NOTCH-BASED FUSION PROTEINS AND USES THEREOF

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

[0001] This application is a continuation of U.S. Ser. No. 12/657,573, filed Jan. 21, 2010, now allowed, which is a continuation of U.S. Ser. No. 11/114,962, filed Apr. 16, 2005, now U.S. Pat. No. 7,662,919, issued Feb. 16, 2010, which claims the benefit of U.S. Provisional Application No. 60/566,877, filed Apr. 29, 2004, the contents of each of which are hereby incorporated by reference into this application.

[0002] 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.

[0003] 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.

[0004] This application incorporates-by-reference nucleotide and/or amino acid sequences which are present in the filed named "150325_--0575_--71308-AAA_Substitute_Sequence_Lisiting_AHC.txt," which is 86.9 kilobytes in size, and which was created Mar. 25, 2015 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is contained in the text file filed Mar. 25, 2015 as part of this application.

BACKGROUND OF THE INVENTION

[0005] Vascular Development

[0006] 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.

[0007] 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.

[0008] 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.

[0009] The Notch Family and Notch Ligands

[0010] Studies of Drosophila, C. Elegans, 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; Weinmaster, 1997). In Drosophila, a single Notch protein is activated by two ligands, Serrate and Delta. In mammals these families have been expanded to four

[0011] Notch genes (Notch1, Notch2, Notch3 and Notch4) and five ligands, 2 Serrate-like (Jagged1-2) and 3 Delta (D11, 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.

[0012] Notch Signaling and Function

[0013] 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.

[0014] Consistent with Notch regulating cell-fate decisions, both the receptors and ligands are cell surface proteins with single transmembrane domains (FIG. 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 Notch1, 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).

[0015] The intracellular domain of Notch (NotchICD, FIG. 1) rescues loss-of-function Notch phenotypes indicating that this form of Notch signals constitutively (Fortini and

[0016] Artavanis-Tsakonas, 1993; Lyman and Young, 1993; Rebay et al., 1993; Struhl et al., 1993).

[0017] The cytoplasmic domain of Notch contains three identifiable domains: the RAM domain, the ankyrin repeat domain and the C-terminal PEST domain (FIG. 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 vitro 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).

[0018] 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 (FIG. 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).

[0019] Notch Signaling and Vascular Development

[0020] 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.

[0021] Notch1, Notch4, Jagged1 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 Jagged1 are embryonic lethal and have severe vascular defects (Xue et al., 1999). Mice nullizygous for Notch1 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 Notch1 and Notch4 die at E9.5 of severe hemorrhaging and vascular patterning defects indicating Notch1 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 Notch1/Notch4 nullizygous mice, suggesting that appropriate levels of Notch signaling is critical for proper development of the embryonic vasculature (Uyttendaele et al., 2001).

[0022] 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.

[0023] Recent experiments have implicated Notch signaling in arterial/venous endothelial cell specification. In situ analysis of E13.5 embryos found that Notch1, Notch3, Notch4, D14, 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.

[0024] 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.

[0025] 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: vasculogenesis, 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.

[0026] Significance

[0027] Shimizu et al. (J. Biol. Chem. 274(46): 32961-32969 (1999)) describe the use of Notch1ECD/Fc, Notch2ECD/Fc and Notch3ECD/Fc in binding studies. However, Shimizu et al. do not mention the use of such proteins for inhibiting angiogenesis.

[0028] U.S. Pat. No. 6,379,925 issued Apr. 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.

[0029] 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.

[0030] 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

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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

[0039] FIG. 1

[0040] This Figure shows the schematic structure of Notch and Notch ligands: Notch1, Notch2, Notch3, Notch4, Jagged-1, Jagged-2, Delta-like 1, Delta-like 3, Delta-like 4.

[0041] FIG. 2

[0042] This Figure shows the schematic design of Notch-based fusion proteins (NotchECD/Fc). The extracellular domain of Notch1, Notch2, Notch3, or Notch4 containing the EGF-repeats is fused to the Fc portion of an antibody.

[0043] FIG. 3

[0044] 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.

[0045] FIG. 4

[0046] 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 Notch1- and 3 types of Notch ligand-expressing cells and these inductions were inhibited by co-transfection of Notch-based fusion protein-expressing vector into Notch1-expressing cells. Therefore, Notch-based fusion proteins can be used as Notch inhibitor based on inhibition of interaction between Notch and Notch ligand.

[0047] FIG. 5

[0048] This Figure shows the expression of Notch1-based fusion protein (Notch1ECD/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.

[0049] FIG. 6

[0050] This Figure shows activation of Notch signaling in HUVEC infected with adenoviral-encoding VEGF-165. Activation of Notch signaling can be detected by using CBF1 promoter activity. Transcriptional activity of CBF1 promoter is activated by binding of Notch-IC to CBF1. We measured CBF1 promoter activity in HUVEC which was infected with adenovirus-encoding VEGF-165 at different MOI. Induction of CBF1 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.

[0051] FIG. 7

[0052] 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 CBF1 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.

[0053] FIG. 8

[0054] 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.

[0055] FIG. 9

[0056] 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.

[0057] FIG. 10

[0058] This Figure shows the amino acid sequence of the extracellular domain of the rat Notch1 protein (SEQ ID NO:1) and a linker sequence (SEQ ID NO:2).

[0059] FIG. 11

[0060] This Figure shows the amino acid sequence of the extracellular domain of the rat Notch2 protein (SEQ ID NO:3) and a linker sequence (SEQ ID NO:2).

[0061] FIG. 12

[0062] This Figure shows the amino acid sequence of the extracellular domain of the mouse Notch3 protein (SEQ ID NO:4).

[0063] FIG. 13

[0064] This Figure shows the amino acid sequence of the extracellular domain of the mouse Notch4 protein (SEQ ID NO:5) and a linker sequence (SEQ ID NO:2).

[0065] FIGS. 14A and 14B

[0066] This Figure shows the nucleic acid sequence of the extracellular domain of the rat Notch1 gene (SEQ ID NO:6).

[0067] FIGS. 15A and 15B

[0068] This Figure shows the nucleic acid sequence of the extracellular domain of the rat Notch2 gene (SEQ ID NO:7).

[0069] FIGS. 16A and 16B

[0070] This Figure shows the nucleic acid sequence of the extracellular domain of the mouse Notch3 gene (SEQ ID NO:8).

[0071] FIGS. 17A and 17B

[0072] This Figure shows the nucleic acid sequence of the extracellular domain of the mouse Notch4 gene (SEQ ID NO:9) and the nucleic acid sequence (SEQ ID NO:10) and the amino acid sequence (SEQ ID NO:2) of a linker sequence.

[0073] FIGS. 18A and 18B

[0074] This Figure shows the nucleic acid sequence of the extracellular domain of the human Notch1 gene (SEQ ID NO:11).

[0075] FIGS. 19A and 19B

[0076] This Figure shows the nucleic acid sequence of the extracellular domain of the human Notch2 gene (SEQ ID NO:12).

[0077] FIGS. 20A and 20B

[0078] This Figure shows the nucleic acid sequence of the extracellular domain of the human Notch3 gene (SEQ ID NO:13).

[0079] FIGS. 21A and 21B

[0080] This Figure shows the nucleic acid sequence of the extracellular domain of the human Notch4 gene (SEQ ID NO:14).

[0081] FIGS. 22A-22I

[0082] 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). FIG. 22A shows RT-PCR analysis of Notch and Notch ligand expression. Numbers show PCR cycles. FIG. 22B shows the effect of transduced VEGF on CSL reporter activity. FIG. 22C shows the effect of SU5416 on CSL reporter activity transactivated by Ad-VEGF. FIG. 22D shows the construct of Notch decoy (N1ECDFc). FIG. 22E shows secretion of N1ECDFc from HUVEC trasduced with Ad-N1ECDFc. FIG. 22F shows the effect of N1ECDFc against ligand-induced CSL reporter activity in a co-culture assay (quadrature: (-); .box-solid.: 0.33 ng pHyTC-N1ECDFc; .box-solid.: 0.67 ng pHyTC-N1ECDFc). FIGS. 22G-I show the effect of N1ECDFc 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-N1ECDFc at indicated dosage. FIG. 22G shows the effect of N1ECDFc on CSL reporter activity transactivated by Ad-VEGF. FIG. 22H shows inhibition of budding of Ad-VEGF-transduced HUVEC with co-transduction of Ad-N1ECDFc at 40 MOI. FIG. 22I shows quantification of the effect of N1ECDFc on budding of Ad-VEGF-transduced HUVEC (quadrature: bud; .box-solid.: cell number).

[0083] FIGS. 23A-23J

[0084] These Figures show that Notch signaling up-regulates Flt1 expression to induce HUVEC budding. HUVEC were transduced with either Ad-LacZ or Ad-N1IC at 40 MOI. FIGS. 23A-23C show the effect of inhibitors for receptor tyrosine kinases on Notch-induced HUVEC budding. FIG. 23A is a photograph of budding of Ad-N1IC-transduced HUVEC treated with PD166866, ZD1893 at 1 gM and SU5416 at 0.5 M. FIG. 23B shows quantification of the effect of inhibitors at 1 μM (quadrature: bud; .box-solid.: cell number). FIG. 23C shows dose-dependency of the effect of SU5416 (quadrature: bud; .box-solid.: cell number). FIGS. 23D-E show induction of Flt-1 expression in Ad-N1IC-transduced HUVEC. FIG. 23D shows RT-PCR analysis of Flt-1 mRNA expression. FIG. 23E shows W.B. analysis of Flt-1 protein expression. FIGS. 23F-G show promotion of Notch-induced HUVEC budding with PlGF stimulation. Ad-N1IC-transduced HUVEC were cultured on collagen gel with SFM, instead of complete medium, in the absence or presence of 50 ng/ml PlGF. FIG. 23F shows PlGF-induced budding of Ad-N1IC-transducec HUVEC (arrow head: buds with single filopodia; arrow: buds with multiple filopodia). FIG. 23G shows the quantification of the effect of PlGF on budding of Ad-N1IC-transduced HUVEC (quadrature: multi; .box-solid.: total). FIGS. 23H-I show the effect of Flt-1 siRNA transfection on Flt1 expression. Ad-N1IC-transduced HUVEC were transfected with 200 pmol of either control (CT) or Flt-1 siRNA. FIG. 23H shows the reduction of Flt-1 mRNA expression. FIG. 23I shows the reduction of Flt-1 protein expression. FIG. 23J shows the effect of Flt-1 siRNA transfection on Notch-induced HUVEC budding. Ad-N1IC-transduced HUVEC were transfected with either 100 or 200 pmol of siRNA and cultured on collagen gel for 2 days.

[0085] FIGS. 24A-24E

[0086] These Figures show that VEGF regulates gelatinase activity via Notch signaling by up-regulation of both MMP-9 and MT1-MMP. FIGS. 24A-B show gelatin zymography analysis of MMP-9 and MMP-2 activity stimulated by VEGF in HUVEC. FIG. 24A shows the effect of N1ECDFc 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). FIG. 24B shows the effect of N1ECDFc on MMP-2 activity. HUVEC were transduced with Ad-N1ECDFc at the indicated doses and condition medium was collected from HUVEC cultured on collagen gel at day 4. FIGS. 24C-D show up-regulation of MMP-9 and MT1-MMP with Notch signaling. HUVEC were transduced with either Ad-LacZ or Ad-N1IC at 40 MOI. Numbers show PCR cycles. FIG. 24C shows RT-PCR analysis of the effect of Notch signaling on expression of MMP-9 and MMP-2. FIG. 24D shows the induction of MT1-MMP expression of both transcript and protein with Notch signaling. FIG. 24E shows RT-PCR analysis of MMP-9 and MT1-MMP expression in Ad-VEGF-HUVEC with co-transduction of Ad-N1ECDFc. HUVEC were transduced with Ad-VEGF in the absence or presence of co-transduction of Ad-N1ECDFc at 40 MOI each. Ad-LacZ was co-transduced to make the same total amount of adenovirus at 80 MOI.

[0087] FIGS. 25A-25D

[0088] These Figures show the role of Notch signaling in VEGF-dependent in vivo angiogenesis. FIGS. 25A-25D show inhibition of VEGF-induced angiogenesis with N1ECDFc in mouse DAS assay. Representative photographs are shown. FIG. 25A show subcutaneous induced angiogenesis with 293/VEGF transfectant versus 293/VEGF also expressing Notch decoy (Notch-based fusion protein) N1ECDFc. FIG. 25B shows the quantitation of degree of vascularization induced by 293/VEGF in control versus 293 expressing Notch decoy (Notch-based fusion protein)-N1ECDFc. FIG. 25C shows subcutaneous induced angiogenesis with Ad-LacZ infected MDA-MB-231 cells versus Ad-N1ECDFc (Notch-based fusion protein) infected MDA-MB-231 cells. MDA-MB-231 breast cancer cells produce VEGF (data not shown). FIG. 25D shows quantitation of degree of vascularization induced by Ad-LacZ infected MDA-MB-231 cells versus Ad-N1ECDFc (Notch-based fusion protein) infected MDA-MB-231 cells.

[0089] FIGS. 26A and 26B

[0090] 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 1×10⁴ 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. FIG. 26A shows the effect of transduced VEGF on proliferation. FIG. 263 shows the inhibitory effect of SU5416. Ad-VEGF-transduced HUVEC were treated with SU5416 at the indicated dosages.

[0091] FIGS. 27A and 27B

[0092] 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.

[0093] FIGS. 28A and 28B

[0094] 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. FIG. 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 1×10⁴ cells/well in 24-well multiwell plates with 0.4 ml of indicated medium (quadrature: Ad-N1IC; .box-solid.: Ad-N1ECDFc). FIG. 28B shows the effect of Notch fusion protein on proliferation of KP1/VEGF transfectants. Transduced KP1/VEGF transfectants were suspended in RPMI1640 medium and then plated at 2×10⁴ cells/well in 24-well multiwell plates with 0.5 ml of medium.

[0095] FIG. 29 This Figure shows the RT-PCR analysis of induction of PIGF expression in Ad-N1IC-transduced HUVEC. HUVEC were infected with either Ad-LacZ or Ad-N1IC at a MOI of 40 pfu/cell. Total RNA was isolated from transduced HUVEC cultured on collagen gel for 5 days with complete medium.

[0096] FIGS. 30A-30C

[0097] These Figures show inhibition of budding of either Ad-N1IC- or Ad-VEGF-transduced HUVEC with Flk-1 siRNA transfection. FIG. 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. FIGS. 30B and 30C show the inhibitory effect of Flk-1 siRNA transfection on either VEGF or Notch-induced HUVEC buds. Either Ad-N1IC- 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. FIG. 30B shows the effect of Flk-1 siRNA transfection on HUVEC buds (quadrature: Ad-VEGF; .box-solid.: Ad-N1IC). FIG. 30C shows quantification of the inhibitory effect of Flk-1 siRNA transfection.

[0098] FIGS. 31A and 31B

[0099] These Figures show inhibition of budding of Ad-N1IC-transduced HUVEC with treatment of matrix metallo-proteinase inhibitor GM6001. Either Ad-LacZ or Ad-N1IC-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. FIG. 31A shows the effect of GM6001 on Notch-induced HUVEC buds. FIG. 31B shows quantification of the inhibitory effect of GM6001.

DETAILED DESCRIPTION OF THE INVENTION

[0100] Terms

[0101] As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below.

[0102] "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.

[0103] "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.

[0104] "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.

[0105] "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 IgG1, 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

[0106] 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.

[0107] "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, IgG1, IgG2, IgG3, IgG4, IgA and IgM molecules. Various publications describe how to make humanized antibodies, e.g., U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089 and 5,693,761, and PCT International Publication No. WO 90/07861.

[0108] 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.

[0109] 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 1 μg/kg-10 mg/kg. In another embodiment, the effective amount is between about 10 μg/kg-1 mg/kg. In a further embodiment, the effective amount is 100 μg/kg.

[0110] "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.

[0111] "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.

[0112] "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.

[0113] "Notch", "Notch protein", and "Notch receptor protein" are synonymous. Notch amino acid sequences are known, e.g. Notch1 (rat); Notch2 (rat); Notch3 (mouse); and Notch4 (mouse). Notch nucleic acid sequences are also known, e.g. Notch1 (rat and human); Notch2 (rat and human); Notch3 (mouse and human); and Notch4 (mouse and human).

[0114] 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, N.J., 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.

[0115] "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.

[0116] 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 modifications such as glycosylation, lipid attachment, sulfation, hydroxylation, and ADP-ribosylation.

[0117] 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.

[0118] "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.

[0119] "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.

[0120] 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.

[0121] The following abbreviations are used herein: ECD: extracellular domain; IC: intracellular domain; NECD/Fc:

[0122] Notch-based fusion protein; N1: Notch1; N2: Notch2; N3: Notch3; N4: Notch4.

Embodiments of the Invention

[0123] 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.

[0124] 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-life-increasing moiety, so as to thereby inhibit angiogenesis in the subject.

[0125] In a first embodiment of the above methods, the Notch receptor protein is Notch1 receptor protein. In one embodiment, the Notch1 receptor protein is human Notch1 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] In a fifth embodiment of the above methods, the subject is a mammal. In one embodiment, the mammal is a human.

[0130] In a sixth embodiment of the above methods, the angiogenesis is tumor angiogenesis.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] In a first embodiment of the above article, the Notch receptor protein is Notch1 receptor protein. In one embodiment, the Notch1 receptor protein is human Notch1 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] In another embodiment of the above article, the composition is admixed with a pharmaceutical carrier. In a final embodiment, the subject is a human.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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

[0144] Materials & Methods

[0145] Plasmid Constructs

[0146] 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 Notch1 cDNA fused in frame with 6 myc tags (Kopan et al., 1994) was cloned into the adenovirus expression vector, pAd-lox. Both VEGF165 and N1ECDFc 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, J1, D111 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 Notch1 (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.

[0147] Notch1ECD, Notch2ECD, Notch3ECD and Notch4ECD are engineered into the Fc containing plasmid pCMX-sFR1-IgG using the methods set forth in Clin. Exp. Immunol. (1992) 87(1):105-110 to create the Notch-based fusion proteins, i.e. Notch1ECD/Fc, Notch2ECD/Fc, Notch3ECD/Fc and Notch4ECD/Fc.

[0148] Adenoviral Gene Transfer

[0149] 7.5×10⁵ 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-N1ECDFc was performed at indicated m.o.i., and incubated at 37²C for 1 hr with occasional swirling of plates.

[0150] Luciferase Reporter Assays

[0151] 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 1.5×10⁶ were transfected with 333 ng of pBOS Notch1, 333 ng pGA981-6, and 83 ng pLNC lacZ with either 666 ng pCMV-Fc or pHyTC-N1ECDFc (333 ng for xl, 666 ng for x2). Bosc cells plated 1-day prior in 10-cm plates at 4×10⁶ were transfected with either 680 ng pHyTc-Jagged1, pHyTc-D111, pHyTc-D114, 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 p-galactosidase activity was determined using the Galacto-Light Plus kit (PE Biosystems). All assays were performed in a Berthold dual-injection luminometer.

[0152] To determine VEGF-induced Notch signaling, HUVEC which were infected with adenovirus were used. HUVEC plated 1-day prior in 6 well plates at 8.0×10⁵ were infected with either Ad-LacZ as control or Ad-VEGF at indicated m.o.i. in the presence or absence of Ad-N1ECD/Fc. Two days after infection, infected HUVEC were re-seeded into 24-well plate at 1.5×10⁵ 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).

[0153] Sprouting Assay

[0154] 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 1.3×10⁵ 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.

[0155] Results and Discussion

NOTCHECD/Fc Fusion Proteins

Function as Antagonists of Notch

[0156] Notch Antagonists-NotchECD/Fc Fusion Proteins

[0157] We have made several Notch antagonists (FIG. 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 Notch1-4ECD/Fcs have been made. The Fc domain facilitates affinity purification and protein detection by immunoblotting or immunohistochemistry.

[0158] Testing Notch Antagonists

[0159] An in vitro co-culture system (FIG. 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 Notch1ECD/Fc functions to block ligand-dependent Notch signaling (FIG. 4). The N1ECD/Fc expression vector was co-transfected at different ratios with full-length Notch1 and the CSL-luciferase reporter in HeLa cells, followed by co-culture with ligand expressing 293 cells. We observed that activation of Notch1 signaling by Notch ligands was reduced by N1ECD/Fc expression. This effect displayed concentration-dependency; a 2:1 ratio of N1ECD/Fc to Notch1 was more effective in inhibiting signaling than a 1:1 ratio. Notch1ECD/Fc could block signaling mediated by Jagged1, Delta-like 1 or Delta-like 4.

[0160] Expressing and Purifying Notch Antagonists

[0161] We have made CHO and HeLa cell lines expressing NotchECD/FCs using retroviral vectors for the purpose of protein purification. N1ECD/Fc proteins are secreted (FIG. 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 (FIG. 5, panel A) demonstrating that N1ECD/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 α-Fc antibodies demonstrating that they express NotchECD/Fc(1, 2, 3, 4) proteins (FIG. 5, panel B). We are currently purifying N1ECD/Fc from CHO cell conditioned media using pA-affinity chromatography.

Defining Angiogenic Inhibition Using Notch Fusion Proteins

[0162] Activation of Notch Signaling can be Detected by Using CBF1 Promoter Activity

[0163] One can measure Notch signaling function by measuring transcriptional activity of CBF1 promoter, which is activated by binding of Notch-IC to CBF1. We measured CBF1 promoter activity in HUVEC which was infected with adenovirus encoding VEGF-165 at different MOI (FIG. 6). Induction of CBF1 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.

[0164] 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 CBF1 promoter activity induced by Ad-VEGF infection alone (FIG. 7). In the case of infection at 40 MOI for each adenovirus in FIG. 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.

[0165] Notch fusion proteins block initiation of angiogenic sprouting induced by VEGF

[0166] 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 (FIG. 8). Ad-Notch fusion protein itself had less effect on morphology.

[0167] In FIG. 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.

[0168] References cited in First Series of Experiments

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[0197] 27. Lindner, V., C. Booth, I. Prudovsky, D. Small, T. Maciag, and L. Liaw. 2001. Members of the Jagged/Notch gene families are expressed in injured arteries and regulate cell phenotype via alteration in cell matrix and cell-cell interations. Pathology 159:875-883.

[0198] 28. Lindsell, C. E., C. J. Shawber, J. Boulter, and G. Weinmaster. 1995. Jagged: A mammalian ligand that activates Notch1. Cell 80:909-917.

[0199] 29. Logeat, F., C. Bessia, C. Brou, O. LeBail, S. Jarriault, N. G. Seidah, and A. Israel. 1998. The Notch1 receptor is cleaved constitutively by a furin-like convertase. Proc Natl Acad Sci USA 95:8108-12.

[0200] 30. Lyman, D., and M. W. Young. 1993. Further evidence for function of the Drosophila Notch protein as a transmembrane receptor. Proc Natl Acad Sci USA 90:10395-10399.

[0201] 31. Matsuno, K., M. J. Go, X. Sun, D. S. Eastman, and S. Artavanis-Tsakonas. 1997. Suppressor of Hairless-independent events in Notch signaling imply novel pathway elements. Development 124:4265-4273.

[0202] 32. Nakagawa, O., D. G. McFadden, M. Nakagawa, H.

[0203] Yanagisawa, T. Hu, D. Srivastava, and E. N. Olson. 2000. Members of the HRT family of basic helix-loop-helix proteins act as transcriptional repressors downstream of Notch signaling. Proc Natl Acad Sci USA 97:13655-13660.

[0204] 33. Oberg, C., J. Li, A. Pauley, E. Wolf, M. Gurney, and U. Lendahl. 2001. The Notch intracellular domain is ubiquitinated and negatively regulated by the mammalian Sel-10 homolog. J Biol Chem. 276:35847-35853.

[0205] 34. Owens, G. K. 1995. Regulation of differentiation of vascular smooth muscle cells. Physiol Rev. 75:487-527.

[0206] 35. Rebay, I., R. G. Fehon, and S. Artavanis-Tsakonas. 1993. Specific truncations of Drosophila Notch define dominant activated and dominant negative forms of the receptor. Cell 74:319-29.

[0207] 36. Robey, E. 1997. Notch in vertebrates. Curr Opin Genet Dev. 7:551-7.

[0208] 37. Roehl, H., M. Bosenberg, R. Blelloch, and J. Kimble. 1996. Roles of the RAM and ANK domains in signaling by the C. elegans GLP-1 receptor. Embo J. 15:7002-7012.

[0209] 38. Rogers, S., R. Wells, and M. Rechsteiner. 1986. Amino acid sequences common to rapidly degrade proteins: The PEST hypothesis. Science 234:364-368.

[0210] 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.

[0211] 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.

[0212] 41. Shawber, C., D. Nofziger, J. J. Hsieh, C. Lindsell, O. Bogler, D. Hayward, and G. Weinmaster. 1996b. Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway. Development 122:3765-73.

[0213] 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.

[0214] 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.

[0215] 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.

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[0217] 46. Swiatek, P. J., C. E. Lindsell, F. Franco del Amo, G. Weinmaster, and T. Gridley. 1994. Notch 1 is essential for postimplantation development in mice. Genes & Development 8:707-719.

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[0220] 49. Uyttendaele, H., J. Ho, J. Rossant, and J. Kitajewski. 2001. Vascular patterning defects associated with expression of activated Notch4 in embryonic endothelium. PNAS. 98:5643-5648.

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Second Series of Experiments

[0232] VEGF Initiates angiogenesis Via an Activation of Notch Signaling

[0233] 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 MT1-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.

[0234] 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 morphogenetic 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 (Notch1C) is released by presenilin/y-secretase, translocates to the nucleus, interacts with the transcriptional repressor CSL (CBF1/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

[0235] 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.

[0236] 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 Jagged1 and Notch1 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 (N1ECDFc) to inhibit Notch signaling (FIG. 22D). Western blotting analysis of conditioned medium of Ad-N1ECDFc-transduced HUVEC (Ad-N1ECDFc-HUVEC) demonstrated that N1ECDFc was expressed and secreted well (FIG. 22E). By using a co-culture assay, in which Bosc cells expressing Notch ligands (either J1, D11 or D14) activated Notch signaling in HeLa cells expressing Notch1 compared to control Bosc cells, we determined inhibition of Notch signaling with transfection of a N1ECDFc-expression plasmid (FIG. 22F). Then, we examined whether N1ECDFc inhibited activation of Notch signaling by transduced VEGF in HUVEC (FIG. 22G). Co-transduction of Ad-N1ECDFc 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 Notch1 (Ad-N1IC) also induced HUVEC budding (FIG. 23A & 27B). Since both VEGF and Notch signaling induce HUVEC budding, we examined whether N1ECDFc inhibited VEGF-induced HUVEC budding (FIG. 22H-I). Budding of Ad-VEGF-HUVEC was clearly inhibited by co-transduction of Ad-N1ECDFc. Neither Ad-LacZ or Ad-N1ECDFc-transduced HUVEC formed buds (FIG. 22H). N1ECDFc inhibited VEGF-induced HUVEC budding without affecting cell number (FIG. 22I). Transduced-N1ECDFc did not clearly alter proliferation of HUVEC, while that of Ad-N1IC-transduced HUVEC was inhibited in a dose-dependent manner (FIG. 28A), consistent with the inhibitory efficacy of Notch signaling against endothelial proliferation (13).

[0237] To test whether Notch signaling is down-stream of VEGF, we evaluated three distinct inhibitors for receptor tyrosine kinases, including VEGFR on N1IC-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-N1IC-HUVEC, while SU5416 clearly inhibited it (FIG. 23A-B). SU5416 selectively inhibited budding of Ad-N1IC-HUVEC with less reduction of viability at lower concentrations (FIG. 23C). Since Taylor et al. reported that Notch down-regulated Flk1/KDR/VEGFR2 expression (14), it was unlikely that Notch co-operated with Flk1 to promote budding. Thus, we examined whether activation of Notch signaling affected Flt1/VEGFR1 expression in HUVEC, because SU5416 inhibits both Flt1 and Flk1 kinase activity (15). RT-PCR analysis demonstrated that expression of Flt1 mRNA was up-regulated in Ad-N1IC-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 Flt1 protein was up-regulated in Ad-N1IC-HUVEC (FIG. 23E). Thus, we examined whether PlGF, which is a selective ligand for Flt1, promoted budding of HUVEC in which Flt1 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 Flt1 expression using small interfering RNA (siRNA) for Flt1 inhibited budding of Ad-N1IC-HUVEC (FIG. 23J), transfection of which selectively decreased expression of Flt1 mRNA (FIG. 23H) and that of Flt1 protein (FIG. 23I). Although reduction of Flk1 expression with Flk1 siRNA also inhibited budding of Ad-N1IC-HUVEC (FIG. 30B), the inhibitory efficacy of Flk1 siRNA was less than that of Flt1 siRNA (FIG. 23J). Effects of Flk1 siRNA were more effective on budding of Ad-VEGF-HUVEC than that of Ad-N1IC-HUVEC (FIG. 30B-C). Transfection with Fit' siRNA inhibited budding of both Ad-N1IC- and Ad-VEGF-HUVEC to a similar extent (data not shown).

[0238] 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.

[0239] 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-N1ECDFc 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-N1IC-HUVEC, but expression of MMP2 mRNA was decreased in Ad-N1IC-HUVEC (FIG. 24C). Since induction of MMP2 activity was not detected in gelatin zymography (FIG. 24E), this result was a likely consequence. While expression of MT1-MMP, which is able to activate MMP2 at the cell surface (17), was up-regulated at both the transcript and protein levels in Ad-N1IC-HUVEC (FIG. 24D). As VEGF can regulate both gelatinase and MT1-MMP expression (16), RT-PCR analysis demonstrated that both MMP9 and MT1-MMP were up-regulated in Ad-VEGF-HUVEC, compared to Ad-LacZ-HUVEC and this induction was inhibited with co-transduction of Ad-N1ECDFc (FIG. 24E). Ad-N1ECDFc infection alone did not affect expression of either MMP9 or MT1-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 MT1-MMP (18). GM6001 clearly decreased budding of Ad-N1IC-HUVEC on both collagen (FIG. 31A-B) and fibrin gel (data not shown).

[0240] 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 N1ECDFc, 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/N1ECDFc (FIG. 258).

[0241] 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 N1ECDFc, 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 N1ECDFc.

[0242] Flk1 is a major positive signal transducer for angiogenesis through its strong tyrosine kinase activity in the embryo, while Flt1 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 PlGF2 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 PlGF/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 (Rip1Tag2 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 MT1-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.

[0243] References cited in Second Series of Experiments

[0244] 1. Artavanis-Tsakonas S, Rand M D, Lake R J. Notch Signaling: Cell Fate Control and Signal Integration in Development. Science 1999;284(5415):770-776.

[0245] 2. Shawber C J, J. K. Notch function in the vasculature: insights from zebrafish, mouse and man. Bioessays. 2004;26(3):225-34.

[0246] 10

[0247] 3. 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.

[0248] 4. Lawson N D, Vogel A M, B M. 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.

[0249] 5. Liu Z J, Shirakawa T, Li Y, Soma A, Oka M, Dotto G P, et al. Regulation of Notch1 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.

[0250] 6. Gale N W, Dominguez M G, Noguera I, Pan L, Hughes V, Valenzuela D M, et al. Haploinsufficiency of delta-like 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.

[0251] 7. Montesano R, L. O. Phorbol esters induce angiogenesis in vitro from large-vessel endothelial cells. J Cell Physiol. 1987;130(2):284-91.

[0252] 8. Jarriault S, Brou C, Logeat F, Schroeter E H, Kopan R, A. I. Signalling downstream of activated mammalian Notch. Nature. 1995;377(6547):355-8.

[0253] 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.

[0254] 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.

[0255] 11. Koolwijk P, van Erck M G, de Vree W J, Vermeer M A, Weich H A, 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.

[0256] 12. Das I, Craig C, Funahashi Y, Jung K M, Kim T W, Byers R, et al. Notch oncoproteins depend on gamma-secretase/presenilin activity for processing and function. J Biol Chem 2004;279(29):30771-80.

[0257] 13. Noseda M, Chang L, McLean G, Grim J E, Clurman B E, Smith L L, et al. Notch activation induces endothelial cell cycle arrest and participates in contact inhibition: role of p21Cip1 repression. Mol Cell Biol 2004;24(20):8813-22.

[0258] 14. Taylor K L, Henderson A M, C C. 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.

[0259] 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.

[0260] 16. Pepper M S. Role of the matrix metalloproteinase and plasminogen activator-plasmin systems in angiogenesis. Arterioscler Thromb Vasc Biol 2001;21(7):1104-17.

[0261] 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.

[0262] 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.

[0263] 19. Funahashi Y, Wakabayashi T, Semba T, Sonoda J, Kitoh K, K. Y. Establishment of a quantitative mouse dorsal air sac model and its application to evaluate a , new angiogenesis inhibitor. Oncol Res. 1999;11(7):319-29.

[0264] 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.

[0265] 21. Autiero M, Waltenberger J, Communi D, Kranz A, Moons L, Lambrechts D, et al. Role of PlGF in the intra- and intermolecular cross talk between the VEGF receptors Flt1 and Flk1. Nat Med 2003;9(7):936-43.

[0266] 22. Leong K G, Hu X L L, Noseda M, Larrivee B, Hull C, Hood L, et al. Activated Notch4 inhibits angiogenesis: role of beta 1-integrin activation. Mol Cell Biol 2002;22(8):2830-41.

[0267] 23. Bergers G, Brekken R, McMahon G, Vu T H, Itoh T, Tamaki K, et al. Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat Cell Biol 2000;2(10):737-44.

Sequence CWU 1

1

1411433PRTRattus norvegius 1Met Pro Arg Leu Leu Ala Pro Leu Leu Cys Leu Thr Leu Leu Pro Ala 1 5 10 15 Leu Ala Ala Arg Gly Leu Arg Cys Ser Gln Pro Ser Gly Thr Cys Leu 20 25 30 Asn Gly Gly Arg Cys Glu Val Ala Asn Gly Thr Glu Ala Cys Val Cys 35 40 45 Ser Gly Ala Phe Val Gly Gln Arg Cys Gln Asp Pro Ser Pro Cys Leu 50 55 60 Ser Thr Pro Cys Lys Asn Ala Gly Thr Cys Tyr Val Val Asp His Gly 65 70 75 80 Gly Ile Val Asp Tyr Ala Cys Ser Cys Pro Leu Gly Phe Ser Gly Pro 85 90 95 Leu Cys Leu Thr Pro Leu Ala Asn Ala Cys Leu Ala Asn Pro Cys Arg 100 105 110 Asn Gly Gly Thr Cys Asp Leu Leu Thr Leu Thr Glu Tyr Lys Cys Arg 115 120 125 Cys Pro Pro Gly Trp Ser Gly Lys Ser Cys Gln Gln Ala Asp Pro Cys 130 135 140 Ala Ser Asn Pro Cys Ala Asn Gly Gly Gln Cys Leu Pro Phe Glu Ser 145 150 155 160 Ser Tyr Ile Cys Gly Cys Pro Pro Gly Phe His Gly Pro Thr Cys Arg 165 170 175 Gln Asp Val Asn Glu Cys Ser Gln Asn Pro Gly Leu Cys Arg His Gly 180 185 190 Gly Thr Cys His Asn Glu Ile Gly Ser Tyr Arg Cys Ala Cys Arg Ala 195 200 205 Thr His Thr Gly Pro His Cys Glu Leu Pro Tyr Val Pro Cys Ser Pro 210 215 220 Ser Pro Cys Gln Asn Gly Gly Thr Cys Arg Pro Thr Gly Asp Thr Thr 225 230 235 240 His Glu Cys Ala Cys Leu Pro Gly Phe Ala Gly Gln Asn Cys Glu Glu 245 250 255 Asn Val Asp Asp Cys Pro Gly Asn Asn Cys Lys Asn Gly Gly Ala Cys 260 265 270 Val Asp Gly Val Asn Thr Tyr Asn Cys Arg Cys Pro Pro Glu Trp Thr 275 280 285 Gly Gln Tyr Cys Thr Glu Asp Val Asp Glu Cys Gln Leu Met Pro Asn 290 295 300 Ala Cys Gln Asn Ala Gly Thr Cys His Asn Ser His Gly Gly Tyr Asn 305 310 315 320 Cys Val Cys Val Asn Gly Trp Thr Gly Glu Asp Cys Ser Asp Asn Ile 325 330 335 Asp Asp Cys Ala Ser Ala Ala Cys Phe Gln Gly Ala Thr Cys His Asp 340 345 350 Arg Val Ala Ser Phe Tyr Cys Glu Cys Pro His Gly Arg Thr Gly Leu 355 360 365 Leu Cys His Leu Asn Asp Ala Cys Ile Ser Asn Pro Cys Asn Glu Gly 370 375 380 Ser Asn Cys Asp Thr Asn Pro Val Asn Gly Lys Ala Ile Cys Thr Cys 385 390 395 400 Pro Arg Gly Tyr Thr Gly Pro Ala Cys Ser Gln Asp Val Asp Glu Cys 405 410 415 Ala Leu Gly Ala Asn Pro Cys Glu His Ala Gly Lys Cys Leu Asn Thr 420 425 430 Leu Gly Ser Phe Glu Cys Gln Cys Leu Gln Gly Tyr Thr Gly Pro Arg 435 440 445 Cys Glu Ile Asp Val Asn Glu Cys Ile Ser Asn Pro Cys Gln Asn Asp 450 455 460 Ala Thr Cys Leu Asp Gln Ile Gly Glu Phe Gln Cys Ile Cys Met Pro 465 470 475 480 Gly Tyr Glu Gly Val Tyr Cys Glu Ile Asn Thr Asp Glu Cys Ala Ser 485 490 495 Ser Pro Cys Leu His Asn Gly Arg Cys Val Asp Lys Ile Asn Glu Phe 500 505 510 Leu Cys Gln Cys Pro Lys Gly Phe Ser Gly His Leu Cys Gln Tyr Asp 515 520 525 Val Asp Glu Cys Ala Ser Thr Pro Cys Lys Asn Gly Ala Lys Cys Leu 530 535 540 Asp Gly Pro Asn Thr Tyr Thr Cys Val Cys Thr Glu Gly Tyr Thr Gly 545 550 555 560 Thr His Cys Glu Val Asp Ile Asp Glu Cys Asp Pro Asp Pro Cys His 565 570 575 Ile Gly Leu Cys Lys Asp Gly Val Ala Thr Phe Thr Cys Leu Cys Gln 580 585 590 Pro Gly Tyr Thr Gly His His Cys Glu Thr Asn Ile Asn Glu Cys His 595 600 605 Ser Gln Pro Cys Arg His Gly Gly Thr Cys Gln Asp Arg Asp Asn Tyr 610 615 620 Tyr Leu Cys Leu Cys Leu Lys Gly Thr Thr Gly Pro Asn Cys Glu Ile 625 630 635 640 Asn Leu Asp Asp Cys Ala Ser Asn Pro Cys Asp Ser Gly Thr Cys Leu 645 650 655 Asp Lys Ile Asp Gly Tyr Glu Cys Ala Cys Glu Pro Gly Tyr Thr Gly 660 665 670 Ser Met Cys Asn Val Asn Ile Asp Glu Cys Ala Gly Ser Pro Cys His 675 680 685 Asn Gly Gly Thr Cys Glu Asp Gly Ile Ala Gly Phe Thr Cys Arg Cys 690 695 700 Pro Glu Gly Tyr His Asp Pro Thr Cys Leu Ser Glu Val Asn Glu Cys 705 710 715 720 Asn Ser Asn Pro Cys Ile His Gly Ala Cys Arg Asp Gly Leu Asn Gly 725 730 735 Tyr Lys Cys Asp Cys Ala Pro Gly Trp Ser Gly Thr Asn Cys Asp Ile 740 745 750 Asn Asn Asn Glu Cys Glu Ser Asn Pro Cys Val Asn Gly Gly Thr Cys 755 760 765 Lys Asp Met Thr Ser Gly Tyr Val Cys Thr Cys Arg Glu Gly Phe Ser 770 775 780 Gly Pro Asn Cys Gln Thr Asn Ile Asn Glu Cys Ala Ser Asn Pro Cys 785 790 795 800 Leu Asn Gln Gly Thr Cys Ile Asp Asp Val Ala Gly Tyr Lys Cys Asn 805 810 815 Cys Pro Leu Pro Tyr Thr Gly Ala Thr Cys Glu Val Val Leu Ala Pro 820 825 830 Cys Ala Thr Ser Pro Cys Lys Asn Ser Gly Val Cys Lys Glu Ser Glu 835 840 845 Asp Tyr Glu Ser Phe Ser Cys Val Cys Pro Thr Gly Trp Gln Gly Gln 850 855 860 Thr Cys Glu Ile Asp Ile Asn Glu Cys Val Lys Ser Pro Cys Arg His 865 870 875 880 Gly Ala Ser Cys Gln Asn Thr Asn Gly Ser Tyr Arg Cys Leu Cys Gln 885 890 895 Ala Gly Tyr Thr Gly Arg Asn Cys Glu Ser Asp Ile Asp Asp Cys Arg 900 905 910 Pro Asn Pro Cys His Asn Gly Gly Ser Cys Thr Asp Gly Val Asn Ala 915 920 925 Ala Phe Cys Asp Cys Leu Pro Gly Phe Gln Gly Ala Phe Cys Glu Glu 930 935 940 Asp Ile Asn Glu Cys Ala Thr Asn Pro Cys Gln Asn Gly Ala Asn Cys 945 950 955 960 Thr Asp Cys Val Asp Ser Tyr Thr Cys Thr Cys Pro Thr Gly Phe Asn 965 970 975 Gly Ile His Cys Glu Asn Asn Thr Pro Asp Cys Thr Glu Ser Ser Cys 980 985 990 Phe Asn Gly Gly Thr Cys Val Asp Gly Ile Asn Ser Phe Thr Cys Leu 995 1000 1005 Cys Pro Pro Gly Phe Thr Gly Ser Tyr Cys Gln Tyr Asp Val Asn 1010 1015 1020 Glu Cys Asp Ser Arg Pro Cys Leu His Gly Gly Thr Cys Gln Asp 1025 1030 1035 Ser Tyr Gly Thr Tyr Lys Cys Thr Cys Pro Gln Gly Tyr Thr Gly 1040 1045 1050 Leu Asn Cys Gln Asn Leu Val Arg Trp Cys Asp Ser Ala Pro Cys 1055 1060 1065 Lys Asn Gly Gly Lys Cys Trp Gln Thr Asn Thr Gln Tyr His Cys 1070 1075 1080 Glu Cys Arg Ser Gly Trp Thr Gly Phe Asn Cys Asp Val Leu Ser 1085 1090 1095 Val Ser Cys Glu Val Ala Ala Gln Lys Arg Gly Ile Asp Val Thr 1100 1105 1110 Leu Leu Cys Gln His Gly Gly Leu Cys Val Asp Glu Glu Asp Lys 1115 1120 1125 His Tyr Cys His Cys Gln Ala Gly Tyr Thr Gly Ser Tyr Cys Glu 1130 1135 1140 Asp Glu Val Asp Glu Cys Ser Pro Asn Pro Cys Gln Asn Gly Ala 1145 1150 1155 Thr Cys Thr Asp Tyr Leu Gly Gly Phe Ser Cys Lys Cys Val Ala 1160 1165 1170 Gly Tyr His Gly Ser Asn Cys Ser Glu Glu Ile Asn Glu Cys Leu 1175 1180 1185 Ser Gln Pro Cys Gln Asn Gly Gly Thr Cys Ile Asp Leu Thr Asn 1190 1195 1200 Thr Tyr Lys Cys Ser Cys Pro Arg Gly Thr Gln Gly Val His Cys 1205 1210 1215 Glu Ile Asn Val Asp Asp Cys His Pro Pro Leu Asp Pro Ala Ser 1220 1225 1230 Arg Ser Pro Lys Cys Phe Asn Asn Gly Thr Cys Val Asp Gln Val 1235 1240 1245 Gly Gly Tyr Thr Cys Thr Cys Pro Pro Gly Phe Val Gly Glu Arg 1250 1255 1260 Cys Glu Gly Asp Val Asn Glu Cys Leu Ser Asn Pro Cys Asp Pro 1265 1270 1275 Arg Gly Thr Gln Asn Cys Val Gln Arg Val Asn Asp Phe His Cys 1280 1285 1290 Glu Cys Arg Ala Gly His Thr Gly Arg Arg Cys Glu Ser Val Ile 1295 1300 1305 Asn Gly Cys Arg Gly Lys Pro Cys Arg Asn Gly Gly Val Cys Ala 1310 1315 1320 Val Ala Ser Asn Thr Ala Arg Gly Phe Ile Cys Arg Cys Pro Ala 1325 1330 1335 Arg Phe Glu Gly Ala Thr Cys Glu Asn Asp Ala Arg Thr Cys Gly 1340 1345 1350 Ser Leu Arg Cys Leu Asn Gly Gly Thr Cys Ile Ser Gly Pro Arg 1355 1360 1365 Ser Pro Thr Cys Leu Cys Leu Gly Ser Phe Thr Gly Pro Glu Cys 1370 1375 1380 Gln Phe Pro Ala Ser Ser Pro Cys Val Gly Ser Asn Pro Cys Tyr 1385 1390 1395 Asn Gln Gly Thr Cys Glu Pro Thr Ser Glu Ser Pro Phe Tyr Arg 1400 1405 1410 Cys Leu Cys Pro Ala Lys Phe Asn Gly Leu Leu Cys His Ile Leu 1415 1420 1425 Asp Tyr Ser Phe Thr 1430 25PRTRattus norvegius 2Asp Leu Gly Pro Gly 1 5 31419PRTRattus norvegius 3Met Pro Ala Leu Arg Pro Ala Ala Leu Arg Ala Leu Leu Trp Leu Trp 1 5 10 15 Leu Cys Gly Ala Gly Pro Ala His Ala Leu Gln Cys Arg Gly Gly Gln 20 25 30 Glu Pro Cys Val Asn Glu Gly Thr Cys Val Thr Tyr His Asn Gly Thr 35 40 45 Gly Tyr Cys Arg Cys Pro Glu Gly Phe Leu Gly Glu Tyr Cys Gln His 50 55 60 Arg Asp Pro Cys Glu Lys Asn Arg Cys Gln Asn Gly Gly Thr Cys Val 65 70 75 80 Thr Gln Ala Met Leu Gly Lys Ala Thr Cys Arg Cys Ala Pro Gly Phe 85 90 95 Thr Gly Glu Asp Cys Gln Tyr Ser Thr Ser His Pro Cys Phe Val Ser 100 105 110 Arg Pro Cys Gln Asn Gly Gly Thr Cys His Met Leu Ser Trp Asp Thr 115 120 125 Tyr Glu Cys Thr Cys Gln Val Gly Phe Thr Gly Lys Gln Cys Gln Trp 130 135 140 Thr Asp Val Cys Leu Ser His Pro Cys Glu Asn Gly Ser Thr Cys Ser 145 150 155 160 Ser Val Ala Asn Gln Phe Ser Cys Arg Cys Pro Ala Gly Ile Thr Gly 165 170 175 Gln Lys Cys Asp Ala Asp Ile Asn Glu Cys Asp Ile Pro Gly Arg Cys 180 185 190 Gln His Gly Gly Thr Cys Leu Asn Leu Pro Gly Ser Tyr Arg Cys Gln 195 200 205 Cys Pro Gln Arg Phe Thr Gly Gln His Cys Asp Ser Pro Tyr Val Pro 210 215 220 Cys Ala Pro Ser Pro Cys Val Asn Gly Gly Thr Cys Arg Gln Thr Gly 225 230 235 240 Asp Phe Thr Ser Glu Cys His Cys Leu Pro Gly Phe Glu Gly Ser Asn 245 250 255 Cys Glu Arg Asn Ile Asp Asp Cys Pro Asn His Lys Cys Gln Asn Gly 260 265 270 Gly Val Cys Val Asp Gly Val Asn Thr Tyr Asn Cys Arg Cys Pro Pro 275 280 285 Gln Trp Thr Gly Gln Phe Cys Thr Glu Asp Val Asp Glu Cys Leu Leu 290 295 300 Gln Pro Asn Ala Cys Gln Asn Gly Gly Thr Cys Thr Asn Arg Asn Gly 305 310 315 320 Gly Tyr Gly Cys Val Cys Val Asn Gly Trp Ser Gly Asp Asp Cys Ser 325 330 335 Glu Asn Ile Asp Asp Cys Ala Phe Ala Ser Cys Thr Pro Gly Ser Thr 340 345 350 Cys Ile Asp Arg Val Ala Ser Phe Ser Cys Leu Cys Pro Glu Gly Lys 355 360 365 Ala Gly Leu Leu Cys His Leu Asp Asp Ala Cys Ile Ser Asn Pro Cys 370 375 380 His Lys Gly Ala Leu Cys Asp Thr Asn Pro Leu Asn Gly Gln Tyr Ile 385 390 395 400 Cys Thr Cys Pro Gln Ala Tyr Lys Gly Ala Asp Cys Thr Glu Asp Val 405 410 415 Asp Glu Cys Ala Met Ala Asn Ser Asn Pro Cys Glu His Ala Gly Lys 420 425 430 Cys Val Asn Thr Asp Gly Ala Phe His Cys Glu Cys Leu Lys Gly Tyr 435 440 445 Ala Gly Pro Arg Cys Glu Met Asp Ile Asn Glu Cys His Ser Asp Pro 450 455 460 Cys Gln Asn Asp Ala Thr Cys Leu Asp Lys Ile Gly Gly Phe Thr Cys 465 470 475 480 Leu Cys Met Pro Gly Phe Lys Gly Val His Cys Glu Leu Glu Val Asn 485 490 495 Glu Cys Gln Ser Asn Pro Cys Val Asn Asn Gly Gln Cys Val Asp Lys 500 505 510 Val Asn Arg Phe Gln Cys Leu Cys Pro Pro Gly Phe Thr Gly Pro Val 515 520 525 Cys Gln Ile Asp Ile Asp Asp Cys Ser Ser Thr Pro Cys Leu Asn Gly 530 535 540 Ala Lys Cys Ile Asp His Pro Asn Gly Tyr Glu Cys Gln Cys Ala Thr 545 550 555 560 Gly Phe Thr Gly Thr Leu Cys Asp Glu Asn Ile Asp Asn Cys Asp Pro 565 570 575 Asp Pro Cys His His Gly Gln Cys Gln Asp Gly Ile Asp Ser Tyr Thr 580 585 590 Cys Ile Cys Asn Pro Gly Tyr Met Gly Ala Ile Cys Ser Asp Gln Ile 595 600 605 Asp Glu Cys Tyr Ser Ser Pro Cys Leu Asn Asp Gly Arg Cys Ile Asp 610 615 620 Leu Val Asn Gly Tyr Gln Cys Asn Cys Gln Pro Gly Thr Ser Gly Leu 625 630 635 640 Asn Cys Glu Ile Asn Phe Asp Asp Cys Ala Ser Asn Pro Cys Leu His 645 650 655 Gly Ala Cys Val Asp Gly Ile Asn Arg Tyr Ser Cys Val Cys Ser Pro 660 665 670 Gly Phe Thr Gly Gln Arg Cys Asn Ile Asp Ile Asp Glu Cys Ala Ser 675 680 685 Asn Pro Cys Arg Lys Asp Ala Thr Cys Ile Asn Asp Val Asn Gly Phe 690 695 700 Arg Cys Met Cys Pro Glu Gly Pro His His Pro Ser Cys Tyr Ser Gln 705 710 715 720 Val Asn Glu Cys Leu Ser Ser Pro Cys Ile His Gly Asn Cys Thr Gly 725 730 735 Gly Leu Ser Gly Tyr Lys Cys Leu Cys Asp Ala Gly Trp Val Gly Ile 740 745 750 Asn Cys Glu Val Asp Lys Asn Glu Cys Leu Ser Asn Pro Cys Gln Asn 755 760 765 Gly Gly Thr Cys Asn Asn Leu Val Asn Gly Tyr Arg Cys Thr Cys Lys 770 775 780 Lys Gly Phe Lys Gly Tyr Asn Cys Gln Val Asn Ile Asp Glu Cys Ala 785 790 795 800 Ser Asn Pro Cys Leu Asn Gln Gly Thr Cys Leu Asp Asp Val Ser Gly 805 810 815 Tyr Thr Cys His Cys Met Leu Pro Tyr Thr Gly Lys Asn Cys Gln Thr 820 825 830 Val Leu Ala Pro Cys Ser Pro Asn Pro

Cys Glu Asn Ala Ala Val Cys 835 840 845 Lys Glu Ala Pro Asn Phe Glu Ser Phe Thr Cys Leu Cys Ala Pro Gly 850 855 860 Trp Gln Gly Gln Arg Cys Thr Val Asp Val Asp Glu Cys Val Ser Lys 865 870 875 880 Pro Cys Met Asn Asn Gly Ile Cys His Asn Thr Gln Gly Ser Tyr Met 885 890 895 Cys Glu Cys Pro Pro Gly Phe Ser Gly Met Asp Cys Glu Glu Asp Ile 900 905 910 Asn Asp Cys Leu Ala Asn Pro Cys Gln Asn Gly Gly Ser Cys Val Asp 915 920 925 Lys Val Asn Thr Phe Ser Cys Leu Cys Leu Pro Gly Phe Val Gly Asp 930 935 940 Lys Cys Gln Thr Asp Met Asn Glu Cys Leu Ser Glu Pro Cys Lys Asn 945 950 955 960 Gly Gly Thr Cys Ser Asp Tyr Val Asn Ser Tyr Thr Cys Thr Cys Pro 965 970 975 Ala Gly Phe His Gly Val His Cys Glu Asn Asn Ile Asp Glu Cys Thr 980 985 990 Glu Ser Ser Cys Phe Asn Gly Gly Thr Cys Val Asp Gly Ile Asn Ser 995 1000 1005 Phe Ser Cys Leu Cys Pro Val Gly Phe Thr Gly Pro Phe Cys Leu 1010 1015 1020 His Asp Ile Asn Glu Cys Ser Ser Asn Pro Cys Leu Asn Ser Gly 1025 1030 1035 Thr Cys Val Asp Gly Leu Gly Thr Tyr Arg Cys Thr Cys Pro Leu 1040 1045 1050 Gly Tyr Thr Gly Lys Asn Cys Gln Thr Leu Val Asn Leu Cys Ser 1055 1060 1065 Pro Ser Pro Cys Lys Asn Lys Gly Thr Cys Ala Gln Glu Lys Ala 1070 1075 1080 Arg Pro Arg Cys Leu Cys Pro Pro Gly Trp Asp Gly Ala Tyr Cys 1085 1090 1095 Asp Val Leu Asn Val Ser Cys Lys Ala Ala Ala Leu Gln Lys Gly 1100 1105 1110 Val Pro Val Glu His Leu Cys Gln His Ser Gly Ile Cys Ile Asn 1115 1120 1125 Ala Gly Asn Thr His His Cys Gln Cys Pro Leu Gly Tyr Thr Gly 1130 1135 1140 Ser Tyr Cys Glu Glu Gln Leu Asp Glu Cys Ala Ser Asn Pro Cys 1145 1150 1155 Gln His Gly Ala Thr Cys Ser Asp Phe Ile Gly Gly Tyr Arg Cys 1160 1165 1170 Glu Cys Val Pro Gly Tyr Gln Gly Val Asn Cys Glu Tyr Glu Val 1175 1180 1185 Asp Glu Cys Gln Asn Gln Pro Cys Gln Asn Gly Gly Thr Cys Ile 1190 1195 1200 Asp Leu Val Asn His Phe Lys Cys Ser Cys Pro Pro Gly Thr Arg 1205 1210 1215 Gly Leu Leu Cys Glu Glu Asn Ile Asp Asp Cys Ala Gly Ala Pro 1220 1225 1230 His Cys Leu Asn Gly Gly Gln Cys Val Asp Arg Ile Gly Gly Tyr 1235 1240 1245 Ser Cys Arg Cys Leu Pro Gly Phe Ala Gly Glu Arg Cys Glu Gly 1250 1255 1260 Asp Ile Asn Glu Cys Leu Ser Asn Pro Cys Ser Ser Glu Gly Ser 1265 1270 1275 Leu Asp Cys Ile Gln Leu Lys Asn Asn Tyr Gln Cys Val Cys Arg 1280 1285 1290 Ser Ala Phe Thr Gly Arg His Cys Glu Thr Phe Leu Asp Val Cys 1295 1300 1305 Pro Gln Lys Pro Cys Leu Asn Gly Gly Thr Cys Ala Val Ala Ser 1310 1315 1320 Asn Val Pro Asp Gly Phe Ile Cys Arg Cys Pro Pro Gly Phe Ser 1325 1330 1335 Gly Ala Arg Cys Gln Ser Ser Cys Gly Gln Val Lys Cys Arg Arg 1340 1345 1350 Gly Glu Gln Cys Val His Thr Ala Ser Gly Pro His Cys Phe Cys 1355 1360 1365 Pro Asn His Lys Asp Cys Glu Ser Gly Cys Ala Ser Asn Pro Cys 1370 1375 1380 Gln His Gly Gly Thr Cys Tyr Pro Gln Arg Gln Pro Pro Tyr Tyr 1385 1390 1395 Ser Cys Arg Cys Ser Pro Pro Phe Trp Gly Ser His Cys Glu Ser 1400 1405 1410 Tyr Thr Ala Pro Thr Ser 1415 4 1379PRTMus musculus 4Met Gly Leu Gly Ala Arg Gly Arg Arg Arg Arg Arg Arg Leu Met Ala 1 5 10 15 Leu Pro Pro Pro Pro Pro Pro Met Arg Ala Leu Pro Leu Leu Leu Leu 20 25 30 Leu Ala Gly Leu Gly Ala Ala Ala Pro Pro Cys Leu Asp Gly Ser Pro 35 40 45 Cys Ala Asn Gly Gly Arg Cys Thr His Gln Gln Pro Ser Leu Glu Ala 50 55 60 Ala Cys Leu Cys Leu Pro Gly Trp Val Gly Glu Arg Cys Gln Leu Glu 65 70 75 80 Asp Pro Cys His Ser Gly Pro Cys Ala Gly Arg Gly Val Cys Gln Ser 85 90 95 Ser Val Val Ala Gly Thr Ala Arg Phe Ser Cys Arg Cys Leu Arg Gly 100 105 110 Phe Gln Gly Pro Asp Cys Ser Gln Pro Asp Pro Cys Val Ser Arg Pro 115 120 125 Cys Val His Gly Ala Pro Cys Ser Val Gly Pro Asp Gly Arg Phe Ala 130 135 140 Cys Ala Cys Pro Pro Gly Tyr Gln Gly Gln Ser Cys Gln Ser Asp Ile 145 150 155 160 Asp Glu Cys Arg Ser Gly Thr Thr Cys Arg His Gly Gly Thr Cys Leu 165 170 175 Asn Thr Pro Gly Ser Phe Arg Cys Gln Cys Pro Leu Gly Tyr Thr Gly 180 185 190 Leu Leu Cys Glu Asn Pro Val Val Pro Cys Ala Pro Ser Pro Cys Arg 195 200 205 Asn Gly Gly Thr Cys Arg Gln Ser Ser Asp Val Thr Tyr Asp Cys Ala 210 215 220 Cys Leu Pro Gly Phe Glu Gly Gln Asn Cys Glu Val Asn Val Asp Asp 225 230 235 240 Cys Pro Gly His Arg Cys Leu Asn Gly Gly Thr Cys Val Asp Gly Val 245 250 255 Asn Thr Tyr Asn Cys Gln Cys Pro Pro Glu Trp Thr Gly Gln Phe Cys 260 265 270 Thr Glu Asp Val Asp Glu Cys Gln Leu Gln Pro Asn Ala Cys His Asn 275 280 285 Gly Gly Thr Cys Phe Asn Leu Leu Gly Gly His Ser Cys Val Cys Val 290 295 300 Asn Gly Trp Thr Gly Glu Ser Cys Ser Gln Asn Ile Asp Asp Cys Ala 305 310 315 320 Thr Ala Val Cys Phe His Gly Ala Thr Cys His Asp Arg Val Ala Ser 325 330 335 Phe Tyr Cys Ala Cys Pro Met Gly Lys Thr Gly Leu Leu Cys His Leu 340 345 350 Asp Asp Ala Cys Val Ser Asn Pro Cys His Glu Asp Ala Ile Cys Asp 355 360 365 Thr Asn Pro Val Ser Gly Arg Ala Ile Cys Thr Cys Pro Pro Gly Phe 370 375 380 Thr Gly Gly Ala Cys Asp Gln Asp Val Asp Glu Cys Ser Ile Gly Ala 385 390 395 400 Asn Pro Cys Glu His Leu Gly Arg Cys Val Asn Thr Gln Gly Ser Phe 405 410 415 Leu Cys Gln Cys Gly Arg Gly Tyr Thr Gly Pro Arg Cys Glu Thr Asp 420 425 430 Val Asn Glu Cys Leu Ser Gly Pro Cys Arg Asn Gln Ala Thr Cys Leu 435 440 445 Asp Arg Ile Gly Gln Phe Thr Cys Ile Cys Met Ala Gly Phe Thr Gly 450 455 460 Thr Tyr Cys Glu Val Asp Ile Asp Glu Cys Gln Ser Ser Pro Cys Val 465 470 475 480 Asn Gly Gly Val Cys Lys Asp Arg Val Asn Gly Phe Ser Cys Thr Cys 485 490 495 Pro Ser Gly Phe Ser Gly Ser Met Cys Gln Leu Asp Val Asp Glu Cys 500 505 510 Ala Ser Thr Pro Cys Arg Asn Gly Ala Lys Cys Val Asp Gln Pro Asp 515 520 525 Gly Tyr Glu Cys Arg Cys Ala Glu Gly Phe Glu Gly Thr Leu Cys Glu 530 535 540 Arg Asn Val Asp Asp Cys Ser Pro Asp Pro Cys His His Gly Arg Cys 545 550 555 560 Val Asp Gly Ile Ala Ser Phe Ser Cys Ala Cys Ala Pro Gly Tyr Thr 565 570 575 Gly Ile Arg Cys Glu Ser Gln Val Asp Glu Cys Arg Ser Gln Pro Cys 580 585 590 Arg Tyr Gly Gly Lys Cys Leu Asp Leu Val Asp Lys Tyr Leu Cys Arg 595 600 605 Cys Pro Pro Gly Thr Thr Gly Val Asn Cys Glu Val Asn Ile Asp Asp 610 615 620 Cys Ala Ser Asn Pro Cys Thr Phe Gly Val Cys Arg Asp Gly Ile Asn 625 630 635 640 Arg Tyr Asp Cys Val Cys Gln Pro Gly Phe Thr Gly Pro Leu Cys Asn 645 650 655 Val Glu Ile Asn Glu Cys Ala Ser Ser Pro Cys Gly Glu Gly Gly Ser 660 665 670 Cys Val Asp Gly Glu Asn Gly Phe His Cys Leu Cys Pro Pro Gly Ser 675 680 685 Leu Pro Pro Leu Cys Leu Pro Ala Asn His Pro Cys Ala His Lys Pro 690 695 700 Cys Ser His Gly Val Cys His Asp Ala Pro Gly Gly Phe Arg Cys Val 705 710 715 720 Cys Glu Pro Gly Trp Ser Gly Pro Arg Cys Ser Gln Ser Leu Ala Pro 725 730 735 Asp Ala Cys Glu Ser Gln Pro Cys Gln Ala Gly Gly Thr Cys Thr Ser 740 745 750 Asp Gly Ile Gly Phe Arg Cys Thr Cys Ala Pro Gly Phe Gln Gly His 755 760 765 Gln Cys Glu Val Leu Ser Pro Cys Thr Pro Ser Leu Cys Glu His Gly 770 775 780 Gly His Cys Glu Ser Asp Pro Asp Arg Leu Thr Val Cys Ser Cys Pro 785 790 795 800 Pro Gly Trp Gln Gly Pro Arg Cys Gln Gln Asp Val Asp Glu Cys Ala 805 810 815 Gly Ala Ser Pro Cys Gly Pro His Gly Thr Cys Thr Asn Leu Pro Gly 820 825 830 Asn Phe Arg Cys Ile Cys His Arg Gly Tyr Thr Gly Pro Phe Cys Asp 835 840 845 Gln Asp Ile Asp Asp Cys Asp Pro Asn Pro Cys Leu His Gly Gly Ser 850 855 860 Cys Gln Asp Gly Val Gly Ser Phe Ser Cys Ser Cys Leu Asp Gly Phe 865 870 875 880 Ala Gly Pro Arg Cys Ala Arg Asp Val Asp Glu Cys Leu Ser Ser Pro 885 890 895 Cys Gly Pro Gly Thr Cys Thr Asp His Val Ala Ser Phe Thr Cys Ala 900 905 910 Cys Pro Pro Gly Tyr Gly Gly Phe His Cys Glu Ile Asp Leu Pro Asp 915 920 925 Cys Ser Pro Ser Ser Cys Phe Asn Gly Gly Thr Cys Val Asp Gly Val 930 935 940 Ser Ser Phe Ser Cys Leu Cys Arg Pro Gly Tyr Thr Gly Thr His Cys 945 950 955 960 Gln Tyr Glu Ala Asp Pro Cys Phe Ser Arg Pro Cys Leu His Gly Gly 965 970 975 Ile Cys Asn Pro Thr His Pro Gly Phe Glu Cys Thr Cys Arg Glu Gly 980 985 990 Phe Thr Gly Ser Gln Cys Gln Asn Pro Val Asp Trp Cys Ser Gln Ala 995 1000 1005 Pro Cys Gln Asn Gly Gly Arg Cys Val Gln Thr Gly Ala Tyr Cys 1010 1015 1020 Ile Cys Pro Pro Gly Trp Ser Gly Arg Leu Cys Asp Ile Gln Ser 1025 1030 1035 Leu Pro Cys Thr Glu Ala Ala Ala Gln Met Gly Val Arg Leu Glu 1040 1045 1050 Gln Leu Cys Gln Glu Gly Gly Lys Cys Ile Asp Lys Gly Arg Ser 1055 1060 1065 His Tyr Cys Val Cys Pro Glu Gly Arg Thr Gly Ser His Cys Glu 1070 1075 1080 His Glu Val Asp Pro Cys Thr Ala Gln Pro Cys Gln His Gly Gly 1085 1090 1095 Thr Cys Arg Gly Tyr Met Gly Gly Tyr Val Cys Glu Cys Pro Ala 1100 1105 1110 Gly Tyr Ala Gly Asp Ser Cys Glu Asp Asn Ile Asp Glu Cys Ala 1115 1120 1125 Ser Gln Pro Cys Gln Asn Gly Gly Ser Cys Ile Asp Leu Val Ala 1130 1135 1140 Arg Tyr Leu Cys Ser Cys Pro Pro Gly Thr Leu Gly Val Leu Cys 1145 1150 1155 Glu Ile Asn Glu Asp Asp Cys Asp Leu Gly Pro Ser Leu Asp Ser 1160 1165 1170 Gly Val Gln Cys Leu His Asn Gly Thr Cys Val Asp Leu Val Gly 1175 1180 1185 Gly Phe Arg Cys Asn Cys Pro Pro Gly Tyr Thr Gly Leu His Cys 1190 1195 1200 Glu Ala Asp Ile Asn Glu Cys Arg Pro Gly Ala Cys His Ala Ala 1205 1210 1215 His Thr Arg Asp Cys Leu Gln Asp Pro Gly Gly His Phe Arg Cys 1220 1225 1230 Val Cys His Pro Gly Phe Thr Gly Pro Arg Cys Gln Ile Ala Leu 1235 1240 1245 Ser Pro Cys Glu Ser Gln Pro Cys Gln His Gly Gly Gln Cys Arg 1250 1255 1260 His Ser Leu Gly Arg Gly Gly Gly Leu Thr Phe Thr Cys His Cys 1265 1270 1275 Val Pro Pro Phe Trp Gly Leu Arg Cys Glu Arg Val Ala Arg Ser 1280 1285 1290 Cys Arg Glu Leu Gln Cys Pro Val Gly Ile Pro Cys Gln Gln Thr 1295 1300 1305 Ala Arg Gly Pro Arg Cys Ala Cys Pro Pro Gly Leu Ser Gly Pro 1310 1315 1320 Ser Cys Arg Val Ser Arg Ala Ser Pro Ser Gly Ala Thr Asn Ala 1325 1330 1335 Ser Cys Ala Ser Ala Pro Cys Leu His Gly Gly Ser Cys Leu Pro 1340 1345 1350 Val Gln Ser Val Pro Phe Phe Arg Cys Val Cys Ala Pro Gly Trp 1355 1360 1365 Gly Gly Pro Arg Cys Glu Thr Pro Ser Ala Ala 1370 1375 5 1170PRTMus musculus 5Met Gln Pro Gln Leu Leu Leu Leu Leu Leu Leu Pro Leu Asn Phe Pro 1 5 10 15 Val Ile Leu Thr Arg Glu Leu Leu Cys Gly Gly Ser Pro Glu Pro Cys 20 25 30 Ala Asn Gly Gly Thr Cys Leu Arg Leu Ser Arg Gly Gln Gly Ile Cys 35 40 45 Gln Cys Ala Pro Gly Phe Leu Gly Glu Thr Cys Gln Phe Pro Asp Pro 50 55 60 Cys Arg Asp Thr Gln Leu Cys Lys Asn Gly Gly Ser Cys Gln Ala Leu 65 70 75 80 Leu Pro Thr Pro Pro Ser Ser Arg Ser Pro Thr Ser Pro Leu Thr Pro 85 90 95 His Phe Ser Cys Thr Cys Pro Ser Gly Phe Thr Gly Asp Arg Cys Gln 100 105 110 Thr His Leu Glu Glu Leu Cys Pro Pro Ser Phe Cys Ser Asn Gly Gly 115 120 125 His Cys Tyr Val Gln Ala Ser Gly Arg Pro Gln Cys Ser Cys Glu Pro 130 135 140 Gly Trp Thr Gly Glu Gln Cys Gln Leu Arg Asp Phe Cys Ser Ala Asn 145 150 155 160 Pro Cys Ala Asn Gly Gly Val Cys Leu Ala Thr Tyr Pro Gln Ile Gln 165 170 175 Cys Arg Cys Pro Pro Gly Phe Glu Gly His Thr Cys Glu Arg Asp Ile 180 185 190 Asn Glu Cys Phe Leu Glu Pro Gly Pro Cys Pro Gln Gly Thr Ser Cys 195 200 205 His Asn Thr Leu Gly Ser Tyr Gln Cys Leu Cys Pro Val Gly Gln Glu 210 215 220 Gly Pro Gln Cys Lys Leu Arg Lys Gly Ala Cys Pro Pro Gly Ser Cys 225 230 235 240 Leu Asn Gly Gly Thr Cys Gln Leu Val Pro Glu Gly His Ser Thr Phe 245 250 255 His Leu Cys Leu Cys Pro Pro Gly Phe Thr Gly Leu Asp Cys Glu Met 260 265 270 Asn Pro Asp Asp Cys Val Arg His Gln Cys Gln Asn Gly Ala Thr Cys 275 280 285 Leu Asp Gly Leu Asp Thr Tyr Thr Cys Leu Cys Pro Lys Thr Trp Lys 290

295 300 Gly Trp Asp Cys Ser Glu Asp Ile Asp Glu Cys Glu Ala Arg Gly Pro 305 310 315 320 Pro Arg Cys Arg Asn Gly Gly Thr Cys Gln Asn Thr Ala Gly Ser Phe 325 330 335 His Cys Val Cys Val Ser Gly Trp Gly Gly Ala Gly Cys Glu Glu Asn 340 345 350 Leu Asp Asp Cys Ala Ala Ala Thr Cys Ala Pro Gly Ser Thr Cys Ile 355 360 365 Asp Arg Val Gly Ser Phe Ser Cys Leu Cys Pro Pro Gly Arg Thr Gly 370 375 380 Leu Leu Cys His Leu Glu Asp Met Cys Leu Ser Gln Pro Cys His Val 385 390 395 400 Asn Ala Gln Cys Ser Thr Asn Pro Leu Thr Gly Ser Thr Leu Cys Ile 405 410 415 Cys Gln Pro Gly Tyr Ser Gly Ser Thr Cys His Gln Asp Leu Asp Glu 420 425 430 Cys Gln Met Ala Gln Gln Gly Pro Ser Pro Cys Glu His Gly Gly Ser 435 440 445 Cys Ile Asn Thr Pro Gly Ser Phe Asn Cys Leu Cys Leu Pro Gly Tyr 450 455 460 Thr Gly Ser Arg Cys Glu Ala Asp His Asn Glu Cys Leu Ser Gln Pro 465 470 475 480 Cys His Pro Gly Ser Thr Cys Leu Asp Leu Leu Ala Thr Phe His Cys 485 490 495 Leu Cys Pro Pro Gly Leu Glu Gly Arg Leu Cys Glu Val Glu Val Asn 500 505 510 Glu Cys Thr Ser Asn Pro Cys Leu Asn Gln Ala Ala Cys His Asp Leu 515 520 525 Leu Asn Gly Phe Gln Cys Leu Cys Leu Pro Gly Phe Thr Gly Ala Arg 530 535 540 Cys Glu Lys Asp Met Asp Glu Cys Ser Ser Thr Pro Cys Ala Asn Gly 545 550 555 560 Gly Arg Cys Arg Asp Gln Pro Gly Ala Phe Tyr Cys Glu Cys Leu Pro 565 570 575 Gly Phe Glu Gly Pro His Cys Glu Lys Glu Val Asp Glu Cys Leu Ser 580 585 590 Asp Pro Cys Pro Val Gly Ala Ser Cys Leu Asp Leu Pro Gly Ala Phe 595 600 605 Phe Cys Leu Cys Arg Pro Gly Phe Thr Gly Gln Leu Cys Glu Val Pro 610 615 620 Leu Cys Thr Pro Asn Met Cys Gln Pro Gly Gln Gln Cys Gln Gly Gln 625 630 635 640 Glu His Arg Ala Pro Cys Leu Cys Pro Asp Gly Ser Pro Gly Cys Val 645 650 655 Pro Ala Glu Asp Asn Cys Pro Cys His His Gly His Cys Gln Arg Ser 660 665 670 Leu Cys Val Cys Asp Glu Gly Trp Thr Gly Pro Glu Cys Glu Thr Glu 675 680 685 Leu Gly Gly Cys Ile Ser Thr Pro Cys Ala His Gly Gly Thr Cys His 690 695 700 Pro Gln Pro Ser Gly Tyr Asn Cys Thr Cys Pro Ala Gly Tyr Met Gly 705 710 715 720 Leu Thr Cys Ser Glu Glu Val Thr Ala Cys His Ser Gly Pro Cys Leu 725 730 735 Asn Gly Gly Ser Cys Ser Ile Arg Pro Glu Gly Tyr Ser Cys Thr Cys 740 745 750 Leu Pro Ser His Thr Gly Arg His Cys Gln Thr Ala Val Asp His Cys 755 760 765 Val Ser Ala Ser Cys Leu Asn Gly Gly Thr Cys Val Asn Lys Pro Gly 770 775 780 Thr Phe Phe Cys Leu Cys Ala Thr Gly Phe Gln Gly Leu His Cys Glu 785 790 795 800 Glu Lys Thr Asn Pro Ser Cys Ala Asp Ser Pro Cys Arg Asn Lys Ala 805 810 815 Thr Cys Gln Asp Thr Pro Arg Gly Ala Arg Cys Leu Cys Ser Pro Gly 820 825 830 Tyr Thr Gly Ser Ser Cys Gln Thr Leu Ile Asp Leu Cys Ala Arg Lys 835 840 845 Pro Cys Pro His Thr Ala Arg Cys Leu Gln Ser Gly Pro Ser Phe Gln 850 855 860 Cys Leu Cys Leu Gln Gly Trp Thr Gly Ala Leu Cys Asp Phe Pro Leu 865 870 875 880 Ser Cys Gln Lys Ala Ala Met Ser Gln Gly Ile Glu Ile Ser Gly Leu 885 890 895 Cys Gln Asn Gly Gly Leu Cys Ile Asp Thr Gly Ser Ser Tyr Phe Cys 900 905 910 Arg Cys Pro Pro Gly Phe Gln Gly Lys Leu Cys Gln Asp Asn Val Asn 915 920 925 Pro Cys Glu Pro Asn Pro Cys His His Gly Ser Thr Cys Val Pro Gln 930 935 940 Pro Ser Gly Tyr Val Cys Gln Cys Ala Pro Gly Tyr Glu Gly Gln Asn 945 950 955 960 Cys Ser Lys Val Leu Asp Ala Cys Gln Ser Gln Pro Cys His Asn His 965 970 975 Gly Thr Cys Thr Ser Arg Pro Gly Gly Phe His Cys Ala Cys Pro Pro 980 985 990 Gly Phe Val Gly Leu Arg Cys Glu Gly Asp Val Asp Glu Cys Leu Asp 995 1000 1005 Arg Pro Cys His Pro Ser Gly Thr Ala Ala Cys His Ser Leu Ala 1010 1015 1020 Asn Ala Phe Tyr Cys Gln Cys Leu Pro Gly His Thr Gly Gln Arg 1025 1030 1035 Cys Glu Val Glu Met Asp Leu Cys Gln Ser Gln Pro Cys Ser Asn 1040 1045 1050 Gly Gly Ser Cys Glu Ile Thr Thr Gly Pro Pro Pro Gly Phe Thr 1055 1060 1065 Cys His Cys Pro Lys Gly Phe Glu Gly Pro Thr Cys Ser His Lys 1070 1075 1080 Ala Leu Ser Cys Gly Ile His His Cys His Asn Gly Gly Leu Cys 1085 1090 1095 Leu Pro Ser Pro Lys Pro Gly Ser Pro Pro Leu Cys Ala Cys Leu 1100 1105 1110 Ser Gly Phe Gly Gly Pro Asp Cys Leu Thr Pro Pro Ala Pro Pro 1115 1120 1125 Gly Cys Gly Pro Pro Ser Pro Cys Leu His Asn Gly Thr Cys Thr 1130 1135 1140 Glu Thr Pro Gly Leu Gly Asn Pro Gly Phe Gln Cys Thr Cys Pro 1145 1150 1155 Pro Asp Ser Pro Gly Pro Arg Cys Gln Arg Pro Gly 1160 1165 1170 64293DNARattus norvegius 6atgacaggct tccagggctg ccaggccctg ctgcatctgg ccaaggccgt ggttcgcttg 60agatgctccc agccaagtgg gacctgcctg aatggaggga ggtgcgaagt ggccaacggc 120actgaagcct gtgtctgcag cggagcgttc gtgggccagc gatgccagga ccccagccct 180tgcctcagca caccatgtaa gaatgctgga acgtgctatg ttgtggacca tggcggcatc 240gtggactatg cctgcagttg ccccctgggt ttctctgggc ccctctgcct gacacctctg 300gccaatgcct gcctggccaa cccctgccgc aacgggggga cctgtgacct gctcactctc 360acagaataca agtgccggtg cccgccaggg tggtcaggaa agtcatgtca gcaagccgac 420ccctgtgcct ccaacccctg tgccaatggt ggccagtgcc tgccctttga gtcttcatac 480atctgtggct gcccgcccgg cttccatggc cccacctgca gacaagatgt taacgagtgc 540agccagaacc ctgggttgtg ccgtcatggc ggcacgtgcc acaatgagat tggctcctat 600cgctgtgcct gccgtgccac ccacactggt ccccactgcg agctgcccta cgtgccctgc 660agcccctcac cctgccagaa cggaggcacc tgccgcccta cgggggacac cacccacgag 720tgtgcctgcc tgccaggctt tgctggacag aactgtgaag aaaatgtgga tgactgccca 780ggaaacaact gcaagaacgg gggtgcctgt gtggacggtg tgaataccta caattgccgc 840tgcccaccgg agtggacagg tcagtactgc acagaggatg tggacgagtg tcagctcatg 900cccaacgcct gccagaatgg cggaacctgc cacaactccc acggtggcta caactgcgtg 960tgtgtcaatg gctggactgg tgaggactgc agtgagaaca ttgatgactg tgccagtgcc 1020gcctgttttc agggtgccac ctgccatgac cgtgtggctt ccttctactg cgagtgtcca 1080catgggcgca caggcctgct gtgccacctg aacgatgcgt gtatcagcaa cccctgcaac 1140gagggctcca actgcgacac caaccctgtc aacggcaagg ccatctgcac ttgcccctcg 1200gggtacacgg ggccagcctg cagccaggac gtggatgagt gcgctctagg tgccaacccg 1260tgtgagcacg cgggcaagtg cctcaacaca ctgggctctt tcgagtgtca gtgtctacag 1320ggctacactg ggccccgctg tgagattgat gtcaacgagt gcatctccaa cccatgtcag 1380aatgatgcca cgtgcctgga ccagattggg gagtttcagt gtatatgtat gccaggttat 1440gagggtgtat actgtgagat caacacggac gagtgtgcca gcagcccctg tctacacaat 1500ggccgctgcg tggacaagat caacgagttc ctgtgtcagt gtcccaaagg cttcagcggg 1560cacctgtgcc agtatgacgt ggatgagtgc gccagcacac catgcaagaa cggcgccaag 1620tgcctggatg ggcccaacac ctacacctgc gtgtgcacag aaggttacac ggggacccac 1680tgcgaggtgg acattgacga gtgtgaccct gacccctgtc actatggttt gtgcaaggat 1740ggtgtggcca cctttacctg cctctgccag ccaggctaca caggccatca ctgtgagacc 1800aacattaatg agtgtcacag ccagccgtgc cgccatggcg gcacctgcca ggaccgtgac 1860aactactacc tctgcttatg cctcaagggg accacaggac ccaactgtga gatcaatctg 1920gatgactgtg cgagcaaccc ctgtgactct ggcacgtgtc tggacaagat cgatggctac 1980gagtgtgcgt gcgagccagg ctacacaggg agcatgtgta atgtcaacat tgacgaatgt 2040gcgggcagcc cctgccacaa cgggggcacc tgtgaggatg gcatcgccgg cttcacttgc 2100cgctgccccg agggctacca cgaccctacg tgcctgtctg aggtcaacga gtgcaacagt 2160aacccctgca tccatggagc ttgccgggat ggcctcaatg gatacaaatg tgactgtgcc 2220cctgggtgga gtgggacaaa ctgtgacatc aacaacaatg agtgtgagtc caacccttgt 2280gtcaacggtg gcacctgcaa agacatgacc agtggctacg tatgcacctg ccgagaaggc 2340ttcagtggcc ctaactgcca gaccaacatt aacgaatgtg cttccaaccc ctgcctgaac 2400cagggcacct gcattgatga tgtcgctggg tacaaatgca actgccctct gccctataca 2460ggagccacat gtgaggtggt gttggcccca tgtgccacca gcccctgcaa aaacagtggg 2520gtatgcaagg agtctgagga ctatgagagc ttttcctgtg tctgtcccac aggctggcaa 2580ggtcaaacct gcgagatcga catcaatgag tgtgtgaaaa gcccgtgtcg ccatggtgcc 2640tcttgccaga acaccaatgg cagctaccgc tgcctctgcc aggctggcta cacgggtcgc 2700aactgcgaga gtgacatcga tgactgccga cccaacccat gtcacaacgg gggttcctgc 2760actgacgggg tcaacgcggc cttctgcgac tgcctgcccg gcttccaggg tgccttctgt 2820gaggaggaca tcaacgaatg cgccagcaat ccatgccaaa atggcgccaa ctgcactgac 2880tgcgtggaca gctacacgtg cacctgcccc acgggcttca atggcatcca ttgcgagaac 2940aacacacctg actgtaccga gagctcctgt ttcaatggtg gcacctgtgt ggatggtatc 3000aactccttca cctgtctgtg cccacctggc ttcacgggca gctactgcca gtatgacgtc 3060aatgagtgtg actcacggcc ctgtctgcat ggtggcacct gccaagacag ctatggtacc 3120tataagtgta cctgcccaca gggctacact ggtctcaact gccagaacct tgtgcgctgg 3180tgtgactcag ctccctgcaa gaatggcggc aagtgctggc agaccaacac acagtaccac 3240tgcgagtgcc gcagcggctg gactggcttc aactgcgacg tgctcagtgt gtcctgcgag 3300gtggctgcac agaagcgagg catcgatgtc actctcctat gccagcacgg agggctctgt 3360gtggatgagg aagacaagca ttactgccac tgccaggcag gatacacggg cagctactgt 3420gaggacgagg tggacgagtg ctcacctaat ccctgccaga acggagccac ctgcactgac 3480tatctcggtg gcttttcctg caagtgtgtg gctgggtacc atggctctaa ctgctctgag 3540gagatcaacg agtgcctatc ccaaccctgc cagaatgggg gtacctgcat tgatctgacc 3600aacacctaca agtgctcctg ccccaggggc acacagggtg tacactgtga gatcaacgtc 3660gatgactgcc atcctcccct agaccctgct tcccgaagcc ccaaatgctt caataatggc 3720acctgcgtgg accaggtggg tggctatacc tgcacctgcc cgccaggctt cgtcggggag 3780cggtgcgagg gcgatgtcaa tgagtgtctc tccaacccct gtgacccacg tggcacccag 3840aactgcgtgc agcgtgttaa tgacttccac tgcgagtgcc gggctggcca cactggacgc 3900cgctgtgagt cggtcattaa tggctgcagg ggcaaaccat gcaggaatgg aggtgtctgt 3960gctgtggcct ccaacaccgc ccgtggattc atctgtaggt gccctgcggg cttcgagggt 4020gccacttgtg aaaatgacgc ccgcacttgt ggcagtttgc gctgcctcaa cggtggtacg 4080tgcatctcag gcccacgcag tcccacctgc ctatgcctgg gctccttcac tggccctgaa 4140tgccagttcc cagccagcag cccctgtgtg ggtagcaacc cctgctacaa tcagggcacc 4200tgtgagccca catccgagag ccctttctac cgctgtctat gccctgccaa attcaacggg 4260ctgctgtgcc acatcctgga ctacagcttc aca 429374257DNARattus norvegius 7atgcccgctc tgcgtcccgc cgcgctgcgg gcgctgctgt ggctctggct gtgcggcgcg 60ggccccgcgc acgctttgca gtgtcgaggt ggtcaagagc cctgtgtaaa tgaggggacc 120tgtgttacct accacaacgg cacaggctac tgccgatgtc cagagggctt cttgggagaa 180tattgtcaac atcgagaccc ttgtgagaag aaccgctgtc agaatggtgg tacttgtgtg 240acgcaggcca tgttgggaaa agccacctgt cgatgtgctc cagggttcac aggggaggac 300tgccaatact cgacctctca cccctgtttt gtttcccgcc cctgtcagaa tggaggtacc 360tgccacatgc tcagctggga cacctatgag tgcacctgtc aagttggctt cacaggaaag 420cagtgtcagt ggacagatgt ctgtctgtct catccctgtg aaaatggaag cacctgtagc 480tctgtggcca accagttctc ctgcagatgt cctgcaggca tcacaggcca gaagtgtgac 540gccgacatca atgaatgtga cattccagga cgctgccaac atggtggcac ctgcctcaac 600cttcctgggt cctaccgatg ccaatgccct cagcggttca caggccagca ctgtgacagc 660ccttacgtgc cctgtgcacc ctcaccctgc gtcaatggag gcacctgccg tcagactgga 720gacttcactt ctgaatgcca ttgcctgcca ggctttgaag ggagcaactg cgagcggaat 780atcgacgact gccctaacca caagtgtcag aatggagggg tgtgtgtgga tggcgtcaat 840acttacaact gccgctgccc ccctcagtgg actgggcagt tctgcacaga agacgtggat 900gagtgtctgc tgcagcccaa tgcttgtcag aatggaggca cttgcaccaa ccgcaacgga 960ggctacggct gcgtgtgcgt gaacggctgg agtggggatg actgcagcga gaacatcgat 1020gactgtgcct tcgcttcctg cacgccaggc tccacctgta ttgaccgtgt ggcctccttc 1080tcctgccttt gtccagaggg aaaggcaggg ctcctgtgtc atctggatga tgcctgtatc 1140agcaaccctt gtcacaaggg ggcgctgtgt gataccaacc ccctgaatgg gcagtacatt 1200tgcacctgcc cacaggcgta caagggcgct gactgcacag aagacgtgga tgagtgtgct 1260atggccaaca gtaacccttg tgagcatgca ggaaagtgtg tgaatacaga tggcgccttc 1320cactgcgagt gtctgaaggg ctacgcaggg cctcgctgtg agatggacat caacgagtgt 1380cactcagacc cctgtcagaa cgacgccacc tgcctggata agattggagg cttcacctgt 1440ctctgcatgc cgggtttcaa aggtgtgcat tgtgaactgg aggtgaatga atgccagagc 1500aacccgtgtg taaacaatgg gcagtgtgtg gacaaagtca atcgcttcca gtgtctgtgt 1560ccccctggtt tcacaggacc agtgtgccag atcgacattg acgactgctc cagtactccc 1620tgcctgaatg gggccaagtg catcgatcac ccgaatggct atgaatgcca gtgtgccaca 1680ggattcactg gcacactgtg tgatgagaac atcgacaact gtgacccgga tccttgccac 1740catggccagt gccaggatgg gattgactcc tacacctgca tctgcaaccc cgggtacatg 1800ggagccatct gtagtgacca gattgatgaa tgctacagca gcccctgcct gaatgatgga 1860cgctgcatcg acctggtgaa cggctaccag tgcaactgcc aaccgggtac ctcaggcctt 1920aattgtgaaa ttaattttga tgactgtgcc agcaaccctt gtctgcacgg agcctgtgtg 1980gacggcatca accgttacag ttgtgtgtgc tctccgggat tcacagggca gaggtgcaac 2040atagacattg atgagtgtgc ctccaacccc tgtcgcaagg atgcgacgtg catcaatgac 2100gtgaatggtt tccggtgtat gtgccctgag ggaccacacc atcccagctg ctactcacag 2160gtgaacgagt gtttgagcag tccctgcatc catggaaact gtactggagg tctcagtggc 2220tataagtgcc tctgcgatgc aggctgggtt ggtatcaact gcgaagtgga caaaaatgag 2280tgtctttcta acccgtgcca gaatggaggg acatgtaata acctggtgaa tggctacagg 2340tgtacatgca agaaggggtt caaaggctat aactgccagg tgaacataga tgagtgtgcc 2400tcgaacccgt gtctgaacca agggacctgc ctcgatgacg tcagtggcta cacctgccac 2460tgcatgctgc cttacacagg caagaattgt caaacggtgt tggcgccctg ctcccctaac 2520ccgtgtgaga acgctgcagt ttgtaaagag gcacccaact ttgagagctt cacctgcctg 2580tgtgcccctg gctggcaagg tcagcgctgt acagttgacg ttgatgagtg tgtctccaag 2640ccgtgtatga acaatggcat ctgccataat actcagggca gctacatgtg cgagtgccct 2700cccggcttca gtggtatgga ctgtgaggag gacatcaatg actgccttgc caacccctgc 2760cagaacggag gctcctgtgt ggacaaagtg aacaccttct cctgcctgtg ccttcctggc 2820ttcgtagggg acaagtgcca aacagacatg aatgaatgtc tgagcgagcc ctgtaagaat 2880ggggggacct gctctgacta cgtcaacagc tacacctgca cgtgccctgc gggcttccat 2940ggagtccact gtgaaaacaa catcgatgag tgcactgaga gctcctgttt caatggcggc 3000acgtgtgttg atgggatcaa ctctttctct tgcttatgcc ctgtgggttt cactggtccc 3060ttctgcctcc atgatatcaa tgagtgcagc tctaacccgt gcctgaattc gggaacgtgt 3120gttgatggcc tgggtaccta ccgatgcacc tgtcccttgg gctacactgg gaaaaactgt 3180cagaccctgg tgaacctctg cagcccctct ccatgtaaaa acaaaggaac ttgtgctcag 3240gaaaaggcaa ggccacgctg cctgtgtccg cctggatggg atggcgcata ctgtgatgtg 3300ctcaatgtgt cctgtaaggc ggcagccttg cagaaaggag tacctgttga acacttgtgc 3360cagcactcgg gtatctgtat caatgctggc aacacgcatc actgccagtg ccccctgggc 3420tacacgggga gctactgcga ggaacagctt gacgagtgtg cgtccaatcc atgccagcat 3480ggtgccacct gcagtgactt catcggagga tacagatgtg agtgtgttcc agggtatcag 3540ggtgtcaact gtgagtatga agtggacgag tgccagaacc agccctgtca gaacggaggc 3600acctgcatcg acctcgtgaa ccatttcaag tgctcgtgcc caccaggcac ccggggcctg 3660ctttgtgaag agaacattga tgactgtgct ggggcccccc actgccttaa tggtggccag 3720tgtgtggacc ggattggagg ctacagttgt cgctgtttgc ctggctttgc tggggagcgg 3780tgtgaggggg acatcaatga atgcctgtcc aatccttgca gctcagaggg cagcctggac 3840tgcattcagc tcaaaaataa ctaccagtgt gtctgccgca gcgccttcac aggccgacac 3900tgcgaaacct tcctagatgt gtgtccccag aagccttgcc tgaatggagg gacttgtgct 3960gtggctagca acgtgcctga tggcttcatt tgtcgttgtc ccccagggtt ctccggggca 4020agatgccaga gcagctgtgg acaagtgaag tgcagaagag gggagcagtg tgtgcacacc 4080gcctcgggac cccactgctt ctgcccgaac cacaaggact gcgagtcagg ttgcgctagt 4140aacccctgcc agcacggagg cacctgctac cctcagcgcc agcctcctta ctactcttgc 4200cgctgctccc caccgttctg gggcagccac tgcgagagct acacagcccc caccagc 425784137DNAMus musculus 8atggggctgg gggcccgggg ccgccgccgc cgtcgtcgcc tgatggcctt gccaccgcca 60ccaccgccca tgcgggcgct gcccctgctg ctgctgctag cggggctggg ggctgcagca 120cccccttgtc tggatggaag cccatgtgca aatggaggtc ggtgcaccca ccagcagccc 180tccctggagg ctgcttgcct gtgcctgcca ggctgggtgg gtgagcggtg ccagctggaa 240gacccttgcc actcaggccc ttgtgctggc cgaggcgttt gccagagttc agtggtggcg 300ggcaccgccc gattctcctg tcgttgtctc cgtggcttcc aaggcccaga ctgctcccag 360ccagacccct gcgtcagcag gccctgtgtt catggtgccc cctgctcagt ggggccggat 420ggccgatttg cctgtgcctg cccacctggc taccagggtc aaagctgcca aagtgacata 480gatgagtgcc gatctggtac aacttgccgt catggtggta cctgtctcaa tacacctgga 540tccttccgct gccagtgtcc tcttggttat acagggctgc tgtgtgagaa ccccgtagtg 600ccctgtgccc cttccccgtg tcgtaatggt ggcacctgta ggcagagcag tgatgtcaca

660tatgactgtg cttgccttcc tggcttcgag ggccagaact gtgaagtcaa cgtggatgac 720tgtcctggac atcggtgtct caatggggga acgtgtgtag acggtgtcaa tacttacaac 780tgccagtgcc ctccggagtg gacaggccag ttctgtacag aagatgtgga tgagtgtcag 840ctgcagccca atgcctgcca caatgggggt acctgcttca acctactggg tggccacagc 900tgtgtatgtg tcaatggctg gacgggtgag agctgcagtc agaatatcga tgactgtgct 960acagccgtgt gtttccatgg ggccacctgc catgaccgtg tggcctcttt ctactgtgcc 1020tgccctatgg ggaagacagg cctcttgtgt catctggatg atgcatgtgt cagcaacccc 1080tgccatgagg atgctatctg tgacacaaac cctgtgagtg gccgggccat ctgcacctgc 1140ccacctggct tcactggagg ggcatgtgac caggatgtgg atgagtgctc gattggtgcc 1200aacccctgtg aacatttggg tcggtgtgtg aatacacagg gctcattctt gtgccaatgt 1260ggccgtggct atactggacc tcgctgtgag actgatgtca atgagtgtct ctccgggccc 1320tgccgcaacc aggccacgtg tcttgaccga attggccagt ttacttgcat ctgcatggca 1380ggcttcacag ggacctactg tgaggtggac atcgacgaat gtcagagcag cccatgtgtc 1440aatggtggtg tctgcaagga cagagtcaat ggcttcagct gcacctgccc atcaggattc 1500agtgggtcca tgtgtcagct ggatgtggat gagtgtgcaa gcactccctg ccggaatggt 1560gccaagtgtg tggaccagcc tgacggctat gagtgtcgct gtgcagaggg ctttgagggc 1620actttgtgtg agcgaaacgt ggatgactgc tctccggatc cctgccacca cgggcgctgt 1680gtcgatggca ttgctagctt ctcgtgtgct tgtgccccag gctatacggg catacgctgt 1740gagagccagg tggatgagtg ccgcagccag ccctgtcgat atgggggcaa atgtctagac 1800ttggtggaca agtacctctg ccgttgtcct cccggaacca caggtgtgaa ctgtgaagtc 1860aacattgatg actgtgccag taacccctgt acctttggag tttgccgtga tggcatcaac 1920cgttatgact gtgtctgtca gcctggattc acagggcccc tctgcaacgt ggagatcaat 1980gagtgtgcat ccagcccatg tggagagggt ggctcctgtg tggatgggga aaatggcttc 2040cactgcctct gtccacctgg ctccctgcct ccactttgcc tacctgcgaa ccatccctgt 2100gcccacaagc cctgtagtca tggagtctgc catgatgcac caggcgggtt ccgctgtgtt 2160tgtgagcccg ggtggagtgg ccctcgctgt agccagagcc tggctccaga tgcctgtgag 2220tcccagccct gccaggctgg tggcacctgc accagtgatg gaataggctt tcgctgcacc 2280tgtgcccctg gattccaggg ccatcagtgt gaggtgctgt ccccctgtac tccaagcctc 2340tgtgagcacg gaggccactg tgagtctgac cctgaccggc tgactgtctg ttcctgtccc 2400ccaggctggc aaggcccacg atgccagcag gatgtggatg aatgtgccgg tgcctcaccc 2460tgcggccccc atggtacctg caccaacctg ccagggaatt tcaggtgcat ctgccacagg 2520ggatacactg gccccttctg tgatcaagac attgacgact gtgaccccaa cccgtgcctc 2580catggtggct cctgccagga tggcgtgggc tccttttcct gttcttgcct cgacggcttt 2640gctggtcctc gctgtgcccg agatgtggac gaatgtctga gcagcccctg tggccctggc 2700acctgtactg atcacgtggc ctccttcacc tgtgcctgtc cacctggtta tggaggcttc 2760cactgtgaga ttgacttgcc ggactgcagc cccagttcct gcttcaatgg agggacctgt 2820gtggatggcg tgagctcctt cagctgtctg tgtcgccccg gctacacagg cacacactgc 2880caatacgagg ctgacccctg cttttcccgg ccctgtctgc acgggggcat ctgcaacccc 2940acccacccag gatttgaatg cacctgccgg gagggcttca ctgggagtca gtgtcagaac 3000ccagtggact ggtgcagcca ggcaccctgt cagaatgggg gtcgctgtgt ccagactggg 3060gcttactgca tttgtccacc tggatggagt ggccgcctgt gcgacataca aagcctgccc 3120tgcacggagg ccgcagccca gatgggggtg aggttggagc agctgtgtca ggaaggtgga 3180aagtgcatag acaagggccg ctcccactac tgtgtgtgtc cagagggccg tacgggtagt 3240cactgtgaac acgaggtgga tccctgcacg gcccagcctt gccagcacgg gggcacttgc 3300cgtggttaca tggggggcta tgtgtgtgag tgtccagctg gctatgctgg tgacagttgt 3360gaggataata tagatgagtg tgcttcccag ccctgccaga acggaggctc ctgtatcgat 3420cttgtggccc gctatctctg ttcctgtccc cctggcacac tgggagttct ctgtgagatc 3480aatgaggacg actgtgacct aggcccatcc ttggactcag gcgttcagtg cctacacaat 3540ggcacctgtg tggacctggt gggtggcttc cgctgtaact gtcccccagg atacacaggt 3600ctgcactgtg aggcagacat caatgagtgt cgcccgggtg cctgccatgc agcgcatact 3660cgggactgcc tacaagatcc aggtgggcat ttccgctgcg tctgccatcc tggcttcaca 3720gggcctcgct gtcagattgc tctgtccccc tgtgagtccc agccatgtca gcatggaggc 3780cagtgccgtc acagcctagg ccgtggaggt gggctgacct tcacctgtca ctgtgtcccg 3840ccattctggg gtctgcgttg tgagcgggtg gcacgctctt gccgagagct gcagtgccca 3900gtgggtatcc catgccagca gacagcccgt ggaccacgct gcgcttgtcc tccggggctg 3960tccgggccct cctgccgggt ttctagggcg tcaccctcag gagctactaa cgccagctgc 4020gcctctgccc cttgtctgca tgggggctca tgcctacctg tacagagtgt ccctttcttc 4080cgctgtgtgt gcgctccggg ctggggcggc ccgcgttgtg agaccccttc cgcagcc 413793510DNAMus musculus 9atgcagcccc agttgctgct gctgctgctc ttgccactca atttccctgt catcctgacc 60agagagcttc tgtgtggagg atccccagag ccctgtgcca acggaggcac ctgcctgagg 120ctatctcagg gacaagggat ctgccagtgt gcccctggat ttctgggtga gacttgccag 180tttcctgacc cctgcaggga tacccaactc tgcaagaatg gtggcagctg ccaagccctg 240ctccccacac ccccaagctc ccgtagtcct acttctccac tgacccctca cttctcctgc 300acctgcccct ctggcttcac cggtgatcga tgccaaaccc atctggaaga gctctgtcca 360ccttctttct gttccaacgg gggtcactgc tatgttcagg cctcaggccg cccacagtgc 420tcctgcgagc ctgggtggac aggtgagcaa tgccagctcc gagacttctg ctcagccaac 480ccctgtgcca acggaggcgt gtgcctggcc acataccccc agatccagtg ccgctgtcca 540cctgggttcg agggtcacac ctgtgaacgc gacatcaacg agtgcttcct ggagccggga 600ccctgccctc agggcacctc ctgccataac accttgggtt cctaccagtg tctctgccct 660gtggggcagg aaggtcccca gtgcaagctc aggaagggag cctgccctcc tggaagctgt 720ctcaatgggg gcacctgcca gctggtccca gagggacact ccacctttca tctctgcctc 780tgtcccccag gtttcacggg gctggactgt gagatgaacc cagatgactg tgtcaggcac 840cagtgtcaga acggggccac ctgtctggat gggctggata cctacacctg cctctgcccc 900aagacatgga agggctggga ctgctctgaa gatatagatg aatgtgaagc ccggggtccc 960cctcgctgca ggaacggtgg cacctgccag aacacagctg gcagctttca ctgtgtgtgc 1020gtgagtggct ggggcggtgc aggttgtgag gagaacctgg atgactgtgc agctgccacc 1080tgtgccccgg gatccacctg catcgaccgt gtgggctctt tctcctgcct ctgcccacct 1140ggacgcacag gcctcctgtg ccacctggaa gacatgtgtt tgagtcagcc gtgccacgtg 1200aatgcccagt gcagcaccaa ccctctgaca ggctccaccc tctgcatatg ccagcctggc 1260tactcaggat ccacctgtca ccaagatctg gatgagtgcc aaatggccca gcaaggaccc 1320agtccctgcg aacatggcgg ctcctgcatc aacacccctg gctccttcaa ctgcctctgc 1380ctgcctggtt acacgggctc ccgctgtgaa gctgaccaca atgagtgcct gtcacagccc 1440tgccacccag gcagcacctg cctggacctg cttgcaacct tccactgcct ctgcccacca 1500ggcttggaag ggaggctctg tgaggtggag gtcaatgagt gcacctctaa tccctgcctg 1560aaccaagctg cctgccatga cctgctcaac ggcttccagt gcctctgcct tcctggattc 1620accggcgccc gatgtgagaa agacatggac gagtgtagca gcaccccctg tgccaatggg 1680gggcgctgcc gagaccagcc tggagccttc tactgcgagt gtctcccagg ctttgaaggg 1740ccacactgtg agaaagaagt ggacgaatgt ctgagtgacc cctgccccgt gggagccagc 1800tgccttgatc tccccggagc attcttctgc ctctgccgtc ctggtttcac aggtcaactt 1860tgtgaggttc ccttgtgcac ccccaacatg tgccaacctg gacagcaatg ccaaggtcag 1920gaacacagag ccccctgcct ctgccctgac ggaagtcctg gctgtgttcc tgccgaggac 1980aactgcccct gtcaccatgg ccattgccag agatccttgt gtgtgtgtga tgagggctgg 2040actggaccag aatgcgagac agaactgggt ggctgcatct ccacaccctg tgcccatggg 2100gggacctgcc acccacagcc gtctggctac aactgtacct gccctgcagg ctacatgggg 2160ttgacctgta gtgaggaggt gacagcttgt cactcagggc cctgtctcaa tggtggctct 2220tgcagcatcc gtcctgaggg ctattcctgc acctgccttc caagtcacac aggtcgccac 2280tgccagactg ccgtggacca ctgtgtgtct gcctcgtgcc tcaatggggg tacctgtgtg 2340aacaagcctg gcactttctt ctgcctctgt gccactggct tccaggggct gcactgtgag 2400gagaagacta accccagctg tgcagacagc ccctgcagga acaaggcaac ctgccaagac 2460acacctcgag gggcccgctg cctctgcagc cctggctata caggaagcag ctgccagact 2520ctgatagact tgtgtgcccg gaagccctgt ccacacactg ctcgatgcct ccagagtggg 2580ccctcgttcc agtgcctgtg cctccaggga tggacagggg ctctctgtga cttcccactg 2640tcctgccaga tggccgcaat gagccaaggc atagagatct ctggcctgtg ccagaatgga 2700ggcctctgta ttgacacggg ctcctcctat ttctgccgct gccctcctgg attccaaggc 2760aagttatgcc aggataatat gaacccctgc gagcccaatc cctgccatca cgggtctacc 2820tgtgtgcctc agcccagtgg ctatgtctgc cagtgtgccc caggctatga gggacagaac 2880tgctcaaaag tacttgaagc ttgtcagtcc cagccctgcc acaaccacgg aacctgtacc 2940tccaggcctg gaggcttcca ctgtgcctgc cctccaggct tcgtgggact gcgctgtgag 3000ggagatgtgg atgagtgtct ggaccggccc tgtcacccct cgggcactgc agcttgccac 3060tctttagcca acgccttcta ctgccagtgt ctgcctgggc acacaggcca gcggtgtgag 3120gtggagatgg acctctgtca gagccaaccc tgctccaatg gaggatcctg tgagatcaca 3180acagggccac cccctggctt cacctgtcac tgccccaagg gttttgaagg ccccacctgc 3240agccacaaag ccctttcctg cggcatccat cactgccaca atggaggcct atgtctgccc 3300tcccctaagc cagggtcacc accactctgt gcctgcctca gtggttttgg gggccctgac 3360tgtctgacac ctccagctcc accgggctgc ggtcccccct caccctgcct gcacaatggt 3420acctgcactg agacccctgg gttgggcaac ccgggctttc aatgcacctg ccctcctgac 3480tctccagggc cccggtgtca aaggccaggg 35101015DNAMus musculus 10gatctgggcc cgggc 15114299DNAHomo sapien 11atgccgccgc tcctggcgcc cctgctctgc ctggcgctgc tgcccgcgct cgccgcacga 60ggcccgcgat gctcccagcc cggtgagacc tgcctgaatg gcgggaagtg tgaagcggcc 120aatggcacgg aggcctgcgt ctgtggcggg gccttcgtgg gcccgcgatg ccaggacccc 180aacccgtgcc tcagcacccc ctgcaagaac gccgggacat gccacgtggt ggaccgcaga 240ggcgtggcag actatgcctg cagctgtgcc ctgggcttct ctgggcccct ctgcctgaca 300cccctggaca atgcctgcct caccaacccc tgccgcaacg ggggcacctg cgacctgctc 360acgctgacgg agtacaagtg ccgctgcccg cccggctggt cagggaaatc gtgccagcag 420gctgacccgt gcgcctccaa cccctgcgcc aacggtggcc agtgcctgcc cttcgaggcc 480tcctacatct gccactgccc acccagcttc catggcccca cctgccggca ggatgtcaac 540gagtgtggcc agaagcccgg gctttgccgc cacggaggca cctgccacaa cgaggtcggc 600tcctaccgct gcgtctgccg cgccacccac actggcccca actgcgagcg gccctacgtg 660ccctgcagcc cctcgccctg ccagaacggg ggcacctgcc gccccacggg cgacgtcacc 720cacgagtgtg cctgcctgcc aggcttcacc ggccagaact gtgaggaaaa tatcgacgat 780tgtccaggaa acaactgcaa gaacgggggt gcctgtgtgg acggcgtgaa cacctacaac 840tgccgctgcc cgccagagtg gacaggtcag tactgtaccg aggatgtgga cgagtgccag 900ctgatgccaa atgcctgcca gaacggcggg acctgccaca acacccacgg tggctacaac 960tgcgtgtgtg tcaacggctg gactggtgag gactgcagcg agaacattga tgactgtgcc 1020agcgccgcct gcttccacgg cgccacctgc catgaccgtg tggcctcctt ctactgcgag 1080tgtccccatg gccgcacagg tctgctgtgc cacctcaacg acgcatgcat cagcaacccc 1140tgtaacgagg gctccaactg cgacaccaac cctgtcaatg gcaaggccat ctgcacctgc 1200ccctcggggt acacgggccc ggcctgcagc caggacgtgg atgagtgctc gctgggtgcc 1260aacccctgcg agcatgcggg caagtgcatc aacacgctgg gctccttcga gtgccagtgt 1320ctgcagggct acacgggccc ccgatgcgag atcgacgtca acgagtgcgt ctcgaacccg 1380tgccagaacg acgccacctg cctggaccag attggggagt tccagtgcat ctgcatgccc 1440ggctacgagg gtgtgcactg cgaggtcaac acagacgagt gtgccagcag cccctgcctg 1500cacaatggcc gctgcctgga caagatcaat gagttccagt gcgagtgccc cacgggcttc 1560actgggcatc tgtgccagta cgatgtggac gagtgtgcca gcaccccctg caagaatggt 1620gccaagtgcc tggacggacc caacacttac acctgtgtgt gcacggaagg gtacacgggg 1680acgcactgcg aggtggacat cgatgagtgc gaccccgacc cctgccacta cggctcctgc 1740aaggacggcg tcgccacctt cacctgcctc tgccgcccag gctacacggg ccaccactgc 1800gagaccaaca tcaacgagtg ctccagccag ccctgccgcc acgggggcac ctgccaggac 1860cgcgacaacg cctacctctg cttctgcctg aaggggacca caggacccaa ctgcgagatc 1920aacctggatg actgtgccag cagcccctgc gactcgggca cctgtctgga caagatcgat 1980ggctacgagt gtgcctgtga gccgggctac acagggagca tgtgtaacat caacatcgat 2040gagtgtgcgg gcaacccctg ccacaacggg ggcacctgcg aggacggcat caatggcttc 2100acctgccgct gccccgaggg ctaccacgac cccacctgcc tgtctgaggt caatgagtgc 2160aacagcaacc cctgcgtcca cggggcctgc cgggacagcc tcaacgggta caagtgcgac 2220tgtgaccctg ggtggagtgg gaccaactgt gacatcaaca acaatgagtg tgaatccaac 2280ccttgtgtca acggcggcac ctgcaaagac atgaccagtg gctacgtgtg cacctgccgg 2340gagggcttca gcggtcccaa ctgccagacc aacatcaacg agtgtgcgtc caacccatgt 2400ctgaaccagg gcacgtgtat tgacgacgtt gccgggtaca agtgcaactg cctgctgccc 2460tacacaggtg ccacgtgtga ggtggtgctg gccccgtgtg cccccagccc ctgcagaaac 2520ggcggggagt gcaggcaatc cgaggactat gagagcttct cctgtgtctg ccccacgggc 2580tggcaagcag ggcagacctg tgaggtcgac atcaacgagt gcgttctgag cccgtgccgg 2640cacggcgcat cctgccagaa cacccacggc ggctaccgct gccactgcca ggccggctac 2700agtgggcgca actgcgagac cgacatcgac gactgccggc ccaacccgtg tcacaacggg 2760ggctcctgca cagacggcat caacacggcc ttctgcgact gcctgcccgg cttccggggc 2820actttctgtg aggaggacat caacgagtgt gccagtgacc cctgccgcaa cggggccaac 2880tgcacggact gcgtggacag ctacacgtgc acctgccccg caggcttcag cgggatccac 2940tgtgagaaca acacgcctga ctgcacagag agctcctgct tcaacggtgg cacctgcgtg 3000gacggcatca actcgttcac ctgcctgtgt ccacccggct tcacgggcag ctactgccag 3060cacgatgtca atgagtgcga ctcacagccc tgcctgcatg gcggcacctg tcaggacggc 3120tgcggctcct acaggtgcac ctgcccccag ggctacactg gccccaactg ccagaacctt 3180gtgcactggt gtgactcctc gccctgcaag aacggcggca aatgctggca gacccacacc 3240cagtaccgct gcgagtgccc cagcggctgg accggccttt actgcgacgt gcccagcgtg 3300tcctgtgagg tggctgcgca gcgacaaggt gttgacgttg cccgcctgtg ccagcatgga 3360gggctctgtg tggacgcggg caacacgcac cactgccgct gccaggcggg ctacacaggc 3420agctactgtg aggacctggt ggacgagtgc tcacccagcc cctgccagaa cggggccacc 3480tgcacggact acctgggcgg ctactcctgc aagtgcgtgg ccggctacca cggggtgaac 3540tgctctgagg agatcgacga gtgcctctcc cacccctgcc agaacggggg cacctgcctc 3600gacctcccca acacctacaa gtgctcctgc ccacggggca ctcagggtgt gcactgtgag 3660atcaacgtgg acgactgcaa tccccccgtt gaccccgtgt cccggagccc caagtgcttt 3720aacaacggca cctgcgtgga ccaggtgggc ggctacagct gcacctgccc gccgggcttc 3780gtgggtgagc gctgtgaggg ggatgtcaac gagtgcctgt ccaatccctg cgacgcccgt 3840ggcacccaga actgcgtgca gcgcgtcaat gacttccact gcgagtgccg tgctggtcac 3900accgggcgcc gctgcgagtc cgtcatcaat ggctgcaaag gcaagccctg caagaatggg 3960ggcacctgcg ccgtggcctc caacaccgcc cgcgggttca tctgcaagtg ccctgcgggc 4020ttcgagggcg ccacgtgtga gaatgacgct cgtacctgcg gcagcctgcg ctgcctcaac 4080ggcggcacat gcatctccgg cccgcgcagc cccacctgcc tgtgcctggg ccccttcacg 4140ggccccgaat gccagttccc ggccagcagc ccctgcctgg gcggcaaccc ctgctacaac 4200caggggacct gtgagcccac atccgagagc cccttctacc gttgcctgtg ccccgccaaa 4260ttcaacgggc tcttgtgcca catcctggac tacagcttc 4299124213DNAHomo sapien 12tcatctggaa ttatgcccgc cctgcgcccc gctctgctgt gggcgctgct ggcgctctgg 60ctgtgctgcg cggcccccgc gcatgcattg cagtgtcgag atggctatga accctgtgta 120aatgaaggaa tgtgtgttac ctaccacaat ggcacaggat actgcaaatg tccagaaggc 180ttcttggggg aatattgtca acatcgagac ccctgtgaga agaaccgctg ccagaatggt 240gggacttgtg tggcccaggc catgctgggg aaagccacgt gccgatgtgc ctcagggttt 300acaggagagg actgccagta ctcaacatct catccatgct ttgtgtctcg accctgcctg 360aatggcggca catgccatat gctcagccgg gatacctatg agtgcacctg tcaagtcggg 420tttacaggta aggagtgcca atggacggat gcctgcctgt ctcatccctg tgcaaatgga 480agtacctgta ccactgtggc caaccagttc tcctgcaaat gcctcacagg cttcacaggg 540cagaaatgtg agactgatgt caatgagtgt gacattccag gacactgcca gcatggtggc 600acctgcctca acctgcctgg ttcctaccag tgccagtgcc ctcagggctt cacaggccag 660tactgtgaca gcctgtatgt gccctgtgca ccctcacctt gtgtcaatgg aggcacctgt 720cggcagactg gtgacttcac ttttgagtgc aactgccttc caggttttga agggagcacc 780tgtgagagga atattgatga ctgccctaac cacaggtgtc agaatggagg ggtttgtgtg 840gatggggtca acacttacaa ctgccgctgt cccccacaat ggacaggaca gttctgcaca 900gaggatgtgg atgaatgcct gctgcagccc aatgcctgtc aaaatggggg cacctgtgcc 960aaccgcaatg gaggctatgg ctgtgtatgt gtcaacggct ggagtggaga tgactgcagt 1020gagaacattg atgattgtgc cttcgcctcc tgtactccag gctccacctg catcgaccgt 1080gtggcctcct tctcttgcat gtgcccagag gggaaggcag gtctcctgtg tcatctggat 1140gatgcatgca tcagcaatcc ttgccacaag ggggcactgt gtgacaccaa ccccctaaat 1200gggcaatata tttgcacctg cccacaaggc tacaaagggg ctgactgcac agaagatgtg 1260gatgaatgtg ccatggccaa tagcaatcct tgtgagcatg caggaaaatg tgtgaacacg 1320gatggcgcct tccactgtga gtgtctgaag ggttatgcag gacctcgttg tgagatggac 1380atcaatgagt gccattcaga cccctgccag aatgatgcta cctgtctgga taagattgga 1440ggcttcacat gtctgtgcat gccaggtttc aaaggtgtgc attgtgaatt agaaataaat 1500gaatgtcaga gcaacccttg tgtgaacaat gggcagtgtg tggataaagt caatcgtttc 1560cagtgcctgt gtcctcctgg tttcactggg ccagtttgcc agattgatat tgatgactgt 1620tccagtactc cgtgtctgaa tggggcaaag tgtatcgatc acccgaatgg ctatgaatgc 1680cagtgtgcca caggtttcac tggtgtgttg tgtgaggaga acattgacaa ctgtgacccc 1740gatccttgcc accatggtca gtgtcaggat ggtattgatt cctacacctg catctgcaat 1800cccgggtaca tgggcgccat ctgcagtgac cagattgatg aatgttacag cagcccttgc 1860ctgaacgatg gtcgctgcat tgacctggtc aatggctacc agtgcaactg ccagccaggc 1920acgtcagggg ttaattgtga aattaatttt gatgactgtg caagtaaccc ttgtatccat 1980ggaatctgta tggatggcat taatcgctac agttgtgtct gctcaccagg attcacaggg 2040cagagatgta acattgacat tgatgagtgt gcctccaatc cctgtcgcaa gggtgcaaca 2100tgtatcaacg gtgtgaatgg tttccgctgt atatgccccg agggacccca tcaccccagc 2160tgctactcac aggtgaacga atgcctgagc aatccctgca tccatggaaa ctgtactgga 2220ggtctcagtg gatataagtg tctctgtgat gcaggctggg ttggcatcaa ctgtgaagtg 2280gacaaaaatg aatgcctttc gaatccatgc cagaatggag gaacttgtga caatctggtg 2340aatggataca ggtgtacttg caagaagggc tttaaaggct ataactgcca ggtgaatatt 2400gatgaatgtg cctcaaatcc atgcctgaac caaggaacct gctttgatga cataagtggc 2460tacacttgcc actgtgtgct gccatacaca ggcaagaatt gtcagacagt attggctccc 2520tgttccccaa acccttgtga gaatgctgct gtttgcaaag agtcaccaaa ttttgagagt 2580tatacttgct tgtgtgctcc tggctggcaa ggtcagcggt gtaccattga cattgacgag 2640tgtatctcca agccctgcat gaaccatggt ctctgccata acacccaggg cagctacatg 2700tgtgaatgtc caccaggctt cagtggtatg gactgtgagg aggacattga tgactgcctt 2760gccaatcctt gccagaatgg aggttcctgt atggatggag tgaatacttt ctcctgcctc 2820tgccttccgg gtttcactgg ggataagtgc cagacagaca tgaatgagtg tctgagtgaa 2880ccctgtaaga atggagggac ctgctctgac tacgtcaaca gttacacttg caagtgccag 2940gcaggatttg atggagtcca ttgtgagaac aacatcaatg agtgcactga gagctcctgt 3000ttcaatggtg gcacatgtgt tgatgggatt aactccttct cttgcttgtg ccctgtgggt 3060ttcactggat ccttctgcct ccatgagatc aatgaatgca gctctcatcc atgcctgaat 3120gagggaacgt gtgttgatgg cctgggtacc taccgctgca gctgccccct gggctacact 3180gggaaaaact gtcagaccct ggtgaatctc tgcagtcggt ctccatgtaa aaacaaaggt 3240acttgtgttc agaaaaaagc agagtcccag tgcctatgtc catctggatg ggctggtgcc 3300tattgtgacg tgcccaatgt ctcttgtgac atagcagcct ccaggagagg tgtgcttgtt 3360gaacacttgt gccagcactc aggtgtctgc atcaatgctg gcaacacgca ttactgtcag 3420tgccccctgg gctatactgg gagctactgt gaggagcaac tcgatgagtg tgcgtccaac 3480ccctgccagc acggggcaac atgcagtgac ttcattggtg gatacagatg cgagtgtgtc 3540ccaggctatc agggtgtcaa ctgtgagtat gaagtggatg

agtgccagaa tcagccctgc 3600cagaatggag gcacctgtat tgaccttgtg aaccatttca agtgctcttg cccaccaggc 3660actcggggcc tactctgtga agagaacatt gatgactgtg cccggggtcc ccattgcctt 3720aatggtggtc agtgcatgga taggattgga ggctacagtt gtcgctgctt gcctggcttt 3780gctggggagc gttgtgaggg agacatcaac gagtgcctct ccaacccctg cagctctgag 3840ggcagcctgg actgtataca gctcaccaat gactacctgt gtgtttgccg tagtgccttt 3900actggccggc actgtgaaac cttcgtcgat gtgtgtcccc agatgccctg cctgaatgga 3960gggacttgtg ctgtggccag taacatgcct gatggtttca tttgccgttg tcccccggga 4020ttttccgggg caaggtgcca gagcagctgt ggacaagtga aatgtaggaa gggggagcag 4080tgtgtgcaca ccgcctctgg accccgctgc ttctgcccca gtccccggga ctgcgagtca 4140ggctgtgcca gtagcccctg ccagcacggg ggcagctgcc accctcagcg ccagcctcct 4200tattactcct gcc 4213133974DNAHomo sapien 13atggggccgg gggcccgtgg ccgccgccgc cgccgtcgcc cgatgtcgcc gccaccgcca 60ccgccacccg tgcgggcgct gcccctgctg ctgctgctag cggggccggg ggctgcagcc 120cccccttgcc tggacggaag cccgtgtgca aatggaggtc gttgcaccca gctgccctcc 180cgggaggctg cctgcctgtg cccgcctggc tgggtgggtg agcggtgtca gctggaggac 240ccctgtcact caggcccctg tgctggccgt ggtgtctgcc agagttcagt ggtggctggc 300accgcccgat tctcatgccg gtgcccccgt ggcttccgag gccctgactg ctccctgcca 360gatccctgcc tcagcagccc ttgtgcccac ggtgcccgct gctcagtggg gcccgatgga 420cgcttcctct gctcctgccc acctggctac cagggccgca gctgccgaag cgacgtggat 480gagtgccggg tgggtgagcc ctgccgccat ggtggcacct gcctcaacac acctggctcc 540ttccgctgcc agtgtccagc tggctacaca gggccactat gtgagaaccc cgcggtgccc 600tgtgcaccct caccatgccg taacgggggc acctgcaggc agagtggcga cctcacttac 660gactgtgcct gtcttcctgg gtttgagggt cagaattgtg aagtgaacgt ggacgactgt 720ccaggacacc gatgtctcaa tggggggaca tgcgtggatg gcgtcaacac ctataactgc 780cagtgccctc ctgagtggac aggccagttc tgcacggagg acgtggatga gtgtcagctg 840cagcccaacg cctgccacaa tgggggtacc tgcttcaaca cgctgggtgg ccacagctgc 900gtgtgtgtca atggctggac aggcgagagc tgcagtcaga atatcgatga ctgtgccaca 960gccgtgtgct tccatggggc cacctgccat gaccgcgtgg cttctttcta ctgtgcctgc 1020cccatgggca agactggcct cctgtgtcac ctggatgacg cctgtgtcag caacccctgc 1080cacgaggatg ctatctgtga cacaaatccg gtgaacggcc gggccatttg cacctgtcct 1140cccggcttca cgggtggggc atgtgaccag gatgtggacg agtgctctat cggcgccaac 1200ccctgcgagc acttgggcag gtgcgtgaac acgcagggct ccttcctgtg ccagtgcggt 1260cgtggctaca ctggacctcg ctgtgagacc gatgtcaacg agtgtctgtc ggggccctgc 1320cgaaaccagg ccacgtgcct cgaccgcata ggccagttca cctgtatctg tatggcaggc 1380ttcacaggaa cctattgcga ggtggacatt gacgagtgtc agagtagccc ctgtgtcaac 1440ggtggggtct gcaaggaccg agtcaatggc ttcagctgca cctgcccctc gggcttcagc 1500ggctccacgt gtcagctgga cgtggacgaa tgcgccagca cgccctgcag gaatggcgcc 1560aaatgcgtgg accagcccga tggctacgag tgccgctgtg ccgagggctt tgagggcacg 1620ctgtgtgatc gcaacgtgga cgactgctcc cctgacccat gccaccatgg tcgctgcgtg 1680gatggcatcg ccagcttctc atgtgcctgt gctcctggct acacgggcac acgctgcgag 1740agccaggtgg acgaatgccg cagccagccc tgccgccatg gcggcaaatg cctagacctg 1800gtggacaagt acctctgccg ctgcccttct gggaccacag gtgtgaactg cgaagtgaac 1860attgacgact gtgccagcaa cccctgcacc tttggagtct gccgtgatgg catcaaccgc 1920tacgactgtg tctgccaacc tggcttcaca gggccccttt gtaacgtgga gatcaatgag 1980tgtgcttcca gcccatgcgg cgagggaggt tcctgtgtgg atggggaaaa tggcttccgc 2040tgcctctgcc cgcctggctc cttgccccca ctctgcctcc ccccgagcca tccctgtgcc 2100catgagccct gcagtcacgg catctgctat gatgcacctg gcgggttccg ctgtgtgtgt 2160gagcctggct ggagtggccc ccgctgcagc cagagcctgg cccgagacgc ctgtgagtcc 2220cagccgtgca gggccggtgg gacatgcagc agcgatggaa tgggtttcca ctgcacctgc 2280ccgcctggtg tccagggacg tcagtgtgaa ctcctctccc cctgcacccc gaacccctgt 2340gagcatgggg gccgctgcga gtctgcccct ggccagctgc ctgtctgctc ctgcccccag 2400ggctggcaag gcccacgatg ccagcaggat gtggacgagt gtgctggccc cgcaccctgt 2460ggccctcatg gtatctgcac caacctggca gggagtttca gctgcacctg ccatggaggg 2520tacactggcc cttcctgcga tcaggacatc aatgactgtg accccaaccc atgcctgaac 2580ggtggctcgt gccaagacgg cgtgggctcc ttttcctgct cctgcctccc tggtttcgcc 2640ggcccacgat gcgcccgcga tgtggatgag tgcctgagca acccctgcgg cccgggcacc 2700tgtaccgacc acgtggcctc cttcacctgc acctgcccgc caggctacgg aggcttccac 2760tgcgaacagg acctgcccga ctgcagcccc agctcctgct tcaatggcgg gacctgtgtg 2820gacggcgtga actcgttcag ctgcctgtgc cgtcccggct acacaggagc ccactgccaa 2880catgaggcag acccctgcct ctcgcggccc tgcctacacg ggggcgtctg cagcgccgcc 2940caccctggct tccgctgcac ctgcctcgag agcttcacgg gcccgcagtg ccagacgctg 3000gtggattggt gcagccgcca gccttgtcaa aacgggggtc gctgcgtcca gactggggcc 3060tattgccttt gtccccctgg atggagcgga cgcctctgtg acatccgaag cttgccctgc 3120agggaggccg cagcccagat cggggtgcgg ctggagcagc tgtgtcaggc gggtgggcag 3180tgtgtggatg aagacagctc ccactactgc gtgtgcccag agggccgtac tggtagccac 3240tgtgagcagg aggtggaccc ctgcttggcc cagccctgcc agcatggggg gacctgccgt 3300ggctatatgg ggggctacat gtgtgagtgt cttcctggct acaatggtga taactgtgag 3360gacgacgtgg acgagtgtgc ctcccagccc tgccagcacg ggggttcatg cattgacctc 3420gtggcccgct atctctgctc ctgtccccca ggaacgctgg gggtgctctg cgagattaat 3480gaggatgact gcggcccagg cccaccgctg gactcagggc cccggtgcct acacaatggc 3540acctgcgtgg acctggtggg tggtttccgc tgcacctgtc ccccaggata cactggtttg 3600cgctgcgagg cagacatcaa tgagtgtcgc tcaggtgcct gccacgcggc acacacccgg 3660gactgcctgc aggacccagg cggaggtttc cgttgccttt gtcatgctgg cttctcaggt 3720cctcgctgtc agactgtcct gtctccctgc gagtcccagc catgccagca tggaggccag 3780tgccgtccta gcccgggtcc tgggggtggg ctgaccttca cctgtcactg tgcccagccg 3840ttctggggtc cgcgttgcga gcgggtggcg cgctcctgcc gggagctgca gtgcccggtg 3900ggcgtcccat gccagcagac gccccgcggg ccgcgctgcg cctgcccccc agggttgtcg 3960ggaccctcct gccg 3974143522DNAHomo sapien 14atgcagcccc cttcactgct gctgctgctg ctgctgctgc tgctgctatg tgtctcagtg 60gtcagaccca gagggctgct gtgtgggagt ttcccagaac cctgtgccaa tggaggcacc 120tgcctgagcc tgtctctggg acaagggacc tgccagtgtg cccctggctt cctgggtgag 180acgtgccagt ttcctgaccc ctgccagaac gcccagctct gccaaaatgg aggcagctgc 240caagccctgc ttcccgctcc cctagggctc cccagctctc cctctccatt gacacccagc 300ttcttgtgca cttgcctccc tggcttcact ggtgagagat gccaggccaa gcttgaagac 360ccttgtcctc cctccttctg ttccaaaagg ggccgctgcc acatccaggc ctcgggccgc 420ccacagtgct cctgcatgcc tggatggaca ggtgagcagt gccagcttcg ggacttctgt 480tcagccaacc catgtgttaa tggaggggtg tgtctggcca cataccccca gatccagtgc 540cactgcccac cgggcttcga gggccatgcc tgtgaacgtg atgtcaacga gtgcttccag 600gacccaggac cctgccccaa aggcacctcc tgccataaca ccctgggctc cttccagtgc 660ctctgccctg tggggcagga gggtccacgt tgtgagctgc gggcaggacc ctgccctcct 720aggggctgtt cgaatggggg cacctgccag ctgatgccag agaaagactc cacctttcac 780ctctgcctct gtcccccagg tttcataggc ccagactgtg aggtgaatcc agacaactgt 840gtcagccacc agtgtcagaa tgggggcact tgccaggatg ggctggacac ctacacctgc 900ctctgcccag aaacctggac aggctgggac tgctccgaag atgtggatga gtgtgagacc 960cagggtcccc ctcactgcag aaacgggggc acctgccaga actctgctgg tagctttcac 1020tgcgtgtgtg tgagtggctg gggcggcaca agctgtgagg agaacctgga tgactgtatt 1080gctgccacct gtgccccggg atccacctgc attgaccggg tgggctcttt ctcctgcctc 1140tgcccacctg gacgcacagg actcctgtgc cacttggaag acatgtgtct gagccagccg 1200tgccatgggg atgcccaatg cagcaccaac cccctcacag gctccacact ctgcctgtgt 1260cagcctggct attcggggcc cacctgccac caggacctgg acgagtgtct gatggcccag 1320caaggcccaa gtccctgtga acatggcggt tcctgcctca acactcctgg ctccttcaac 1380tgcctctgtc cacctggcta cacaggctcc cgttgtgagg ctgatcacaa tgagtgcctc 1440tcccagccct gccacccagg aagcacctgt ctggacctac ttgccacctt ccactgcctc 1500tgcccgccag gcttagaagg gcagctctgt gaggtggaga ccaacgagtg tgcctcagct 1560ccctgcctga accacgcgga ttgccatgac ctgctcaacg gcttccagtg catctgcctg 1620cctggattct ccggcacccg atgtgaggag gatatcgatg agtgcagaag ctctccctgt 1680gccaatggtg ggcagtgcca ggaccagcct ggagccttcc actgcaagtg tctcccaggc 1740tttgaagggc cacgctgtca aacagaggtg gatgagtgcc tgagtgaccc atgtcccgtt 1800ggagccagct gccttgatct tccaggagcc ttcttttgcc tctgcccctc tggtttcaca 1860ggccagctct gtgaggttcc cctgtgtgct cccaacctgt gccagcccaa gcagatatgt 1920aaggaccaga aagacaaggc caactgcctc tgtcctgatg gaagccctgg ctgtgcccca 1980cctgaggaca actgcacctg ccaccacggg cactgccaga gatcctcatg tgtgtgtgac 2040gtgggttgga cggggccaga gtgtgaggca gagctagggg gctgcatctc tgcaccctgt 2100gcccatgggg ggacctgcta cccccagccc tctggctaca actgcacctg ccctacaggc 2160tacacaggac ccacctgtag tgaggagatg acagcttgtc actcagggcc atgtctcaat 2220ggcggctcct gcaaccctag ccctggaggc tactactgca cctgccctcc aagccacaca 2280gggccccagt gccaaaccag cactgactac tgtgtgtctg ccccgtgctt caatgggggt 2340acctgtgtga acaggcctgg caccttctcc tgcctctgtg ccatgggctt ccagggcccg 2400cgctgtgagg gaaagctccg ccccagctgt gcagacagcc cctgtaggaa tagggcaacc 2460tgccaggaca gccctcaggg tccccgctgc ctctgcccca ctggctacac cggaggcagc 2520tgccagactc tgatggactt atgtgcccag aagccctgcc cacgcaattc ccactgcctc 2580cagactgggc cctccttcca ctgcttgtgc ctccagggat ggaccgggcc tctctgcaac 2640cttccactgt cctcctgcca gaaggctgca ctgagccaag gcatagacgt ctcttccctt 2700tgccacaatg gaggcctctg tgtcgacagc ggcccctcct atttctgcca ctgcccccct 2760ggattccaag gcagcctgtg ccaggatcac gtgaacccat gtgagtccag gccttgccag 2820aacggggcca cctgcatggc ccagcccagt gggtatctct gccagtgtgc cccaggctac 2880gatggacaga actgctcaaa ggaactcgat gcttgtcagt cccaaccctg tcacaaccat 2940ggaacctgta ctcccaaacc tggaggattc cactgtgcct gccctccagg ctttgtgggg 3000ctacgctgtg agggagacgt ggacgagtgt ctggaccagc cctgccaccc cacaggcact 3060gcagcctgcc actctctggc caatgccttc tactgccagt gtctgcctgg acacacaggc 3120cagtggtgtg aggtggagat agacccctgc cacagccaac cctgctttca tggagggacc 3180tgtgaggcca cagcaggatc acccctgggt ttcatctgcc actgccccaa gggttttgaa 3240ggccccacct gcagccacag ggccccttcc tgcggcttcc atcactgcca ccacggaggc 3300ctgtgtctgc cctcccctaa gccaggcttc ccaccacgct gtgcctgcct cagtggctat 3360gggggtcctg actgcctgac cccaccagct cctaaaggct gtggccctcc ctccccatgc 3420ctatacaatg gcagctgctc agagaccacg ggcttggggg gcccaggctt tcgatgctcc 3480tgccctcaca gctctccagg gccccggtgt cagaaacccg ga 3522

Claims

1-90. (canceled)

91. A method for treating a subject having a tumor comprising administering to the subject an effective amount of a fusion protein comprising an extracellular domain of a human Notch receptor protein and an Fc portion of an antibody, wherein the extracellular domain of the Notch receptor protein comprises the EGF repeats of such Notch receptor protein and the extracellular domain is linked at its carboxyl terminus to the amino terminus of the Fc portion of an antibody, wherein the Notch receptor protein is a human Notch1, Notch2, Notch3, or Notch4 receptor protein, so as to thereby treat the subject.

92. The method of claim 91, wherein the fusion protein comprises an extracellular domain of a human Notch receptor protein and an Fc portion of an antibody, wherein the extracellular domain of the Notch receptor protein comprises the EGF repeats of such Notch receptor protein and the extracellular domain is linked at its carboxyl terminus to the amino terminus of the Fc portion of an antibody by means of a peptide linker, wherein the Notch receptor protein is a human Notch1, Notch2, Notch3, or Notch4 receptor protein, the amino acid sequence of which is encoded by the nucleic acid sequence set forth in SEQ ID NOs. 11, 12, 13 and 14, respectively, and wherein the extracellular domain is bound to the Fc portion of the antibody by means of a peptide linker which comprises the consecutive amino acids DLGPG (SEQ ID NO:2).

93. The method of claim 91, wherein the fusion protein consists of consecutive amino acids, the amino acid sequence of which, commencing at the amino terminus, is identical to the amino acid sequence of the EGF repeats of a human Notch receptor protein, followed by an amino acid sequence identical to the sequence of an Fc portion of an antibody, wherein the amino acid sequence of the EGF repeats of the human Notch receptor protein is selected from the group consisting of: (a) the amino acid sequence of EGF repeats 1-36 of human Notch1 encoded by the nucleic acid coding sequence of Notch1 EGF repeats 1-36 set forth in SEQ ID NO: 11; (b) the amino acid sequence of EGF repeats 1-36 of human Notch2 encoded by the nucleic acid coding sequence of Notch2 EGF repeats 1-36 set forth in SEQ ID NO: 12; (c) the amino acid sequence of EGF repeats 1-34 of human Notch3 encoded by the nucleic acid coding sequence of Notch3 EGF repeats 1-34 set forth in SEQ ID NO: 13; and (d) the amino acid sequence of EGF repeats 1-29 of human Notch4 is encoded by the nucleic acid coding sequence of Notch4 EGF repeats 1-29 set forth in SEQ ID NO: 14.

94. The method of claim 91, wherein the fusion protein consists of consecutive amino acids, the amino acid sequence of which, commencing at the amino terminus, is identical to the amino acid sequence of the EGF repeats of a human Notch receptor protein, followed by a linker sequence of amino acids which is followed by an amino acid sequence identical to the sequence of an Fc portion of an antibody, wherein the amino acid sequence of the EGF repeats of the human Notch receptor protein is selected from the group consisting of: (a) the amino acid sequence of EGF repeats 1-36 of human Notch1 encoded by the nucleic acid coding sequence of Notch1 EGF repeats 1-36 set forth in SEQ ID NO: 11; (b) the amino acid sequence of EGF repeats 1-36 of human Notch2 encoded by the nucleic acid coding sequence of Notch2 EGF repeats 1-36 set forth in SEQ ID NO: 12; (c) the amino acid sequence of EGF repeats 1-34 of human Notch3 encoded by the nucleic acid coding sequence of Notch3 EGF repeats 1-34 set forth in SEQ ID NO: 13; and (d) the amino acid sequence of EGF repeats 1-29 of human Notch4 is encoded by the nucleic acid coding sequence of Notch4 EGF repeats 1-29 set forth in SEQ ID NO: 14.

95. The method of claim 94, wherein the amino acid sequence of the EGF repeats of the human Notch receptor protein is identical to the amino acid sequence of EGF repeats 1-36 of human Notch1 encoded by the nucleic acid coding sequence of Notch1 EGF repeats 1-36 set forth in SEQ ID NO: 11.

96. The method of claim 94, wherein the amino acid sequence of the EGF repeats of the human Notch receptor protein is identical to the amino acid sequence of EGF repeats 1-34 of human Notch3 encoded by the nucleic acid coding sequence of Notch3 EGF repeats 1-34 set forth in SEQ ID NO: 13.

97. The method of claim 94, wherein the amino acid sequence of the EGF repeats of the human Notch receptor protein is identical to the amino acid sequence of EGF repeats 1-29 of human Notch4 is encoded by the nucleic acid coding sequence of Notch4 EGF repeats 1-29 set forth in SEQ ID NO: 14.

98. The method of claim 94, wherein the Fc portion of the antibody is the Fc portion of a human antibody.

99. A method of inhibiting angiogenesis in a subject comprising administering to the subject an effective amount of a fusion protein comprising an extracellular domain of a human Notch receptor protein and an Fc portion of an antibody, wherein the extracellular domain of the Notch receptor protein comprises the EGF repeats of such Notch receptor protein and the extracellular domain is linked at its carboxyl terminus to the amino terminus of the Fc portion of an antibody, wherein the Notch receptor protein is a human Notch1, Notch2, Notch3, or Notch4 receptor protein, so as to thereby treat the subject.

100. The method of claim 99, wherein the fusion protein comprises an extracellular domain of a human Notch receptor protein and an Fc portion of an antibody, wherein the extracellular domain of the Notch receptor protein comprises the EGF repeats of such Notch receptor protein and the extracellular domain is linked at its carboxyl terminus to the amino terminus of the Fc portion of an antibody by means of a peptide linker, wherein the Notch receptor protein is a human Notch1, Notch2, Notch3, or Notch4 receptor protein, the amino acid sequence of which is encoded by the nucleic acid sequence set forth in SEQ ID NOs. 11, 12, 13 and 14, respectively, and wherein the extracellular domain is bound to the Fc portion of the antibody by means of a peptide linker which comprises the consecutive amino acids DLGPG (SEQ ID NO:2).

101. The method of claim 99, wherein the fusion protein consists of consecutive amino acids, the amino acid sequence of which, commencing at the amino terminus, is identical to the amino acid sequence of the EGF repeats of a human Notch receptor protein, followed by an amino acid sequence identical to the sequence of an Fc portion of an antibody, wherein the amino acid sequence of the EGF repeats of the human Notch receptor protein is selected from the group consisting of: (a) the amino acid sequence of EGF repeats 1-36 of human Notch1 encoded by the nucleic acid coding sequence of Notch1 EGF repeats 1-36 set forth in SEQ ID NO: 11; (b) the amino acid sequence of EGF repeats 1-36 of human Notch2 encoded by the nucleic acid coding sequence of Notch2 EGF repeats 1-36 set forth in SEQ ID NO: 12; (c) the amino acid sequence of EGF repeats 1-34 of human Notch3 encoded by the nucleic acid coding sequence of Notch3 EGF repeats 1-34 set forth in SEQ ID NO: 13; and (d) the amino acid sequence of EGF repeats 1-29 of human Notch4 is encoded by the nucleic acid coding sequence of Notch4 EGF repeats 1-29 set forth in SEQ ID NO: 14.

102. The method of claim 99, wherein the fusion protein consists of consecutive amino acids, the amino acid sequence of which, commencing at the amino terminus, is identical to the amino acid sequence of the EGF repeats of a human Notch receptor protein, followed by a linker sequence of amino acids which is followed by an amino acid sequence identical to the sequence of an Fc portion of an antibody, wherein the amino acid sequence of the EGF repeats of the human Notch receptor protein is selected from the group consisting of: (a) the amino acid sequence of EGF repeats 1-36 of human Notch1 encoded by the nucleic acid coding sequence of Notch1 EGF repeats 1-36 set forth in SEQ ID NO: 11; (b) the amino acid sequence of EGF repeats 1-36 of human Notch2 encoded by the nucleic acid coding sequence of Notch2 EGF repeats 1-36 set forth in SEQ ID NO: 12; (c) the amino acid sequence of EGF repeats 1-34 of human Notch3 encoded by the nucleic acid coding sequence of Notch3 EGF repeats 1-34 set forth in SEQ ID NO: 13; and (d) the amino acid sequence of EGF repeats 1-29 of human Notch4 is encoded by the nucleic acid coding sequence of Notch4 EGF repeats 1-29 set forth in SEQ ID NO: 14.

103. The method of claim 102, wherein the amino acid sequence of the EGF repeats of the human Notch receptor protein is identical to the amino acid sequence of EGF repeats 1-29 of human Notch4 is encoded by the nucleic acid coding sequence of Notch4 EGF repeats 1-29 set forth in SEQ ID NO: 14.

104. The method of claim 102, wherein the Fc portion of the antibody is the Fc portion of a human antibody.

105. The method of claim 99, wherein the angiogenesis is tumor angiogenesis.

106. The method of claim 99, wherein the subject is afflicted with a pathologic vascular hyperplasia or lymphatic vascular proliferative disease.

Images