WO2005111072A2 - Notch-based fusion proteins and uses thereof - Google Patents

Abstract

This invention provides a method for treating a subject having a tumor and a method for inhibiting angiogenesis in a subject, both comprising administering to the subject an effective amount of a composition of matter comprising the extracellular domain of a Notch receptor protein operably affixed to a half-life-increasing moiety. This invention also provides a composition of matter comprising the extracellular domain of Notch4 receptor protein operably affixed to a half-life-increasing moiety. This invention further provides an article of manufacture. Finally, this invention provides a replicable vector which encodes a polypeptide comprising the extracellular domain of a Notch receptor protein operably affixed to a half-life-increasing moiety, a host vector system which comprises such replicable vector and a method of producing such polypeptide.

Description

NOTCH-BASED FUSION PROTEINS AND USES THEREOF

The invention disclosed herein was made with United States government support under grant number R01 HL62454 from the National Institutes of Health. Accordingly, the United States government has certain rights in this invention.

This application claims priority of U.S. Provisional Application No. 60/566,877, filed April 29, 2004, the contents of which are hereby incorporated herein by reference .

Throughout this application, various publications are referenced by arabic numbers within parentheses or by author and publication date within parentheses. Full citations for these publications may be found at the end of the specification. The disclosures of these publications are hereby incorporated by reference into this application to describe more fully the art to which this invention pertains.

Background of the Invention

Vascular Development

During mammalian embryogenesis, formation of the vascular system is an early and essential process. In the embryo, vascular development initiates with the pluripotent hemangioblast derived from the paraxial and lateral plate mesoderm. The hemangioblast has the potential to differentiate into either a hematopoietic progenitor or an endothelial cell progenitor, known as the angioblast . Vascular development begins with a process known as vasculogenesis whereby angioblasts differentiate into endothelial cells and migrate together to form the primitive vascular plexus. This initial vascular network consists of vessels that are homogenous in size and made up wholly of endothelial cells. The vascular plexus is then remodeled via angiogenesis .

Angiogenesis involves the sprouting of new vessels, the migration of these vessels into avascular regions, and the recruitment of accessory cells, pericytes and smooth muscle cells (Gale and Yancopoulos, 1999). The smooth muscle cells that differentiate and form the contractile vessel walls originate from multiple progenitors including neural crest cells, mesenchymal cells and even endothelial cells (Owens, 1995) . In adults, angiogenesis is involved in follicular development, wound healing, and pathological processes such as tumor angiogenesis and heart disease.

The Notch Family and Notch Ligands

Studies of Drosophila, C. Blegans, zebrafish and mammals have demonstrated that the Notch pathway is an evolutionarily conserved signaling mechanism that functions to modulate numerous cell-fate decisions. Notch signaling is required for the proper patterning of cells originating from all three germ layers. Depending on the cellular context, Notch signaling may both inhibit and induce differentiation, induce proliferation, and promote cell survival (Artavanis-Tsakonas et al . , 1995; Lewis, 1998; einmaster, 1997) . In Drosophila, a single Notch protein is activated by two ligands, Serrate and Delta. In mammals these families have been expanded to four Notch genes (Notchl, Notch2, Notch3 and Notch4) and five ligands, 2 Serrate-like (Jaggedl-2) and 3 Delta (Dll, 3, 4) (Bettenhausen et al . , 1995; Dunwoodie et al . , 1997; Gallahan and Callahan, 1997; Lardelli et al . , 1994; Lindsell et al . , 1995; Shawber et al . , 1996a; Shutter et al . , 2000a; Uyttendaele et al . , 1996; Weinmaster et al . , 1992; Weinmaster et al . , 1991). During embryogenesis, Notch receptors and ligands are expressed in dynamic spatial and temporal patterns. However, it is not known if all ligands activate all receptors.

Notch Signaling and Function

Notch signaling influences many different types of cell- fate decisions by providing inhibitory, inductive or proliferative signals depending on the environmental context (reviewed in Artavanis-Tsakonas et al . , 1995; Greenwald, 1998; Robey, 1997; Vervoort et al . , 1997). This pleiotropic function suggests that Notch modulates multiple signaling pathways in a spatio-temporal manner.

Consistent with Notch regulating cell-fate decisions, both the receptors and ligands are cell surface proteins with single transmembrane domains (Figure 1) . The regulatory extracellular domain of Notch proteins consists largely of tandemly arranged EGF-like repeats that are required for ligand binding (Artavanis-Tsakonas et al., 1995; Weinmaster, 1998). C-terminal to the EGF- like repeats are an additional three cysteine-rich repeats, designated the LIN12/Notch repeats (LNR) (Greenwald, 1994) . Downstream of the LNR lies the proteolytic cleavage sequence (RXRR) that is recognized by a furin-like convertase. For Notchl, cleavage at this site yields a 180 kilodalton extracellular peptide and a 120 kilodalton intracellular peptide that are held together to generate a heterodimeric receptor at the cell surface (Blaumueller et al . , 1997; Kopan et al . , 1996; Logeat et al . , 1998) .

The intracellular domain of Notch (NotchlCD, Figure 1) rescues loss-of-function Notch phenotypes indicating that this form of Notch signals constitutively (Fortini and Artavanis-Tsakonas, 1993; Lyman and Young, 1993; Rebay et al., 1993; Struhl et al . , 1993).

The cytoplasmic domain of Notch contains three identifiable domains: the RAM domain, the ankyrin repeat domain and the C-terminal PEST domain (Figure 1) . Upon ligand-activation Notch undergoes two additional proteolytic cleavages which results in the release of the cytoplasmic domain (Weinmaster, 1998) . This Notch peptide translocates to the nucleus and interacts with transcriptional repressors known as CSL (CBF, Su (H) , Lag-2) and converts it to transcriptional activator. The CSL/Notch interaction is dependent on the presence of the RAM domain of Notch; while, transcriptional activity also requires the presence of the ankyrin repeats (Hsieh et al., 1996; Hsieh et al . , 1997; Roehl et al . , 1996; Tamura et al., 1995; Wettstein et al . , 1997). Both in vivo and in vi tro studies indicate that the HES and Hey genes are the direct targets of Notch/CSL-dependent signaling (Bailey and Posakony, 1995; Eastman et al . , 1997; Henderson et al . , 2001; Jarriault et al . , 1995; Nakagawa et al., 2000; Wettstein et al . , 1997). The HES and Hey genes are bHLH transcriptional repressor that bind DNA at N-boxes (Nakagawa et al . , 2000; Sasai et al . , 1992; Tietze et al . , 1992) . Notch has also been proposed to signal by a CSL-independent pathway. In fact, expression of just the ankyrin repeat domain is necessary and sufficient for some forms of Notch signaling (Lieber et al., 1993; Matsuno et al . , 1997; Shawber et al . , 1996b).

Finally, the PEST domain has been implicated in protein turnover by a SEL-10/ubiquitin-dependent pathway (Greenwald, 1994; Oberg et al . , 2001; Rogers et al . , 1986; Wu et al . , 1998; Wu et al . , 2001). Similar to the receptors, the extracellular domain of the Notch ligands also consist mostly of tandemly arranged EGF-like repeats (Figure 1) . Upstream of these repeats is a divergent EGF-like repeat known as the DSL (Delta, Serrate, Lag-2) that is required for ligand binding and activation of the receptors (Artavanis-Tsakonas et al . , 1995).

Notch Signaling and Vascular Development

Although many of the genes that function to induce vasculogenesis and angiogenesis have been identified, little is known about how cell-fate decisions are specified during vascular development . A number of observations suggest that the Notch signaling pathway may play a role in cell fate determination and patterning of the vascular system.

Notchl, Notch4, Jaggedl and D114 are all expressed in the developing vasculature, while Notch3 is expressed in the accessory smooth muscle cells (Krebs et al . , 2000; Shutter et al . , 2000b; Uyttendaele et al . , 1996; Villa et al., 2001; Xue et al . , 1999). Mice lacking Jaggedl are embryonic lethal and have severe vascular defects (Xue et al . , 1999). Mice nullizygous for Notchl are embryonic lethal and die of severe neuronal defects, but also have defects in angiogenesis (Krebs et al . , 2000; Swiatek et al . , 1994) . Mice lacking Notch4 are born and appear to be normal, but embryos that have lost both Notchl and Notch4 die at E9.5 of severe hemorrhaging and vascular patterning defects indicating Notchl and Notch4 may be functionally redundant during vascular development (Krebs et al . , 2000) . Exogenous expression of an activated form of Notch4 in endothelium also resulted in vascular defects similar to those seen for the double

Notchl/Notch4 nullizygous mice, suggesting that appropriate levels of Notch signaling is critical for proper development of the embryonic vasculature

(Uyttendaele et al . , 2001).

Taken together, the data from mice mutant for Notch/Notch signaling components uncover several processes dependent on Notch including vascular remodeling, arterial venous specification, vascular smooth muscle cell recruitment and heart/heart outflow vessel development.

Recent experiments have implicated Notch signaling in arterial/venous endothelial cell specification. In si tu analysis of E13.5 embryos found that Notchl, Notch3, Notch4, Dl , Jaggedl and Jagged2 expression was restricted to the arteries and absent in the veins (Villa et al . , 2001). Consistent with expression data, disruption of Notch signaling in Zebrafish was associated with loss of the arterial marker ephrinB2; while, ectopic expression of an activated form of Notch lead to a loss in the venous cell marker EphB4 within the dorsal aorta (Lawson et al . , 2001). These data suggest that Notch signaling may help to specify arterial and venous cell fates during angiogenesis.

Taken together, the data from mice mutant for Notch/Notch signaling components uncover several processes dependent on Notch including vascular remodeling, arterial venous specification, vascular smooth muscle cell recruitment and heart/heart outflow vessel development.

Notch signaling has also been suggested to function in the adult vascular system. In humans, missense mutations in the extracellular domain of Notch3 correlate with the development of the degenerative vascular disease, CADASIL (Caronti et al . , 1998; Desmond et al . , 1998; Joutel et al . , 2000; Joutel et al . , 1996) . In a wound healing model, an increase in Jaggedl expression was observed at the regenerating endothelial wound edge, suggesting Notch signaling may function during processes of adult angiogenesis (Lindner et al . , 2001) . Taken together these data support Notch signaling functions at a number of critical steps during vascular development: vasculόgenesis, vascular patterning/angiogenesis, and arterial/venous specification. However, the molecular mechanism (s) by which the Notch signaling pathways influence these different steps has yet to be elucidated. Significance

Shimizu et al . (J. Biol . Chem. 274(46): 32961-32969 (1999)) describe the use of NotchlECD/Fc, Notch2ECD/Fc and Notch3ECD/Fc in binding studies. However, Shimizu et al . do not mention the use of such proteins for inhibiting angiogenesis.

U.S. Patent No. 6,379,925 issued April 30, 2002 to Kitajewsky et al . describes murine Notch4. However, it does not describe Notch-based fusion proteins as set forth in the subject application.

This invention differs from the prior art because it is the first study using Notch-based fusion proteins comprising the extracellular domain of Notch operably affixed to a half-life-increasing moiety to inhibit angiogenesis. This invention therefore provides an advantage over the prior art in that it provides evidence that such Notch-based fusion proteins are capable of inhibiting angiogenesis.

Notch proteins play key roles in developmental decisions involving the vasculature, the hematopoietic system, and the nervous system. As such, an understanding of their function is key to understanding how cell-fate decisions and commitment are controlled during development and in adult tissues. To date, several reports on Notch or Notch ligand gene disruptions have described vascular phenotypes providing emphasis that this pathway is a fundamental part of the machinery that guides vascular development. Aberrant Notch activity has been linked to human pathologies; including both cancer and vascular disorders (CADASIL) . The analysis of Notch in tumor angiogenesis has only recently begun; however, our discovery of potential downstream targets of Notch suggests a roles in pathological processes associated with angiogenesis. For instance, VEGFR-3 has been linked to both tumor angiogenesis and tumor lymphangiogenesis . The expression or function of several other potential Notch targets has also been linked to tumor angiogenesis; including ephrinB2 , Id3 , Angiopoietin 1, and PDGF-B. Insights on the role of these targets in Notch gene function will clearly facilitate future analysis of Notch in human pathologies .

Summary of the Invention

This invention provides a method for treating a subject having a tumor comprising administering to the subject an effective amount of a composition of matter comprising the extracellular domain of a Notch receptor protein operably affixed to a half-life-increasing moiety, so as to thereby treat the subject.

This invention also provides a method for inhibiting angiogenesis in a subject comprising administering to the subject an effective amount of a composition of matter comprising the extracellular domain of a Notch receptor protein operably affixed to a half-life-increasing moiety, so as to thereby inhibit angiogenesis in the subject .

This invention further provides a composition of matter comprising the extracellular domain of Notch4 receptor protein operably affixed to a half-life-increasing moiety. In one embodiment, the extracellular domain is covalently bound to the half-life-increasing moiety. In another embodiment, the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.

This invention further provides a composition of matter comprising the extracellular domain of Notch4 receptor protein operably affixed to a half-life-increasing moiety and a pharmaceutically acceptable carrier.

This invention further provides an article of manufacture comprising (i) a packaging material having therein a composition of matter comprising the extracellular domain of a Notch receptor protein operably affixed to a half- life-increasing moiety and (ii) a label indicating that the composition is intended for use in treating a subject having a tumor or other disorder treatable by inhibiting angiogenesis in the subject.

This invention further provides a replicable vector which encodes a polypeptide comprising the extracellular domain of a Notch receptor protein operably affixed to a half- life-increasing moiety.

This invention further provides a host vector system which comprises a replicable vector which encodes a polypeptide comprising the extracellular domain of a Notch receptor protein operably affixed to a half-life- increasing moiety and a suitable host cell.

Finally, this invention provides a method of producing a polypeptide which comprises growing a host vector system which comprises a replicable vector which encodes a polypeptide comprising the extracellular domain of a Notch receptor protein operably affixed to a half-life- increasing moiety and a suitable host cell under conditions permitting production of the polypeptide, and recovering the polypeptide so produced. Brief Description of the Figures

Figure 1

This Figure shows the schematic structure of Notch and Notch ligands: Notchl, Notch2, Notch3, Notch4, Jagged-1, Jagged-2, Delta-like 1, Delta-like 3, Delta-like 4.

Figure 2

This Figure shows the schematic design of Notch-based fusion proteins (NotchECD/Fc) . The extracellular domain of Notchl, Notch2 , Notch3 , or Notch4 containing the EGF- repeats is fused to the Fc portion of an antibody.

Figure 3 This Figure shows a co-culture assay for testing the activity of Notch-based fusion proteins. Notch and Notch responsive transcriptional reporters are expressed in a "Notch-responsive" cell, HeLa. Notch ligands, Jagged-1, Delta-like 1, or Delta-like 4 are expressed in a "ligand- presenting" cell, 293. Expression is mediated by transfection of individual cell populations, cells are co-cultured, and then assayed for Notch-dependent reporter activity.

Figure 4

This Figure shows the inhibitory activity of Notch-based fusion protein against activation of Notch signaling by interaction between Notch and Notch ligand. Induction of Notch signaling was detected by co-cultivating both Notchl- and 3 types of Notch ligand-expressing cells and these inductions were inhibited by co-transfection of Notch-based fusion protein-expressing vector into Notchl- expressing cells. Therefore, Notch-based fusion proteins can be used as Notch inhibitor based on inhibition of interaction between Notch and Notch ligand.

Figure 5

This Figure shows the expression of Notchl-based fusion protein (NotchlECD/Fc) in 293. Panel A: expression in cell lystates (lys) or secreted into media (sup) . Panel B : expression in 293 lysates of NECD/Fcs, as listed.

Figure 6

This Figure shows activation of Notch signaling in HUVEC infected with adenoviral-encoding VEGF-165. Activation of Notch signaling can be detected by using CBFl promoter activity. Transcriptional activity of CBFl promoter is activated by binding of Notch-IC to CBFl. We measured CBFl promoter activity in HUVEC which was infected with adenovirus-encoding VEGF-165 at different MOI . Induction of CBFl promoter was clearly detected in Ad-VEGF-infected HUVEC, compared to Ad-LacZ-infected cells in the MOI dependent manner. This data showed overexpression of VEGF could activate Notch signaling in HUVEC.

Figure 7 This Figure shows the effect of Notch-based fusion proteins on VEGF-induced activation of Notch signaling. Co-infection of Ad-Notch-based fusion protein with Ad- VEGF clearly reduced activation of CBFl promoter activity induced by Ad-VEGF infection alone. In the case of infection at 40 MOI for each adenovirus in panel A, 60% inhibition at 24 hour and 90% inhibition at 48 hour after reporter gene transfection was detected. This inhibitory activity of Notch trap was dependent on MOI of Ad-Notch- based fusion protein.

Figure 8 This Figure shows an experiment in which we evaluated the effect of Notch-based fusion proteins on induction of budding by overexpressed VEGF-165 in HUVEC. When Ad-VEGF- infected HUVEC were cultured on type collagen gel for 8 days, budding was induced into collagen gel. This induction of budding by overexpressed VEGF was clearly inhibited by coinfection of adenoviral-encoding Notch- based fusion proteins. Ad-Notch-based fusion protein itself had less effect on morphology.

Figure 9

This Figure shows the result of counting buds per field under microscope. Ad-VEGF-infection into HUVEC increased the number of buds depending on used MOI . Even though a half MOI of Notch-based fusion protein was used, compared to Ad-VEGF, Ad-VEGF-induced budding was clearly inhibited. These data suggested that VEGF induced budding of HUVEC through activation of Notch signaling and Notch- based fusion protein could inhibit VEGF-induced budding.

Figure 10

This Figure shows the amino acid sequence of the extracellular domain of the rat Notchl protein and a linker sequence.

Figure 11

This Figure shows the amino acid sequence of the extracellular domain of the rat Notch2 protein and a linker sequence. Figure 12

This Figure shows the amino acid sequence of the extracellular domain of the mouse Notch3 protein.

Figure 13

This Figure shows the amino acid sequence of the extracellular domain of the mouse Notch4 protein and a linker sequence.

Figures 14A and 14B

This Figure shows the nucleic acid sequence of the extracellular domain of the rat Notchl gene.

Figures 15A and 15B

This Figure shows the nucleic acid sequence of the extracellular domain of the rat Notch2 gene.

Figures 16A and 16B This Figure shows the nucleic acid sequence of the extracellular domain of the mouse Notch3 gene .

Figures 17A and 17B

This Figure shows the nucleic acid sequence of the extracellular domain of the mouse Notch4 gene.

Figures 18A and 18B

This Figure shows the nucleic acid sequence of the extracellular domain of the human Notchl gene.

Figures 19A and 19B

This Figure shows the nucleic acid sequence of the extracellular domain of the human Notch2 gene. Figures 20A and 2OB

This Figure shows the nucleic acid sequence of the extracellular domain of the human Notch3 gene.

Figures 21A and 2IB

This Figure shows the nucleic acid sequence of the extracellular domain of the human Notch4 gene.

Figures 22A - 221

These Figures show that VEGF activates Notch signaling to induce HUVEC budding. HUVEC were transduced with Ad-VEGF at 40 MOI (Figs. 22A, 22H, 221) or 20 MOI (Figs. 22C, 22G) . Ad-LacZ was co-transduced to HUVEC to make the same total amount of adenovirus 60 MOI (Fig. 22G) , 80 MOI (Fig. 22A) and 100 MOI (Figs. 22H, 221) . Figure 22A shows

RT-PCR analysis of Notch and Notch ligand expression.

Numbers show PCR cycles. Figure 22B shows the effect of transduced VEGF on CSL reporter activity. Figure 22C shows the effect of SU5416 on CSL reporter activity transactivated by Ad-VEGF. Figure 22D shows the construct of Notch decoy (NlECDFc) . Figure 22E shows secretion of NlECDFc from HUVEC trasduced with Ad-NlECDFc. Figure 22F shows the effect of NlECDFc against ligand-induced CSL reporter activity in a co-culture assay (D: (-) ; ■: 0.33 ng pHyTC-NlECDFc,- ■: 0.67 ng pHyTC-NlECDFc) . Figures 22G- I show the effect of NlECDFc against Ad-VEGF-transduced HUVEC. Notch signaling was activated with transduction of Ad-VEGF in HUVEC in the absence or presence of co- transduction of Ad-NlECDFc at indicated dosage. Figure 22G shows the effect of NlECDFc on CSL reporter activity transactivated by Ad-VEGF. Figure 22H shows inhibition of budding of Ad-VEGF-transduced HUVEC with co-transduction of Ad-NlECDFc at 40 MOI. Figure 221 shows quantification of the effect of NlECDFc on budding of Ad-VEGF-transduced HUVEC (D: bud; ■: cell number) .

Figures 23A - 23J

These Figures show that Notch signaling up-regulates Fltl expression to induce HUVEC budding. HUVEC were transduced with either Ad-LacZ or Ad-NlIC at 40 MOI. Figures 23A-23C show the effect of inhibitors for receptor tyrosine kinases on Notch-induced HUVEC budding. Figure 23A is a photograph of budding of Ad-NlIC-transduced HUVEC treated with PD166866, ZD1893 at 1 μM and SU5416 at 0.5 μM. Figure 23B shows quantification of the effect of inhibitors at 1 μM (D: bud; ■: cell number) . Figure 23C shows dose- dependency of the effect of SU5416 (D: bud; ■: cell number) . Figures 23D-E show induction of Flt-1 expression in Ad-NlIC-transduced HUVEC. Figure 23D shows RT-PCR analysis of Flt-1 mRNA expression. Figure 23E shows W.B. analysis of Flt-1 protein expression. Figures 23F-G show promotion of Notch-induced HUVEC budding with PlGF stimulation. Ad-NlIC-transduced HUVEC were cultured on collagen gel with SFM, instead of complete medium, in the absence or presence of 50 ng/ml PlGF. Figure 23F shows PlGF-induced budding of Ad-NlIC-transducec HUVEC (arrow head: buds with single filopodia; arrow, buds with multiple filopodia) . Figure 23G shows the quantification of the effect of PlGF on budding of Ad-NlIC-transduced HUVEC (□: multi; ■: total) . Figures 23H-I show the effect of Flt-1 siRNA transfection on Fltl expression. Ad-NlIC-transduced HUVEC were transfected with 200 pmol of either control (CT) or Flt-1 siRNA. Figure 23H shows the reduction of Flt-1 mRNA expression. Figure 231 shows the reduction of Flt-1 protein expression. Figure 23J shows the effect of Flt-1 siRNA transfection on Notch- induced HUVEC budding. Ad-NlIC-transduced HUVEC were transfected with either 100 or 200 pmol of siRNA and cultured on collagen gel for 2 days .

Figures 24A - 24E

These Figures show that VEGF regulates gelatinase activity via Notch signaling by up-regulation of both MMP-9 and MTl-MMP. Figures 24A-B show gelatin zymography analysis of MMP-9 and MMP-2 activity stimulated by VEGF in HUVEC. Figure 24A shows the effect of NlECDFc on MMP-9 activity. Transduced HUVEC were cultured on fibrin gel on the indicated day (i.e. D2 , D4, D6, D8) . Similar results were also obtained by using collagen gel, although induction of MMP-9 was stronger on fibrin gel than collagen gel (data not shown) . Figure 24B shows the effect of NlECDFc on MMP-2 activity. HUVEC were transduced with Ad-NlECDFc at the indicated doses and condition medium was collected from HUVEC cultured on collagen gel at day 4. Figures 24C-D show up-regulation of MMP-9 and MTl-MMP with Notch signaling. HUVEC were transduced with either Ad-LacZ or Ad-NlIC at 40 MOI. Numbers show PCR cycles. Figure 24C shows RT-PCR analysis of the effect of Notch signaling on expression of MMP-9 and MMP-2. Figure 24D shows the induction of MTl-MMP expression of both transcript and protein with Notch signaling. Figure 24E shows RT-PCR analysis of MMP-9 and MTl-MMP expression in Ad-VEGF-HUVEC with co-transduction of Ad-NlECDFc. HUVEC were transduced with Ad-VEGF in the absence or presence of co-transduction of Ad-NlECDFc at 40 MOI each. Ad-LacZ was co-transduced to make the same total amount of adenovirus at 80 MOI. Figures 25A - 25D

These Figures show the role of Notch signaling in VEGF- dependent in vivo angiogenesis. Figures 25A-25D show inhibition of VEGF-induced angiogenesis with NlECDFc in mouse DAS assay. Representative photographs are shown. Figure 25A show subcutaneous induced angiogenesis with 293/VEGF transfectant versus 293/VEGF also expressing Notch decoy (Notch-based fusion protein) NlECDFc. Figure 25B shows the quantitation of degree of vascularization induced by 293/VEGF in control versus 293 expressing Notch decoy (Notch-based fusion protein)- NlECDFc. Figure 25C shows subcutaneous induced angiogenesis with Ad-LacZ infected MDA-MB-231 cells versus Ad-NlECDFc (Notch-based fusion protein) infected MDA-MB-231 cells. MDA-MB-231 breast cancer cells produce VEGF (data not shown) . Figure 25D shows quantitation of degree of vascularization induced by Ad-LacZ infected MDA-MB-231 cells versus Ad- NlECDFc (Notch-based fusion protein) infected MDA-MB-231 cells.

Figures 26A and 26B

These Figures show proliferation of Ad-VEGF165-transduced

HUVEC. HUVEC were transduced with Ad-VEGF165 at the indicated dosages. Ad-LacZ was also co-infected to make the same total amount of adenovirus at a MOI of 40 pfu/cell. HUVEC were suspended in SFM supplemented with 1% FBS and then plated at 1 x 10⁴ cells/well in 24-well multi-wll plates with 0.4 ml of medium. After 4 days, cell numbers were determined using the CCK-8 kit and the results are indicated as the ratio of cell numbers determined to the number of control cells, which were transduced with Ad-GFP at a MOI of 40 pfu/cell. Figure 26A shows the effect of transduced VEGF on proliferation. Figure 26B shows the inhibitory effect of SU5416. Ad- VEGF-transduced HUVEC were treated with SU5416 at the indicated dosages .

Figures 27A and 27B

These Figures show the induction of HUVEC buds on type I collagen gel. HUVEC were transduced with either Ad- VEGF165 or AD-N1IC at the indicated dosages. Ad-LacZ was also co-infected to make the same total amount of adenovirus at a MOI of 40 pfu/cell. Transduced HUVEC were cultured on collagen gel with complete medium. The amount of budding was evaluated under microscopy at day 7.

Figures 28A and 28B

These Figures show the effect of alteration of Notch signaling on cell proliferation. The cells were transduced with the indicated adenoviruses . Ad-GFP was also co-infected to make the same total amount of adenovirus at a MOI of 60 pfu/cell. After 4 days, cell numbers were determined using the CCK-8 kit and results are indicated as the ratio of cell numbers determined to the number of control cells, which were transduced with AD-GFP at MOI of 60 pfu/cell. Figure 28A shows the effect of transduced N1IC and Notch fusion protein on the proliferation of HUVEC. Transduced HUVEC were suspended in complete medium and then plated at 1 x 10⁴ cells/well in 24-well multiwell plates with 0.4 ml of indicated medium (□: Ad-NlIC; ■: Ad-NlECDFc) . Figure 28B shows the effect of Notch fusion protein on proliferation of KP1/VEGF transfectants. Transduced KPl/VEGF transfectants were suspended in RPMI1640 medium and then plated at 2x 10⁴ cells/well in 24-well multiwell plates with 0.5 ml of medium.

Figure 29

This Figure shows the RT-PCR analysis of induction of PlGF expression in Ad-NlIC-transduced HUVEC. HUVEC were infected with either Ad-LacZ or Ad-NlIC at a MOI of 40 pfu/cell. Total RNA was isolated from transduced HUVEC cultured on collagen gel for 5 days with complete medium.

Figures 30A - 3PC

These Figures show inhibition of budding of either Ad- N1IC- or Ad-VEGF-transduced HUVEC with Flk-1 siRNA transfection. Figure 30A shows reduction of Flk-1 mRNA and protein expression in Ad-VEGF-HUVEC with transfection of 200 pmol Flk-1 siRNA. Ad-VEGF-HUVEC at a MOI of 40 pfu/cell were transfected with 200 pmol of either control (CT) or Flk-1 siRNA. Total RNA was isolated 48 hours after transfection. Total cell lysate was collected from serum starved cells with SFM for 48 hours after transfection. Figures 30B and 30C show the inhibitory effect of Flk-1 siRNA transfection on either VEGF or Notch-induced HUVEC buds. Either Ad-NlIC- or Ad-VEGF- HUVEC at a MOI of 40 pfu/cell were transfected with 200 pmol of siRNA as indicated and cultured on collagen gel for 5 days. Figure 30B shows the effect of Flk-1 siRNA transfection on HUVEC buds (D: Ad-VEGF; ■: Ad-NlIC) . Figure 30C shows quantification of the inhibitory effect of Flk-1 siRNA transfection.

Figures 31A and 3IB

These Figures show inhibition of budding of Ad-NlIC- transduced HUVEC with treatment of matrix metallo- proteinase inhibitor GM6001. Either Ad-LacZ or Ad-NlIC- HUVEC at a MOI of 40 pfu/cell were cultured on collagen gel for 5 days in the absence or presence of GM6001 at 50 μm. Figure 31A shows the effect of GM6001 on Notch- induced HUVEC buds. Figure 3IB shows quantification of the inhibitory effect of GM6001.

Detailed Description of the Invention

Terms

As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below.

"Administering" may be effected or performed using any of the methods known to one skilled in the art. The methods comprise, for example, intralesional, intramuscular, subcutaneous, intravenous, intraperitoneal, liposome- mediated, transmucosal , intestinal, topical, nasal, oral, anal, ocular or otic means of delivery.

"Affixed" shall mean attached by any means. In one embodiment, affixed means attached by a covalent bond. In another embodiment, affixed means attached non- covalently.

"Amino acid, " "amino acid residue" and "residue" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide or peptide. The amino acid can be, for example, a naturally occurring amino acid or an analog of a natural amino acid that can function in a manner similar to that of the naturally occurring amino acid.

"Antibody" shall include, without limitation, (a) an immunoglobulin molecule comprising two heavy chains and two light chains and which recognizes an antigen; (b) a polyclonal or monoclonal immunoglobulin molecule; and (c) a monovalent or divalent fragment thereof. Immunoglobulin molecules may derive from any of the commonly known classes, including but not limited to IgA, secretory IgA, IgG, IgE and IgM. IgG subclasses are well known to those in the art and include, but are not limited to, human IgGl, IgG2, IgG3 and IgG4. Antibodies can be both naturally occurring and non-naturally occurring. Furthermore, antibodies include chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. Antibodies may be human or nonhuman. Nonhuman antibodies may be humanized by recombinant methods to reduce their immunogenicity in humans . Antibody fragments include, without limitation, Fab and F_c fragments. The "Fc portion of an antibody", in one embodiment, is a crystallizable fragment obtained by papain digestion of immunoglobulin that consists of the C-terminal half of two heavy chains linked by disulfide bonds and known as the "effector region" of the immunoglobulin. In another embodiment, "Fc portion of an antibody" means all, or substantially all, of one C- terminal half of a heavy chain.

"Humanized", with respect to an antibody, means an antibody wherein some, most or all of the amino acids outside the CDR region are replaced with corresponding amino acids derived from a human immunoglobulin molecule. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not abrogate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules include, without limitation, IgGl, IgG2, IgG3, IgG4, IgA and IgM molecules. Various publications describe how to make humanized antibodies, e.g., United States Patent Nos. 4,816,567, 5,225,539, 5,585,089 and 5,693,761, and PCT International Publication No. WO 90/07861.

As used herein, the term "composition", as in pharmaceutical composition, is intended to encompass a product comprising the active ingredient (s) and the inert ingredient (s) that make up the carrier, as well as any product which results, directly or indirectly from combination, complexation, or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients.

As used herein, "effective amount" refers to an amount which is capable of treating a subject having a tumor or inhibiting angiogenesis in a subject. Accordingly, the effective amount will vary with the subject being treated, as well as the condition to be treated. A person of ordinary skill in the art can perform routine titration experiments to determine such sufficient amount. The effective amount of a compound will vary depending on the subject and upon the particular route of administration used. Based upon the compound, the amount can be delivered continuously, such as by continuous pump, or at periodic intervals (for example, on one or more separate occasions) . Desired time intervals of multiple amounts of a particular compound can be determined without undue experimentation by one skilled in the art. In one embodiment, the effective amount is between about lμg/kg - 10 mg/kg. In another embodiment, the effective amount is beteen about lO g/kg - 1 mg/kg. In a further embodiment, the effective amount is lOOμg/kg. "Extracellular domain" as used in connection with Notch receptor protein means all or a portion of Notch which (i) exists extracellularly (i.e. exists neither as a transmembrane portion or an intracellular portion) and (ii) binds to extracellular ligands to which intact Notch receptor protein binds . The extracellular domain of Notch may optionally include a signal peptide. "Extracellular domain" and "ECD" are synonymous.

"Half -life-increasing moiety" means a moiety which, when operably affixed to a second moiety, increases the in vivo half-life of the second moiety. Half-life-increasing moieties include, for example, Fc portions of antibodies, glycosylation tags (i.e. glycosylated polypeptides), polyethylene glycol (PEG) , polypeptides having PEG affixed thereto, and lipid-modified polypeptides.

"Inhibiting" the onset of a disorder or undesirable biological process shall mean either lessening the likelihood of the disorder's or process' onset, or preventing the onset of the disorder or process entirely. In the preferred embodiment, inhibiting the onset of a disorder or process means preventing its onset entirely.

"Notch", "Notch protein", and "Notch receptor protein" are s nonymous. The following Notch amino acid sequences are known and hereby incorporated by reference: Notchl (Genbank accession no. S18188 (rat)); Notch2 (Genbank accession no. NP__077334 (rat)); Notch3 (Genbank accession no. Q61982 (mouse) ) ; and Notch4 (Genbank accession no. T09059 (mouse) ) . The following Notch nucleic acid sequences are known and hereby incorporated by reference : Notchl (Genbank accession no. XM_342392 (rat) and NM_017617 (human)); Notch2 (Genbank accession no. NM_024358 (rat), M99437 (human and AF308601 (human)); Notch3 (Genbank accession no. NM__008716 (mouse) and XM_009303 (human)); and Notch4 (Genbank accession no. NM_010929 (mouse) and NM_004557 (human) ) .

The terms "nucleic acid", "polynucleotide" and "nucleic acid sequence" are used interchangeably herein, and each refers to a polymer of deoxyribonucleotides and/or ribonucleotides . The deoxyribonucleotides and ribonucleotides can be naturally occurring or synthetic analogues thereof. "Nucleic acid" shall mean any nucleic acid, including, without limitation, DNA, RNA and hybrids thereof. The nucleic acid bases that form nucleic acid molecules can be the bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art, and are exemplified in PCR Systems, Reagents and Consumables (Perkin Elmer Catalogue 1996- 1997, Roche Molecular Systems, Inc., Branchburg, New Jersey, USA) . Nucleic acids include, without limitation, anti-sense molecules and catalytic nucleic acid molecules such as ribozymes and DNAzymes . Nucleic acids also include nucleic acids coding for peptide analogs, fragments or derivatives which differ from the naturally- occurring forms in terms of the identity of one or more amino acid residues (deletion analogs containing less than all of the specified residues; substitution analogs wherein one or more residues are replaced by one or more residues; and addition analogs, wherein one or more resides are added to a terminal or medial portion of the peptide) which share some or all of the properties of the naturally-occurring forms . "Operably affixed" means, with respect to a first moiety affixed to a second moiety, affixed in a manner permitting the first moiety to function (e.g. binding properties) as it would were it not so affixed.

The terms "polypeptide," "peptide" and "protein" are used interchangeably herein, and each means a polymer of amino acid residues. The amino acid residues can be naturally occurring or chemical analogues thereof. Polypeptides, peptides and proteins can also include modi ications such as glycosylation, lipid attachment, sulfation, hydroxylation, and ADP-ribosylation.

As used herein, "pharmaceutically acceptable carrier" means that the carrier is compatible with the other ingredients of the formulation and is not deleterious to the recipient thereof, and encompasses any of the standard pharmaceutically accepted carriers. Such carriers include, for example, 0.01-0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions and suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer' s dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like.

"Subject" shall mean any organism including, without limitation, a mammal such as a mouse, a rat, a dog, a guinea pig, a ferret, a rabbit and a primate. In the preferred embodiment, the subject is a human being.

"Treating" means either slowing, stopping or reversing the progression of a disorder. As used herein, "treating" also means the amelioration of symptoms associated with the disorder.

Units, prefixes and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acid sequences are written left to right in 5 'to 3 'orientation and amino acid sequences are written left to right in amino- to carboxy-terminal orientation. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

The following abbreviations are used herein: ECD: extracellular domain; IC: intracellular domain; NECD/Fc: Notch-based fusion protein; Nl : Notchl; N2 : Notch2; N3 : Notch3; N4 : Notch4. Embodiments of the Invention

This invention provides a first method for treating a subject having a tumor comprising administering to the subject an effective amount of a composition of matter comprising the extracellular domain of a Notch receptor protein operably affixed to a half-life-increasing moiety, so as to thereby treat the subject.

This invention also provides a second method for inhibiting angiogenesis in a subject comprising administering to the subject an effective amount of a composition of matter comprising the extracellular domain of a Notch receptor protein operably affixed to a half- li e-increasing moiety, so as to thereby inhibit angiogenesis in the subject.

In a first embodiment of the above methods, the Notch receptor protein is Notchl receptor protein. In one embodiment, the Notchl receptor protein is human Notchl receptor protein. In another embodiment, the half-life- increasing moiety is an Fc portion of an antibody. In another embodiment, the Fc portion of the antibody is the Fc portion of a human antibody. In a further embodiment, the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.

In a second embodiment of the above methods, the Notch receptor protein is Notch2 receptor protein. In one embodiment, the Notch2 receptor protein is human Notch2 receptor protein. In another embodiment, the half-life- increasing moiety is an Fc portion of an antibody. In another embodiment, the Fc portion of the antibody is the Fc portion of a human antibody. In a further embodiment, the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.

In a third embodiment of the above methods, the Notch receptor protein is Notch3 receptor protein. In one embodiment, the Notch3 receptor protein is human Notch3 receptor protein. In another embodiment, the half-life- increasing moiety is an Fc portion of an antibody. In another embodiment, the Fc portion of the antibody is the Fc portion of a human antibody. In a further embodiment, the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.

In a fourth embodiment of the above methods, the Notch receptor protein is Notch4 receptor protein. In one embodiment, the Notch4 receptor protein is human Notch4 receptor protein. In another embodiment, the half-life- increasing moiety is an Fc portion of an antibody. In another embodiment, the Fc portion of the antibody is the Fc portion of a human antibody. In a further embodiment, the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.

In a fifth embodiment of the above methods, the subject is a mammal. In one embodiment, the mammal is a human.

In a sixth embodiment of the above methods, the angiogenesis is tumor angiogenesis.

In a further embodiment of the second method, the subject has a tumor. In another embodiment, the subject is afflicted with a pathologic vascular hyperplasia. In one embodiment, the pathologic vascular hyperplasia is a benign hemagioma. In a further embodiment, the subject is afflicted with a lymphatic vascular proliferative disease.

This invention provides a first composition of matter comprising the extracellular domain of Notch4 receptor protein operably affixed to a half-life-increasing moiety. In one embodiment, the extracellular domain is covalently bound to the half-life-increasing moiety. In another embodiment, the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.

This invention also provides a second composition of matter comprising the extracellular domain of Notch4 receptor protein operably affixed to a half-life- increasing moiety and a pharmaceutically acceptable carrier.

This invention further provides an article of manufacture comprising (i) a packaging material having therein a composition of matter comprising the extracellular domain of a Notch receptor protein operably affixed to a half- life-increasing moiety and (ii) a label indicating that the composition is intended for use in treating a subject having a tumor or other disorder treatable by inhibiting angiogenesis in the subject.

In a first embodiment of the above article, the Notch receptor protein is Notchl receptor protein. In one embodiment, the Notchl receptor protein is human Notchl receptor protein. In another embodiment, the half-life- increasing moiety is an Fc portion of an antibody. In another embodiment, the Fc portion of the antibody is the Fc portion of a human antibody. In a further embodiment, the extracellular domain and the Half-life-increasing moiety are within the same polypeptide chain.

In a second embodiment of the above article, the Notch receptor protein is Notch2 receptor protein. In one embodiment, the Notch2 receptor protein is human Notch2 receptor protein. In another embodiment, the half-life- increasing moiety is an Fc portion of an antibody. In another embodiment, the Fc portion of the antibody is the Fc portion of a human antibody. In a further embodiment, the extracellular domain and the Half-life-increasing moiety are within the same polypeptide chain.

In a third embodiment of the above article, the Notch receptor protein is Notch3 receptor protein. In one embodiment, the Notch3 receptor protein is human Notch3 receptor protein. In another embodiment, the half-life- increasing moiety is an Fc portion of an antibody. In another embodiment, the Fc portion of the antibody is the Fc portion of a human antibody. In a further embodiment, the extracellular domain and the Half-life-increasing moiety are within the same polypeptide chain.

In a fourth embodiment of the above article, the Notch receptor protein is Notch4 receptor protein. In one embodiment, the Notch4 receptor protein is human Notch4 receptor protein. In another embodiment, the half-life- increasing moiety is an Fc portion of an antibody. In another embodiment, the Fc portion of the antibody is the Fc portion of a human antibody. In a further embodiment, the extracellular domain and the Half-life-increasing moiety are within the same polypeptide chain.

In another embodiment of the above article, the composition is admixed with a pharmaceutical carrier. In a final embodiment, the subject is a human.

This invention provides a replicable vector which encodes a polypeptide comprising the extracellular domain of a Notch4 receptor protein operably affixed to a half-life- increasing moiety. In one embodiment, the half-life- increasing moiety is an Fc portion of an antibody. In another embodiment, the vector includes, without limitation, a plasmid, a cosmid, a retrovirus, an adenovirus, a lambda phage or a YAC.

This invention also provides a host vector system which comprises a replicable vector which encodes a polypeptide comprising the extracellular domain of a Notch receptor protein operably affixed to a half-life-increasing moiety and a suitable host cell. In one embodiment, the host cell is a eukaryotic cell. In another embodiment, the eukaryotic cell is a CHO cell. In a another embodiment, the eukaryotic cell is a HeLa cell. In a further embodiment, the host cell is a bacterial cell.

Finally, this invention provides a third method of producing a polypeptide which comprises growing a host vector system which comprises a replicable vector which encodes a polypeptide comprising the extracellular domain of a Notch receptor protein operably affixed to a half- life-increasing moiety and a suitable host cell under conditions permitting production of the polypeptide, and recovering the polypeptide so produced.

This invention is illustrated in the Experimental Details section which follows. This section is set forth to aid in an understanding of the invention but is not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow thereafter.

EXPERIMENTAL DETAILS

First Series of Experiments

Materials & Methods

^'Plasmid Constructs

Adenovirus constructs encoding LacZ, full-length Notch4, or the activated form of Notch4/int3 have been previously described (Shawber et al . , 2003). An activated form of Notchl cDNA fused in frame with 6 myc tags (Kopan et al . , 1994) was cloned into the adenovirus expression vector, pAd-lox. Both VEGF165 and NlECDFc was also cloned into the pAd-lox. Adenoviral stocks were generated and titered as previously described (Hardy et al . , 1997) . The retroviral expression vector pHyTc encoding either LacZ, the activated form of Notch4/int3, Jl, Dill and D114 have been previously described (Uyttendaele et al . , 2000, Shawber et al . , 2003, Das et al . , 2004 in print). Plasmids encoding the intracellular domain of Notchl (bp 5479-7833, Genbank accession# X57405) and the extracellular domain of D114 (bp 1-1545, Genbank accession# AF253468, provided by Chiron) fused in frame with a myc/His tag, were engineered into pHyTC.

NotchlECD, Notch2ECD, Notch3ECD and Notch4ECD are engineered into the Fc containing plasmid pCMX-sFRl-IgG using the methods set forth in Clin . Exp . Immunol . (1992) 87(1) : 105-110 to create the Notch-based fusion proteins, i.e. NotchlECD/Fc, Notch2ECD/Fc, Notch3ECD/Fc and Notch4ECD/Fc. Adenoviral Gene Transfer

7.5xl0⁵ cells of HUVEC at passage 3 were seeded into type I collagen-coated 6 well plates on the day before adenoviral infection, Adenoviral infection with Ad-lacZ, Ad-VEGF165 or Ad-NlECDFc was performed at indicated m.o.i., and incubated at 37 °C for 1 hr with occasional swirling of plates. ucif erase reporter assays

To determine ligand-induced Notch signaling, co-culture assays were performed using HeLa and 293-derived Bosc cells. Transient transfections were performed by calcium phosphate precipitation. Hela cells plated 1-day prior in 10-cm plates at 1.5xl0⁶ were transfected with 333 ng of pBOS Notchl, 333 ng pGA981-6, and 83 ng pLNC lacZ with either 666 ng pCMV-Fc or pHyTC-NlECDFc (333 ng for xl, 666 ng for x2) . Bosc cells plated 1-day prior in 10-cm plates at 4x10^s were transfected with either 680 ng pHyTc- Jaggedl, pHyTc-Dlll, pHyTc-DH4, or pHyTc-x (empty vector) . One day after transfection, the cells were co- cultured in triplicate (HeLa:Bosc, 1:2) on 12-well plates for 24 hours. Cells were harvested and luciferase activity was determined 2-days post-transfection using the Enhanced Luciferase assay kit (BD PharMingen) , and β- galactosidase activity was determined using the Galacto- Light Plus kit (PE Biosystems) . All assays were performed in a Berthold dual-injection luminometer.

To determine VEGF-induced Notch signaling, HUVEC which were infected with adenovirus were used. HUVEC plated 1- day prior in 6 well plates at 8.0x10^s were infected with either Ad-LacZ as control or Ad-VEGF at indicated m.o.i. in the presence or absence of Ad-NIECD/Fc . Two days after infection, infected HUVEC were re-seeded into 24-well plate at 1.5 x 10^s cell in triplicate and cultured for 24 hours, and then transfected with 12.5 ng pRL-SV40 (Promega) and 137.5 ng pGA981-6 using Effectene transfection reagent (Qiagen) . Cells were harvested either 1 or 2 days post-transfection and luciferase activity was determined by using the Dual-Luciferase Reporter Assay System (Promega) .

Sprouting assay

For making collagen gels, an ice-cold solution of porcine type I collagen (Nitta gelatin, Tokyo, Japan) was mixed with 10xRPMI1640 medium and neutralization buffer at the ratio of 8:1:1. 400 μl aliquots of collagen gel were then added to 24-well plates and allowed to gel for at least 1 hour at 37°C. Following adenoviral infection (above), HUVEC was harvested and plated at 1.3xl0⁵ cells per well onto the top of the collagen gel in 24-well plates in 0.8 ml of EGM2 medium. HUVEC became nearly confluent 48 hours after plating. After seeding, medium was changed every 2 days for 1 week. Sprouting was observed and photographs taken after 8 days with an Olympus digital camera mounted to a microscope. For quantification of the number of sprouts, 5 fields per each well were randomly selected and sprouting was counted under microscopy in a blind manner by two investigators. Results and Discussion

NOTCHECD/Fc Fusion Proteins Function As Antagonists Of Notch

Notch Antagonists -No tchECD/Fc Fusion Proteins

We have made several Notch antagonists (Figure 2) . Our strategy was to fuse the coding sequence of Notch EGF repeats in the Extracellular Domain (ECD) to the human or mouse Fc domain. This design makes a secreted protein without signaling function but which retains the ligand- binding domain and thus should bind to and inhibit ligand function. We refer to these proteins as "NotchECD/Fc" and all four Notchl-4ECD/Fcs have been made. The Fc domain facilitates affinity purification and protein detection by immunoblotting or immunohistochemistry.

Testing Notch Antagonists

An in vi tro co-culture system (Figure 3) with ligands expressed on one cell and Notch receptor activation scored in another cell was used to measure transcriptional activation of the Notch pathway. We used this co-culture assay to show that NotchlECD/Fc functions to block ligand-dependent Notch signaling (Figure 4) . The NIECD/Fc expression vector was co-transfected at different ratios with full-length Notchl and the CSL- luciferase reporter in HeLa cells, followed by co-culture with ligand expressing 293 cells. We observed that activation of Notchl signaling by Notch ligands was reduced by NIECD/Fc expression. This effect displayed concentration-dependency; a 2:1 ratio of NIECD/Fc to Notchl was more effective in inhibiting signaling than a 1:1 ratio. NotchlECD/Fc could block signaling mediated by Jaggedl, Delta-like 1 or Delta-like 4.

Expressing and Purifying Notch Antagonists

We have made CHO and HeLa cell lines expressing NotchECD/FCs using retroviral vectors for the purpose of protein purification. NIECD/Fc proteins are secreted (Figure 5) ; as shown in conditioned media collected from

HeLa-NotchECD/Fc lines and purified with Protein-A(pA) agarose. The pA purified sample (Sup) and whole cell lysates (Lys) were immunoblotted with α-Fc antibody (Figure 5, panel A) demonstrating that NIECD/Fc is secreted into the media. Adenovirus vectors for NotchECD/Fc were used to infect HeLa cells and lysates from these cells were immunoblotted with -Fc antibodies demonstrating that they express NotchECD/Fc (1, 2, 3, 4) proteins (Figure 5, panel B) . We are currently purifying

NIECD/Fc from CHO cell conditioned media using pA- affinity chromatography.

Defining Angiogenic Inhibi tion Using Notch Fusion Proteins

Activation of Notch signaling can be detected by using CBFl promoter activi ty

One can measure Notch signaling function by measuring transcriptional activity of CBFl promoter, which is activated by binding of Notch-IC to CBFl. We measured CBF1 promoter activity in HUVEC which was infected with adenovirus encoding VEGF-165 at different MOI (Figure 6) . Induction of CBFl promoter was clearly detected in Ad- VEGF-infected HUVEC, compared to Ad-LacZ-infected cells in the MOI dependent manner. This data showed over- expression of VEGF could activate Notch signaling in HUVEC. Thus VEGF induced Notch signaling activity.

We asked whether Notch fusion proteins could block VEGF- induced activation of Notch signaling. Co-infection of Ad-Notch fusion protein with Ad-VEGF clearly reduced activation of CBFl promoter activity induced by Ad-VEGF infection alone (Figure 7) . In the case of infection at 40 MOI for each adenovirus in Figure 7 (panel A) , 60% inhibition at 24 hr and 90% inhibition at 48 hr after reporter gene transfection were detected also the inhibitory activity of Notch decoy was dependent on MOI of Ad-Notch fusion protein.

Notch fusion proteins block initiation of angiogenic sprouting induced by VEGF

In this experiment, we evaluated the effect of Notch decoy on induction of budding (initiation of sprouting) by over-expressed VEGF-165 in HUVEC. When Ad-VEGF- infected HUVEC were cultured on type collagen gel for 8 days, budding was induced into collagen gel. This induction of budding by overexpressed VEGF was clearly inhibited by coinfection of adenoviral encoding Notch fusion protein (Figure 8) . Ad-Notch fusion protein itself had less effect on morphology.

In Figure 9 we counted buds per field using the microscope. Ad-VEGF-infection into HUVEC increased the number of buds depending on the MOI used. Ad-VEGF-induced budding was clearly inhibited. These data suggest that VEGF induced budding of HUVEC through activation of Notch signaling and that the Notch fusion protein could inhibit VEGF-induced budding.

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39. Sasai, Y. , R. Kageyama, Y. Tagawa, R. Shigemoto, and S. Nakanishi. 1992. Two mammalian helix-loop-helix factors structurally related to Drosophila hairy and Enhancer of split. Genes & Dev. 6:2620-2634.

40. Shawber, C, J. Boulter, C.E. Lindsell, and G. Weinmaster. 1996a. Jagged2 : a serrate-like gene expressed during rat embryogenesis . Dev Biol . 180:370-6.

41. Shawber, C. , D. Nofziger, J.J. Hsieh, C. Lindsell, 0. Bogler, D. Hayward, and G. Weinmaster. 1996b. Notch signaling inhibits muscle cell differentiation through a CBFl- independent pathway. Development 122:3765-73. 42. Shimizu, K. , S. Chiba, T. Saito, T. Takahashi, K. Kumano, H. Hamada, and H. Hirai. 2002. Integrity of intracellular domain of Notch ligand is indespensable for cleavage required for the release of the Notch2 intracellular domain. Embo J. 21:294- 302.

43. Shutter, J.R., S. Scully, W. Fan, W.G. Richards, J. Kitajewski, G.A. Deblandre, C.R. Kintner, and K.L. Stark. 2000a. D114, a novel Notch ligand expressed in arterial endothelium. Genes Dev. 14:1313-1318.

44. Shutter, J.R., S. Scully, W. Fan, W.G. Richards, J. Kitajewski, G.A. Deblandre, C.R. Kitner, and K.L. Stark. 2000b. D114, a novel Notch ligand expressed in arterial endothelium. Genes and Development 14:1313-1318.

45. Struhl, G., K. Fitzgerald, and I. Greenwald. 1993. Intrinsic activity of the Lin-12 and Notch intracellular domains in vivo. Cell 74:331-45.

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50. Uyttendaele, H., G. Marazzi, G. Wu, Q. Yan, D. Sassoon, and J. Kitajewski. 1996. Notch4/int-3 , a mammary proto-oncogene, is an endothelial cell- specific mammalian Notch gene. Development 122:2251- 9.

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Second Series of Experiments

VEGF ini tiates angiogenesis via an activation of Notch signaling

Both the VEGF and Notch signaling pathways are critical for vascular development . Here we show that VEGF activates Notch signaling to initiate angiogenesis. VEGF increased the expression of Delta4 and Notch4 causing Notch signal activation and inducing filopodia in cultured primary endothelial cells. Studies using VEGF Receptor inhibitors show that Notch signal activation in turn enhances VEGF action by inducing VEGFR-1 (Flt-1) expression. Other elements of VEGF action, including the induction of MMP-9 and MTl-MMP, are mediated by Notch. Using in vivo assays to model VEGF-induced skin neovascularization, we found that a secreted Notch inhibitor (Notch-based fusion protein) blocks VEGF- induced neo-vascularization and induction of VEGFR-1 expression. Thus, Notch signaling is requisite for angiogenesis regulated by VEGF, likely at the level of initiation.

VEGF is a key regulator of angiogenesis progression consisting of multiple processes, such as degradation of ECM, budding (filopodia formation) , proliferation, survival, and migration of endothelial cells. Although most of the steps might be co-operated with downstream molecules of VEGF signaling, it is not known how these steps are coordinately regulated to result in more complex orphogenetic events, such as angiogenic sprouting. Notch signaling is an evolutionarily conserved signaling mechanism that functions to regulate cell fate decisions (1) . Upon binding by a ligand, such as Jagged and Delta-like, the cytoplasmic domain of Notch (NotchIC) is released by presenilin/γ-secretase, translocates to the nucleus, interacts with the transcriptional repressor CSL (CBFl/Su (H) /lag2) , and converts it to a transcriptional activator (1) . Roles of Notch signaling in vascular development were suggested by studies of mice with targeted mutation (2) . Since Notch activation within the endothelium also disrupts vascular remodeling, proper Notch signaling is essential for vascular development (3) . Although relevance of Notch to VEGF signaling is suggested (4-6) , it is still unclear how Notch signaling has a role in VEGF-regulated angiogenesis and whether Notch signaling participates in physiological and pathological angiogenesis in the adult vasculature.

HUVEC (Human Umbilical Vein Endothelial cells) growth are dependent on VEGF (Figs. 26A and 26B) and differentiation-related biological responses, such as sprouting, and can be evaluated at an early stage (7) . At first, we examined whether adenovirally transduced VEGF induced both Notch and Notch ligand expression in HUVEC cultured with complete medium containing bFGF (Fig. 22A) , as reported (5) . RT-PCR analysis showed that both D14 and Notch4 mRNA was up-regulated in adenovirally-transduced VEGF HUVEC (Ad-VEGF-HUVEC) , compared to adenovirally- transduced LacZ HUVEC (Ad-LacZ-HUVEC) (Fig. 22A) . Transduced VEGF did not appear to induce Jaggedl and Notchl expression. Transduced-VEGF also activated Notch signaling in a dose-dependent manner by measuring CSL- luciferase reporter activity (Fig. 22B) , which was transactivated with Notch signaling (8) . Notch signaling was activated at a higher dosage of Ad-VEGF, compared to proliferation (Fig. 26A) . Since SU5416, which is an inhibitor of VEGFR kinases, decreased VEGF-induced CSL- luciferase reporter activity (Fig. 22C) , VEGF .induced Notch signaling through activation of receptor kinase. Since Notch mutants lacking both transmembrane and cytoplasmic domains functioned as dominant negative inhibitors against Notch signaling (9) , we made a Notch- based fusion protein or decoy (NlECDFc) to inhibit Notch signaling (Fig. 22D) . Western blotting analysis of conditioned medium of Ad-NlECDFc-transduced HUVEC (Ad- NlECDFc-HUVEC) demonstrated that NlECDFc was expressed and secreted well (Fig. 22E) . By using a co-culture assay, in which Bosc cells expressing Notch ligands

(either Jl, Dll or D14) activated Notch signaling in HeLa cells expressing Notchl compared to control Bosc cells, we determined inhibition of Notch signaling with transfection of a NlECDFc-expression plasmid (Fig. 22F) . Then, we examined whether NlECDFc inhibited activation of Notch signaling by transduced VEGF in HUVEC (Fig. 22G) . Co-transduction of Ad-NlECDFc with Ad-VEGF into HUVEC clearly decreased CSL luciferase activity induced by VEGF. Gerhardt et al . reported that VEGF controlled angiogenesis in the early postnatal retina by guiding filopodia extension at the tips of the vascular sprouts (10) . During angiogenic sprouting, the formation of a specialized endothelial cell making filopodia projections among quiescent endothelial cells, might be one of the early events. Here we mean formation of a single endothelial cell making filopodia protrusions as budding. Budding of the primary endothelial cells is induced by cultivating them 3-dimensionally on either fibrin or collagen gel (11) . In the case where Ad-VEGF-HUVEC were cultured on collagen gel with complete medium, transduced-HUVEC made filopodia extensions into the collagen gel for 5 days (Fig. 22H) and the number of buds was increased in a dose-dependent manner (Fig. 27A) . Activation of Notch signaling by adenovirus encoding the activated form of Notch4 (Ad-Notch4/int3) induced HUVEC budding (12) and that of Notchl (Ad-NlIC) also induced HUVEC budding (Fig. 23A & 27B) . Since both VEGF and Notch signaling induce HUVEC budding, we examined whether NlECDFc inhibited VEGF-induced HUVEC budding (Fig. 22H- I) . Budding of Ad-VEGF-HUVEC was clearly inhibited by co- transduction of Ad-NlECDFc . Neither Ad-LacZ or Ad- NlECDFc-transduced HUVEC formed buds (Fig. 22H) . NlECDFc inhibited VEGF-induced HUVEC budding without affecting cell number (Fig. 221) . Transduced-NlECDFc did not clearly alter proliferation of HUVEC, while that of Ad- NllC-transduced HUVEC was inhibited in a dose-dependent manner (Fig. 28A) , consistent with the inhibitory efficacy of Notch signaling against endothelial proliferation (13).

To test whether Notch signaling is down-stream of VEGF, we evaluated three distinct inhibitors for receptor tyrosine kinases, including VEGFR on NllC-induced HUVEC budding, because three growth factors existed in complete medium (Fig. 23A-C) . At a concentration of 1 μM, each compound showed selective inhibition against each kinase (data not shown) . Neither PD166866 or ZD1893 affected budding of Ad-NlIC-HUVEC, while SU5416 clearly inhibited it (Fig. 23A-B) . SU5416 selectively inhibited budding of Ad-NlIC-HUVEC with less reduction of viability at lower concentrations (Fig. 23C) . Since Taylor et al . reported that Notch down-regulated Flkl/KDR/VEGFR2 expression (14) , it was unlikely that Notch co-operated with Flkl to promote budding. Thus, we examined whether activation of Notch signaling affected Fltl/VEGFRl expression in HUVEC, because SU5416 inhibits both Fltl and Flkl kinase activity (15) . RT-PCR analysis demonstrated that expression of Fltl mRNA was up-regulated in Ad-NlIC- HUVEC, while expression of endothelial cell maker, CD31 mRNA, was not compared to that in Ad-LacZ-HUVEC (Fig. 23D) . Western blotting analysis also showed that expression of Fltl protein was up-regulated in Ad-NlIC- HUVEC (Fig. 23E) . Thus, we examined whether PlGF, which is a selective ligand for Fltl, promoted budding of HUVEC in which Fltl was up-regulated via activation of Notch signaling (Fig. 23F-G) . PlGF increased the number of Ad-

N1IC-HUVEC buds by 150%, compared to the absence of PlGF (Fig. 23F) . Moreover, PlGF increased HUVEC buds containing multiple filopodia by 250% (Fig. 23G) . While reduction of Fltl expression using small interfering RNA (siRNA) for Fltl inhibited budding of Ad-NlIC-HUVEC (Fig.

23J) , transfection of which selectively decreased expression of Fltl mRNA (Fig. 23H) and that of Fltl protein (Fig. 231) . Although reduction of Flkl expression with Flkl siRNA also inhibited budding of Ad-NlIC-HUVEC (Fig. 30B) , the inhibitory efficacy of Flkl siRNA was less than that of Fltl siRNA (Fig. 23J) . Effects of Flkl siRNA were more effective on budding of Ad-VEGF-HUVEC than that of Ad-NlIC-HUVEC (Fig. 30B-C) . Transfection with Fltl siRNA inhibited budding of both Ad-NlIC- and Ad-VEGF-HUVEC to a similar extent (data not shown) .

Several studies demonstrated that VEGF regulated gelatinase activities in endothelial cells and the significance of gelatinase activity like MMP-2 and MMP-9 has been firmly established to induce angiogenic sprouting (16) . We examined whether VEGF regulated gelatinase acitivity via Notch signaling in HUVEC.

In Gelatin zymography, conditioned medium of Ad-VEGF- HUVEC showed both induction and activation of MMP9, which started to be detected at day 6 (Fig. 24A) and activation of MMP2, which was detected at day 4 (Fig. 24B) , compared to those of Ad-LacZ-HUVEC. Co-transduction of Ad-NlECDFc with Ad-VEGF showed inhibition of both induction and activation of MMP9 (Fig. 24A) and an activation of MMP2 (Fig. 24B) . RT-PCR analysis demonstrated that expression of MMP9 mRNA was up-regulated in Ad-NlIC-HUVEC, but expression of MMP2 mRNA was decreased in Ad-NlIC-HUVEC (Fig. 24C) . Since induction of MMP2 activity was not detected in gelatin zymography (Fig. 24B) , this result was a likely consequence. While expression of MTl-MMP, which is able to activate MMP2 at the cell surface (17) , was up-regulated at both the transcript and protein levels in Ad-NlIC-HUVEC (Fig. 24D) . As VEGF can regulate both gelatinase and MTl-MMP expression (16) , RT-PCR analysis demonstrated that both MMP9 and MTl-MMP were up- regulated in Ad-VEGF-HUVEC, compared to Ad-LacZ-HUVEC and this induction was inhibited with co-transduction of Ad- NlECDFc (Fig. 24E) . Ad-NlECDFc infection alone did not affect expression of either MMP9 or MTl-MMP in Ad-LacZ infected HUVEC (data not shown) . Requisition of MMPs for angiogenic sprouting has been established by synthetic MMP inhibitors (16) . GM6001 is one broad inhibitor against MMPs including MMP2 , MMP9 and MTl-MMP (18) . GM6001 clearly decreased budding of Ad-NlIC-HUVEC on both collagen (Fig. 31A-B) and fibrin gel (data not shown) .

In the mouse Dorsa Air Sac (DAS) assay (19) , stable transfectant of 293 cells over-expressing VEGF121 (293/VEGF) significantly induced in vivo angiogenesis (Fig. 25A, left panel) . This VEGF-induced angiogenesis was clearly inhibited by coexpression of NlECDFc, compared to 293/VEGF alone (Fig. 25A) . Vessel density was measured and an index of angiogenesis given in Fig. 25B, demonstrating the 293/VEGF induced angiogenesis is inhibited by co-expression of 293/NlECDFc (Fig. 25B) .

Also, in the mouse Dorsa Air Sac (DAS) assay (19) , ^"the human breast cancer cell line, MDA-MB-231 significantly induced in vivo angiogenesis, presumably via the secretion of VEGF (Fig. 25C, left panel) . This VEGF- induced angiogenesis was clearly inhibited by adenovirus mediated expression of NlECDFc, compared to adenovirus expressing LacZ. (Fig. 25C) . Vessel density was measured and an index of angiogenesis given in Fig. 25D, demonstrating the MDA-MB-231 induced angiogenesis is inhibited by expression of NlECDFc.

Flkl is a major positive signal transducer for angiogenesis through its strong tyrosine kinase activity in the embryo, while Fltl is thought to be a negative signal transducer for angiogenesis. However, a positive role for Flt-1 was demonstrated in adult mice, as in vivo growth of LLC over-expressing P1GF2 was severely compromised in mice lacking the cytoplasmic Flt-1 kinase domain (20) . Notch might function to alter VEGF signaling by inducing Flt-1 signaling and moderate Flk-1 signaling either to induce filopodia extension or potentiate angiogenic sprouting, since P1GF/Flt-1 signaling altered the phospholyration site of Flk-1 and potentiated ischemic myocardial angiogenesis (21) . Interestingly, Notch signaling also up-regulated PlGF expression (Fig. 29) . However, continuous activation of Notch signaling inhibits formation of multi-cellular lumen-containing angiogenic sprouts, as previously reported (22) . Notch signaling should be turned off after budding/filopodia formation and transient activation of the Notch pathway might be required. In a transgenic mouse model of pancreatic beta-cell carcinogenesis

(RiplTag2 mice) in which tumor angiogenesis is VEGF dependent, the level of VEGF expression is not increased, but mobilization of extracellular VEGF stored in the matrix to VEGF receptors occurs. MMP-9 is responsible for this mobilization and tumor progression was inhibited in RiplTag23MMP-9-null double-transgenic mice (23) . Notch up-regulated MMP-9 expression and might increase local VEGF level at the site for angiogenic sprouting. While Notch also up-regulates MTl-MMP expression, extracellular MMP-2 might be targeted to the cell membrane of Notch- activated endothelial cells. Notch might determine the site for angiogenic sprouting by regulating gelatinase activity and VEGF concentration. Since endothelial MMP-9 was regulated by Flt-1 in lung specific metastasis (20) , Flt-1 might participate in induction of MMP-9 indirectly. References cited in Second Series of Experiments

1. Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch Signaling: Cell Fate Control and Signal Integration in Development. Science 1999;284 (5415) : 770-776.

2. Shawber CJ, J. K. Notch function in the vasculature: insights from zebrafish, mouse and man. Bioessays. 2004;26(3) :225-34.

Uyttendaele H, Ho J, Rossant J, J. K. Vascular patterning defects associated with expression of activated Notch4 in embryonic endothelium. Proc Natl Acad Sci U S A. 2001 ; 98 (10) : 5643-8.

Lawson ND, Vogel AM, BM . W. sonic hedgehog and vascular endothelial growth factor act upstream of the Notch pathway during arterial endothelial differentiation. Dev Cell 2002 ,-3 (1) : 127-36.

5. Liu ZJ, Shirakawa T, Li Y, Soma A, Oka M, Dotto GP, et al . Regulation of Notchl and D114 by vascular endothelial growth factor in arterial endothelial cells: implications for modulating arteriogenesis and angiogenesis. Mol Cell Biol. 2003 ,-23 (1) : 14-25.

6. Gale NW, Dominguez MG, Noguera I, Pan L, Hughes V, Valenzuela DM, et al . Haploinsufficiency of deltalike 4 ligand results in embryonic lethality due to major defects in arterial and vascular development. Proc Natl Acad Sci U S A. 2004,-101 (45) .-5949-54. 7. Montesano R, L. 0. Phorbol esters induce angiogenesis in vitro from large-vessel endothelial cells. J Cell Physiol. 1987; 130 (2) :284-91.

8. Jarriault S, Brou C, Logeat F, Schroeter EH, Kopan R, A. I. Signalling downstream of activated mammalian Notch. Nature. 1995; 377 (6547) : 355-8.

9. Small D, Kovalenko D, Kacer D, Liaw L, Landriscina M, Di Serio C, et al . Soluble Jagged 1 represses the function of its transmembrane form to induce the formation of the Src-dependent chord-like phenotype. J Biol Chem 2001;276 (34) : 32022-30.

10. Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A, et al . VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 2003 ; 161 (6) : 1163-77.

11. Koolwijk P, van Erck MG, de Vree WJ, Vermeer MA, Weich HA, Hanemaaijer R, et al . Cooperative effect of TNFalpha, bFGF, and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix. Role of urokinase activity. J Cell Biol 1996; 132 (6) : 1177-88.

12. Das I, Craig C, Funahashi Y, Jung KM, Kim TW, Byers R, et al . Notch oncoproteins depend on gamma- secretase/presenilin activity for processing and function. J Biol Chem 2004 ;279 (29) :30771-80.

13. Noseda M, Chang L, McLean G, Grim JE, Clurman BE, Smith LL, et al . Notch activation induces endothelial cell cycle arrest and participates in contact inhibition: role of p21Cipl repression. Mol Cell Biol 2004;24(20) :8813-22.

14. Taylor KL, Henderson AM, CC. H. Notch activation during endothelial cell network formation in vitro targets the basic HLH transcription factor HESR-1 and downregulates VEGFR-2/KDR expression. Microvasc Res 2002;64(3) :372-83.

15. Itokawa T, Nokihara H, Nishioka Y, Sone S, Iwamoto Y, Yamada Y, et al . Antiangiogenic effect by SU5416 is partly attributable to inhibition of Flt-1 receptor signaling. Mol Cancer Ther 2002 ; 1 (5) :295-302.

16. Pepper MS. Role of the matrix metalloproteinase and plasminogen activator-plasmin systems in angiogenesis . Arterioscler Thromb Vase Biol 2001;21 (7) :1104-17.

17. Seiki M, Koshikawa N, I. Y. Role of pericellular proteolysis by membrane-type 1 matrix metalloproteinase in cancer invasion and angiogenesis. Cancer Metastasis Rev 2003; 22 (2- 3) :129-43.

18. Yamamoto M, Tsujishita H, Hori N, Ohishi Y, Inoue S, Ikeda S, et al . Inhibition of membrane-type 1 matrix metalloproteinase by hydroxamate inhibitors: an examination of the subsite pocket. J Med Chem 1998;41 (8) .-1209-17.

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20. Hiratsuka S, Nakamura K, Iwai S, Murakami M, Itoh T, Kijima H, et al . MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lung-specific metastasis. Cancer Cell 2002;2 (4) :289- 300.

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Claims

What is claimed is:

1. A method for treating a subject having a tumor comprising administering to the subject an effective amount of a composition of matter comprising the extracellular domain of a Notch receptor protein operably affixed to a half-life-increasing moiety, so as to thereby treat the subject.

2. The method of claim 1, wherein the Notch receptor protein is Notchl receptor protein.

3. The method of claim 2, wherein the Notchl receptor protein is human Notchl receptor protein.

4. The method of claim 2, wherein the half-life- increasing moiety is an Fc portion of an antibody.

5. The method of claim 2, wherein the half-life- increasing moiety is the Fc portion of a human antibody.

6. The method of claim 2, wherein the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.

7. The method of claim 1, wherein the Notch receptor protein is Notch2 receptor protein.

8. The method of claim 7, wherein the Notch2 receptor protein is human Notch2 receptor protein.

9. The method of claim 7, wherein the half-life- increasing moiety is an Fc portion of an antibody.

10. The method of claim 7, wherein the Fc portion of the antibody is the Fc portion of a human antibody.

11. The method of claim 7, wherein the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.

12. The method of claim 1, wherein the Notch receptor protein is Notch3 receptor protein.

13. The method of claim 1 , wherein the Notch3 receptor protein is human Notch3 receptor protein.

14. The method of claim 12, wherein the half-life- increasing moiety is an Fc portion of an antibody.

15. The method of claim 12, wherein the Fc portion of the antibody is the Fc portion of a human antibody.

16. The method of claim 12, wherein the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.

17. The method of claim 1, wherein the Notch receptor protein is Notch4 receptor protein.

18. The method of claim 17, wherein the Notch4 receptor protein is human Notch4 receptor protein.

19. The method of claim 17, wherein the half-life- increasing moiety is an Fc portion of an antibody.

20. The method of claim 17, wherein the Fc portion of the antibody is the Fc portion of a human antibody.

21. The method of claim 17, wherein the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.

22. The method of claim 1, wherein the subject is a mammal .

23. The method of claim 22, wherein the mammal is a human.

24. A method for inhibiting angiogenesis in a subject comprising administering to the subject an effective amount of a composition of matter comprising the extracellular domain of a Notch receptor protein operably affixed to a half-life-increasing moiety, so as to thereby inhibit angiogenesis in the subject .

25. The method of claim 24, wherein the Notch receptor protein is Notchl receptor protein.

26. The method of claim 25, wherein the Notchl receptor protein is human Notchl receptor protein.

27. The method of claim 25, wherein the half-life- increasing moiety is an Fc portion of an antibody.

28. The method of claim 25, wherein the Fc portion of the antibody is the Fc portion of a human antibody.

29. The method of claim 25, wherein the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.

30. The method of claim 24, wherein the Notch receptor protein is Notch2 receptor protein.

31. The method of claim 30, wherein the Notch2 receptor protein is human Notch2 receptor protein.

32. The method of claim 30, wherein the half-life- increasing moiety is an Fc portion of an antibody.

33. The method of claim 30, wherein the Fc portion of the antibody is the Fc portion of a human antibody.

34. The method of claim 30, wherein the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.

35. The method of claim 24, wherein the Notch receptor protein is Notch3 receptor protein.

36. The method of claim 35, wherein the Notch3 receptor protein is human Notch3 receptor protein.

37. The method of claim 35, wherein the half-life- increasing moiety is an Fc portion of an antibody.

38. The method of claim 35, wherein the Fc portion of the antibody is the Fc portion of a human antibody.

39. The method of claim 35, wherein the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.

40. The method of claim 24, wherein the Notch receptor protein is Notch4 receptor protein.

41. The method of claim 40, wherein the Notch4 receptor protein is human Notch4 receptor protein.

42. The method of claim 40, wherein the half-life- increasing moiety is an Fc portion of an antibody.

43. The method of claim 40, wherein the Fc portion of the antibody is the Fc portion of a human antibody.

44. The method of claim 40, wherein the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.

45. The method of claim 24, wherein the subject is a mammal .

46. The method of claim 45, wherein the mammal is a human.

47. The method of claim 24, wherein the angiogenesis is tumor angiogenesis.

48. The method of claim 24, wherein the subject has a tumor.

49. The method of claim 24, wherein the subject is afflicted with a pathologic vascular hyperplasia.

50. The method of claim 49, which the pathologic vascular hyperplasia is a benign hemagioma.

51. The method of claim 24, wherein the subject is afflicted with a lymphatic vascular proliferative disease .

52. A composition of matter comprising the extracellular domain of Notch4 receptor protein operably affixed to a half-life-increasing moiety.

53. The composition of claim 52, wherein the extracellular domain is covalently bound to the half-life-increasing moiety.

54. The composition of claim 52, wherein the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.

55. A composition comprising the composition of matter of claim 52 and a pharmaceutically acceptable carrier.

56. An article of manufacture comprising (i) a packaging material having therein a composition of matter comprising the extracellular domain of a Notch receptor protein operably affixed to a half-life- increasing moiety and (ii) a label indicating that the composition is intended for use in treating a subject having a tumor or other disorder treatable by inhibiting angiogenesis in the subject.

57. The article of claim 56, wherein the Notch receptor protein is Notchl receptor protein.

58. The article of claim 57, wherein the Notchl receptor protein is human Notchl receptor protein.

59. The article of claim 57, wherein the half-life- increasing moiety is an Fc portion of an antibody.

60. The article of claim 57, wherein the Fc portion of the antibody is the Fc portion of a human antibody.

61. The article of claim 57, wherein the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.

62. The article of claim 56, wherein the Notch receptor protein is Notch2 receptor protein.

63. The article of claim 62, wherein the Notch2 receptor protein is human Notch2 receptor protein.

64. The article of claim 62, wherein the half-life- increasing moiety is an Fc portion of an antibody.

65. The article of claim 62, wherein the Fc portion of the antibody is the Fc portion of a human antibody.

66 . The article of claim 62, wherein the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.

67. The article of claim 56, wherein the Notch receptor protein is Notch3 receptor protein.

68. The article of claim 67, wherein the Notch3 receptor protein is human Notch3 receptor protein.

69. The article of claim 67, wherein the half-life- increasing moiety is an Fc portion of an antibody.

70. The article of claim 67, wherein the Fc portion of the antibody is the Fc portion of a human antibody.

71. The article of claim 67, wherein the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.

72. The article of claim 56, wherein the Notch receptor protein is Notch4 receptor protein.

73. The article of claim 72, wherein the Notch4 receptor protein is human Notch4 receptor protein.

74. The article of claim 72, wherein the half-life- increasing moiety is an Fc portion of an antibody.

75. The article of claim 72, wherein the Fc portion of the antibody is the Fc portion of a human antibody.

76. The article of claim 72, wherein the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.

77. The article of claim 56, wherein the composition is admixed with a pharmaceutical carrier.

78. The article of claim 56, wherein the subject is a human.

79. A replicable vector which encodes a polypeptide comprising the extracellular domain of a Notch4 receptor protein operably affixed to a half-life- increasing moiety.

80. The vector of claim 79, wherein the half-life- increasing moiety is an Fc portion of an antibody.

81. The vector of claim 79, wherein the vector is a plasmid.

82. The vector of claim 79, wherein the vector is a cosmid.

83. The vector of claim 79, wherein the vector is a retrovirus .

84. The vector of claim 79, wherein the vector is an adenovirus.

85. A host vector system which comprises the vector of claim 79 and a suitable host cell.

86. The host vector system of claim 85 wherein the host cell is a eukaryotic cell.

87. The host vector system of claim 86, wherein the eukaryotic cell is a CHO cell.

88. The host vector system of claim 86, wherein the eukaryotic cell is a HeLa cell.

89. The host vector system of claim 85, wherein the host cell is a bacterial cell.

90. A method of producing a polypeptide which comprises growing the host vector system of claim 85 under conditions permitting production of the polypeptide, and recovering the polypeptide so produced.