Compositions of humanised notch fused proteins and methods of treating

FIELD: medicine.

SUBSTANCE: what is presented is a fused protein that is a Notch1 antagonist, which consists of a human Fc region fused with the EGF-like repeat 1-13 of Notch1 or the EGF-like repeat 1-24 of Notch1. Fc-portion is localised on a carboxy-terminal portion of the EGF-repeat. There are described a pharmaceutical composition for the protein-based Notch signal transmission inhibition and using it for preparing the pharmaceutical composition for treating an individual suffering from: tumour; ovarian cancer; metabolic disorder; vascular proliferative retinopathy. What is presented is using the fused protein for producing the pharmaceutical composition for inhibition: angiogenesis in the individual; physiological lymphangiogenesis or pathological lymphangiogenesis in the individual; tumour deposits in the individual.

EFFECT: using the invention provides the proteins expressed in a supernatant at a level by several times more than the fused protein containing the EGF-like repeats 1-36 of Notch1; they penetrate into the tumour better, maintain a ligand-binding ability with the fused protein containing the repeats 1-24, binds to DLL4 and JAG1, whereas the fused protein containing the repeats 1-13 only binds to DLL4, but not to JAG1 that can find application in therapy of various diseases related to the Notch1 activity.

18 cl, 124 dwg, 10 ex

 

This application claims the priority of provisional application U.S. No. 60/966052, filed August 23, 2007, the contents of which are incorporated herein by reference.

The invention described herein was made with U.S. government support under grant R01 HL62454 from the National Institutes of Health and grant number DAMRDCW81XWH-04-1-054 and grant number DAMD17-03-1-0218 from the Department of Defense. Thus, the U.S. government has certain rights in this invention.

Throughout the application, various publications are marked by Arabic numerals in parentheses or by author and date of publication in parentheses. Full references for these publications are presented in the end of the description. Descriptions of these publications are included as references in this application for a more complete description of the field to which this invention relates.

The prior art to which the invention relates

The development of blood vessels

During mammalian embryogenesis, the formation of the vascular system is an early and necessary process. The embryonic development of blood vessels begins with IPS hemangioblasts derived from the paraxial mesoderm and the lateral plate. Hemangioblast has the potential to differentiation or hematopoietic precursor or precursor endothelial cells, known as ang is the sphere.

The development of blood vessels begins with a process known as vasculogenesis where angioplasty differentiate into endothelial cells and migrate together with the formation of the embryonic vascular plexus. This initial vascular network consists of vessels that are uniform in size and are composed of endothelial cells. Then there is remodeling of the vascular plexus through angiogenesis.

Angiogenesis involves the growth of new vessels, the migration of these vessels in the avascular region and attraction of auxiliary cells, pericytes and smooth muscle cells (Gale and Yancopoulos, 1999). The smooth muscle cells that differentiate to form the contractile blood vessels originate from a variety of precursors, including cells of the nervous roller, mesenchymal cells, and even endothelial cells (Owens, 1995). In adults, angiogenesis is involved in follicular development, wound healing and pathological processes such as angiogenesis in cancer and heart disease.

The family of Notch and Notch ligands

Studies in Drosophila, C. Elegans, Danio and mammals have shown that the Notch cascade is an evolutionary conservative mechanism of signal transmission that is operated, modulating many choices of cell fate. Signal transmission Notch is required for the proper article is actuarialy cells, originating from all three germ layers. Depending on the cellular environment, the signal transmission Notch can both inhibit and induce differentiation and to induce proliferation and survival of cells (Artavanis-Tsakonas et al., 1995; Lewis, 1998; Weinmaster, 1997). In Drosophila, one Notch protein is activated by two ligands, Serrate and Delta. In mammals, this family expanded to four Notch genes (Notch1, notch2 is, Notch3 and Notch4) and five ligands, 2 Serrate-like (Jagged1-2) and (3 Delta (Dll, 3, 4) (Bettenhausen et al., 1995; Dunwoodie et al., 1997; Gallahan and Callahan, 1997; Lardelli et al., 1994; Lindsell et al., 1995; Shawber et al., 1996a; Shutter et al., 2000a; Uyttendaele et al., 1996; Weinmaster et al., 1992; Weinmaster et al., 1991). In the process of embryogenesis receptors and Notch ligands are expressed in dynamic spatial and temporal profiles. However, it is not known to activate if all the ligands all receptors.

Signal transmission and the function of Notch

Signal transmission Notch affects many different choices of cell fate by providing inhibitor, inductive or proliferative signals, depending on environmental conditions (reviewed in Artavanis-Tsakonas et al., 1995; Greenwald, 1998; Robey, 1997; Vervoort et al., 1997). This pleiotropic function indicates that Notch modulates the many cascades of signal transmission space-time image.

In accordance with the regulation of Notch selection of cell fate, as receptors and ligands to depict ablaut a cell surface proteins with single transmembrane domains (figure 1). Regulatory extracellular domain of Notch proteins is mainly composed of tandem EGF-like repeats that are required for binding of ligand (Artavanis-Tsakonas et al., 1995; Weinmaster, 1998). From C-Terminus of EGF-like repeats are three additional cysteine-rich repeat, denoted as replays LIN12/Notch (LNR) (Greenwald, 1994). Below LNR is a sequence of proteolytic cleavage (RXRR), which recognized furin-like convertases. In the case of Notch1 cleavage at this site leads to extracellular peptide size 180 kilodaltons and intracellular peptide size 120 kilodaltons, which are held together with the formation of a heterodimeric receptor on the cell surface (Blaumueller et al., 1997; Kopan et al., 1996; Logeat et al., 1998).

The intracellular domain of Notch (NotchICD, figure 1) restores the phenotypes after loss of Notch function, which indicates that this form of Notch signals and constitutive (Fortini and Artavanis-Tsakonas, 1993; Lyman and Young, 1993; Rebay et al., 1993; Struhl et al., 1993).

The cytoplasmic domain of Notch contains three identifiable domain, RAM domain, domain ankerinho repeat and C-terminal PEST domain (figure 1). Upon activation of the Notch ligand undergoes two additional proteolytic cleavage, which leads to the release of the cytoplasmic domain (Weinmaster, 1998). This peptide Notch is transferred to the nucleus and interaction of the light with the transcription repressor substances, known as CSL (CBF, Su (H), Lag-2) and turns them into an activator of transcription. Interaction CSL/Notch depends on the availability of RAM-domain Notch; although transcriptional activity also requires the presence anchirinah repeats (Hsieh et al., 1996; Hsieh et al., 1997; Roehl et al., 1996; Tamura et al., 1995; Wettstein et al., 1997). Research asin vivoandin vitroindicate that genesHESandHeyare direct targets dependent on Notch/CSL signal (Bailey and Posakony, 1995; Eastman et al., 1997; Henderson et al., 2001; Jarriault et al., 1995; Nakagawa et al., 2000; Wettstein et al., 1997). GenesHESandHeyrepresent the bHLH repressor of transcription that binds DNA at the N-boxes (Nakagawa et al., 2000; Sasai et al., 1992; Tietze et al., 1992). It was also hypothesized that Notch transmits a signal through independent CSL cascade. In fact, expression of only the domain ankerinho repetition is necessary and sufficient for some forms of signal transmission Notch (Lieber et al., 1993; Matsuno et al., 1997; Shawber et al., 1996b).

In conclusion, PEST-domain involved in protein turnover through dependent SEL-10/ubiquitin cascade (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 Notch ligands consists, mainly, of the tandem EGF-like repeats (figure 1). Above these repetitions is divergent EGF-like repeat, known as DSL (Delta, Serrate, Lag-2), which is required on the I-binding ligand and activation receptor (Artavanis-Tsakonas et al., 1995).

Signal transmission Notch and the development of blood vessels

Although identified many of the genes that function induction vasculogenesis and angiogenesis, little known about how to choose the fate of cells during the development of blood vessels. The number of observations indicates that the cascade signal transmission Notch may be involved in determining the fate of cells and the formation of the vascular system.

Notch1, Notch4, Jagged1 and Dll4 is expressed in the developing vessels, and Notch3 is expressed in the supporting smooth muscle cells (Krebs et al., 2000; Shutter et al., 2000b; Uyttendaele et al., 1996; Villa et al., 2001; Xue et al., 1999). Mice deprived of Jagged1 are lethal at the embryonic stage and have several severe vascular defects (Xue et al., 1999). The mouse, which is the zero-zygote in Notch1 are lethal at the embryonic stage and die from severe neuronal defects, but they also have defects in angiogenesis (Krebs et al., 2000; Swiatek et al., 1994). Mice deprived of Notch4, born and have a normal appearance, but the embryos have lost both Notch1 and Notch4 die on stage E9.5 from severe hemorrhage and defects in the structure of blood vessels, which indicates that Notch1 and Notch4 may functionally overlap during the development of blood vessels (Krebs et al., 2000). Exogenous expression of an activated form of Notch4 in the endothelium also leads to vascular defects, the initial defects, observed for double null zygote mice by Notch1/Notch4, indicating that the respective signal transmission Notch are critical for the proper development of blood vessels of the embryo (Uyttendaele et al., 2001).

Taken together, the data for mice mutant for components of the signal transmission Notch/Notch, explain some of the processes that are dependent on Notch, including vascular remodeling, arterial-venous characteristics, involvement of smooth muscle cells of blood vessels and the development of vessels of the heart/cardiac outflow.

Recent experiments indicate the involvement of signal transmission Notch in the characteristics of arterial/venous endothelial cells. Analysis ofin situof E13 embryos.5 revealed that the expression of Notch1, Notch3, Notch4, D14, Jagged1 and Jagged2 limited to arteries and veins (Villa et al., 2001). In accordance with the data about the expression, disruption of signal transmission Notch at Danio associated with loss of arterial marker ephrinB2; and ectopic expression of an activated form of Notch leads to loss of venous marker EphB4 cells in the dorsal aorta (Lawson et al., 2001). These data indicate that signaling Notch can aid in the determination of the fate of the arterial and venous cells during angiogenesis.

Taken together, the data for mice mutant for components of the signal transmission Notch/Notch, explain some of the processes related to the s from the Notch, including vascular remodeling, arterial-venous characteristics, involvement of smooth muscle cells of blood vessels and the development of vessels of the heart/cardiac outflow.

It was also assumed that the signal transmission Notch functions in the vascular system of adults. In humans, missense mutations in the extracellular domain of the Notch3 correlate with the development of degenerative vascular diseases CADASIL (Caronti et al., 1998; Desmond et al., 1998; Joutel et al., 2000; Joutel et al., 1996). In a model of wound healing increased expression of Jagged1 was observed during regeneration of endothelial wound edges, indicating that the signal transmission Notch may function in the processes of angiogenesis in adults (Lindner et al., 2001). Taken together, these data indicate transfer functions of the signal Notch on the number of critical stages in the development process vessels: vasculogenesis, the formation of vessels/angiogenesis and arterial/venous feature. However, the molecular mechanism(s)by which cascades of signal transmission Notch affect these different stages, remains to be seen.

Value

Shimizu et al. (J. Biol. Chem. 274(46): 32961-32969 (1999)) describe the application of Notch1ECD/Fc, Notch2ECD/Fc and Notch3ECD/Fc research associate. However, Shimizu et al. do not mention the use of such proteins for inhibition of angiogenesis.

In U.S. patent No. 6379925, issued April 30, 2002, Kitajewski et al., described Notch4 mouse. But N. is described fused proteins on the basis of the Notch, as shown in the specified application.

Proteins Notch play a key role in development-related choices involving vascular, hematopoietic system and the nervous system. Essentially, the understanding of their function is key to understanding how the choice of cell fate and commitirovannah controlled during development and in adult tissues. To date, few reports of violation of the genes of the Notch ligand or Notch described phenotypes vessels and draws attention to the fact that this cascade is a fundamental part of the device that directs the development of blood vessels. Aberrant Notch activity is associated with pathologies in humans, including both malignant tumor, and vascular disorders (CADASIL). Analysis of Notch in angiogenesis in the tumor started only recently; however, the discovery of the inventors of potential targets for Notch below the cascade, indicates a role in pathological processes associated with angiogenesis. For example, VEGFR-3 is associated with tumor angiogenesis and lymphangiogenesis tumors. The expression or the function of several other potential targets of Notch is also associated with angiogenesis in tumors, including ephrinB2, Id3, angiopoietin-1 and PDGF-B.

To elucidate the role of these targets in gene function Notch, obviously, can facilitate future analysis of the Notch at pathologies in humans.

This invention relates to fused protein containing the signal peptide, extracellular domain of the receptor protein Notch person and the Fc part of the antibodies associated with them.

This invention relates to a method of treatment of a subject having a tumor, comprising an introduction to the principal amount specified above, fused protein, is effective for the treatment of the subject, thereby effecting treatment of a subject having a tumor.

This invention relates to a method of inhibiting angiogenesis in a subject comprising administration to the subject of the amount specified above, fused protein, effective to inhibit angiogenesis in the subject, thereby inhibiting angiogenesis in the subject.

This invention relates to a method of treatment of a subject having ovarian cancer comprising administration to the subject of the amount specified above, fused protein, is effective for the treatment of the subject, thereby effecting treatment of a subject having ovarian cancer.

This invention relates to a method of treatment of a subject having a metabolic disorder, comprising administration to the subject of the amount specified above, fused protein, is effective for the treatment of the subject, thereby effecting treatment of a subject having a metabolic disorder.

This invention relates to the use of the above fused protein to obtain the pharmaceutical companies who stand to treatment of the subject, having a tumor.

This invention relates to the use of the above fused protein to obtain a pharmaceutical composition for inhibiting angiogenesis in the subject.

This invention relates to the use of the above fused protein to obtain a pharmaceutical composition for the treatment of a subject having ovarian cancer.

This invention relates to the use of the above fused protein to obtain a pharmaceutical composition for the treatment of a subject having a metabolic disorder.

This invention relates to a method of inhibiting the physiological lymphangiogenesis or pathological lymphangiogenesis the subject, including the introduction of the principal amount specified above, fused protein, effective for the inhibition of physiological lymphangiogenesis or pathological lymphangiogenesis the subject.

This invention relates to a method of inhibiting metastasis of a tumor in a subject comprising administration to the subject of the amount specified above, fused protein, effective for inhibiting metastasis of a tumor in the subject.

This invention relates to a method of inhibiting the growth of a secondary tumor in a subject comprising administration to the subject of the amount specified above, fused protein, effective to inhibit the growth of secondary tumors in su is the target.

This invention relates to a method for inhibiting the co-opting of the blood vessels of the tumour in a subject, comprising administration to the subject of the amount specified above, fused protein, effective for the inhibition of the co-opting of the blood vessels of the tumor in the subject.

This invention relates to a method of treating a malignant tumor in a subject comprising administration to the subject of the above fused protein and inhibitor of growth factor vascular endothelial (VEGF), VEGF-A, P1GF, VEGF-B, VEGF-C or VEGF-D, each of them in a quantity effective for the treatment of malignant tumors in the subject.

This invention relates to a method of treating a malignant tumor in a subject comprising administration to the subject of the above fused protein and the VEGF receptor antagonist, an antagonist of VEGFR-1, an antagonist of VEGFR-2 or VEGFR antagonist-3, each of them in a quantity effective for the treatment of malignant tumors in the subject.

This invention relates to a method of treating a malignant tumor in a subject comprising administration to the subject of the above fused protein and inhibitor of platelet-derived growth factor (PDGF), PDGF-A or PDGF-B, each of them in a quantity effective for the treatment of malignant tumors in the subject.

This invention relates to a method of treating a malignant tumor in a subject comprising administration to the subject is shown above the fused protein and the PDGF receptor antagonist, each of them in a quantity effective for the treatment of malignant tumors in the subject.

This invention relates to a method of treating a malignant tumor in a subject comprising administration to the subject of the above fused protein and an inhibitor of HER2/neu, each of them in a quantity effective for the treatment of malignant tumors in the subject.

This invention relates to a method for the treatment of vascular proliferative retinopathy, including introduction to the subject of the above fused protein in an amount effective for the treatment of vascular proliferative retinopathy.

Brief description of drawings

Figure 1

This figure presents a schematic structure of Notch and Notch ligands: Notch1, notch2 is, Notch3, Notch4, Jagged-1, Jagged-2, Delta-like 1, Delta-like 3, Delta-like 4.

Figure 2 :

This figure shows a schematic representation of the fused protein on the basis of the Notch (NotchECD/Fc). The extracellular domain of Notch1, notch2 is, Notch3 or Notch4 containing EGF-repeats, is fused with the Fc part of antibodies.

Figure 3

This figure presents the analysis of coculture to test the activity of the fused protein on the basis of the Notch. Notch and Notch transcriptional reporters expressed in "answering Notch" cells, HeLa. The Notch ligands, Jagged-1, Delta-like 1 and Delta-like 4 is expressed in "predstavila ligand" cell, 293. Carry out the expression by t is inspectie separate cell populations, cells will cocultured, and then analyze in respect of Notch-dependent activity of the reporter.

Figure 4

This figure presents the inhibitory activity of the fused protein on the basis of the Notch against the activation signal transmission Notch by the interaction between Notch and Notch ligand. Induction signal transmission Notch was detected by cocultivation cells expressing the Notch ligand of both types, Notch1 and 3, and this induction is inhibited by cotransfection fused protein on the basis of expressing the Notch of the vector in cells expressing Notch1. Thus, based on the inhibition of the interaction between Notch and Notch ligand, fused proteins on the basis of the Notch can be used as an inhibitor of the Notch.

Figure 5

This figure presents the expression of the fused protein on the basis of Notch1 (Notch1ECD/Fc) 293. Panel A: expression in cell lysates (lys) or secretion into the medium (sup). Panel B: expression of NECD/Fc in lysates of 293 as specified.

Figure 6

This figure presents the activation signal transmission Notch in HUVEC infected with adenovirus encoding VEGF-165. Activation of signal transmission Notch can be identified using activity CBF1 promoter. Transcriptional activity of CBF1 promoter is stimulated by binding of Notch-IC with CBF1. The authors of the present invention measured the activity of the promoter of CBF1 in HUVEC, which were infection is ofany adenovirus, encoding VEGF-165 at different MOI. Induction of CBF1 promoter was clearly detected in HUVEC infected with Ad-VEGF compared with those infected with Ad-LacZ cells dependent on MOI way. These data showed that overexpression of VEGF can activate the signal transmission Notch in HUVEC.

Figure 7

This figure presents the effect of the fused protein on the basis of the Notch on inducible VEGF activation signal transmission Notch. Confezione design Ad-fused protein on the basis of the Notch and Ad-VEGF clearly reduced the stimulation of the activity of CBF1 promoter induced by infection with only Ad-VEGF. In the case of infection with an MOI of 40 for each of adenovirus in panel A, were detected in 60% inhibition after 24 hours and 90% inhibition at 48 hours after transfection reporter gene. This inhibitory activity traps Notch depended on MOI design Ad-fused protein on the basis of the Notch.

Figure 8

This figure presents an experiment in which the inventors evaluated the effect of the fused protein on the basis of the Notch on the induction of budding through sverhagressivnomu VEGF-165 in HUVEC. When HUVEC infected with Ad-VEGF, were cultured on collagen gel type for 8 days in collagen gel were induced budding. This induction of budding sverhagressivnym VEGF clearly inhibited by Confucianism adenovirus coding for fused be the key based on the Notch. Independently design Ad-fused protein on the basis of the Notch has a lesser effect on the morphology.

Figure 9

This figure presents the result of counting under a microscope the number of buds on the field. Infection of HUVEC by Ad-VEGF increased the number of buds, depending on the MOI. Even despite the fact that he used half of the MOI for the fused protein on the basis of the Notch, compared with Ad-VEGF-induced Ad-VEGF budding clearly inhibited. These data indicate that VEGF induces the formation HUVEC through activation of signal transmission Notch, and protein on the basis of the Notch may inhibit induced VEGF budding.

Figure 10

This figure presents the amino acid sequence of the extracellular domain of Notch1 protein rat (SEQ ID NO:1) and the linker sequence (SEQ ID NO:2).

Figure 11

This figure presents the amino acid sequence of the extracellular domain of the protein notch2 is rat (SEQ ID NO:3) and the linker sequence (SEQ ID NO:2).

Figure 12

This figure presents the amino acid sequence of the extracellular domain of the Notch3 protein mouse (SEQ ID NO:4).

Figure 13

This figure presents the amino acid sequence of the extracellular domain of Notch4 protein mouse (SEQ ID NO:5) and the linker sequence (SEQ ID NO:2).

Figures 14A and 14B

This is the figure presents the nucleic acid sequence of the extracellular domain of Notch1 gene rat (SEQ ID NO:6).

Figures 15A and 15B

This figure presents the nucleic acid sequence of the extracellular domain of the gene notch2 is rat (SEQ ID NO:7).

Figures 16A and 16B

This figure presents the nucleic acid sequence of the extracellular domain of the Notch3 gene mouse (SEQ ID NO:8).

Figures 17A and 17B

This figure presents the nucleic acid sequence of the extracellular domain of Notch4 gene mouse (SEQ ID NO:9) and nucleic acid sequence (SEQ ID NO:10) and amino acid sequence (SEQ ID NO:2) linker sequence.

Figures 18A and 18B

This figure presents the nucleic acid sequence of the extracellular domain of Notch1 gene of human (SEQ ID NO:11).

Figures 19A and 19B

This figure presents the nucleic acid sequence of the extracellular domain of the gene notch2 is human (SEQ ID NO:12).

Figures 20A and 20B

This figure presents the nucleic acid sequence of the extracellular domain of the Notch3 gene of human (SEQ ID NO:13).

Figures 21A and 21B

This figure presents the nucleic acid sequence of the extracellular domain of Notch4 gene of human (SEQ ID NO:14).

Figures 22A-22I

On these figures it is shown that VEGF activates the signal transmission Notch inducyruya budding HUVEC. HUVEC were transducible Ad-VEGF at MOI 40 (Fig. 22A, 22H, 22I) or MOI 20 (Fig. 22C, 22G). Ad-LacZ to what was transducible in HUVEC so, that the total number of adenovirus was MOI 60 (Fig. 22G), MOI 80 (Fig. 22A) and MOI 100 (Fig. 22H, 22I). In the figure 22A presents the analysis by RT-PCR the expression of Notch and Notch ligand. Numbers indicate the number of PCR cycles. In the figure 22B presents the effect transducing VEGF on reporter activity CSL. In the figure 22C presents the effect of SU5416 on reporter activity CSL, transactionmanager Ad-VEGF. Figure 22D shows the design of the trap Notch (N1ECDFc). In the figure 22E presents secretion N1ECDFc from HUVEC, transduced by Ad-N1ECDFc. In the figure 22F presents the effect N1ECDFc against induced by ligand reporter activity CSL analysis coculture(□: (-); ■: 0,33 ng pHyTC-N1ECDFc; ■: 0,67 ng pHyTC-N1ECDFc). On the figures 22G-I presents the effect N1ECDFc against HUVEC, transduced by Ad-VEGF. Signal transmission Notch enforced during transduction with Ad-VEGF in HUVEC in the absence or presence of atransducer Ad-N1ECDFc in the specified dosage. In the figure 22G presents the effect N1ECDFc on reporter activity CSL, transactionmanager Ad-VEGF. In the figure 22H presents the inhibition of budding HUVEC, transduced by Ad-VEGF, if atransducer Ad-N1ECDFc at MOI of 40. In the figure 221 presents quantitative determination of the effect N1ECDFc on the split in HUVEC, transduced by Ad-VEGF (□: kidney; ■: number of cells).

Figures 23A-23J

On these figures it is shown that the signal transmission Notch increases the expression of Flt1, and utiroa budding HUVEC. HUVEC were transducible either Ad-LacZ or Ad-N1IC at MOI of 40. In figures 23A-23C presents the effect of inhibitors of receptor tyrosinekinase on induced Notch budding HUVEC. Figure 23A shows a photograph of budding HUVEC, transduced by Ad-N1IC-treated PD166866, ZD1893 in quantities of 1 μm and SU5416 in the amount of 0.5 microns. In the figure 23B presents quantitative determination of the effect of inhibitors in quantities of 1 μm (□: kidney; ■: number of cells). Figure 23C shows the dependence of the dose effect of SU5416 (□: kidney; ■: number of cells). On the figures 23D-E presents the induction of the expression of Flt-1 in HUVEC, transduced by Ad-N1IC. In the figure 23D presents an analysis by RT-PCR the mRNA expression of Flt-1. In the figure 23E presents W. B.-analysis of gene protein Flt-1. On the figures 23F-G presents stimulation induced Notch of budding HUVEC by stimulation of P1GF. HUVEC transduced with Ad-N1IC, were cultured on collagen gel with SFM, instead of the full environment, in the absence or in the presence of 50 ng/ml P1GF. In the figure 23F presents induced P1GF budding HUVEC, transduced by Ad-N1IC (the end of the arrow: kidney with one filopodia; arrows: buds with a variety of filopodia). In the figure 23G presents quantitative determination of the effect of P1GF in the budding HUVEC, transduced by Ad-N1IC (□: many;■:. On the figures 23H-I presents the effect of siRNA transfection for Flt-1 on the expression of Flt1. HUVEC, razdolbannye Ad-N1IC, was transfusional 200 pmol of either the control (CT) siRNA or siRNA for Flt-1. In the figure 23H presents the decrease in the mRNA expression of Flt-1. In the figure 23I presents the decrease in the expression of the protein Flt-1. In the figure 23J presents the effect of siRNA transfection for Flt-1 induced Notch budding HUVEC. HUVEC transduced with Ad-N1IC, transfusional either 100 or 200 pmol siRNA and cultured on collagen gel within 2 days.

Figures 24A-24E

On these figures it is shown that VEGF regulates the activity gelatinase through the signal transmission by activating Notch as MMP-9 and MT1-MMP. In figures 24A-B presents the analysis gelatinases demografia activity of MMP-9 and MMP-2, stimulated VEGF in HUVEC. In the figure 24A presents the effect N1ECDFc on the activity of MMP-9. Transduced HUVEC were cultured on fibrin gel on the specified day (i.e., D2, D4, D6, D8). Similar results were also obtained with the use of collagen gel, although the induction of MMP-9 was stronger on fibrin gel than on collagen gel (data not shown). In the figure 24B presents the effect N1ECDFc on the activity of MMP-2. HUVEC were transducible Ad-N1ECDFc these doses and collected conditioned medium from HUVEC, cultured on collagen gel on the 4th day. In figures 24C-D presents the activation of MMP-9 and MT1-MMP by sending a signal Notch. HUVEC were transducible through either Ad-LacZ or Ad-N1IC at MOI of 40. The numbers of the criminal code which shows the number of PCR cycles. In the figure 24C presents an analysis by RT-PCR effect signal transmission Notch on the expression of MMP-9 and MMP-2. In the figure 24D presents the induction of expression of both transcript and protein MT1-MMP during signal transmission Notch. In the figure 24E presents an analysis by RT-PCR the expression of MMP-9 and MT1-MMP in Ad-VEGF HUVEC if atransducer Ad-N1ECDFc. HUVEC were transducible Ad-VEGF in the absence or presence of atransducer Ad-N1ECDFc at MOI of 40 for each of them. Ad-LacZ was cotranslationally with obtaining the total number of adenovirus comprising MOI 80.

Figures 25A - 25D

In these figures shows the role of signal transmission Notch dependent on VEGF angiogenesisin vivo. In figures 25A-25D presents inhibition induced VEGF angiogenesis through N1ECDFc in the analysis of the DAS in the mouse. Presents representative pictures. In the figure 25A presents subcutaneous induced angiogenesis for transfectant 293/VEGF against 293/VEGF expressing the trap Notch (protein-based Notch) N1ECDFc. In the figure 25B presents the quantitative determination of the degree of vascularization induced 293/VEGF in control against 293 expressing trap Notch (protein-based Notch) N1ECDFc. In the figure 25C presents subcutaneous induced angiogenesis for infected with Ad-LacZ cells MDA-MB-231 against infected with Ad-N1ECDFc (protein-based Notch) cells MDA-MB-231. The breast cancer cells MDA-MB-231 is reducyruet VEGF (data not shown). In the figure 25D presents the quantitative determination of the degree of vascularization induced infected with Ad-LacZ cells MDA-MB-231 against infected with Ad-N1ECDFc (protein-based Notch) cells MDA-MB-231.

Figures 26A and 26B

In these figures shows the proliferation of HUVEC, transduced by Ad-VEGF165. HUVEC were transducible Ad-VEGF165 in these dosages. Also were co-infected with Ad-LacZ, that the total number of adenovirus was MOI 40 b.about.E./cell. HUVEC suspended in SFM supplemented with 1% FBS, and then were sown in the amount of 1×104cells/well in 24-hole advance tablets with 0.4 ml of medium. After 4 days to determine the number of cells using the set of CCK-8, and the results are presented as the ratio of specific numbers of cells and the number of control cells, which were transpulmonary Ad-GFP at MOI 40 b.about.E./cell. In the figure 26A presents the effect transducing VEGF on the proliferation. In the figure 26B presents the inhibitory effect of SU5416. HUVEC transduced with Ad-VEGF, was treated with SU5416 in these dosages.

Figures 27A and 27B

In these figures shows the induction of kidney HUVEC on collagen gel type I. HUVEC were transducible or Ad-VEGF165 or AD-N1IC in these dosages. Also conditionaly Ad-LacZ to the total number of adenovirus was MOI 40 b.about.E./cell. Transduced HUVEC were cultured on the collagen is m the gel with a full environment. The value of budding was evaluated under the microscope on day 7.

Figures 28A and 28B

In these figures shows the effect of changing the signal transmission Notch on cell proliferation. The cells were transducible the indicated adenoviruses. Also spent confezione Ad-GFP to the total number of adenovirus was MOI 60 b.about.E./cell. After 4 days was determined by the number of cells using the set of CCK-8, and the results are presented as the ratio of specific numbers of cells and the number of control cells, which were transpulmonary Ad-GFP at MOI 60 b.about.E./cell. In figure 28A presents the effect transducing N1IC and merged Notch protein on the proliferation of HUVEC. Transduced HUVEC suspended in complete medium, and then were sown in the amount of 1×104cells/well in 24-hole advance tablets with 0.4 ml of the specified environment (□: Ad-N1IC; ■: Ad-N1ECDFc). In the figure 28B presents the effect of the slit Notch protein on the proliferation of transfectants KP1/VEGF. Transduced the transfectants KP1/VEGF suspended in RPMI1640 medium, and then were sown in the amount of 2×104cells/well in 24-hole advance tablets with 0.5 ml of medium.

Figure 29

This figure presents the analysis by RT-PCR induction of expression of PIGF in HUVEC, transduced by Ad-N1IC. HUVEC were infected with either Ad-LacZ or Ad-N1IC at MOI 40 b.about.E./cell. Total RNA was isolated from transducers the data HUVEC, cultured on collagen gel for 5 days with complete medium.

Figures 30A-30C

In these figures shows the inhibition of budding HUVEC, transduced with either Ad-N1IC or Ad-VEGF by transfection of siRNA for Flk-1. Figure 30A shows a decrease in the expression of mRNA and protein Flk-1 in Ad-VEGF-when HUVEC transfection with 200 pmol of siRNA for Flk-1. Ad-VEGF HUVEC at MOI 40 b.about.E./the cell was transfusional 200 pmol of either control siRNA (CT)or siRNA for Flk-1. 48 hours after transfection were isolated total RNA. Have collected a total cell lysate from cells that are not fed serum with SFM within 48 hours after transfection. In figures 30B and 30C presents the inhibitory effect of siRNA transfection for Flk-1 in kidney HUVEC induced by either VEGF or Notch. Or Ad-N1IC-or Ad-VEGF HUVEC at MOI 40 b.about.E./the cell was transfusional 200 pmol siRNA, as indicated, and were cultured on collagen gel for 5 days. In the figure 30B presents the effect of siRNA transfection for Flk-1 in kidney HUVEC (□: Ad-VEGF; ■: Ad-N1IC). In the figure 30C presents quantitative determination of the inhibitory effect of siRNA transfection for Flk-1.

Figures 31A and 31B

In these figures shows the inhibition of budding HUVEC, transduced by Ad-N1IC, when processing inhibitor of matrix metalloproteinase GM6001. Either Ad-LacZ or Ad-N1IC-HUVEC at MOI 40 b.about.E./cell were cultured on collagen gel for 5 days in the absence or risotti GM6001 in the amount of 50 μm. In the figure 31A presents the effect of GM6001 on induced Notch kidney HUVEC. In the figure 31B presents quantitative determination of the inhibitory effect of GM6001.

Figures 32A-32D

On these figures it is shown that the trap Notch1 inhibits the activation of signal transmission Notch stimulated by Notch ligands. Figure 32A shows a diagram of the trap Notch1 (N1ECDFc) and Western blotting for detection of secreted traps Notch1 in air-conditioned environment. HUVEC were tranditional adenovirus coding for the trap of Notch1 (Ad-N1ECDFc), for a given m.o.i. Figure 32B shows that the trap Notch1 inhibits ligand-induced reporter activity CSL in the analysis of signal transmission in coculture. The activation signal transmission Notch was measured in HeLa cells expressing Notch1, coculturing with 293 cells expressing Notch ligands. Data are presented as mean value ± SD. Figure 32C shows that ectopic expression of Notch4 induces morphogenesis HUVEC, cultured on fibrin gel. HUVEC were transducible adenovirus coding for Notch4 (Ad-Notch4) at m.o.i. 30 and Ad-GFP m.o.i. at 10, for labeling of infected cells. Two days HUVEC were cocultivated stable transfectants HUVEC on fibrin gel and documented morphological changes using fluorescence microscopy. Notch 4 induces elongation of cells (top following is a, white arrows), and the processing of 200 nm compound E blocks induced Notch4 elongation (top right). The expression of trap blocking Notch1 induced Notch4 elongation of cells. Transduced with adenovirus HUVEC were cocultivated on fibrin gels with a stable transfectants HUVEC expressing either Fc (bottom left), or the trap of Notch1 (top right), and photographed two days. Scale = 200 ám. In the figure 32D presents quantitative determination of the effect of inhibition of Notch signal induced on Notch4 elongation. The reduction in germination of sprouts was statistically significant after treatment with compound E and transduction N1ECDFc (p<0,0001, in both cases; data are presented as mean value ± SD).

Figures 33A-33D

On these figures it is shown that FGF4 induces the expression of Notch ligands in the cells of breast carcinoma mouse Mm5MT. Stable transfectants Mm5MT received retroviral gene transfer. In the figure 33A presents quantitative analysis by RT-PCR the expression of Notch ligands, demonstrating the induction of Jagged1 and Dll1 in the transfectants Mm5MT expressing FGF4 (Mm5MT-FGF4), compared to simulating the transfectants (Mm5MT-X). Figure 33B shows that the level of Jagged1 protein increased in Mm5MT-FGF4 relatively Mm5MT-X, when determined by Western blotting. Figure 33C shows the decrease in expressionand Notch in cells Mm5MT-FGF4 with PD166866, kinase inhibitor of the FGF receptor. In the figure 33DB shows immunohistochemical analysis of the staining of Jagged1 in the transfectants Mm5MT. Scale = 50 ám.

Figures 34A-34C

On these figures it is shown that the trap Notch1 inhibits angiogenesis and subcutaneous tumor growth of tumors Mm5MT-FGF4 in mice. Figure 34A shows that the volume of tumors Mm5MT-FGF4-X and Mm5MT-FGF4-Fc significantly different from transfectants Mm5MT-FGF4-N1ECDFC mice (day 21, P=0,037 and P=0.008, Mm5MT-FGF4-X and Mm5MT-FGF4-Fc against Mm5MT-FGF4-N1ECDFc, respectively; data are presented as mean value ± SD). In the figure 34B presents immunohistochemical analysis of new blood vessels by staining of CD31 in tumor transfectants Mm5MT-FGF4. Top panel, scale = 100 µm, bottom panel, scale = 50 µm. In the figure 34C presents quantitative analysis showing the reduction of new blood vessels CD31(+) transfectants Mm5MT-FGF4-N1ECDFc compared with tumors, transfitsirovannykh Fc or imitation (P<0,001, for Mm5MT-FGF4-X and Mm5MT-FGF4-Fc against Mm5MT-FGF4-N1ECDFc; data are presented as mean value ± SD). The xenografts were collected 22 days after inoculation and stained with antibody against CD31.

Figures 35A-35D

On these figures it is shown that the expression of trap Notch1 disrupts angiogenesis and reduces the viability of a tumor xenografts NGP person. The inventors have previously reported that these Sinotrans entity human neuroblastoma in mice have Mature hierarchical vascular network, which is relatively resistant to VEGF blockade (16). To determine whether activation of the Notch receptor for angiogenesis NGP, the authors present invention has transfusional cells NGP design traps Notch1, which did not affect their ability to grow in culture (data not shown). However, there was marked reduction in the viability of thein vivo(figure 35A) TUNEL = red fluorescence, erythrocytes = green fluorescence, scale = 100 µm) (figure 35B) significantly increased apoptosis of tumor cells (P=0.0002, TUNEL-positive cells in tumors NGP-N1ECDFc against NGP-LacZ), and (figure 35C) increased intratumoral haemorrhage (p<0,0001, quantification of parenchymal signal of erythrocytes). In addition, the vascular network of tumors in xenografts NGP-N1ECDFc look physically destroyed compared with controls NGP-LacZ, (figure 35D) immunological staining of EC and VMC (using antibodies against CD31 and αSMA, respectively), demonstrating the lack of integrity of these layers of vascular cells (scale = 50 ám). Individual cells of blood vessels appear separated from each other. Taken together, these results indicate that the expression of trap Notch1 impairs EC and VMC to form stable vascular tubes, causing the breaking of the vessels, hemorrhage and ischemia tumor tissues.

Figo is a 36

This figure shows that SKOV3 tumor cells with the programmed expression of traps Notch1 block the growth of xenografts of ovarian cancer.

Figure 37

This figure shows the regulation of the differentiation of cultured myoblasts by Foxo and Notch. The C2C12 cells were subjected to immune staining with an antibody against myosin (green) and DAPI (blue). Cm. the text for the description panel. Each experiment was repeated ≥ six times.

Figures 38A-38C

In these figures shows a quantitative analysis of C2C12 differentiation. Figure 38A shows a Western blot analysis of the expression of myosin in C2C12 cells. In the figure 38B presents morphometric analysis of myosin-positive cells. The results of experiments on the differentiation was analyzed by estimating the number of immunologically stained myosin cells as a percent of all DAPI-positive cells. Figure 38C shows the analyses of reporter gene DBD-Foxo1ADA. The inventors have conducted tests with reporter gene using the canonical answer Foxo1 promoterIgfbp1(left panel) and promoterHes1(right panel) in cells, cotransfection Foxo1-ADA or DBD-Foxo1ADA. The Western blot demonstrates (box)that the levels of expression of the two proteins are similar. The asterisk indicated P<0,01 through ANOVA.

Figure 39A-39E

These figures pok what are coimmunoprecipitate Foxo1 with Csl. a. Coimmunoprecipitate endogenous Foxo1 and Csl in C2C12 cells, coculturing with HEK293 cells expressing LacZ (marked with "-") or Jagged1 (marked with "+"). b-c. Experiments with coimmunoprecipitate in C2C12 cells, cotransfection FLAG-Csl and HA-Foxo1. d-e. Experiments with coimmunoprecipitate in C2C12 cells, cotransfection FLAG-Csl and transduced labeled mutant Myc or HA Δ256 Foxo1.

Figures 40A-40D

On these figures it is shown that Foxo1 binds directly with Csl. a. The analysis with the "lowering" for GST fused protein GST-Foxo1 with Csl, immunoprecipitated from HEK293 cells. b-c. The binding of GST-Foxo1 and GST-FLAG-Csl in a cell-free system and the mapping of domain interaction Csl. Full-size and truncated fragments of GST-Foxo1 and GST-FLAG/Csl was purified from bacteria and consumerable. Then allocated Csl using antibodies against FLAG, and immunoprecipitates analyzed by Western blot turns with antibodies against Foxo1 or against the FLAG. d. The promoterHes1ChIP, covering the Csl binding site in C2C12 cells for detection of endogenous Foxo1, Csl and Notch1 (Endog) or after transduction Foxo1-ADA (Foxo1-ADA) during differentiation of cultured myoblasts. The source material is a DNA extracted from chromatin before immunoprecipitation. Shows the expressionHes1(semiquantitative RT-PCR) and myosin (Western blot), corresponding to each IOM is NTU time. 0 day is defined as the time when cells are deprived of serum for the induction of fusion of cultured myoblasts. Abbreviations: IP: immunoprecipitation; IB: Western blot turns; TCL, total cell lysate.

Figures 41A-41B

On these figures it is shown that Foxo1 regulates induced Notch expression of Hes1, Hes5 and Hey1. a. The expressionHes1,Hes5andHey1measured by semiquantitative RT-PCR in C2C12 cells, transduced Foxo1-ADA or Notch1-IC after transfection with Gfp siRNA, Foxo1 or Csl, as indicated. b. Analysis of reporter geneHes1in HEK293 cells, transduced siRNAs for Foxo1-ADA, Notch1-IC, Foxo1 siRNA, GFP siRNA or a control plasmid. The authors of the present invention was measured by the luciferase activity and normalizability its activity of β-galactosidase. Data represent arbitrary units relative to the control empty vector.

Figures 42A-42F

On these figures it is shown that Foxo1 is required for Notch binding with the Hes1 promoter and activation of target genes Hes1. a. Analyses ChIP endogenous Foxo1 and Notch1 in C2C12 cells, coculturing with HEK293 cells expressing LacZ (marked with "-") or Jagged1 (marked with "+") in the absence (lanes 1-2) and presence (lanes 3-4) siRNA for Csl. b. Analyses ChIP endogenous Notch1 in the system coculture in the absence (lanes 1-2) and presence (lanes 3-4) siRNA for Foxo1. c. Analyses of the Hes1 promoter after coculture the absence and in the presence of siRNA for Foxo1 or Gfp. d. Analyses ChIP binding of Ncor and Smrt and Maml1 withHes1in the system coculture in the absence (lanes 1-2) and in the presence (lanes 3-4) siRNA for Foxo1. e. The expressionMyoD,Myf5andβ-actinin C2C12 cells by semiquantitative RT-PCR. f. Model of the regulation of Foxo1 Notch and Hes1 promoter.

Figures 43A-43D

These pieces are shown dependent on conditions, the elimination of Foxo1 in skeletal muscle. a. Western blot analysis of the levels of expression of Foxo1 and Foxo4 in different types of muscles. b. Metachromatic and immunohistochemical analysis of soleus and plantar muscles from mice Myog-Foxo1 and control (lox/lox) odnopolnyh animals. c. Analysis of gene expression in Myog-Foxo1 (shaded columns) and control mice (empty columns); TropC: troponin-C; TropT: troponin-T; Mlc: light chain of myosin; Myog: myogenin; Mck: creatine kinase muscle type. Data represent mean values ± SEM for three independent measurements (n=6 for each genotype). The asterisk is specified P<0.05 using ANOVA. d. Functional test on the "treadmill" of mice Myog-Foxo1 at the age of 8 weeks and odnopolnyh animalslox/lox(n=6 for each genotype). The asterisk indicated P<0.05 using ANOVA.

Figure 44

This figure presents the efficiency of adenoviral transduction in C2C12 cells. The authors present invention has transducible cells by adenovirus HA-Foxo1-ADA or HA-Notch1-IC and conducted immunohistochemistry the antibody against HA (red) and DAPI (blue).

Figure 45

This figure presents the inhibition of expression of transfitsirovannykh Foxo1. The authors of the present invention tested the ability of siRNA to Foxo1 to inhibit the expression of endogenous (left panel) and transfitsirovannykh (right panel) Foxo1 after adenoviral transduction.

Figure 46

This figure presents the specificity of siRNA for Foxo1. Western blot analysis of the expression of Foxo1, Foxo3 and Foxo4 in C2C12 cells, transfected with siRNA for Foxo1.

Figure 47

In this figure it is shown that Foxo1-ADA and Notch1-IC does not affect cell proliferation. The authors present invention has transducible C2C12 cells by adenovirus LacZ, Foxo1-ADA or Notch1-IC was performed immunohistochemistry with an antibody against Ki67 and DAPI and calculated the index of Ki67 labeling as a percentage of Ki67-positive cells by counting at least 1000 cells.

Figure 48

This figure presents siRNA-resistant Foxo1-ADA. Western blot Foxo1-ADA and siRNA-resistant Foxo1-ADA in cells transfected with siRNA for Foxo1.

Figure 49

This figure presents the specificity of coimmunoprecipitate Foxo1-Csl. After transfection expressing vectors Foxo3 or Foxo4, the authors of the present invention conducted experiments on coimmunoprecipitate with Csl.

Figure 50

This figure presents the analysis of the Hes1 promoter. The authors present invention COI is litovali synthetic reporter gene Hes1containing four tandem repeat of binding sites for Csl in the analysis of promoter with Foxo1 and Notch1-IC in C2C12 cells.

Figure 51

This figure presents the inhibition of Csl expression by siRNA. The authors of the present invention measured the levels of Csl using Western blot after transfection of C2C12 cells by siRNA for Csl in various concentrations.

Figure 52

This figure presents the scheme of the eleven compositions traps Notch1 person.

Figure 53

This figure presents the analysis of the signal sequence of Notch1 to determine the end of the signal peptide Notch1. Presents the results of the prediction analysis using the Signal I 3.0 Server provided online Technical Univeristy of Denmark. The results predict the main cleavage site located between alanine 18 (A19) and A19, and minor cleavage site between the A19 and arginine 20 (R20). These two sites of cleavage are indicated by "/" in the amino acid sequence 1-20 Notch1 person: MPPLLAPLLCLALLPALA/A/R (SEQ ID NO:15). The nucleotide sequence encoding amino acids 1-23 Notch1 man represents atgccgccgc tcctggcgcc cctgctctgc ctggcgctgc tgcccgcgct cgccgcacga ggcccgcga (SEQ ID NO:16).

Figure 54

This figure presents the analysis of the signal sequence Hc person to determine the end of the signal peptide Hc man. MWGWKCLLFWAVLVTATLCTA/R (EQ ID NO:17). The results of the prediction analysis using SignalIP 3.0 Server provided online Technical University of Denmark, described above. These results predict the main cleavage site located between alanine 21 (A21) and arginine 22 (R22). This cleavage site is indicated by "/" in the amino acid sequence 1-22 Hc human (SEQ ID NO:17), given above. The nucleotide sequence encoding amino acids 1-22 Hc man represents atgtggggct ggaagtgcct cctcttctgg gctgtgctgg tcacagccac tctctgcact gccagg (SEQ ID NO:18).

Figure 55

This figure presents the merged sequence of Notch1/Fc man for all designs that end after repeat 36 EGF in human Notch1.

Figure 56

This figure presents the merged sequence of Notch1/Fc man for all designs that end after repeat 13 EGF in human Notch1.

Figure 57

This figure presents the merged sequence of Notch1/Fc man for all designs that end after repeat 23 Notch1 EGF person.

Figure 58

This figure presents the merged sequence of Notch1/Fc man for all designs that end after repeat 24 EGF in human Notch1.

Figure 59A and 59B

This figure presents full amino acid (A. K.) the sequence of human Notch1, consisting of the OS is Atkov with and.to. 1 (M = methionine).to. 2555 (K = lysine) (SEQ ID NO:52). The signal peptide and the first 36 EGF-like domain present in A. to. 1-1433 this sequence. Amino acids 1-1433, or a subset of these.K. used to construct proteins traps Notch1 man, described in the following sections. Amino acids, covering the EGF-repeats 1-36, underlined.

Figure 60

This figure presents the amino acid sequence of the Fc man used to produce Fc-labels on proteins traps Notch1 (SEQ ID NO:53). 237 amino acids Fc man was subjected to fusion with the C-end of all designs of traps Notch1 directly below EGF-like repeats of Notch1. This area Fc of the person allows the detection and cleaning traps Notch and serves to stabilize secreted slit proteins human Notch1-Fc man.

Figure 61

This figure presents the amino acid sequence of the protein trap h-Notch1(1-36)(SEQ ID NO:54). Protein traps h-Notch1(1-36)consists of the following three components: (1) the signal sequence of human Notch1, consisting of amino acids 1-23 Notch1 person, then (2) amino acids encoding the EGF-like repeats 1-36 human Notch1, consisting of amino acids 24-1433, and then (3) amino acids 1434-1670 that contain HC-label person. The predicted sequence of the signal peptide is underlined, and the label Fc man p is darknut and italics. This composition contains 1670 amino acids.

Figure 62

This figure presents the amino acid sequence of the protein trap h-Notch1(1-13)(SEQ ID NO:55). Protein traps h-Notch1(1-13)consists of the following three components: (1) the signal sequence of human Notch1, consisting of amino acids 1-23 Notch1 person, then (2) amino acids encoding the EGF-like repeats 1-13 Notch1 person, consisting of amino acids 24-531, and then (3) amino acids 532-768 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 768 amino acids.

Figure 63

This figure presents the amino acid sequence of the protein trap h-Notch1(1-24)(SEQ ID NO:56). Protein traps h-Notch1(1-24)consists of the following three components: (1) the signal sequence of human Notch1, consisting of amino acids 1-23 Notch1 person, then (2) amino acids encoding the EGF-like repeats 1-24 human Notch1, consisting of amino acids 24-948, and then (3) amino acids 949-1185 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 1185 amino acids.

Figure 64

This figure presents aminokislot the I sequence of the protein trap h-Sp NNotch1(9-23)(SEQ ID NO:57). Protein traps h-spNNotch1(9-23)consists of the following three components: (1) the signal sequence of human Notch1, consisting of amino acids 1-23 Notch1 person, then (2) amino acids encoding the EGF-like repeats 9-23 human Notch1, consisting of amino acids 24-594, and then (3) amino acids 595-831 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 829 amino acids.

Figure 65

This figure presents the amino acid sequence of the protein trap h-spHCNotch1(9-23)(SEQ ID NO:58). Protein traps h-spHCNotch1(9-23)consists of the following three components: (1) the signal sequence of the HC of a person, consisting of amino acids 1-22 HC person, then (2) amino acids encoding the EGF-like repeats 9-23 human Notch1, consisting of amino acids 23-593, and then (3) amino acids 594-830 that contain HC-label person. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 829 amino acids.

Figure 66

This figure presents the amino acid sequence of the protein trap h-spNNotch1(9-36)(SEQ ID NO:59). Reduce spNindicates that this is the left uses the signal peptide human Notch1. Protein traps h-spNNotch1(9-36)consists of the following three components: (1) the signal sequence of human Notch1, consisting of amino acids 1-23 Notch1 person, then (2) amino acids encoding the EGF-like repeats 9-36 human Notch1, consisting of amino acids 24-1118, and then (3) amino acids 1119-1355 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 1355 amino acids.

Figure 67

This figure presents the amino acid sequence of the protein trap h-spHCNotch1(9-36)(SEQ ID NO:60). Reduce spHCindicates that this compound is used, signal peptide HC man. Protein traps h-spHCNotch1(9-36)consists of the following three components: (1) the signal sequence of the HC of a person, consisting of amino acids 1-22 HC person, then (2) amino acids encoding the EGF-like repeats 9-36 human Notch1, consisting of amino acids 23-1117, and then (3) amino acids 1118-1354 that contain HC-label person. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 1354 amino acids.

Figure 68

This figure presents the amino acid sequence of the protein trap h-sp NNotch1(13-24)(SEQ ID NO:61). Reduce spNindicates that this compound is used, signal peptide human Notch1. Protein traps h-spNNotch1(13-24)consists of the following three components: (1) the signal sequence of human Notch1, consisting of amino acids 1-23 Notch1 person, then (2) amino acids encoding the EGF-like repeats 13-24 human Notch1, consisting of amino acids 24-478, and then (3) amino acids 479-715 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 715 amino acids.

Figure 69

This figure presents the amino acid sequence of the protein trap h-spHCNotch1(13-24)(SEQ ID NO:62). Reduce spHCindicates that this compound is used, signal peptide HC man. Protein traps h-spHCNotch1(13-24)consists of the following three components: (1) the signal sequence of the HC of a person, consisting of amino acids 1-22 HC person, then (2) amino acids encoding the EGF-like repeats 13-24 human Notch1, consisting of amino acids 23-477, and then (3) amino acids 478-714 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains what it 714 amino acids.

Figure 70

This figure presents the amino acid sequence of the protein trap h-spNNotch1(25-36)(SEQ ID NO:63). Reduce spNindicates that this compound is used, signal peptide human Notch1. Protein traps h-spNNotch1(25-36)consists of the following three components: (1) the signal sequence of human Notch1, consisting of amino acids 1-23 Notch1 person, then (2) amino acids encoding the EGF-like repeats 25-36 human Notch1, consisting of amino acids 24-508, and then (3) amino acids 509-745 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 745 amino acids.

Figure 71

This figure presents the amino acid sequence of the protein trap h-spHCNotch1(25-36)(SEQ ID NO:64). Reduce spHCindicates that this compound is used, signal peptide HC man. Protein traps h-spHCNotch1(25-36)consists of the following three components: (1) the signal sequence of the HC of a person, consisting of amino acids 1-22 HC person, then (2) amino acids encoding the EGF-like repeats 25-36 human Notch1, consisting of amino acids 23-507, and then (3) amino acids 508-744 that contain HC-label person. The predicted sequence is the signal peptide is underlined, and Fc-label human underlined and in italics. This composition contains 744 amino acids.

Figure 72A and 72B

This figure presents the sequence of the nucleic acid, which encodes a protein trap h-Notch1(1-36)indicated on figure 61 (SEQ ID NO:65).

Figure 73

This figure presents the sequence of the nucleic acid, which encodes a protein trap h-Notch1(1-13)indicated on figure 62 (SEQ ID NO:66).

Figure 74A and 74B

This figure presents the sequence of the nucleic acid, which encodes a protein trap h-Notch1(1-24)indicated on figure 63 (SEQ ID NO:67).

Figure 75

This figure presents the sequence of the nucleic acid, which encodes a protein trap h-spNNotch1(9-23)indicated on figure 64 (SEQ ID NO:68).

Figure 76

This figure presents the sequence of the nucleic acid, which encodes a protein trap h-spHCNotch1(9-23)indicated on figure 65 (SEQ ID NO:69).

Figure 77A and 77B

This figure presents the sequence of the nucleic acid, which encodes a protein trap h-spNNotch1(9-36)indicated on figure 66 (SEQ ID NO:70).

Figure 78A and 78B

This figure presents the sequence of the nucleic acid, which encodes a protein trap h-spHCNotch1(9-36)that decree is hydrated in figure 67 (SEQ ID NO:71).

Figure 79

This figure presents the sequence of the nucleic acid, which encodes a protein trap h-spNNotch1(13-24)indicated on figure 68 (SEQ ID NO:72).

Figure 80

This figure presents the sequence of the nucleic acid, which encodes a protein trap h-spHCNotch1(13-24)indicated on figure 69 (SEQ ID NO:73).

Figure 81

This figure presents the sequence of the nucleic acid, which encodes a protein trap h-spNNotch1(25-36)indicated on figure 70 (SEQ ID NO:74).

Figure 82

This figure presents the sequence of the nucleic acid, which encodes a protein trap h-spHCNotch1(25-36)indicated on figure 71 (SEQ ID NO:75).

Figure 83

This figure presents full amino acid (A. K.) the sequence of human Notch4, consisting of residues.to. 1 (M = methionine).to. 2003 (K = lysine) (SEQ ID NO:76). The signal peptide and the first 29 domains, EGF-like repeats are present in A. to. 1-1174 this sequence. Amino acids 1-1174, or a subset of these.K. used to construct proteins traps human Notch4, described in the following sections. Amino acids, covering the EGF-repeats 1-29, underlined.

Figure 84

This figure presents the sequence of the Fc man used to on the teachings of the Fc-tag on the protein traps Notch4 (SEQ ID NO:77). 237 amino acids Fc man, shown here, was subjected to fusion with the C-end designs of traps Notch4, directly below the EGF-like repeats Notch4. This area Fc of the person allows the detection and cleaning traps Notch and serves to stabilize secreted slit proteins Notch4 human Fc human.

Figure 85

This figure presents the amino acid sequence of the protein trap h-Notch4(1-29)(SEQ ID NO:78). Protein traps h-Notch4(1-29)consists of the following three components:(1) the signal sequence of human Notch4, consisting of amino acids 1-27 Notch4 person, then (2) amino acids encoding the EGF-like repeats 1-29 Notch4 person, consisting of amino acids 28-1173, and then (3) amino acids 1174-1410 that contain HC-label person. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 1410 amino acids.

Figure 86

This figure presents the amino acid sequence of the protein trap h-Notch4(1-13)(SEQ ID NO:79). Protein traps h-Notch4(1-13)consists of the following three components: (1) the signal sequence of human Notch4, consisting of amino acids 1-27 Notch4 person, then (2) amino acids encoding the EGF-like repeats 1-13 Notch4 person, consisting of amino acids 28-554, and then (3) amino acids 555-791, the cat is who contain the Fc-tag man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 791 amino acid.

Figure 87

This figure presents the amino acid sequence of the protein trap h-Notch4(1-23)(SEQ ID NO:80). Protein traps h-Notch4(1-23)consists of the following three components: (1) the signal sequence of human Notch4, consisting of amino acids 1-27 Notch4 person, then (2) amino acids encoding the EGF-like repeats 1-23 Notch4 person, consisting of amino acids 28-933, and then (3) amino acids 934-1170 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 1170 amino acids.

Figure 88

This figure presents the amino acid sequence of the protein trap h-spNNotch4(9-23)(SEQ ID NO:81). Reduce spNindicates that this compound is used, signal peptide human Notch4. Protein traps h-spNNotch4(9-23)consists of the following three components: (1) the signal sequence of human Notch4, consisting of amino acids 1-27 Notch4 person, then (2) amino acids encoding the EGF-like repeats 9-23 Notch4 person, consisting of amino acids 28-602, and then (3) amino acids 603-839 that contain Fc-m is woven of man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 839 amino acids.

Figure 89

This figure presents the amino acid sequence of the protein trap h-spHCNotch4(9-23)(SEQ ID NO:82). Reduce spHCindicates that this compound is used, signal peptide HC man. Protein traps h-spHCNotch4(9-23)consists of the following three components: (1) the signal sequence of the HC of a person, consisting of amino acids 1-22 HC person, then (2) amino acids encoding the EGF-like repeats 9-23 Notch4 person, consisting of amino acids 23-597, and then (3) amino acids 598-834 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 834 amino acids.

Figure 90

This figure presents the amino acid sequence of the protein trap h-spNNotch4(9-29)(SEQ ID NO:83). Reduce spNindicates that this compound is used, signal peptide human Notch4. Protein traps h-spNNotch4(9-23)consists of the following three components: (1) the signal sequence of human Notch4, consisting of amino acids 1-27 Notch4 person, then (2) amino acids encoding the EGF under the nye repetitions 9-29 Notch4 person, consisting of amino acids 28-843, and then (3) amino acids 844-1080 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 1080 amino acids.

Figure 91

This figure presents the amino acid sequence of the protein trap h-spHCNotch4(9-29)(SEQ ID NO:84). Reduce spHCindicates that this compound is used, signal peptide HC man. Protein traps h-spHCNotch4(9-29)consists of the following three components: (1) the signal sequence of the HC of a person, consisting of amino acids 1-22 HC person, then (2) amino acids encoding the EGF-like repeats 9-29 Notch4 person, consisting of amino acids 23-838, and then (3) amino acids 839-1075 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 1075 amino acids.

Figure 92

This figure presents the amino acid sequence of the protein trap h-spNNotch4(13-23)(SEQ ID NO:85). Reduce spNindicates that this compound is used, signal peptide human Notch4. Protein traps h-spNNotch4(13-23)consists of the following three components: (1) the signal sequence Ntch4 person, consisting of amino acids 1-27 Notch4 person, then (2) amino acids encoding the EGF-like repeats 13-23 Notch4 person, consisting of amino acids 28-444, and then (3) amino acids 445-681 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 681 amino acid.

Figure 93

This figure presents the amino acid sequence of the protein trap h-spHCNotch4(13-23)(SEQ ID NO:86). Reduce spHCindicates that this compound is used, signal peptide HC man. Protein traps h-spHCNotch4(13-23)consists of the following three components: (1) the signal sequence of the HC of a person, consisting of amino acids 1-22 HC person, then (2) amino acids encoding the EGF-like repeats 13-23 Notch4 person, consisting of amino acids 23-439, and then (3) amino acids 440-676 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 676 amino acids.

Figure 94

This figure presents the amino acid sequence of the protein trap h-spNNotch4(21-29)(SEQ ID NO:87). Reduce spNindicates that this compound is used, signal peptide human Notch4.Protein traps h-sp NNotch4(21-29)consists of the following three components: (1) the signal sequence of human Notch4, consisting of amino acids 1-27 Notch4 person, then (2) amino acids encoding the EGF-like repeats 21-29 Notch4 person, consisting of amino acids 28-392, and then (3) amino acids 393-629 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 629 amino acids.

Figure 95

This figure presents the amino acid sequence of the protein trap h-spHCNotch4(21-29)(SEQ ID NO:88). Reduce spHCindicates that this compound is used, signal peptide HC man. Protein traps h-spHCNotch4(21-29)consists of the following three components: (1) the signal sequence of the HC of a person, consisting of amino acids 1-22 HC person, then (2) amino acids encoding the EGF-like repeats 21-29 Notch4 person, consisting of amino acids 23-387, and then (3) amino acids 388-624 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 624 amino acids.

Figure 96A and 96B

This figure presents the sequence of the nucleic acid Notch4 human (SEQ ID NO:89).

is igura 97

This figure presents the sequence of the signal peptide human Notch4 (SEQ ID NO:90). Underlined sequence encodes a signal peptide.

Figure 98

This figure presents the nucleic acid sequence of the signal peptide HC man (N. 1-66) (SEQ ID NO:91).

Figure 99

This figure presents the sequence of the nucleic acid FC-label human (SEQ ID NO:92).

Figure 100

This figure presents the sequence of the nucleic acid, which encodes a protein trap h-Notch4(1-29)(SEQ ID NO:93). Trap Notch4 person (EGF-like repeats 1-29) [N. 1-3522].

Figure 101

This figure presents the sequence of the nucleic acid, which encodes a protein trap h-Notch4(1-13)(SEQ ID NO:94). Trap Notch4 person (EGF-like repeats 1-13) [N. 1-1662].

Figure 102

This figure presents the sequence of the nucleic acid, which encodes a protein trap h-Notch4(1-23)(SEQ ID NO:95). Trap Notch4 person (EGF-like repeats 1-23) [N. 1-2799].

Figure 103

This figure presents the sequence of the nucleic acid, which encodes a protein trap h-spNNotch4(9-29)(SEQ ID NO:96). Trap Notch4 person (EGF-like repeats 9-29) [N. 1-81, 1075-3522].

Figure 104

This figure presents the sequence nuclein the howling acid, which encodes a protein trap h-spHCNotch4(9-29)(SEQ ID NO:97). Trap Notch4 person (EGF-like repeats 9-29) [N. 1075-3522] and signal peptide HC [N. 1-66].

Figure 105

This figure presents the sequence of the nucleic acid, which encodes a protein trap h-spNNotch4(9-23)(SEQ ID NO:98). Trap Notch4 person (EGF-like repeats 9-23) [N. 1-81, 1075-2799].

Figure 106

This figure presents the sequence of the nucleic acid, which encodes a protein trap h-spHCNotch4(9-23)(SEQ ID NO:99). Trap Notch4 person (EGF-like repeats 9-23) [N. 1075-2799] and signal peptide HC [N. 1-66].

Figure 107

This figure presents the sequence of the nucleic acid, which encodes a protein trap h-spNNotch4(13-23)(SEQ ID NO:100). Trap Notch4 person (EGF-like repeats 13-23) [N. 1-81, 1549-2799].

Figure 108

This figure presents the sequence of the nucleic acid, which encodes a protein trap h-spHCNotch4(13-23)(SEQ ID NO:101). Trap Notch4 person (EGF-like repeats 13-23) [N. 1549-2799] and signal peptide HC [N. 1-66].

Figure 109

This figure presents the sequence of the nucleic acid, which encodes a protein trap h-spNNotch4(21-29)(SEQ ID NO:102). Trap Notch4 person (EGF-like repeats 21-29) [N. 1-81, 2428-3522].

Figure 110

This shit is f presents the sequence of the nucleic acid, which encodes a protein trap h-spHCNotch4(21-29)(SEQ ID NO:103). Trap Notch4 person (EGF-like repeats 21-29) [N. 2428-3522] and signal peptide HC [N. 1-66].

Figure 111

This figure presents the scheme of the eleven compositions traps Notch4 person.

Figure 112

This figure presents the analysis of the signal sequence Notch4 to determine the end of the signal peptide Notch4.

Figure 113

This figure presents the merged sequence Notch4/Fc man for all designs that end after EGF-repeat 29 in human Notch4.

Figure 114

This figure presents the merged sequence Notch4/Fc man for all designs that end after EGF-repeat 13 Notch4 person.

Figure 115

This figure presents the merged sequence Notch4/Fc man for all designs that end after EGF-repeat 23 Notch4 person.

Figure 116

This figure shows that lack of Notch4 reduces circulating levels of glucose and insulin. The authors of the present invention are presently analyzing the levels of glucose and insulin in the blood in the mutant by Notch4 (N4) mice. Upon weaning from the mother (P21) many litters resulting from crosses N4+/-, were fed either a normal diet or a diet high in fat, contain Asim 45% of calories from fat. At the age of 5 months were collected and blood was determined by the levels of glucose and insulin in mice with any diet or mice after 6 hours of fasting. Females have lower allele N4 correlated with a significant reduction in circulating glucose levels regardless of diet. On the contrary, the failure N4 was associated with reduced levels of glucose only in males fed a normal diet. Insulin levels all females were unchanged (data not shown). This may be due to the fact that female mice are genetically protected from resistance to insulin, and thus, metabolic abnormalities are largely soft. Under normal diet there was no difference in insulin levels in male mice. However, the removal N4 correlated with a significant decrease in the levels of insulin in any diet in male mice fed a diet high in fat. A similar trend was observed for males with knockout N4, which were subjected to fasting for 6 hours. Thus, the loss of N4 correlated with a significant decrease of glucose levels in the blood, as in males and females fed a normal diet. Females have lower levels of glucose, but not insulin levels was observed in females mutant for N4 fed a diet high in fat. In contrast, levels and glucose were unchanged in mice with knockout N4, while insulin levels were reduced. These results are consistent with the failure Notch4, protecting against genetic and environmental forms of hyperglycemia due to impaired transmission of the insulin signal.

Figure 117

This figure shows that the loss of expression of Notch4 suppressed weight gain in mice fed a diet high in fat.

Figure 118

This figure shows that the trap rat Notch1 is present in the serum of a mouse. Tested the stability of the trap rat Notch1 in the bloodstream of mammals. Western blot analysis demonstrated that the full-sized protein can be expressed in mice and is amenable to detection levels with little signs of degradation.

Figure 119

This figure shows that the trap Notch1 person (trap-n-Notch(1-36)and trap rat Notch1 block the growth of tumors in the mammary gland of the mouse. Growth curve presented here shows that as the trap rat Notch1 and trap Notch1 person reduced the growth of xenografts tumors in nude mice.

Figure 120

This figure shows that the trap rat Notch1 inhibits metastasis SKNEP1 in the tissue of the lungs. Cell sarcoma Ewing SKNEP1 was programmed for the expression of a control Fc protein or trap rat Notch1 s1 (type 2) or traps rat Notch1 s4 (type 4). the t cell line SKNEP1 orthotopic implanted in the kidney of nude mice. After tumor growth in a period of 6 weeks was performed histological evaluation of metastases in the lung. In cells SKNEP1 expressing trap rat Notch1 was shown the lower part of the lung, which was positive for metastases. The authors of the present invention concluded that the expression of trap rat Notch1 in nude mice reduces the ability of cells SKNEP1 metastasize to the lung.

Figure 121

In this figure it is shown that Notch1 and Notch4 coexpressed with VEGFR-3 and PRONOUNCED-1 in lymphatic vessels of the skin of the mouse. The expression of Notch1 and Notch4 were analyzed in the blood vessels of the skin on the back of the mouse P4. At this point in time, lymphatic vessels of the skin actively remodelers in lymphatic capillaries near the surface and collecting ducts in the lower layers of the skin. Transverse sections of skin the size of 5 μm were co-stained with antibodies against Notch1 or Notch4 (red) and PECAM, VEGFR-3 or PRONOUNCED-1 (green). Notch1 and Notch4 have overlapping expression pattern with markers of blood and lymphatic endothelial cells, PECAM (top panel). Notch1 and Notch4 were coexpressions as with VEGFR-3 (middle panel), and with PRONOUNCED-1 in the blood vessels of the skin (bottom panel). This pattern of expression demonstrates that Notch1 and Notch4 are expressed and can function in the lymph vessels of neonatal skin.

Figure 122

This figure shows that the lymphatic capillaries of the skin changed from th is shotnik mice with knockout of Notch4. The authors of the present invention investigated the lymph vessels in the skin of mice P4. Secrets wild-type mice and mice zero-zygote by Notch4, were subjected to immune staining with antibodies against PECAM and PRONOUNCED-1 (green). Analysis of the staining of PECAM looked similar between mutant skin and skin of wild-type (upper panel). On the contrary, positive PRONOUNCED on-1 vessels in the skin of mutants by Notch4 had a morphology that is different from the morphology of wild-type mice (middle panel). Mutant by Notch4 vessels PRONOUNCED-1 were often extended, and staining PRONOUNCED-1 was intermittent (bottom panel). These results indicate that the Notch4 signaling may be involved in remodeling of lymphatic vascular plexus.

Figure 123

This figure shows that the loss of Notch4 correlates with reduced expression of LYVE1 in the lymph vessels in the skin of mice. Mice heterozygous for Notch4 (N4+/-), were crossed, and back skin obtained calves were removed and placed on 14 day after birth. Transverse sections of skin were subjected to immune staining for a marker of endothelial cells, PECAM (data not shown), or a marker of lymphatic endothelial cells, LYVE1 (A). In each case, registered five areas through microscopy and staining of PECAM and LYVE1 was subjected to quantitative determination using software provided with the I for visualization (B, C). The expression of PECAM was reduced by approximately 25% in the skin N4-/- compared with skin of wild-type (WT) (B). Staining PRONOUNCED-1 was changed more than PECAM staining, and staining LYVE1 was reduced almost by 50% in mice N4-/- relative to WT mice (C). Also there was a decrease in the intensity of staining LYVE1 in the lymph vessels N4-/- relative to WT (A).

Figure 124

In this figure it is shown that Notch1 and Notch4 are expressed in the lymph vessels of the breast cancer people. The authors of the present invention conducted a double immunohistochemistry with antibodies against VEGFR-3 or PRONOUNCED-1 (green) and Notch1 or Notch4 (red) breast cancer. Notch1 and Notch4 expressibility in neophilia the endothelium of the blood and limfaticeskih of microcapillaries carcinomas of the breast of man. To determine whether activation signal Notch1 in lymphatic endothelium of the tumor, the authors of the present invention conducted a double staining with an antibody against podoplanin (green) and antibody N1Val (red; cell signaling), which provides specific detection of activated Notch1 peptide. Expression of activated Notch1 peptide was observed in the majority (white arrows), but not all (yellow arrows) from the nuclei of lymphatic endothelium (bottom panel). These results demonstrate that Notch1 actively re the avala signal in pathological lymph vessels.

Detailed description of the invention

Terms

In this application, except as expressly provided herein, each of the following terms has the meaning specified below.

"Introduction" can be implemented or performed using any methods known to the person skilled in the art. The methods include, for example, vnutrizonovoy, intramuscular, subcutaneous, intravenous, intraperitoneal, mediated by liposomes through the mucosa, intestinal, local, nasal, oral, anal, ocular or auricular shipping method.

"Fixed" means connected in any way. In one embodiment, a fixed means attached covalent bond. In another embodiment, a fixed means attached ecovalence.

The terms "amino acid", "amino acid residue" and "residue" are used herein interchangeably, and they refer to the amino acid which is incorporated into a protein, polypeptide or peptide. The amino acid may represent, for example, naturally occurring amino acid or analog of the natural amino acids that can function similar to the function of naturally occurring amino acids.

"Antibody" includes, but is not limited to, (a) they say the Kullu immunoglobulin, containing two heavy chains and two light chains, and which recognizes the antigen; (b) the molecule polyclonal or monoclonal immunoglobulin; and (c) their monovalent or bivalent fragment. Of the immunoglobulin molecule can be any of the widely known classes, including, but not limited to, IgA, secretory IgA, IgG, IgE and IgM. The IgG subclasses is well known to specialists in this field and include, but are not limited to, IgG1, IgG2, IgG3 and human IgG4. Antibodies can be found in nature, and not found in nature. Moreover, antibodies include chimeric antibodies, synthetic antibodies, single-chain antibodies, and fragments thereof. The antibody can be a human antibodies and antibodies are not human. Antibodies can be humanitarian recombinant ways to reduce their immunogenicity in humans. Fragments of antibodies include, but are not limited to, Fab and Fc-fragments. "Fc part of antibodies, in one embodiment, is kristallizuetsya fragment obtained by papain cleavage of immunoglobulin, which consists of the C-terminal half of the two heavy chains connected by disulfide bonds, and is known as "effector region of the immunoglobulin. In another embodiment, "Fc part of the antibody" means all, or there is actually an entire, one C-terminal half of the heavy chain.

"Humanitarianly", in relation to an antibody means an antibody in which some, most or all of the amino acids outside the scope of the CDR replaced with the corresponding amino acids of the molecule of the immunoglobulin. Small insertions, deletions, insertions, or substitutions or amino acid modifications are acceptable, provided that they do not eliminate the ability of the antibody to bind the antigen. Suitable molecules of the immunoglobulin include, but are not limited to, molecules IgG1, IgG2, IgG3, IgG4, IgA, and IgM. Obtaining humanized antibodies are described in various publications, for example, in U.S. patent No. 4816567, 5225539, 5585089 and 5693761 and international PCT publication no WO 90/07861.

In this document, the term "composition", as in the case of pharmaceutical compositions, covers the product containing the active ingredient(s) and the inert ingredient(s), which forms the carrier, as well as any product which is formed due to, directly or indirectly, combining, complex formation or aggregation of two or more ingredients, or due to the dissociation of two or more ingredients, or due to other types of reactions or interactions of one or more ingredients.

Herein "effective amount" refers to an amount which is capable of Les the giving of the subject, having a tumor, the disease or disorder. Thus, the effective amount varies depending on the subject being treated, and the condition to be treated. The person skilled in the art can conduct conventional titration experiments to determine such sufficient. An effective amount of the compound may vary depending on the subject and the specific method used in the introduction. Based on the connection number can be delivered continuously, for example, by means of a continuous pump, or at periodic intervals (for example, one or more separate times). The required time intervals for different quantities of specific compounds can be determined by a person skilled in the art without undue experimentation. In one embodiment, the effective amount is between about 1 μg/kg to 10 mg/kg In another embodiment, the effective amount is between about 10 μg/kg to 1 mg/kg In the following embodiment, an effective amount is 100 ág/kg

"Extracellular domain", as used in connection with the receptor protein Notch, Notch means all or part of that

(i) there is extracellular (i.e., do not exist as transmembrane part or as wew is kletochnoi part) and

(ii) binds to extracellular ligands bound to the intact receptor protein Notch. The extracellular domain of the Notch may not necessarily include the signal peptide. "Extracellular domain", "ECD" and "ectodomain" are synonyms.

"The group that increases the half-life" means a group which, when it is functionally connected with the second group, increases the half-life ofin vivothe second group. Groups that increase the half-life include, for example, the Fc portion of antibodies, glycosylceramide labels (i.e., glycosylated polypeptides), polyethylene glycol (PEG), polypeptides having a PEG associated with them, and modified lipids polypeptides.

"Inhibition" of the violations occurred or undesirable biological process means either reducing the likelihood of violations or process, or the prevention of the occurrence of the violation or process completely.

In a preferred embodiment, the inhibition of the occurrence of the violation or process means the prevention of its occurrence altogether.

"Notch", "Notch protein and receptor protein Notch" are synonyms. In addition, the terms "protein-based Notch" and "trap Notch" are synonyms. The following amino acid sequence Notch known and incorporated herein as references: Notch1 (registration the first number Genbank No. S18188 (rat)); Notch2 is (Genbank registration number № NP_077334 (rat)); Notch3 (Genbank registration number № Q61982 (mouse)); and Notch4 (Genbank registration number № T09059 (mouse)). The following nucleic acid sequences Notch known and included as references: Notch1 (Genbank registration number № XM_342392 (rat) and NM_017617 (person)); notch2 is (Genbank registration number № NM_024358 (rat), M99437 (man) and AF308601 (person)); Notch3 (Genbank registration number № NM_008716 (mouse) and XM_009303 (person)); and Notch4 (Genbank registration number № NM_010929 (mouse) and NM_004557 (people)).

The terms "nucleic acid", "polynucleotide" and "nucleic acid sequence" are used herein interchangeably, and they refer to a polymer of deoxyribonucleotides and/or ribonucleotides. The deoxyribonucleotides and ribonucleotides can be found in nature, or they can represent their synthetic analogues. "Nucleic acid" means any nucleic acid, including, but not limited to, DNA, RNA, and their hybrids. Bases of nucleic acids, which form a molecule of nucleic acids may constitute grounds A, C, G, T and U and their derivatives. Derivatives of these bases are well known in the field and illustrated in PCR Systems, Reagents and Consumables (Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc., Branchburg, New Jersey, USA). Nucleic acids include, but is s limited to, antisense molecules and catalytic molecules of nucleic acids such as ribozymes and Decimi. Also nucleic acids include nucleic acids encoding peptide analogs, fragments or derivatives, which differ from naturally occurring forms one or more amino acid residues (analogues with a deletion of the containing less than all of the above residues; analogues with replacement, where one or more residues substituted by one or more residues; and analogues with insert, where one or more residues are added to the end or the middle part of the peptide), which share some or all properties of naturally occurring forms.

"Functionally linked" means, in respect of the first group associated with the second group, linked in a manner that allows the first group to function (e.g., binding properties), as if it was not connected.

The terms "polypeptide", "peptide" and "protein" are used herein interchangeably, and they all mean a polymer of amino acid residues. Amino acid residues can be found in nature, or they can represent their chemical counterparts. Polypeptides, peptides and proteins may also have modifications, such as glycosylation, the accession of lipids, sulfation, hydroxylean the tion and ADP-ribosylating.

Herein "pharmaceutically acceptable carrier" means the carrier is compatible with other ingredients of the composition and is not harmful to the recipient, and encompasses any of the standard pharmaceutically acceptable carriers. Such media include, for example, 0.01 to 0.1 M, and preferably of 0.05 M phosphate buffer or 0.8% saline. In addition, such pharmaceutically acceptable carriers may be an 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 etiloleat. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered environment. Parenteral carriers include sodium chloride, dextrose ringer's solution, dextrose and sodium chloride, lactate ringer's solution and fatty oils. Intravenous carriers include liquid and compensating solutions of nutrients, compensating electrolyte solutions, such as solutions on the basis of dextrose ringer, etc. can Also contain preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, Artie gases, etc.

"Subject" means any organism, including, but not limited to, a mammal, such as mouse, rat, dog, Guinea pig, ferret, rabbit, and Primate. In a preferred embodiment, the subject is the man.

"Treatment" means either slowing or stopping or reversal of the progression of the disease or disorders. Herein "treatment" also means the mitigation of symptoms associated with a disease or disorder. Diseases include, but are not limited to, tumor angiogenesis, atherosclerosis, wound healing, macular degeneration, retrolental fibroplasia, pre-eclampsia, diabetic retinopathy, ischemia, stroke, cardiovascular disease, psoriasis, lymphedema, the formation of tumors and lymphangiogenesis tumors.

Angiogenesis occurs during the process of wound healing, the menstrual cycle in women and remodeling of the endometrium, as well as during embryonic development and growth of organs. In pathological conditions, angiogenesis plays an important role in various diseases, such as rheumatoid arthritis, psoriasis, macular degeneration, diabetic retinopathy and tumor growth.

There are convincing datain vivoincluding clinical observations that abnormal angiogenesis is involved in a number of painful conditions, which include revmatoidnyi arthritis, inflammation, cancer, psoriasis, degenerative eye condition and other.

Other diseases for use merged Notch proteins are metabolic disorders, such as, but not limited to, diabetes, obesity, pre-diabetic condition, atherosclerosis, ischemia, stroke, cardiovascular disease, regulation of expression of insulin and regulating functions of the insulin.

The use of fused proteins Notch also shown in the case of metabolic syndrome, which refers to the combination of medical disorders that increase the risk of a person of cardiovascular disease and diabetes. Other known names for this syndrome is syndrome X, syndrome of resistance to insulin, the syndrome Riven. Some signs of the syndrome include: hyperglycemia on an empty stomach, high blood pressure, obesity, Central type (also known as visceral obesity), reduced levels of high-density lipoprotein (LDL), increased levels of triglycerides, increased levels of uric acid. Hyperglycemia on an empty stomach, above, includes diabetes mellitus type 2 or reduced fasting glucose and impaired glucose tolerance or insulin resistance. In addition to the metabolic syndrome, trap Notch may have indications for pre-diabetic conditions.

The unit is risteski and symbols can be identified in their accepted form SI. Unless otherwise indicated, nucleic acid sequences are presented from left to right in the orientation from the 5' to 3', and the amino acid sequence represented from left to right in the orientation from N - to C-Termini. Amino acids can be identified in this document by using either their commonly known three letter symbols or by the one-letter symbols recommended by the Commission on biochemical nomenclature of the IUPAC-IUB. Similarly, the nucleotides can be labeled by their accepted single-letter codes.

This document uses the following abbreviations: ECD: extracellular domain; IC: intracellular domain; NECD/Fc: protein-based Notch; N1: Notch1; N2: notch2 is; N3: Notch3; N4: Notch4; Dll: Delta-like; EC: endothelial cells; FGF: fibroblast growth factor; FGFR: receptor fibroblast growth factor; HUVEC: endothelial cell umbilical vein of man; m.o.i.: the multiplicity of infection; VMC: cell walls of blood vessels; VEGF: a growth factor for cells of the vascular endothelium; VEGFR: growth factor receptor vascular endothelial cells; sp: signal peptide; PDGF: platelet-derived growth factor; DERIVED: the receptor for platelet-derived growth factor; P1GF: placental growth factor.

Embodiments of the invention

This invention relates to fused protein containing the signal peptide, extracellular domain of the receptor protein Notch person and Fc-part of the tetela, related.

In the first embodiment, the slit protein receptor protein Notch is a receptor protein Notch1. In one embodiment, the extracellular domain of the receptor Notch1 protein contains EGF-like repeats 1-36. In another embodiment, the extracellular domain of the receptor Notch1 protein contains EGF-like repeats 1-13. In another embodiment, the extracellular domain of the receptor Notch1 protein contains EGF-like repeats 1-24. In another embodiment, the extracellular domain of the receptor Notch1 protein contains EGF-like repeats 9-23. In another embodiment, the extracellular domain of the receptor Notch1 protein contains EGF-like repeats 9-36. In another embodiment, the extracellular domain of the receptor Notch1 protein contains EGF-like repeats 13-24. In the following embodiment, the extracellular domain of the receptor Notch1 protein contains EGF-like repeats 25-36.

In the second embodiment, the slit protein receptor protein Notch is a receptor protein notch2 is.

In the third embodiment, the slit protein receptor protein Notch is a receptor Notch3 protein.

In the fourth embodiment, the slit protein receptor protein Notch is a receptor protein Notch4. In one embodiment, the extracellular domain receptornegative Notch4 contains EGF-like repeats 1-29. In another embodiment, the extracellular domain of the receptor protein Notch4 contains EGF-like repeats 1-13. In another embodiment, the extracellular domain of the receptor protein Notch4 contains EGF-like repeats 1-23. In another embodiment, the extracellular domain of the receptor protein Notch4 contains EGF-like repeats 9-23. In another embodiment, the extracellular domain of the receptor protein Notch4 contains EGF-like repeats 9-29. In another embodiment, the extracellular domain of the receptor protein Notch4 contains EGF-like repeats 13-23. In the following embodiment, the extracellular domain of the receptor protein Notch4 contains EGF-like repeats 21-29.

In one embodiment, fused protein Fc part of the antibody is an Fc-portion of human antibodies.

In one embodiment, fused protein, signal peptide is a signal peptide Notch1, notch2 is, Notch3, or Notch4 Hc-part (HC, heavy chain) antibody.

In one embodiment, a protein contains consistently located the amino acid sequence shown in SEQ ID NO:54. In another embodiment, a protein contains consistently located the amino acid sequence shown in SEQ ID NO:55. In another embodiment, a protein contains consistently located the amino acids, the sequence listed in SEQ ID NO:56. In another embodiment, a protein contains consistently located the amino acid sequence shown in SEQ ID NO:57. In another embodiment, a protein contains consistently located the amino acid sequence shown in SEQ ID NO:58. In another embodiment, a protein contains consistently located the amino acid sequence shown in SEQ ID NO:59. In another embodiment, a protein contains consistently located the amino acid sequence shown in SEQ ID NO:60. In another embodiment, a protein contains consistently located the amino acid sequence shown in SEQ ID NO:61. In another embodiment, a protein contains consistently located the amino acid sequence shown in SEQ ID NO:62. In another embodiment, a protein contains consistently located the amino acid sequence shown in SEQ ID NO:63. In the following embodiment, a protein contains consistently located the amino acid sequence shown in SEQ ID NO:64.

In one embodiment, a protein is encoded sequentially location is nymi nucleotides, the sequence listed in SEQ ID NO:65. In another embodiment, a protein encoded successive nucleotides of the sequence listed in SEQ ID NO:66. In another embodiment, a protein encoded successive nucleotides of the sequence listed in SEQ ID NO:67. In another embodiment, a protein encoded successive nucleotides of the sequence listed in SEQ ID NO:68. In another embodiment, a protein encoded successive nucleotides of the sequence listed in SEQ ID NO:69. In another embodiment, a protein encoded successive nucleotides of the sequence listed in SEQ ID NO:70. In another embodiment, a protein encoded successive nucleotides of the sequence listed in SEQ ID NO:71. In another embodiment, a protein encoded successive nucleotides of the sequence listed in SEQ ID NO:72. In another embodiment, a protein encoded successive nucleotides of the sequence listed in SEQ ID NO:73. In another embodiment, a protein is encoded after avatele spaced nucleotides, the sequence listed in SEQ ID NO:74. In the following embodiment, a protein encoded successive nucleotides of the sequence listed in SEQ ID NO:75.

In one embodiment, a protein contains consistently located the amino acid sequence shown in SEQ ID NO:78. In another embodiment, a protein contains consistently located the amino acid sequence shown in SEQ ID NO:79. In another embodiment, a protein contains consistently located the amino acid sequence shown in SEQ ID NO:80. In another embodiment, a protein contains consistently located the amino acid sequence shown in SEQ ID NO:81. In another embodiment, a protein contains consistently located the amino acid sequence shown in SEQ ID NO:82. In another embodiment, a protein contains consistently located the amino acid sequence shown in SEQ ID NO:83. In another embodiment, a protein contains consistently located the amino acid sequence shown in SEQ ID NO:84. In another embodiment, a protein contains consistently located the military amino acids, the sequence listed in SEQ ID NO:85. In another embodiment, a protein contains consistently located the amino acid sequence shown in SEQ ID NO:86. In another embodiment, a protein contains consistently located the amino acid sequence shown in SEQ ID NO:87. In another embodiment, a protein contains consistently located the amino acid sequence shown in SEQ ID NO:88.

In one embodiment, a protein encoded successive nucleotides of the sequence listed in SEQ ID NO:89. In another embodiment, a protein encoded successive nucleotides of the sequence listed in SEQ ID NO:90. In another embodiment, a protein encoded successive nucleotides of the sequence listed in SEQ ID NO:91. In another embodiment, a protein encoded successive nucleotides of the sequence listed in SEQ ID NO:92. In another embodiment, a protein encoded successive nucleotides of the sequence listed in SEQ ID NO:93. In another embodiment, a protein encoded serial is Ino spaced nucleotides, the sequence listed in SEQ ID NO:94. In another embodiment, a protein encoded successive nucleotides of the sequence listed in SEQ ID NO:95. In another embodiment, a protein encoded successive nucleotides of the sequence listed in SEQ ID NO:96. In another embodiment, a protein encoded successive nucleotides of the sequence listed in SEQ ID NO:97. In another embodiment, a protein encoded successive nucleotides of the sequence listed in SEQ ID NO:98. In another embodiment, a protein encoded successive nucleotides of the sequence listed in SEQ ID NO:99. In another embodiment, a protein encoded successive nucleotides of the sequence listed in SEQ ID NO:100. In another embodiment, a protein encoded successive nucleotides of the sequence listed in SEQ ID NO:101. In another embodiment, a protein encoded successive nucleotides of the sequence listed in SEQ ID NO:102. In another embodiment, a protein encoded PEFC is therefore spaced nucleotides, the sequence listed in SEQ ID NO:103.

This invention relates to a method of treatment of a subject having a tumor, comprising an introduction to the principal amount specified above, fused protein, is effective for the treatment of the subject, thereby effecting treatment of a subject having a tumor.

This invention relates to a method of inhibiting angiogenesis in a subject comprising administration to the subject of the amount specified above, fused protein, effective to inhibit angiogenesis in the subject, thereby inhibiting angiogenesis in the subject.

This invention relates to a method of treatment of a subject having ovarian cancer comprising administration to the subject of the amount specified above, fused protein, is effective for the treatment of the subject, thereby effecting treatment of a subject having ovarian cancer.

This invention relates to a method of treatment of a subject having a metabolic disorder, comprising administration to the subject of the amount specified above, fused protein, is effective for the treatment of the subject, thereby effecting treatment of a subject having a metabolic disorder. In one embodiment, a metabolic disorder is a diabetes, obesity, atherosclerosis, ischemia, stroke or cardiovascular disease.

This invention relates to the use of the above merged the th protein to obtain a pharmaceutical composition for the treatment of the subject, having a tumor.

This invention relates to the use of the above fused protein to obtain a pharmaceutical composition for inhibiting angiogenesis in the subject.

This invention relates to the use of the above fused protein to obtain a pharmaceutical composition for the treatment of a subject having ovarian cancer.

This invention relates to the use of the above fused protein to obtain a pharmaceutical composition for the treatment of a subject having a metabolic disorder. In one embodiment, a metabolic disorder is a diabetes, obesity, atherosclerosis, ischemia, stroke or cardiovascular disease.

This invention relates to a method of inhibiting the physiological lymphangiogenesis or pathological lymphangiogenesis the subject, including the introduction of the principal amount specified above, fused protein, effective for the inhibition of physiological lymphangiogenesis or pathological lymphangiogenesis the subject. In one embodiment, the pathological lymphangiogenesis is lymphangiogenesis tumor or metastasis in the lymph nodes, which may depend on lymphangiogenesis tumors.

This invention relates to a method of inhibiting metastasis of a tumor in the subject, including the surrounding introduction to the subject of the amount above the slit molecules, effective for inhibiting metastasis of a tumor in the subject. In one embodiment, metastasis occurs through a blood vessel, lymphatic vessel or a lymph node. Tumour metastasis is the spread of a malignant tumor from one organ to another non-adjacent organ.

This invention relates to a method of inhibiting the growth of a secondary tumor in a subject comprising administration to the subject of the amount specified above, fused protein, effective to inhibit the growth of secondary tumors in the subject. Inhibition may also be an inhibition of tumor angiogenesis associated with secondary or metastatic tumor. In one embodiment, the secondary tumor growth is inhibited by inhibiting angiogenesis associated with a secondary tumor.

This invention relates to a method for inhibiting the co-opting of the blood vessels of the tumour in a subject, comprising administration to the subject of the amount specified above, fused protein, effective for the inhibition of the co-opting of the blood vessels of the tumor in the subject. The process of cooptation of vessels is a process in which tumor cells are associated with the existing vessels and grow with the help of co-opted vessels. This is the growth of tumors on co-opted vessels can occur in the absence of tumor angiogenesis, it may precede tumor angiogenesis, or it may occur together with it.

The present invention relates to a method of treating a malignant tumor in a subject comprising administration to the subject of the above fused protein and inhibitor of growth factor vascular endothelial (VEGF), each of them in a quantity effective for the treatment of malignant tumors in the subject. In one embodiment, the VEGF inhibitor is an inhibitor of VEGF-A, an inhibitor of P1GF, VEGF inhibitor-B, an inhibitor of VEGF-C or VEGF inhibitor-D. Examples of VEGF inhibitors include, but are not limited to, bevacizumab, PTK787, Bay43-9006, SU11248, AG013676, ZD6474, a VEGF trap, and antibody against VEGFR2. Examples of such inhibitors are more fully described in Ferrara et al., (2004) Nature Reviews Drug Discovery, Vol. 3:391-400 and Ellis et al. (2008) Nature Reviews Cancer Vol. 8:579-591, the contents of each of which are incorporated herein as references.

This invention relates to a method of treating a malignant tumor in a subject comprising administration to the subject of the above fused protein and inhibitor of VEGFR-1, VEGFR-2 or VEGFR-3, each of them in a quantity effective for the treatment of malignant tumors in the subject. In one embodiment, the inhibitor is focused on one or more of the VEGFR.

This invention relates to a method of treating a malignant tumor in a subject, comprising the introduction subjectivizing above the slit protein and inhibitor of platelet-derived growth factor (PDGF), each of them in a quantity effective for the treatment of malignant tumors in the subject. In one embodiment, the inhibitor of platelet-derived growth factor is an inhibitor of PDGF-a or PDGF inhibitor-B.

The present invention relates to a method of treating a malignant tumor in a subject comprising administration to the subject of the above fused protein and the PDGF receptor antagonist, each of them in a quantity effective for the treatment of malignant tumors in the subject. In one embodiment, the PDGF receptor antagonist is a receptor antagonist-B PDGF.

This invention relates to a method of treating a malignant tumor in a subject comprising administration to the subject of the above fused protein and an inhibitor of HER2/neu, each of them in a quantity effective for the treatment of malignant tumors in the subject.

This invention relates to a method for the treatment of vascular proliferative retinopathy, including introduction to the subject of the above fused protein in an amount effective for the treatment of vascular proliferative retinopathy.

101. The method according to p. 100, where vascular proliferative retinopathy is a diabetic retinopathy, macular degeneration or retrolental fibroplasia.

The present invention also relates to a first method is treatment of the subject, having a tumor, comprising an introduction to the subject an effective amount of the composition containing the extracellular domain of the receptor protein Notch, functionally associated with the group, increasing the half-life, to thereby treat the subject.

This invention also relates to the second method of inhibiting angiogenesis in a subject comprising administration to the subject an effective amount of the composition containing the extracellular domain of the receptor protein Notch, functionally associated with the group, increasing the half-life in order to inhibit angiogenesis in the subject.

In the first embodiment, the above methods of the receptor protein Notch is a receptor protein Notch1. In one embodiment, the receptor Notch1 protein is a receptor protein Notch1 person. In another embodiment, the group that increases the half-life represents the Fc portion of antibodies. In another embodiment, the Fc part of the antibody is an Fc-portion of human antibodies. In the following embodiment, the extracellular domain and the group, increasing the half-life, are in the same polypeptide chain.

In the second embodiment, the above methods of the receptor protein Notch is a receptor protein notch2 is. In one embodiment, R is septory protein notch2 is is a receptor protein notch2 is human. In another embodiment, the group that increases the half-life represents the Fc portion of antibodies. In another embodiment, the Fc part of the antibody is an Fc-portion of human antibodies. In the following embodiment, the extracellular domain and the group, increasing the half-life, are in the same polypeptide chain.

In the third embodiment, the above methods of the receptor protein Notch is a receptor Notch3 protein. In one embodiment, the receptor Notch3 protein is a receptor protein of human Notch3. In another embodiment, the group that increases the half-life represents the Fc portion of antibodies. In another embodiment, the Fc part of the antibody is an Fc-portion of human antibodies. In the following embodiment, the extracellular domain and the group, increasing the half-life, are in the same polypeptide chain.

In the fourth embodiment, the above methods of the receptor protein Notch is a receptor protein Notch4. In one embodiment, the receptor Notch4 protein is a receptor protein of human Notch4. In another embodiment, the group that increases the half-life represents the Fc portion of antibodies. In another embodiment, the Fc part of the antibody Ave is dstanley an Fc-portion of human antibodies. In the following embodiment, the extracellular domain and the group, increasing the half-life, are in the same polypeptide chain.

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

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

In the following embodiment, the second method, the subject has a tumor. In another embodiment, the subject suffers from pathological vascular hyperplasia. In one embodiment, the pathological vascular hyperplasia is a benign hemangioma. In the following embodiment, the subject suffers lymphatic vascular proliferative disease.

The present invention relates to the first composition containing the extracellular domain of the receptor protein Notch4, functionally associated with the group, increasing the half-life. In one embodiment, the extracellular domain covalently linked to a group that increases the half-life. In another embodiment, the extracellular domain and the group, increasing the half-life, are in the same polypeptide chain.

This invention also relates to the second composition containing vneck mocny domain receptor protein Notch4, functionally associated with the group, increasing the half-life, and a pharmaceutically acceptable carrier.

In addition, this invention relates to a product containing (i) a packaging material having therein a composition comprising the extracellular domain of the receptor protein Notch, functionally associated with the group, increasing the half-life, and (ii) a label indicating that the composition is intended for use for the treatment of a subject having a tumor or other violation amenable to treatment by inhibiting angiogenesis in the subject.

In the first embodiment, the above articles receptor protein Notch is a receptor protein Notch1. In one embodiment, the receptor Notch1 protein is a receptor protein Notch1 person. In another embodiment, the group that increases the half-life represents the Fc portion of antibodies. In another embodiment, the Fc part of the antibody is an Fc-portion of human antibodies. In the following embodiment, the extracellular domain and the group, increasing the half-life, are in the same polypeptide chain.

In the second embodiment, the above articles receptor protein Notch is a receptor protein notch2 is. In one embodiment, the receptor protein notch2 is receptory the protein notch2 is human. In another embodiment, the group that increases the half-life represents the Fc portion of antibodies. In another embodiment, the Fc part of the antibody is an Fc-portion of human antibodies. In the following embodiment, the extracellular domain and the group, increasing the half-life, are in the same polypeptide chain.

In the third embodiment, the above articles receptor protein Notch is a receptor Notch3 protein. In one embodiment, the receptor Notch3 protein is a receptor protein of human Notch3. In another embodiment, the group that increases the half-life represents the Fc portion of antibodies. In another embodiment, the Fc part of the antibody is an Fc-portion of human antibodies. In the following embodiment, the extracellular domain and the group, increasing the half-life, are in the same polypeptide chain.

In the fourth embodiment, the above articles receptor protein Notch is a receptor protein Notch4. In one embodiment, the receptor Notch4 protein is a receptor protein of human Notch4. In another embodiment, the group that increases the half-life represents the Fc portion of antibodies. In another embodiment, the Fc part of the antibody Ave is dstanley an Fc-portion of human antibodies. In the following embodiment, the extracellular domain and the group, increasing the half-life, are in the same polypeptide chain.

In another embodiment, the above product composition is mixed with a pharmaceutical carrier. In the final embodiment, the subject is the man.

The present invention relates to a replicable vector which encodes a polypeptide containing the extracellular domain of the receptor protein Notch4, functionally associated with the group, increasing the half-life. In one embodiment, the group that increases the half-life represents the Fc portion of antibodies. In another embodiment, the vector includes, but is not limited to, plasmids, kosmidou, retrovirus, adenovirus, phage lambda or YAC.

This invention also relates to the system of vector-host that contains the replicated vector that encodes a polypeptide containing the extracellular domain of the receptor protein Notch, functionally associated with the group, increasing the half-life, and is suitable cell host. In one embodiment, a host cell is a eukaryotic cell. In another embodiment, the eukaryotic cell is a CHO cell. In another embodiment, the eukaryotic cell is a tile is at HeLa. In the following embodiment, a host cell is a bacterial cell.

In conclusion, this invention relates to a third method of obtaining a polypeptide, which includes the cultivation system, the vector-host that contains the replicated vector that encodes a polypeptide containing the extracellular domain of the receptor protein Notch, functionally associated with the group, increasing the half-life, and is suitable cell host, under conditions permitting production of the polypeptide and isolating the polypeptide produced in this way.

The present invention is illustrated in the experimental details, which are presented below. This section is specified to facilitate understanding of the invention, but it is not intended, and should not be construed to limit the invention in any way, as defined in the claims below.

EXPERIMENTAL DETAILS

The first series of experiments

Fused proteins of human Notch1 (traps Notch)

Traps Notch1 collected using the sequences encoding the signal peptide part of the extracellular domain of Notch1, covering all or a subset of domains, EGF-like repeats, and a part of the Fc-human protein (amino acids 1-237). Full full sequence of the human Notch1 presented in figure 59.

<> Used signal peptides are either native signal peptide Notch1 or signal peptide Hc man, each of which is fused with a plot of Notch1. Signal peptide provides for secretion of proteins traps Notch.

Used extracellular domains of Notch1 designed for binding of Notch ligands and consist of all or a subgroup of 36 domains, EGF-like repeats of Notch1 protein man.

C-the end of this EGF-like repeat Notch1 man fused Fc-tag, and it serves to provide purification, detection and stabilization of proteins traps Notch1.

The overall design of the traps of human Notch1, eleven compounds is such that it encodes; (1) a signal peptide that provides secretion of proteins traps Notch1 in the extracellular environment of eukaryotic cells, which are used for production of proteins, (2) part of the extracellular domain of all or part of the EGF-like repeats of Notch1 person to ensure binding to Notch ligands, and (3) the portion of the Fc-protein person, ensuring the detection.

Describes the following eleven compositions traps Notch1 person, and they are schematically presented in figure 52.

1) trap h-Notch1(1-36)(N1-1 figure 52)

2) trap h-Notch1(1-13)(N1-2 figure 52)

3) trap h-Notch1(1-24)(N1-3 figure 52)

4) trap h-spNNotch1(9-23)(N1-4 figure 52)

5) trap h-spHCNotch1(9-23) (N1-5 figure 52)

6) trap h-spNNotch1(9-36)(N1-6 figure 52)

7) trap h-spHCNotch1(9-36)(N1-7 figure 52)

8) trap h-spNNotch1(13-24)(N1-8 figure 52)

9) trap h-spHCNotch1(13-24)(N1-9 figure 52)

10) trap h-spNNotch1(25-36)(N1-10 figure 52)

11) trap h-spHCNotch1(25-36)(N1-11 figure 52)

The sequence of Notch1 person

Full amino acid (A. K.) the sequence of human Notch1, consisting of residues.to. 1 (M = methionine).to. 2555 (K = lysine)shown in figure 59. The signal peptide and the first 36 domains, EGF-like repeats are and.to. 1-1433 this sequence. Amino acids 1-1433, or a subset of these.K. used to construct proteins traps Notch1, described in the following sections. Amino acids, covering the EGF-repeats 1-36, underlined.

Sequence Fc man used to generate Fc-labels on proteins traps Notch1

237 amino acids Fc man presented on figure 60, subjected to fusion with the C-end of all designs of traps Notch1 directly below EGF-like repeats of Notch1. This section Fc man allows detection and cleaning traps Notch and serves to stabilize secreted slit proteins human Notch1-Fc man.

Signal peptides used in proteins traps Notch1

In the structure, the s protein traps Notch1 person included two different sequences of the signal peptide. The first is the signal peptide Notch1 man who, as predicted, comprises amino acids 1-20 human Notch1. This determination was performed using a program Signal IP Server 3.0 provided Technical University of Denmark. The second is the signal peptide Hc of a man who, as predicted, encompasses amino acids 1-22 of the signal peptide of the heavy chain (HC) human IgG.

1. Signal peptide human Notch1 (and.to. 1-20)

MPPLLAPLLCLALLPALA/A/R (SEQ ID NO:16)

Amino acid sequence of the predicted signal peptide Notch1 person is schematically represented in figure 53. The results of the prediction analysis using Signal IP Server 3.0 provided online Technical University of Denmark, presented in figure 53. These results predict the main cleavage site located between alanine 18 (A18) and alanine 19 (A19), and minor cleavage site between the A19 and arginine 20 (R20). These two sites of cleavage are indicated by "/" in the amino acid sequence 1-20 Notch1 person listed above.

2. Signal peptide Notch1 person to merge (and.to. 1-23)used in the traps of Notch1, which uses this signal sequence.

In order to ensure that the signal peptide Notch1 is used effectively in the traps of human Notch1 are three additional amino acids after pre is said the main site of cleavage. Thus, the amino acid sequence used in traps Notch1 person, which includes the signal peptide Notch1, contains a glycine-Proline-arginine (GPR - bold/underlined) between the sites of cleavage of the predicted signal peptide and EGF-like repeats of Notch1, as shown below.

MPPLLAPLLCLALLPALAARGPR(SEQ ID NO:130)

3. Signal peptide HC man (and.to. 1-22)

Amino acid sequence of the predicted signal peptide Hc person is a

MWGWKCLLFWAVLVTATLCTA/R (SEQ ID NO:18)

The results of the prediction analysis using Signal IP Server 3.0 provided online Technical University of Denmark, described above. These results predict the main cleavage site located between alanine 21 (A21) and arginine 22 (22). This cleavage site is indicated by "/" in the amino acid sequence 1-22 Hc person listed above.

Trap h-Notch1(1-36)

Trap h-Notch1(1-36)indicates the trap of human Notch1, which covers the EGF-like repeats 1-36 Notch1 (N1-1 figure 52).

Protein traps h-Notch1(1-36)that is represented in the figure 61 consists of the following three components: (1) the signal sequence of human Notch1, consisting of amino acids 1-23 Notch1 person, then (2) amino acids encoding the EGF-like repeats 1-36 Notch1 person, state the substance of the amino acids 24-1433, and then (3) amino acids 1434-1670 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 1670 amino acids.

Trap h-Notch1(1-13)

Trap h-Notch1(l-13)indicates the trap of human Notch1, which covers the EGF-like repeats 1-13 Notch1 (N1-2 figure 52).

Protein traps h-Notch1(1-13)which is represented on figure 62, consists of the following three components: (1) the signal sequence of human Notch1, consisting of amino acids 1-23 Notch1 person, then (2) amino acids encoding the EGF-like repeats 1-13 Notch1 person, consisting of amino acids 24-531, and then (3) amino acids 532-768 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 768 amino acids.

Trap h-Notch1(1-24)

Trap h-Notch1(1-24)indicates the trap of human Notch1, which covers the EGF-like repeats 1-24 Notch1 (N1-3 figure 52).

Protein traps h-Notch1(1-24)which is represented in figure 63, consists of the following three components: (1) the signal sequence of human Notch1, consisting of amino acids 1-23 Notch1 person, then (2) amino acids encoding the EGF-like repeats 1-24 Notch1 person who astasia of amino acids 24-948, and then (3) amino acids 949-1185 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 1185 amino acids.

Trap h-spNNotch1(9-23)

Trap h-spNNotch1(9-23)indicates the trap of human Notch1, which covers the EGF-like repeats 9-23 Notch1 (N1-4 figure 52). Reduce spNindicates that this compound is used, signal peptide Notch1 person.

Protein traps h-spNNotch1(9-23)which is represented in figure 64, consists of the following three components: (1) the signal sequence of human Notch1, consisting of amino acids 1-23 Notch1 person, then (2) amino acids encoding the EGF-like repeats 9-23 human Notch1, consisting of amino acids 24-593, and then (3) amino acids 594-830 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 830 amino acids.

Trap h-spHCNotch1(9-23)

Trap h-spHCNotch1(9-23)indicates the trap of human Notch1, which covers the EGF-like repeats 9-23 Notch1 (N1-5 figure 52). Reduce spHCindicates that this compound is used, signal peptide HC man.

Protein traps hsp HCNotch1(9-23)which is presented in figure 65, consists of the following three components: (1) the signal sequence of the HC of a person, consisting of amino acids 1-22 Hc person, then (2) amino acids encoding the EGF-like repeats 9-23 human Notch1, consisting of amino acids 23-592, and then (3) amino acids 593-829 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 829 amino acids.

Trap h-spNNotch1(9-36)

Trap h-spNNotch1(9-36)indicates the trap of human Notch1, which covers the EGF-like repeats 9-36 Notch1 (N1-6 figure 52). Reduce spNindicates that this compound is used, signal peptide Notch1 person.

Protein traps h-spNNotch1(9-36)which is represented in figure 66, consists of the following three components: (1) the signal sequence of human Notch1, consisting of amino acids 1-23 Notch1 person, then (2) amino acids encoding the EGF-like repeats 9-36 human Notch1, consisting of amino acids 24-1118, and then (3) amino acids 1119-1355 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 1355 amino acids.

Loves the h-sp HCNotch1(9-36)

Trap h-spHCNotch1(9-36)indicates the trap of human Notch1, which covers the EGF-like repeats 9-36 Notch1 (N1-7 figure 52). Reduce spHCindicates that this compound is used, signal peptide HC man.

Protein traps h-spHCNotch1(9-36)which is represented in figure 67, consists of the following three components: (1) the signal sequence of the HC of a person, consisting of amino acids 1-22 Hc person, then (2) amino acids encoding the EGF-like repeats 9-36 human Notch1, consisting of amino acids 23-1117, and then (3) amino acids 1118-1354 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 1354 amino acids.

Trap h-spNNotch1(13-24)

Trap h-spNNotch1(13-24)indicates the trap of human Notch1, which covers the EGF-like repeats 13-24 Notch1 (N1-8 figure 52). Reduce spNindicates that this compound is used, signal peptide Notch1 person.

Protein traps h-spNNotch1(13-24)that is represented in the figure 68 consists of the following three components: (1) the signal sequence of human Notch1, consisting of amino acids 1-23 Notch1 person, then (2) amino acids encoding the EGF-like repeats 1-24 human Notch1, consisting of amino acids 24-478, and then (3) amino acids 479-715 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 715 amino acids.

Trap h-spHCNotch1(13-24)

Trap h-spHCNotch1(13-24)indicates the trap of human Notch1, which covers the EGF-like repeats 13-24 Notch1 (N1-9 figure 52). Reduce spHCindicates that this compound is used, signal peptide HC man.

Protein traps h-spHCNotch1(13-24)which is represented in figure 69, consists of the following three components: (1) the signal sequence of the HC of a person, consisting of amino acids 1-22 Hc person, then (2) amino acids encoding the EGF-like repeats 13-24 human Notch1, consisting of amino acids 23-477, and then (3) amino acids 478-714 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 714 amino acids.

Trap h-spNNotch1(25-36)

Trap h-spNNotch1(25-36)indicates the trap of human Notch1, which covers the EGF-like repeats 25-36 Notch1 (N1-10 figure 52). Reduce spNindicates that this compound is used, signal peptide Notch person.

Protein traps h-spNNotch1(25-36)which is represented in figure 70, consists of the following three components: (1) the signal sequence of human Notch1, consisting of amino acids 1-23 Notch1 person, then (2) amino acids encoding the EGF-like repeats 25-36 human Notch1, consisting of amino acids 24-508, and then (3) amino acids 509-745 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 745 amino acids.

Trap h-spHCNotch1(25-36)

Trap h-spHCNotch1(25-36)indicates the trap of human Notch1, which covers the EGF-like repeats 25-36 Notch1 (N1-11 figure 52). Reduce spHCindicates that this compound is used, signal peptide HC man.

Protein traps h-spHCNotch1(25-36)that is represented in the figure 71, consists of the following three components: (1) the signal sequence of the HC of a person, consisting of amino acids 1-22 Hc person, then (2) amino acids encoding the EGF-like repeats 25-36 human Notch1, consisting of amino acids 23-507, and then (3) amino acids 508-744 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 744 s is necessaty.

Ways

Designing traps Notch1 person

To obtain variants traps Notch1 person used total RNA endothelial cells of the umbilical vein of a person (HUVEC). Total RNA was subjected to reverse transcription using reverse transcriptase M-MLV and either a random review of the primers or specific primers Notch1. Then the synthesized cDNA amplified using specific to the trap Notch1 upstream (sense) and downstream (antisense) primers.

The underlying primers encode the restriction sites either BamHI or BglII at the 5'-end, which with the subsequent ligation with the BglII site in the Fc sequence with the formation of chimeras Notch1/Fc man in the box read.

In the case of traps Notch1, which form a fused molecule after the nucleotide sequence encoding the EGF-like repeat 36, creates a BglII site for the education section of the merge, and this merged sequence provided (Notch1, figure 4). This applies to the compositions: trap h-Notch1(1-36)trap h-spNNotch1(9-36)trap h-spHCNotch1(9-36)trap h-spNNotch1(25-36)trap h-spHCNotch1(25-36).

In the case of traps Notch1, which form a fused molecule after the nucleotide sequence encoding the EGF-like repeat 13, creates a BamHI site for education participation is ka merge and this merged sequence provided (Notch1, figure 5). This applies to the composition of the trap h-Notch1(1-13).

In the case of traps Notch1, which form a fused molecule after the nucleotide sequence encoding the EGF-like repeat 23, creates a BglII site for the education section of the merge, and this merged sequence provided (Notch1, figure 6). This applies to the compounds of the trap h-spNNotch1(9-23)trap h-spHCNotch1(9-23).

In the case of traps Notch1, which form a fused molecule after the nucleotide sequence encoding the EGF-like repeat 24, creates a BglII site for the education section of the merge, and this merged sequence provided (Notch1, figure 7). This applies to the compounds of the trap h-Notch1(1-24)trap h-spNNotch1(13-24)trap h-spHCNotch1(13-24).

Amplificatory the PCR product subcloning in pBluescript SK II Fc for various chimeras Notch1/Fc man. Then the sequence of traps Notch1/Fc human embed in expressing vectors mammals (pAd-lox, pCCL, pcDNA3 for expression and purification of proteins traps Notch1 person.

Slit proteins human Notch4 (traps Notch)

Traps Notch4 collected using the sequences encoding the signal peptide part of the extracellular domain of Notch4,coveringall or a subset of the house is a new EGF-like repeats, and part of the Fc-human protein (amino acids 1-237). Full full sequence of human Notch4 presented in figure 83.

Used signal peptides are either native signal peptide Notch4 or signal peptide Hc man, each of which is fused with a plot of Notch4. Signal peptide provides for secretion of proteins traps Notch.

Used extracellular domains Notch4 designed so that they linked the Notch ligands, and they consist of all or a subgroup of 29 domain EGF-like repeats of the protein human Notch4.

Fc-tag is fused to the C end of the EGF-like repeat Notch4 person and serves to provide purification, detection and stabilization of proteins traps Notch4.

The overall design of the traps of human Notch4, eleven compounds is such that it encodes; (1) a signal peptide that provides secretion of proteins traps Notch4 in the extracellular environment of eukaryotic cells, which are used for production of proteins, (2) part of the extracellular domain of all or part of the EGF-like repeats of human Notch4 to ensure binding to Notch ligands, and (3) the portion of the Fc-protein person, ensuring the detection.

Describes the following eleven compositions traps Notch4 person, and they are schematically represented in figure 111.

1) trap h-Notch4(1-29)(N4-1 figure 111)

2) trap h-Notch4 (1-13)(N4-2 figure 111)

3) trap h-Notch4(1-23)(N4-3 figure 111)

4) trap h-spNNotch4(9-23)(N4-4 figure 111)

5) trap h-spHCNotch4(9-23)(N4-5 figure 111)

6) trap h-spNNotch4(9-29)(N4-6 figure 111)

7) trap h-spHCNotch4(9-29)(N4-7 figure 111)

8) trap h-spNNotch4(13-23)(N4-8 figure 111)

9) trap h-spHCNotch4(13-23)(N4-9 figure 111)

10) trap h-spNNotch4(21-29)(N4-10 figure 111)

11) trap h-spHCNotch4(21-29)(N4-11 figure 111)

The sequence of human Notch4

Full amino acid (A. K.) the sequence of human Notch4, consisting of residues.to. 1 (M = methionine).to. 2003 (K = lysine)shown in figure 83. The signal peptide and the first 29 domains, EGF-like repeats are and.to. 1-1174 this sequence. Amino acids 1-1174, or a subset of these.K. used to construct proteins traps Notch4, described in the following sections. Amino acids, covering the EGF-repeats 1-29, underlined.

Sequence Fc man used to generate Fc-labels on proteins traps Notch1

237 amino acids Fc man presented on figure 84 were subjected to fusion with the C-end of all designs of traps Notch4 directly below EGF-like repeats of Notch1. This section Fc man allows detection and cleaning traps Notch and serve the stabilization of secreted slit proteins Notch4 human Fc human.

Signal peptides used in proteins traps Notch4

In the design of proteins traps Notch4 person included two different sequences of the signal peptide. The first is the signal peptide Notch4 man who, as predicted, encompasses amino acids 1-24 human Notch4. This determination was performed using a program Signal IP Server 3.0 provided Technical University of Denmark. The second is the signal peptide Hc of a man who, as predicted, encompasses amino acids 1-22 of the signal peptide Hc man.

1. Signal peptide human Notch4.to. 1-24)

MQPPSLLLLLLLLLLLCVSVVRP/R (SEQ ID NO:104)

Amino acid sequence of the predicted signal peptide Notch4 person is schematically represented in figure 112. The results of the prediction analysis using Signal IP Server 3.0 provided online Technical University of Denmark, presented in figure 112. These results predict the cleavage site located between the Proline 23 (A23) and arginine 24 (R24). The site of cleavage is indicated by "/" in the amino acid sequence 1-24 Notch4 person listed above.

2. Signal peptide Notch4 person to merge (and.to. 1-27)used in the traps Notch4 that use this signal sequence.

In order to ensure that the signal peptide Notch4 used the SJ effectively, in the traps of human Notch4 are three additional amino acids after the predicted primary site of cleavage. Thus, the amino acid sequence used in traps Notch4 person, which includes the signal peptide Notch4, contains a glycine-Proline-arginine (GPR - bold/underlined) between the sites of cleavage of the predicted signal peptide and EGF-like repeats Notch4, as shown below.

MQPPSLLLLLLLLLLLCVSVVRPRGLL(SEQ ID NO:131)

3. Signal peptide HC man (and.to. 1-22)

Amino acid sequence of the predicted signal peptide Hc person is a

MWGWKCLLFWAVLVTATLCTA/R (SEQ ID NO:17)

The results of the prediction analysis using Signal IP Server 3.0 provided online Technical University of Denmark, described above. These results predict the main cleavage site located between alanine 21 (A21) and arginine 22 (22). This cleavage site is indicated by "/" in the amino acid sequence 1-22 Hc person provided above.

Trap h-Notch4(1-29)

Trap h-Notch4(1-29)indicates the trap Notch4 person, which covers the EGF-like repeats 1-29 Notch4 (N4-1 figure 111).

Protein traps h-Notch4(1-29)consists of the following three components: (1) the signal sequence of human Notch4, consisting of amine the acids 1-27 Notch4 person, then (2) amino acids encoding the EGF-like repeats 1-29 Notch4 person, consisting of amino acids 28-1173, and then (3) amino acids 1174-1410 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 1410 amino acids.

Trap h-Notch4(1-13)

Trap h-Notch4(1-13)indicates the trap Notch4 person, which covers the EGF-like repeats 1-13 Notch4 (N4-2 figure 111).

Protein traps h-Notch4(1-13)consists of the following three components: (1) the signal sequence of human Notch4, consisting of amino acids 1-27 Notch4 person, then (2) amino acids encoding the EGF-like repeats 1-13 Notch4 person, consisting of amino acids 28-554, and then (3) amino acids 555-791 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 791 amino acid.

Trap h-Notch4(1-23)

Trap h-Notch4(1-23)indicates the trap Notch4 person, which covers the EGF-like repeats 1-23 Notch4 (N4-3 figure 111).

Protein traps h-Notch4(1-23)consists of the following three components: (1) the signal sequence of human Notch4, consisting of amino acids 1-27 Notch4 person, then (2) amino acids encoding GF-like repeats 1-23 Notch4 person, consisting of amino acids 28-933, and then (3) amino acids 934-1170 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 1170 amino acids.

Trap h-spNNotch4(9-23)

Trap h-spNNotch4(9-23)indicates the trap Notch4 person, which covers the EGF-like repeats 9-23 Notch4 (N4-4 figure 111). Reduce spNindicates that this compound is used, signal peptide Notch4.

Protein traps h-spNNotch4(9-23)consists of the following three components: (1) the signal sequence of human Notch4, consisting of amino acids 1-27 Notch4 person, then (2) amino acids encoding the EGF-like repeats 9-23 Notch4 person, consisting of amino acids 28-602, and then (3) amino acids 603-839 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 839 amino acids.

Trap h-spHCNotch4(9-23)

Trap h-spHCNotch4(9-23)indicates the trap Notch4 person, which covers the EGF-like repeats 9-23 Notch4 (N4-5 figure 111). Reduce spHCindicates that this compound is used, signal peptide HC man.

Protein traps h-spHCNoth4 (9-23)consists of the following three components: (1) the signal sequence of the HC of a person, consisting of amino acids 1-22 Hc person, then (2) amino acids encoding the EGF-like repeats 9-23 Notch4 person, consisting of amino acids 23-597, and then (3) amino acids 598-834 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 834 amino acids.

Trap h-spNNotch4(9-29)

Trap h-spNNotch4(9-29)indicates the trap Notch4 person, which covers the EGF-like repeats 9-29 Notch4 (N4-6 figure 111). Reduce spNindicates that this compound is used, signal peptide human Notch4.

Protein traps h-spNNotch4(9-29)consists of the following three components: (1) the signal sequence of human Notch4, consisting of amino acids 1-27 Notch4 person, then (2) amino acids encoding the EGF-like repeats 9-29 Notch4 person, consisting of amino acids 28-843, and then (3) amino acids 844-1080 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 1080 amino acids.

Trap h-spHCNotch4(9-29)

Trap h-spHCNotch4(9-29)putting the AET trap Notch4 person, which covers the EGF-like repeats 9-29 Notch4 (N4-7 figure 111). Reduce spHCindicates that this compound is used, signal peptide HC man.

Protein traps h-spHCNotch4(9-29)consists of the following three components: (1) the signal sequence of the HC of a person, consisting of amino acids 1-22 Hc person, then (2) amino acids encoding the EGF-like repeats 9-29 Notch4 person, consisting of amino acids 23-838, and then (3) amino acids 839-1075 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 1075 amino acids.

Trap h-spNNotch4(13-23)

Trap h-spNNotch4(13-23)indicates the trap Notch4 person, which covers the EGF-like repeats 13-23 Notch4 (N4-8 figure 111). Reduce spNindicates that this compound is used, signal peptide human Notch4.

Protein traps h-spNNotch4(13-23)consists of the following three components: (1) the signal sequence of human Notch4, consisting of amino acids 1-27 Notch4 person, then (2) amino acids encoding the EGF-like repeats 13-23 Notch4 person, consisting of amino acids 28-444, and then (3) amino acids 445-681 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is podcherknuto, and Fc-label human underlined and in italics. This composition contains 681 amino acid.

Trap h-spHCNotch4(13-23)

Trap h-spHCNotch4(13-23)indicates the trap Notch4 person, which covers the EGF-like repeats 13-23 Notch4 (N4-9 figure 111). Reduce spHCindicates that this compound is used, signal peptide HC man.

Protein traps h-spHCNotch4(13-23)consists of the following three components: (1) the signal sequence of the HC of a person, consisting of amino acids 1-22 Hc person, then (2) amino acids encoding the EGF-like repeats 13-23 Notch4 person, consisting of amino acids 23-439, and then (3) amino acids 440-676 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 676 amino acids.

Trap h-spNNotch4(21-29)

Trap h-spNNotch4(21-29)indicates the trap Notch4 person, which covers the EGF-like repeats 21-29 Notch4 (N4-10 figure 111). Reduce spNindicates that this compound is used, signal peptide human Notch4.

Protein traps h-spNNotch4(21-29)consists of the following three components: (1) the signal sequence of human Notch4, consisting of amino acids 1-27 Notch4 person, then (2) s is necessaty, encoding the EGF-like repeats 21-29 Notch4 person, consisting of amino acids 28-392, and then (3) amino acids 393-629 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 629 amino acids.

Trap h-spHCNotch4(21-29)

Trap h-spHCNotch4(21-29)indicates the trap Notch4, person which covers the EGF-like repeats 21-29 Notch4 (N4-11 figure 111). Reduce spHCindicates that this compound is used, signal peptide HC man.

Protein traps h-spHCNotch4(21-29)consists of the following three components: (1) the signal sequence of the HC of a person, consisting of amino acids 1-22 Hc person, then (2) amino acids encoding the EGF-like repeats 21-29 Notch4 person, consisting of amino acids 23-387, and then (3) amino acids 388-624 that contain Fc-the mark of the man. The predicted sequence of the signal peptide is underlined, and Fc is the mark of a man is underlined and in italics. This composition contains 624 amino acids.

Ways

Designing traps Notch4 person

To obtain variants traps Notch4 person used total RNA endothelial cells of the umbilical vein of a person (HUVEC). Total RNA was subjected to reverse transcripti is using reverse transcriptase M-MLV and either a random review of the primers, specific primer Notch4. Then the synthesized cDNA amplified using specific to the trap Notch4 upstream (sense) and downstream (antisense) primers.

The underlying primer encodes the restriction sites either BamHI or BglII at the 5'-end, which with the subsequent ligation with the BglII site in the Fc sequence with the formation of chimeras Notch4/Fc man in the box read.

In the case of traps Notch4, which form a fused molecule after the nucleotide sequence encoding the EGF-like repeat 29, creates a BglII site for the education section of the merge, and this merged sequence provided (Notch4, figure 113). This applies to the compositions: trap h-Notch4(1-29)trap h-spNNotch4(9-29)trap h-spHCNotch4(9-29)trap h-spNNotch4(21-29)trap h-spHCNotch4(21-29).

In the case of traps Notch4, which form a fused molecule after the nucleotide sequence encoding the EGF-like repeat 13, creates a BamHI site for the education section of the merge, and this merged sequence provided (Notch4, figure 114). This applies to the composition of the trap h-Notch4(1-13).

In the case of traps Notch4, which form a fused molecule after the nucleotide sequence encoding the EGF-like repeat 23, creates a BglII site for the education section of the merge, and this merged p is the sequence provided (Notch4, figure 115). This applies to the compounds of the trap h-Notch4(1-23)trap h-spNNotch4(9-23)trap h-spHCNotch4(9-23)trap h-spNNotch4(13-23)trap h-spHCNotch4(13-23).

Amplificatory the PCR product subcloning in pBluescript SK II Fc for various chimeras Notch4/Fc man. Then the sequence of traps Notch4/Fc human embed in expressing vectors mammals (pAd-lox, pCCL) for expression and purification of proteins traps Notch4 person.

The second series of experiments

Materials and methods

Plasmid construction

Adenoviral constructs encoding LacZ, full-Notch4 or the activated form of Notch4/int3 described previously (Shawber et al., 2003). The activated form of Notch1 cDNA, fused in reading frame with 6 labels myc (Kopan et al., 1994) cloned into adenovirus expressing vector, pAd-lox. Also in pAd-lox cloned as VEGF165, and N1ECDFc. Received source solutions with adenovirus and was titrated as described previously (Hardy et al., 1997). Retroviral expressing vector pHyTc encoding any of LacZ, the activated form of Notch4/int3, J1, Dll1 and Dll4 described previously (Uyttendaele et al., 2000, Shawber et al., 2003, Das et al., 2004 in press). Plasmids encoding the intracellular domain of Notch1 (p. N. 5479-7833, registration number Genbank # X57405) and the extracellular domain of Dll4 (p. N. 1-1545, registration number Genbank # AF253468 provided by Chiron), SL is th frame read tagged with myc/His, built in pHyTC.

Notch1ECD, Notch2ECD, Notch3ECD and Notch4ECD design using Fc sequences contained in the plasmid pCMX-sFR1-IgG using the methods described in Clin. Exp. Immunol. (1992) 87 (1):105-110, to create a fused protein on the basis of the Notch, i.e., Notch1ECD/Fc, Notch2ECD/Fc, Notch3ECD/Fc and Notch4ECD/Fc.

Adenoviral gene transfer

7,5×105the HUVEC cells after 3 passirovanny were sown in coated with collagen type I 6-hole tablets per day up to adenoviral infection. Adenoviral infection by Ad-lacZ, Ad-VEGF165 or Ad-N1ECDFc was carried out at the specified m.o.i. and incubated at 37°C for 1 h with periodic rotation of the tablets.

Assays with luciferase reporter

To determine induced by ligand signal transmission Notch tested coculture using HeLa cells and originating from 293 cells Bosc. Temporary transfection was performed by precipitation with calcium phosphate. HeLa cells, seeded 1 day in 10-cm plates in quantities of 1,5x106was transfusional 333 ng pBOS Notch1, 333 ng pGA981-6 and 83 ng pLNC with lacZ or 666 ng pCMV-Fc, or pHyTC-N1ECDFc (333 ng for x1, 666 ng for x2). The Bosc cells, seeded 1 day in 10-cm plates in the number of 4×106was transfusional 680 ng or pHyTc-Jagged1 or pHyTc-Dll1 or pHyTc-Dll4 or pHyTc-x (empty vector). Through one day after transfection cells were cocultivated in triplicate (HeLa: Bosc, 1:2) 12-hole the tablets within 24 hours. The cells were collected and determined the luciferase activity in 2 days after transfection using the test kit Enhanced Luciferase (BD PharMingen), and the activity of β-galactosidase activity was determined using a set of Galacto-Light Plus (PE Biosystems). All analyses were performed with a luminometer with dual injection Berthold.

To determine the induced VEGF signal transmission Notch, used HUVEC that were infected with adenovirus. HUVEC, seeded 1 day in 6-well plates in the number 8,0x105were infected with either Ad-LacZ as a control or Ad-VEGF for a given m.o.i. in the presence or absence of Ad-N1ECD/Fc. Two days after infection, infected HUVEC were perseval in a 24-well plate in an amount of 1.5×105cells in triplicate and cultured for 24 hours, and then was transfusional of 12.5 ng pRL-SV40 (Promega) and 137.5 (ng pGA981-6 using the reagent for transfection with Effectene (Qiagen). Cells were collected either in 1 or in 2 days after transfection and was determined by the luciferase activity using the system for the analysis of Dual-Luciferase® Reporter Assay System (Promega).

Analysis of germination

To obtain collagen gels ice-cold solution of collagen pig type I (Nitta gelatin, Tokyo, Japan) was mixed with 10x RPMI1640 medium and neutralizing buffer in the ratio of 8:1:1. Then in 24-hole tablets were added to a 400-μl aliquot of the collagen gel and allowed them to form the gel for at least 1 hour at 37°C. After adenoviral infection (above) HUVEC were collected and sown in the amount of 1.3×105cells per well on top of the collagen gel in a 24-hole tablets in 0.8 ml of EGM2 medium. HUVEC were achieved almost complete closure of the monolayer after 48 hours after sowing. After sowing the medium was replaced every 2 days for 1 week. Germination was observed, and pictures taken after 8 days using a digital camera Olympus, was placed under the microscope. For the quantitative determination of the number of shoots randomly chose 5 fields per well, and two researchers were calculated germination under a microscope blind method.

Results and discussion

Slit proteins NOTCHECD/Fc function as antagonists of Notch

Antagonists of Notch - fused proteins NotchECD/Fc

The authors of the present invention has been several antagonists of Notch (figure 2). The strategy of the authors of the present invention was to merge the coding sequence of the EGF-repeats of Notch extracellular domain (ECD) with the Fc-domain of human or mouse. This design leads to Sekretareva squirrel without a transfer feature of a signal, but which retains the ligand-binding domain, and, thus, should contact the ligand and inhibit its function. The authors of the present invention indicate these proteins as "NotchECD/Fc", and received all four the e Notch1-4ECD/Fc. Fc-domain facilitates affinity purification and detection of the protein by Western blot turns or immunohistochemistry.

Testing of antagonists of Notch

To measure the transcriptional activation of the cascade Notch used the system coculturein vitro(figure 3) with ligands expressed on the same cell, and activation of the Notch receptor being evaluated in another cell. The authors of the present invention used this analysis coculture in order to show that Notch1ECD/Fc operates by blocking the ligand-dependent signal transmission Notch (figure 4). Expressing the vector N1ECD/Fc was cotranslationally in different ratios with a full Notch1 and CSL-luciferase reporter in HeLa cells, with subsequent cocultivation ligand-expressing 293 cells. The authors of the present invention have observed that the activation signal Notch1 Notch ligands decreased by the expression N1ECD/Fc. This effect showed a dependence on the concentration; the ratio of 2:1 for N1ECD/Fc and Notch1 was more effective in inhibiting the transmission of the signal than the ratio of 1:1. Notch1ECD/Fc can block the signal transmission mediated Jagged1, Delta-like 1 and Delta-like 4.

Expression and purification of Notch antagonists

The authors of the present invention has been cell lines CHO and HeLa expressing NotchECD/FC using retroviral vectors DL the protein purification. Proteins N1ECD/Fc are secreted (figure 5), as indicated, in conditioned medium collected from lines HeLa-NotchECD/Fc and purified using agarose protein A (pA). Purified using pA sample (Sup) and lysates of whole cells (Lys) were subjected to immunoblotting with antibody to α-Fc (figure 5, panel A), demonstrating that N1ECD/Fc secreted into the environment. Adenoviral vectors for NotchECD/Fc was used to infect HeLa cells, and lysates of these cells were subjected to Western blot turns with antibodies to α-Fc, demonstrating that they Express proteins NotchECD/Fc(1, 2, 3, 4) (figure 5, panel B). The authors of the present invention at the present time clear N1ECD/Fc environment, conditioned by CHO cells using affinity chromatography with pA.

Determination of inhibition of angiogenesis using fused proteins Notch

The detection of the activation signal transmission Notch can be performed using promoter activity CBF1

The transfer function of the Notch signal can be measured by measuring the transcriptional activity of CBF1 promoter, which is activated upon binding of Notch-IC with CBF1. The authors of the present invention measured the activity of the promoter of CBF1 in HUVEC that were infected with adenovirus encoding VEGF-165 at different MOI (figure 6). Induction of CBF1 promoter was clearly detected in HUVEC infected with Ad-VEGF compared with cells infected with Ad-LacZ, dependent on the MOI. These data show that overexpression of VEGF can activate the signal transmission Notch in HUVEC. Thus, VEGF induced activity signal transmission Notch.

The authors of the present invention were interested in whether the fused proteins Notch block induced VEGF activation signal transmission Notch. Confezione fused protein Ad-Notch with Ad-VEGF clearly reduced activation activity of CBF1 promoter induced by infection by Ad-VEGF separately (figure 7). In the case of infection at MOI of 40 for each adenovirus, as shown in figure 7 (panel A), was identified in 60% inhibition after 24 h and 90% inhibition after 48 after transfection reporter gene, and that the inhibitory activity of traps Notch depended on MOI fused protein Ad-Notch.

Merged Notch proteins block the initiation of angiogenic sprouting induced VEGF

In this experiment, the authors present invention was evaluated the effect of traps Notch on the induction of budding (initiation of germination) sverhagressivnym VEGF-165 in HUVEC. When HUVEC infected with Ad-VEGF, were cultured on collagen gel type for 8 days, there was induction of budding in collagen gel. This induction of budding sverhagressivnym VEGF clearly inhibited by Confucianism adenovirus coding for a protein Notch (figure 8). Protein Ad-Notch records of the tion had less effect on the morphology.

The figure 9 shows the calculation of the kidneys on the field with a microscope. Infection of HUVEC by Ad-VEGF increased the number of buds, depending on the MOI. Induced by Ad-VEGF budding clearly inhibited. These data indicate that VEGF induces the formation HUVEC through activation of signal transmission Notch and that Notch protein can inhibit induced VEGF budding.

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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 rquired for the release of the notch2 is intracellular domain. Embo J. 21:294-302.

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

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

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

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.

47. Tamura, K., Y. Taniguchi, S. Minoguchi, T. Sakai, T. Tun, T. Furukawa, and T. Honjo. 1995. Physical interaction between a novel domain of the receptor Notch and the reduced factor RBP-J kappa/Su (H). Curr Biol. 5:1416-1423.

48. Tietze, K., N. Oellers, and E. Knust. 1992. Enhancer of splitD, a dominant mutation of Drosophila, and its use in the study of functional domains of a helix-loop-helix protein. Proc Natl Acad Sci USA 89:6152-6156.

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.

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

51. Vervoort, M., C. Dambly-Chaudiere, and A. Ghysen. 1997. Cell fate determination in Drosophila. Curr Opin Neurobiol. 7:21-28.

52. Villa, N., L. Walker, C. E. Lindsell, J. Gasson, M. L. Iruela-Arispe, and G. Weinmaster. 2001. Vascular expression of Notch pathway receptors and ligands is restricted to arterial vessels. Mechanisms of Development 108:161-164.

53. Weinmaster, G. 1997. The Ins and Outs of Notch Signaling. Mol. Cel. Neurosci. 9:91-102.

54. Weinmaster, G. 1998. Notch signaling: direct or what? Curr Opin Genet Dev. 8:436-42.

55. Weinmaster, G., V. J. Roberts, and G. Lemke. 1992. Notch 2: a second mammalian Notch gene. Development 116:931-941.

56. Weinmaster, G., V. J. Roberts, and G. A. Lemke. 1991. A homolog of Drosophila Notch expressed during mammalian development. Development 113:199-205.

57. Wettstein, D. A., D. L. Turner, and C. Kintner. 1997. The Xenopus homolog of Drosophila Suppressor of Hairless mediates Notch signaling during primary neurogenesis. Development 124:693-702.

58. Wu, G., E. J. Hubbard, Kitajewski J. K., and I. Greenwald. 1998. Evidence for functional and physical association between Caenorhabditis elegans SEL-10, a Cdc4p-related protein, and SEL-12 presenilin. Proc Natl Acad Sci USA 95:15787-91.

59. Wu, G., S. A. Lyapina, I. Das, J. Li, M. Gurney, A. Pauley, I. Chui, R. J. Deshaies, and J. Kitajewski. 2001. SEL-10 is an inhibitor of notch signaling that targets notch for ubiquitin-mediated protein degradation. Mol. Cell. Biol. 21:7403-7015.

60. Xue, Y., X. Gao, C. E. Lindsell, C. R., Norton, B. Chang, C. Hicks, M. Gendron-Maguire, E. B. Rand, G. Weinmaster, and T. Gridley. 1999. Embryonic lethality and vascular defects in mice lacking the Notch ligand Jaggedl. Hum Mol. Genet. 8:723-30

The third series of experiments

VEGF initiates angiogenesis through activation of signal transmission Notch

Cascades of signal transmission as VEGF and Notch important for the development of blood vessels. Here the authors present invention show that VEGF activates the signal transmission Notch to initiate angiogenesis. VEGF increased the expression of Delta4 and Notch4, causing the activation of signal transmission Notch and inducyruya filopodia in cultured primary endothelial cells. Studies using inhibitors of VEGF receptor show that the activation of signal transmission Notch, in turn, enhances the action of VEGF by induction of expression of VEGFR-1 (Flt-1). Other elements of the actions of VEGF, including the induction of MMP-9 and MT1-MMP, aposre the described Notch. Using analysisin vivofor modeling induced VEGF neovascularization of the skin, the authors present invention revealed that the secretory inhibitor of Notch (protein-based Notch) blocks induced VEGF the neovascularization and the induction of expression of VEGFR-1. Thus, the signal transmission Notch is required for angiogenesis, which is regulated by VEGF, probably at the level of initiation.

VEGF is a key regulator of angiogenesis, consisting of many processes, such as degradation of ECM, formation (education filopodia), proliferation, survival and migration of endothelial cells. Although most stages can interact with later in the cascade molecules signal transmission VEGF, it is not known how these stages coordination regulated, resulting in more complex morphogenetic events, such as angiogenic sprouting. Signal transmission Notch is evolutionarily conservative mechanism of signal transmission, which operates by adjusting the choice of cell fate (1). Upon binding ligand, such as Jagged and Delta-like, cytoplasmic domain of Notch (NotchIC) released presenilin/γ secretases, translocases in the nucleus, interacts with the transcriptional repressor CSL (CBF1/Su(H)/lag2) and turns it into a transcriptional activator (1). The assumption about the role of the transmission signal is Ala Notch in the development of vessels were made through studies of mice with targeted mutation (2). Because activation of Notch in the endothelium also interrupts vascular remodeling, proper signal transmission Notch is required for the development of blood vessels (3). Although an assumption was made about the importance of Notch for signal transmission VEGF (4-6), it is still unclear how the signal transmission Notch is involved in the regulated VEGF angiogenesis, and then whether the signal transmission Notch in physiological and pathological angiogenesis in vascular adults.

The growth of HUVEC (endothelial cells of the umbilical vein of a person depends on VEGF (Fig. 26A and 26B) and associated with the differentiation of biological responses, such as germination, and can be assessed at an early stage (7). First, the authors present invention was evaluated, induces whether translationally using adenovirus VEGF expression as Notch and Notch ligand in HUVEC, cultured with complete medium containing bFGF (Fig. 22A), as described (5). Analysis by RT-PCR showed that there was activation of RNA as the Dl4, and Notch4 in HUVEC, transduced VEGF using adenovirus (Ad-VEGF HUVEC), compared to HUVEC, translotsirovannoi LacZ using adenovirus (Ad-LacZ HUVEC) (Fig. 22A). Translationally VEGF, as it turned out, no induces the expression of Jagged1 and Notch1. Translationally VEGF is also activated by the signal transmission Notch dose-dependent manner when measuring the activity of CSL-luciferase reporter (Fig. 22B), which Transact who was virvalsya by transmitting a signal Notch (8). Signal transmission Notch was activated at a higher dose of Ad-VEGF compared with proliferation (Fig. 26A). As SU5416, which is an inhibitor of VEGFR kinase, reduced induced VEGF activity CSL-luciferase reporter (Fig. 22C), VEGF induced signal transmission by the activation of Notch receptor kinase. Since the Notch mutants lacking both the transmembrane and cytoplasmic domains, functioned as dominant-negative inhibitors of signal transmission Notch (9), the authors of the present invention has been fused protein on the basis of the Notch or hook Notch (N1ECDFc) for inhibition of signal transmission Notch (Fig. 22D). Analysis of Western blot testing air-conditioned environment HUVEC, transduced by Ad-N1ECDFc (Ad-N1ECDFc-HUVEC)showed that N1ECDFc well expressed and secreted (Fig. 22E). Using the analysis in coculture in which the Bosc cells expressing Notch ligands (any of J1, D11 or D14), activated signal transmission Notch in HeLa cells expressing Notch1, compared with control cells Bosc, the authors present invention was determined by inhibition of signal transmission Notch at transfection expressing N1ECDFc plasmids (Fig. 22F). Then the authors present invention investigated, inhibits whether N1ECDFc activation signal transmission Notch through transducing VEGF in HUVEC (Fig. 22G). Totranslate Ad-N1ECDFc with d-VEGF in HUVEC clearly lowered activity of CSL-luciferase, induced by VEGF. Gerhardt et al. described that VEGF controls angiogenesis in the retina in the early postnatal period by controlling the elongation of filopodia on the tops of the shoots vessels (10). During angiogenic sprouting education specialized endothelial cells forming the protrusions filopodia, among resting endothelial cells, can be one of the early events. Here the authors present invention understand the formation of endothelial cells forming the protrusion filopodia as budding. The formation of primary endothelial cells induce their 3-dimensional cultivation of either fibrin or collagen gel (11). In the case where Ad-VEGF HUVEC were cultured on collagen gel with a full environment, transduced HUVEC formed lengthening filopodia in collagen gel within 5 days (Fig. 22H), and the number of kidneys was increased in a dose-dependent manner (Fig. 27A). Activation of signal transmission Notch by adenovirus encoding the activated form of Notch4 (Ad-Notch4/int3), induced budding HUVEC (12) and the activated form of Notch1 (Ad-N1IC), also induced the split in HUVEC (Fig. 23A and 27B). Because the signal transmission as VEGF and Notch induces the split in HUVEC, the authors of the present invention investigated, inhibits whether N1ECDFc induced VEGF budding HUVEC (Fig. 22H-I). Budding Ad-VEGF HUVEC clearly inhibi who was Avalos atransducer Ad-N1ECDFc. No HUVEC transduced with Ad-LacZ or HUVEC transduced with Ad-N1ECDFc, do not form buds (Fig. 22H). N1ECDFc inhibited induced VEGF budding HUVEC without affecting the number of cells (Fig. 22I). Translationally N1ECDFc did not change clearly the proliferation of HUVEC, although the proliferation of transduced with Ad-N1IC HUVEC inhibited dose-dependent manner (Fig. 28A), which is consistent with the inhibitory efficiency of signal transmission Notch on the proliferation of the endothelium (13).

To test whether signal transmission Notch directed to VEGF, the authors of the present invention were evaluated by three different inhibitor receptor tyrosinekinase, including VEGFR, induced N1IC budding HUVEC, because these three growth factor was in complete medium (Fig. 23A-C). At a concentration of 1 μm each compound showed selective inhibition of each kinase (data not shown). No PD166866, nor ZD1893 did not affect the formation of Ad-N1IC-HUVEC and SU5416 clearly inhibited it (Fig. 23A-B). SU5416 selectively inhibited the formation of Ad-N1IC-HUVEC with a smaller decrease in viability at lower concentrations (Fig. 23C). Since then, when Taylor et al. described what Notch inhibited the expression of Flk1/KDR/VEGFR2 (14), it was unlikely that Notch combined with Flk1 to provide budding. Thus, the authors of the present invention investigated whether activation of signal transmission Notch on the expression Ft1/VEGFR1 in HUVEC, as SU5416 inhibits kinase activity as Flt1 and Flk1 (15). Analysis by RT-PCR showed that the expression of Flt1 mRNA is increased in Ad-N1IC-HUVEC, and the expression of a marker of endothelial cells, CD31 mRNA, compared with expression in Ad-LacZ-HUVEC (Fig. 23D). Analysis of Western blotting showed that the expression of Flt1 protein was increased in Ad-N1IC-HUVEC (Fig. 23E). Thus, the authors of the present invention investigated, runs whether P1GF, which is a selective ligand Flt1, budding HUVEC in which Flt1 activated by the activation signal transmission Notch (Fig. 23F-G). P1GF increased the number of kidneys Ad-N1IC-HUVEC by 150%, compared with the absence of P1GF (Fig. 23F). Moreover, P1GF increased the number of kidney HUVEC containing many filopodia, 250% (Fig. 23G). While the reduction of Flt1 expression using small interfering RNA (siRNA) for Flt1 inhibited the formation of Ad-N1IC-HUVEC (Fig. 23J), transfection selectively reduced expression of Flt1 mRNA (Fig. 23H) and Flt1 protein (Fig. 23I). Although the decline of Flk1 expression by siRNA for Flk1 also inhibited the formation of Ad-N1IC-HUVEC (Fig. 30B), the inhibitory efficiency of siRNA for Flk1 was less than the inhibitory efficiency of siRNA for Flt1 (Fig. 23J). Effects of siRNA for Flk1 were higher in respect of budding Ad-VEGF HUVEC than in respect of Ad-N1IC-HUVEC (Fig. 30B-C). Transfection by siRNA for Flt1 inhibited budding as Ad-N1IC-and Ad-VEGF-HVEC at similar levels (data not shown).

Some studies have shown that VEGF regulated activity gelatinase in endothelial cells and firmly set activity gelatins, such as MMP-2 and MMP-9, for the induction of angiogenic sprouting (16). The authors of the present invention investigated, regulates whether VEGF activity gelatinase through the signal transmission Notch in HUVEC.

In gelatinases demografie air-conditioned environment of Ad-VEGF HUVEC showed how the induction and activation of MMP9, which began to be detected at 6 days (Fig. 24A), and activation of MMP2, which was detected at 4 days (Fig. 24B), compared with conditioned medium from Ad-LacZ-HUVEC. Totranslate Ad-N1ECDFc with Ad-VEGF showed inhibition of both induction and activation of MMP9 (Fig. 24A), and activation of MMP2 (Fig. 24B). Analysis of RT-PCR showed that the expression of MMP9 mRNA was increased in Ad-N1IC-HUVEC, but the expression of MMP2 mRNA was decreased in Ad-N1IC-HUVEC (Fig. 24C). Since the induction of MMP2 activity was not detected in gelatinases demografie (Fig. 24B), this result was plausible consequence. At the same time, the expression of MT1-MMP, which is able to activate MMP2 on the cell surface (17), were increased at both the protein and transcript in Ad-N1IC-HUVEC (Fig. 24D). Because VEGF may regulate the expression as gelatinase and MT1-MMP (16), analysis of RT-PCR showed that both MMP9 and MT1-MMP are activated in Ad-VEGF HUVEC compared with Ad-LacZ HUVEC, and this induction is inhibited to what restuccia Ad-N1ECDFc (Fig. 24E). Infection with Ad-N1ECDFc separately did not affect the expression of both MMP9, MT1-MMP in HUVEC infected with Ad-LacZ (data not shown). The need for MMP for angiogenic sprouting was installed using synthetic MMP inhibitors (16). GM6001 is a broad inhibitor of MMPs, including MMP2, MMP9 and MT1-MMP (18). GM6001 was clearly reduced the formation of Ad-N1IC-HUVEC, as on collagen (Fig. 31A-B)and fibrin gel (data not shown).

In the analysis of the dorsal air SAC (DAS) in mice (19), stable transfectant 293 cells, sverkhekspressiya VEGF121 (293/VEGF), significantly induced angiogenesisin vivo(Fig. 25A, left panel). This induced VEGF angiogenesis clearly inhibited by coexpressing N1ECDFc, compared with 293/VEGF separately (Fig. 25A). Conducted to measure the density of vessels, and the index of angiogenesis, shown in Fig. 25B shows that the induced 293/VEGF angiogenesis inhibited by co-expression of 293/N1ECDFc (Fig. 25B).

Also, in the analysis of the dorsal air SAC (DAS) mice (19), cell line breast cancer human MDA-MB-231, significantly induced angiogenesisin vivopresumably by the secretion of VEGF (Fig. 25C, left panel). This induced VEGF angiogenesis clearly inhibited mediated by adenovirus expression N1ECDFc compared with adenovirus expressing LacZ. (Fig. 25C). Density with the perception was measured, and the index of angiogenesis, shown in Fig. 25D shows that induced MDA-MB-231 angiogenesis inhibited by expression N1ECDFc.

Flk1 is a major positive signal transmitter for angiogenesis due to its pronounced tyrosinekinase activity in the embryo, whereas Flt1 is considered a negative signal transmitter for angiogenesis. However, the positive role of Flt-1 was demonstrated in adult mice, because the growth ofin vivoLLC, sverkhekspressiya P1GF2, was significantly decreased in mice lacking the cytoplasmic kinase domain of Flt-1 (20). Notch may function by altering signaling by VEGF induction signal Flt-1 and reduce transmission of the signal Flk-1, or for the induction of elongation of filopodia, or to enhance angiogenic sprouting, since the signal transmission P1GF/Flt-1 changes the site of phosphorylation of Flk-1 and potentional ischemic angiogenesis in the myocardium (21). Interestingly, the signal transmission Notch also increases the expression of P1GF (Fig. 29). However, a continuous activation signal transmission Notch inhibits formation of multicellular containing the lumen of the processes, as described previously (22). Signal transmission Notch should turn off after budding/education filopodia, and may require temporary activation of the Notch cascade. In models of carcinogenesis beta-cells POG lodochnoy cancer in transgenic mice (Rip1Tag2 mice), in which the tumor angiogenesis is dependent on VEGF, the expression level of VEGF did not increase, however, is the mobilization of extracellular VEGF, in the matrix, to the VEGF receptors. MMP-9 is responsible for this mobilization, and the progression of the tumor is inhibited from RiplTag23MMP-9-zero-double transgenic mice (23). Notch activated the expression of MMP-9, and it can increase the local level of VEGF in the field of angiogenic sprouting. Although Notch also increases the expression of MT1-MMP, extracellular MMP-2 may be targeted to the cell membrane of activated Notch endothelial cells. Notch can determine the site for angiogenic sprouting by regulation gelatinase activity and the concentration of VEGF. Because MMP-9 is regulated via Flt-1 is specific to the lung metastases (20), Flt-1 may participate in the induction of MMP-9 indirect.

References cited in the third series of experiments

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.

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

3. Uyttendaele, H., J. Ho, J. Rossant, 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.

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.

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

6. Gale, N. W., Dominguez, M. G., I. Noguera, 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.

7. Montesano R., L. 0. Phorbol esters induce angiogenesis in vitro from large-vessel endothelial cells. J. Cell. Physiol. 1987; 130 (2):284-91.

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

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

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

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

12. Das I., Craig C., Y. Funahashi, K. Jung, 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.

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

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

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

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

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

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

19. Funahashi Y., Wakabayashi, T., Semba, T., J. Sonoda, K. Kitoh, 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.

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.

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 Fltl and Flkl. Nat Med 2003; 9(7):936-43.

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

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.

Fourth series of experiments

New design, created of ectodomain Notch1, inhibits signal transmission Notch, morphogenesis of endothelial and tumor angiogenesis

Signal transmission Notch is required for the development of blood vessels, but also it functions in tumor angiogenesis. Inhibition of growth factor vascular endothelial (VEGF) is a proven antiangiogenic therapy, and VEGF can induce the expression of both Notch and ligand Delta-like 4(Dll4) for Notch, in endothelial cells (EC). Although inhibition of Dll4 may limit the growth of the tumor and destroy new blood vessels, the effect of inhibiting the function of the Notch receptor in tumor angiogenesis remains to be determined. In this study, the authors of the present invention has been soluble protein receptor Notch1 (N1ECDFc, or traps Notch1) to block this cascade and evaluated its effect on angiogenesisin vitroandin vivo.

Expression of the trap is reduced Notch1 signaling stimulated by binding of three different Notch ligands with Notch1 and inhibited morphogenesis EC, sverkhekspressiya Notch4. The authors of the present invention tested the effects of expression of traps Notch1 on tumor angiogenesis using two models: the tumor cells are breast cancer mouse Mm5MT, sverkhekspressiya fibroblastic growth factor 4 (Mm5MT-FGF4), and neuroblastoma cells NGP person. Exogenously expressed FGF4 induced expression of Notch ligands Jagged1 and Delta-like 1 (Dll1) in cells Mm5MT-FGF4, and Jagged1 expressively in xenografts Mm5MT-FGF4. Overexpression of traps Notch1 did not affect the ability of cells Mm5MT-FGF4 to the formation of tumorsin vitrohowever , limited the growth of xenograft Mm5MT-FGF4 in mice, while significantly reducing neoangiogenesis. Similarly, the expression of trap Notch1 did not affect cell NGPin vitrohowever destroyed vessels and reduced LM is the inability of a tumor xenografts NGP. These results strictly indicate that the signal transmission Notch is required for tumor neoangiogenesis, and provide new targets for therapy of tumors.

Angiogenesis is finely regulated by numerous cascades of signal transmission, including VEGF, fibroblast growth factors (FGF) and a growth factor for hepatocytes (HGF). Among them, VEGF critically affects nearly every stage of angiogenesis, including the proliferation of endothelial survival of the endothelium and the formation of tube (1). In accordance with this versatile role, VEGF inhibitors reduce angiogenesis in preclinical models, and they have been clinically tested as a therapy of malignant tumors (2). Despite this established the effectiveness of different types of tumors show widely varying susceptibility to blockade of VEGF (2). The underlying causes of this variability are not clear. One possibility is that alternative signals retain the tumor vessels, providing perfusion, despite the inhibition of VEGF. Identification of these stages, therefore, has obvious therapeutic value.

Highly conservative family of Notch genes encode transmembrane receptors (Notch1, -2, -3, -4) and ligands (Jagged1, -2; Delta-like or Dll1, -3, -4), also transmembrane proteins. When the binding of the ligand of the cytoplasmic domain of Notch (NotchIC) released presenilin/γ-secret of the Oh (3). The defect signal transmission Notch cause severe defects in blood vessels in embryos (4) haploidentical Dll4, causing mortality. The potential role of signal transmission Notch in tumor angiogenesis, thus, has recently generated a lot of interest. Mouse on transgenic reporter construct Dll4 demonstrate expression in endothelial tumor cells (EC) (5), and increased expression of Dll4 was detected in malignant human tumors (6, 7). Two recent reports confirm that this role is critical, by demonstrating that blockade of Dll4 inhibits the growth and perfusion in experimental tumors (8, 9). Interestingly, in these studies, inhibition of Dll4 was led to disruption of tumor vessels, and not to simply impeding the proliferation of blood vessels, suggesting that Dll4 is required for the functional Assembly of vessels.

Recent data indicate that Notch receptors also function in tumor vessels. For example, it was recently shown that in squamous cell carcinoma of the head and neck (HNSCC) HGF increases the expression of Jagged1 in the tumor cells, but not in the endothelium. Increased expression of Jagged1 activated signal transmission Notch in neighboring EC, stimulating angiogenesis and tumor growth in mice (10). Thus, these data indicate that there are at least two different activation mechanism PE is Adachi signal Notch in EC tumors.

In these studies, the authors of the present invention evaluated the role of activation of the Notch receptor during angiogenesis using the new instant design based on the extracellular domain of Notch1 (N1ECDFc, or trap Notch1).In vitrotrap Notch1 inhibited as induced by ligand activation signal transmission Notch and morphogenesis EC, sverkhekspressiya Notch4 using adenovirus.In vivoexpression traps Notch1 slowed the growth of xenografts Mm5MT mouse in which expression of Jagged1 was increased by transduction FGF4, and broke the vessels and the viability of the tumor in the tumor neuroblastoma NGP. These data support the need for the function of the Notch receptor during tumor neoangiogenesis and indicate that inhibition of this cascade can provide an effective new anticancer strategy.

Materials and methods

Reagents and expressing vectors

Compound E was purchased from Calbiochem (San Diego, CA), and PD166866 from Eisai (Tokyo, Japan). Trap Notch1 (N1ECDFc) encodes ectodomain Notch1 rat (p. N. 241-4229, registration number Genbank #X57405), fused in reading frame with the Fc of IgG person. Retroviral pHyTC-Jagged1, -Dll1, -Dll4 and pBos-Notch1 have been described (11). The trap of Notch1 and Fc have been built into the retroviral vector pHyTCX, and FGF4 was built in pQNCX. Adenoviral constructs encoding LacZ and Notch4 mouse and pAdlox-GFP were described(12), (13).

HUVEC, INFI is new adenovirus and retrovirus

HUVEC were isolated as described (14), and carcinoma of the breast mouse Mm5MT was purchased (ATCC, Manassas, VA). For infection, the authors of the present invention used the adenovirus with the indicated multiplicity of infection (m.o.i.) and retroviral supernatant from cells GP2-293 (BD Biosciences, Bedford, MA). Selection of HUVEC was performed using 300 μg/ml of hygromycin B (Invitrogen, Carlsbad, CA), and selection Mm5MT-FGF4 was performed in 1 mg/ml G418 (Gibco-Invitrogen, Grand Island, NY), with the double transfectants 300 μg/ml of hygromycin B.

Western blotting

HUVEC transduced with Ad-N1ECDFc, were cultured in serum-free medium for endothelial (GIBCO-Invitrogen) for 48 h, and the transfectants Mm5MT-FGF4 were cultured in DMEM. Western blots were obtained using antibodies against Fc man (Pierce, Rockford, IL).

Quantitative RT-PCR

The transfectants Mm5MT were cultured for 7 days with medium or 1 μm PD166866 (kinase inhibitor of FGF receptor), total RNA was isolated (RNeasy kit mini-kit, Qiagen, Valencia, CA)and synthesized the first chain cDNA (SuperscriptTMFirst-Strand Synthesis System, Invitrogen). Quantitative RT-PCR for β-actin, FGF4, Jagged1, Dll1 and Dll4 (SYBER Green PCR Master Mix, 7300 Real Time PCR; Applied Biosystems, Foster City, CA) was performed in triplicate, and values normalizability for β-actin. Values are shown for the ratio of induction compared to controls (sequences of primers available on request).

Analysis the signal transmission in coculture

Inhibition of trap Notch1 ligand-induced signal was performed as described (11). HeLa cells were transfusional 333 ng pBOS-Notch1, 333 ng pGA981-6 and 83 ng pLNC-LacZ with either 666 ng pCMV-Fc, or pHyTC-N1ECDFc (333 ng for x1, 666 ng for x2). The 293 cells were transfusional 680 ng pHyTc-Jagged1, pHyTc-Dll1, pHyTc-Dll4 or pHyTc-X (empty vector). Cells were collected, was determined by the luciferase activity 48 h after transfection (set for analysis of the Enhanced Luciferase assay kit, BD PharMingen, San Diego, CA), and determined the activity of β-galactosidase (set Galacto-Light Plus kit, Applied Biosystems). Analyses were performed in triplicate.

Analysis of the morphogenesis of endothelium in coculture

HUVEC morphogenesis was assessed as described (11), with the modification of adding cocultivation HUVEC, transduced by Ad-Notch4, with transfectants trap Notch1-HUVEC or Fc-HUVEC. Ad-GFP at m.o.i. 10 was cotranslationally in HUVEC with Ad-LacZ or Ad-Notch4 at m.o.i. 30 and after 48 h were sown on fibrin gels (24-hole tablets of 1.5×104cells/well). Stable transfectants HUVEC-imitation (HUVEC-X), HUVEC-Fc or HUVEC-N1ECDFc were sown in the amount of 1.35×105cells/well, and after 3 h was added to the media or 200 nm of compound E. in seven days HUVEC morphogenesis was calculated as the number of GFP-positive cells with processes in comparison with the total number of GFP-positive cells/field.

Model tumor Mm5MT

Female C3H mice aged 6-8 weeks, (Taconic, Hudson, NY) under the Yergali subcutaneous implant 10 6the transfectants Mm5MT (N=10 each). The diameters of tumors were measured with calipers and calculated volume (length (mm) × width (mm)2× 1/2). Tumors were collected at 22 days and analyzed. Experiments were performed three times.

Immunohistochemistry

5 μm frozen sections of tissue Mm5MT plunged immune staining (15) (see supplementary data for a list of antibodies). Quantification of CD31 was performed using a microscope Eclipse E800 and ImagePro Plus v.4.01 (Silver Spring, MD). A measurement was performed 20 different fields/glass slide, and calculated density (area-specific staining)/(total area, each field). Data are presented as a ratio of the average values of the ratios of average densities for each transfectant Mm5MT regarding imitation transfectant Mm5MT.

Model tumor NGP

Model NGP tumors was previously described (16). The NGP cells were transfusional LacZ or N1ECDFc, as described above, and 106cells NGP-LacZ or NGP-N1ECDFc implanted vnutriyazychno mice NCR nude at the age of 4-6 weeks, (Taconic, Germantown, NY; NGP-LacZ (n=11, NGP-N1ECDFc n=13). After 6 weeks, tumors were collected for analysis. 5-μm paraffin sections were subjected to immune staining for CD-31/PECAM and α-smooth muscle actin (αSMA). For detection of apoptosis (TUNEL analysis) the authors of the present invention used the kit Apoptag Red in situ Kit (Chemcon). The signal was subjected to quantitative determination by photographing 20-23 randomly selected fields for each tissue, excluding the area of the normal kidney. Each frame photographed in the red (signal TUNEL), and green channels. Using Adobe Photoshop signals green channels are subtracted to eliminate the autofluorescence of the cells. Arbitrarily chose a uniform threshold red channel, and the total area of the signal was measured in 4 tumors NGP-N1ECDFc and 3 tumors NGP-LacZ. Similarly performed quantitative determination of erythrocytes.

Statistical analysis

Significance in quantitative studies were assessed using tests Tukey-Kramer (quantification of CD31) and analysis of Well-Wallis (all others).

Results

Trap Notch1 inhibits induced by ligand signal transmission Notch in cells expressing Notch1

Trap Notch1 based on ectodomain Notch1, fused with the Fc of IgG person, and it is secreted, as determined by blotting environment, air-conditioned HUVEC infected with adenovirus with trap Notch1 (Ad-N1ECDFc) (Fig. 32A). The authors of the present invention were evaluated by activity traps Notch1 using analysis of signal transmission in coculture (11). The 293 cells expressing Notch ligands (Jagged1, Dll1, Dll4), activated signal transmission Notch when the cult is the cultivation with HeLa cells, expressing Notch1, which was measured by the activity of CSL-luciferase reporter (Fig. 32B). The expression of trap Notch1 or HeLa cells (Fig. 32B)or 293 cells (data not shown) blocked the signal transmission Notch1 in the analysis of coculture, indicating that the trap Notch1 prevents activation by Jagged1, Dll1 or Dll4.

Trap blocking Notch1 HUVEC morphogenesis induced Notch4

Expression of Notch4 induced elongation of HUVEC cells, cultured on fibrin gels (Fig. 32C), similar to the morphological changes induced VEGF and FGF2 (17, 18). Fibrin can be induced Jagged1 in EC (19). The authors of the present invention tested the hypothesis that this extension reflects the endogenous activation of the Notch ligand Notch4, transducing in HUVEC using either connection E (CE) (-inhibitor secretase (GSI)), or traps Notch1. Compared to the media processing 200 nm CE clearly inhibited elongation in Notch4-HUVEC, (Fig. 32C, the top panel, 32D). The decrease in germination was significant (Fig. 32D, p<0,0001) how to process connection E, and transduction N1ECDFc; data are presented as mean value ± SD). Coexpressed Notch4 + trap Notch1 in HUVEC similarly blocked the extension of the endothelium relative to the control in the form of Notch4 + Fc (Fig. 32C, the bottom panel; 32D). Collectively, these data indicate that activation of prescriptions is ora Notch is required, and sufficient for the induction of movements HUVEC in this analysis, and that the trap Notch1 functions similarly with GSI, further confirming its activity as an inhibitor of the Notch receptor.

FGF4 induced expression of Notch ligands, Jagged1 and Dll1 in tumor cells of breast cancer mouse Mm5MT

Overexpression of FGF4 in Mm5MT cells provided the ability to form tumors in the count of clonogenic assays and analyses xenografts (data not shown). Because the signal transmission HGF/MAPK induced the expression of Jagged1 in HNSCC (10), the authors present invention are interested, does FGF4 expression of Notch ligands in Mm5MT cells. The authors of the present invention revealed the activation of Jagged1 and Dll1 in the transfectants Mm5MT-FGF4 using quantitative PCR (expression of Dll4 was not changed) (Fig. 33A). Inhibitor of FGFR kinase PD166866 inhibited the induction of both Jagged1 and Dll1 in the transfectants Mm5MT-FGF4 (Fig. 33C), indicating that induced FGF4 expression of Jagged1 and Dll1 requires signal transmission FGFR. The Western blot turns confirmed the activation of Jagged1 protein in cells Mm5MT-FGF4 (Fig. 33B), and immune staining showed surprisingly high levels of Jagged1 in tumors Mm5MT-FGF4 (Fig. 33D). In addition, Notch4 was detected in the endothelium of tumor Mm5MT-FGF4 (not shown).

The expression of trap Notch1 inhibited angiogenesis and tumor growth Mm5MT-FGF4 in mice

<> The authors of the present invention hypothesized that tumor Mm5MT-FGF4, expressing Jagged1, trigger angiogenesis by passing the signal through an endothelial receptor Notch. Thus, the authors of the present invention evaluated the effect of expression of traps Notch1 on the growth of xenograft Mm5MT-FGF4 in mice. The ability to form tumors in Mm5MT-FGF4, stable sverkhekspressiya either Fc or trap Notch1, was unchanged in the count of clonogenic assay (data not shown). However, the growth of xenograft Mm5MT-FGF4-N1ECDFc was considerably slowed down compared with simulating the transfectants Mm5MT-FGF4, and Fc-transfectants Mm5MT-FGF4, indicating that inhibition of Notch broke a critical element in the formation of tumors (Fig. 34A). Immune staining for the endothelial marker CD31/PECAM demonstrated significant inhibition of angiogenesis in tumors Mm5MT-FGF4-N1ECDFc (Fig. 34B). In accordance with the need in the Notch in the Assembly of vessels, EC looked as detached single cells or small groups, with a small number of identified organized vessels. Quantitative analysis of staining with antibody against CD31 demonstrated a reduction of 58% of the density of microvessels in tumors expressing the trap of Notch1 (P<0,001, for Mm5MT-FGF4-X and Mm5MT-FGF4-Fc relative to Mm5MT-FGF4-N1ECDFc; data are presented as mean value ± SD; Fig.34C).

The expression of trap Notch1 violated angiogenesis in neuroblastoma xenografts NGP person

The xenografts NGP mice ordered form Mature vascular network, which is relatively resistant to VEGF blockade (16). To determine whether activation of the Notch receptor for angiogenesis NGP, the authors present invention has transfusional NGP cells through N1ECDFc, as described above. Similar to the results observed for cells Mm5MT-FGF4-N1ECDFc, cell growth NGP-N1ECDFc were not affected by transfection (not shown). However, the viability of xenograft significantly disrupted (Fig. 35A), with significantly increased apoptosis of tumor cells (P=0.0002, TUNEL-positive cells in tumors NGP-N1ECDFc relatively NGP-LacZ, Fig. 35B). Intratumoral hemorrhage was significantly increased in tumors NGP-N1ECDFc, indicating that the vessels were physically destroyed (P<0,0001, Fig. 36C). Immune staining component of the basal membrane of blood vessels, collagen IV, showed an overall decrease in vessels with reduced branching, although the rest of the collagen membrane was smooth and intact (not shown). However, immune staining of EC cells and blood vessels (VMC) (using antibodies against CD31 and antibodies against αSMA, respectively) showed disruption of these normal continuous cell layers. On the individual cells of the vessels looked wrong, and would randomly ukreplenii from each other, with the loss of continuity of the vessels (Fig. 35D). Taken together, these results indicate that the expression of trap Notch1 violated the interaction between endothelium and VMC in tumor vessels, causing instability, hemorrhage and defective perfusion of tumor tissues.

Discussion

In recent reports confirmed the critical role of Notch ligand Dll4 in angiogenesis and demonstrates that blockade of Dll4 can effectively suppress tumor growth by destruction of the vascular network (8, 9). In this study, the authors present invention have shown that blockade of receptor function Notch with the new design, formed from ectodomain Notch1, also effectively reduces perfusion of the tumor, although its effects on vasculature diverse. Trap Notch1 inhibited the transmission of a signal induced by the binding of ligands Jagged1, Dll1 and Dll4 with Notch1. In accordance with the role of activation of the Notch receptor in neoangiogenesis, overexpression of Notch4 induced elongation of endothelial cells, which can be prevented by blocking the transmission of the signal Notch either through traps Notch1 or GSI. Although the trap Notch1 not inhibited tumor cell growthin vitroexpression traps Notch1 inhibited growth and angiogenesis xenografts Mm5MT-FGF4, in which increased expression of Jagged1. Similarly, the former is Russia traps Notch1 had no effect on the proliferation of tumor cells NGP in vitrobut violated the vessels and viability of tumorin vivo.

Overexpression of Notch4 in HUVEC was sufficient for the induction of elongation of the endothelium on fibrin gel without exogenous expression of Notch ligands. Since it is known that fibrin induces the expression of Jagged1 in the EC and, therefore, can function by triggering the expression of Jagged1 in HUVEC in this analysis, the authors of the present invention is believed that this causes the activation of Notch4. In the analysis of signal transmission in coculture HeLa trap Notch1 inhibited signaling through the interaction of the ligand-receptor Notch1. The authors of the present invention were unable similarly to assess the activity of Notch4, because Notch4 bad processionals and presentials in HeLa cells (not shown). However processionary Notch4 detected in HUVEC after adenoviral transduction Notch4 (not shown), indicating that the trap Notch1 can block induced by ligand activation of Notch4.

Many roles, recently demonstrated for signal transmission Notch in the formation of tumors, increase the attractiveness of this cascade as a potential target for therapy of malignant tumors. Although activation of Notch, probably operates directly with malignant transformation in human tumors (20, 21), it can also be required for angiogenesis (8, 9). the nteresno induction of the Notch ligand may be regulated by signals from growth factors. For example, Jagged1 induced in tumor cells by HGF (10), and Dll4 induced in EC by VEGF (22). Here, the authors present invention show that FGF4 can similarly stimulate the expression of Jagged1 and Dll1 in Mm5MT cells of the mouse. Trap Notch1 reduced tumor growth Mm5MT-FGF4 and angiogenesisin vivobut did not affect the ability to form tumorsin vitro. Thus, these results suggest that neoplastic growth in this system requires activation of the Notch receptor in vascular Mm5MT, but not in tumor cells.

Although xenografts as Mm5MT-FGF4 and NGP showed a significant violation of a tumor vascular network after expression traps Notch1, differences in the phenotype of vessels observed in these models indicate that ofwholesale patterns of Notch function can accurately regulate the Assembly of vessels. Tumor Mm5MT-FGF4 proliferate rapidly and form dense, disordered endothelial network, relatively deprived attracted VMC. These immature vasculature Express Dll4 (data not shown) and to a large extent eliminated by expression of the trap Notch1, leaving a small group or individual EC in the parenchyma of the tumor. According to this source the inability to form vascular CE and, expressing the trap of residual tumor are not necrotic and are significantly lower than the controls. In contrast, in tumors NGP develops a Mature vascular plexus with almost uniform coating of endothelial VMC. Vessels NGP Express Notch1 and Dll4 (not shown). The expression of trap Notch1 in the NGP tumors induces intratumoral hemorrhage and necrosis with loss of continuity of the vessels, indicating that perfezione vessels become unstable after happens tumor growth to some extent.

Collectively, these data provide the basis for a model in which the signal transmission Notch controls many aspects of tumor angiogenesis. Although activation of Notch is widely required for neoangiogenesis, a separate Notch proteins can also regulate vascular remodeling. Obtained by the authors of the present invention results confirm the importance of the interactions of Notch ligand-receptor in tumor vessels and acknowledge that violation of function of Notch receptor may provide a new and effective way of violation of tumor angiogenesis.

References cited in the fourth series of experiments

1. Ferrara N. Vascular Endothelial Growth Factor: Basic Science and Clinical Progress. Endocr. Rev., 25:581-611, 2004.

2. Jain, R., Duda, D., Clark, J., and Loeffler, J. Lessons from phase III clinical trials on anti-VEGF therapy for cancer. at Clin Pract Oncol. 2006 Jan; 3 (1):24-40., 3:24-40, 2006.

3. Kopan, R. Notch: a membrane-bound factor reduced. J. Cell Sci., 115:1095-1097, 2002.

4. Shawber, C. and Kitajewski, J. Notch function in the vasculature: insights from zebrafish, mouse and man. Bioessays., 26:225-234, 2004.

5. Gale, N. W., Dominguez, M. G., Noguera, I., Pan, L., Hughes, V., Valenzuela, D. M., Murphy, A. J., Adams, N. C, Lin, H. C, Holash, J., Thurston, G., and Yancopoulos, G. D. Haploinsufficiency of delta-like 4 ligand results in embryonic lethality due to major defects in arterial and vascular development. Proc Natl Acad Sci 101:15949-15954, 2004.

6. Patel, N. S., Li, J.-L., Generali, D., Poulsom, R., Cranston, D. W., and Harris, A. L. Up-regulation of Delta-like 4 Ligand in Human Tumor Vasculature and the Role of Basal Expression in Endothelial Cell Function. Cancer Res., 65:8690-8697, 2005.

7. Patel, N. S., Dobbie, M. S., Rochester, M., Steers, G., Poulsom, R., Le Monnier, K., Cranston, D. W., Li, J.-L., and Harris, A. L. Up-Regulation of Endothelial Delta-like 4 Expression Correlates with Vessel Maturation in Bladder Cancer. Clin Cancer Res, 12:4836-4844, 2006.

8. Ridgway, J., Zhang, G., Wu, Y., Stawicki, S., Liang, W. C., Chanthery, Y., Kowalski, J., Watts, R. J., Callahan, C., Kasman, I., Singh, M., Chien, M., Tan, C., Hongo, J. A., de Sauvage, F., Plowman, G., and Yan, M. Inhibition of D114 signalling inhibits tumour growth by deregulating angiogenesis. Nature, 444:1083-1087, 2006.

9. Noguera-Troise, I., Daly, C., Papadopoulos, N. J., Coetzee, S., Boland, P., Gale, N. W., Lin, H. C., Yancopoulos, G. D., and Thurston, G. Blockade of D114 inhibits tumour growth by promoting nonproductive angiogenesis. Nature, 444:1032-1037, 2006.

10. Zeng, Q., Li, S., Chepeha, D. B., Giordano, T. J., Li, J., Zhang, H., Polverini, P. J., Nor, J. Kitajewski, J., and Wang, C-Y. Crosstalk between tumor and endothelial cells promotes tumor angiogenesis by MAPK activation of Notch signaling. Cancer Cell 8:13-23, 2005.

11. Das, I., Craig, C, Funahashi, Y., Jung, K.-M., Kim, T.-W., Byers, R., Weng, A. P., Kutok, J. L., Aster, J. C., and Kitajewski, J. Notch Oncoproteins Depend on {gamma}-Secretase/Presenilin Activity for Processing and Function. J. Biol Chem., 279:30771-30780, 2004.

12. Shawber, C. J., Das, I., Francisco, E., and Ktajewski, J. A. N. Notch Signaling in Primary Endothelial Cells. Ann NY Acad Sci, 995:162-170, 2003.

13. Hardy, S., Kitamura, M., Harris-Stansil, T., Dai, Y., and Phipps, M. L. Construction of adenovirus vectors through Cre-lox recombination. J. Virol., 71:1842-1849, 1997.

14. Jaffe, E., Nachman, R., Becker, C., and Minick, C. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J. Clin. Invest., 52:2745-2756, 1973.

15. Vorontchikhina, M. A., Zimmermann, R. C., Shawber, C. J., Tang, H., and Kitajewski, J. Unique patterns of Notch1, Notch4 and Jagged1 expression in ovarian vessels during folliculogenesis and corpus luteum formation. Gene Expression Patterns, 5:701-709, 2005.

16. Kim, E. S., Serur, A., Huang, J., Manley, C. A., McCrudden, K. W., Frischer, J. S., Soffer, S. Z., Ring, L., New, T., Zabski, S., Rudge, J. S., Holash, J., Yancopoulos, G. D., Kandel, J. J., and Yamashiro, D. J. Potent VEGF blockade causes regression of coopted vessels in a model of neuroblastoma. Proc Natl Acad Sci U S A, 99:11399-11404, 2002.

17. Montesano R. and L. O. Phorbol esters induce angiogenesis in vitro from large-vessel endothelial cells. J. Cell Physiol., 130:284-291, 1987.

18. Koolwijk p, van Erck M. G., de Vree, W. J., Vermeer, M. A., Weich, H. A., R. Hanemaaijer, and V. W., v. H. 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, 132:1177-1188, 1996.

19. Zimrin, A. B., Pepper, M. S., McMahon, G. A., Nguyen, F., Montesano, R., and Maciag, T. An Antisense Oligonucleotide to the Notch Ligand Jagged Enhances Fibroblast Growth Factor-induced Angiogenesis in Vitro. J Biol Chem, 271:32499-32502, 1996.

20. Nickoloff, B., Osborne, B., and L. M. Notch signaling as a therapeutic target in cancer: a new approach to the development of cell fate modifying agents. Oncogene, 22:6598-6608, 2003.

21. Radtke, F. and Raj, K. THE ROLE OF NOTCH IN TUMORIGENESIS: ONCOGENE OR TUMOUR SUPPRESSOR? Nature Reviews Cancer, 3:756-767, 2003.

22. Liu, Z.-J., Shirakawa, T., Li, Y., Soma, A., Oka, M., Dotto, G. P., Fairman, R. M., Velazquez, O. C., and Herlyn, M. Regulation of Notchl and D114 by Vascular Endothelial Growth Factor inArterial Endothelial Cells: Implications for Modulating Arteriogenesis and Angiogenesis. Mol. Cell. Biol. 23:14-25, 2003.

Fifth series of experiments

Notch as a therapeutic target for ovarian cancer

Epithelial carcinoma of the ovary is the main cause of death from gynecologic malignancies in the United States. Patients suffer as a result of failure to diagnose the disease at an early stage and lack of new drugs directed against this tumor. Growth factor vascular endothelial (VEGF) is produced by malignant epithelial cells of ovarian cancer, and he, in turn, starts the tumor angiogenesis, which nourishes the tumor [1]. Recent clinical trials demonstrate that targeting VEGF therapeutic intervention may in some cases improve the outcome [2]. Thus, antiangiogenic drugs have been approved as an approach for the treatment of ovarian cancer, but to achieve this requires more work. The authors of the present invention identified a cascade of signal transmission Notch as a new angiogenic cascade as in physiological and pathological angiogenesis [3, 4]. Notch is a receptor found on the cell surface (figure 1 - Notch1), which provides for the determination of cell fate, survival of cells and proliferation. In the laboratory of the authors of nastoyascheevremya developed inhibitor of Notch, called trap Notch (schematically represented in figure 1), a protein containing the extracellular domain of Notch1, fused with the Fc-labeled (N1ECDFc), which can inhibit dependent on the ligand signal transmission Notch (data not shown). The authors of the present invention demonstrated that the trap Notch can block induced VEGF angiogenesis and tumor angiogenesis in a model of Wilms tumor in mice (data not shown). The authors of the present invention has been cells of ovarian cancer SKOV3 that sverkhekspressiya trap Notch, SKOV3-N1ECDFc. The authors of the present invention revealed that the expression of trap Notch in SKOV3 cells significantly inhibited tumor growth in xenotransplantation mice (figure 36). Thus, the trap Notch inhibits the growth of ovarian cancer. The authors of the present invention believe that the targeted inhibition on angiogenesis in the tumor. In addition, the authors of the present invention have studied samples of ovarian cancer man and found that some of the Notch ligands and Notch receptors highly expressed in tumor vessels associated with cancer of the ovary, thus, supports the hypothesis that it is possible to conduct targeting Notch in vascular tumors of the ovary of a person and, ultimately, to block their growth.

This study tested the hypothesis that lane is giving a signal Notch triggers angiogenesis in tumors in cancer of the ovary. In addition, the authors of the present invention is believed that inhibition of Notch using "traps Notch" will block tumor growth. The authors will determine whether directed action traps Notch against tumor vessels or against the growth of tumor cells, or against both of them. The General objective is to establish traps Notch as a therapeutic agent for the treatment of ovarian cancer.

Specific objectives:

Specific aim 1: to Determine blocked trap Notch growth of xenograft tumors from cells SKOV3 or OVCAR3, and to determine whether aimed trap Notch on tumor angiogenesis.

Specific objective 2: to Modify the design of the trap Notch to obtain variants with improved performance/stability.

Specific objective 3: to Use treated traps Notch to block xenografts tumors of the ovary in mice, also in combination with standard chemotherapeutics.

Strategy research:

Specific objective 1:To determine blocked trap Notch growth of xenograft tumors from cells SKOV3 or OVCAR3, and to determine whether aimed trap Notch on tumor angiogenesis.

Preliminary results of the authors of the present invention demonstrate that tumor cells SKOV3 with the programmed expression of traps Notch1 block the growth of xenotransplant the tats cancer of the ovary (Fig. 36). Play this opening will be held on different cell lines of ovarian cancer, OVCAR3. Trap Notch can inhibit the growth of tumor cells, tumor vessels, or both. To determine whether aimed trap Notch on tumor cells, the authors will determine whether the growth lines of ovarian cancer expressing the trap Notch, weaker analyses on soft agar. To determine whether aimed trap on tumor angiogenesis, the authors present invention will assess xenotransplantion tumors in relation to characteristics such inhibition. Indicators of tumor angiogenesis to control xenografts will include an assessment of the density of microvessels, proliferative index of endothelial tumor cells and visualization of tumor vessels by lectin perfusion. Indicators of inhibiting angiogenesis of a tumor xenografts expressing the trap Notch, will include an assessment of apoptotic endothelial cells, reducing tumor microvessels and the decrease in the accumulation of ascitic fluid.

Specific objective 2:To modify the design of the trap Notch to obtain variants with improved performance/stability.

The existing trap Notch1, although it is effective as a means of blocking Notch, can not easily poddavalis the cleaning, because it is a relatively large protein. The authors of the present invention propose to develop ways to trap Notch1, which cover smaller portions of the extracellular domain. These options will be subjected to screening as inhibitors of signal transmission Notch in assays based on cell cultures. Inhibitory options will also be subjected to screening for their effectiveness secretion from producing cells, their stability after secretion, their solubility and ease of purification using affinity tags Fc. Refined options will be injected mice to assess the potential toxicity.

Specific objective 3:To use treated traps Notch to block xenografts tumors of the ovary in mice, also in combination with standard chemotherapeutics.

Cleaned the trap, according to specific objective 2 will use either as individual items or in combination with chemotherapy to assess their effectiveness against xenografts of ovarian cancer (SKOV3/OVCAR3). Using purified traps Notch as the sole means, the authors of the present invention will attempt to reproduce the unit growth of xenograft tumors observed in experiments under specific aims 1 and to determine the optimal Konz is Tracey for inhibition. For this purpose, the authors of this definition will determine the optimal dose, dosing scheme and the duration of treatment required to block the growth of SKOV3 xenograft/OVCAR3 in mice. Further, the authors of the present invention will use the trap Notch in combination with paclitaxel. The optimal dose traps Notch will be used with paclitaxel or without it to determine the effectiveness of blocking the growth of the xenograft.

Effect:This study aimed directly advance in the treatment of ovarian cancer. In clinical studies have previously installed the successful treatment of ovarian cancer blocking VEGF means. Few new drugs have been developed to complement chemotherapy treatment. Despite this success, targeting VEGF to block tumor growth may only restrict the growth to some extent. Also it can lead to growth of new blood vessels in tumors that are resistant to VEGF blockade. The authors of the present invention suggested that the targeting of alternative angiogenic cascade in the tumor, involved in ovarian cancer, a cascade of signal transmission Notch. This study will lead to progress in treatment through the development of new therapeutic molecules, traps Notch. In the preliminary research of the authors in the present invention has already been installed, trap Notch may block the growth of ovarian cancer model in mice. Thus, the effect of development traps Notch as therapeutic molecules can be highly significant.

Innovation:The recent development of the authors of the present invention traps Notch represents a new and previously untried model for the treatment of ovarian cancer. No other published study has yet involves the cascade in the treatment of ovarian cancer, and at the same time, preliminary data of the authors of the present invention define it as a key area for research. In addition, despite the recent success of the blockade of VEGF as a new therapeutic approach for the treatment of ovarian cancer, the authors of the present invention believe that this approach may require additions to the inhibition of other angiogenic cascades, such as the Notch.

References cited in the fifth series of experiments

1. Abu-Jawdeh, G. M., et al., Strong expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in ovarian borderline and malignant neoplasms. Lab Invest 1996. 74(6): p. 1105-15.

2. Monk, B. J., et al., Activity of bevacizumab (rhuMAB VEGF) in advanced refractory epithelial ovarian cancer. Gynecol Oncol, 2005. 96(3): p. 902-5.

3. Shawber, C., J. J. Kandel, and J. Kitajewski, Notch: cell fate determination from vascular development to human vasculopathy. Drug Discovery Today: Disease Models, 2004. 1(3): p. 351-8.

4. Zeng, Q., et al., Crosstalk between tumor and endothelial cells promotes tumor angiogenesis by MAPK activation of Notch signaling. Cancer Cell, 2005. 8(1): p. 13-23.

the Estai series of experiments

Cascade Foxo/Notch control myogenic differentiation and specification for fibre type

Transcription factors Foxo regulates metabolism and cell differentiation. Unlike Foxo-dependent metabolic cascades and gene targets, the mechanisms by which these proteins regulate differentiation, were not studied. Activation of signal transmission Notch simulates the effects of an increase in Foxo function in cell differentiation. Using differentiation of muscle as a model system, the authors of the present invention have shown that Foxo physically and functionally interacts with Notch by providing correct corepressor from the Notch effector Csl, leading to activation of target genes Notch. The inhibition of differentiation of cultured myoblasts constitutively active Foxo1 is partially preserved when the inhibition signal transmission Notch, while the loss of function of Foxo1 prevents inhibition by Notch myogenesis and increases the expression of MyoD.

Thus, dependent on conditions, the elimination of Foxo1 in skeletal muscle, leading to increased formation MyoD-containing (fast-twitch) muscle fibers and modified type distribution of fibers due to containing myogenin (slow-twitch) fibers. Cooperation Notch/Foxo1 can integrate external stimuli through the Notch with the metabolic stimulus the mi via Foxo1 to regulate the maintenance and differentiation of progenitor cells.

The Central question of regenerative medicine is the understanding of how a highly specialized cell types arise from undifferentiated stem cells or progenitor cells (1). This question is closely related to the question of how biochemical signals generated by the signal microenvironment and endocrine/nutritional signals, integrate transcriptional activation of the processes of cellular differentiation.

The O subfamily of protein forkhead (Fox) regulates the response to hormones, nutrients and stress, ensuring the survival and metabolism of cells. The ability to accurately regulate the transcription of Foxo is required for control of these cellular functions and to a large extent depends on posttranscriptional modifications, including phosphorylation and acetylation (2). In addition to their role in terminal differentiated cells, Foxo proteins are also involved in the differentiation of cultured myoblasts (3), preadipocytes (4) and endothelial cells (5). Moreover, Foxo4 regulates the differentiation of smooth muscle cells of blood vessels through interactions with myocardium (6). Mouse Foxo3 knockout show premature loss of ovarian function, which is consistent with a role for this gene in the maturation of the ovarian follicles (7). The mechanisms by which Foxo proteins in control of cell differentiation, the OS which are stated unclear and recent studies with conditionally elimination are consistent with a significant degree of functional overlap among the three Foxo isoforms in hematopoietic Rostock (8, 9).

The Notch cascade plays an important role in the differentiation of nerves, blood vessels, muscles and endocrine systems during embryogenesis (10). When induced by ligand cleavage of the intracellular domain of the Notch receptor moves into the nucleus, where it interacts with the DNA binding protein Csl, changing its transcriptional properties with suppressor transcription on activator of transcription (11). Target Csl include genes Hairy and Enhancer of Split (Hes, Hey). Hes1 controls the differentiation of endoderm intestine (12), preadipocytes (13) and neurogenic differentiation (14). Active signal transmission Notch, or enhancing the function of Notch1 receptor, inhibits the differentiation of cultured myoblasts C2C12 and 10T/2 by suppressing the transcription of MyoD (15-21).

It is noteworthy that improves the function of Foxo1 (3-5) phenocopies aktivacii Notch1 (13, 17, 22, 23) in each context of cellular differentiation. Moreover, the elimination of Foxo1 (24) phenocopies elimination of Notch1 (25) in mice. Despite these interesting similarities, Foxo and Notch transmit a signal through two apparently different mechanism, cascade phosphatidylinositol-3-kinase (Foxo) and cascade Hes/Hey (Notch). In this study, the authors present invention have shown that Foxo physically and functionality is but interacts with Notch by providing correct corepressor from Csl, thus, controlling myogenic program.

Myogenic precursors originate from stem cells in the mesoderm (26) and turn into a muscular tube through a multistage process that culminates in the expression of myogenic transcription factors of the MRF family (MyoD, myogenin, MRF4 and Myf5) (27). Myogenic transcription factors heterodimerize with E proteins and provide the expression of specific muscle genes, acting in close cooperation with specific myocytes factors enhance MEF2 (28).

Adult muscle is a heterogeneous tissue, mainly determined by the content of the muscle fibers (29). Different subtypes of the heavy chains of myosin (MyHC) describe different muscle fibers. Fiber type I Express mainly slow-twitch MyHC, and fiber type II Express MyHC fast-twitch (29). Process specifications type fiber is controlled at multiple stages. First, there appears to be heterogeneity between myogenic cells predecessors, and the experimental data for cross-transplantation of embryos of birds indicate that early precursors of lead, mainly to the slow muscle fibers, and late predecessors lead to fast fibers (29). In the postnatal period on the specification of the type of fiber also is Lieut Autonomous factors of the cells, including innervation and endocrine signals/signals nutrients (28). Coactivator Foxo Pgc1α plays a critical role in ensuring the formation of slow-twitch fibers (30), and recent data also showed that this process involved deacetilaza Foxo Sirt1 (31). Using dependent conditions mutagenesis in mice, the authors of the present invention showed that the role of Foxo1 in suppressing MyoD-dependent of myogenesis in C2C12 cells is reflected by the increase in containing MyoD muscle fibers in Foxo1 deficient skeletal muscle, consistent with a key function in the specification of the germ of cultured myoblasts.

Results

The interaction signal Foxo1 and Notch during differentiation of C2C12

To understand, interact whether Notch and Foxo control of muscle development, the authors of the present invention used a model of cellular differentiation. The C2C12 cells undergo myogenic conversion and merging muscle tubes when removing growth factors, the process associated with the translocation of Foxo1 in the nucleus (3). Thus, transduction of adenovirus encoding constitutively active mutant of Foxo1 (Foxo1-ADA) (4), blocked the effect of eliminating serum on the induction of differentiation of C2C12, which is reflected by the inhibition of fusion of cultured myoblasts (Fig. 37a-c). In contrast, inhibition of Foxo1 by siRNA had no effect on these processes is s (Fig. 37d).

Similarly, constitutively active Notch (Notch1-IC) was Prokopieva Foxo1-ADA in relation to block the differentiation of cultured myoblasts (Fig. 37e). Almost all cells were transnacionales adenovirus (Fig. 44). siRNA for Foxo1 effectively inhibited the expression of endogenous Foxo1 and transfitsirovannykh FLAG-Foxo1 (Fig. 45) a dose-dependent manner, without affecting the control proteins or other Foxo isoforms (Fig. 46). No Foxo1-ADA or Notch1-IC had no effect on the proliferation of C2C12 (Fig. 47).

The authors of the present invention are interested in whether they can eliminate the effect of Foxo1-ADA by inhibiting endogenous signal transmission Notch. In this regard, the authors of the present invention used a truncated Notch1 receptor, lacking the transmembrane anchor and intracellular domain, which acts as a receptor traps by binding of Notch ligands (32, 33) (Y. F. and J. K., unpublished observation). The trap did not affect the ability of C2C12 to undergo differentiation in response to the removal of growth factors (Fig. 37f), but partially preserved inhibition through Foxo1-ADA differentiation of cultured myoblasts (Fig. 37g). As an alternative probe for blocking signal transmission Notch, the inhibitor presenilin (PSI), the compound E (34), also maintained inhibition of Foxo1-ADA during the differentiation of cultured myoblasts (Fig. 37h).

To study the effect of Foxo1 on the transmission signal is Notch and the authors of the present invention has cotranslational siRNA for Foxo1 and Notch-IC. siRNA for Foxo1 kept the inhibition of differentiation of cultured myoblasts and expression of myosin by Notch1-IC (Fig. 37i indigenous), whereas control siRNA had no effect (data not shown). To exclude non-specific effects of siRNA for Foxo1 on differentiation of cultured myoblasts, the authors present invention has created resistant to siRNA for Foxo1-ADA (Fig. 48). siRNA for Foxo1 caused a reversion of the effects of Foxo1-ADA (Fig. 37j), but didn't save the inhibition of differentiation of C2C12 caused by resistant siRNA for Foxo1-ADA (Fig. 37k). The authors of the present invention presented a quantitative analysis of these data in Fig. 38a, indicating that Foxo1 and Notch1-IC reduced the levels of myosin >80%, and trap-Notch and siRNA for Foxo1 was restored them to ~70% of the fully differentiated cells. The authors of the present invention has been similar data by performing morphometric analysis positive for myosin cells (Fig. 38b). These data indicate that Foxo1 is required for Notch effect on the differentiation of cultured myoblasts.

Next, the authors present invention was tested, does Foxo1 on differentiation through its transcriptional function. To do this, the authors of the present invention has been mutant, deficient in DNA binding, mutant skeleton ADA, by replacement N208A and H212R (DBD-Foxo1ADA) (6, 35). The authors of the present invention have confirmed that this mutant is unable to bind the NC, by measuring the activity of the promoterIgfbp1canonical targets Foxo1. Foxo1-ADA increased the activity of promoterIgfbp110 times, and DBD-Foxo1ADA was not able to do so (Fig. 38c, left panel). Surprisingly, in relation to the inhibition of differentiation of this mutant was as effective as competent against DNA binding Foxo1-ADA (Fig. 37l). These data indicate that Foxo1 controls the differentiation independently of its ability to bind DNA specific for the sequence.

Foxo1 binds to Csl and is required for the Hes1 promoter

Notch1-IC binds to Csl and activates it, providing the expressionHesandHey(11). Based on the results for the mutant DBD-Foxo1ADA, the authors present invention was tested, interacts whether Foxo1 with Csl-dependent Notch way using coculture C2C12 cells expressing the Notch1 receptor, with HEK293 cells expressing the Notch ligand Jagged1 (denoted by "+"), or LacZ as a negative control (denoted by "-"). The authors present invention has provided several lines of evidence that Foxo1 and Csl interact in cultured cells. The authors of the present invention revealed endogenous Foxo1 in immunoprecipitated endogenous Csl, and coimmunoprecipitate was significantly increased by the activation signal transmission Notch (Fig. 39a). For the otverzhdenija specificity of the interaction between the authors of the present invention expressed labeled HA Foxo1 and labeled FLAG Csl in C2C12 cells. After thus with anticorodal against HA (Foxo1), the authors present invention conducted deterio FLAG-Csl in immunoblot (Fig. 39b). On the contrary, after thus with anticorodal against FLAG (Csl), the authors of the present invention carried out the detection of HA-Foxo1 in immunoblot (Fig. 39c). The ability to coimmunoprecipitate with Csl apparently is specific to Foxo1, because the authors of the present invention showed no other Foxo isoforms in immunoprecipitated Csl (Fig. 42). The truncated mutant Foxo1 (Δ256 encoding amino acids 1-256) (36) retained the ability to interact with Csl. The authors of the present invention carried out the detection of FLAG-Csl in immunoprecipitated Myc-Δ256 (Fig. 39d), and HA-Δ256 in immunoprecipitated FLAG-Csl (Fig. 39e), indicating that Csl interacts with the N-terminal domain of Foxo1.

To determine whether this protein-protein interaction is direct and mapping domain(s) of interaction, the authors present invention was first tested with the "lowering" with affinity purified GST-Foxo1 produced in bacteria and expressed FLAG-Csl in HEK293 cells. The authors of the present invention conducted the detection of Association with Csl full-and N-terminal Foxo1 (amino acids 1-300), but not with C-terminal Foxo1 (and.to. 290-655) or GST (Fig. 40a). Further, the authors of the present invention mapped domain Csl, which interacts with Foxo1, using the m cell-free system with GST-Foxo1 and GST-Flag/Csl, purified from bacterial cultures. Also, the authors of the present invention have identified full (1-655) and N-terminal (1-300), but not C-terminal (290-655) Foxo1 in immunoprecipitated Csl. In contrast, N-terminal Foxo1 interacts with the N-terminal Csl (Fig. 40b).

The authors of the present invention used mutants with a deletion of the Csl for mapping Foxo1 binding domain in Csl. These studies indicate that Foxo1 binds to the domain spanning amino acids 172-279 (Fig. 40c), which is contained in the domain NTD Csl (37) (Fig. 40c, chart). Interestingly, this domain is required for DNA binding and corepressor, but does not lead to binding of Notch (38, 39).

Csl binds to a consensus sequence in the promoter ofHes1(40)that, therefore, provides a suitable analysis with the read data on the interaction of Foxo/Csl. If the latter is required for the regulation of differentiation of C2C12 should be satisfied with three predictions: (a) Foxo1 should be identified in the analysis thus with chromatin (ChIP), covering the element Csl in the promoterHes1(b) interaction should be dependent on differentiation and (c) inhibition of differentiation Foxo1-ADA must be accompanied by constitutive binding element Csl in the promoterHes1. In the figure 40d shown that all predictions are satisfied. First, the authors present invention conducted ChIP with ISOE what Itanium primers, covering the binding site CslHes1in differentiating C2C12 cells. The authors of the present invention conducted the detection of endogenous Foxo1, Notch1 and Csl in immunoprecipitated from undifferentiated cells (Fig. 40d. track Endog, 0 day). Because amplificatory PCR sequence does not contain forkhead binding sites, the authors of the present invention concluded that Foxo1 binds to this DNA fragment through Csl. Moreover, the binding of both Foxo1 and Notch1 decreased as cells become differentiated (1 and 2 days). When the authors of the present invention has transducible cells constitutively nuclear Foxo1-ADA, differentiation, inhibited (Fig. 37c) and mutant Foxo1 consistently been associated with promoterHes1as Csl and Notch1 (Fig. 40d, track Foxo1-ADA).

Further, the authors of the present invention analyzed the expressionHes1. It was predicted that the levels of Hes1 should correlate with the occupation promoterHes1by Foxo1 and Notch1. Indeed, the expression of mRNAHes1decreased as decreased binding of Foxo1 and Notch1 with Csl, while levels of the protein myosin increased (Fig. 40d). To exclude a direct effect of Foxo1 transcription Csl, the authors of the present invention have conducted analyses of reporter genes with promoter Csl. Foxo1 is not activated reporter gene expression Csl, despite the presence of ten is Ovcharov forkhead binding site in the promoter Csl (41) (data not shown). Moreover, the Csl expression was not changed in C2C12 cells expressing Foxo1-ADA (not shown). These data indicate that Foxo1 regulates Notch-dependent differentiation through protein/protein interactions with Csl.

Foxo1 is required long induction through Notch genes of the Hes and Hey through Csl

The authors of the present invention investigated the ability of Foxo1-ADA to ensure the expression of endogenousHes1,Hes5andHey1in C2C12 cells. As Foxo1-ADA, and Notch1-IC was increased expression of these three genes, whereas siRNA for Foxo1 inhibited the expressionHes1,Hes5andHey1induced Notch1-IC (Fig. 41a). siRNA for Foxo1 had no effect on the expressionHes1,Hes5andHey1in cells deprived of growth factors (Fig. 41a).

The authors of the present invention has focused the next group of experiments onHes1as a prototype gene target Notch. The authors of the present invention tested the ability of Foxo1 to regulate the transcription ofHes1using reporter assays with promoterHes1and measuring the expressionHes1. Foxo1-ADA and Notch1-IC induced the promoter activityHes11.8 and 2.5 times, respectively. Cotransfected Foxo1-ADA with Notch1-IC was caused increased 2.5 times (Fig. 41b). Cotransfected siRNA for inhibited Foxo1 induced Notch activityHes1a dose-dependent manner, whereas control siRNA had no effect (Phi is. 41b). The authors of the present invention obtained similar results for the synthetic reporterHes1containing four tandem repeat Csl-binding motif (Fig. 50). Moreover, DBD-Foxo1ADA was able to induce the activity of the reporter geneHes1even more than Foxo1-ADA, confirming that DNA binding is not required for activationHes1through Foxo1 (Fig. 38c, right panel).

The inability of Notch1-IC to induce the expression ofHes1in cells expressing siRNA for Foxo1, indicates that Foxo1 is required for interaction Csl/Notch. Thus, the authors of the present invention investigated the binding of Foxo1 and Notch1 with promoterHes1in the system coculture. The authors present invention has acultural C2C12 cells expressing Notch1 with HEK293 cells expressing the Notch ligand Jagged1, for the induction of activation of the endogenous signal transmission Notch. Cocultivated in the presence of expressing Jagged1 cells increased the binding of endogenous Foxo1 (Fig. 42a, lanes 1-2) and with Notch1 promoterHes1in the analysis ChIP (Fig. 42a, b, lanes 1-2) (42). These data are consistent with the observation that coimmunoprecipitate Foxo1 with Csl was increased in cocultivation (Fig. 39a). To test whether the binding of Foxo1 with promoterHes1dependent on the Csl, the authors of the present invention inhibited the expression of Csl by siRNA (Fig. 1) Transfection of siRNA for Csl inhibited binding of both Foxo1, and with Notch1 promoterHes1(Fig. 42a, tracks 3-4), indicating that they are dependent on Csl. Moreover, Foxo1-ADA is not induced the expressionHes1in the presence of siRNA for Csl (Fig. 41a, track 5). The results of ChIP experiments was confirmed by analyses of promoterHes1. The expression of Jagged1 or Notch1 separately had no effect on activityHes1but cocultivation has increased 3.7 times the activity of the reporter geneHes1(Fig. 42c). siRNA for Foxo1 eliminated binding of Notch with promoterHes1in the analysis ChIP (Fig. 42b, tracks 3-4) and the induction of promoter activityHes1(Fig. 42c). These results suggest that Foxo1 is required for binding with Notch1 promoterHes1and provide a mechanism by which inhibition of the expression of Foxo1 restores differentiation of cultured myoblasts expressing Notch1-IC. The ability of siRNA to Foxo1 to inhibit the induction by NotchHes1in the system coculture exclude the possibility that the effects observed in experiments on differentiation with Notch1-IC, are the result of non-physiological activation signal transmission Notch shortened intracellular mutant Notch1 (15).

Foxo1 removes corepressor and linking with Maml1 Csl

To identify the molecular mechanism of Foxo1-dependent activation of expression ofHes1the authors present invention studies which followed an exchange of corepressor/coactivator in the promoter Hes1. Activation of Notch removed corepressor Ncor and Smrt (43) and attracted coactivator Maml1 (42) to the promoterHes1. siRNA for Foxo1 prevented induced Notch sharing corepressors (Fig. 42d). These data are consistent with the observation that Foxo1 binds to the region 172-279 in Csl (Fig. 40c), which, as shown, contains plots of the binding of Ncor/Smrt (38, 39).

In order to show that the observed changes in the transcriptional complex lead to changes in activityHes1the authors of the present invention investigated the expression of target genesHes1involved in migenes. It has been suggested thatHes1suppresses the differentiation of cultured myoblasts by inhibiting the transcription factor MyoD bHLH without affecting Myf5 (16, 17). The expression analysis showed that Notch1-IC or Foxo1-ADA inhibited MyoD, whereas Myf5 was unchanged. Trap Notch or siRNA for Foxo1 was partially restored the expression of MyoD (Fig. 42e).

A modified type of the composition of the fibers in skeletal muscle lacking Foxo1

Based on the cell data, the authors of the present invention conducted a study of the function of Foxo1 in the differentiation of musclein vivousing dependent conditions inactivation of genes. The predicted outcome of this experiment is to Express differentiation containing MyoD, but not containing Myf5 of cultured myoblasts. Because MyoD is the predominant myogenic the actor in fast fibers, while myogenin is the predominant factor in slow fibers (44), eliminating inhibition of Foxo/Notch expression of MyoD should lead to increased formation of fast fibers, potentially at the expense of slow fibers.

There are three Foxo isoforms in mice: Foxo1, 3, and 4 (8, 9). The latter prevails in most types of muscles (45), except soleus, where the most common is the Foxo1 (Fig. 43a). By coincidence, the soleus muscle is also physiologically enriched with slow-twitch fibers, and thus, allowed the authors of the present invention to easily test their hypothesis. The authors present invention iactiveaware the expression of Foxo1 in skeletal muscle by crossing mice homozygous for the floxed allele Foxo1 transgenic mice Myogenin-cre. Analysis of mRNA showed that the knockout occurred as planned (data not shown). Histological analysis showed a decrease in fiber type I (slow-twitch) in the soleus muscle of mice Myog-Foxo1, while the muscle-enriched type II fibers were unchanged (figure 43b). In accordance with the histological data, the expression of markers of fibers of type I has been reduced, while the expression of markers of type II fibers was increased in mice Myog-Foxo1 (figure 43c). Then the authors present invention analyzed expr is the Russia of myogenic transcription factors MyoD, Myf5 and myogenin. MyoD is a predominant factor in fast fibers, and myogenin in slow fibers (44). In accordance with the histopathology, the authors of the present invention showed a twofold increased expression of MyoD and ~80% decrease in the expression myogenin, while the expression of Myf5 was unchanged (Fig. 43c). Moreover, expression of coactivator Foxo1 Pgc1α, which regulates the determination of fiber type I (30), was unchanged, indicating that the phenotype of mice Myog-Foxo1 could explain the decrease in Foxo1-dependent transcription Pgc1α (Fig. 43c) (46). As a functional correlate of the observed switching type fibers, the authors of the present invention investigated the ability to run on a "treadmill". Indeed, mouse Myog-Foxo1 showed reduced ability to run, as predicted, on the basis of reduction of fiber type I (endurance) (Fig. 43d).

In conclusion, to determine, to reflect these changes associated with development, changes in specifications fiber type, as opposed to adaptive or cell-non-Autonomous factors, the authors present invention was determined by the expression of MyoD in embryos with Foxo1 knockout (24) and Notch1 (25) at E9,5. In embryos of Foxo1-/-the levels of MyoD increased 3.1±1.1 times, and in embryos of Notch1-/-they grew 7.3±2.9 times compared with controls (P<0,05 in both mutants FR is in wild-type, n=4). The increased expression of MyoD observedin vivoconsistent with the physical and functional interactions between Foxo1 and Notch in this key link in the transmission signal during differentiation of cultured myoblasts. Thus, the authors of the present invention believe that switching types of fibers in mice Myog-Foxo1 is the result of accelerated differentiation containing MyoD of cultured myoblasts during embryonic development.

Discussion

In this study provided biochemical, cellular and genetic evidence that the cascades of signal transmission Foxo and Notch cooperate in the regulation of muscle differentiation. The data reveal a new way of Foxo1 action to ensure exchange of corepressors on the promoterHes1through direct binding region of the NTD Csl (Fig. 42f). The authors of the present invention is believed that binding of Foxo1 with this domain stabilizes the complex Notch/Csl and starts removing corepressor and attraction of Maml1, which is consistent with the proposed role of NTD, based on studies of the structure (37). Also, the data provide a mechanism by which two main biochemical cascade, cascade phosphoinositol-3-kinase/Akt and the cascade Notch/Hes, combined synergistic manner to control cell differentiationin vivo.

Suggested role for Foxo1 does not depend on its transcription is uncle and involves direct interaction with Csl. While the study authors present invention focuses on Hes-1 as the prototype of the effector signal Notch1, these authors present invention should not be construed as indicating that Hes-1 is the only mediator of the interaction of Notch/Foxo. For example, the authors of the present invention have observed similar epistasis Foxo/Notch in the differentiation of preadipocytes, PC-12 and HUVEC, indicating that Foxo interacts with Notch in different cellular contexts (data not shown). The authors of the present invention believe that the interaction of Notch/Foxo integrates environmental signals through Notch with metabolic signals via Foxo1, regulating the maintenance and differentiation of progenitor cells. This bunk mechanism allows comitiorum cells-the precursors in various tissues to avoid differentiation in response to developmental signals (Notch)when active Foxo1, i.e. in the absence of growth factors. These cells can then be maintained in a quiescent state in adult tissues, where they can undergo terminal differentiation in response to the sequence of the Notch ligand and hormonal signals/signals nutrients, which leads to inhibition of Foxo1. This interpretation is consistent with switching types of fibers observed in deficient Foo1 muscle, and this observation, apparently, sets Foxo1 as a factor in deciding the fate in myogenic Rostock, in contrast to the inductor myogenic program. It remains to identify, communicate whether the Foxo isoforms and Notch, and how they participate in the process.

To demonstrate that Foxo1 is coregulation gene expression provides a potential explanation for the diverse functions of this transcription factor. An interesting question arising from the research of the authors of the present invention is the manner achieved by switching from one function to another, and how complex posttranslational modification of Foxo1 in response to growth factors, hormones and nutrients associated with this process. These data play a wide role in the pathophysiology of disease processes that involve the transmission signal Foxo1. The potential role of observation of the authors of the present invention lies in the ability to study the application of funds, which inhibit the signal transmission Notch (47) as a method of treatment of metabolic disorders characterized by excessive Foxo function (48).

Materials and methods

Getting animals and analysis

Mouse Myogenin-cre (49) and Foxo1floxdescribed (9). Detection of wild-type alleles, null alleles and Foxo1floxwere used in the by PCR with primers 5'-GCT TAG AGC AGA GAT GTT CTC ACA TT-3', 5'-CCA GAG TCT TTG TAT CAG GCA AAT AA-3' and 5'-CAA GTC CAT TAA TTC AGC ACA TTG A-3'. Before the effectiveness test on a treadmill mice were trained for 2 days (Columbus Instruments). The test was carried out at 15 m/min for the first 30 min, then increased to 1 m/min at intervals of 10 min until exhaustion. Samples of skeletal muscle were rapidly frozen in a matrix of OCT, and received a 7-μm serial sections. Muscle fibers were identified by using metachromatic ATPase (50) or immune staining with antibody against slow myosin in skeletal muscle (Sigma). For research on embryos, the authors of the present invention carried out within the stipulated time crossing heterozygous for Foxo1 (24) or Notch1 (25) mice and were isolated embryos at E9,5. mRNA was isolated from whole embryos and performed RT-PCR with detection in real time, as described below.

Studies of viral expression

The C2C12 cells were subjected to differentiation, as described (3, 4). Adenoviral vectors and expressing vectors mammals with Foxo1-ADA, Notch1-IC, Jagged1, Csl and trap Notch described (36, 51). The authors of the present invention has been retroviruses expressing Foxo1-ADA and Notch1-IC using vector pQCXIH. To receive traps Notch (pAdlox Notch1ECD-Fc) of the extracellular domain of Notch1 (p. N. 241-4229, registration number GenBank # X57405) was subjected to a merger in reading frame with the Fc-labeled human IgG and cloned into pAdlox. The floor is Ali retroviral supernatant from cells GP2-293 (BD Bioscience), temporarily cotransfection vector pVSV-G and constructed vector pQCXIH. To obtain defective Foxo1 DNA binding authors present invention has spent replacement N208 and H212 to alanine and arginine, respectively, using the kit QuikChange Mutagenesis Kit (Stratagene). Then the mutation was cloned in frame mutant Foxo1-ADA.

Analysis of luciferase and analysis coculture

The authors present invention has transfusional cells HEK293 reporter genes Hes1-luciferase (-194 to 160 from the site of transcription initiation) (Hes1/pGL2 basic), synthetic Hes1-luciferase (containing the binding site of 4× Csl, 4× Csl/pGL2 basic) or Csl-luciferase (-1536 to 22, Csl/pGL2 basic) together with pCMV5, pCMV5-Foxo1-ADA, pQNC-Notch1-IC, trap Notch pHyTc or siRNA for Foxo1. The authors of the present invention used plasmid pRSV-β-galactosidase as a control of transfection efficiency (51). For the analysis in coculture the authors of the present invention expressed Notch1 in C2C12 cells and Jagged1 or LacZ in HEK293 cells by transfection. Then the authors present invention has collected cells HEK293 and sowed them on C2C12 cells. After incubation for 1 h, the authors present invention has cocultivated cells for experiments.

Western blotting and immunoprecipitation

The authors of the present invention have conducted these tests in accordance with standard SP is ways using antibodies against myosin (MF-20), against HA (12CA5, Boehringer Mannheim), against FLAG (M2, Sigma), against Foxo1 (H128 and N20, Santa Cruz), against Notch1 (C-20, Santa-Cruz), anti Csl (Chemicon and Santa-Cruz), anti NcoR (Santa-Cruz), against SMRT (Santa-Cruz) or anti MAML1 (Chemicon). For coimmunoprecipitation Foxo/Csl authors of the present invention used purified nuclear fraction (52). Because Csl migrates near the heavy chain of IgG by SDS-PAGE, the authors of the present invention used dimethylpyrimidin (DMP from Pierce) for cross-linking of antibodies with beads with protein A and avoid contamination of IgG in buervenich protein complexes (52).

The analyses thus with chromatin

The authors of the present invention conducted an analysis ChIP in C2C12 cells, as described previously (4), and in coculturing cells, as described Fryer (42). Pairs of primers used for amplification of binding sites for Csl promoterHes1,are: 5'-GCAAAGCCCAGAGGAAAGAGTTAG-3' and 5'-AGGAGAGAGGTAGACAGGGGATTC-3'.

Transfection of siRNA-resistant and Foxo1 siRNA

Specific Foxo1 siRNA sequence is a 5'-ACGGAGGATTGAACCAGTATA-3'. Specific to Csl siRNA sequence is a 5'-TAGGGAAGCTATGCGAAATTA-3'. siRNA was transfusional using reagent Lipofectamin plus (Invitrogen). The authors of the present invention has been resistant to siRNA Foxo1 by replacing three residues (underlined) in the sequence 5'-ACGGCGGTCTGAACCAGTATA-3'. The sequence the primers, used for studies of RT-PCR detection in real time, available on request.

Recombinant proteins and the analysis of interaction

The authors of the present invention has been GST-FLAG-Csl covering fragments of amino acids 1-527, 1-279, 1-172 and 279-527, through cloning in PGEX6P-1. The design of GST-Foxo1 were described (53). After bacterial culture and induction of IPTG, the authors present invention was purified fused protein GST and incubated them together. After that, the authors present invention has allocated GST-FLAG/Csl through thus with antibody against FLAG, carefully washed immune precipitate and conducted Western blot turns with anticorodal against Foxo1.

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21. Conboy, I. M., and Rando, T. A. 2002. The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis. Dev Cell 3:397-409.

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31. Lagouge, M., Argmann, C., Gerhart-Hines, Z., Meziane, H., Lerin, C., Daussin, F., Messadeq, N., Milne, J., Lambert, P., Elliott, P., et al. 2006. Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRTl and PGC-lalpha. Cell 127:1109-1122.

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33. Nickoloff, B. J., Qin, J. Z., Chaturvedi, V., Denning, M. F., Bonish, B., and Miele, L. 2002. Jagged-1 mediated activation of notch signaling dosage complete maturation of human keratinocytes through NF-kappaB and PPARgamma. Cell Death Differ 9:842-855.

34. Pan, Y., Lin, M. H., Tian, X., Cheng, H. T., Gridley, T., Shen, J., and Kopan, R. 2004. gamma-seeretase functions through Notch signaling to maintain skin appendages but is not required for their patterning or initial morphogenesis. Dev Cell 7:731-743.

35. Dowell, P., Otto, T. C., Adi, S., and Lane, M. D. 2003. Convergence of peroxisome proliferator-activated receptor gamma and Foxol signaling pathways. J Biol Chem 278:45485-45491.

36. Nakae, J., Kitamura, T., black, D. L., and Accili, D. 2001. The forkhead factor reduced Foxol (Fkhr) confers insulin sensitivity onto glucose-6-phosphatase expression. J. Clin Invest 108:1359-1367.

37. Kovall, R. A., and Hendrickson, W. A. 2004. Crystal structure of the nuclear effector of Notch signaling, CSL, bound to DNA. Embo J. 23:3441-3451.

38. Hsieh, J. J., and Hayward, S. D. 1995. Masking of the CBFl/RBPJ kappa transcriptional repression domain by Epstein-Barr virus EBNA2. Science 268:560-563.

39. Kao, H. Y., Ordentlich, P., Koyano-Nakagawa, N., Tang, Z., Downes, M., Kintner, C. R., Evans, R. M., and Kadesch, T. 1998. A histone deacetylase corepressor complex regulates the Notch signal transduction pathway. Genes Dev 12:2269-2277.

40. Tun, T., Hamaguchi, Y., Matsunami, N., Furukawa, T., Honjo, T., and Kawaichi, M. 1994. Recognition sequence of a highly conserved DNA binding protein RBP-J kappa. Nucleic Acids Res 22:965-971.

41. Kawaichi, M., Oka, C., Shibayama, S., Kormilas, A. E., Matsunami, N., Hamaguchi, Y., and Honjo, T. 1992. Genomic organization of mouse J kappa recombination signal binding protein (RBP-J kappa) gene. J. Biol Chem 267:4016-4022.

42. Fryer, C. J., White, J. B., and Jones, K. A. 2004. Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover. Mol. Cell 16:509-520.

43. Liang, Y., Chang, J., Lynch, S. J., Lukac, D. M., and Ganem, D. 2002. The lytic switch protein of KSHV activates gene expression via functional interaction with RBP-Jkappa (CSL), the target of the Notch signaling pathway. Genes Dev 16:1977-1989.

44. Hughes, S. M., Taylor, J. M., Tapscott, S. J., Gurley, C. M., Carter, W. J., and Peterson, C. A. 1993. Selective accumulation of MyoD and myogenin mRNAs in fast and slow adult skeletal muscle is controlled by innervation and hormones. Development 118:1137-1147.

45. Kitamura, T., Nakae, J., Kitamura, Y., Kido, Y., Biggs, W. H., 3rd, Wright, C. V., White, M. F., Arden, K. C., and Accili, D. 2002. The forkhead factor reduced Foxol links insulin signaling to Pdxl regulation of pancreatic beta cell growth. J. Clin Invest 110:1839-1847.

46. Daitoku, H., Yamagata, K., Matsuzaki, H., Hatta, M., and Fukamizu, A. 2003. Regulation of PGC-1 promoter activity by protein kinase B and the forkhead reduced factor FKHR. Diabetes 52:642-649.

47. Miele, L., Miao, H., and Nickoloff, B. J. 2006. NOTCH signaling as a novel cancer therapeutic target. Curr Cancer Drug Targets 6:313-323.

48. Accili, D. 2004. Lilly lecture 2003: the struggle for mastery in insulin action: from triumvirate to republic. Diabetes 53:1633-1642.

49. Knapp, J. R., Davie, J. K., Myer, A., Meadows, E., Olson, E. N., and Klein, W. H. 2006. Loss of myogenin in postnatal life leads to normal skeletal muscle but reduced body size. Development 133:601-610.

50. Ogilvie, R. W., and Feeback, D. L. 1990. A metachromatic dye-ATPase method for the simultaneous identification of skeletal muscle fiber types I, IIA, IIB and IIC Stain Technol 65:231-241.

51. Das, I., Craig, C., Funahashi, Y., Jung, K. M., Kim, T. W., Byers, R., Weng, A. P., Kutok, J. L., Aster, J. C., and Kitajewski, J. 2004. Notch oncoproteins depend on gamma-secretase/presenilin activity for processing and function. J. Biol Chem 279:30771-30780.

52. Chi ., Yan, Z., Xue, Y., and Wang, W. 2004. Purification and functional analysis of the mammalian SWI/SNF family of chromatin-remodeling complexes. Methods Enzymol 377:299-316.

53. Puigserver, P., Rhee, J., Donovan, J., Walkey, C. J., Yoon, J. C., Oriente, F., Kitamura, Y., Altomonte, J., Dong, H., Accili, D., et al. 2003. Insulin-regulated hepatic gluconeogenesis through FOXOl-PGC-lalpha interaction. Nature 423:550-555.

Seventh series of experiments

Patients with diabetes often develops obesity and cardiovascular diseases. The molecular mechanisms that lead to diabetes complications, remains to be seen. Over the past two years the author of the present invention was evaluated in mice with knockout of Notch4 in relation to postnatal defects. These studies showed that the mutant by Notch4 mice develop signs of diabetes: 1) the early emergence of obesity observed a significant increase in subcutaneous fat, and 2) reduced the content of pericytes in retinal vessels, reminiscent of diabetic retinopathy. The authors of the present invention found that Notch and Foxo1, a transcription regulator of the transmission of the insulin signal, are combined to regulation adipogenesis and angiogenesis. Mouse Notch1 deficient, Notch1/Notch4 or Foxo1, die in utero with angiogenic defects. These data led to the fact that the authors of the present invention hypothesized that impaired regulation of signal transmission Notch leads to diabetes obesity and vascular pathologies. The proposed task is to study what s this hypothesis and definition of the roles of the interactions of the signal transmission Notch and insulin in adipogenesis and angiogenesis. For changing the activity of Notch, Foxo1 and receptors of insulin through genetic manipulation will use the model in mice. Adipokines and metabolic dysfunction will be assessed in mice with knockout of Notch4 and insulin receptors in embryonic fibroblasts derived from these mice. Angiogenesis embryo and retina will evaluate haploidisation mice by Notch1, Notch4 and/or Foxo1. In conclusion, the function of signal transmission Notch and Foxo1 in proliferative retinopathy will be evaluated in the model triggered by hypoxia angiogenesis of the retina in mice. Career goal of the author of this is to become an independent researcher in the field of diabetes research.

Evaluation of the function of Notch in the metabolism.

The authors of the present invention found that Notch and Foxo1, a transcription regulator of signal transmission insulin, form a transcriptional complex with CSL for the regulation of the choice of cell fate. The loss of one allele Foxo1 saves hyperglycemia and hyperinsulinemia in haploidisation the insulin receptor mice. Similarly, the loss of Notch4 in mice correlated with lower levels of blood glucose compared with odnopolnymi wild-type animals. Foxo1 and Notch4 have overlapping expression pattern in β-cells of the pancreas of adult mice, Notch1 is expressed in α-and β-cells. In the connection with this task, the authors of the present invention will be further oharakterizovat metabolism mutant for Notch mice. Also, the authors of the present invention will determine whether there are defects in the pancreas of mice mutant for Notch and Notch/Foxo1.

Evaluation of the function of Notch4 in adipogenesis.

The authors of the present invention found that mice with knockout of Notch4 have larger deposits of adipose tissue. In the skin Notch4 is expressed in adipocytes and blood vessels. Notch4 can adjust adipokines either through cell-Autonomous or cell-nonautonomous mechanism. In cell-Autonomous model of Notch function in adipocytes by regulating the differentiation of commiteeman of preadipocytes in stromal-vascular fraction. Alternative Notch may regulate angiogenesis in adipose tissue, which then affects adipogenesis. The authors of the present invention have found that Notch and Foxo1 are combined to inhibition induced by hormones adipogenesis cultured fibroblasts. In the mutant for the insulin receptor mice adipokines was broken, and differentiation was partially restored when the inhibition of Foxo1. However, it is unknown whether the dysfunction of the Notch to affect insulin-dependent differentiation and function of adipocytesin vivo. To begin clarify this issue, the authors of the present invention will complement the correctly characterize the adipose phenotype in mutant for Notch mice with focus on subcutaneous and visceral fat depots. Then the authors present invention will determine restores whether failure Notch4 subcutaneous adipose phenotype in mutant on Insr mice. In conclusion, the authors of the present invention will appreciate the Notch function during differentiation into adipocytes embryonic fibroblasts derived from mice deficient in Notch4 and Insr. The intent of the authors of the present invention is the definition of the role of interactions signal transmission Notch and insulin in the differentiation of adipocytes.

Diabetes and obesity

Obesity is a major risk factor for resistance to insulin, hyperglycemia and the development of type 2 diabetes {Eckel, 2005 #769}. It is also associated with cardiovascular dysfunction. Adipose tissue has an important metabolic function in the conservation of triacylglyceride during excess energy and release of free fatty acids and glycerol during depletion of energy. In addition, adipocytes regulate metabolic homeostasis through the production of a number of biologically active substances called adipokines. Adipokines are composed of hormones, cytokines, growth factors and other biologically active compounds. These include leptin, tumor necrosis factor alpha, angiotensin II, interleukin-6, interleukin-1, adiponectin, resistin, and prostaglandins. These secreted factors play a major role in the regulation as metabolic is, and vascular biology and essentially it has been suggested that they are the link between insulin resistance and cardiovascular disease.

The development of obesity, appears to be regulated by the signaling of insulin and angiogenesis dependent. In adipocytes transfer signal induces insulin growth factor vascular endothelial (VEGF) {Mick, 2002 #767}. VEGF is a potent inducer of angiogenesis, which can run the proliferation, migration and differentiation of endothelial cells and increased permeability of the vascular wall {Yancopoulos, 2000 #65}. In models of obesity in mice VEGF antagonists violate not only angiogenesis, but prevent adipogenesis {Rupnick, 2002 #766; Fukumura, 2003 #768}. Thus, adipose tissue is reciprocal paracrine regulation of differentiation of adipocytes and angiogenesis.

The function of Notch in adipogenesis

The role of Notch in the differentiation of adipocytes are only beginning to clarify. Using analysisin vitroit was shown that the signal transmission Notch as launches and inhibits induced by hormone adipogenic fibroblasts. In stromal cell lines Notch violated the differentiation of osteoblasts, which then led to increased differentiation of adipocytes {Sciaudone, 2003 #772}. On the contrary, as mediated by ligand signaling Notch, and ectopic expression of constitute the but active Notch1 inhibited adipogenic fibroblasts 3T3-L1 {Garces, 1997 #177; Ross, 2004 #773}. Similarly, overexpression of the target genes Notch, HES1, inhibited differentiation into adipocyte fibroblast {Ross, 2004 #773}. Violation of HES1 expression by siRNA also blocked the differentiation of fibroblasts into adipocytes. Thus, both inhibition and activation of Notch violates adipokines that indicates that adipokines is sensitive to the dose signal transmission Notch.

Mediated Notch inhibition adipogenesis fibroblasts correlated with the loss of adipocyte-specific genes of C/EBPα and PPARγ. Differentiation into adipocytes fibroblasts, in which the signal transmission Notch was activated, it was restored by the expression of either C/EBPα, or PPARγ, suggesting that Notch inhibits adipogenesis by suppressing the expression of these two genes. In accordance with the data for fibroblasts induced by retinoic acid adipokines was slightly enhanced in cells of embryos with a knockout of Notch1 {Nichols, 2004 #770}. In conclusion, it was found that the expression of Notch1 is reduced in adipose tissue obtained from patients with insulin resistance are relatively sensitive to insulin subjects, indicating the role of Notch in the associated with diabetes differentiation of adipocytes {Yang, 2003 #777}.

Vascular complications in diabetes

In patients with diabetes there are many vascular complications, including the Aya arterial hypertension, stroke, ischemia, retinopathy, atherosclerosis and heart attacks. However, little is known about the molecular mechanisms leading to diabetic vascular complications. Blindness is one of these complications are vascular in origin, but it is little understood. Within 20 years after diagnosis in a quarter of diabetics develop proliferative retinopathy leading to blindness. Diabetic retinopathy begins with increasing the permeability of blood vessels, thickening of the basal membrane and loss of pericytes in the microvasculature of the retina with subsequent proliferative phase neovascularization {Cukiernik, 2004 #749}. In the development of proliferative diabetic retinopathy involved in vascular growth factor (VEGF). Signal transmission VEGF can run the proliferation, migration, differentiation of endothelial cells and increased permeability of blood vessels {Yancopoulos, 2000 #65}. In models in mice for diabetes type I and type II revealed that the level sensitive to hypoxia transcription factor, HIF-1α, and VEGF is increased in the eyes {Kondo, 2004 #744}, indicating that hypoxia is the initiating event in the development of diabetic retinopathy. Induction of VEGF, probably mediated by HIF-1α, because VEGF is a direct transcriptional target of HIF-1α {Yancopoulos, 2000 #65}. As confirmation of the role of VEGF in diabetic pet is noptii, ectopic expression of VEGF in the eyes of primates leads to the rapid development of proliferative retinopathy and macular edema {Lebherz, 2005 #748}. In model rats with diabetes subcutaneous injection of the inhibitor signal transmission VEGF SU5416 inhibits induced VEGF permeability and narrowing of the retinal microvessels {Cukiernik, 2004 #749}. In conclusion, the injection into the vitreous body of an inhibitor of all three VEGFR, PTK/ZK, reduces neovascularization of the retina in a model of hypoxia in mice {Maier, 2005 #750}. Thus, dysregulation of signal transmission VEGF plays a critical role in the development of proliferative retinopathy and can also lead to other diabetic vascular complications.

Evaluation of the function of NOTCH IN METABOLISM

The authors found that Notch and Foxo1, a transcription regulator of signal transmission insulin, form a transcriptional complex with CSL for the regulation of the choice of cell fate. The loss of one allele Foxo1 restores hyperglycemia and hyperinsulinemia in mice with deficiency of signal transmission insulin (Nakae ng 2002). In mice, the loss of N4 correlated with a significant decrease of glucose levels in the blood compared to odnopolnymi wild-type animals. Like Foxo1 Notch4 may interfere with the signaling of insulin in the regulation of metabolism. The levels of glucose and insulin in the circulation are regulated producing glucose by tissues such as the liver and about useraudit insulin islet β-cells of the pancreas. Increased signal transmission Foxo1 in β-cells transgenic for Foxo1 mice leads to failure of β-cells and the development of diabetes (Nakea ng 2002). Foxo1 and Notch4 have overlapping expression pattern in β-cells of the pancreas of adult mice, whereas Notch1 is expressed both in α and in β-cells. Thus, Notch4 and/or Notch1 may have a function in the endocrine cells of the pancreatic islets. Because of this task, the authors will further characterize the metabolism and pancreatic cancer in mice mutant for Notch. Mutant for Notch mice will breed with mice L2 Ttr-Insr-/-(Fig. 21) and used to determine suppresses whether the failure N4 and/or N1 diabetic defects and defects of the pancreas observed in this deficit byInsrbackground.

Evaluation of the function of NOTCH IN ADIPOGENESIS

The authors of the present invention have found that mice with knockout N4 have hypertrophy of the adipose tissue of the skin. This phenotype of adipose tissue in the mutant on N4 mice may occur due to cell-Autonomous defect in adipocyte or as a result of non cell-Autonomous angiogenic defect. In contrast to the zero-zygote on N4 mice, mice defective in Insr, showing hypertrophy of adipose tissue {Cinti know in common, 1998 #774; Kitamura, 2004 #745} (Okamoto JCI 2004). Ectopic expression of dominant-negative Foxo1 restores ADI is agent embryonic fibroblasts Insr -/-{Nakae, 2003 #765}. The authors of the present invention found that Notch and Foxo1 act synergistically to inhibit induced hormone adipogenesis fibroblasts.

Since the function of Foxo1 are in epistasis interactions with signaling of insulin (Fig. 3), Notch in angiogenesis can also have the opposite function of the signal transmission insulin. In accordance with the cell-Autonomous function of adipose tissue for Notch4, Notch4 is expressed in subcutaneous adipocytes. Thus, the authors of the present invention will assess the function of Notch in adipogenesis embryonic fibroblasts derived from mice deficient in Notch and Notch:Insr. The authors of the present invention will conduct additional characterizatio defect of subcutaneous fat and will appreciate the visceral fat depot in the mutant for Notch mice. Finally, the authors present invention will determine restores whether the failure Notch4 defect in adipose tissue of the skin in mice mutant for Insr.

In models of obesity in mice VEGF antagonists inhibit both angiogenesis and adipogenesis {Rupnick, 2002 #766; Fukumura, 2003 #768}, indicating that there is a one-to-back regulation of adipogenesis and angiogenesis. Since the mouse, the zero-zygote on the N4 also show defects in angiogenesis of the retina, the observed increase in subcutaneous adipose tissue may be due to dysfunction of CN is telilng cells. Thus, the authors of the present invention will also determine whether there are differences in the vessels of adipose tissue of mice mutant for Notch.

Eighth series of experiments

Trap rat Notch1 present in mouse serum

Tested the stability of the trap rat Notch1 in the bloodstream of the mammal. As shown in figure 118, traps Notch are stable in the circulatory system of mammals.

The Nude mice were injected with control adenovirus or adenovirus expressing the trap of Notch1 rat (trap rN1). 2 weeks after injection, serum was collected and 4 microliters were evaluated by Western blot analysis. This analysis demonstrates that the full-sized protein trap rat Notch1 (see arrow on figure 118) can be expressed in mice and is amenable to detection levels with little signs of degradation.

Ninth series of experiments

Trap Notch1 person (trap h-Notch (1-36)and trap Notch 1 rat block the growth of tumors in the mammary gland of the mouse

Activity traps Notch1 person and traps Notch1 rats were compared against the growth of cell lines of breast cancer Mm5MT-FGF4. As shown in figure 119, as a trap hNotch1 and trap rNotchl reduce growth rate Mm5Mt-FGF4.

The authors of the present invention have developed a model of the tumor, which uses CL the TCI Mm5MT-FGF4, grown in nude mice. In this experiment, 2×105cells Mm5MT-FGF4 implanted nude mice, and after four days adenovirus encoding control in the form of Fc, the trap rat Notch1 or trap Notch1 man, were injected with in ophthalmic vein. Traps Notch produced infected with adenovirus liver of mice and secreted into the bloodstream (the example in the figure 118). Growth curve presented in figure 119 shows that as the trap rat Notch1 and trap Notch1 person reduced the growth of tumor xenografts in nude mice.

Trap rat Notch1 inhibits metastasis SKNEP1 in lung tissue

Trap Notch1 may block the metastasis model in mice. The authors of the present invention tested the activity trap rat Notch1 against tumor growth and metastasis cell lines sarcoma Ewing's sarcoma, SKNEP. In this model, tumor tumor cells SKNEP orthotopic implanted in the kidney, where the tumor grows, and then spreads into the lung. The expression of trap rat Notch1 in tumor cells SKNEP reduced tumor growth and metastasis in the lung, as shown in figure 120.

Cell sarcoma Ewing SKNEP1 was programmed for the expression of a control Fc protein or trap rat Notch1 s1 (type 2) or traps rat Notch1 s4 (type 4). These cell lines SKNEP1 orthotopic implanted in the kidney of nude mice. After tumor growth within 6 weeks were histologic the massive evaluation of metastases in the lung. Cells SKNEP1 expressing the trap of Notch1 rats showed lower part of the lung, which was positive for metastases. The authors of the present invention concluded that the expression of trap rat Notch1 in nude mice reduces the ability of cells SKNEP1 to metastasis in the lung.

The tenth series of experiments

Notch1 and Notch4 coexpressed with VEGFR-3 and PRONOUNCED-1 in lymphatic vessels of the skin of the mouse

Notch1 and Notch4 are expressed in the lymph vessels of the skin of the mouse

The authors of the present invention have analyzed the expression of Notch1 and Notch4 in the blood vessels of the skin on the back of the mouse P4. At this point in time, lymphatic vessels of the skin actively remodelers in lymphatic capillaries near the surface and collecting ducts in the lower layers of the skin. Transverse sections of skin the size of 5 μm were co-stained with antibodies against Notch1 or Notch4 (red)and PECAM, VEGFR-3 or PRONOUNCED-1 (green). Notch1 and Notch4 have overlapping expression pattern with a marker of endothelial cells of blood and lymphatic vessels, PECAM (top panel, figure 121). Notch1 and Notch4 were coexpressions as with VEGFR-3 (middle panel, figure 121)and PRONOUNCED-4 in the skin vessels (bottom panel, figure 121). This pattern of expression demonstrates that Notch1 and Notch4 are expressed and can function in lymphatic vessels neonatal dermis.

Lymphatic capillaries de what we changed in mice mutant by Notch4

The authors of the present invention were evaluated by the lymphatic vessels of the dermis of mice P4. Slices from wild-type mice and zero-zygote by Notch4 were stained with antibodies against PECAM and PRONOUNCED-1 (green). Analysis of the staining of PECAM looked similar between mutant skin and skin of wild-type (top panel, figure 122). On the contrary, positive PRONOUNCED on-1 vessels in the skin of mutants by Notch4 had a morphology that is different from the vessels of wild-type (middle panel, figure 122). Mutant by Notch4 vessels PRONOUNCED-1 were often extended, and staining PRONOUNCED-1 was intermittent (bottom panel, figure 122). These results indicate that the Notch4 signaling may be involved in remodeling of lymphatic vascular plexus.

The loss of Notch4 leads to a reduction in the number of positive PRONOUNCED on-1 cell

Heterozygous for Notch4 (N4+/-) mice were crossed, and back skin obtained calves were removed and placed on 14 day after birth. The results are shown in figure 123. Transverse sections of skin were subjected to immune staining for a marker of endothelial cells, PECAM (data not shown), or a marker of lymphatic endothelial cells, LYVE1 (A). In each case, registered five areas through microscopy and staining of PECAM and LYVE1 was subjected to quantitative determination using software in which solitarii (B, C). The expression of PECAM was reduced by approximately 25% in the skin N4-/-compared with the skin of wild-type (WT) (B). Staining PRONOUNCED-1 was changed more than PECAM staining, and staining LYVE1 was reduced almost by 50% in mice N4-/-relative to WT mice (C). Also there was a decrease in the intensity of staining LYVE1 in the lymph vessels N4-/-relative to WT (A).

The loss of function of Notch4 in mice disrupts the development of the lymphatic vessels of the skin, indicating a role in lymphangiogenesis.

Notch1 and Notch4 expressibility in breast cancer man

The authors of the present invention conducted a double immunohistochemistry with antibodies against VEGFR-3 or PRONOUNCED-1 (green) and Notch1 or Notch4 (red) breast cancer. The results are shown in figure 124. Notch1 and Notch4 expressibility in neophilia blood and lymphatic endothelium of microcapillaries carcinomas of the breast of man. To determine whether activation signal Notch1 in lymphatic endothelium of the tumor, the authors of the present invention conducted a double staining with an antibody against podoplanin (green) and antibody N1Val (red; cell signaling), which provides specific detection of activated Notch1 peptide. Expression of activated Notch1 peptide was observed in the majority (white arrows), but not all (yellow the arrows) from the nuclei of lymphatic endothelium (bottom panel). These results demonstrate that Notch1 actively transmit a signal in pathological lymph vessels. These results also demonstrate that Notch1 and Notch4 can function lymphangiogenesis tumors.

1. Protein, which is an antagonist of Notch1, containing
(a) amino acids, the sequence of which is identical to the sequence of part of the extracellular domain of the receptor Notch person, and
(b) amino acids, the sequence of which is identical to the sequence of the Fc portion of antibodies, where part of the extracellular domain of the receptor protein Notch person selected from the group consisting of EGF-like repeats 1-13 Notch1 and EGF-like repeats 1-24 Notch1, where Fc part of the antibody is an Fc-portion of human antibodies, where (b) localized on carboxykinase parts (a) and (b) attached to (a) directly or through a linker sequence.

2. Protein in p. 1, where part of the extracellular domain of the receptor protein Notch man is an EGF-like repeats 1-13 Notch1.

3. Protein in p. 1, where part of the extracellular domain of the receptor protein Notch man is an EGF-like repeats 1-24 Notch1.

4. Protein in p. 1, where (b) is attached directly to (a).

5. Protein in p. 1, where (b) is attached to (a) via a linker sequence.

6. Protein in p. 1, where h is a R extracellular domain of the receptor protein Notch person is a consistently located amino acids, sequence which is an amino acid 24-531 specified in SEQ ID NO:55, or amino acids 24-948 specified in SEQ ID NO:56.

7. Pharmaceutical composition for inhibiting the transmission of Notch signal containing an effective amount of the fused protein, which is an antagonist of Notch, according to any one of paragraphs.1-6 and a pharmaceutically acceptable carrier.

8. The use of fused protein according to any one of paragraphs.1-6 to obtain a pharmaceutical composition for the treatment of a subject having a tumor.

9. The use of fused protein according to any one of paragraphs.1-6 to obtain a pharmaceutical composition for inhibiting angiogenesis in the subject.

10. The use of fused protein according to any one of paragraphs.1-6 to obtain a pharmaceutical composition for the treatment of a subject having ovarian cancer.

11. The use of fused protein according to any one of paragraphs.1-6 to obtain a pharmaceutical composition for the treatment of a subject having a metabolic disorder.

12. Application under item 11, where the metabolic disorder is a diabetes, obesity, atherosclerosis, ischemia, stroke or cardiovascular disease.

13. The use of fused protein according to any one of paragraphs.1-6 to obtain a pharmaceutical composition for the inhibition of physiological lymphangiogenesis or pathological lymphangiogenesis the subject.

14. Application under item 13, where pathological lymphangiogenesis represent lymphangiogenesis tumors or metastases in the lymph nodes.

15. The use of fused protein according to any one of paragraphs.1-6 to obtain a pharmaceutical composition for inhibiting metastasis of a tumor in the subject.

16. Application under item 15, where metastasis occurs through a blood vessel, lymphatic vessels or lymph node.

17. The use of fused protein according to any one of paragraphs.1-6 to obtain a pharmaceutical composition for the treatment of vascular proliferative retinopathy in a subject.

18. Application under item 17, where vascular proliferative retinopathy is a diabetic retinopathy, macular degeneration or retrolental fibroplasia.



 

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SUBSTANCE: inventions relate to the field of immunology. Claimed are a single-chain antibody, specific to a carcinoembryonic antigen, a chimeric mononuclear T-cell receptor, a vector, a host cell and a method of diagnostics or treatment of diseases, characterised by the presence of antigens, capable of binding with the chimeric receptor. Described is a genetic construction, coding chimeric monomolecular T-cell receptors, in which an effector fragment of the T-cell receptor is combined with an antigen-recognising part, which represents variable fragments of two different antibodies to the carcinoembryonic antigen (CEA).

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SUBSTANCE: invention refers to biotechnology and immunology. What is presented is an antibody representing a neutralising VEGFR-2/KDR antibody with its hypervariable regions being identical to the hypervariable regions of TTAC 0001 of VEGFR-2/KDR antibody fused with a binding domain of angiopoietin 2 which is Tie-2 ligand for treating cancer by angiogenesis inhibition. A DNA coding the above antibody, an expression vector containing the above DNA, and a CHO host cell transformed by the above vector for preparing the antibody are also described. What is also presented is a method for preparing the antibody involving: host cell incubation, and the antibody recovery from a culture fluid of CHO cell. What is described is a pharmaceutical composition for treating an angiogenesis-related disease, containing an effective amount of the above antibody and at least one pharmaceutically acceptable carrier.

EFFECT: invention enables preparing the VEGFR-2/KDR antibody fused with the binding domain of angiopoietin 2 which may be used for effective treatment of a disease related to excessive angiogenesis.

13 cl, 10 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: claimed invention relates to immunology and biotechnology. Claimed is binding protein for binding one or more targets, which contains four polypeptide chains forming four functional antigen-binding sites. Four polypeptide chains contain VD1-(X1)n-VD2-C-(X2)n. VD1 stands for first variable domain of heavy chain, VD2 stands for second variable domain of heavy chain, C stands for CH1 domain, X1 stands for polypeptide linker, on condition that it is not constant domain, and X2 stands for Fc-region, and n equals 0 or 1. Two polypeptide chains contain VD1-(X1)n-VD2-C. VD1 stands for first variable domain of light chain, VD2 stands for second variable domain of light chain, C stands for CL domain, X1 stands for linker, on condition that it is not constant domain; and n equals 0 or 1. Conjugate of binding protein with visualising detecting cytotoxic or therapeutic agent is described. Disclosed are: nucleic acids (NA), coding polypeptide chains, as well as expressing vectors, vectors for replication, host cells which contain them, and method of obtaining antibody applying cells. Described is pharmaceutical composition for treatment or preventing target-associated disease or disorder based on binding protein. Method of treatment by introduction of binding protein is described.

EFFECT: application of invention provides new format (DVD-Ig) of antigen-binding molecules, which in the same dosage possess higher activity with respect to target than respective full-size antibodies, which can be applied in medicine for prevention and treatment of various diseases.

45 cl, 27 tbl, 5 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to a method for obtaining an antibody, the pharmacokinetic properties of which have been changed at maintaining antigen-binding activity of a variable area, which provides for the following stages: (a) obtaining antibodies in which there has been modified a charge of amino-acid residues chosen from amino-acid residues in positions 31, 61, 62, 64 and 65 of the variable area of a heavy chain and in positions 24, 27, 53, 54 and 55 of the variable area of a light chain in compliance with numbering as per Kabat system, where modification of the charge of amino-acid residues leads to the change of 1.0 or more at a theoretical isoelectric point of the variable area of the antibody, and (b) extracting an antibody with stored antigen-binding activity from antibodies obtained at stage (a).

EFFECT: invention allows effective change in pharmacokinetic properties of an antibody, thus maintaining its antigen-binding activity.

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biotechnology and immunology. Claimed is antibody, specifically binding with form A FcγRIII (CD16) (FcγRIIIA, CD16A) and not-binding specifically with form B (FcγRIIIB, CD16B), its antigen-binding fragment and multi-specific antibody, which includes antigen-binding fragment of antibody by invention. Compositions, which contain antibody by invention or its antigen-binding fragment, and their application in treatment of autoimmune, inflammatory, inflectious diseases, allergy and cancer, as well as set for detection of FcγRIIIA are described. Polynucleotides, vectors and host cells and method of obtaining antibody by invention or its antigen-binding fragment are described.

EFFECT: claimed invention provides novel antibodies to FcγRIIIA and, in that way, can find further application in therapy of FcγRIIIA-mediated diseases.

51 cl, 24 ex, 8 dwg, 5 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. There are prepared monomer and dimer domains VHH produced of camelids' antibodies able to bind the anti-rotavirus group A antigen VP6, as well as a method of detecting rotavirus group A, a composition for eliciting a passive immunity against rotavirus group A, a vector, cells and a method for producing the domain VHH of the invention, and a immunoassay kit for detecting rotavirus group A.

EFFECT: invention can find further application in preventing and treating rotaviral infections.

14 cl, 7 dwg, 5 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a single-chain multi-specific binding protein for binding or modulating activity of one or more biding partner molecules. The protein contains from the N-terminal to the C-terminal: a first binding domain derived from variable regions of a light and a heavy immunoglobulin chain; a constant sub-region derived from immunoglobulin containing a hinge or hinge-like domain, a CH2-domain and a CH3-domain; scorpion linker peptide, where said peptide contains an amino acid sequence from the immunoglobulin hinge or a type II C-lectin protein stalk region; and a second binding domain derived from variable regions of a light and a heavy immunoglobulin chain. The invention describes an encoding nucleic acid, as well as an expression vector based thereon and a host cell for vector based protein expression. The invention discloses a pharmaceutical composition for treating cell proliferation disorders based on a single-chain protein and a protein dimer for binding or modulating activity of one or more binding partner molecules based on two single-chain binding proteins.

EFFECT: invention provides multi-specific molecules which retain effector functions, insensitivity to proteolytic cleavage, and can be used in therapy of different diseases.

31 cl, 61 dwg, 14 tbl, 19 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology and biotechnology. What is presented is an antibody specifically bound to human CD26. There are described versions of coding NC, expression vector, and versions of a host cell for producing the antibodies. There are disclosed methods of inhibiting (cell proliferation, tumour growth) and a method of treating a condition caused by CD26 expression, as well as a pharmaceutical composition using the antibody of cl. 1. There are described methods of inhibiting (tumour growth, metastasis proliferation) using the pharmaceutical composition. What is disclosed is E.coli for producing the antibody, deposited under accession number PTA-7695 in ATCC (American typical culture collection).

EFFECT: use of the invention can find application in treating the immune disorders associated with CD26 expression, including cancer.

29 cl, 42 dwg, 14 tbl, 14 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented invention refers to immunology. There are presented versions of recovered monoclonal antibodies and an antigen-binding portion thereof specific to human IP-10. There are described: an immunoconjugate, bispecific molecule thereof, as well as versions of a composition of the antibody, immunoconjugate or bispecific molecule - for treating autoimmune and inflammatory diseases. There are also disclosed: a coding nucleic acid, an expression vector thereof and a host cell carrying this vector to produce the antibody. What is described is using the antibody or antigen-binding portion thereof for preparing a medicine for treating: either a viral or bacterial infection entailing undesired IP-10 activity, or autoimmune and inflammatory diseases caused by undesired IP-10 activity. What is presented is a hybridoma producing the antibody, derived from a transgenic mouse splenocyte cross-linked to an immortalised cell.

EFFECT: use of the invention provides the novel antibodies that can be find application in medicine to treat a variety of diseases associated with IP-10 activity.

22 cl, 30 dwg, 9 tbl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention discloses CD19-binding agents representing an antibody or an antigen-binding fragment containing an amino acid sequence of a variable region of a heavy chain, and an amino acid sequence of a variable region of a light chain which are bound with human CD 19 with a dissociation constant equal or less 1x10 -7M. The amino acid sequences are presented in the description. There are disclosed nucleic acid coding the heavy and/or light chain of the antibody or the antigen-binding fragment and a ligand conjugate - a therapeutic agent, or its pharmaceutically acceptable salt or solvate for treating a CD19-associated disorder in a mammal specified in a group involving CD19-expressing cancer, chronic leukaemia, B-cell lymphoma, multiple myeloma and a number of the other oncological diseases. The conjugate for treating the disorder in the mammal is used in an effective amount.

EFFECT: use of the presented antibodies or conjugate enables higher survival rate of the patients with oncological diseases expressing CD19, as well as in treating immunopathological diseases.

18 cl, 26 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: invention refers to biochemistry, particularly to artificial immunogenic proteins having the properties of melanoma antigens. What is declared is an artificial gene coding MEL-TCI-A0201 polyepitope immunogenic protein containing multiple cytotoxic restricted HLA-A*0201 and T-helper epitopes of NY-ESO-1, MART1, MAGE-A1, MAGE-A3, MAGE-A11, MAGE-C1 melanoma antigens, having a sequence of 1,535 base pairs presented in Fig. 3. There are also declared a recombinant plasmid DNA containing the above artificial gene, and MEL-TCI-A0201 immunogenic protein with the properties of the melanoma antigens.

EFFECT: invention enables providing higher immunogenicity of the artificial polyepitope T-cell immunogen inducing a higher level of cytotoxic T-lymphocyte response.

3 cl, 11 dwg, 3 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to biotechnology and provides humanised synthetic analogues (R6313/G2) of variable domains of the anti-angiotensin II type 1 receptor monoclonal scFv antibody 6313/G2, i.e. which specifically bind the molecule which specifically binds with a peptide, having the amino acid sequence EDGIKRIQDD. The invention also discloses a method of treating cancer, use of specifically binding molecules according to the invention when preparing a drug for treating cancer, a combined preparation and pharmaceutical compositions containing a specifically binding molecule according to the invention and angiotensin II.

EFFECT: present invention enables to block the harmful effect of angiotensin II and use said analogues to treat cancer in humans.

9 cl, 15 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly to recombinant fused protein dimers intended to inhibit or suppress immune response in a mammal, which bind human CD80 or human CD86 or the extracellular domain of any thereof, and has higher capacity for suppressing immune response than a dimer of the fused protein LEA29Y-Ig. Also disclosed are nucleic acids which code said dimers, expression vectors containing said nucleic acids, as well as recombinant host cells containing said nucleic acids and/or said vectors. Disclosed are pharmaceutical compositions for inhibiting or suppressing immune response in a mammal, which contain said fused protein dimers, as well as use of said dimers to produce drugs for inhibiting or suppressing immune response in a mammal, treating diseases or disorders of the immune system or treating organ or tissue transplant rejection in a mammal. Methods of producing said fused protein dimers are also disclosed.

EFFECT: invention provides effective inhibition or suppression of immune response in a mammal.

9 cl, 15 dwg, 11 tbl, 12 ex

FIELD: biotechnologies.

SUBSTANCE: invention describes creation of an artificial conformational antigen including peptide fragments consisting of two extracellular loops of muscarine M2-receptor, which are cross-linked with disulphide bond, and which is able to fix autoantibodies to this receptor with sensitivity exceeding the sensitivity for a linear prototype or mechanical mixture of data of linear peptide fragments. Synthetic antigen represents an individual chemical compound containing peptide corresponding to the sequence of the 1-st extracellular loop and peptide of the 2-nd extracellular loop, which are linked with disulphide connection between Cys96 and Cys176 residues (structure of antigen is disclosed in the formula and description). As per the invention, there described are methods for obtaining and using an antigen for creation of a diagnostics set for reveal of antibodies to muscarine M2-receptor in blood of patients with idiopathic arrhythmia using an enzyme-linked immunosorbent assay (ELISA) method.

EFFECT: improvement of the method.

5 cl, 1 dwg, 3 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: obtained is protein complex, possessing GPCRα1L affinity to ligand, which includes GPCRα1A and polypeptide with sequence of amino acids SEQ ID NO:1. Binding of said G-protein-conjugated receptor with polypeptide alters ligand affinity of the receptor. Also claimed are methods of screening agonists or antagonists of G-protein-conjugated receptor with application of transformant, in which said altered G-protein-conjugated receptor is expressed.

EFFECT: carrying out analysis of many supposed G-protein-conjugated receptors with still unknown structure.

12 cl, 3 dwg, 2 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, particularly to such binding elements specific for IgE as antibody molecules or its antigen-binding fragments.

EFFECT: antibody or its versions specific for IgE are effective for treating IgE-associated disorders, such as allergy and asthma.

17 cl, 17 dwg, 8 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: what is presented is a method for making a panel of serums for the purpose of quality control of diagnosing hepatitis B with the certified low concentration of HBsAg subtypes AD and AY which involves the analysis of donor serums for the presence of anti-HIV, anti-HCV and anti-HBs antibodies and the nonspecific HBsAg binding; the selection of donor serums with said parameters not found; the use of the selected serums to prepare diluting solutions with a stabilising additive introduced; the certification of the monopreparations of HBsAg subtypes AD and AY by titration dilutions of international standards and the preparation of a reference panel from the certified monopreparations of HBsAg subtypes AD and AY within the range of 0.01 to 0.5 IU/ml; the lyophilisation of the serums followed by thermal degradation panel testing to determine a shelf life. The use of a new formulation of the stabilising additive containing proline 200-250 mM and benzoic acid 0.05-0.08 wt %, and the selection of an optimum temperature storage conditions promote the prolonged activity preservation at annual loss (at +4°C) making less than 0.5%.

EFFECT: higher shelf life.

3 cl, 5 tbl, 1 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to peptides of general formula Rr-AA-R2 which can regulate neuron exocytosis, their mixtures and their cosmetically and pharmaceutically acceptable salts wherein AA represents a sequence of 6-40 neighbouring amino acids contained in the amino acid sequence of the protein SNAP-25 R1 is specified in a group consisting of H or acetyl, or saturated or unsaturated, linear, branched or cyclic C3-C24acyl, or a polyethylene glycol polymer; R2 is specified in a group consisting of amino, unsubstituted or substituted by saturated linear or branched, or cyclic C2-C24aliphatic groups provided when R1 represents H or acetyl, R2 is other tuhan unsubstituted amino. The invention also refers to cosmetic or pharmaceutical compositions containing such peptides, and use of such for treating the conditions which require regulation of neuron exocytosis, preferentially for skin treatment.

EFFECT: higher clinical effectiveness.

12 cl, 1 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: invention discloses a method of solid-phase immune-enzyme assay wherein as an antigen, an equimolar mixture of synthetic peptides is immobilised on plastic with the mixture containing: nonapeptide (position 125-133), tridecapeptide (position 208-218) of an amino acid sequence of a human β1 adrenoreceptor molecule and a chimeric construct of the same peptides jointed by a disulphide bond. The presented method shows substantially greater sensitivity in detecting the presence of β1-adrenoreceptor autoimmune antibodies in blood plasma and serum in the patients with dilatation cardiomyopathy.

EFFECT: invention is also applicable for diagnosing the patients with various cardiovascular diseases.

1 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to biotechnology. Described is a peptide capable to induce cytotoxic (killer)T-cells. Presented are medications containing the described peptide that are intended for induction of immunity against malignant tumours, for tumours treatment and/or prevention, for induction of antigen-presenting cells highly capable to induce tumour-reactive T-cells and for induction of tumour-reactive T-cells. Presented is an antibody specifically bonded to the described peptide. Described are an extracted tumour-reactive killer T-cell that is induced in vitro by way of stimulation by the described peptide, a tumour-reactive helper T-cell induced in vitro by way of stimulation by the described peptide, an antigen-presenting cell representing a complex of an HLA molecule and the described peptide.

EFFECT: invention allows to intensify immunity with a patient suffering from metastasis cancer or resistant cancer when surgery treatment, chemotherapy and radiation therapy are practically inapplicable.

10 cl, 4 dwg, 1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of general formula (1), which possess an affinity to the µ-opiod receptor and the ORL1-receptor. The invention also relates to the application of the said compounds for obtaining medications, which can be used in treatment of fear, stress and associated with stress syndromes, depressions, epilepsy, Alzheimer's disease, senile dementia, general cognitive dysfunctions, learning and memory disorders (as nootropic), withdrawal syndromes, alcohol and/or drug abuse and/or abuse of medications and/or alcohol, narcotic and medication addiction, etc. In general formula (1) (1) Y1, Y1', Y2, Y2', Y3, Y3', Y4 and Y4' in each case stand for -H; Q stands for -R0, -C(=O)-R0, -C(=O)OR0, -C(=O)NHR0, -C(=O)N(R0)2 or-C(=NH)-R0; R0 in each case stands for -C1-8-aliphate, -C3-12-cycloaliphate, -aryl, -heteroaryl, -C1-8-aliphate-C3-12-cycloaliphate, -C1-8-aliphate-aryl, -C1-8-aliphate-heteroaryl, -C3-8-cycloaliphate-C1-8-aliphate, -C3-8-cycloaliphate-aryl or -C3-8-cycloaliphate-heteroaryl; R1 and R2 independently on each other stand for -C-1-8-aliphate; R3 stands for -C1-8-aliphate, -aryl, -heteroaryl or -C1-8-aliphate-C3-12-cycloaliphate; n stands for 0; X stands for -NRA-;RA stands for -C1-8-aliphate; RB stands for -C1-8-aliphate; on condition that R1, R2, RA and RB simultaneously do not stand for the non-substituted-C1-8-aliphate.

EFFECT: increased efficiency of the application of the compounds.

9 cl, 11 tbl, 164 ex

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