a characteristic feature of Notch ligands and is important for protein function; thus, point mutation of the DSL domain in LAG-2 results in a loss of activity. Although the Delta and Jagged (Serrate) proteins of vertebrates exhibit similar structures, each group of proteins also possesses several distinct features. Thus, whereas vertebrate Delta proteins contain eight EGF-like repeats, Jagged proteins contain 16 such repeats. Furthermore, the EGF domains are followed by a cysteine-rich domain in Jagged proteins but not in Delta proteins. However, the consequences of these structural differences remain unclear.

Uyttendaele et al. (1996) cloned cDNAs corresponding to the complete coding region of the mouse Notch4 gene. In situ hybridization revealed that Notch4 transcripts are primarily restricted to endothelial cells in embryonic and adult life, suggesting a role for Notch4 during development of vertebrate endothelium. Li et al. (Genomics. 1998 Jul l;51(l):45-58) reported that the human NOTCH4 gene contains 30 exons and spans approximately 30 kb. They isolated cDNAs corresponding to 6.7-kb NOTCH4(S) and 9.3-kb NOTCH4(L) mRNA isoforms. The predicted protein encoded by NOTCH4(S) is 2,003 amino acids long and contains the characteristic Notch motifs: a signal peptide, 29 epidermal growth factor (EGF)-like repeats, 3 Notch/lin-12 repeats, a transmembrane region, 6 cdclO (603151)/ankyrin repeats, and the PEST conserved region at the C terminus. The sequences of the mouse and human NOTCH4 proteins are 82% identical. The incompletely spliced NOTCH4(L) cDNA potentially encodes 2 different proteins. One consists of the first 7 EGF repeats. The second contains the transmembrane domain and intracellular region and is similar to the mouse int3 protooncoprotein. Northern blot analysis revealed that NOTCH4(S) is the major transcript and is expressed in a wide variety of tissues.

Krebs et al. (2000) generated Notch4-deficient mice by gene targeting. Embryos homozygous for this mutation developed normally, and homozygous mutant adults were viable and fertile. However, the Notch4 mutation displayed genetic interactions with a targeted mutation of the related Notchl gene. Both Notchl mutant and Notchl/Notch4 double mutant embryos displayed severe defects in angiogenic vascular remodeling. Analysis of the expression patterns of genes encoding ligands for Notch family receptors indicated that only the D114 gene is expressed in a pattern consistent with that expected for a gene encoding a ligand for the Notchl and Notch4 receptors in the early embryonic vasculature. Therefore, there is an essential role for the Notch signaling pathway in regulating vascular

morphogenesis and remodeling, and indicate that whereas the Notch4 gene is not essential during embryonic development, the Notch4 and Notchl genes have partially overlapping roles during embryogenesis in mice.

As noted above, the disclosure provides compositions and methods for using antagonists of D114 signaling. Examples of D114 antagonists are described in US Patent Application Publication Nos. 2007-0213266 and 2009-0035308, and US Patent Nos.

6,664,098 and 7,022,499, and PCT Publication Nos. WO96/01839 and W098/51799, incorporated by reference herein. Candidate antagonists will generally be any antibody that binds to, or soluble portions of, proteins involved in the D114 signaling pathway, including, for example, D114, Notchl, Notch4 and presenilin. Candidate antagonists may also be small molecules or other agents that bind to or effect members of the pathway. Antisense or RNAi nucleic acids may be used as antagonists of D114, Notchl, Notch4 or presenilin or other members of the signaling pathway.

Examples of agents include:

(a) an antibody that binds selectively to D114;

(b) an antibody that binds selectively to Notchl;

(c) an antibody that binds selectively to Notch4;

(d) an antibody that binds to Notchl and Notch4;

(e) a polypeptide monomer comprising a Notch-receptor binding portion of

D114;

(f) a polypeptide dimer comprising a Notch-receptor binding portion of D114;

(g) a polypeptide multimer comprising two or more polypeptides comprising a Notch-receptor binding portion of D114;

(h) a polypeptide monomer comprising a D114-binding portion of Notchl or

Notch4;

(i) a polypeptide multimer comprising two or more polypeptides comprising a D114-binding portion of Notchl or Notch4.

Agents that interfere with presenilin activity or other metalloproteinases (e.g., kuzbanian) are expected to modulate D114 signaling. Each of these agents may be assessed for antagonist activity as described herein.

A. DU4 Polypeptides

In certain aspects, the agent for use as a D114/Notch antagonist in the disclosed methods and compositions is a soluble polypeptide comprising an extracellular domain of a D114 protein, e.g., as shown in amino acids 27-531 of SEQ ID NO: 1. In a specific embodiment, the D114 soluble polypeptide comprises a DSL domain of a D114 protein. In another embodiment, the D114 soluble polypeptide is a truncate comprising at least domains 5 or 6 of the EGF-like domains. As used herein, the soluble polypeptides include fragments, functional variants, and modified forms of D114 soluble polypeptide. These fragments, functional variants, and modified forms of the soluble polypeptides may be tested for activity as antagonists of D114 by assessing effects on arterial or venous phenotype in endothelial cells. In certain embodiments, isolated fragments of the soluble polypeptides can be obtained by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding a D114. 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 modulate D114 signaling.

In certain embodiments, a functional variant of a D114 soluble polypeptide comprises an amino acid sequence that is at least 90%, 95%, 97%, 99% or 100% identical to residues 27-531 of the amino acid sequence of SEQ ID NO: 1.

In certain embodiments, the present invention contemplates using functional variants by modifying the structure of the 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). 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.

This invention further contemplates the use of sets of combinatorial mutants of the D114 polypeptides, as well as truncation mutants, and 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 D114. 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 soluble polypeptide levels by modulating their half-life. 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.

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, SA (1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; 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. Patent Nos: 5,223,409, 5,198,346, and 5,096,815).

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, NY; 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 a soluble polypeptide.

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 soluble polypeptides. The most widely used techniques for screening large gene libraries typically comprise 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.

In certain embodiments, the soluble polypeptides of the invention may further comprise post-translational modifications. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. As a result, the modified soluble polypeptides may contain non-amino acid elements, such as polyethylene glycols, 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 antagonist effects on D114.

In certain aspects, functional variants or modified forms of the 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. In certain embodiments, the soluble polypeptides of the present invention contain one or more modifications that are capable of stabilizing the soluble polypeptides. For example, such modifications may enhance the in vitro half life of the soluble polypeptides, enhance circulatory half life of the soluble polypeptides or reduce proteolytic degradation of the soluble polypeptides.

In certain embodiments, soluble polypeptides (unmodified or modified) of the invention can be produced by a variety of suitable 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).

B. Gene therapy

In certain aspects, the D114/Notch antagonists are isolated and/or recombinant nucleic acids encoding a D114 polypeptide. The 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.

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 NO:2. One of ordinary skill in the art will appreciate that nucleic acid sequences complementary to the nucleic acids, and variants of the 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. 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 NO:2, or sequences complementary thereto. As discussed above, one of ordinary skill in the art will readily understand that appropriate stringency conditions which promote DNA hybridization can be varied. For example, one could perform the

hybridization at 6.0 x sodium chloride/sodium citrate (SSC) at about 45 °C, followed by a wash of 2.0 x SSC at 50 °C. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50 °C to a high stringency of about 0.2 x 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 x SSC at room temperature followed by a wash at 2 x SSC at room temperature.

Isolated nucleic acids which differ 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 lead to changes in the amino acid sequences of the 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. 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 certain embodiments, 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.

In certain aspect of the invention, the nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding a D114 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, CA (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 a-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.

This invention also pertains to a host cell transfected with a recombinant gene including a coding sequence for one or more of the 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.

C. Antibodies In certain aspects, the D114/Notch antagonist comprises antibodies that have antagonist effects on D114 signaling. Such antibodies may bind to antigens such as D114, Notch 1 or Notch4. In certain embodiments, the antibody binds to an extracellular domain of such antigens. It is understood that antibodies may be polyclonal or monoclonal; intact or truncated, e.g., F(ab')2, Fab, Fv; xenogeneic, allogeneic, syngeneic, fully human or modified forms thereof, e.g., humanized, chimeric. Fully human antibodies may be selected from transgenic animals that express human immunoglobulin genes or assembled from

recombinant libraries expressing antibody fragments.

For example, by using immunogens derived from D114, Notchl or Notch4, anti- protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (see, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal, such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide (e.g., a polypeptide or an antigenic fragment which is capable of eliciting an antibody response, or a fusion protein). Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. An immunogenic portion of an antigen can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.

Following immunization of an animal with an antigenic preparation, antisera can be obtained and, if desired, polyclonal antibodies can be isolated from the serum. To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells. Such techniques are well known in the art, and include, for example, the hybridoma technique (originally developed by Kohler and Milstein, (1975) Nature, 256: 495-497), the human B cell hybridoma technique (Kozbar et al, (1983) Immunology Today, 4: 72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with D114, Notchl, Notch4 or other target polypeptide and monoclonal antibodies isolated from a culture comprising such hybridoma cells. In certain embodiments, a suitable antibody is a monoclonal antibody. A method for generating a monoclonal antibody that binds specifically to D114, Notchl or Notch4 may comprise administering to a mouse an amount of an immunogenic composition comprising the antigen polypeptide effective to stimulate a detectable immune response, obtaining antibody-producing cells (e.g., cells from the spleen) from the mouse and fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody-producing hybridomas to identify a hybridoma that produces a monocolonal antibody that binds specifically to the antigen. Once obtained, a hybridoma can be propagated in a cell culture, optionally in culture conditions where the hybridoma-derived cells produce the monoclonal antibody. The monoclonal antibody may be purified from the cell culture.

In certain embodiments, the methods utilize humanized and/or deimmunized versions of any of the antibodies disclosed herein, and/or antibodies and antigen-binding portions thereof that comprise at least one CDR portion derived from an antibody disclosed herein, particularly the CDR3. In certain embodiments, the antibody is a monoclonal antibody that is immunocompatible with the subject to which it is to be administered, and may be clinically acceptable for administration to a human.

In certain embodiments, single chain antibodies, and chimeric, humanized or primatized (CDR-grafted) antibodies, as well as chimeric or CDR-grafted single chain antibodies, comprising portions derived from different species, are also encompassed by the present invention as antigen binding portions of an antibody. The various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., Cabilly et al, U.S. Pat. No. 4,816,567; Cabilly et al, European Patent No.

0,125,023 Bl; Boss et al, U.S. Pat. No. 4,816,397; Boss et al, European Patent No.

0,120,694 Bl; Neuberger, M. S. et al, WO 86/01533; Neuberger, M. S. et al, European Patent No. 0,194,276 Bl; Winter, U.S. Pat. No. 5,225,539; and Winter, European Patent No. 0,239,400 Bl . See also, Newman, R. et al, BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody. See, e.g., Ladner et al, U.S. Pat. No. 4,946,778; and Bird, R. E. et al, Science, 242: 423-426 (1988)), regarding single chain antibodies.

In addition, functional fragments of antibodies, including fragments of chimeric, humanized, primatized or single chain antibodies, can also be produced. Functional fragments of the antibodies retain at least one binding function and/or modulation function of the full-length antibody from which they are derived. In certain embodiments, functional fragments retain an antigen binding function of a corresponding full-length antibody (e.g., specificity for D114). Certain functional fragments retain the ability to inhibit one or more functions characteristic of D114, such as a binding activity, a signaling activity, and/or stimulation of a cellular response.

In addition, the techniques used to screen antibodies in order to identify a desirable antibody may influence the properties of the antibody obtained. For example, an antibody to be used for certain therapeutic purposes may be able to target a particular cell type.

Accordingly, to obtain antibodies of this type, it may be desirable to screen for antibodies that bind to cells that express the antigen of interest (e.g., by fluorescence activated cell sorting). Likewise, if an antibody is to be used for binding an antigen in solution, it may be desirable to test solution binding. A variety of different techniques are available for testing

antibody:antigen interactions to identify particularly desirable antibodies. Such techniques include ELISAs, surface plasmon resonance binding assays (e.g. the Biacore binding assay, Bia-core AB, Uppsala, Sweden), sandwich assays (e.g. the paramagnetic bead system of IGEN International, Inc., Gaithersburg, Maryland), western blots, immunoprecipitation assays and immunohistochemistry.

D. Antisense and RNAi In certain aspects, the D114/Notch antagonist is an isolated nucleic acid compound comprising at least a portion that hybridizes to a D114 transcript under physiological conditions and decreases the expression of D114 in a cell. Such nucleic acids may be used as D114 antagonists, as described herein. The D114 transcript may be any pre-splicing transcript (i.e., including introns), post-splicing transcript, as well as any splice variant. In certain embodiments, the D114 transcript has a sequence corresponding to the cDNA set forth in SEQ ID NO:2, and particularly the coding portion thereof. In certain aspects, the disclosure provides isolated nucleic acid compounds comprising at least a portion that hybridizes to a Notchl or Notch4 transcript under physiological conditions and decreases the expression of Notchl or Notch4 in a cell. These may be used as D114 antagonists also. The Notchl or Notch4 transcript may be any pre-splicing transcript (i.e., including introns), post-splicing transcript, as well as any splice variant.

Examples of categories of nucleic acid compounds include antisense nucleic acids, RNAi constructs and catalytic nucleic acid constructs. A nucleic acid compound may be single or double stranded. A double stranded compound may also include regions of overhang or non-complementarity, where one or the other of the strands is single stranded. A single stranded compound may include regions of self-complementarity, meaning that the compound forms a so-called "hairpin" or "stem-loop" structure, with a region of double helical structure. A nucleic acid compound may comprise a nucleotide sequence that is complementary to a region consisting of no more than 1000, no more than 500, no more than 250, no more than 100 or no more than 50 nucleotides of the D114, Notchl or Notch4 nucleic acid sequence. The region of complementarity may be at least 8 nucleotides, and optionally at least 10 or at least 15 nucleotides. A region of complementarity may fall within an intron, a coding sequence or a noncoding sequence of the target transcript, such as the coding sequence portion. Generally, a nucleic acid compound will have a length of about 8 to about 500 nucleotides or base pairs in length, and optionally the length will be about 14 to about 50 nucleotides. A nucleic acid may be a DNA (particularly for use as an antisense), RNA or RNA:DNA hybrid. Any one strand may include a mixture of DNA and RNA, as well as modified forms that cannot readily be classified as either DNA or RNA. Likewise, a double stranded compound may be DNA:DNA, DNA:RNA or RNA:RNA, and any one strand may also include a mixture of DNA and RNA, as well as modified forms that cannot readily be classified as either DNA or RNA. A nucleic acid compound may include any of a variety of modifications, including one or modifications to the backbone (the sugar-phosphate portion in a natural nucleic acid, including internucleotide linkages) or the base portion (the purine or pyrimidine portion of a natural nucleic acid). An antisense nucleic acid compound will may have a length of about 15 to about 30 nucleotides and will often contain one or more modifications to improve characteristics such as stability in the serum, in a cell or in a place where the compound is likely to be delivered, such as the stomach in the case of orally delivered compounds and the lung for inhaled compounds. In the case of an RNAi construct, the strand complementary to the target transcript will generally be RNA or modifications thereof. The other strand may be RNA, DNA or any other variation. The duplex portion of double stranded or single stranded "hairpin" RNAi construct may have a length of 18 to 40 nucleotides in length and optionally about 21 to 23 nucleotides in length, so long as it serves as a Dicer substrate. Catalytic or enzymatic nucleic acids may be ribozymes or DNA enzymes and may also contain modified forms. Nucleic acid compounds may inhibit expression of the target by about 50%, 75%, 90%> or more when contacted with cells under physiological conditions and at a concentration where a nonsense or sense control has little or no effect. In certain embodiments, concentrations for testing the effect of nucleic acid compounds are 1, 5 and 10 micromolar. Nucleic acid compounds may also be tested for effects on cellular phenotypes, such as arterial or venous identity.

III. Eph/Ephrin pathway antagonists

Examples of Eph and Ephrin proteins and their binding specificities are known in the art and are described in WO 9952541, incorporated by reference herein. Although specific examples of Ephrin B2 and EphB4 antagonists are described below, other Eph and Ephrin antagonists are contemplated for used in the claimed compositions and methods. Eph/Ephrin pathway antagonists may be of any type including polypeptides, nucleic acids and small molecules. Examples of Eph/Ephrin pathway antagonists are are known in the art and are described in US Patent Application Publication Nos. 2005-0084873, 2010-0261653, and 2009-0196880, and US Patent Nos. 6,864,227, 6,887,674, and 7,381,410, incorporated by reference herein.

A. Polypeptides

In certain aspects, the methods employ 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). In certain embodiments, a soluble polypeptide is a monomer and is capable of binding with high affinity to Ephrin B2 or EphB4. In a specific embodiment, an EphB4 soluble polypeptide comprises a globular domain of an EphB4 protein.

As used herein, the 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 soluble polypeptides antagonize function of EphB4, Ephrin B2 or both.

In certain embodiments, isolated fragments of the 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. 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 SEQ ID NO: 12. 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 SEQ ID NO: 12.

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 SEQ ID NO: 13 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 SEQ ID NO: 13.

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 may be 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 SEQ ID NO: 12. A soluble EphB4 polypeptide may comprise a sequence at least 90% identical to residues 1-412 of the amino acid sequence defined by SEQ ID NO: 12. A soluble EphB4 polypeptide may comprise a sequence at least 90%) identical to residues 1-312 of the amino acid sequence defined by SEQ ID NO: 12. A soluble EphB4 polypeptide may comprise a sequence encompassing the globular (G) domain (amino acids 29-197 of SEQ ID NO: 12, and optionally additional domains, such as the cysteine-rich domain (amino acids 239-321 of SEQ ID NO: 12), the first fibronectin type 3 domain (amino acids 324-429 of SEQ ID NO: 12) and the second fibronectin type 3 domain (amino acids 434-526 of SEQ ID NO: 12). Certain 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 SEQ ID NO:12. A soluble EphB4 polypeptide may comprise a sequence as set forth in SEQ ID Nos. 5 or 6. 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 SEQ ID NO: 12. 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 SEQ ID NO: 12): 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 SEQ ID NO: 12): 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. In certain embodiments, any variations in the amino acid sequence from the sequence shown in SEQ ID NO: 12) 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. 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 may be 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 SEQ ID NO: 13. A soluble EphrinB2 polypeptide may comprise a sequence defined by SEQ ID NO:5. 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 SEQ ID NO: 13. 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 SEQ ID NO: 13. 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 SEQ ID NO: 13): 26-225. In certain embodiments, the soluble EphrinB2 polypeptide may inhibit the interaction between Ephrin B2 and EphB4. The soluble EphrinB2 polypeptide may inhibit clustering of or

phosphorylation of EphrinB2 or EphB4. As noted above, the soluble EphrinB2 polypeptide may be prepared as a monomeric or multimeric fusion protein. The soluble polypeptide may include one or more modified amino acids. Such amino acids may contribute to desirable properties, such as increased resistance to protease digestion. In certain embodiments, the present invention contemplates making functional variants by modifying the structure of the 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 polypeptides 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.

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 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.

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, SA (1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; 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. Patent Nos: 5,223,409, 5,198,346, and 5,096,815).

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, NY; 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 a soluble polypeptide. 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 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.

To illustrate, by employing scanning mutagenesis to map the amino acid residues of a soluble polypeptidewhich are involved in binding to another protein, peptidomimetic compounds can be generated which mimic those residues involved in binding. For instance, non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al, in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al, in Peptides:

Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma lactam rings (Garvey et al, in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al, (1986) J. Med. Chem. 29:295; and Ewenson et al, in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co.

Rockland, IL, 1985), b-turn dipeptide cores (Nagai et al, (1985) Tetrahedron Lett 26:647; and Sato et al., (1986) J Chem Soc Perkin Trans 1 : 1231), and b-aminoalcohols (Gordon et al., (1985) Biochem Biophys Res Commun 126:419; and Dann et al., (1986) Biochem Biophys Res Commun 134:71). In certain embodiments, the soluble polypeptides of the invention may further comprise post-translational modifications. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. As a result, the modified soluble polypeptides may contain non-amino acid elements, such as polyethylene glycols, 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 antagozing role in EphB4 or Ephrin B2 function, e.g, it inhibitory effect on angiogenesis or on tumor growth.

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

The one or more PEG may have a molecular weight ranging from about 1 kDa to about 100 kDa, and may 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 certain embodiments, the soluble, monomeric Eph/Ephrin conjugate comprises an

Eph/Ephrin polypeptide covalently linked to one PEG group of from about 10 to about 40 kDa (e.g., monoPEGylated EphB4), or from about 15 to 30 kDa, such as via an ε-amino group of lysine or the N-terminal amino group. In certain embodiments, the Eph/Ephrin is randomly PEGylated at one amino group out of the group consisting of the ε-amino groups of lysine and the N-terminal amino group. In one embodiment, the pegylated polypeptides provided by the invention have a serum half-life in vivo at least 50%, 75%, 100%, 150% or 200% greater than that of an unmodified EphB4 polypeptide. In another embodiment, the pegylated EphB4 polypeptides provided by the invention inhibit EphrinB2 activity. In a specific embodiment, they inhibit EphrinB2 receptor clustering, EphrinB2 phosphorylation, and/or EphrinB2 kinase activity. Surprisingly, it has been found that monoPEGylated EphB4 according to the invention has superior properties in regard to the therapeutic applicability of unmodified soluble EphB4 polypeptides and poly-PEGylated EphB4. Nonetheless, the disclosure also provides poly-PEGylated EphB4 having PEG at more than one position. Such

polyPEGylated forms provide improved serum-half life relative to the unmodified form. In certain embodiments, a soluble Eph/Ephrin polypeptide is stably associated with a second stabilizing polypeptide that confers improved half-life without substantially diminishing binding. A stabilizing polypeptide may be immunocompatible with human patients (or animal patients, where veterinary uses are contemplated) and have little or no significant biological activity. In certain embodiments, the stabilizing polypeptide is a human serum albumin, or a portion thereof. A human serum albumin may be stably associated with the Eph/Ephrin polypeptide covalently or non-covalently. Covalent attachment may be achieved by expression of the Eph/Ephrin 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 Eph/Ephrin polypeptide. Exposed loops of the Eph/Ephrin would be appropriate positions for insertion of an albumin sequence. Albumin may also be post-translationally attached to the Eph/Ephrin polypeptide by, for example, chemical cross-linking. An Eph/Ephrin 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 embodiment, the EphB4-HSA fusion inhibits the interaction between Ephrin B2 and EphB4, the clustering of Ephrin B2 or EphB4, the phosphorylation of Ephrin B2 or EphB4, or combinations thereof. In other embodiments, the EphB4-HSA fusion has enhanced in vivo stability relative to the unmodified wildtype polypeptide. In certain 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.

B. Nucleic acids encoding soluble polypeptides In certain aspects, the invention relates to isolated and/or recombinant nucleic acids encoding an EphB4 or Ephrin B2 soluble polypeptide. The 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. 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: 9 or 10. One of ordinary skill in the art will appreciate that nucleic acid sequences complementary to the nucleic acids, and variants of the 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.

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: 9 or 10, 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 x sodium

chloride/sodium citrate (SSC) at about 45 °C, followed by a wash of 2.0 x SSC at 50 °C. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50 °C to a high stringency of about 0.2 x 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 x SSC at room temperature followed by a wash at 2 x SSC at room temperature.

Isolated nucleic acids which differ 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 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.

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 one 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.

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).

C. Antibodies

In certain aspects, the Eph/Ephrin pathway antagonist may be a polypeptide binding agent. The disclosure provides, in part, defined portions of the Eph/Ephrin molecule, such as EphB4, that can be effectively targeted by polypeptide binding agents, such as antibodies, antigen binding portions of antibodies, and non-immunoglobulin antigen binding scaffolds. The Eph/Eprhin polypeptide binding agents described herein may be used to treat a variety of disorders, particularly cancers and disorders related to unwanted angiogenesis. The disclosure provides antibodies and antigen binding portions thereof that inhibit one or more Eph/Ephrin mediated functions, such as EphrinB2 binding or EphB4 kinase activity. Such binding agents may be used to inhibit function in vitro and in vivo, and may be used for treating cancer or disorders associated with unwanted angiogenesis. The disclosure also provides antibodies and antigen binding portions thereof that activate EphB4 kinase activity (typically assessed by evaluating EphB4 phosphorylation state). Surprisingly, such antibodies also inhibit EphB4 functions in cell based and in vivo assays. Accordingly, such binding agents may be used to inhibit EphB4 function in vitro and in vivo, and for may be used treating cancer or disorders associated with unwanted angiogenesis. While not wishing to be limited to any particular mechanism, it is expected that these antibodies stimulate not only EphB4 kinase activity, but also EphB4 removal from the membrane, thus decreasing overall EphB4 levels.

A number of monoclonal antibodies against EphB4 as well as hybridoma cell lines producing EphB4 monoclonal antibodies may be used in the disclosed methods and compositions. These antibodies were further characterized in many ways, such as, their ability to inhibit interaction between EphB4 and its ligand (e.g., Ephrin B2), their ability to inhibit dimerization or multimerization of EphB4 receptor, their ability to induce tyrosine phosphorylation of EphB4, their cross-reactivity with other Eph family members, their ability to inhibit angiogenesis, and their ability to inhibit tumor growth. Further, epitope mapping studies reveals that these EphB4 antibodies may specifically bind to one or more regions of EphB4 (e.g., a globular domain, a cystein-rich domain, or a fibronectin type III domain). For example, an EphB4 antibody may bind to both fibronectin type 3 domains.

In certain aspects, antibodies of the invention specifically bind to an extracellular domain (ECD) of an EphB4 protein (also referred to herein as a soluble EphB4 polypeptide). A soluble EphB4 polypeptide may comprise a sequence encompassing the globular (G) domain (amino acids 29-197 of SEQ ID NO: 12), and optionally additional domains, such as the cysteine -rich domain (amino acids 239-321 of SEQ ID NO: 12), the first fibronectin type 3 domain (amino acids 324-429 of SEQ ID NO: 12) and the second fibronectin type 3 domain (amino acids 434-526 of SEQ ID NO: 12). As used herein, the EphB4 soluble polypeptides include fragments, functional variants, and modified forms of EphB4 soluble polypeptide.

In certain aspects, the present invention provides antibodies (anti-EphB4) having binding specificity for an EphB4 or a portion of EphB4. Examples of these antibodies include, but are not limited to, EphB4 antibody Nos. 1, 23, 35, 47, 57, 79, 85L, 85H, 91, 98, 121, 131, and 138. Optionally, the immunoglobulins can bind to EphB4 with an affinity of at least about 1 10 ^"6 , 1 x 10 ^"7 , 1 x 10 ^"8 , 1 x 10 ^"9 M or less. Optionally, antibodies and portions thereof bind to EphrinB2 with an affinity that is roughly equivalent to that of a soluble extracellular EphB4 polypeptide comprising the globular ligand binding domain. Antibodies disclosed herein may be specific for EphB4, with minimal binding to other members of the Eph or Ephrin families.

In certain embodiments, antibodies of the present invention bind to one or more specific domains of EphB4. For example, an antibody binds to one or more extracellular domains of EphB4 (such as the globular domain, the cystein-rich domain, and the first fibronectin type 3 domain, and the second fibronectin type 3 domain). For example, EphB4 antibody Nos. 1, 23, 35, and 79 bind to an epitope spanning the globular domain. EphB4 antibody Nos. 85L, 85H, 91, and 131 bind to an epitope in the region spanning amino acids 327-427, including the first fibronectin type 3 domain. EphB4 antibody Nos. 47, 57, 85H, 98, 121, and 138 bind to an epitope in the region spanning amino acids 428-537, including the second fibronectin type 3 domain. Optionally, the subject antibody (e.g., EphB4 antibody No. 85H) can bind to at least two domains of an EphB4. Antibodies 23, 91, 98, 131 and 138 were deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209 under Accession Numbers PTA-6208, PTA-6209, PTA-6210, PTA-6211 and PTA-6214, respectively, on October 7, 2004. Antibody 47 was deposited with the ATCC under Accession Number PTA-11338 on October 11, 2010.

In certain embodiments, antibodies of the present invention are modified antibodies, or antigen-binding fragments thereof that bind to the extracellular domain of EphB4. The modified anti-EphB4 antibodies, or antigen-binding fragments thereof, are less immunogenic compared to their unmodified parent antibodies in a given species, e.g., a human. The antibodies and antigen binding fragments are useful in therapeutic treatments for affecting EphB4 function in order to inhibit angiogenesis and tumor growth. In one embodiment, the deimmunized antibody or antigen binding fragment thereof that binds the extracellular domain of EphB4, including a heavy chain variable region and a light chain variable region, wherein each variable region has between 2 to 20 amino acid substitutions in the framework region in comparison to a nonhuman or parent antibody that binds the extracellular domain of EphB4.

In one embodiment, the deimmunized antibody or antigen binding fragment thereof has one or more complementarity determining regions (CDRs) from a nonhuman or parent antibody that binds the extracellular domain of EphB4. In one embodiment, between 1-5 substitutions are present in the complementarity determining regions (CDRs). In certain embodiments, the parent antibodies are antibodies 47 or 131. In certain embodiments, the antibodies comprise the six CDRs of a heavy and light chain selected from the sequences of SEQ ID NOs: 22 -73. In certain embodiments, the antibodies comprise a heavy and light chain selected from the sequences of SEQ ID NOs: 22 -73.

In certain embodiments, single chain antibodies, and chimeric, humanized or primatized (CDR-grafted) antibodies, as well as chimeric or CDR-grafted single chain antibodies, comprising portions derived from different species, are also encompassed by the present invention as antigen binding portions of an antibody. The various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., Cabilly et al, U.S. Pat. No. 4,816,567; Cabilly et al, European Patent No.

0,125,023 Bl; Boss et al, U.S. Pat. No. 4,816,397; Boss et al, European Patent No.

0,120,694 Bl; Neuberger, M. S. et al, WO 86/01533; Neuberger, M. S. et al, European Patent No. 0,194,276 Bl; Winter, U.S. Pat. No. 5,225,539; and Winter, European Patent No. 0,239,400 Bl . See also, Newman, R. et al, BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody. See, e.g., Ladner et al, U.S. Pat. No. 4,946,778; and Bird, R. E. et al, Science, 242: 423-426 (1988)), regarding single chain antibodies.

In addition, functional fragments of antibodies, including fragments of chimeric, humanized, primatized or single chain antibodies, can also be produced. Functional fragments of the subject antibodies retain at least one binding function and/or modulation function of the full-length antibody from which they are derived. In certain embodiments, functional fragments retain an antigen binding function of a corresponding full-length antibody (e.g., specificity for an EphB4). Certain functional fragments retain the ability to inhibit one or more functions characteristic of an EphB4, such as a binding activity, a signaling activity, and/or stimulation of a cellular response. For example, in one

embodiment, a functional fragment of an EphB4 antibody can inhibit the interaction of EphB4 with one or more of its ligands (e.g., Ephrin B2) and/or can inhibit one or more receptor-mediated functions, such as cell migration, cell proliferation, angiogenesis, and/or tumor growth.

For example, antibody fragments capable of binding to an EphB4 receptor or portion thereof, including, but not limited to, Fv, Fab, Fab' and F(ab') ₂ fragments are encompassed by the invention. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For instance, papain or pepsin cleavage can generate Fab or F(ab') ₂ fragments, respectively. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons has been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab') ₂ heavy chain portion can be designed to include DNA sequences encoding the CHi domain and hinge region of the heavy chain.

The term "humanized immunoglobulin" as used herein refers to an immunoglobulin comprising portions of immunoglobulins of different origin, wherein at least one portion is of human origin. Accordingly, the present invention relates to a humanized immunoglobulin having binding specificity for an EphB4 (e.g., human EphB4), said immunoglobulin comprising an antigen binding region of nonhuman origin (e.g., rodent) and at least a portion of an immunoglobulin of human origin (e.g., a human framework region, a human constant region or portion thereof). For example, the humanized antibody can comprise portions derived from an immunoglobulin of nonhuman origin with the requisite specificity, such as a mouse, and from immunoglobulin sequences of human origin (e.g., a chimeric

immunoglobulin), joined together chemically by conventional techniques (e.g., synthetic) or prepared as a contiguous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portions of the chimeric antibody can be expressed to produce a contiguous polypeptide chain).

Another example of a humanized immunoglobulin of the present invention is an immunoglobulin containing one or more immunoglobulin chains comprising a CDR of nonhuman origin (e.g., one or more CDRs derived from an antibody of nonhuman origin) and a framework region derived from a light and/or heavy chain of human origin (e.g., CDR- grafted antibodies with or without framework changes). In one embodiment, the humanized immunoglobulin can compete with murine monoclonal antibody for binding to an EphB4 polypeptide. Chimeric or CDR-grafted single chain antibodies are also encompassed by the term humanized immunoglobulin. In certain embodiments, the present invention provides Eph/Ephrin antagonist antibodies. As described herein, the term "antagonist antibody" refers to an antibody that can inhibit one or more functions of an Eph/Ephrin, such as a binding activity (e.g., ligand binding) and a signaling activity (e.g., clustering or phosphorylation of EphB4, stimulation of a cellular response, such as stimulation of cell migration or cell proliferation). For example, an antagonist antibody can inhibit (reduce or prevent) the interaction of an EphB4 receptor with a natural ligand (e.g., Ephrin B2 or fragments thereof). In certain embodiments, antagonist antibodies directed against EphB4 can inhibit functions mediated by EphB4, including endothelial cell migration, cell proliferation, angiogenesis, and/or tumor growth. Optionally, the antagonist antibody binds to an extracellular domain of EphB4. In certain embodiments, anti-idiotypic antibodies are also useful. Anti-idiotypic antibodies recognize antigenic determinants associated with the antigen-binding site of another antibody. Anti-idiotypic antibodies can be prepared against a second antibody by immunizing an animal of the same species, and may be of the same strain, as the animal used to produce the second antibody. See e.g., U.S. Pat. No. 4,699,880. In one embodiment, antibodies are raised against receptor or a portion thereof, and these antibodies are used in turn to produce an anti-idiotypic antibody. The anti-idiotypic antibodies produced thereby can bind compounds which bind receptor, such as ligands of receptor function, and can be used in an immunoassay to detect or identify or quantitate such compounds. Such an anti- idotypic antibody can also be an inhibitor of an EphB4 receptor function, although it does not bind receptor itself. Such an anti-idotypic antibody can also be called an antagonist antibody.

In certain embodiments, suitable antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab)2 fragments can be generated by treating antibody with pepsin. The resulting F(ab)2 fragment can be treated to reduce disulfide bridges to produce Fab fragments. In certain embodiments, suitable antibodies are further intended to include bispecific, single-chain, and chimeric and humanized molecules having affinity for an EphB4

polypeptide conferred by at least one CDR region of the antibody. Techniques for the production of single chain antibodies (US Patent No. 4,946,778) can also be adapted to produce single chain antibodies. Also, transgenic mice or other organisms including other mammals, may be used to express humanized antibodies. Methods of generating these antibodies are known in the art. See, e.g., Cabilly et al, U.S. Pat. No. 4,816,567; Cabilly et al, European Patent No. 0,125,023 Bl; Queen et al, European Patent No. 0,451,216 Bl; Boss et al, U.S. Pat. No. 4,816,397; Boss et al, European Patent No. 0,120,694 El;

Neuberger, M. S. et al, WO 86/01533; Neuberger, M. S. et al, European Patent No.

0,194,276 Bl; Winter, U.S. Pat. No. 5,225,539; winter, European Patent No. 0,239,400 Bl; Padlan, E. A. et al., European Patent Application No. 0,519,596 Al . See also, Ladner et al., U.S. Pat. No. 4,946,778; Huston, U.S. Pat. No. 5,476,786; and Bird, R. E. et al, Science, 242: 423-426 (1988)). Such humanized immunoglobulins can be produced using synthetic and/or

recombinant nucleic acids to prepare genes (e.g., cDNA) encoding the desired humanized chain. For example, nucleic acid (e.g., DNA) sequences coding for humanized variable regions can be constructed using PCR mutagenesis methods to alter DNA sequences encoding a human or humanized chain, such as a DNA template from a previously

humanized variable region (see e.g., Kamman, M., et al, Nucl. Acids Res., 17: 5404 (1989)); Sato, K., et al, Cancer Research, 53: 851-856 (1993); Daugherty, B. L. et al, Nucleic Acids Res., 19(9): 2471-2476 (1991); and Lewis, A. P. and J. S. Crowe, Gene, 101 : 297-302 (1991)). Using these or other suitable methods, variants can also be readily produced. In one embodiment, cloned variable regions can be mutagenized, and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see e.g., Krebber et al, U.S. Pat. No. 5,514,548; Hoogenboom et al, WO 93/06213, published Apr. 1, 1993)).

In certain embodiments, an antibody is a monoclonal antibody. For example, a method for generating a monoclonal antibody that binds specifically to an EphB4 polypeptide may comprise administering to a mouse an amount of an immunogenic composition comprising the EphB4 polypeptide effective to stimulate a detectable immune response, obtaining antibody-producing cells (e.g., cells from the spleen) from the mouse and fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody-producing hybridomas to identify a hybridoma that produces a monocolonal antibody that binds specifically to the EphB4 polypeptide. Once obtained, a hybridoma can be propagated in a cell culture, optionally in culture conditions where the hybridoma-derived cells produce the monoclonal antibody that binds specifically to EphB4 polypeptide. The monoclonal antibody may be purified from the cell culture.

In addition, the techniques used to screen antibodies in order to identify a desirable antibody may influence the properties of the antibody obtained. For example, an antibody to be used for certain therapeutic purposes may be able to target a particular cell type.

Accordingly, to obtain antibodies of this type, it may be desirable to screen for antibodies that bind to cells that express the antigen of interest (e.g., by fluorescence activated cell sorting). Likewise, if an antibody is to be used for binding an antigen in solution, it may be desirable to test solution binding. A variety of different techniques are available for testing

antibody:antigen interactions to identify particularly desirable antibodies. Such techniques include ELISAs, surface plasmon resonance binding assays (e.g., the Biacore binding assay, Bia-core AB, Uppsala, Sweden), sandwich assays (e.g., the paramagnetic bead system of IGEN International, Inc., Gaithersburg, Maryland), western blots, immunoprecipitation assays and immunohistochemistry.

D. Nucleic Acid Therapeutic Agents

This disclosure relates to nucleic acid therapeutic agents and methods for inhibiting or reducing gene expression of ephrin and/or ephrin receptor (Eph). By "inhibit" or "reduce," it is meant that the expression of the gene, or level of nucleic acids or equivalent nucleic acids encoding one or more proteins or protein subunits, such as Ephrin B2 and/or EphB4, is reduced below that observed in the absence of the nucleic acid therapeutic agents of the disclosure. By "gene," it is meant a nucleic acid that encodes a RNA, for example, nucleic acid sequences including but not limited to structural genes encoding a polypeptide.

As used herein, the term "nucleic acid therapeutic agent" or "nucleic acid agent" or "nucleic acid compound" refers to any nucleic acid-based compound that contains nucleotides and has a desired effect on a target gene. The nucleic acid therapeutic agents can be single-, double-, or multiple-stranded, and can comprise modified or unmodified nucleotides or non-nucleotides or various mixtures, and combinations thereof. Examples of nucleic acid therapeutic agents of the disclosure include, but are not limited to, antisense nucleic acids, dsRNA, siRNA, and enzymatic nucleic acid compounds.

In one embodiment, the disclosure features one or more nucleic acid therapeutic agents that independently or in combination modulate expression of the Ephrin B2 gene encoding an Ephrin B2 protein (e.g., Genbank Accession No.: NP 004084) or the EphB4 receptor gene which encodes an EphB4 protein (e.g., Genbank Accession No.: NP 004435). i. Antisense nucleic acids

In certain embodiments, the disclosure relates to antisense nucleic acids. By

"antisense nucleic acid," it is meant a non-enzymatic nucleic acid compound that binds to a target nucleic acid by means of RNA-RNA, RNA-DNA or RNA-PNA (protein nucleic acid) interactions and alters the activity of the target nucleic acid (for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al, U.S. Pat. No. 5,849,902). Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule can form a loop and binds to a substrate nucleic acid which forms a loop. Thus, an antisense molecule can be complementary to two (or more) non-contiguous substrate sequences, or two (or more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence, or both. For a review of current antisense strategies, see Schmajuk et al, 1999, J. Biol. Chem., 274, 21783-21789, Delihas et al, 1997, Nature, 15, 751-753, Stein et al, 1997, Antisense N. A. Drug Dev., 7, 151, Crooke, 2000, Methods

EnzymoL, 313, 3-45; Crooke, 1998, Biotech. Genet. Eng. Rev., 15, 121-157, Crooke, 1997, Ad. Pharmacol, 40, 1-49.

In addition, antisense DNA can be used to target nucleic acid by means of DNA-RNA interactions, thereby activating RNase H, which digests the target nucleic acid in the duplex. The antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H to cleave a target nucleic acid. Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof. By "RNase H activating region" is meant a region (generally greater than or equal to 4-25 nucleotides in length, or from 5-11 nucleotides in length) of a nucleic acid compound capable of binding to a target nucleic acid to form a non-covalent complex that is recognized by cellular RNase H enzyme (see for example Arrow et al., U.S. Pat. No. 5,849,902; Arrow et al, U.S. Pat. No. 5,989,912). The RNase H enzyme binds to a nucleic acid compound-target nucleic acid complex and cleaves the target nucleic acid sequence.

The RNase H activating region comprises, for example, phosphodiester,

phosphorothioate, phosphorodithioate, 5'-thiophosphate, phosphoramidate or

methylphosphonate backbone chemistry, or a combination thereof. In addition to one or more backbone chemistries described above, the RNase H activating region can also comprise a variety of sugar chemistries. For example, the RNase H activating region can comprise deoxyribose, arabino, fluoroarabino or a combination thereof, nucleotide sugar chemistry. Those skilled in the art will recognize that the foregoing are non-limiting examples and that any combination of phosphate, sugar and base chemistry of a nucleic acid that supports the activity of RNase H enzyme is within the scope of the definition of the RNase H activating region and the instant disclosure.

Thus, the antisense nucleic acids of the disclosure include natural-type

oligonucleotides and modified oligonucleotides including phosphorothioate-type oligodeoxyribonucleotides, phosphorodithioate-type oligodeoxyribonucleotides, methylphosphonate-type oligodeoxyribonucleotides, phosphoramidate-type

oligodeoxyribonucleotides, H-phosphonate-type oligodeoxyribonucleotides, triester-type oligodeoxyribonucleotides, alpha-anomer-type oligodeoxyribonucleotides, peptide nucleic acids, other artificial nucleic acids, and nucleic acid-modified compounds. Other modifications include those which are internal or at the end(s) of the oligonucleotide molecule and include additions to the molecule of the internucleoside phosphate linkages, such as cholesterol, cholesteryl, or diamine compounds with varying numbers of carbon residues between the amino groups and terminal ribose, deoxyribose and phosphate modifications which cleave, or crosslink to the opposite chains or to associated enzymes or other proteins which bind to the genome. Examples of such modified oligonucleotides include oligonucleotides with a modified base and/or sugar such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide having a sugar which, at both its 3' and 5' positions is attached to a chemical group other than a hydroxyl group (at its 3' position) and other than a phosphate group (at its 5' position). Other examples of modifications to sugars include modifications to the 2' position of the ribose moiety which include but are not limited to 2'-0-substituted with an --0— lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an --O-aryl, or allyl group having 2-6 carbon atoms wherein such— O-alkyl, aryl or allyl group may be unsubstituted or may be substituted, (e.g., with halo, hydroxy, trifluoromethyl cyano, nitro acyl acyloxy, alkoxy, carboxy, carbalkoxyl, or amino groups), or with an amino, or halo group. Nonlimiting examples of particularly useful oligonucleotides of the disclosure have 2'-0-alkylated ribonucleotides at their 3', 5', or 31 and 5' termini, with at least four or five contiguous nucleotides being so modified. Examples of 2'-0-alkylated groups include, but are not limited to, 2'-0-methyl, 2'-0-ethyl, 2'-0-propyl, and 2'-0-butyls.

In certain embodiments, the antisense nucleic acids of the disclosure can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes an ephrin B2 or EphB4 polypeptide. Alternatively, the construct is an oligonucleotide which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences encoding an ephrin B2 or EphB4 polypeptide. Such oligonucleotide probes are optionally modified oligonucleotide which are resistant to endogenous nucleases, e.g., exonucleases and/or endonucleases, and are therefore stable in vivo. Exemplary nucleic acid compounds for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Patent Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in nucleic acid therapy have been reviewed, for example, by van der Krol et al, (1988) Biotechniques 6:958-976; and Stein et al, (1988) Cancer Res 48:2659-2668. ii. dsRNA and RNAi Constructss

In certain embodiments, the disclosure relates to double stranded RNA (dsRNA) and RNAi constructs. The term "dsRNA" as used herein refers to a double stranded RNA molecule capable of RNA interference (RNAi), including siRNA (see for example, Bass, 2001, Nature, 411, 428-429; Elbashir et al, 2001, Nature, 411, 494-498; and Kreutzer et al, PCT Publication No. WO 00/44895; Zernicka-Goetz et al, PCT Publication No. WO

01/36646; Fire, PCT Publication No. WO 99/32619; Plaetinck et al, PCT Publication No. WO 00/01846; Mello and Fire, PCT Publication No. WO 01/29058; Deschamps-Depaillette, PCT Publication No. WO 99/07409; and Li et al, PCT Publication No. WO 00/44914). In addition, RNAi is a term initially applied to a phenomenon observed in plants and worms where double-stranded R A (dsRNA) blocks gene expression in a specific and post- transcriptional manner. RNAi provides a useful method of inhibiting gene expression in vitro or in vivo.

The term "short interfering RNA," "siRNA," or "short interfering nucleic acid," as used herein, refers to any nucleic acid compound capable of mediating RNAi or gene silencing when processed appropriately be a cell. For example, the siRNA can be a double- stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises complementarity to a target nucleic acid compound (e.g., Ephrin B2 or EphB4). The siRNA can be a single-stranded hairpin polynucleotide having self-complementary sense and antisense regions, wherein the antisense region comprises complementarity to a target nucleic acid compound. The siRNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises complementarity to a target nucleic acid compound, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA capable of mediating RNAi. The siRNA can also comprise a single stranded polynucleotide having complementarity to a target nucleic acid compound, wherein the single stranded polynucleotide can further comprise a terminal phosphate group, such as a 5'-phosphate (see for example Martinez et al, 2002, Cell, 110, 563-574), or 5',3'-diphosphate. Optionally, the siRNAs of the disclosure contain a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the mRNA transcript for the gene to be inhibited (the "target" gene). The double- stranded RNA need only be sufficiently similar to natural RNA that it has the ability to mediate RNAi. Thus, the disclosure has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism or evolutionary divergence. The number of tolerated nucleotide mismatches between the target sequence and the siRNA sequence is no more than 1 in 5 basepairs, or 1 in 10 basepairs, or 1 in 20 basepairs, or 1 in 50 basepairs. Mismatches in the center of the siRNA duplex are most critical and may essentially abolish cleavage of the target RNA. In contrast, nucleotides at the 3' end of the siRNA strand that is complementary to the target RNA do not significantly contribute to specificity of the target recognition. Sequence identity may be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith- Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Sequence identity between the siR A and the portion of the target gene may be greater than 90% sequence identity, or even 100%. Alternatively, the duplex region of the R A may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 °C or 70 °C hybridization for 12-16 hours; followed by washing). The double-stranded structure of dsR A may be formed by a single self- complementary RNA strand, two complementary RNA strands, or a DNA strand and a complementary RNA strand. Optionally, RNA duplex formation may be initiated either inside or outside the cell. The RNA may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of double-stranded material may yield more effective inhibition, while lower doses may also be useful for specific applications. Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition.

As described herein, the subject siRNAs are around 19-30 nucleotides in length, or 21-23 nucleotides in length. The siRNAs are understood to recruit nuclease complexes and guide the complexes to the target mRNA by pairing to the specific sequences. As a result, the target mRNA is degraded by the nucleases in the protein complex. In a particular embodiment, the 21-23 nucleotides siRNA molecules comprise a 3' hydroxyl group. In certain embodiments, the siRNA constructs can be generated by processing of longer double- stranded RNAs, for example, in the presence of the enzyme dicer. In one embodiment, the Drosophila in vitro system is used. In this embodiment, dsRNA is combined with a soluble extract derived from Drosophila embryo, thereby producing a combination. The combination is maintained under conditions in which the dsRNA is processed to RNA molecules of about 21 to about 23 nucleotides. The siRNA molecules can be purified using a number of techniques known to those of skill in the art. For example, gel electrophoresis can be used to purify siRNAs. Alternatively, non-denaturing methods, such as non-denaturing column chromatography, can be used to purify the siRNA. In addition, chromatography (e.g., size exclusion chromatography), glycerol gradient centrifugation, affinity purification with antibody can be used to purify siRNAs.

As used herein, dsRNA or siRNA molecules of the disclosure need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides. For example, the dsRNAs may include modifications to either the phosphate-sugar backbone or the nucleoside, e.g., to reduce susceptibility to cellular nucleases, improve bioavailability, improve formulation characteristics, and/or change other pharmacokinetic properties. To illustrate, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. Modifications in RNA structure may be tailored to allow specific genetic inhibition while avoiding a general response to dsRNA. Likewise, bases may be modified to block the activity of adenosine deaminase. The dsRNAs may be produced enzymatically or by partial/total organic synthesis, any modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis. Methods of chemically modifying RNA molecules can be adapted for modifying dsRNAs (see, e.g., Heidenreich et al. (1997) Nucleic Acids Res,

25:776-780; Wilson et al. (1994) J Mol Recog 7:89-98; Chen et al. (1995) Nucleic Acids Res 23:2661-2668; Hirschbein et al. (1997) Antisense Nucleic Acid Drug Dev 7:55-61). Merely to illustrate, the backbone of an dsRNA can be modified with phosphorothioates,

phosphoramidate, phosphodithioates, chimeric methylphosphonate-phosphodiesters, peptide nucleic acids, 5-propynyl-pyrimidine containing oligomers or sugar modifications (e.g., 2'- substituted ribonucleosides, a-configuration). In certain cases, the dsRNAs of the disclosure lack 2'-hydroxy (2'-OH) containing nucleotides.

In a specific embodiment, at least one strand of the siRNA molecules has a 3' overhang from about 1 to about 6 nucleotides in length, though may be from 2 to 4 nucleotides in length. The 3' overhangs may be 1-3 nucleotides in length. In certain embodiments, one strand having a 3' overhang and the other strand being blunt-ended or also having an overhang. The length of the overhangs may be the same or different for each strand. In order to further enhance the stability of the siRNA, the 3' overhangs can be stabilized against degradation. In one embodiment, the RNA is stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine nucleotide 3' overhangs by 2'-deoxythyinidine is tolerated and does not affect the efficiency of RNAi. The absence of a 2' hydroxyl significantly enhances the nuclease resistance of the overhang in tissue culture medium and may be beneficial in vivo.

In another specific embodiment, the subject dsRNA can also be in the form of a long double-stranded RNA. For example, the dsRNA is at least 25, 50, 100, 200, 300 or 400 bases. In some cases, the dsRNA is 400-800 bases in length. Optionally, the dsRNAs are digested intracellularly, e.g., to produce siRNA sequences in the cell. However, use of long double-stranded RNAs in vivo is not always practical, presumably because of deleterious effects which may be caused by the sequence-independent dsRNA response. In such embodiments, local delivery systems and/or agents which reduce the effects of interferon or PKR may be used.

In a further specific embodiment, the dsRNA is in the form of a hairpin structure (named as hairpin RNA). The hairpin RNAs can be synthesized exogenously or can be formed by transcribing from RNA polymerase III promoters in vivo. Examples of making and using such hairpin RNAs for gene silencing in mammalian cells are described in, for example, Paddison et al, Genes Dev, 2002, 16:948-58; McCaffrey et al, Nature, 2002,

418:38-9; McManus et al, RNA, 2002, 8:842-50; Yu et al, Proc Natl Acad Sci U S A, 2002, 99:6047-52). Such hairpin RNAs may be engineered in cells or in an animal to ensure continuous and stable suppression of a desired gene. It is known in the art that siRNAs can be produced by processing a hairpin RNA in the cell. PCT application WO 01/77350 describes an exemplary vector for bi-directional transcription of a transgene to yield both sense and antisense RNA transcripts of the same transgene in a eukaryotic cell. Accordingly, in certain embodiments, the present disclosure provides a recombinant vector having the following unique characteristics: it comprises a viral replicon having two overlapping transcription units arranged in an opposing orientation and flanking a transgene for a dsRNA of interest, wherein the two overlapping transcription units yield both sense and antisense RNA transcripts from the same transgene fragment in a host cell. iii. Enzymatic Nucleic Acid Compounds

In certain embodiments, the disclosure relates to enzymatic nucleic acid compounds. By "enzymatic nucleic acid compound," it is meant a nucleic acid compound which has complementarity in a substrate binding region to a specified target gene, and also has an enzymatic activity which is active to specifically cleave a target nucleic acid. It is understood that the enzymatic nucleic acid compound is able to intermolecularly cleave a nucleic acid and thereby inactivate a target nucleic acid compound. These complementary regions allow sufficient hybridization of the enzymatic nucleic acid compound to the target nucleic acid and thus permit cleavage. One hundred percent complementarity (identity) is preferred, but complementarity as low as 50-75% can also be useful in this disclosure (see for example Werner and Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al, 1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31). The enzymatic nucleic acids can be modified at the base, sugar, and/or phosphate groups. As described herein, the term

"enzymatic nucleic acid" is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamer-binding ribozyme, regulatable ribozyme, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme,

endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of these terminologies describe nucleic acid compounds with enzymatic activity. The specific enzymatic nucleic acid compounds described in the instant application are not limiting in the disclosure and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid compound of this disclosure is that it has a specific substrate binding site which is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving and/or ligation activity to the molecule (Cech et al, U.S. Pat. No. 4,987,071; Cech et al, 1988, 260 JAMA 3030).

Several varieties of naturally-occurring enzymatic nucleic acids are currently known. Each can catalyze the hydrolysis of nucleic acid phosphodiester bonds in trans (and thus can cleave other nucleic acid compounds) under physiological conditions. In general, enzymatic nucleic acids act by first binding to a target nucleic acid. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target nucleic acid. Thus, the enzymatic nucleic acid first recognizes and then binds a target nucleic acid through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target nucleic acid. Strategic cleavage of such a target nucleic acid will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its nucleic acid target, it is released from that nucleic acid to search for another target and can repeatedly bind and cleave new targets. In a specific embodiment, the enzymatic nucleic acid is a ribozyme designed to catalytically cleave mR A transcripts to prevent translation of mRNA (see, e.g., PCT International Publication WO90/11364, published October 4, 1990; Sarver et al, 1990, Science 247: 1222-1225; and U.S. Patent No. 5,093,246). While ribozymes that cleave mRNA at site-specific recognition sequences can be used to destroy particular mRNAs, hammerhead ribozymes may also be used. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNAs have the following sequence of two bases: 5 -UG-3'. The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, 1988, Nature, 334:585-591. The ribozymes of the present disclosure also include RNA endoribonucleases (hereinafter "Cech-type ribozymes") such as the one which occurs naturally in Tetrahymena thermophila (known as the IVS or L-19 IVS RNA) and which has been extensively described (see, e.g., Zaug, et al, 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231 :470-475; Zaug, et al., 1986, Nature, 324:429-433; published International patent application No.

WO88/04300 by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216).

In another specific embodiment, the enzymatic nucleic acid is a DNA enzyme. DNA enzymes incorporate some of the mechanistic features of both antisense and ribozyme technologies. DNA enzymes are designed so that they recognize a particular target nucleic acid sequence, much like an antisense oligonucleotide, however much like a ribozyme they are catalytic and specifically cleave the target nucleic acid. Briefly, to design an ideal DNA enzyme that specifically recognizes and cleaves a target nucleic acid, one of skill in the art must first identify the unique target sequence. In certain embodiments, the unique or substantially sequence is a G/C rich of approximately 18 to 22 nucleotides. High G/C content helps insure a stronger interaction between the DNA enzyme and the target sequence. When synthesizing the DNA enzyme, the specific antisense recognition sequence that will target the enzyme to the message is divided so that it comprises the two arms of the DNA enzyme, and the DNA enzyme loop is placed between the two specific arms. Methods of making and administering DNA enzymes can be found, for example, in U.S. Patent No. 6,110,462. In certain embodiments, the nucleic acid therapeutic agents of the disclosure can be between 12 and 200 nucleotides in length. In one embodiment, exemplary enzymatic nucleic acid compounds of the disclosure are between 15 and 50 nucleotides in length, including, for example, between 25 and 40 nucleotides in length (for example see Jarvis et al, 1996, J. Biol. Chem., 271, 29107-29112). In another embodiment, exemplary antisense molecules of the disclosure are between 15 and 75 nucleotides in length, including, for example, between 20 and 35 nucleotides in length (see for example Woolf et al, 1992, PNAS., 89, 7305-7309; Milner et al., 1997, Nature Biotechnology, 15, 537-541). In another embodiment, exemplary siRNAs of the disclosure are between 20 and 27 nucleotides in length, including, for example, between 21 and 23 nucleotides in length. Those skilled in the art will recognize that all that is required is that the subject nucleic acid therapeutic agent be of length and conformation sufficient and suitable for catalyzing a reaction contemplated herein. The length of the nucleic acid therapeutic agents of the instant disclosure is not limiting within the general limits stated.

IV. Optimizing Activity of the Nucleic acid compounds

Nucleic acid compounds with modifications (e.g., base, sugar and/or phosphate) can prevent their degradation by serum ribonucleases and thereby increase their potency. There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid compounds with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'- amino, 2'-C-allyl, 2'-flouro, 2'-0-methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al, 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al, 1996, Biochemistry, 35, 14090). Sugar modification of nucleic acid compounds have been extensively described in the art (see Eckstein et al., PCT Publication No. WO 92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. PCT Publication No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al, 1995, J. Biol. Chem., 270, 25702; Beigelman et al, PCT publication No. WO 97/26270; Beigelman et al, U.S. Pat. No. 5,716,824; Usman et al, U.S. Pat. No. 5,627,053; Woolf et al, PCT Publication No. WO 98/13526; Thompson et al, U.S. S No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al, 1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al, 1997, Bioorg. Med. Chem., 5, 1999- 2010). Similar modifications can be used to modify the nucleic acid compounds of the instant disclosure.

While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorothioate, and/or 5'-methylphosphonate linkages improves stability, an over-abundance of these modifications can cause toxicity. Therefore, the amount of these internucleotide linkages should be evaluated and appropriately minimized when designing the nucleic acid compounds. The reduction in the concentration of these linkages should lower toxicity resulting in increased efficacy and higher specificity of these molecules. In one embodiment, nucleic acid compounds of the disclosure include one or more G- clamp nucleotides. A G-clamp nucleotide is a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine within a duplex, see for example, Lin and Matteucci, 1998, J. Am. Chem. Soc, 120, 8531-8532. A single G-clamp analog substitution within an

oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides. The inclusion of such nucleotides in nucleic acid compounds of the disclosure results in both enhanced affinity and specificity to nucleic acid targets. In another embodiment, nucleic acid compounds of the disclosure include one or more LNA (locked nucleic acid) nucleotides such as a 2', 4'-C mythylene bicyclo nucleotide (see for example Wengel et al., PCT Publication Nos. WO 00/66604 and WO 99/14226).

In another embodiment, the disclosure features conjugates and/or complexes of nucleic acid compounds targeting an Eph/Ephrin pathway molecule or a D114/Notch pathway molecule. Such conjugates and/or complexes can be used to facilitate delivery of nucleic acid compounds into a biological system, such as cells. The conjugates and complexes provided by the instant disclosure can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid compounds of the disclosure.

The present disclosure encompasses the design and synthesis of novel conjugates and complexes for the delivery of molecules, including, but not limited to, small molecules, lipids, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes. In general, the transporters described are designed to be used either individually or as part of a multi- component system, with or without degradable linkers. These compounds are expected to improve delivery and/or localization of nucleic acid compounds of the disclosure into a number of cell types originating from different tissues, in the presence or absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of the molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules. The term "biodegradable nucleic acid linker molecule" as used herein, refers to a nucleic acid compound that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule. The stability of the biodegradable nucleic acid linker molecule can be modulated by using various combinations of ribonucleotides, deoxyribonucleotides, and chemically modified nucleotides, for example, 2'-0-methyl, 2'-fluoro, 2'-amino, 2'-0-amino, 2'-C-allyl, 2'-0-allyl, and other 2'-modified or base modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid compound, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus based linkage, for example, a

phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base

modifications. The term "biodegradable" as used herein, refers to degradation in a biological system, for example enzymatic degradation or chemical degradation.

Therapeutic nucleic acid compounds, such as the molecules described herein, delivered exogenously are optimally stable within cells until translation of the target RNA has been inhibited long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. These nucleic acid compounds should be resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of nucleic acid compounds described in the instant disclosure and in the art have expanded the ability to modify nucleic acid compounds by introducing nucleotide modifications to enhance their nuclease stability as described above. In another aspect the nucleic acid compounds comprise a 5' and/or a 3'-cap structure. By "cap structure," it is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see for example Wincott et al., WO 97/26270). These terminal modifications protect the nucleic acid compound from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5'- terminus (5 '-cap) or at the 3 '-terminus (3 '-cap) or can be present on both terminus. In non- limiting examples, the 5'-cap includes inverted abasic residue (moiety), 4',5'-methylene nucleotide; l-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3, 4-dihydroxybutyl nucleotide; acyclic 3, 5 -dihydroxypentyl nucleotide, 3 '-3 '-inverted nucleotide moiety; 3 '-3 '-inverted abasic moiety; 3'-2'-inverted nucleotide moiety; 3 -2'- inverted abasic moiety; 1 ,4-butanediol phosphate; 3'-phosphoramidate; hexylphosphate;

aminohexyl phosphate; 3 '-phosphate; 3'-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety (for more details see Wincott et al, PCT publication No. WO 97/26270). In other non-limiting examples, the 3'-cap includes, for example, 4',5'- methylene nucleotide; l-(bela-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5 '-amino-alkyl phosphate; l,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1 ,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threopentofuranosy nucleotide; acyclic 3',4'-seco nucleotide; 3,4- dihydroxybutyl nucleotide; 3, 5 -dihydroxypentyl nucleotide, 5 '-5 '-inverted nucleotide moiety; 5 '-5 '-inverted abasic moiety; 5'-phosphoramidate; 5'-phosphorothioate; 1 ,4-butanediol phosphate; 5'-amino; bridging and/or non-bridging 5'-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5'-mercapto moieties (for more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925).

V. Methods of Treatment

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 at least one Eph/Ephrin pathway antagonist and at least one D114/Notch pathway antagonist as described above.

These methods are particularly aimed at therapeutic and prophylactic treatments of animals, and more particularly, humans. One of ordinary skill in the art will be able to measure the effectiveness of the disclosed methods using known assays and assays disclosed in the specification such as measuring tumor volume and survival rate.

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; Osier- Webber Syndrome; myocardial angiogenesis; plaque neovascularization;

telangiectasia; hemophiliac joints; angiofibroma; and wound granulation and wound healing; telangiectasia psoriasis scleroderma, pyogenic granuloma, coronary collaterals, ischemic limb angiogenesis, corneal diseases, rubeosis, arthritis, diabetic neovascularization, fractures, vasculogenesis, hematopoiesis.

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.

In particular, 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.

In certain embodiments of such methods, one or more Eph/Ephrin pathway antagonists and one or more D114/Notch pathway antagonists can be conjointly administered, e.g., together (simultaneously (whether in a single composition or in separate compositions) or at different times (sequentially, e.g., within). The use of the term "in combination" or "conjoint" does not restrict the order in which therapies (e.g., agents and/or other therapeutic modalities) are administered to a subject. A first therapy (e.g., a first agent and/or other therapeutic modality) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy (e.g., a second agent and/or other therapeutic modality) to a subject. By way of example, "other therapeutic modalities" include, but are not limited to, surgery, radiation therapy, dialysis, stem cell transplant, ventilatory support, diet, physical therapy, etc. In certain embodiments, one or more Eph/Ephrin pathway antagonists is the first agent and one or more D114/Notch pathway antagonists is the second. In certain embodiments, one or more Eph/Ephrin pathway antagonist is the second agent and one or more D114/Notch pathway antagonist is the first. In certain embodiments, one or more Eph/Ephrin pathway antagonist and one or more D114/Notch pathway antagonist are administered together in a single pharmaceutical composition. Regardless of the order of administration, the use of an Eph/Ephrin pathway antagonist in combination with a D114/Notch pathway antagonist is referred to herein as a combination therapeutic.

In certain embodiments, the conjoint administration of at least one Eph/Ephrin pathway antagonist and at least one D114/Notch pathway antagonist provides cumulative efficacy over antagonists to either pathway alone. In certain embodiments, the conjoint administration of at least one Eph/Ephrin pathway antagonist and at least one D114/Notch pathway antagonist has an additive effect. In certain embodiments, the doses of at least one Eph/Ephrin pathway antagonist and at least one D114/Notch pathway antagonist are designed to produce an additive effect. In certain embodiments, the timing of treatment of at least one Eph/Ephrin pathway antagonist and at least one D114/Notch pathway antagonist are designed to produce an additive effect. In certain embodiments, the conjoint administration of at least one Eph/Ephrin pathway antagonist and at least one D114/Notch pathway antagonist has a synergistic effect. In certain embodiments, the doses of at least one Eph/Ephrin pathway antagonist and at least one D114/Notch pathway antagonist are designed to produce a synergistic effect. In certain embodiments, the timing of treatment of at least one Eph/Ephrin pathway antagonist and at least one D114/Notch pathway antagonist are designed to produce a synergistic effect. The Eph/Ephrin pathway antagonist may be any of the various Eph/Ephrin pathway antagonists described herein, and preferably antagonises EphB4/Ephrin B2 signaling. In certain embodiments, the D114/Notch pathway antagonist may be any

D114/Notch pathway antagonist described herein, such as a soluble extracellular portion of D114 or an antibody that binds D114.

In certain embodiments, the conjoint administration of at least one Eph/Ephrin pathway antagonist and at least one D114/Notch pathway antagonist provides increased safety over a D114/Notch pathway antagonist. In certain embodiments, the conjoint administration of at least one Eph/Ephrin pathway antagonist and at least one D114/Notch pathway antagonist decreases the incidence and/or severity of D114/Notch pathway antagonist-related toxicity. In certain embodiments, the conjoint administration of at least one Eph/Ephrin pathway antagonist and at least one D114/Notch pathway antagonist decreases the incidence and/or severity of D114/Notch pathway antagonist-related vascular alterations. In certain embodiments, the conjoint administration of at least one Eph/Ephrin pathway antagonist and at least one D114/Notch pathway antagonist decreases the incidence and/or severity of D114/Notch pathway antagonist-related hepatic vascular alterations. In certain embodiments, the Eph/Ephrin pathway antagonist is administered before D114/Notch pathway antagonist- related toxicity, such as hepatic vascular alterations, occurs or is diagnosed. In certain embodiments, the Eph/Ephrin pathway antagonist is administered after D114/Notch pathway antagonist-related toxicity, such as hepatic vascular alterations, occurs or is diagnosed. In certain embodiments, the Eph/Ephrin pathway antagonist is administered at the time

D114/Notch pathway antagonist-related toxicity, such as hepatic vascular alterations, may occur. The Eph/Ephrin pathway antagonist may be any of the various Eph/Ephrin pathway antagonists described herein, and preferably antagonises EphB4/Ephrin B2 signaling. In certain embodiments, the D114/Notch pathway antagonist may be any D114/Notch pathway antagonist described herein, such as a soluble extracellular portion of D114 or an antibody that binds D114.

In addition, the at least one Eph/Ephrin pathway antagonist and at least one

D114/Notch pathway antagonist can be administered conjointly with another type of compound for treating cancer or for inhibiting angiogenesis.

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 anticancer 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 combination therapeutic agent.

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.

When a combination 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 antineoplastic 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.

Pharmaceutical compounds that may be used for combinatory anti-tumor therapy include, merely to illustrate: aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, 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. 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 (VP 16)); 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.

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) neutralizes of angiogenic molecules, such as an anti-pbFGF 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 D ₃ 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 6573256. 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 _ν β ₃ , 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. Administration of the therapeutic agents may be made in a single dose, or in multiple doses. In some instances, administration of the 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

In certain embodiments, the at least one Eph/Ephrin pathway antagonist and at least one D114/Notch pathway antagonist 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.

Formulations of the subject 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.

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.

Pharmaceutical compositions suitable for parenteral administration may comprise one or more 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.

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.

Injectable depot forms are made by forming microencapsule matrices of one or more 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. In other embodiments, polypeptide therapeutic agents of the instant invention can be expressed within cells from eukaryotic promoters. For example, a soluble polypeptide of EphB4 and a soluble polypeptide of D114 can be expressed in eukaryotic cells from an appropriate vector. The vectors may be DNA plasmids or viral vectors. Viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. In certain embodiments, the vectors are 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

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 - Targeted DU4 allele deletion reduces tumor growth in RT2 mice.

To assess the effect of impaired signaling through the D114/Notch pathway in an autochthonous tumor model, we crossed RT2 transgenic mice with DU4 ^+/~ mice and compared tumor burden between RT2 DU4 ^+/+ and RT2 DU4 ^+/~ offspring. The two groups were humanely sacrificed at 13.5 weeks of age. Average tumor numbers per mouse were similar in RT2 DU4 ^+/+ and RT2 DU4 ^+/~ littermates (Fig. I A). In contrast, a significant decrease in tumor growth was observed in RT2 DU4 ^+/~ mice when compared to the RT2 DU4 ^+/+ control group. On average, RT2 DU4 ^+/~ insulinoma volume and overall tumor burden, calculated as a sum of tumor volumes per mouse, were reduced by approximately 50% compared to the control animals (Fig. I A). Example 2 - Partial DU4/Notch suppression due to DU4 allelic deletion promotes immature and non-functional vessel proliferation in RT2 insulinomas.

We also examined the vascular morphology of tumors derived from both RT2 DU4 ^+/+ and RT2 Dll4 ^+/~ mice at week 13.5. Tumor endothelium was visualized by immunostaining of PECAM while PECAM/a-SMA co-localization was used to evaluate mural cell recruitment and thereby vessel maturity. In addition, subgroups of RT2 DU4 ^+/+ and RT2 DU4 ^+/~ mice were perfused with endothelium-binding lectin to visualize the perfused regions of the tumor vasculature. The RT2 DU4 ^+/+ tumors had irregular vessel formation and absence of a distinct branching pattern (Fig. IB) as well as a high degree of vessel maturity with a mean a-SMA- positive cell coverage of approximately 75% (Fig. 1C). Furthermore, the lectin-perfusion study indicated that nearly 80%> (78 ± 4%) of the vessels displayed appropriate lumen formation and were perfused (Fig. ID). In comparison, RT2 DU4 ^~ insulinoma showed a 1.8- fold increase in the density of PECAM-positive areas (p < 0.05) and formed atypical networks lacking vessel hierarchy or symmetry but displaying pronounced branching and multiple interconnections (Fig. IB). The number of a-SMA-positive cells lining PECAM- positive endothelial cells was greatly reduced (45% reduction, p < 0.05, Fig. \C). As anticipated, a significant reduction in the proportion of functional vessels was demonstrated by lectin-perfusion (2-fold decrease, p < 0.05, Fig. ID).

Example 3 - Reduced DU4/Notch signaling in the RT2 tumor affects expression of vessel growth regulators and/or their receptors. Quantitative RT-PCR was used to analyze RT2 DU4 ^+/+ and RT2 DU4 ^+/~ tumors for putative differences in the expression of genes known to be involved in physiological and tumor angiogenesis (Fig. 2). Relative to DU4 ^+/+ insulinomas, DU4 ^+/~ tumors showed an approximately 50%> reduction in DU4 mRNA levels, as expected, as well as reduced Hey2 expression, consistent with a reduction in Notch signaling. Furthermore, there was increased Vegf-a expression and an over 8-fold increase in the VEGFR2/VEGFR1 ratio in the DU4 ^+/~ tumors. Similarly, both Vegf-c and Vegfr3 were up-regulated in these tumors. In addition, there was an increase in PDGFR-β, and decrease in Ephrin-B2 and Tie2 mRNA levels. The RNA level of PEC AM was not significantly increased (1.2 fold), different from 40% increase of the PEC AM signal in Figure IB. One explanation is that quantitative PCR was done in whole tumor tissue and not microdissected vessels, which likely resulted in failure to document PECAM modulation in mRNA level.

Example 4 - sEphB4-Alb potentiates the effects ofDU4 allelic deletion on RT2 insulinoma growth and survival rate by further antagonizing tumor vessel maturation. sEphB4-Alb has been shown to be a potent inhibitor of tumor angiogenesis by blocking Ephrin-B2 and EphB4 signaling. To examine the effect of sEphB4-Alb in the RT2 system, RT2 mice were treated with sEphB4-Alb or PBS for 3.5 weeks beginning at 10 weeks of age. At 13.5 weeks of age, sEphB4-Alb treated RT2 mice showed reduced mean tumor volumes relative to PBS-treated control mice by approximately 50% (p < 0.05) (Fig. 3A). At this point tumors were harvested and RNA was isolated and used for global gene expression analysis. Genes with expression changes between the two groups higher than 2- fold and P value smaller than 0.05 can be found in the NCBI-GEO database. Selected genes with known role in vascular biology were evaluated by quantitative RT-PCR for validation studies. Of these genes, Rgs5 and Psenen are of special interest. They are upregulated in sEphB4-Alb treated group compared to the untreated wild type mice and their changes were validated by quantitative RT-PCR (Fig. 6A). Rgs5 is a marker of pericytes and is a negative regulator of pericyte maturation. Rgs5 was also upregulated in umbilical artery smooth muscle cell co-cultured with human umbilical artery endothelial cell when treated with sEphB4-Alb (Fig. 6B). Psenen is a critical component of presenilin complex that is required for Notch receptor processing.

To examine whether synchronous suppression of D114/Notch and EphB4/Ephrin-B2 signaling pathways might result in greater inhibition of RT2 tumor development, we treated RT2 DU4 ^+/~ mice with sEphB4-Alb. The reduction of the average tumor volume was even more pronounced in sEphB4-Alb treated RT2 DU4 ^+/~ group (approximately 90% reduction, p < 0.01) where D114/Notch and Ephrin-B2/EphB4 signaling were simultaneously inhibited (Fig. ). To test whether these tumor-suppressive effects are reflected on longevity, both

DU4 ^+/+ and DU4 ^+/~ RT2 mice were treated with PBS or half dose of sEphB4-Alb (5 mg/kg) beginning at 10 weeks of age and continuously monitored for survival benefit estimation. DU4 allelic deletion just as sEphB4-Alb treatment were found to extend the mean lifespan of RT2 mice by 2 weeks (15 ± 1 wk in PBS treated RT2 DU4 ^+/+ versus 17 ± 1 wk in PBS treated RT2 DU4 ^+/~ and sEphB4-Alb treated RT2 DU4 ^+/+ mice, p < 0.05 Fig. 3B).

Significantly, an additional survival benefit of 2 weeks, on average, was provided by combinatorial D114/Notch and Ephrin-B2/EphB4 inhibition (15 ± 1 wk in PBS-treated RT2 DU4 ^+/+ versus 19 ± 1 wk in sEphB4-treated RT2 DU4 ^+/~ , p < 0.01, Fig. 3B).

Histologicaly, microvessel density assessed by immunostaining of PEC AM was significantly increased in PBS treated DU4 ^+/~ tumors compared to PBS treated DU4 ^+/+ insulinomas, while sEphB4-Alb treatment in RT2 DU4 ^+/+ mice caused a statistically significant decrease of tumor vessel density (Fig. 3Q. Notably, in both PBS treated DU4 ^+/~ tumors and sEphB4-Alb treated RT2 DU4 ^+/+ tumors, average vessel caliber was reduced, and pericyte recruitment and vessel wall formation were markedly impaired, as indicated by PECAM/NG2 and PECAM/a-SMA immunostaining (Fig. 3C and D). Furthermore, the simultaneous suppression of D114/Notch and Ephrin-B2/EphB4 signaling in sEphB4-Alb treated RT2 DU4 ^+/~ mice drastically reduced the vessel diameters and their mural cell coverage, even though the total PECAM-positive area apparently similar to PBS-treated DU4 ^+/+ insulinomas (Fig. 3C and 3D). Therefore, combinational targeting D114/Notch signaling EphrinB2-EphB4 has greater efficacy in blocking vessel maturation and perfusion of the tumor. Example 5 - Both sDU4 and sEphB4-Alb inhibit RT2 tumor growth and act in a cumulative manner. sD114 is a potent inhibitor of Notch signaling and has been shown to reduce tumor growth in various tumor models [7, 8, 21]. We sought to determine the effect of combined blockade of D114/Notch pathway (by sD114) and Ephrin-B2/EphB4 pathway (by sEphB4-Alb) on tumor angiogenesis. RT2 mice were treated from 10 to 13.5 weeks of age. Compared to control mice treated with the vehicle (PBS), tumor volumes were reduced in both sD114 treated (81% reduction, p < 0.01) and sEphB4-Alb treated animals (60%, p < 0.05). As presented in Fig. 4A, the combination therapy caused an even more significant tumor suppression (92%, p < 0.01), which has significant difference when compared to the therapy with sD114 or sEphB4-Alb alone (p < 0.05).

Harvested tumors were double immunostained for PECAM/a-SMA to analyze microvessel density and maturity and PECAM/lectin to estimate tumor perfusion and vessel functionality (Fig. 4B-D). Compared to PBS treated group, sD114 treated tumors showed an increase in total tumor vessel density of 41% on average (p < 0.01), reduced mural cell coverage of 47% (p < 0.05), and reduced lectin perfusion of 32% (p < 0.05) sEphB4-Alb treatment in contrast caused reduced microvessel density (39% reduction, p < 0.01), pericyte recruitment (39%) and lectin perfusion (21%). Finally, the small sized tumors observed in mice treated with both sD114 and sEphB4-Alb were characterized by decreased vessel diameters (Fig. 4B) and markedly impaired vessel maturation with 73% reduction in mural cell coverage compared to PBS-treated insulinomas (p < 0.01) (Fig. 4Q. Meanwhile, lectin perfusion was drastically decreased (79%, p < 0.01) (Fig. 4D). These results indicate that sD114/sEphB4-Alb combination therapy has at least a cumulative effect on minimizing tumor vessel competency and blood delivery to neoplasic cells, consequently reducing tumor growth. Example 6 - Endothelial DU4/Notch inhibition results in hepatic vascular alterations that can be prevented by concomitant inhibition of Ephrin-B2/EphB4. Chronic D114 blockade results in benign vascular proliferative lesions in the liver [4]. In contrast, DU4 haploinsufficiency and intermittent administration of sD114 (3 times a week for 3.5 weeks) in RT2 mice significantly reduced tumor growth, but did not cause any vascular lesions in the liver or other vital organs such as heart, brain, lung, kidney, and intestine (data not shown). To confirm if complete and persistent loss of D114 causes organ toxicity, we used a conditional, endothelial-specific DU4 knock-out mouse line (Dll4 ^lox/lox Cre+), in which DU4 deletion is dependent on tamoxifen administration. Comparative histological analyses were performed with cardiac, cerebral, pulmonary, renal, intestinal and hepatic samples obtained from mice 10 weeks after tamoxifen-induction or vehicle (PBS)- administration. While control animals presented normal organ histology, the endothelial D114 knock-out mice showed alterations in liver architecture. Macroscopically, the liver surface of tamoxifen-induced D114 ^lox/lox Cre+ mice had a characteristic micro-nodule. Although general lobular architecture and portal spaces were preserved, we observed some areas with markedly dilated sinusoids. However, the most prominent feature was excessive subcapsular vessel proliferation with a few hemangioma-like structures (Fig. 5 A and B). To assess whether and to which extent these alterations can be influenced by concomitant Ephrin-B2/EphB4 inhibition, we treated tamoxifen-induced D114 ^lox/lox Cre+ mice with PBS or sEphB4-Alb (10 mg/kg). While PBS treated mice developed previously described subcapsular vascular alterations (Fig. 5E), sEphB4-Alb treated animals showed a few dilated sinusoids but no evidence of vascular proliferative lesions (Fig. 5F).

Example 7 - Materials and Methods.

Experimental animals. All animal-involving procedures in this study were approved by the Faculty of Veterinary Medicine of Lisbon Ethics and Animal Welfare Committee. The generation of DU4 ^+/~ (Dll4/LacZ) mice on CD1 background has been reported previously [4]. The transgenic RIPl-Tag2 (RT2) mice of CD1 and C57/BL6 backgrounds, used for breeding with the DU4 ^+/~ line and in experimental drug trials, respectively. D114 conditional knockout mice (Dll4 ^lox/lox ) were generated as previously described [2] and crossed with VE-cadherin- Cre-ERT2 mice to produce a tamoxifen-inducible endothelial-specific D114 loss-of- function line (Dll4 ^lox/lox Cre+). The animals were housed in well ventilated propylene cages with sawdust as bedding, in a room with controlled temperature between 22°C and 25°C and a 12- hours-light/12-hours-dark cycle. The mice were fed with standard laboratory diet and water ad libitum. From 12 weeks of age, all RT2 mice received 5% sugar in their water to relieve the hypoglycemia induced by the insulin-secreting tumors.

Experimental design, tumor burden analyses and therapeutic trials. To study the effects of impaired D114/Notch signaling on RT2 insulinoma growth, RT2 DU4 ^+/+ and RT2 DU4 ^+/~ littermates (CD1 background, n=8 for each group) were sacrificed for tumor measurement, histological analysis of vascular morphology and gene expression analysis at

13.5 weeks of age. The pancreas glands were dissected and the macroscopic tumors (≥ l x l mm) were excised. Tumor volume was calculated using the formula V= 0.52 x a x b ² where a and b equal the longer and shorter diameter of the tumor, respectively. The volumes of all tumors from each mouse were added to give the overall tumor burden per animal.

The effect of D114 allelic deletion in combination with Ephrin-B2/EphB4 signaling inhibition on the growth of the RT2 insulinoma was assessed by the administration of the soluble extracellular domain of EphB4 fused with albumin (sEphB4-Alb), which was produced as previously described [17]. Both RT2 D114 ^+/+ and RT2 D114 ^+/" mice (CD1 background, n=12 for each group) were separated in equal subgroups, treated

intraperitoneally (i.p.) with vehicle (PBS) or sEphB4-Alb (lOmg/kg) 3x/wk for 3.5 weeks beginning at the age of 10 weeks and finally sacrificed for tumor measurement and histological analysis.

In the therapeutical trials we assessed the efficacy of a systemically administered D114/Notch-inhibitor, soluble D114 extracellular domain fused to Fc (sD114), both alone and in combination with sEphB4-Alb. sD114 was produced as previously described [8]. Vehicle (PBS, i.p. 3x/wk), sD114 (10 mg/kg/day, i.p., 3x/wk), sEphB4-Alb (10 mg/kg/day, i.p., 3x/wk), and the combination of sD114 (10 mg/kg/day, i.p., 3x/wk) with sEphB4-Alb (10 mg/kg/day, i.p., 3x/wk) treatments were started when RT2 mice (C57BL6 background) reached the age of 10 weeks and continued until mice were 13.5 week-old. Two independent experiments involved 6 animals per treatment group.

Longevity study. To evaluate the effect of D114 allelic deletion in combination with Ephrin-B2/EphB4 signaling inhibition on longevity, the RT2 DU4 ^+/+ and RT2 DU4 ^+/~ mice were separated in two equal groups (n=10 for each group), treated i.p. with vehicle (PBS) or sEphB4-Alb (5 mg/kg, 3x/wk), beginning at the age of 10 weeks and continuously monitored for signs of hypoglycemic shock. The mice were sacrificed if found moribund or if body weight loss exceeded 15%. Survival rate was calculated as the percentage of live mice at the end of each week relative to the initial number of animals in the experimental group.

Assessment of toxicity. Heart, lung, liver, brain, kidney and intestines were collected from sD114 and sEphB4-Alb treated mice used in the therapeutical trials, fixed in 10% formalin solution for 48 h, dehydrated in alcohol, cleared in xylene, embedded in paraffin, sectioned at ΙΟμιη and stained with hematoxylin (Fluka AG Buchs SG Switzerland) and eosin Y (Sigma Chemicals, St. Louis, MO) to study eventual histopatho logical alterations. To assess the side effects that might arise from total (100%) inhibition of endothelial-specific D114 signaling, 8 week-old DU4 ^lox/lox Cre+ (n=10) were treated with tamoxifen (50mg/kg daily for 5 days) to produce endothelial-specific D114 null individuals, while a group of 8 week-old DU4 ^lox/lox Cre+ (n=10) were left uninduced (control mice with constitutive DU4 expression). Ten weeks later, the mice were sacrificed and heart, lung, liver, brain, kidney and intestines were collected, processed and examined as described above. Since DU4 endothelial loss of function has been associated with hepatic lesions, we decided to determine the potential toxico logical effect of a combination of D114/Notch and Ephrin-B2/EphB4 targeted therapy. Therefore another group of 8-week-old DU4 ^lox/lox Cre+ (n=10) were treated with tamoxifen (50mg/kg daily) for 5 days, subsequently divided in two equal subgroups that were injected with vehicle (PBS) or sEphB4-Alb (lOmg/kg) for ten weeks and then sacrificed. Liver samples were processed and examined as described above. Immunohistochemistry. RT2 insulinomas obtained from tumor burden studies and therapeutical trials were fixed in a 4% paraformaldehyde (PFA) solution at 4°C for lh, cryoprotected in 15% sucrose, embedded in 7.5% gelatin, snap frozen in liquid nitrogen and cryosectioned at 10 and 20μιη. Double fluorescent immunostaining to the platelet endothelial cell adhesion molecule (PECAM) and the peri-vascular cell marker alpha smooth muscle actin (a-SMA) was performed on tissue sections to examine tumor vascular density and vessel maturity while double fluorescent immunostaining to PECAM and the pericyte marker neurogenin 2 chondroitin sulfate proteoglycan (NG2) was used to visualize pericyte recruitment. Rat monoclonal anti-mouse PECAM (BD Pharmingen, San Jose, CA), and rabbit polyclonal anti-mouse a-SMA (Abeam, Cambridge, UK) or rabbit polyclonal anti- mouse NG2 (Millipore, Billerica, MA) were used as primary antibodies. Species-specific secondary antibodies conjugated with Alexa Fluor 488 and 555 were from Invitrogen (Carlsbad, CA). Tissue sections were incubated with primary antibody overnight at 4°C and with secondary antibody for 1 hour at room temperature. Nuclei were counterstained with 4 ^' ,6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI; Molecular Probes, Eugene, OR). Fluorescent immunostained sections were examined under a Leica DMRA2

fluorescence microscope with Leica HC PL Fluotar 10 and 20X/0.5 NA dry objective, captured using Photometries CoolSNAP HQ, (Photometries, Friedland, Denmark), and processed with Metamorph 4.6-5 (Molecular Devices, Sunnyvale, CA). Morphometric analyses were performed using the NIH ImageJ 1.37v program. Vessel density corresponds to the percentage of each tumor section field occupied by a PECAM-positive signal. As a measure of vascular maturity, vessel wall assembly was assessed by quantifying the percentage of PECAM-positive structures lined by a-SMA-positive coverage while pericyte recruitment was assessed by quantifying the percentage of PECAM-positive structures lined by NG2 -positive coverage.

Vessel perfusion study. To mark vessel perfusion, mice were anesthetized and biotin- conjugated lectin from Lycopersicon esculentum (100μg in ΙΟΟμΙ of PBS; Sigma, St. Luis, MO) was injected via caudal vein and allowed to circulate for 5 minutes before the vasculature was transcardially perfused with 4% PFA in PBS for 3 minutes. Tumor samples were collected and processed as described above. Endothelial cells were stained with

PECAM antibody and perfused vessels were visualised by streptavidin-Alexa 488

(Invitrogen, Carlsbad, CA), which binds to biotinylated lectin. The images were obtained and processed as described above. Tumor perfusion was quantified by determining the percentage of PECAM-positive structures that were colocalized with Alexa 488 signals.

Global gene expression and quantitative transcriptional analysis. Tumors from sEphB4-Alb or PBS treated RT2 DU4 ^+/+ mice were harvested at week 13.5. RNA was then isolated and used for global gene expression analysis with Illumina MouseRef-8 v2.0 Expression BeadChip (Illumina, San Diego, CA). The genearray data were deposited to

NCBI-GEO database (on the world wide web at ncbi.nlm.nih.gov/geo/query/acc.cgi). Genes with expression change between two groups higher than 2 fold and P value smaller than 0.05 were selected and the changes were validated by quantitative RT-PCR.

Using a Superscript III FirstStrand Synthesis Supermix (Invitrogen, Carlsbad, CA), first-strand cDNA was synthesized from total RT2 DU4 ^+/+ and RT2 DU4 ^+/~ insulinoma RNA. Real-time PCR analysis was performed as described [4] using specific primers for β-actin, GAPDH, PECAM, DU4, Hey2, VEGF-A, VEGFR1, VEGFR2, VEGF-C, VEGFR3, PDGF-β, Ephrin-B2, and Tie2. Primer pair sequences are available on request. Gene expression was normalized to β-actin and GAPDH.

Statistical analyses. Data processing was carried out using the Statistical Package for the Social Sciences version 15.0 (SPSS v. 15.0; Chicago, IL). Kaplan-Meier product-limit estimation with Breslow generalized Wilcoxon test was used for survival analyses. All other statistical analyses were performed using the Mann- Whitney- Wilcoxon test. All results are presented as mean ± SEM or mean ± SD when more appropriate. -values < 0.05 and <0.01 were considered significant (indicated in the figures with *) and highly significant (indicated with **), respectively. REFERENCES

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INCORPORATION BY REFERENCE 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.

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.

SEQUENCES SEQ ID NO: : 1 - D114 amino acid sequence

SEQ ID NO: :2 - D114 nucleic acid sequence

SEQ ID NO: :3 - Recombinant B4ECv3 protein

SEQ ID NO: :4 - Recombinant B4ECv3NT protein

SEQ ID NO: :5 - Recombinant B2EC protein

SEQ ID NO: :6 - Recombinant B4ECv3-FC protein

SEQ ID NO: :7 - Recombinant B2EC-FC protein

SEQ ID NO: :8 - Genomic nucleotide sequence of human EphB4

SEQ ID NO: :9 - cDNA nucleotide sequence of human EphB4

SEQ ID NO: : 10 - Genomic nucleotide sequence of human Ephrin B2

SEQ ID NO: : 11 - cDNA nucleotide sequence of human Ephrin B2

SEQ ID NO : 12 - amino acid sequence of human EphB4

SEQ ID NO: : 13 - amino acid sequence of human Ephrin B2

SEQ ID NO: : 14 - Recombinant B4EC-GC

SEQ ID NO: : 15 - Recombinant GCF

SEQ ID NO: : 16 - Recombinant FL-hB4EC

SEQ ID NO: : 17 - Recombinant B4-CF2

SEQ ID NO: : 18 - Recombinant B4-GCF2F

SEQ ID NO: : 19 - Recombinant processed B4-GCF2F

SEQ ID NO: :20 - Recombinant HSA-EphB4 precursor protein

SEQ ID NO: ^■ 21 - Recombinant HSA-EphB4 mature protein

SEQ ID NO: 22 - Heavy chain variable region deimmunized 47 variant 1

SEQ ID NO: 23 - Heavy chain variable region deimmunized 47 variant 2

SEQ ID NO: 24 - Heavy chain variable region deimmunized 47 variant 3

SEQ ID NO: 25 -Heavy chain variable region deimmunized 47 variant 4

SEQ ID NO: 26 - Heavy chain variable region deimmunized 47 variant 5

SEQ ID NO: 21 - Light chain variable region deimmunized 47 variant 1

SEQ ID NO: :28 - Light chain variable region deimmunized 47 variant 2

SEQ ID NO: 29 - Light chain variable region deimmunized 47 variant 3

SEQ ID NO: :30 - Light chain variable region deimmunized 47 variant 4

SEQ ID NO: :31 - Heavy chain variable region deimmunized 131 variant 1

SEQ ID NO: :32 - Heavy chain variable region deimmunized 131 variant 2

SEQ ID NO: :33 - Heavy chain variable region deimmunized 131 variant 3

SEQ ID NO: :34 - Heavy chain variable region deimmunized 131 variant 4 SEQ ID NO :35 - Heavy chain variable region deimmunized 131 variant 5

SEQ ID NO :36 - Light chain variable region deimmunized 131 variant 1

SEQ ID NO :37 - Light chain variable region deimmunized 131 variant 2

SEQ ID NO :38 - Light chain variable region deimmunized 131 variant 3

SEQ ID NO :39 - Light chain variable region deimmunized 131 variant 4

SEQ ID NO AO - Mouse monoclonal antibody #47 heavy chain CDR1

SEQ ID NO :41 - Mouse monoclonal antibody #47 heavy chain CDR2

SEQ ID NO :42 - Mouse monoclonal antibody #47 heavy chain CDR3

SEQ ID NO :43 - Mouse monoclonal antibody #47 light chain CDR1

SEQ ID NO :44 - Mouse monoclonal antibody #47 light chain CDR2

SEQ ID NO :45 - Mouse monoclonal antibody #47 light chain CDR3

SEQ ID NO :46 - Mouse monoclonal antibody #131 heavy chain CDR1

SEQ ID NO :47 - Mouse monoclonal antibody #131 heavy chain CDR2

SEQ ID NO :48 - Mouse monoclonal antibody #131 heavy chain CDR3

SEQ ID NO :49 - Mouse monoclonal antibody #131 light chain CDR1

SEQ ID NO :50 - Mouse monoclonal antibody #131 light chain CDR2

SEQ ID NO :51 - Mouse monoclonal antibody #131 light chain CDR3

SEQ ID NO :52 - Heavy chain variable region deimmunized 47 variant 1

SEQ ID NO :53 - Heavy chain variable region deimmunized 47 variant 2

SEQ ID NO :54 - Heavy chain variable region deimmunized 47 variant 3

SEQ ID NO :55 - Heavy chain variable region deimmunized 47 variant 4

SEQ ID NO :56 - Heavy chain variable region deimmunized 47 variant 5

SEQ ID NO :57 - Light chain variable region deimmunized 47 variant 1

SEQ ID NO :58 - Light chain variable region deimmunized 47 variant 2

SEQ ID NO :59 - Light chain variable region deimmunized 47 variant 3

SEQ ID NO :60 - Light chain variable region deimmunized 47 variant 4

SEQ ID NO :61 - Heavy chain variable region deimmunized 131 variant 1

SEQ ID NO :62 - Heavy chain variable region deimmunized 131 variant 2

SEQ ID NO :63 - Heavy chain variable region deimmunized 131 variant 3

SEQ ID NO :64 - Heavy chain variable region deimmunized 131 variant 4

SEQ ID NO :65 - Heavy chain variable region deimmunized 131 variant 5

SEQ ID NO :66 - Light chain variable region deimmunized 131 variant 1

SEQ ID NO :67 - Light chain variable region deimmunized 131 variant 2

SEQ ID NO :68 - Light chain variable region deimmunized 131 variant 3 SEQ ID NO: :69

SEQ ID NO: :70

SEQ ID NO: :71

SEQ ID NO: :72

SEQ ID NO: :73

SEQ ID NO: :74

SEQ ID NO: :75

SEQ ID NO: :76

SEQ ID NO: :77