US 2013/0143320 A1 Li Et Al

US 2013 O143320A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0143320 A1 Li et al. (43) Pub. Date: Jun. 6, 2013

(54) METHODS AND COMPOSITIONS FOR (60) Provisional application No. 60/392,142, filed on Jun. MANIPULATING THE GUIDED NAVIGATION 27, 2002. OF ENDOTHELLALTUBES DURING ANGIOGENESIS Publication Classification (71) Applicants: Dean Y. Li, Salt Lake City, UT (US); (51) Int. Cl Kye Won Park, Salt Lake City, UT (US) CI2N5/071 (2006.01) (72) Inventors: Dean Y. Li, Salt Lake City, UT (US); C07K 4/705 (2006.01) Kye Won Park, Salt Lake City, UT (US) (52) U.S. Cl CPC ...... CI2N5/0691 (2013.01); C07K 14/705 (73) Assignee: University of Utah Research (2013.01) Foundation, Salt Lake City, UT (US) USPC ...... 435/375:536/23.5; 530/350 (21) Appl. No.: 13/658,526 (57) ABSTRACT (22) Filed: Oct. 23, 2012 Methods and compositions for manipulating the directed Related U.S. Application Data navigation of physiological tracking tubular structures are .S. App provided. A novel cell-bound receptor, roundabout-4 (Robo (63) Continuation of application No. 12/975,160, filed on 4), is described. The Robo-4 receptor shows sequence and Dec. 21, 2010, now abandoned, which is a continu structural similarity to members of the roundabout family of ation of application No. 10/519,342, filed on Sep. 21, receptors. Also, the Robo-4 receptor binds Slit ligand, a 2005, now abandoned, filed as application No. PCT/ known receptor of the roundabout receptors. Polynucleotides US03/20508 on Jun. 27, 2003. and polypeptides of the Robo-4 receptor are described. Patent Application Publication Jun. 6, 2013 Sheet 1 of 6 US 2013/0143320 A1

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None VEGF HEKCM Slit-myc CMSi-myc CMSlit-myc CMSt-myc CM Anti HA Anti-cmyc N-Robot -- depletion Figure 7 US 2013/0143320 A1 Jun. 6, 2013

METHODS AND COMPOSITIONS FOR 0006. Therefore, there is a need for compositions and MANIPULATING THE GUIDED NAVIGATION methods that are able to manipulate the navigation of physi OF ENDOTHELLALTUBES DURING ological tracking tubular structures, such as endothelial tubes, ANGOGENESIS such that the structures can be directed towards a desired tissue, or prevented from reaching a target tissue. FIELD OF THE INVENTION SUMMARY OF THE INVENTION 0001. The present invention relates to methods and com positions that are useful in manipulating the guidance of 0007. The present invention is directed at a guidance sys physiological tracking tubular structures, such as endothelial tem and methods that function to direct navigation of physi tubes. In preferred embodiments, the invention relates to ological tubular structures, such as endothelial tubes. The methods and compositions that are useful in manipulating the invention includes a novel cell-bound receptor, Roundabout 4 directed navigation of endothelial tubes, such as during (Robo-4), that is expressed in endothelial cells and interacts angiogenesis, during embryonic development and in the with a known ligand to affect directed navigation of endothe neovascularization of tumors and other cell masses and/or lial tubes during vascular development. Together, the Robo-4 tissues. More specifically, the present invention relates to a receptor and the ligand, the slit ligand, inhibit the directed guidance system that can be used to both direct endothelial navigation of endothelial tubes to target cell masses and/or tubes toward a target, such as ischemic tissue, and direct tubes tissues. Thus, the interaction between the Robo-4 receptor away from a target, Such as a solid tumor. and the slit ligand provides a repulsive cue that affects the guidance of tubular structures, such as endothelial tubes. As BACKGROUND OF THE INVENTION described herein, the repulsive cue provided by Robo-4/slit interactions can be used to direct tubular structure both 0002 The vasculature provides a network of vessels that toward and away from a tissue or other cell mass of interest. efficiently delivers nutrients to and removes waste from tis 0008. The present invention is useful for a variety of pur sues of the body. This network extends throughout most of the poses. For example, the polynucleotides of the present inven body, reaching all major tissues, and consists of two distinct tion can be used for gene therapy, such as replacement of types of structures—arteries and veins. The arterial and defective copies of naturally occurring genes or provision of venous systems are parallel networks that function to deliver Supplemental genes. Furthermore, the polypeptides of the blood to a tissue and carry blood and waste away from tissues, present invention can be used in therapeutic procedures. For respectively. These two systems are anatomically distinct and example, the polypeptide encoding the receptor, or a frag connect at distal capillary beds. ment thereof, can be supplied to an environment in order to 0003. The network of vessels that comprise the arterial and compete with cell-bound receptors, thereby effectively low venous systems develops by a process of directed movement ering or preventing activation of the cell-bound receptors. of endothelial tubes to desired cell masses and/or tissues. Also, the various methods of the present invention are useful During embryogenesis, the initial vascular framework is in studying and treating conditions related to angiogenesis, defined by the de novo formation of the dorsal aortae and Such as ischemia and tumor growth. cardinal veins. Mature circulatory networks are formed when 0009. The inventors have identified and sequenced the endothelial tubes sprout from central vessels, navigate gene that encodes the receptor, identified at least one ligand through the embryo, and reach their target cell mass and/or for the receptor (the slit ligand), and identified sequence and tissue. Upon reaching the target, the tubes are able to Supply structural similarities between the novel receptor and a family blood as nourishment for the tissue. of existing receptors—the Roundabout family of receptors. 0004. This navigation of endothelial tubes is important not 0010. Also, the inventors have identified a function for the only during embryonic development when the vasculature is Robo-4 receptor. The receptor, following interaction with the first forming, but also in all physiological processes that slit ligand, inhibits the migration of endothelial tubes. The include the introduction of a blood Supply to a cell mass repulsive cue provided by Robo-4/slit interaction contributes and/or tissue. These processes include various disease condi to the directed navigation of endothelial tubes by steering the tions that are sustainable only because of the introduction of tubes away from a location having the ligand, such as a cell a blood Supply to a cell mass. Such as the Survival of a Solid expressing the ligand. cancerous tumor. The continued growth of a Solid tumor 0011. The Robo-4 receptor is expressed on sprouting requires the presence of a blood Supply that nourishes the endothelial tubes that form the perineural vasculature beds. cells of the tumor mass. Angiogenesis, a physiological pro The neural tubes produce and secrete the slit ligand. This cess in which new blood vessels are formed and directed into enables the directed navigation of the endothelial tubes away a target cell mass and/or tissue, is a critical step in tumor from the neural tubes. As a result of this negative cue and development and Survival. Accordingly, methods and com likely in combination with attractive cues, the endothelial positions that are able to interfere with this process could be tubes, through slit-Robo-4 binding interactions that result in useful in preventing the growth and/or Survival of tumors. directed navigation, form a vasculature network around the 0005. Other disease conditions exist in which the blood developing central nervous system. This interaction with the Supply to a particular tissue is blocked or otherwise impeded, central nervous system leads to the close association between thereby diminishing the Supply of nutrients to that particular the nervous and vasculature systems that is evident in both tissue. For example, ischemia is a condition in which a local macro and micro anatomies. ized anemia occurs in a tissue due to an obstruction of the 0012. Thus, the present invention includes the isolated inflow of arterial blood. This condition can be corrected by cDNA and polypeptides of the Robo-4 receptor. Also, the removal of the obstruction, or development of new vessels invention includes methods of manipulating the guided navi that are capable of Supplying the required nourishment to the gation of endothelial tubes based on interactions between the affected tissues. Robo-4 receptor and the slit ligand. Further, the invention US 2013/0143320 A1 Jun. 6, 2013

includes methods of inducing and preventing angiogenesis by Marchuk D A. (1997), The activin-like kinase 1 gene: inhibiting and activating, respectively, the Robo-4 receptor. genomic structure and mutations in hereditary hemorrhagic 0013. In a preferred embodiment, the method of the telangiectasia type 2. Am J Hum Genet. 61 (1):60-7). present invention comprises a method of directing the navi 0024. The inventors have generated and characterized gation of endothelial tubes away from a target by allowing genetic knockout mice that lack functional Alk-1. (Urness, L. binding between the slit ligand and the Robo-4 receptor on the D., Sorenson, L. K. Li, D.Y. (2000) Arteriousvenous mal endothelial cells of the tubes. The directed navigation of formations in mice lacking activin receptor-like kinase-1. endothelial tubes away from target tissue in this method can Nature Genetics. 26:328-331). These mice are described in be accomplished by expressing slit ligand in cells of the target our U.S. patent application Ser. No. 09/578,553, which is tissue. hereby incorporated by reference in its entirety. In homozy 0014. In a second preferred embodiment, the method of gous Alk-1-/- embryos, the distinct anatomical, structural, the present invention comprises a method of inducing the molecular, and functional properties of arteries and veins are directed navigation of endothelial tubes toward a first target lost. As a result, the development of these embryos is arrested cell mass and/or tissue by repelling the endothelial cells away at about day 10.5. Based on these studies, the inventors have from a second target through Robo-4/slit binding. This can be discovered that Alk-1 regulates molecular programs that accomplished by expressing the slit ligand in the second instruct sprouting arteries and veins to remain distinct as they target and exposing the endothelial tubes to the second target. are guided along parallel pathways to common distal target In a particularly preferred embodiment, a Substantially con Organs. tinuous second target, such as a tissue Surface or vessel, is 0025 To further characterize the role of Alk-1 in vascular lined with slit ligand, thereby providing a continuous repul development, the inventors have investigated genes that are sive force away from the second target. differentially expressed in Alk-1+/+ and Alk-1-/- cells. A screen of these differentially expressed genes revealed a BRIEF DESCRIPTION OF THE DRAWINGS novel receptor, which the inventors have termed the Round 0015 FIG. 1 represents results of a Northern Blot analysis about 4 (Robo-4) receptor. These differential expression stud of Robo4 expression in Alk1 +/+ and Alk1-/- tissues. The ies showed that Robo-4 is expressed in Alk-1-/- mice at bottom panel shows loading controls, 28S and 18S RNA. levels that are approximately 4 to 5 fold higher than those in 0016 FIG. 2 represents visualization of staining of Robo4 wild-type mice (See FIG. 1). anti-sense cFNA at days 9.0 and 9.5 of embryonic develop 0026. Also, in situ hybridization of Robo-4 shows the ment. temporal and spatial expression of Robo-4 in vascular tissues 0017 FIG.3 is a schematic comparing various domains of (See FIG. 2). Between E8.0 to 8.5 Robo-4 was expressed in various members of the Robo family of receptors. the central vessels, the dorsal aortae and cardinal veins. 0018 FIG. 4 is a schematic illustrating the phylogeny of Between E8.5 and E 10.0, intersomitic vessels sprout and a some members of the Robo family of receptors. capillary plexus forms around the neural tube. Robo-4 0019 FIG. 5 represents visualization of an immunoblot expression was detected throughout the endothelium of these ting assay in which Human Slit 2-myc was coimmunopre structures during this critical periodofangiogenesis. In cross cipitated with Robe 4-HA using anti-HA antibodies. sections, the expression of Robo-4 was more prominent in 0020 FIGS. 6A-6F graphically represent data from vari Smaller vessels and capillary beds than in large vessels such as ous cell migration assays. the dorsal aortae and cardinal veins. Robo-4 expression was 0021 FIG.7 graphically represents data from a cell migra detected in the endothelial cells that invaded the neural tube, but never in the neural tissue proper. This is in contrast with tion assay utilizing human microvascular endothelial cells Robo-1, Robo-2, and Robo-3 which are highly expressed in (HMVECs). the nervous system of mice, chick and Zebrafish consistent with their roles in neuronal migration and axonal guidance DETAILED DESCRIPTION OF PREFERRED (13-18). During Zebrafish development, ZfRobo4, with its EMBODIMENTS OF THE INVENTION unique extracellular domain structure of three IgG and two 0022. The following description of preferred embodi FN domains, was expressed in both the developing neural ments provides examples of the present invention. The tube and vascular system. Northern blot analysis indicated embodiments discussed herein are merely exemplary in that Robo-4 is expressed throughout embryogenesis and dur nature, and are not intended to limit the scope of the invention ing adulthood. Robo-4 expression was highest in the heart in any manner. Rather, the description of these preferred and was undetectable within the brain, spleen, and testis. embodiments serves to enable a person of ordinary skill in the Interestingly, Robo-4 was expressed at intermediate levels in relevant art to make and use the present invention. tracking tubular structures in the liver, kidney, and lung, Such 0023 Activin receptor-like kinase 1 (Alk-1) is a receptor as bronchioles. Northern blot analysis for Robo-1 expression that plays a role in vascular development. Loss of function demonstrated prominent brain expression consistent with mutations in the Alk-1 receptor are responsible for Hereditary previously published reports (13). These results demonstrate Hemorrhagic Telangiectasia (HHT), an autosomal dominant that during development, Robo-4 differs from other Robe vascular dysplasia. (Johnson D. W. Berg JN, Baldwin MA, family member in its prominent endothelial expression pat Gallione C J, Marondel I, Yoon SJ, Stenzel T T. Speer M, tern Perciak-Vance MA, Diamond A, Guttmacher A E, Jackson C 0027. The inventors have cloned and sequenced both the E, Attisano L. Kucherlapati R, Porteous ME, Marchuk D A. human and mouse Robo-4 genes. The mouse Robo-4 cDNA (1996), Mutations in the activin receptor-like kinase 1 gene in sequence appears as SEQID 1, and the human Robo-4 cDNA hereditary hemorrhagic telangiectasia type 2. Nat Genet. appears as SEQID 2. Also, the deduced amino acid sequence 13(2):189-95; Berg JN, Gallione CJ, Stenzel TT, Johnson D for the mouse Robo-4 receptor appears as SEQID 3 and the W. Allen W P Schwartz CE, Jackson C E, Porteous ME, deduced amino acid sequence for the human Robo-4 receptor US 2013/0143320 A1 Jun. 6, 2013

appears as SEQID 4. The invention includes isolated poly cal activity. A conservative amino acid Substitution is a nucleotides that encode a Robo-4 receptor, complimentary change in the amino acid sequence that does not affect bio polynucleotide sequences, and fragments and portions logical activity of the receptor. thereof, including the polynucleotides listed herein as SEQ 0032. In addition to the polynucleotide sequences shown ID 1 and SEQ ID 2 and complimentary nucleic acid mol in SEQID NOS 1 and 2, DNA sequence polymorphisms that ecules of these polynucleotides. change the amino acid sequences of the Robo-4 receptor may 0028. As used herein, the term "isolated” refers to a mol exist within a population. For example, allelic variation ecule that is purified from the setting in which it is found in among individuals will exhibit genetic polymorphism in the nature and is separated from at least one contaminant mol Robo-4 receptor. As used herein, a “variant polynucleotide' is ecule of the same class of molecules. Thus, an isolated poly a nucleic acid molecule, or a complementary nucleic acid nucleotide comprises a polynucleotide that is purified from its molecule, which encodes an active Robo-4 receptor that has natural setting and separated from at least one contaminant at least about 80% nucleic acid sequence identity with a polynucleotide. Similarly, an isolated polypeptide comprises nucleic acid sequence encoding a full-length native Robo-4 a polypeptide that is purified from its natural setting and receptor, or any other fragment of a full-length Robo-4 separated from at least on contaminant polypeptide. As used nucleic acid or complementary nucleic acid. Ordinarily, a herein, the term “complementary nucleic acid molecule' variant polynucleotide will have at least about 80% nucleic refers to a polynucleotide that is sufficiently complementary acid sequence identity, more preferably at least about 81%, to a sequence, e.g., SEQID NOS 1 and 2, such that hydrogen 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, bonds are formed with few mismatches, forming a stable 92%.93%,94%,95%,96%.97%.98% nucleic acid sequence duplex. As used herein, the term “complementary” refers to identity and yet more preferably at least about 99% nucleic Watson-Crick or Hoogsteen base pairing between nucle acid sequence identity with a nucleic acid sequence encoding otides. a full-length native Robo-4 receptor, or complimentary 0029. The invention also includes derivative, analog, and nucleic acid molecule. Variant polynucleotides do not encom homolog nucleic acid molecules of the polynucleotides of the pass the native nucleotide sequence. invention, including the polynucleotides listed hereinas SEQ 0033. The invention also includes isolated polypeptides ID 1 and SEQ ID 2. As used herein, the term "derivative comprising a Robo-4 receptor, including the polypeptides nucleic acid molecule' refers to a nucleic acid sequences having the amino acid sequences listed herein as SEQ ID 3 formed from native compounds either directly or by modifi and SEQID 4. cation or partial substitution. As used herein, the term “analog nucleic acid molecule” refers to nucleic acid sequences that 0034. The invention also includes derivative, analog, and have a structure similar, but not identical, to the native com homolog polypeptides of those listed herein as SEQID NOS pound but differ from it in respect to certain components or 3, 4, 5, and 6. As used herein, the terms derivative amino acid side chains. Analogs may be synthesized or from a different sequence, analog amino acid sequence, and homolog amino evolutionary origin. As used herein, the term "homolog acid sequence have the same meaning as for the nucleic acid nucleic acid molecule” refers to nucleic acid sequences of a terms, described above, applied to polypeptides. particular gene that are derived from different species. 0035. As a preliminary matter, the inventors determined 0030 Derivatives and analogs may be full length or other whether the identified genes share any sequence homology than full length, if the derivative or analog contains a modified with any known families of receptors. Analysis of the Robo-4 nucleic acid or amino acid. Derivatives or analogs of the gene sequences revealed significant homology with members polynucleotides of the invention include, but are not limited of the Roundabout (Robo) family of receptors, which func to, molecules comprising regions that are substantially tion in the guidance of neural tubes during development. The homologous to the polynucleotides of the invention, includ results of this analysis revealed that the Robo-4 gene shares ing the polynucleotides listed hereinas SEQID 1 and SEQID 45% sequence similarity and 31% identity to members of the 2 by at least about 70%, 80%, or 95% identity over a nucleic Robo family. Also, the inventors determined that the Robo-4 acid of identical size or when compared to an aligned gene has a chromosomal position adjacent that of the Robo-3 sequence in which the alignment is done by a homology gene, Rig-1. Accordingly, the inventors named the novel algorithm, or whose encoding nucleic acid is capable of receptor Robo-4 due to this sequence homology as well as hybridizing to the complement of a sequence encoding a structural homology and functional similarities, as described Robo-4 receptor. below. 0031) “Homologous nucleotide sequences encode those 0036. The human and mouse Robo-4 cDNA’s encode pro sequences coding for isoforms of the Robo-4 receptor. teins of 1007 and 1012 amino acids, respectively. The Homologous nucleotide sequences include nucleotide deduced polypeptide sequence includes a signal sequence of sequences encoding a polynucleotide for a Robo-4 receptor 20 amino acids and a single transmembrane domain. Further, of species other than humans, such as vertebrates, e.g., frog, structural analysis of the polypeptide sequence revealed the mouse, rat, rabbit, dog, cat, cow and horse. The polynucle presence of two IgG domains as a well as two fibronectin otide listed herein as SEQID 1 is a cDNA sequence for the domains. The IgG and fibronectin domains are all located to mouse Robo-4 receptor. Homologous nucleotide sequences one side of the transmembrane domain. This arrangement is a also include naturally occurring allelic variations and muta structural feature shared by all members of the Robo family tions of the nucleotide sequences. A homologous nucleotide (See FIG. 3). Also, the Robo-4 polypeptide includes two sequence does not, however, include the exact nucleotide cytoplasmic domains that are partially conserved (See FIG.3 sequence encoding the human Robo-4 receptor. Homologous in which the partially conserved domains are labeled as nucleic acid sequences also include those nucleic acid domains 0 and 2). sequences that encode conservative amino acid Substitutions 0037 FIG. 4 illustrates the phylogeny of the Robo family as well as a polypeptide possessing Robo-4 receptor biologi of receptors. In the Figure, the length of lines is proportional US 2013/0143320 A1 Jun. 6, 2013

to the evolutionary distance between branch points. As the (2001), Curr Biol 17; 11 (8):R300-3: Battye R, Stevens A, Figure shows, Robo-4 is closely associated with the Robo Jacobs J. R. (1999), Axon repulsion from the midline of the family of receptors. Drosophila CNS requires slit function. Development. 126 0038 Based on the observed sequence and structural simi (11):2475-81: Li HS, Chen J. H. Wu W. Fagaly T. Zhou L, larities between the novel Robo-4 receptor and the Robo Yuan W. Dupuis S. Jiang Z H, Nash W. Gick C, Omitz, OM, family of receptors, the inventors hypothesized that the Wu JY. Rao Y. (1999), Vertebrate slit, a secreted ligand for the Robo-4 receptor is a member of the Robo family. To confirm transmembrane protein roundabout, is a repellant for olfac this hypothesis, the inventors evaluated the ability of the tory bulb axons. Cell 96(6):807-18; Brose K, Bland KS, Robo-4 receptor to bind Slit2, a known ligand of receptors of Wang K H. Arnott D. Henzel W. Goodman C. S. Tessier the Robo family. Lavigne M. Kidd T. (1999), Slit proteins bind Robo receptors 0039. The Slit ligands show promiscuous binding to and have an evolutionary conserved role in repulsive axon receptors of the Robo family. (Johnson D. W. Berg JN, Bald guidance. Cell 96(6):795-806). In the neural system, a series win Ma, Gallione C J, Marondel I, Yoon SJ, Stenzel T T. of repulsive and attractive cues provide a guidance system for Speer M. Perciak-Vance MA, Diamond A. Guttmacher A E, directing the navigation of axons to and/or away from targets. Jackson C E, Attisano L. Kucheerlapati R, Porteous ME, The Robo receptors, in conjunction with the Slit ligands, are Marchuk D.A. (1996), Mutations in the activin receptor-like critical for guiding axons to synapse with the appropriate kinase 1 gene hereditary hemorrhagic telangiectasia type 2. distal targets through repulsive cues. Thus, in the neural sys Nat Genet. 13(2):189-95; Berg JN, Gallione CJ, Stenzel TT, tem, the Slit ligands and some of the previously known Robo Johnson D. W. Allen W. P. Schwartz CE, Jackson CE, Porte receptors direct the navigation of neurons during develop ous ME, Marchuk D.A. (1997), The activin-like kinase gene: ment of the neural system. genomic structure and mutations in hereditary hemorrhagic 0042 Considering the sequence and structural similarities telangiectasia type 2. Am J Hum Genet. 61 (1):60-7: Urness, between the novel Robo-4 receptor and the Robo family of L. O., Sorenson, L. K. Li, D. Y. (2000), Arteriousvenous receptors, and also the expression of the Robo-4 receptor in malformations in mice lacking activin receptor-like kinase-1. vascular cells, such as endothelial tubes, the inventors Nature Genetics. 26:328-331). The inventors investigated the hypothesized that the novel receptor has a function in direct ability of mouse Robo-4 receptor to bind human Slit2 ligand. ing the navigation of the vasculature during its development For these experiments, stable cells line expressing full length by way of a repulsive cue. To investigate this proposed func hemagglutinin-tagged Robo-4 receptors (Robo-4-HA) were tion, the inventors have evaluated the ability of the Slit ligand generated. These cells also expressed a secreted form of to affect behavior of endothelial tubes via interaction with the hemagglutinin-tagged Robo-4. All constructs were con Robo-4 receptor. firmed by sequencing and western blotting. Human Slit2 0043. To confirm the function of the Robo-4 receptor ligand tagged with the Myc Epitope was stably transfected observed in vitro, the inventors examined the function of the into HEK 293 cells. Immunoprecipitation studies indicated receptor in Vivo. In addition to their role in neuronal guidance, that Robo-4-HA and Human Slit2-Myc complexes could be it has recently been shown that Slit inhibits the migration of coprecipitated by antibodies against HA (See FIG. 5, particu HEK cells that express Robo-1 (22). The inventors have larly gel lane 7). This demonstrates that the Robo-4 receptor found that Slit had a similar effect in cells expressing Robo-4. binds the Slit2 ligand. For these studies, standard transfilter assay were performed in 0040. The binding of Human Slit2 to the mouse Robo-4 which test factors were placed in the lower chamber and cells receptor is saturable. Thus, the Robo-4 receptor specifically were placed in the upper chamber. The number of cells that binds the Slit2 ligand, confirming the identity of the novel migrated to the lower chamber after 2 hours was determined. receptor as a member of the roundabout family of receptors In these experiments, the migration of Robo-4-HEK and Con (the Robo receptors). Immunoprecipitation data was con trol-HEK cells to Slit-myc conditioned media (CM) as well as firmed by determining whether Slit protein bound to mem to media collected from control HEK cells, i.e., media lacking branes of cells expressing Robo-4. HEK cells expressing slit, was observed. Slit specifically inhibited the migration of Robo-4 (Robo-4-HEK) or Control-HEK cells were incubated Robo-4 expressing HEK cells. As expected, fibroblast growth with conditioned media from Slit-expressing cells (Slit-myc factor and HEK-CM induced both Robo-4-HEK and Control CM). Binding of Slit-myc proteins to the cell surfaces was HEK cells to migrate at a rate of three to four-fold greater than detected by indirect immunofluoresence using a murine anti background (FIG. 6A). Slit-myc CM induced comparable myc antibody and an Alexa 594 conjugated anti-mouse anti levels of migration of Control-HEK cells (FIG. 6B). How body. Fluorescence was detected on the surface of Robo-4- ever, when applied to Robo-4-HEK cells, Slit-myc CM inhib HEK cells and not Control-HEK cells. Together, the ited migration to baseline levels (FIG. 6A). immunoprecipitation and immunofluorescence data provide 0044) This inhibitory effect of Slit-myc CM was specific strong evidence that Slit binds to Robo-4 on the cell surface. for the Slit protein (FIG. 6C-6F). Conditioned media from 0041. The Robo receptors have a well-defined function in HEK cells that expressed the soluble ligand binding neural guidance. (Song, H. Poo M. (2001), The cell biology of ectodomain of Robo-1 (NRobo-1-HA) was incubated with neuronal navigation. Nat Cell Biol (3):E81-8; Brose K. Slit-myc CM. The binding of NRobo-1 to Slit-myc is an Tessier-Lavigne M. (2000), Slit proteins: key regulators of effective method for removing Slit protein from conditioned axon guidance, axonal branching, and cell migration. Curr media (21-23). The inhibitory effect of Slit-myc CM on the Opin Neurobiol. 10(1):95-102; Wong K, Ren X R, Huang Y migration of Robo-4-HEK cells was lost following depletion Z, Xie Y. Liu G. Saito H. Tang H. Wen L. Brady-Kalnay S M, with NRobo-1 (FIG. 6C, 6D). Similarly, Slit-myc CM pre Mei L. Wu JY,Xiong WC, Rao Y. (2001), Signal transduction treated with an anti-myc antibody lost its inhibitory effect on in neuoronal migration roles ofgtpase activating proteinc and Robo-4 HEK cell migration (FIG. 6E, 6F). Mock depletions the small gtpase cdc42 in the slit-robo pathway. Cell, 107(2): with an anti-HA antibody did not reduce the inhibitory effect 209-21; Guthrie S. Axon guidance: Robos make the rules of Slit-myc CM. US 2013/0143320 A1 Jun. 6, 2013

0045 Slit modulates endothelial cell migration via Robo target by directing endothelial tubes away from the target. 4. To demonstrate that Robo-4 was present on the cell surface The presence of a blood supply is vital to survival of cell of primary endothelial cells, the inventors generated a poly masses and/or tissues. In some instances, it may be desirable clonal antibody to its cytoplasmic region (amino acids 964 to remove the blood supply or prevent its formation and/or 981). This region is highly conserved between human and generation in order to lyse the cell mass and/or tissue by mice, and is specific to Robo-4. Culture media from HEK removing its nutrient Supply. For example, cancerous cell cells induced migration of human microvascular endothelial masses, such as Solid tumors, ensure their long-term Survival cells (HMVECs) at a level comparable to 10 ng/ml of vascular by developing a blood Supply through angiogenesis. By pre endothelial growth factor (VEGF) (FIG. 7). However, with venting this development, the methods of the present inven Slit-myc CM, there was a 70% inhibition of migration (FIG. tion provide techniques for lysing cell masses and/or tissues. 7). Depleting Slit protein from Slit-myc CM with anti-myc 0051. Thus, in one particularly preferred embodiment, the antibody or NRobo-1 blocked in the inhibitory effect of Slit invention includes methods of preventing the guided naviga myc CM on endothelial cell migration (FIG. 7). The inhibi tion of endothelial tubes during angiogenesis to a target cell tory effect of Slit on Robo-4 expressing endothelial cells mass/tissue. The method according to this embodiment mirrored that of Robo-4-HEK cells (FIG. 7). Thus, Slit binds includes exposing the endothelial tubes to a ligand of the and activates Robo-4 in primary endothelial cells. Robo-4 receptor, such as the Slit ligand. The Slit ligand binds 0046. The function of the Robe receptor makes the recep to the Robo-4 receptor on the endothelial tubes and inhibits tor useful in a variety of methods relevant to medicine and their migration which interrupts the directed navigation of the research. Specifically, because the Robo-4 receptor provides endothelial tubes towards the cell mass and/or tissue. Any a repulsive cue in the directed navigation of endothelial tubes suitable technique for allowing binding between the receptor during angiogenesis, the receptor and Slit ligand can be used and ligand can be used. Preferred techniques include express to manipulate this process. Accordingly, the present invention ing the Slit ligand in the target and allowing the expressed Slit also includes methods of manipulating the guided navigation ligand to interact with the Robo-4 receptor on the endothelial of endothelial tubes during angiogenesis. tubes. 0047. In one preferred embodiment, the invention 0.052 Angiogenesis may be inhibited and/or prevented includes methods of directing the navigation of physiological generally, without a directional limitation, in endothelium by tubular structures toward a target tissue. This method is useful activating Robo-4 receptor in the tissue. Activation of the to encourage the directed navigation of developing vascula receptor can be accomplished by any suitable technique, Such ture to a target cell mass and/or tissue. The method can be as by providing a ligand of the Robo-4 receptor to the recep used to provide new vasculature to a cell mass/tissue that is in tor, and allowing the ligand to bind to the receptor. Slit ligand need of a new system of nutrient Supply and waste removal. is a particularly preferred ligand. The ligand can be provided For example, an ischemic tissue Suffers from reduced oxygen in any suitable manner, Such as by providing a soluble form of Supply due to poor blood flow to the tissue. By encouraging the receptor directly to the endothelium, by expressing the angiogenesis to an ischemic tissue, a new blood Supply route ligand in cells of the endothelium or adjacent tissue, or other can be created, effectively providing a new nutrient Supply Suitable techniques. Also, fragments of ligands of the Robo-4 and waste removal system for the tissue, which can help to receptor may be used. The fragment need only retain the correct the condition. ability to bind and activate the receptor. Also, activation of the 0048 Thus, in one particularly preferred embodiment, the Robo-4 receptor can be accomplished by other suitable tech invention comprises a method of directing endothelial tubes niques, such as by using agonosits of the Robo-4 receptor, to a first target cell mass and/or tissue by repelling the endot including monoclonal and polyclonal antibodies that bind helial tubes away from a second target via Robo-4 binding and activate the receptor. interactions with a ligand of the receptor, such as a Slit. The 0053. In another preferred embodiment, the invention pro repelling away from the second target can direct the endot vides methods of disrupting the navigation of tracking tubular helial tubes toward the first target. Due to the presence of the structures, such as endothelial tubes, that express the Robo-4 receptor and the ligand, the endothelial tubes will navigate receptor. The negative cue provided by Slit/Robo-4 binding away from the second target, and toward the first target. likely works in combination with positive cues that, together, 0049 Angiogenesis may be induced by inhibiting Robo-4 provide a navigation system that directs tracking tubular structures toward and away from a series of local targets to activation in endothelium by inhibiting activation of the ultimately direct the structures along a desired path. By inter Robo-4 receptor. The absence of the negative cues provided fering with the negative cue, the entire navigation system will by Robo-4 activation may induce angiogenesis in the tissue, be dysfunctional, and the tracking tubular structures will not which may be independent of directional limitations. The be positioned on the desired path. This may result in the inhibition of activation of the Robo-4 receptor can be accom structures going in several directions, due to the presence of plished in any suitable manner, such as by providing a soluble positive cues, but not in the path naturally desired due to the form of the receptor to the endothelium tissue. The presence lack of counteracting negative cues. This can be useful in of soluble receptor may bind any ligand that is present, which experimental work with and clinical treatment of conditions may prevent ligand bind to and activation of the cell-bound in which an excessive amount of tracking tubular structure receptor. SEQID 5 and SEQID 6 provide mouse and human penetration occurs. For example, in cancer, retinopathy, and soluble receptor forms, respectively. Also, fragments of these inflammatory conditions, excessive neovasculartization sequences may be suitable for use in the methods of the occurs, and disruption of the navigation of endothelial tubes invention, as may sequences with less than 100% homology could interfere with this condition, which may ultimately to these sequences. Particularly preferred sequences have limit the progression of the condition. 80% sequence identity to SEQID 6, or a fragment thereof. 0054. In a preferred embodiment, the methods of disrupt 0050. In a second preferred embodiment, the present ing navigation comprise inhibiting activation of the Robo-4 invention includes methods of preventing angiogenesis to a receptor(s) of the tracking tubular structures. The inhibiting US 2013/0143320 A1 Jun. 6, 2013 can be accomplished using various techniques Suitable for 0055. Other compositions capable of binding the ligand(s) accomplishing inhibition of activation of a cell-bound recep of the receptor, Such as Slit, could also be prepared and used tor, Such as blocking the receptor with a monoclonal antibody to prevent ligand binding to the receptor. Examples of suitable or polyclonal immunoglobulin, or with other agents capable of specifically binding the receptor without activating the Such compositions include polyclonal and monoclonal anti receptor. Also, a soluble receptor or receptor component can bodies capable of binding ligand(s). Further examples be prepared. The inventors have prepared a soluble form of include soluble forms of other receptors capable of binding the mouse Robo-4 receptor, termed N-Robo-4 and listed the Slit ligand, such as other Robo receptors. herein as SEQID 5. The N-Robo-4 composition contains the 0056. The references cited in this disclosure are hereby ectodomain (extracellular), but lacks the transmembrane and incorporated into the disclosure in their entirety, except to any cytoplasmic domains of the cell-bound receptor. The amino extent to which they contradict any statement or definition acid sequence of the human N-Robo-4 composition is listed made herein. hereinas SEQID 6. These soluble compositions will bind the ligand(s) of the receptor, such as Slit, and prevent their bind 0057 The foregoing disclosure includes the best mode ing to and Subsequent activation of the cell-bound receptor. devised by the inventors for practicing the invention. It is These compositions may be engineered to include portions apparent, however, that several variations may be conceivable that enhance the effectiveness of the composition. For by one skilled in the art. Inasmuch as the foregoing disclosure example, an immunoglobulin Fc segment can be added to the is intended to enable Such person to practice the instant inven composition, which can facilitate removal of complexes of tion, it should not be construed to be limited thereby, but the composition and ligand through cells bearing Fc recep should be construed to include such aforementioned varia tors. Such as macrophages. tions.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 6

<21 Os SEQ ID NO 1 &211s LENGTH: 3742 &212s. TYPE: DNA <213> ORGANISM: Mus musculus

<4 OOs SEQUENCE: 1 caaaagtgta toggacaagg agaggagc.cg agagcagcca toggctctgg aggaacgggc 60 CtcCtgggga cqgagtggcc totgcct Ctg Ctgctgcttt to at catggg aggtgaggct 12O

ctggattctic caccc.ca.gat cotagttcac cc cc agg acc agctact tca gggctctggc 18O

Ccagccaaga tigaggtgcag at catc.cggc calaccacct C cc act atc.cg Ctggctgctg 24 O aatgggcagc cc ct cagcat ggccaccc.ca gacctacatt acct tttgcc ggatgggacc 3 OO

ctic ct gttac atcqgcc ct c tdtcc aggga cqgccacaag atgaccagaa catcct ct ca 360 gcaatcc tig gtgtctacac atgtgaggcc agcaa.ccggc tigggcacagc agtgagc.cgg 42O

ggtgctaggc tigtctgtggc tigtcCt c cag gaggact tcc agat.ccaac C. tcgggacaca 48O

gtggc.cgtgg togga.gagag cttggttctt gagtgtggit c ct Coctgggg ct acccaaaa 54 O

Ccct cqgtct catggtggala agacgggaaa cc cctggtcc to cagcCagg gaggcgcaca 6 OO gtatctgggg attcCctgat ggtgtcaaga gcagagalaga atgact cqgg gacctatatg 660

tgt atggc.ca ccaacaatgc tigggcaacgg gaga.gc.cgag cagc.cagggit gt Citat coag 72O gaatcCC agg accacaagga acatctagag cttctggctg. titcgcattca gctggaaaat 78O

gtgaccctgc taalaccc.cga acctgtaaaa ggtc.ccaagc ctgggc catc. c9tgtggctic 84 O

agctggalagg tagcggcc c tectgcacct gctgagt cat acacagctict gttcaggact 9 OO

cagaggit coc cc agggacca aggat ct coa tigacagagg togctgctgcg tdgCttgcag 96.O agtgcaaagc titgggggtct coactggggc caag act atgaattcaaagt gaga.ccgt.cc 102O

to cigc.cggg ct caggc.cc tacagcaat gtgttgctcc taggctgcc talacaggtg 108O

cc.cagtgc.cc cacct Caagg agtgacctta agatctggca acgg tagtgt Ctttgtgagt 114 O

tgggctic cac cacctgctga aagccataat ggtgtcatcc gtggittacca ggtctggagc 12 OO

US 2013/0143320 A1 Jun. 6, 2013 10

- Continued

Lieu. Lieu. Lieu. Lieu. Phe Ile Met Gly Gly Glu Ala Lieu. Asp Ser Pro Pro 2O 25 3O Glin Ile Lieu Val His Pro Glin Asp Gln Lieu. Lieu. Glin Gly Ser Gly Pro 35 4 O 45 Ala Lys Met Arg Cys Arg Ser Ser Gly Glin Pro Pro Pro Thr Ile Arg SO 55 6 O Trp Lieu. Lieu. Asn Gly Glin Pro Lieu. Ser Met Ala Thr Pro Asp Lieu. His 65 70 7s 8O Tyr Lieu. Lieu Pro Asp Gly Thr Lieu. Lieu. Lieu. His Arg Pro Ser Val Glin 85 90 95 Gly Arg Pro Glin Asp Asp Glin Asn. Ile Lieu. Ser Ala Ile Lieu. Gly Val 1OO 105 11 O Tyr Thr Cys Glu Ala Ser Asn Arg Lieu. Gly Thr Ala Val Ser Arg Gly 115 12 O 125 Ala Arg Lieu. Ser Val Ala Val Lieu. Glin Glu Asp Phe Glin Ile Glin Pro 13 O 135 14 O Arg Asp Thr Val Ala Val Val Gly Glu Ser Lieu Val Lieu. Glu. Cys Gly 145 150 155 160 Pro Pro Trp Gly Tyr Pro Llys Pro Ser Val Ser Trp Trp Lys Asp Gly 1.65 17O 17s Llys Pro Lieu Val Lieu. Glin Pro Gly Arg Arg Thr Val Ser Gly Asp Ser 18O 185 19 O Lieu Met Val Ser Arg Ala Glu Lys Asn Asp Ser Gly Thr Tyr Met Cys 195 2OO 2O5 Met Ala Thr Asn. Asn Ala Gly Glin Arg Glu Ser Arg Ala Ala Arg Val 21 O 215 22O Ser Ile Glin Glu Ser Glin Asp His Lys Glu. His Lieu. Glu Lieu. Lieu Ala 225 23 O 235 24 O Val Arg Ile Glin Lieu. Glu Asn Val Thir Lieu. Lieu. Asn Pro Glu Pro Val 245 250 255 Lys Gly Pro Llys Pro Gly Pro Ser Val Trp Leu Ser Trp Llys Val Ser 26 O 265 27 O Gly Pro Ala Ala Pro Ala Glu Ser Tyr Thr Ala Leu Phe Arg Thr Glin 27s 28O 285 Arg Ser Pro Arg Asp Glin Gly Ser Pro Trp Thr Glu Val Lieu. Lieu. Arg 29 O 295 3 OO Gly Lieu. Glin Ser Ala Lys Lieu. Gly Gly Lieu. His Trp Gly Glin Asp Tyr 3. OS 310 315 32O Glu Phe Llys Val Arg Pro Ser Ser Gly Arg Ala Arg Gly Pro Asp Ser 3.25 330 335 Asn Val Lieu. Lieu. Lieu. Arg Lieu Pro Glu Glin Val Pro Ser Ala Pro Pro 34 O 345 35. O Gln Gly Val Thr Lieu. Arg Ser Gly Asn Gly Ser Val Phe Val Ser Trp 355 360 365 Ala Pro Pro Pro Ala Glu Ser His Asn Gly Val Ile Arg Gly Tyr Glin 37 O 375 38O Val Trp Ser Lieu. Gly Asn Ala Ser Lieu Pro Ala Ala Asn Trp Thr Val 385 390 395 4 OO Val Gly Glu Gln Thr Glin Lieu. Glu Ile Ala Thr Arg Lieu Pro Gly Ser 4 OS 41O 415

Tyr Cys Val Glin Val Ala Ala Val Thr Gly Ala Gly Ala Gly Glu Lieu. US 2013/0143320 A1 Jun. 6, 2013 11

- Continued

42O 425 43 O Ser Thr Pro Val Cys Lieu. Lieu. Lieu. Glu Glin Ala Met Glu Glin Ser Ala 435 44 O 445 Arg Asp Pro Arg Llys His Val Pro Trp Thir Lieu. Glu Gln Lieu. Arg Ala 450 45.5 460 Thir Lieu. Arg Arg Pro Glu Val Ile Ala Ser Ser Ala Val Lieu. Lieu. Trp 465 470 47s 48O Lieu. Lieu. Lieu. Lieu. Gly Ile Thr Val Cys Ile Tyr Arg Arg Arg Lys Ala 485 490 495 Gly Val His Lieu. Gly Pro Gly Lieu. Tyr Arg Tyr Thr Ser Glu Asp Ala SOO 505 51O Ile Lieu Lys His Arg Met Asp His Ser Asp Ser Pro Trp Lieu Ala Asp 515 52O 525 Thir Trp Arg Ser Thr Ser Gly Ser Arg Asp Leu Ser Ser Ser Ser Ser 53 O 535 54 O Lieu. Ser Ser Arg Lieu. Gly Lieu. Asp Pro Arg Asp Pro Lieu. Glu Gly Arg 5.45 550 555 560 Arg Ser Lieu. Ile Ser Trp Asp Pro Arg Ser Pro Gly Val Pro Lieu. Lieu 565 st O sts Pro Asp Thr Ser Thr Phe Tyr Gly Ser Lieu. Ile Ala Glu Glin Pro Ser 58O 585 59 O Ser Pro Pro Val Arg Pro Ser Pro Llys Thr Pro Ala Ala Arg Arg Phe 595 6OO 605 Pro Ser Lys Lieu Ala Gly. Thir Ser Ser Pro Trp Ala Ser Ser Asp Ser 610 615 62O Lieu. Cys Ser Arg Arg Gly Lieu. Cys Ser Pro Arg Met Ser Lieu. Thr Pro 625 630 635 64 O Thr Glu Ala Trp Lys Ala Lys Llys Lys Glin Glu Lieu. His Glin Ala Asn 645 650 655 Ser Ser Pro Leu Lieu. Arg Gly Ser His Pro Met Glu Ile Trp Ala Trp 660 665 67 O Glu Lieu. Gly Ser Arg Ala Ser Lys Asn Lieu. Ser Glin Ser Pro Gly Pro 675 68O 685 Asn Ser Gly Ser Pro Gly Glu Ala Pro Arg Ala Val Val Ser Trp Arg 69 O. 695 7 OO Ala Val Gly Pro Gln Lieu. His Arg Asn. Ser Ser Glu Lieu Ala Ser Arg 7 Os 71O 71s 72O Pro Leu Pro Pro Thr Pro Leu Ser Lieu. Arg Gly Ala Ser Ser His Asp 72 73 O 73 Pro Glin Ser Glin Cys Val Glu Lys Lieu. Glin Ala Pro Ser Ser Asp Pro 740 74. 7 O Lieu Pro Ala Ala Pro Lieu. Ser Val Lieu. Asn. Ser Ser Arg Pro Ser Ser 7ss 760 765 Pro Glin Ala Ser Phe Leu Ser Cys Pro Ser Pro Ser Ser Ser Asn Lieu. 770 775 78O Ser Ser Ser Ser Lieu. Ser Ser Lieu. Glu Glu Glu Glu Asp Glin Asp Ser 78s 79 O 79. 8OO Val Lieu. Thr Pro Glu Glu Val Ala Lieu. Cys Lieu. Glu Lieu. Ser Asp Gly 805 810 815 Glu Glu Thr Pro Thr Asn Ser Val Ser Pro Met Pro Arg Ala Pro Ser 82O 825 83 O US 2013/0143320 A1 Jun. 6, 2013 12

- Continued Pro Pro Thr Thr Tyr Gly Tyr Ile Ser Ile Pro Thr Cys Ser Gly Lieu. 835 84 O 845 Ala Asp Met Gly Arg Ala Gly Gly Gly Val Gly Ser Glu Val Gly Asn 850 855 860 Lieu. Leu Tyr Pro Pro Arg Pro Cys Pro Thr Pro Thr Pro Ser Glu Gly 865 87O 87s 88O Ser Lieu Ala Asn Gly Trp Gly Ser Ala Ser Glu Asp Asn Val Pro Ser 885 890 895 Ala Arg Ala Ser Lieu Val Ser Ser Ser Asp Gly Ser Phe Lieu Ala Asp 9 OO 905 91 O Thir His Phe Ala Arg Ala Lieu Ala Val Ala Val Asp Ser Phe Gly Lieu. 915 92 O 925 Ser Lieu. Asp Pro Arg Glu Ala Asp Cys Val Phe Thr Asp Ala Ser Ser 93 O 935 94 O Pro Pro Ser Pro Arg Gly Asp Leu Ser Lieu. Thr Arg Ser Phe Ser Lieu. 945 950 955 96.O Pro Lieu. Trp Glu Trp Arg Pro Asp Trp Lieu. Glu Asp Ala Glu Ile Ser 965 97O 97. His Thr Glin Arg Lieu. Gly Arg Gly Lieu Pro Pro Trp Pro Pro Asp Ser 98O 985 99 O Arg Ala Ser Ser Glin Arg Ser Trp Lieu. Thr Gly Ala Val Pro Lys Ala 995 1OOO 1005 Gly Asp Ser Ser 1010

<210s, SEQ ID NO 4 &211s LENGTH: 1.OO7 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 4 Met Gly Ser Gly Gly Asp Ser Lieu. Lieu. Gly Gly Arg Gly Ser Lieu Pro 1. 5 1O 15 Lieu. Lieu. Lieu. Lieu. Lieu. Ile Met Gly Gly Met Ala Glin Asp Ser Pro Pro 2O 25 3O Glin Ile Leu Val His Pro Glin Asp Gln Leu Phe Glin Gly Pro Gly Pro 35 4 O 45 Ala Arg Met Ser Cys Glin Ala Ser Gly Glin Pro Pro Pro Thr Ile Arg SO 55 6 O Trp Leu Lieu. Asn Gly Glin Pro Leu Ser Met Val Pro Pro Asp Pro His 65 70 7s 8O His Lieu. Lieu Pro Asp Gly. Thir Lieu. Lieu. Lieu. Lieu. Glin Pro Pro Ala Arg 85 90 95 Gly His Ala His Asp Gly Glin Ala Lieu. Ser Thr Asp Lieu. Gly Val Tyr 1OO 105 11 O Thir Cys Glu Ala Ser Asn Arg Lieu. Gly. Thir Ala Val Ser Arg Gly Ala 115 12 O 125 Arg Lieu. Ser Val Ala Val Lieu. Arg Glu Asp Phe Glin Ile Glin Pro Arg 13 O 135 14 O Asp Met Val Ala Val Val Gly Glu Glin Phe Thr Lieu. Glu. Cys Gly Pro 145 150 155 160 Pro Trp Gly His Pro Glu Pro Thr Val Ser Trp Trp Lys Asp Gly Lys 1.65 17O 17s US 2013/0143320 A1 Jun. 6, 2013 13

- Continued Pro Lieu Ala Lieu. Glin Pro Gly Arg His Thr Val Ser Gly Gly Ser Lieu. 18O 185 19 O Lieu Met Ala Arg Ala Glu Lys Ser Asp Glu Gly Thr Tyr Met Cys Val 195 2OO 2O5 Ala Thr Asn. Ser Ala Gly His Arg Glu Ser Arg Ala Ala Arg Val Ser 21 O 215 22O Ile Glin Glu Pro Glin Asp Tyr Thr Glu Pro Val Glu Lieu. Leu Ala Val 225 23 O 235 24 O Arg Ile Glin Lieu. Glu Asn Val Thir Lieu. Lieu. Asn Pro Asp Pro Ala Glu 245 250 255 Gly Pro Llys Pro Arg Pro Ala Val Trp Leu Ser Trp Llys Val Ser Gly 26 O 265 27 O Pro Ala Ala Pro Ala Glin Ser Tyr Thr Ala Leu Phe Arg Thr Glin Thr 27s 28O 285 Ala Pro Gly Gly Glin Gly Ala Pro Trp Ala Glu Glu Lieu. Lieu Ala Gly 29 O 295 3 OO Trp Glin Ser Ala Glu Lieu. Gly Gly Lieu. His Trp Gly Glin Asp Tyr Glu 3. OS 310 315 32O Phe Llys Val Arg Pro Ser Ser Gly Arg Ala Arg Gly Pro Asp Ser Asn 3.25 330 335 Val Lieu. Lieu. Lieu. Arg Lieu Pro Glu Lys Val Pro Ser Ala Pro Pro Glin 34 O 345 35. O Glu Val Thr Lieu Lys Pro Gly Asn Gly Thr Val Phe Val Ser Trp Val 355 360 365 Pro Pro Pro Ala Glu Asn His Asn Gly Ile Ile Arg Gly Tyr Glin Val 37 O 375 38O Trp Ser Leu Gly Asn. Thir Ser Leu Pro Pro Ala Asn Trp Thr Val Val 385 390 395 4 OO Gly Glu Gln Thr Gln Lieu. Glu Ile Ala Thr His Met Pro Gly Ser Tyr 4 OS 41O 415 Cys Val Glin Val Ala Ala Val Thr Gly Ala Gly Ala Gly Glu Pro Ser 42O 425 43 O Arg Pro Val Cys Lieu. Lieu. Lieu. Glu Glin Ala Met Glu Arg Ala Thr Glin 435 44 O 445 Glu Pro Ser Glu. His Gly Pro Trp Thr Lieu. Glu Gln Lieu. Arg Ala Thr 450 45.5 460 Lieu Lys Arg Pro Glu Val Ile Ala Thr Cys Gly Val Ala Lieu. Trp Lieu. 465 470 47s 48O Lieu. Lieu. Lieu. Gly Thr Ala Val Cys Ile His Arg Arg Arg Arg Ala Arg 485 490 495 Val His Lieu. Gly Pro Gly Lieu. Tyr Arg Tyr Thr Ser Glu Asp Ala Ile SOO 505 51O Lieu Lys His Arg Met Asp His Ser Asp Ser Glin Trp Lieu Ala Asp Thr 515 52O 525 Trp Arg Ser Thir Ser Gly Ser Arg Asp Lieu. Ser Ser Ser Ser Ser Lieu. 53 O 535 54 O Ser Ser Arg Lieu. Gly Ala Asp Ala Arg Asp Pro Lieu. Asp Cys Arg Arg 5.45 550 555 560 Ser Lieu. Lieu. Ser Trp Asp Ser Arg Ser Pro Gly Val Pro Lieu. Lieu Pro 565 st O sts

Asp Thr Ser Thr Phe Tyr Gly Ser Lieu. Ile Ala Glu Lieu Pro Ser Ser 58O 585 59 O US 2013/0143320 A1 Jun. 6, 2013 14

- Continued

Thr Pro Ala Arg Pro Ser Pro Glin Val Pro Ala Val Arg Arg Lieu Pro 595 6OO 605 Pro Gln Leu Ala Gln Leu Ser Ser Pro Cys Ser Ser Ser Asp Ser Lieu. 610 615 62O Cys Ser Arg Arg Gly Lieu. Ser Ser Pro Arg Lieu. Ser Lieu Ala Pro Ala 625 630 635 64 O Glu Ala Trp Lys Ala Lys Llys Lys Glin Glu Lieu Gln His Ala Asn. Ser 645 650 655 Ser Pro Lieu. Lieu. Arg Gly Ser His Ser Lieu. Glu Lieu. Arg Ala Cys Glu 660 665 67 O Lieu. Gly Asn Arg Gly Ser Lys Asn Lieu. Ser Glin Ser Pro Gly Ala Val 675 68O 685 Pro Glin Ala Lieu Val Ala Trp Arg Ala Lieu. Gly Pro Llys Lieu. Lieu. Ser 69 O. 695 7 OO Ser Ser Asn Glu Lieu Val Thr Arg His Leu Pro Pro Ala Pro Leu Phe 7 Os 71O 71s 72O

Pro His Glu. Thr Pro Pro Thr Glin Ser Glin Glin Thr Glin Pro Pro Wall 72 73 O 73

Ala Pro Glin Ala Pro Ser Ser Ile Lleu Lleu Pro Ala Ala Pro Ile Pro 740 74. 7 O Ile Leu Ser Pro Cys Ser Pro Pro Ser Pro Glin Ala Ser Ser Leu Ser 7ss 760 765 Gly Pro Ser Pro Ala Ser Ser Arg Lieu Ser Ser Ser Ser Leu Ser Ser 770 775 78O Lieu. Gly Glu Asp Glin Asp Ser Val Lieu. Thr Pro Glu Glu Val Ala Lieu. 78s 79 O 79. 8OO Cys Lieu. Glu Lieu. Ser Glu Gly Glu Glu Thr Pro Arg Asn. Ser Val Ser 805 810 815 Pro Met Pro Arg Ala Pro Ser Pro Pro Thr Thr Tyr Gly Tyr Ile Ser 82O 825 83 O Val Pro Thr Ala Ser Glu Phe Thr Asp Met Gly Arg Thr Gly Gly Gly 835 84 O 845 Val Gly Pro Lys Gly Gly Val Lieu. Lieu. Cys Pro Pro Arg Pro Cys Lieu 850 855 860 Thr Pro Thr Pro Ser Glu Gly Ser Leu Ala Asn Gly Trp Gly Ser Ala 865 87O 87s 88O Ser Glu Asp Asn Ala Ala Ser Ala Arg Ala Ser Lieu Val Ser Ser Ser 885 890 895 Asp Gly Ser Phe Lieu Ala Asp Ala His Phe Ala Arg Ala Lieu Ala Val 9 OO 905 91 O Ala Val Asp Ser Phe Gly Phe Gly Lieu. Glu Pro Arg Glu Ala Asp Cys 915 92 O 925 Val Phe Ile Asp Ala Ser Ser Pro Pro Ser Pro Arg Asp Glu Ile Phe 93 O 935 94 O Lieu. Thr Pro Asn Lieu. Ser Lieu Pro Lieu. Trp Glu Trp Arg Pro Asp Trp 945 950 955 96.O Lieu. Glu Asp Met Glu Val Ser His Thr Glin Arg Lieu. Gly Arg Gly Met 965 97O 97. Pro Pro Trp Pro Pro Asp Ser Glin Ile Ser Ser Glin Arg Ser Gln Leu 98O 985 99 O His Cys Arg Met Pro Lys Ala Gly Ala Ser Pro Val Asp Tyr Ser US 2013/0143320 A1 Jun. 6, 2013 15

- Continued

995 1OOO 1005

<210s, SEQ ID NO 5 &211s LENGTH: 470 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 5 Met Gly Ser Gly Gly Thr Gly Lieu. Leu Gly Thr Glu Trp Pro Leu Pro 1. 5 1O 15 Lieu. Lieu. Lieu. Lieu. Phe Ile Met Gly Gly Glu Ala Lieu. Asp Ser Pro Pro 2O 25 3O Glin Ile Lieu Val His Pro Glin Asp Gln Lieu. Lieu. Glin Gly Ser Gly Pro 35 4 O 45 Ala Lys Met Arg Cys Arg Ser Ser Gly Glin Pro Pro Pro Thr Ile Arg SO 55 6 O Trp Lieu. Lieu. Asn Gly Glin Pro Lieu. Ser Met Ala Thr Pro Asp Lieu. His 65 70 7s 8O Tyr Lieu. Lieu Pro Asp Gly Thr Lieu. Lieu. Lieu. His Arg Pro Ser Val Glin 85 90 95 Gly Arg Pro Glin Asp Asp Glin Asn. Ile Lieu. Ser Ala Ile Lieu. Gly Val 1OO 105 11 O Tyr Thr Cys Glu Ala Ser Asn Arg Lieu. Gly Thr Ala Val Ser Arg Gly 115 12 O 125 Ala Arg Lieu. Ser Val Ala Val Lieu. Glin Glu Asp Phe Glin Ile Glin Pro 13 O 135 14 O Arg Asp Thr Val Ala Val Val Gly Glu Ser Lieu Val Lieu. Glu. Cys Gly 145 150 155 160 Pro Pro Trp Gly Tyr Pro Llys Pro Ser Val Ser Trp Trp Lys Asp Gly 1.65 17O 17s Llys Pro Lieu Val Lieu. Glin Pro Gly Arg Arg Thr Val Ser Gly Asp Ser 18O 185 19 O Lieu Met Val Ser Arg Ala Glu Lys Asn Asp Ser Gly Thr Tyr Met Cys 195 2OO 2O5 Met Ala Thr Asn. Asn Ala Gly Glin Arg Glu Ser Arg Ala Ala Arg Val 21 O 215 22O Ser Ile Glin Glu Ser Glin Asp His Lys Glu. His Lieu. Glu Lieu. Lieu Ala 225 23 O 235 24 O Val Arg Ile Glin Lieu. Glu Asn Val Thir Lieu. Lieu. Asn Pro Glu Pro Val 245 250 255 Lys Gly Pro Llys Pro Gly Pro Ser Val Trp Leu Ser Trp Llys Val Ser 26 O 265 27 O Gly Pro Ala Ala Pro Ala Glu Ser Tyr Thr Ala Leu Phe Arg Thr Glin 27s 28O 285 Arg Ser Pro Arg Asp Glin Gly Ser Pro Trp Thr Glu Val Lieu. Lieu. Arg 29 O 295 3 OO Gly Lieu. Glin Ser Ala Lys Lieu. Gly Gly Lieu. His Trp Gly Glin Asp Tyr 3. OS 310 315 32O Glu Phe Llys Val Arg Pro Ser Ser Gly Arg Ala Arg Gly Pro Asp Ser 3.25 330 335 Asn Val Lieu. Lieu. Lieu. Arg Lieu Pro Glu Glin Val Pro Ser Ala Pro Pro 34 O 345 35. O

Gln Gly Val Thr Lieu. Arg Ser Gly Asn Gly Ser Val Phe Val Ser Trp US 2013/0143320 A1 Jun. 6, 2013 16

- Continued

355 360 365 Ala Pro Pro Pro Ala Glu Ser His Asn Gly Val Ile Arg Gly Tyr Glin 37 O 375 38O Val Trp Ser Lieu. Gly Asn Ala Ser Lieu Pro Ala Ala Asn Trp Thr Val 385 390 395 4 OO Val Gly Glu Gln Thr Glin Lieu. Glu Ile Ala Thr Arg Lieu Pro Gly Ser 4 OS 41O 415 Tyr Cys Val Glin Val Ala Ala Val Thr Gly Ala Gly Ala Gly Glu Lieu. 42O 425 43 O Ser Thr Pro Val Cys Lieu. Lieu. Lieu. Glu Glin Ala Met Glu Glin Ser Ala 435 44 O 445 Arg Asp Pro Arg Llys His Val Pro Trp Thir Lieu. Glu Gln Lieu. Arg Ala 450 45.5 460 Thir Lieu. Arg Arg Pro Glu 465 470

<210s, SEQ ID NO 6 &211s LENGTH: 469 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 6 Met Gly Ser Gly Gly Asp Ser Lieu. Lieu. Gly Gly Arg Gly Ser Lieu Pro 1. 5 1O 15 Lieu. Lieu. Lieu. Lieu. Lieu. Ile Met Gly Gly Met Ala Glin Asp Ser Pro Pro 2O 25 3O Glin Ile Leu Val His Pro Glin Asp Gln Leu Phe Glin Gly Pro Gly Pro 35 4 O 45 Ala Arg Met Ser Cys Glin Ala Ser Gly Glin Pro Pro Pro Thr Ile Arg SO 55 6 O Trp Leu Lieu. Asn Gly Glin Pro Leu Ser Met Val Pro Pro Asp Pro His 65 70 7s 8O His Lieu. Lieu Pro Asp Gly. Thir Lieu. Lieu. Lieu. Lieu. Glin Pro Pro Ala Arg 85 90 95 Gly His Ala His Asp Gly Glin Ala Lieu. Ser Thr Asp Lieu. Gly Val Tyr 1OO 105 11 O Thir Cys Glu Ala Ser Asn Arg Lieu. Gly. Thir Ala Val Ser Arg Gly Ala 115 12 O 125 Arg Lieu. Ser Val Ala Val Lieu. Arg Glu Asp Phe Glin Ile Glin Pro Arg 13 O 135 14 O Asp Met Val Ala Val Val Gly Glu Glin Phe Thr Lieu. Glu. Cys Gly Pro 145 150 155 160 Pro Trp Gly His Pro Glu Pro Thr Val Ser Trp Trp Lys Asp Gly Lys 1.65 17O 17s Pro Lieu Ala Lieu. Glin Pro Gly Arg His Thr Val Ser Gly Gly Ser Lieu. 18O 185 19 O Lieu Met Ala Arg Ala Glu Lys Ser Asp Glu Gly Thr Tyr Met Cys Val 195 2OO 2O5 Ala Thr Asn. Ser Ala Gly His Arg Glu Ser Arg Ala Ala Arg Val Ser 21 O 215 22O Ile Glin Glu Pro Glin Asp Tyr Thr Glu Pro Val Glu Lieu. Leu Ala Val 225 23 O 235 24 O

Arg Ile Glin Lieu. Glu Asn Val Thir Lieu. Lieu. Asn Pro Asp Pro Ala Glu US 2013/0143320 A1 Jun. 6, 2013 17

- Continued

245 250 255

Gly Pro Pro Arg Pro Ala Wall Trp Luell Ser Trp Wall Ser Gly 26 O 265 27 O

Pro Ala Ala Pro Ala Glin Ser Tyr Thr Ala Leu Phe Arg Thir Gn. Thir 285

Ala Pro Gly Gly Glin Gly Ala Pro Trp Ala Glu Glu Lell Luell Ala Gly 29 O 295 3 OO

Trp Glin Ser Ala Glu Lieu. Gly Gly Lieu. His Trp Gly Glin Asp Glu 3. OS 310 315 32O

Phe Wall Arg Pro Ser Ser Gly Arg Ala Arg Gly Pro Asp Ser Asn 3.25 330 335

Wall Lieu. Luell Luell Arg Lieu Pro Glu Lys Wall Pro Ser Ala Pro Pro Glin 34 O 345 35. O

Glu Wall. Thir Luell Pro Gly Asn Gly Thir Wall Phe Wall Ser Trp Val 355 360 365

Pro Pro Pro Ala Glu Asn His Asn Gly Ile Ile Arg Gly Tyr Glin Wall 37 O 375

Trp Ser Lieu. Gly Asn Thir Ser Lieu. Pro Pro Ala Asn Trp Thir Wall Wall 385 390 395 4 OO

Gly Glu Glin. Thir Glin Lell Glu Ile Ala Thr His Met Pro Gly Ser Tyr 4 OS 415

Wall Glin Wall Ala Ala Val Thr Gly Ala Gly Ala Gly Glu Pro Ser 425 43 O

Arg Pro Wall Cys Lieu Lleu Lieu. Glu Glin Ala Met Glu Arg Ala Thr Gin 435 44 O 445

Glu Pro Ser Glu His Gly Pro Trp Thir Luell Glu Glin Lell Arg Ala Thr 450 45.5 460

Lell Lys Arg Pro Glu 465

1. An isolated polynucleotide comprising SEQID 1. 13. A method of disrupting navigation of physiological 2. An isolated polynucleotide comprising SEQID 2. tracking tubular structures that express Robo-4 receptor, 3. An isolated polypeptide comprising SEQID 3. comprising inhibiting activation of said Robo-4 receptor. 14. The method of claim 13, wherein said physiological 4. An isolated polypeptide comprising SEQID 4. tracking tubular structures comprise endothelial tubes. 5. An isolated polypeptide comprising SEQID 5. 15. A method of inducing angiogenesis in endothelium 6. An isolated polypeptide comprising SEQID 6. tissue expressing Robo-4 receptor, comprising inhibiting 7. A method of directing the navigation of physiological activation of said Robo-4 receptor. tracking tubular structures that express Robo-4 receptor away 16. The method of claim 15, wherein inhibiting activation from a target cell mass, comprising expressing a ligand of said of said Robo-4 receptor comprises providing a soluble form Robo-4 receptor in said target cell mass and allowing binding of said Robo-4 receptor to said endothelium tissue. between the ligand and said Robo-4 receptor. 17. The method of claim 16, wherein the soluble form of 8. The method of claim 7, wherein the ligand comprises Slit said Robo-4 receptor comprises SEQID 6. ligand. 18. The method of claim 16, wherein the soluble form of said Robo-4 receptor comprises an amino acid sequence hav 9. The method of claim 7, wherein said physiological track ing at least 80% sequence identity to SEQID 6, or a fragment ing tubular structures comprise endothelial tubes. thereof. 10. A method of directing the navigation of physiological 19. A method of preventing angiogenesis in endothelium tracking tubular structures that express Robo-4 receptor tissue expressing Robo-4 receptor, comprising activating said toward a target cell mass, comprising expressing a ligand of Robo-4 receptor. said Robo-4 receptor in a second cell mass and allowing 20. The method of claim 19, wherein activating said binding between the ligand and said Robo-4 receptor. Robo-4 receptor comprises providing a ligand of said Robo-4 11. The method of claim 10, wherein the ligand comprises receptor and allowing the ligand to bind to said Robo-4 recep Slit ligand tOr. 12. The method of claim 10, wherein said physiological 21. The method of claim 20, wherein the ligand comprises tracking tubular structures comprise endothelial tubes. Slit ligand. US 2013/0143320 A1 Jun. 6, 2013 18

22. The method according to any of claim 7, 10 and 20, wherein the ligand comprises human Slit2 ligand, or a frag ment thereof.