MXPA06000653A - Compositions and methods relating to multimeric and oligomeric soluble fragments of the tweak receptor - Google Patents

Abstract

The present invention provides methods and compositions relating to fusion proteins comprising multimeric soluble TWEAK receptor fragments and an oligomerization domain. Such fusion proteins are useful for antagonizing the TWEAK receptor and for treating diseases or conditions mediated by angiogenesis, such as solid tumors and inflammatory conditions.

Description

COMPOSITION IS AND M ETHODS THAT REFER TO SOLUTIONS MULTIMERICQS AND OLIGOMERICOS OF THE RECEIVER OF TWEAK This application claims the benefit of US Provisional Application 60 / 490,036, filed on July 24, 2003, the description of which is incorporated by reference.

BACKGROUND OF THE INVENTION Angiogenesis is a multistep development process that results in the formation of new blood vessels outside existing vessels. This spatially and temporally regulated process involves loosening matrix contacts and supporting cellular interactions in existing vessels by proteases, followed by coordinated movement, morphological alteration and proliferation of endothelial cells and smooth muscle of the existing vessel. The nascent cells then spread to the target tissue followed by cell-cell interactions in which the endothelial cells form tubes surrounding the soft muscle cells. In a coordinated fashion, the extracellular matrix proteins of the vessel are secreted, the peri-endothelial support cells are recruited to support and maintain the structural integrity (see, for example, Daniel et al., Ann. Rev. Physiol. 2000 (62 ): 649, 2000). Angiogenesis plays important roles in both normal and pathological physiology. Under normal physiological conditions, angiogenesis is involved in fetal and embryonic development, wound healing, organ regeneration and female reproductive remodeling processes, including the formation of the endometrium, corpus luteum and placenta. Angiogenesis is regulated severely under normal conditions, especially in adult animals, and disturbance of regulatory controls can lead to pathological angiogenesis. Pathological angiogenesis has been implicated in the manifestation and / or progression of inflammatory diseases, certain eye disorders and cancer. In particular, several lines of evidence support the concept that angiogenesis is essential for the growth and persistence of solid tumors and their metastases see, for example, Folkman, N. Engl. J. Med. 285: 1 1 82, 1 971; Folkman et al. , Nature 339: 58, 1989; Kim et al. , Nature 362: 841, 1 993; Hori et al. , Cancer Res., 51: 61 80, 1 991). Inhibitors of angiogenesis are therefore useful for the prevention (eg, treatment of premalignant conditions), intervention (eg, treatment of small tumors) and regression (eg, treatment of large tumors) of cancer (see , for example, Bergers et al., Science 284: 808, 1999). There is a need for additional compositions and methods to modulate angiogenesis for the prevention, abrogation and mitigation of disease. The TWEAK protein, which has also been called TREPA and Apo3L, is a member of the tumor necrosis factor (TNF) family and is expressed in a wide variety of human tissues (Chicheportiche et al., J. Biol. Chem. ., 272 (51): 3240, 1997, see also Wiley, PCT publication No. WO 98/35061, August 1, 1998). Like most members of the TNF family, TWEAK is a type I membrane protein with an extracellular C-terminal domain. Although TWEAK was originally described as a weak inducer of apoptosis, this induction of cell death subsequently proved to be indirect (Schneider et al., Eur. J. I m munol., 29: 1 785, 1999). Lynch et al. , demonstrated that TWEAK directly induces endovenous cell proliferation and angiogenesis (J. Biol. Chem., 274 (1 3): 8455, 1999). Picomolar concentrations of recombinant soluble TWEAK induce proliferation in multiple cell lines, endothelial cells and soft aortic muscle cells, and reduce the requirement for growth factors and serum in culture. Furthermore, TWEAK induces a strong angiogenic response in a rat corneal pocket trial. Because members of the TNF family initiate biological responses through signaling through members of the TNF receptor family, there has been great interest in identifying and characterizing the TWEAK receiver. Marsters et al. reported that TWEAK binds to and signals through a death domain containing receptor variously known as DR3, Apo3, WSL-1, TRAMP or LARD (Marsters et al., Current Biology 8 (9): 525, 1998) . However, Schneider et al. , showed that TWEAK binds to and signals in Kym-1 cells but that Kym-1 cells do not exude the DR3 receptor (Schneider et al., Eur. J., Immunol., 29: 1 785, 1999). Wiley subsequently identified the primary TWEAK receptor and described certain soluble fragments and variants of it that antagonize the native Nike TWEAK receptor (Pub. PCT No. WO 01/45730). Because TWEAK induces angiogenesis in vivo, there is a particular need for antagonists of the main functional TWEAK receptor. Such TWEAK receptor antagonists would be useful for reducing angiogenesis and treating human diseases, including cancers and inflammatory diseases.

BRIEF DESCRIPTION OF THE INVENTION The present invention is based on the identification and characterization of polypeptides comprising soluble multimeric fragments of the functional TWEAK receptor (TWEAKR) and an oligomerization domain. Surprisingly, these polypeptides have a higher binding affinity for TWEAK and / or are better competitors for TWEAK binding than would be expected from the binding of TWEAK and competition properties of polypeptides comprising monomeric TWEAKR fragments and an oligomerization domain. or comprising multimeric TWEAKR fragments without an oligomerization domain. The invention provides, for example, compositions and methods for inhibiting angiogenesis in a mammal in need of such a treatment, which comprises administering a therapeutically effective amount of a composition comprising a receptor antagonist.

TWEAK The composition preferably comprises a pharmaceutically acceptable carrier and the mammal is preferably a human.

In one aspect, the present invention provides a polypeptide comprising a first soluble fragment of a TWEAK receptor, a second soluble fragment of a TWEAK receptor, and an oligomerization domain, wherein said polypeptide is linked to TWEAK, and said first Soluble fragment consists of a sequence that is at least 90% identical to a sequence selected from the group consisting of: residues 29 to 70 of the amino acid sequence of S EQ ID NO: 7; residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. In one embodiment, said first fragment consists of a sequence that is at least 95% identical to a sequence selected from the group consisting of: residues 29 to 70 of the amino acid sequence of SEQ I D NO: 7; residues 28 to 79 of the amino acid sequence of SEQ ID NO: 7; residues 30 to 73 of the amino acid sequence of SEQ ID NO: 7; and residues 30 to 70 of the amino acid sequence of SEQ ID NO: 7. In another embodiment, said first soluble fragment consists of a sequence selected from the group consisting of: residues 29 to 70 of the amino acid sequence of SEQ I D NO: 7; residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. In another embodiment, said first soluble fragment and said second soluble fragment each independently consists of a sequence that is at least 90% identical to a sequence selected from the group consisting of: residues 29 to 70 of the amino acid sequence of SEQ ID NO. : 7; residues 28 to 79 of the amino acid sequence of SEQ ID NO: 7; residues 30 to 73 of the amino acid sequence of SEQ ID NO: 7; and residues 30 to 70 of the amino acid sequence of SEQ ID NO: 7. In another embodiment, said first soluble fragment and said second soluble fragment each independently consists of a sequence that is at least 90% identical to a sequence selected from the group consisting of: residues 29 to 70 of the amino acid sequence of SEQ ID NO. : 7; residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. In another embodiment, said first soluble fragment and said second soluble fragment each consist independently of a sequence selected from the group consisting of: residues 29 to 70 of the amino acid sequence of SEQ I D NO: 7; residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. In another embodiment, said polypeptide comprises a third soluble fragment of a TWEAK receptor. In another embodiment, said first soluble fragment, second soluble fragment and third soluble fragment each consist independently of a sequence that is at least 90% identical to a sequence selected from the group consisting of: residues 29 to 70 of the amino acid sequence of SEQ ID NO: 7; residues 28 to 79 of the amino acid sequence of SEQ ID NO: 7; residues 30 to 73 of the amino acid sequence of S EQ I D NO: 7; and residues 30 to 70 of the amino acid sequence of SEQ ID NO: 7. In another embodiment, said first soluble fragment, second soluble fragment and third soluble fragment each consist independently of a sequence that is at least 95% identical to a sequence selected from the group consisting of: residues 29 to 70 of the amino acid sequence of SEQ ID NO: 7; residues 28 to 79 of the amino acid sequence of SEQ ID NO: 7; residues 30 to 73 of the amino acid sequence of SEQ ID NO: 7; and residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. In another embodiment, said first soluble fragment, second soluble fragment and third soluble fragment each independently of a sequence selected from the group consisting of: residues 29 to 70 of the amino acid sequence of SEQ I D NO: 7; residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; residues 30 to 73 of the amino acid sequence of SEQ ID NO: 7; and residues 30 to 70 of the amino acid sequence of S EQ I D NO: 7. In another embodiment, said polypeptide comprises a fourth soluble fragment of a TWEAK receptor. In another embodiment, said first soluble fragment, second soluble fragment, third soluble fragment and fourth soluble fragment each consist independently of a sequence that is at least 90% identical to a sequence selected from the group consisting of: residues 29 to 70 of the amino acid sequence of SEQ ID NO: 7; residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. In another embodiment, said first soluble fragment, second soluble fragment, third soluble fragment and fourth soluble fragment each consist independently of a sequence that is at least 95% identical to a sequence selected from the group consisting of: residues 29 to 70 of the amino acid sequence of SEQ ID NO: 7; residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; residues 30 to 73 of the amino acid sequence of S EQ I D NO: 7; and residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. In another embodiment, said first soluble fragment, second soluble fragment, third soluble fragment and fourth soluble fragment each consist independently of a sequence selected from the group consisting of: residues 29 to 70 of the amino acid sequence of SEQ ID NO. 7; residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. In another embodiment, said polypeptide comprises a fifth soluble fragment of a TWEAK receptor. In another embodiment, said first soluble fragment, second soluble fragment, third soluble fragment, fourth soluble fragment and fifth soluble fragment, each independently of a sequence that is at least 90% identical to a sequence selected from the group consisting of: residues 29 to 70 of the amino acid sequence of SEQ ID NO: 7; residues 28 to 79 of the amino acid sequence of SEQ ID NO: 7; residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. In another embodiment, said first soluble fragment, second soluble fragment, third soluble fragment, fourth soluble fragment and fifth soluble fragment, each independently of a sequence that is at least 95% identical to a sequence selected from the group consisting of: residues 29 to 70 of the amino acid sequence of SEQ ID NO: 7; residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. In another embodiment, said first soluble fragment, second soluble fragment, third soluble fragment, fourth soluble fragment and fifth soluble fragment, each independently of a sequence selected from the group consisting of: residues 29 to 70 of the sequence of amino acids of SEQ ID NO: 7; residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; residues 30 to 73 of the amino acid sequence of SEQ ID NO: 7; and residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. In another embodiment, said polypeptide comprises a sixth soluble fragment of a TWEAK receptor. In another embodiment, said first soluble fragment, second soluble fragment, third soluble fragment, fourth soluble fragment, fifth soluble fragment and sixth soluble fragment, each consists independently of a sequence that is at least 90% identical to a sequence selected from the group consists of: residues 29 to 70 of the amino acid sequence of SEQ ID NO: 7; residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. In another embodiment, said first soluble fragment, according to a soluble fragment, a third soluble fragment, a fourth soluble fragment, a fifth soluble fragment and a sixth soluble fragment, each independently of a sequence that is at least 95% identical to a sequence selected from the group. which consists of: residues 29 to 70 of the amino acid sequence of SEQ ID NO: 7; residues 28 to 79 of the amino acid sequence of SEQ ID NO: 7; residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and residues 30 to 70 of the amino acid sequence of S EQ I D NO: 7. In another embodiment, said first soluble fragment, second soluble fragment, third soluble fragment, fourth soluble fragment, fifth soluble fragment and sixth soluble fragment, each consists independently of a sequence selected from the group consisting of: residues 29 to 70 of the sequence of amino acids of SEQ ID NO: 7; residues 28 to 79 of the amino acid sequence of SEQ ID NO: 7; residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. In another embodiment, said polypeptide comprises a seventh soluble fragment of a TWEAK receptor. In another embodiment, said first soluble fragment, second soluble fragment, third soluble fragment, fourth soluble fragment, fifth soluble fragment, sixth soluble fragment and seventh soluble fragment, each consisting independently of a sequence that is at least 90% identical to a sequence selected from the group consisting of: residues 29 to 70 of the amino acid sequence of SEQ ID NO: 7; residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. In another embodiment, said first soluble fragment, second soluble fragment, third soluble fragment, fourth soluble fragment, fifth soluble fragment, sixth soluble fragment and seventh soluble fragment, each independently of a sequence that is at least 95% identical to a sequence selected from the group consisting of: residues 29 to 70 of the amino acid sequence of SEQ ID NO: 7; residues 28 to 79 of the amino acid sequence of SEQ ID NO: 7; residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. In another embodiment, said first soluble fragment, second soluble fragment, third soluble fragment, fourth soluble fragment, fifth soluble fragment, sixth soluble fragment and seventh soluble fragment, each independently of a sequence selected from the group consisting of: residues 29 a 70 of the amino acid sequence of SEQ ID NO: 7; residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. In another embodiment, said polypeptide comprises a linker. In another embodiment, said linker binds said first fragment of soluble TEWAKR and said second fragment of soluble TWEAKR. In another modalitysaid linker binds to said second fragment of soluble TWEAKR and said oligomerization domain. In another embodiment, said polypeptide comprises a first linker and a second linker, wherein said first linker binds to said first soluble TWEAKR fragment and said second soluble TWEAKR fragment, and said second linker binds to said second soluble TWEAKR fragment. and said oligomerization domain. In another embodiment, said linker comprises the amino acid sequence GGGGG (SEQ I D NO: 45). In another embodiment, said first soluble fragment and said second soluble fragment are joined together without a intervening polypeptide sequence. In another embodiment, said first soluble fragment and said oligomerization domain are joined together without a intervening polypeptide sequence. In another embodiment, said first soluble fragment, said second soluble fragment and said oligomerization domain are joined together, without a intervening polypeptide sequence, in a linear and contiguous polypeptide. In another embodiment, said oligomerization domain is N-terminal to said first soluble TWEAKR fragment and said second soluble TWEAKR fragment. In another embodiment, said oligomerization domain is C-terminal to said first soluble TWEAKR fragment and said second soluble TWEAKR fragment. In another embodiment, said oligomerization domain comprises a leucine lock. In another embodiment, said oligomerization domain comprises a fragment of an antibody. In another embodiment, said fragment of an antibody comprises an Fc domain. In another embodiment, said polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of: SEQ ID NO: 9; SEQ ID NO: 1 1; SEQ ID NO: 13; SEQ ID NO: 15; SEQ I D NO: 1 7; SEQ I D NO: 1 8; SEQ I D NO: 19; SEQ I D NO: 21; SEQ ID NO: 23; SEQ I D NO: 25; SEQ I D NO: 27; SEQ I D NO: 29; SEQ I D NO: 31; SEQ ID NO: 33; SEQ ID NO: 35; SEQ ID NO: 37; SEQ ID NO: 39; SEQ ID NO: 41; SEQ ID NO: 43; and SEQ ID NO: 44. In another embodiment, said polypeptide comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of: SEQ ID NO: 9; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 21; SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO: 27; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 33; SEQ ID NO: 35; SEQ ID NO: 37; SEQ ID NO: 39; SEQ ID NO: 41; SEQ ID NO: 43; and SEQ ID NO: 44. In another embodiment, said polypeptide comprises a sequence selected from the group consisting of: SEQ ID NO: 9; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 21; SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO: 27; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 33; SEQ ID NO: 35; SEQ ID NO: 37; SEQ ID NO: 39; SEQ ID NO: 41; SEQ ID NO: 43; and SEQ ID NO: 44. In another aspect, the present invention provides a protein comprising said first polypeptide and said second polypeptide, wherein said first and second polypeptides are oligomerized to each other. In another embodiment, the amino acid sequence of said first polypeptide is identical to the amino acid sequence of said second polypeptide. In another embodiment, the amino acid sequence of said first polypeptide is not identical to the amino acid sequence of said second polypeptide. In another aspect, the present invention provides a method for inhibiting a TWEAK receptor in a subject comprising administering said polypeptide to said subject.

In another aspect, the present invention provides a method for inhibiting angiogenesis in a subject comprising administering to said subject a therapeutically effective amount of a composition comprising said polypeptide. In another embodiment, said composition further comprises a pharmaceutically acceptable carrier. In another embodiment, said subject is a mammal. In another embodiment, said mammal is a human. In another embodiment, said subject has a disease or condition mediated or exacerbated by angiogenesis. In another embodiment, said disease or condition is characterized by ocular neovascularization. In another embodiment, said disease or condition is a solid tumor. In another embodiment, said method further comprises treating said subject with radiation. In another embodiment, said method further comprises treating said subject with a second chemotherapeutic agent. In another embodiment, said second chemotherapeutic agent is selected from the group consisting of: an alkylating agent, an antimetabolite, a vinca alkaloid, a plant-derived chemotherapeutic, a nitrosourea, an antitumor antibiotic, an antitumor enzyme, a topoisomerase inhibitor. , a platinum analogue, an adrenocorticoid suppressant, a hormone, a hormone agonist, a hormone antagonist, an antibody, an immunotherapeutic, a blood cell factor, a radiotherapeutic and a biological response modifier. In another embodiment, said second chemotherapeutic agent is selected from the group consisting of cisplatin, cyclophosphamide, mechlorethamine, melphalan, bleomycin, carboplatin, fluorouracil, 5-fluorodeoxyuridine, methotrexate, taxol, asparaginase, vincristine, vinblastine, a lymphokine, a cytokine, a interleukin, an interferon, alpha interferon, beta interferon, delta interferon, TNF, chlorambucil, busulfan, carmustine, lomustine, semustine, streptozocin, dacarbazine, cytarabine, mercaptopurine, thioguanine, vindesine, etoposide, terniposide, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, L-asparaginase, hydroxyurea, methylhydrazine, mitotane, tamoxifen, and fluoxymesterone. In another embodiment, said disease or condition is an inflammatory disease or condition. In another embodiment, said method further comprises treating said subject with a second therapeutic agent. In another embodiment, said second therapeutic agent inhibits a cytokine or cytokine receptor that promotes inflammation. In another embodiment, said second therapeutic agent comprises a soluble fragment of said cytokine receptor, an antibody that binds to said cytokine or an antibody that binds to said cytokine receptor. In another embodiment, said second therapeutic agent activates a receptor that inhibits inflammation. In another embodiment, said second therapeutic agent activates a receptor that inhibits inflammation. In another embodiment, said second therapeutic agent is selected from the group consisting of ligand Flt3, ligand CD40, interleukin-2, an antagonist of interleukin-4, an antagonist of 11-1 3, interleukin 12, ligand 4-1 BB, an anti-4-1 BB antibody, a TNF antagonist, a TNF receptor antagonist, TRAI L, a CD148 agonist, a VEGF antagonist, a VEGF receptor antagonist, an IgE antagonist and a Tek antagonist. In another aspect, the present invention provides a nucleic acid, or its complement, comprising a sequence encoding said polypeptide. In another embodiment, said nucleic acid, or its complement, hybrid under conditions of moderately severe hybridization to a second nucleic acid, and said second nucleic acid comprises a sequence selected from the group consisting of: SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 24; SEQ ID NO: 26; SEQ ID NO: 28; SEQ ID NO: 30; SEQ ID NO: 32; SEQ ID NO: 34; SEQ ID NO: 36; SEQ ID NO: 38; SEQ ID NO: 40; and SEQ ID NO: 42. In another embodiment, said nucleic acid, or its complement, comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of: SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 24; SEQ ID NO: 26; SEQ ID NO: 28; SEQ ID NO: 30; SEQ ID NO: 32; SEQ ID NO: 34; SEQ ID NO: 36; SEQ ID NO: 38; SEQ ID NO: 40; and SEQ ID NO: 42. In another embodiment, said nucleic acid, or its complement, comprises a sequence that is at least 95% identical to a sequence selected from! group consisting of: SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 24; SEQ ID NO: 26; SEQ ID NO: 28; SEQ ID NO: 30; SEQ ID NO: 32; SEQ ID NO: 34; -SEQ ID NO: 36; SEQ ID NO: 38; SEQ ID NO: 40; and SEQ ID NO: 42. In another embodiment, said nucleic acid, or its complement, comprises a sequence selected from the group consisting of: SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 24; SEQ ID NO: 26; SEQ ID NO: 28; SEQ ID NO: 30; SEQ ID NO: 32; SEQ ID NO: 34; SEQ ID NO: 36; SEQ ID NO: 38; SEQ ID NO: 40; and SEQ ID NO: 42. In another embodiment, said nucleic acid encodes a polypeptide sequence selected from the group consisting of: SEQ ID NO: 9; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 21; SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO: 27; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 33; SEQ ID NO: 35; SEQ ID NO: 37; SEQ ID NO: 39; SEQ ID NO: 41; SEQ ID NO: 43; and SEQ ID NO: 44. In another aspect, the present invention provides a vector comprising said nucleic acid. In another embodiment, said vector is an expression vector. In another aspect, the present invention provides a host cell comprising said nucleic acid. In another aspect, the present invention provides a method for producing a polypeptide comprising culturing said host cell under conditions that promote the expression of said polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a sequence alignment of the human and murine TWEAK receptor polypeptide sequences. The top sequence is the murine TWEAK receptor polypeptide (SEQ ID NO: 5), and the lower sequence is the human TWEAK receptor polypeptide.

(SEQ ID NO: 4). Figure 2 shows the effect of TWEAKR-Fc on wound closure of HRMEC induced by PMA. Figure 3 shows the effect of TWEAKR-Fc on wound closure of HRMEC induced by EGF.

Figure 4 shows the effect of human TWEAKR-Fc on proliferation of HUVEC induced by TWEAK (100 ng / ml). Figure 5 shows the effect of human TWEAKR-Fc on proliferation of HUVEC induced by FGF-2 (10 ng / ml). Figure 6 shows the binding of TWEAKR: Fc (SEQ ID NO: 7; "monomer"), TWEAKR: DR5: Fc (SEQ ID NO: 9, "DR5"), di-TWEAKR: Fc (SEQ ID NO: 11, "dimer"), and tri-TWEAKR: Fc (SEQ ID NO: 13, "trimer") to TWEAK-FLAG in ELISA binding assay. Figure 7 shows the capacity of TWEAKR: Fc (SEQ ID NO: 7; "hu + eu"), TWEAKR: DR5: Fc (SEQ ID NO: 9, "DR5 + eu"), di-TWEAKR: Fc (SEQ ID NO: 11, "di + eu"), and tri-TWEAKR: Fc (SEQ ID NO: 13, "tri + eu") to compete with TWEAKR labeled with europium to bind to TWEAK in a binding assay of competition. Figure 8 shows the binding of TWEAKR: Gly5: Fc (SEQ ID NO: 15, black circles), TWEAKR: 1 KPEG: Fc (SEQ ID NO: 17, asterisks), TWEAKR: 1KPEG: TWEAKR: Gly5: Fc (SEQ ID NO: 18, black triangles), and TWEAKR: Gly5: TWEAKR: Gly5: Fc (SEQ ID NO: 19, white circles) a TWEAK using an ELISA-style assay. Figure 9 shows a comparison of TweakR-Fc oligomers that bind to soluble TWEAK at 50 ng / ml using huTWEAKR: Fc (SEQ ID NO: 7) (black squares), monomer TWEAKR 43 (SEQ ID NO: 31) ( white squares), TWEAKR 43 dimer (SEQ ID NO: 33) (white triangles), TWEAKR trimer 43 (SEQ ID NO: 35) (crosses), TWEAKR 43 tetramer (SEQ ID NO: 37) (white diamonds), and pentamer TWEAKR 43 (SEQ ID NO: 39) (white circles).

Figure 10 shows a comparison of TweakR-Fc oligomers that bind to soluble TWEAK at 50 ng / ml using TWEAKR tetramer 43 (SEQ ID NO: 37) (white diamonds), pentamer TWEAKR 43 (SEQ ID NO: 39) (white circles), TWEAKR hexamer 43 (SEQ ID NO: 41) (white triangles), and heptamer TWEAKR 43 (SEQ ID NO: 43) (asterisks). Figure 11 shows a comparison of TweakR-Fc oligomers that bind to soluble TWEAK at 33 ng / ml using CHO TANDEM (SEQ ID NO: 19 expressed in stably transfected CHO cells), TWEAKR pentamer 43 (SEQ ID NO: 39 ) (white circles), TWEAKR hexamer 43 (SEQ ID NO: 41) (white triangles), and heptamer TWEAKR 43 (SEQ ID NO: 43) (asterisks). Figure 12 shows a comparison of TweakR-Fc oligomers that bind to soluble TWEAK at 50 ng / ml using huTWEAKR: Fc (SEQ ID NO: 7) (black squares), TWEAKR 40 monomer (SEQ ID NO: 21) (squares) blanks), TWEAKR dimer 40 (SEQ ID NO: 23) (white triangles), TWEAKR trimer 40 (SEQ ID NO: 25) (crosses), TWEAKR 40 tetramer (SEQ ID NO: 27) (white diamonds), and TWEAKR pentamer 40 (SEQ ID NO: 29) (white circles). Figure 13 shows a comparison of TweakR-Fc vs. europium TweakR oligomers binding to TWEAK immobilized at 50 ng / ml using huTWEAKR: Fc (SEQ ID NO: 7) (black squares), monomer TWEAKR 40 monomer (SEQ ID NO. : 21) (white squares), TWEAKR dimer 40 (SEQ ID NO: 23) (white triangles), TWEAKR 40 trimer (SEQ ID NO: 25) (crosses), TWEAKR 40 tetramer (SEQ ID NO: 27) (white diamonds) ), TWEAKR pentamer 40 (SEQ ID NO: 29) (white plum trees).

Figure 14 shows a comparison of TweakR-Fc oligomers vs. europium TweakR oligomers that bind TWEAK immobilized at 50 ng / ml using huTWEAKR: Fc (S EQ ID NO: 7) (black squares), monomer TWEAKR 43 (SEQ ID NO: 31) (white squares), TWEAKR dimer 43 (SEQ ID NO: 33) (white triangles), TWEAKR trimer 43 (SEQ ID NO: 35) (crosses), TWEAKR 43 tetramer (SEQ ID NO: 37) (diamonds) blanks), TWEAKR pentamer 43 (SEQ ID NO: 39) (white circles), TWEAKR hexamer 43 (SEQ ID NO: 41) (vertical lines), and heptamer TWEAKR 43 (SEQ ID NO: 43) (asterisks).

DETAILED DESCRIPTION OF THE INVENTION The present invention provides compositions and methods that relate to polypeptides comprising m ultimers of soluble fragments of TWEAKR and an oligomerization domain.

Abbreviations and terminology used in the specification "4-1 BB" and "ligand 4-1 BB" (4-1 BB-L) are polypeptides described, inter alia, in US Pat. 5,674, 704, including soluble forms thereof. "bFGF" is a basic fibroblast growth factor. "BSA" is bovine serum albumin. "CD40 ligand" (CD40L) is a polypeptide described, inter alia, in U.S. Patent No. 5, 71 6, 805, including soluble forms thereof. "CHO" is a line of Chinese hamster ovarian cells.

"DMEM" is Dulbecco's modified Eagle medium, a commercially available cell culture medium. "ELISA" is a non-absorbent enzyme bound assay. "Flt3L" is ligand Flt3, a polypeptide described, inter alia, in U.S. Patent No. 5,554, 512, including soluble forms thereof. "HRMEC" are primary human renal microvascular endothelial cells. "HUVEC" is a line of human umbilical vein endothelial cells. "PBS" is saline buffered with phosphate. "PMA" is phorbol 1-myristate-1-3-acetate. "RTKs" are receptor tyrosine kinases. "Tek," which is also called Tíe2 and ork, is an RTK that is predominantly expressed in vascular endothelium. Molecular cloning of human Tek (ork) has been described by Ziegler, U.S. Patent No. 5,447, 860. "Antagon istas de Tek" are described, inter alia, in Cerretti er al. , PCT Publication No. WO 00/75323, December 14, 2000. "TNFR" is a tumor necrosis factor receptor, including soluble forms thereof. "TNFR / Fc" is a fusion polypeptide of tumor necrosis factor-Fe receptor. "TRAI L" is a ligand that induces apoptosis related to TNF, a transmembrane polypeptide type I I in the TNF family described, inter alia, in U.S. Patent No. 5,763,223, including soluble forms thereof. "VEGF" is an endothelial growth factor, also known as VPF or vascular permeability factor.

Soluble TWEAK receptor polypeptides The natural human TWEAK receptor cDNA has the sequence SEQ I D NO: 3, which encodes a 129 residue polypeptide (SEQ ID NO: 4). Examination of the DNA sequence predicts a polypeptide having an extracellular domain of about 78 amino acids (residues 1-78 of SEQ ID NO: 4, including the signal peptide), a transmembrane domain of about 23 amino acids (residues 79-101 of SEQ ID NO: 4) and an intracellular domain of approximately 28 amino acids (residues 1 02-129 of SEQ ID NO: 4). The TWEAK receptor sequence has also been reported by Kato et al. , PCT Publication No. WO 98/55508, December 1, 1998, and by Incyte, PCT Publication No. WO 99/61471, December 2, 1999. As used herein, "TWEAKR" includes polypeptides having these sequences, and in particular comprising amino acids 28-79 of S EQ ID NO: 7, as well as naturally occurring variants thereof. In one aspect of the invention, a polypeptide comprising a soluble TWEAK receptor fragment and an oligomerization domain is used as a TWEAKR antagonist to inhibit angiogenesis and / or to inhibit ligand binding of TWEAK to TWEAKR. The soluble polypeptides are capable of being secreted from the cells in which they are expressed. The use of soluble forms of polypeptides is advantageous for certain applications. The purification of the polypeptides from the recombinant host cells is facilitated because the polypeptides are secreted, and the soluble proteins are generally suitable for parenteral administration. A secreted soluble polypeptide can be identified (and distinguished from its non-soulable membrane-bound counterparts) by separating intact cells, which express the desired polypeptide from the culture medium, for example, by centrifugation, and assay the medium (supernatant) for the presence of the desired polypeptide. The presence of the desired polypeptide in the medium indicates that the polypeptide was secreted from the cells and thus, is a soluble form of the polypeptide. The soluble polypeptides can be prepared by any of a variety of conventional techniques. A DNA sequence encoding a desired soluble polypeptide can be subcloned into an expression vector for the production of the polypeptide, or the desired coding DNA fragment can be chemically synthesized. The soluble TWEAKR polypeptides comprise all or part of the extracellular domain of TWEAKR, but generally lack the transmembrane domain or a fragment thereof that will cause retention of the polypeptide on the cell surface. The soluble polypeptides may include part of the transmembrane domain or all or part of the cytoplasmic domain as long as the polypeptide is secreted from the cell in which it is produced. The soluble TWEAKR polypeptides advantageously comprise a natural or heterologous signal peptide when initially synthesized, to promote the secretion of the cell, but the signal sequence is cut off on secretion. The term "TWEAKR extracellular domain" is intended to encompass all or part of the extracellular domain of natural TWEAKR, as well as related forms including but not limited to: (a) fragments, (b) variants, (c) derivatives and (d) polypeptides of fusion. The ability of these related forms to inhibit angiogenesis or other responses mediated by TWEAKR can be determined in vitro or in vivo, using methods such as those exemplified below or using other assays known in the art. Examples of soluble TWEAKR polypeptides are provided below. The multimeric soluble TWEAKR fragments are dimers, trimers, tetramers, pentamers, hexamers, heptamers, ocimers, nonamers, decamers or higher multimers of a soluble fragment of TWEAKR. The multimers can be linked together by any means known in the art. For example, they can be part of a continuous polypeptide chain, wherein each monomer can be linked to its or its neighboring monomers directly via the peptide bond or bonds, or indirectly, through one or more peptide and intervening amino acid bonds. , for example, through a linker. The multimers can also be linked to each other by, for example, other types of covalent bonds, for example, by disulfide bonds formed between cysteine residues in different soluble TWEAKR polypeptides. Oligomerization domes are polypeptides that cause the polypeptides that comprise them to oligomerize, that is, form covalent and / or non-covalent associations with another polypeptide comprising a corresponding oligomerization domain, in this way, two or more polypeptides are "oligomerized" "if they are linked to each other via their oligomerization domains. Any oligomerization domain known in the art can be used. Examples include leucine closures and certain polypeptides derived from antibodies, for example, Fc domains, as described in more detail below. The polypeptides in an oligomer can have identical polypeptide sequences, similar polypeptide sequences or different polypeptide sequences. In particular embodiments, the oligomerized polypeptides of the present invention comprise from four to fourteen soluble TWEAKR fragments. In some embodiments, a polypeptide comprising a multimeric soluble TWEAKR fragment and an oligomerization domain is prepared using polypeptides derived from immunoglobulins. The preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of polypeptides derived from antibodies (including the Fc domain) has been described, for example, by Ashkenazi et al. (Proc. Nati, Acad. Sci. USA 88: 1 0535, 1 991); Byrn et al. (Nature 344: 677, 1 990); and Hollenbaugh and Aruffo ("Construction of I mm unoglobulin Fusion Proteins", in Current Protocols in Immunology, Suppl. 4, pages 1 90.1 9.1.-1 0. 1 9.1 1, 1 992). One embodiment of the present invention is directed to an TWEAKR-Fc oligomer comprising two polypeptides, each polypeptide comprising two soluble TWEAKR fragments and one Fc domain (diTWEAKR-Fc). A polynucleotide encoding the diTWEAKR-Fc polypeptide is inserted into an appropriate expression vector. The diTWEAKR-Fc polypeptides are expressed in host cells transformed with the recombinant expression vector, and allow very similar antibody molecules to be assembled, over which interchain disulfide bonds are formed between the Fc portions to produce tetravalent soluble TWEAKR. The term "Fc polypeptide", as used herein, includes natural and mutein forms of polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides containing the hinge region that promotes dimerization are also included. A suitable Fc polypeptide, described in PCT application WO 93/1 01 51, is a single chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgG 1 antibody. . Another useful Fc polypeptide is the Fc mutein described in US Patent 5,457,035 and by Baum et al. , EMBO J. 1 3: 3992, 1 994. The amino acid sequence of this mutein is identical to that of the natural Fc sequence presented in WO 93/10151, except that amino acid 1 9 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu and amino acid 22 has been changed from Gly to Ala. The mutein exhibits reduced affinity for Fc receptors. Fusion polypeptides comprising Fc moieties, and multimers formed therefrom, offer an advantage of easy purification by affinity chromatography on Protein A or Protein G columns, and Fc fusion polypeptides can provide a longer in vivo half-life. long, which is useful in therapeutic applications, than unmodified polypeptides. In other embodiments, a fragment of multimeric soluble TWEAKR may be substituted for the variable portion of an antibody heavy or light chain. If the fusion proteins are made with both heavy and light chains of an antibody, it is possible to form a soluble TWEAKR oligomer with eight or more soluble TWEAKR fragments. In another embodiment, the oligomerization domain comprises a leucine closure domain. The closing domains of leucine are peptides that promote the oligomerization of the proteins in which they are found. Leucine closures were originally identified in several DNA binding proteins (Landschulz et al., Science 240: 1759, 1988) and have been found in a variety of different proteins. Among the known leucine closures are the naturally occurring peptides and the derivatives thereof which dimerize or trimerize. Examples of leucine closure domains suitable for producing soluble multimeric proteins are described in PCT application WO 94/10308, and closure of leucine derived from lung surfactant protein D (SPD) described in Hoppe et al. FEBS Lett. 344: 191, 1994. The use of a modified leucine lock that allows stable trimerization of a heterologous protein fused to it, is described in Fanslow et al. , Semin. Immunol. 6: 267, 1 994. Recombinant fusion proteins comprising a soluble TWEAKR polypeptide fused to a leucine-closing peptide are expressed in suitable host cells, and the soluble TWEAKR multimer that is formed is recovered from the culture supernatant. Alternatively, the polypeptides of the invention comprise peptide linkers (spacers). A linker is a sequence of one or more amino acids whose amino terminus is a peptide linked to a first polypeptide and whose terminal carboxy terminus is a peptide linked to a second polypeptide., so that the first polypeptide, the linker and the second polypeptide form a contiguous amino acid sequence. It is said that such a linker "binds" the first polypeptide and the second polypeptide, in contrast to a first polypeptide and a second polypeptide that are linked together without an intervening polypeptide sequence (ie, without a linker). Among the suitable peptide linkers are those described in U.S. Patents 4,751, 1 80, 4,935,233 and 5,073,627. A DNA sequence encoding a desired peptide linker can be inserted between, and in the same reading frame as, for example, the DNA sequences encoding TWEAKR fragments, and / or between the polynucleotide sequences encoding the fragments of TWEAKR and the oligomerization domain, using conventional techniques known in the art. For example, a chemically synthesized oligonucleotide encoding the linker can be ligated between sequences encoding soluble TWEAKR fragments. In particular embodiments, a polypeptide of the invention comprises from two to four soluble TWEAKR fragments, separated by peptide linkers and an oligomerization domain.

The present invention encompasses the use of various forms of soluble TWEAKR multimers that inhibit angiogenesis and / or other responses mediated by TWEAKR. The term "oligomerized soluble TWEAKR multimer" is intended to encompass oligomerized multimers containing all or part of the extracellular domain of natural TWEAKR, as well as related forms including, but not limited to, oligomerized multimers of TWEAKR fragments, variants, derivatives and fusion polypeptides. soluble. The ability of these related forms to inhibit angiogenesis or other responses mediated by TWEAKR can be determined in vitro or in vivo, using methods such as those exemplified in the examples or using other assays known in the art. Among the polypeptides, the m ultimers and oligomers useful for practicing the present invention are polypeptides comprising TWEAKR variants that retain the ability to bind ligand and / or inhibit angiogenesis or other TWEAKR mediated responses. Such variants of TWEAKR include polypeptides that are substantially homologous to nautral TWEAKR, but which have an amino acid sequence different from that of a natural TWEAKR due to one or more deletions, insertions or substitutions. Particular embodiments include, but are not limited to, TWEAKR polypeptides comprising from one to ten deletions, insertions or substitutions of amino acid residues, when compared to a natural TWEAKR sequence. Included are variants of TWEAKR polypeptides that naturally occurring variants, such as allelic forms and otherwise spliced forms, as well as variants that have been constructed by modifying the amino acid sequence of a TWEAKR polypeptide or nucleotide sequence. of a nucleic acid encoding a TWEAKR polypeptide. Generally, substitutions of one or more amino acids present in the natural polypeptide should be done conservatively. Examples of conservative substitutions include the substitution of amino acids outside the active domains, and the substitution of amino acids that do not alter the secondary and / or tertiary structure of TWEAKR. Additional examples include replacing an aliphatic residue with another, such as Lie, Val, Leu or Ala for one another, or substitutions of one polar residue with another, such as between Lys and Arg; Glu and Asp; or Gin and Asn, or substitutions of one aromatic residue for another, such as Phe, Trp, or Tyr for one another. Other such conservative substitutions, for example, substitutions of complete regions having similar hydrophobicity characteristics, are known in the art. In some preferred embodiments, the TWEAKR variant is at least about 70% identical in amino acid sequence to the natural TWEAKR amino acid sequence; in some preferred embodiments the TWEAKR variant is at least about 80% identical in an amino acid sequence to the natural TWEAKR amino acid sequence. In some preferred embodiments, the TWEAKR variant is at least about 90% identical in amino acid sequence to the natural TWEAKR amino acid sequence; in some preferred embodiments, the TWEAKR variant is at least about 95% identical in amino acid sequence to the natural TWEAKR amino acid sequence. In some highly preferred embodiments, the TWEAKR variant is at least about 98% identical in amino acid sequence to the natural TWEAKR amino acid sequence; in some highly preferred embodiments the TWEAKR variant is at least about 99% identical in amino acid sequence to the natural TWEAKR amino acid sequence. The percentage of identity, in the case of both polypeptides and nucleic acids can be determined by visual inspection. Percent identity can also be determined using the alignment method of Needleman and Wunsch (J. Mol. Biol. 48: 443, 1 970) as reviewed by Sm ith and Waterman (Adv. Appl. Math 2: 482, 1 981). Preferably, the percentage of identity is determined by using a computer program, for example, the GAP computor program version 10.x available from Genetics Computer Group (GCG).; Madison, Wl, see also Devereux et al. , Nucí. Acids Res. Q2: 387, 1884). The preferred failure parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the heavy comparison matrix of Gribskov and Buergess, Nucí. Acids Res. 14: 6745, 1986, as described by Schwartz and Dayhoff, eds. , Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, p. 353-358, 1979 for amino acids; (2) a penalty of 30 (amino acids) or 50 (nucleotides) for each opening and a penalty of 1 (amino acids) or 3 (nucleotides) additional for each symbol in each opening; (3) without penalty for final openings; and (4) without maximum penalty for long openings. Other programs used by someone skilled in the sequence comparison technique can also be used. For TWEAKR fragments, the identity percentage is calculated based on that portion of TWEAKR that is present in the fragment. The present invention further encompasses the use of soluble TWEAKR polypeptides with or without glycosylation of associated natural standards. TWEAKR expressed in yeast or mammalian expression systems (eg, COS-1 or COS-7 cells) may be similar to or significantly different from a nautral TWEAKR polypeptide in glycosylation pattern and molecular weight, depending on the choice of expression system . The expression of TWEAKR polypeptides in bacterial expression systems, such as E. coli, provides non-glycosylated molecules. Different host cells can also process polypeptides differentially, resulting in heterogeneous mixtures of polypeptides with varying N or C ends. The primary amino acid structure of soluble TWEAKR polypeptides can be modified to create derivatives by forming covalent or aggregation conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like. The covalent derivatives of TWEAKR can be prepared by linking particular functional groups to amino acid side chains of TWEAKR or at the N-terminus or C-terminus of a TWEAKR polypeptide. Soluble TWEAKR fusion polypeptides that are useful for practicing the invention also include covalent or aggregation conjugates of a TWEAKR polypeptide with other added polypeptides to provide novel polyfunctional entities.

Recombinant production of TWEAK receptor polypeptides TWEAKR polypeptides, including soluble TWEAKR polypeptides, fragments and fusion polypeptides, used in the present invention, can be prepared using a recombinant expression system. Host cells transformed with a recombinant expression vector ("recombinant host cells") encoding the TWEAKR polypeptide are cultured under conditions that promote the expression of TWEAKR and the TWEAKR is recovered. TWEAKR polypeptides can also be produced in transgenic plants or animals, or by chemical synthesis. The invention encompasses nucleic acid molecules encoding the TWEAKR polypeptides used in the invention, including: (a) nucleic acids encoding residues 28-79 of S EQ I D NO: 7 and fragments thereof that bind TWEAK; (b) nucleic acids that are at least 70%, 80%, 90% or, 95%, 98% or 99% identical to a nucleic acid of (a), and which encode a polypeptide capable of binding TWEAK; and (c) nucleic acids that hybridize at moderate severity to a nucleic acid of (a), and which encode a polypeptide capable of binding TWEAK. Due to the degeneracy of the genetic code, there may be considerable variation in the nucleotide sequences encoding the same amino acid sequence. Nucleic acid sequences capable of hybridizing under moderately severe conditions are included as embodiments of the invention (eg, 5 X SSC pre-wash solution, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and 50 hybridization conditions. ° C, 5 X SSC, overnight) to the DNA sequences encoding TWEAKR. The skilled artisan can determine additional salt and temperature combinations that constitute moderate hybridization severity (see also, Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Maniatis, Molecular Cloning: A Laboratory Manual (Molecular Cloning: A Laboratory Manual), Cold Spring Harbor Laboratory Press, 1982, and Ausubel, Current Protocols in Molecular Biology, Wiley and Sons, 1989 and later, which are incorporated herein by reference). Higher severity conditions include higher temperatures for hybridization and post-hybridization washes, and / or lower salt concentration. The percentage of identity of nucleic acids can be determined using the methods described above for polypeptides, that is, by methods that include visual inspection and the use of computer programs, such as GAP. Any suitable expression system can be used for the production of recombinant TWEAKR. Recombinant expression vectors include DNA encoding a TWEAKR polypeptide operably linked to suitable transcriptional and translational regulatory nucleotide sequences, such as those derived from a mammalian, microbe, virus or insect gene. The nucleotide sequences are operably linked when the regulatory sequence is functionally related to the TWEAKR DNA sequence. In this manner, a promoter nucleotide sequence is operably linked to a TWEAKR DNA sequence if the promoter nucleotide sequence controls the transcription of the TWEAKR DNA sequence. Examples of regulatory sequences include promoters, transcription enhancers or operators, a ribosomal binding site of mRNA, and appropriate sequences that control the initiation and termination of transcription and translation. A sequence encoding an appropriate signal peptide (natural or heterologous) can be incorporated into expression vectors. A DNA sequence for a signal peptide (referred to by a variety of names including secretory leader, leader peptide or leader) can be fused in frame to the TWEAKR sequence, such that the TWEAKR polypeptide is initially translated as a fusion protein that comprises the signal peptide. A signal peptide that is functional in the intended host cells promotes extracellular secretion of the TWEAKR polypeptide. The signal peptide is cut from the TWEAKR polypeptide on the TWEAKR secretion of the cell. Suitable host cells for expression of TWEAKR polypeptides include prokaryotes, yeast and higher eukaryotic cells, including insect and mammalian cells. Suitable expression and cloning vectors for use with cell hosts of bacteria, fungi, yeasts, insects and mammals are described, for example, in Pouwels et al. Cloning Vectors: A Laboratory Manual (Cloning Vectors: A Laboratory Manual), Elsevier, New York, 1985.

Prokaryotes include gram negative or gram positive organisms, for example E. coli or Bacilli. Prokaryotic host cells suitable for transformation include, for example, E. cli, Bacillus subtilis, Salmonella typhimurium, and several other species within the genera Pseudomonas, Streptomyces and Staphylococcus. In the prokaryotic host cell, such as E. coli, the TWEAKR polypeptides may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal Met can be cut from the expressed recombinant polypeptide. Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker genes. A phenotypic selectable marker gene is, for example, a gene that encodes a protein that confers resistance to antibiotics or that supplies an autotrophic requirement. Examples of useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids, such as the cloning vector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means to identify the transformed cells. An appropriate promoter and a TWEAKR DNA sequence are inserted into the pBR322 vector. Other commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM 1 (Promega Biotec, Madison, Wl, US). Promoter sequences commonly used for expression vectors of recombinant prokaryotic host cells include β-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275: 61 5, 1978; Goeddel et al., Nature 281: 544 , 1979), tryptophan (trp) promoter system (Goeddel et al., Nucí Acids Res. 8: 4057, 1 980; EP-A-36776) and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual (Cloning Molecular: A Laboratory Manual), Cold Spring Harbor Laboratory, p 412, 1 982). A particularly useful prokaryotic host cell expression system employs a phage P promoter. and a thermolabile repressor sequence cl 857ts. The plasmid vectors available from the American Type Culture Collection, which incorporate derivatives of the promoter? PL include the plasmid pHUB2 (resident in E. coli strain JMB9, ATCC 37092) and pPLc28 (resident in E. coli RR1, ATCC 53082). TWEAKR polypeptides can also be expressed in yeast host cells, preferably of the genus Saccharomyces (e.g., S. cerevisiae). Other yeast genera, such as Pichia or Kluyveromyces, may also be employed. Yeast vectors will frequently contain a replication sequence origin of a 2μ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination and a selectable marker gene. Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255: 2073, 1 980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7: 149, 1 968; Holland et al., Biochem. 1 7: 4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyrovate decarboxylase, phosphofructokinase, glucose- 6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phospho-glucose isomerase and glucokinase. Other vectors and promoters suitable for use in yeast expression are further described in Hitzeman, EPA-73,657. Another alternative is the glucose-reprehensible ADH2 promoter described by Russell et al. (J. Biol. Chem. 258: 2674, 1982) and Beier et al. (Nature 300: 724, 1982). Shuttle vectors replicable in both yeast and E. coli can be constructed by inserting DNA sequences from pBR322 for selection and replication in E. coli (Ampr gene and origin of replication) in the yeast vectors described above. The leader sequence of yeast factor a can be used to direct the secretion of recombinant polypeptides. The leader sequence of factor a is frequently inserted between the promoter sequence and the structural gene sequence. See, for example, Kurjan et al. , Cell 30: 933, 1 982; Bitter et al. , Proc. Nati Acad. Sci. USA 81: 5330, 1884. Other suitable leader sequences for facilitating the secretion of recombinant polypeptides from yeast hosts are known to those skilled in the art. A leader sequence can be modified near its 3 'end to contain one or more restriction sites. This will facilitate the fusion of the leader sequence to the structural gene. Yeast transformation protocols are known to those skilled in the art. One such protocol is described by Hinnen et al. , Proc. Nati Acad. Sci. USA 75: 1 929, 1 978. The protocol of Hinnen et al. selects Trp + transformants in a selective medium, where the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 μg / ml adenine and 20 μg / ml uracil. Yeast host cells transformed by vectors containing an ADH2 promoter sequence can be cultured to induce expression in a "rich" medium. An example of a rich medium is one consisting of 1% yeast extract, 2% peptone and 1% glucose supplemented with 80 μg / ml adenine and 80 μg / ml uracil. The derepression of the ADH2 promoter occurs when glucose is depleted from the medium. Insect host cell culture systems can also be used to express recombinant TWEAKR polypeptides, including soluble TWEAKR polypeptides. Bacculovirus systems for the production of heterologous polypeptides in insect cells are reviewed by Luckow and Summers, Bio / Technology 6:47, 1988. Mammalian cells are particularly preferred for use as host cells. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23: 175, 1981), L cells, C127 cells, 3T3 cells ( ATCC CCI 163), Chinese hamster ovary cells (CHO), HeLa cells and BHK cell lines (ATCC CRL 1 0) and CV1 / EBNA cell line derived from the African green monkey kidney cell line CV1 ( ATCC CCL 70) as described by McMahan et al. (EMBO J. 1 0: 2821, 1 991). For the production of therapeutic polypeptides, it is particularly advantageous to use a mammalian host cell line which has been adapted to grow in medium containing no animal proteins. Stable methods for introducing DNA into mammalian cells have been described (Kaufman, R.J., Large Scale Mammalian Cell Culture, 1990, pp. 15-69). Additional protocols using commercially available reagents, such as Lipfectamine (Gibco / BRL) or Lipfectamine-Plus, can be used to transfect cells (Flegner et al., Proc. Nati, Acad. Sci. USA 84: 741 3, 1987). In addition, electroporation can be used to transfect mammalian cells using conventional methods, such as those in Sambrook et al. , Molecular Cloning: A Laboratory manual (Molecular Cloning: A Laboratory Manual), 2 ed. Vol. 1 -3, Cold Spring Harbor Laboratory Press, 1989). The selection of stable transformants can be performed using methods known in the art, such as, for example, resistance to cytotoxic drugs. Kaufman et al. , Meth. In Enzymology 1 85: 487, 1 990, it describes various selection schemes, such as dihydrofolate reductase (DHFR) resistance. A suitable host strain for DHFR selection can be CHO strain DX-B1 1, which is deficient in DHFR (Urlaub and Chasin, Proc. Nati Acad.Sci. US 77: 421 6, 1 980). A plasmid expressing the DH FR cDNA can be introduced into the DX-B1 1 strain, and only cells containing the plasmid can be cultured in appropriate selective media. Other examples of selectable markers that can be incorporated into an expression vector include cDNAs that confer resistance to antibiotics, such as G41 8 and hygromycin B. Cells harboring the vector can be selected on the basis of resistance to these compounds. Transcriptional and translational control sequences for expression vectors of mammalian host cells can be excised from viral genomes. The promoter sequences and intensifying sequences commonly used are derived from polyoma virus, adenovirus 2, simian virus 40 (SV40) and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, eg, SV40 origin, early and late promoter, enhancer, splice and polyadenylation sites, can be used to provide other genetic elements for expression of a structural gene sequence in a host cell of mammal. Early and late viral promoters are particularly useful because both are readily obtained from a viral genome as a fragment, which may also contain a viral origin of replication (Fiers et al., Nature 273: 13, 1778; Kaufman, Meth. In Enzymology, 1 990). Larger or smaller SV40 fragments can also be used, provided that the sequence of approximately 250 bp (base pairs) that extend from the Hind I II site to the Bgl I site located in the replication site SV40 viral origin is included . Additional control sequences shown to enhance the expression of heterologous genes of mammalian expression vectors include such elements as the 8EASE expression enhancer sequence element derived from CHO cells (Morris et al., Animal Cell Technology, 1 997 , pp. 529-534) and the tripartite leader (TPL) and RNA gene RNAs of Adenovirus 2 (Gingeras et al., J .. Biol. Chem. 257: 1 3475, 1982). Internal ribosome entry site (IRES) sequences of viral origin allow dicistronic mRNAs to be translated efficiently (Oh and Sarnow, Current Opinion in Genetics and Development 3: 295, 1993; Ramesh et al., Nucleic Acids Research 24 : 2697, 1996). The expression of a heterologous cDNA as part of a dicistronic mRNA followed by the gene for a selectable marker (e.g., DHFR) has been shown to improve transfectability of the host and expression of the heterologous cDNA (Kaufman, Meth. I n Enzymology, 1 990 ). Exemplary expression vectors employing m dicistronic RNAs are pTR-DC / GFP described by Mosser et al. , Biotechniques 22.1 50, 1 997 and p2A5l described by Morris et al. , Animal Cell Technology, 1 997, pp. 529-534. A useful high expression vector, pCAVNOT, has been described by Mosley et al. , Cell 59: 335, 1989. Other expression vectors for use in mammalian host cells can be constructed as described by Okayama and Berg (Mol Cell Cell Biol 3: 280, 1 983). A useful system for high stable level expression of mammalian cDNAs in murine C1 27 murine epithelial cells can be constructed substantially as described by Cosman et al. (Mol Immunol., 23: 935, 1986). A useful high-expression vector, PMSLV N 1 / N4, described by Cosman et al. , Nature 31 2: 768,1984, has been deposited as ATCC 39890. Additional useful mammalian expression vectors are known in the art.

With respect to the signal peptides that can be employed to produce TWEAKR polypeptides, the natural TWEAKR signal peptide can be used or can be replaced by a heterologous signal peptide or leader sequence, if desired. The choice of signal peptide or leader may depend on factors such as the type of host cells in which the recombinant TWEAKR is to be produced. Examples of heterologous signal peptides that are functional in mammalian host cells include the signal sequence for interleukin-7 (I L-7) described in US Pat. No. 4,965,195, the signal sequence for interleukin receptor. -2 described in Cosman et al. , Nature 312: 768 (1988); the interleukin-4 receptor signal peptide described in EP 367,566; the type I interleukin-1 receptor signal peptide described in US Patent 4, 968, 607; and the interleukin-1 type II receptor signal peptide described in EP 460,846. Using recombinant DNA techniques including mutagenesis and the polymerase chain reaction (PCR), the skilled artisan can produce DNA sequences encoding the TWEAKR polypeptides comprising various additions or substitutions of residues or amino acid sequences, or deletions of terminal or internal residues or sequences, including fragments of TWEAKR, variants, derivatives and fusion polypeptides. Transgenic animals, including mice, goats, sheep and pigs, and transgenic plants, including tobacco, tomato, legumes, grasses and grains, can also be used as bioreactors for the production of TWEAKR polypeptides, including soluble TWEAKR polypeptides. In the case of transgenic animals, it is particularly advantageous to construct a chimeric DNA including a TWEAKR coding sequence operably linked to cis-acting regulatory sequences that promote the expression of soluble TWEAKR in milk and / or other body fluids (see for example, U.S. Patent No. 5,843,705; U.S. Patent No. 5,880,327). In the case of transgenic plants, it is particularly advantageous to produce TWEAKR in a particular cell, tissue or organ type (see, for example, U.S. Patent No. 5,639,947; U.S. Patent No. 5,889,189). The skilled artisan will recognize that the method for purifying expressed soluble TWEAKR polypeptides will vary according to the host system employed and whether the recombinant polypeptide is secreted or not. The soluble TWEAKR polypeptides can be purified using methods known in the art, including one or more steps of concentration, desalting, ion exchange, hydrophobic interaction, affinity purification, HPLC, or size exclusion chromatography. Fusion polypeptides comprising Fc portions (and m ultimers formed therefrom) offer the advantage of easy purification by affinity chromatography on Protein A or Protein G columns.

Methods of treatment Methods and compositions employing the TWEAK receptor or ligand, or the genes encoding the TWEAK receptor or ligand, are described below to promote or suppress angiogenesis in a tissue or group of target cells. The terms "treat", "treating", "treatment", "therapy", "therapeutic" and the like, they intend to include preventive therapy, prophylactic therapy, improvement therapy and curative therapy. The disclosed polypeptides, compositions and methods are used to inhibit angiogenesis or other TWEAKR mediated responses in a mammal in need of such treatment. The term "TWEAKR mediated response" includes any cellular, physiological or other biological response that is caused at least in part by the binding of the TWEAK ligand to TWEAKR, or which can be inhibited or suppressed, in whole or in part, by blocking TWEAK of the union to TWEAKR. The treatment is advantageously administered in order to prevent the onset or recurrence of a disease or condition mediated by angiogenesis, or to treat a mammal having a disease or condition mediated by angiogenesis. Diseases and conditions mediated by angiogenesis include, but are not limited to, ocular disorders, malignant and metastatic conditions and inflammatory diseases. Among the ocular disorders that can be treated according to the present invention are ocular diseases characterized by ocular neovascuiarization including, but not limited to, diabetic retinopathy (a major complication of diabetes), premature retinopathy (this devastating ocular condition, which frequently leads to chronic vision problems and carries a high risk of blindness, is a severe complication during the care of premature infants), neovascular glaucoma, retinoblastoma, retrolental fibroplasia, rubeosis, uveitis, macular degeneration and neovascularization of cornea graft. Other ocular inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization can also be treated according to the present invention. The present invention can also be used to treat malignant and metastatic conditions, such as solid tumors. Solid tumors include both sarcomas and primary and metastatic carcinomas. The present invention can also be used to treat inflammatory diseases including, but not limited to, arthritis, rheumatism and psoriasis. Other diseases and conditions that can be treated in accordance with the present invention include benign tumors and preneoplastic conditions, myocardial angiogenesis, hemophilic joints, scleroderma, vascular adhesions, atherosclerotic plaque neovascularization, telangiectasia, and wound granulation. Disease states that are angiogenic-dependent include coronary or peripheral atherosclerosis and ischemia of any tissue or organ, including the heart, liver, brain and the like. These types of diseases can be treated by compositions that promote angiogenesis. The methods according to the present invention can be tested in animal models in vivo to confirm the desired prophylactic or therapeutic activity, as well as to determine the optical therapeutic dosage, before administration to humans. The amount of a particular TWEAKR antagonist that will be effective in a particular treatment method depends on the age, type and severity of the condition to be treated, body weight, desired duration of treatment, method of administration and other parameters. Effective dosages are determined by a doctor or other qualified medical professional. Normal effective dosages are about 0.01 mg / kg to about 1000 mg / kg of body weight. In some preferred embodiments, the dosage is approximately 0.1-50 mg / kg; in some preferred embodiments the dosage is about 0.5-1 0 mg / kg. The dosage for local administration is usually lower than for systemic administration. In some embodiments, a simple administration is sufficient; in some embodiments, the TWEAKR antagonist is administered as multiple doses over one or more days. The TWEAKR antagonists are normally administered in the form of a pharmaceutical composition comprising one or more pharmacologically acceptable carriers. The pharmaceutically acceptable carriers include diluents, fillers, auxiliaries, excipients and vehicles which are pharmaceutically acceptable for the route of administration and can be aqueous or oleaginous suspensions formulated using suitable dispersing, wetting and suspending agents. The pharmaceutically acceptable carriers are generally sterile and free of pyrogenic agents and can include water, oils, solvents, salts, sugars and other carbohydrates, emulsifying agents, buffering agents, antimicrobial agents and chelating agents. The particular pharmaceutically acceptable carrier and the proportion of active compound to carrier are determined by the solubility and chemical properties of the composition, the mode of administration and standard pharmaceutical practice. The compositions as described herein may be contained in a vial, bottle, tube, syringe, inhaler or other container for single or multiple administrations. Such containers may be made of glass or a polymeric material, such as polypropylene, polyethylene or polyvinyl chloride, for example. Preferred containers may include a seal, or other closure system, such as a rubber stopper that can be penetrated by a needle in order to remove a single dose and then re-seal upon removal of the needle. All such containers for injectable liquids, lyophilized formulations, reconstituted lyophilized formulations or reconstitutable injection powders known in the art or for the administration of aerosol compositions are contemplated for use in the compositions and methods described herein. The TWEAKR antagonists are administered to the patient in a manner appropriate to the indication. Thus, for example, a TWEAKR antagonist, or a pharmaceutical composition thereof, can be administered by intravenous, transdermal, intradermal, intraperitoneal, intramuscular, intranasal, epidural, oral, topical, subcutaneous, intracavity, sustained release from implants, peristaltic routes or by any other suitable technique. Parenteral administration is preferred.

In certain embodiments of the claimed invention, the treatment further comprises treating the mammal with one or more additional chemotherapeutic agents. The additional chemotherapeutic agent (s) may be administered prior to, concurrent with, or following the administration of the TWEAKR antagonist. The use of more than one chemotherapeutic agent is particularly advantageous when the mammal being treated has a solid tumor. In some embodiments of the claimed invention, the treatment further comprises treating the mammal with radiation. The radiation, including brachytherapy and teletherapy, may be administered before, concurrent with, or following the administration of the second chemotherapeutic agent and / or TWEAKR antagonist. When the mammal being treated has a solid tumor, the method preferably includes the administration of, in addition to a TWEAKR antagonist, one or more chemotherapeutic agents selected from the group consisting of alkylating agents, antimetabolites, vinca alkaloids, and others. chemotherapeutics derived from plants, nitrosoureas, antitumor antibiotics, antifumor enzymes, topoisomerase inhibitors, platinum analogs, adrenocorticoid suppressors, hormones, hormone agonists and antagonists, antibodies, - immunotherapeutics, blood cell factors, radiotherapeutics and biological response modifiers. In some preferred embodiments, the method includes the administration of, in addition to a TWEAKR antagonist, one or more chemotherapeutic agents selected from the group consisting of cisplatin, cyclophosphamide, mechlorethamine, melphalan, bleomycin, carboplatin, fluorouracil, 5-fluorodeoxyuridine, methotrexate, taxol, asparaginase, vincristine and vinblastine, lymphokines and cytokines, such as interleukins, intereferons (including alpha, beta or delta), and TNF, chlorambucil, busulfan, carmustine, lomustine, semustine, streptozocin, dacarbazine, cytarabine, mercaptopurine, thioguanine, vindesine , etoposide, teniposide, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, L-asparaginase, hydroxyurea, methylhydrazine, mitotane, tamoxifen, and fluoxymesterone. In some preferred embodiments, the method includes administering, in addition to a TWEAKR antagonist, one or more chemotherapeutic agents, including various soluble forms thereof, selected from the group consisting of ligand Flt3, ligand CD40, interleukin-2, interleukin -12, ligand 4-1 BB, anti-4-1 BB antibodies, TNF antagonists and TNF receptor antagonists, TRAI L, VEGF antagonists, VEGF receptor antagonists (including VEGF-R1 and VEGF-R2, also known as Flt1 and flkl or KDR), Tek antagonists and CD148 (also referred to as DEP-1, ECRTP and PTPRJ, see Takahashi et al., J. Am. Soc. Nephrol. 1 0:21 35-45, 1 999 ). In some preferred embodiments, the TWEAKR antagonists of the invention are used as a component of, or in combination with, "metronomic therapy," such as that described by Browder et al. and Klement et al. (Cancer Research 60: 1878, 2000; J. Clin. Invest. 1 05 (8): R1 5, 2000; see also Barinaga, Science 288: 245, 2000). The polypeptides, compositions and methods of the present invention can be used as a first-line treatment, for the treatment of residual disease following primary therapy, or as an adjunct to other therapies including chemotherapy, surgery, radiation and other known therapeutic methods. in the technique. When the nucleic acid sequences of the present invention are delivered according to the methods described herein, it is advantageous to use a delivery mechanism, so that the sequences will be incorporated into a cell for expression. Delivery systems that can be advantageously employed in the contemplated methods include the use of, for example, viral delivery systems, such as retroviral and adenoviral vectors, as well as non-viral delivery systems. Such delivery systems are well known to those skilled in the art.

Classification Methods The TWEAK receptor as described herein, can be used in a variety of classification methods to isolate, for example, TWEAKR agonists and antagonists. TWEAKR agonists are compounds that promote the biological activity of TWEAKR. The compounds identified via the following classification assays can be used in compositions and methods to modulate angiogenesis to treat a variety of disease states. The present invention provides methods for classifying compounds that (1) modulate TWEAK receptor or ligand gene expression in a tissue or target cell, (2) modulate the TWEAK receptor-ligand interaction to regulate angiogenesis; (3) bind the TWEAK receptor or ligand to influence angiogenesis; or (4) interfering with or regulating the influence of TWEAK-ligand receptor complex bound in downstream events, such as angiogenesis. The present invention contemplates the use of assays that are designed to identify compounds that modulate the activity of a TWEAK ligand or receptor gene (i.e., modulate the gene expression level of TWEAK and / or modulate the level of product activity. of TWEAK gene). The assays can be further used to identify compounds that bind to TWEAK gene regulatory sequences (e.g., promoter sequences; see, e.g., Platt, 1994, J. Biol. Chem. 269, 28558-28562), and that can modulate the gene expression level of TWEAK. Such assay may involve, for example, the use of a control system, in which the transcription and translation of the ligand gene or TWEAK receptor, as compared to a system including test compounds that are suspected to influence normal transcription or translation of a TWEAK gene. For example, one could determine the proportion of TWEAK receptor RNA produced by cardiac cells, and use this to determine if a test compound influences that ratio. To assess the influence of a test compound that is suspected to influence this normal rate of transcription, one would first determine the rate of production of TWEAK receptor RNA in a cardiac cell culture by, for example, Northern Blotting. One could then administer the test compound to a cardiac cell culture under otherwise identical conditions as the control culture. Then the proportion of TWEAK receptor RNA in the culture brought with the test compound could be determined by, for example, Northern blotting, and compared to the proportion of TWEAK receptor RNA produced by the control culture cells. An increase in the TWEAK receptor RNA in cells contacted with the test compound relative to the control cells is indicative of a transcription stimulator and / or translation of the TWEAK receptor gene in cardiac cells, while a decrease is indicative of a transcription inhibitor and / or translation of TWEAK receptor gene in cardiac cells. There are a variety of other methods that can be used to determine the expression level of the ligand or TWEAK receptor gene as well and can also be used in assays to determine the influence of a test compound on the level of ligand gene expression. or TWEAK receiver. For example, RNA of a type of cell or tissue that is known, or is suspected, that expresses the ligand gene or TWEAK receptor, such as cardiac, can be isolated and tested using hybridization or PCR techniques. The isolated cells may be derived from cell culture or from a patient. Analysis of cells taken from culture may be a necessary step in the evaluation of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on gene expression. of ligand or TWEAK receptor. Such analyzes can reveal both quantitative and qualitative aspects of the expression pairing of the TWEAK ligand or receptor gene., including activation or inactivation of ligand gene expression or TWEAK receptor. In one embodiment of the detection scheme, a cDNA molecule is synthesized from an RNA molecule of interest (e.g., by reverse transcription of the RNA molecule in cDNA). A sequence within the cDNA is then used as the template for a nucleic acid amplification reaction, such as a PCR amplification reaction or the like. The nucleic acid reagents used as synthesis initiation reagents (e.g., primers) in the steps of nucleic acid amplification and reverse transcription of this method, are chosen from the nucleic acid segments of the ligand gene or TWEAK receptor. described above. Preferred lengths of such nucleic acid reagents are at least 9-30 nucleotides. For the detection of the amplified product, nucleic acid amplification can be carried out using radioactive or non-radioactive labeled nucleotides. Alternatively, sufficient amplified product can be made so that the product can be visualized by staining with standard ethidium bromide or by using any other suitable nucleic acid staining method. Additionally, it is possible to perform such assays of ligand gene expression or TWEAK receptor "in situ", that is, directly on tissue sections (fixed and / or frozen) of a patient tissue obtained from biopsies or resections, in a manner that no nucleic acid purification is necessary. The TWEAK ligand or receptor gene nucleic acid segments described above can be used as probes and / or primers for such in situ procedures (see, eg, Nuovo, GJ, 1 992, "PCR I n Situ Hybridization: Protocols And Applications "(In situ Hybridization with PCR: Protocol and Applications), Raven Press, NY). Compounds identified via assays such as those described herein may be useful, for example, for modulating angiogenesis influenced by the ligand-receptor interaction of TWEAK. Such methods for stimulating or inhibiting angiogenesis influenced by TWEAK are discussed herein. Alternatively, the systems can be designed to identify compounds capable of binding the TWEAK ligand or receptor polypeptide of the invention and thereby influence the angiogenesis that results from this interaction. The identified compounds may be useful, for example, to modulate the vascularization of target tissues or cells, may be used for screening to identify compounds that break TWEAK ligand-receptor interactions, or may themselves break such interactions. The principle of assays used to identify compounds that bind to the TWEAK ligand or receptor involves preparing a reaction mixture of the TWEAK ligand or receptor and the test compound under conditions and for a sufficient time to allow the two components to interact and join, thus forming a complex that can be removed and / or detected in the reaction mixture. These trials can be conducted in a variety of ways. For example, a method for conducting such an assay that classifies compounds that bind to the TWEAK receptor would involve anchoring the TWEAK receptor or test substance on a solid phase and detecting the TWEAK test compound / receptor complexes anchored at the solid phase at the end of the reaction. In one embodiment of such a method, the TWEAK receiver can be anchored on a solid surface; and the test compound, which is not anchored, can be labeled, either directly or indirectly. Alternatively, these same methods could be used to classify test compounds that bind to the TWEAK ligand instead of the receptor. In practice, microtiter plates can conveniently be used as the solid phase. The anchored component can be immobilized by non-covalent or covalent linkages. Non-covalent binding can be achieved by simply coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein to be immobilized can be used to anchor the protein to the solid surface. The surfaces can be prepared in advance and stored. In order to conduct the test, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is completed, the unreacted components are removed (for example, by washing) under conditions so that any complex formed will remain immobilized on the solid surface. The detection of anchors anchored in the solid surface can be achieved in a variety of ways. Where the previously immobilized component is pre-labeled, the detection of immobilized label on the surface indicates that the complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; for example, using a specific tagged antibody to the previously non-immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Alternatively, a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components and complexes detected; for example, using an immobilized antibody specific for binding or receiving TWEAK or the test compound to anchor any complex formed in solution, and a labeled antibody specific for the other component of the complex possible to detect anchoring complexes. Those compounds identified as binding agents for either the TWEAK receptor or the TWEAK ligand can be further assessed for their ability to interfere with the TWEAK ligand-receptor interaction, as described below, and thereby suppress or promote the angiogenesis that results from the ligand-receptor interaction of TWEAK. Such compounds can then be used therapeutically to stimulate or inhibit angiogenesis. The TWEAK ligand and receptor polypeptides of the present invention can also be used in a classification assay to identify compounds and small molecules, which specifically interact with the described TWEAK ligand or receptor to either inhibit (antagonize) or intensify (agonize ) the interaction between these molecules. Thus, for example, the polypeptides of the invention can be used to identify cell antagonists and agonists, cell-free preparations, chemical libraries and mixtures of natural products. Antagonists and agonists can be natural or modified substrates, ligands, enzymes, receptors, etc. of the polypeptides of the present invention, or they can be structural or functional mimics of the polypeptides. Potential antagonists of the TWEAK ligand-receptor interaction of the present invention may include small molecules, peptide and antibodies that bind to and occupy a binding site of the polypeptides, causing them to be unavailable to interact and thereby prevent its normal ability to modulate angiogenesis. Other potential antagonisms are antisense molecules, which can anneal to mRNA in vivo and block the translation of mRNA in the polypeptides of the present invention. Potential agonists include small molecules, peptides and antibodies which bind to the present TWEAK polypeptides and influence angiogenesis as caused by the described interactions of the TWEAK polypeptides of the present invention. Small molecule agonists and antagonists are usually less than 10K molecular weight and may possess a variety of physiochemical and pharmacological properties that enhance cellular penetration, resist degradation and prolong their physiological half-lives. (Gibbs, "Pharmaceutical Research in Molecular Oncology" (Pharmaceutical Research in Molecular Oncology), Cell, Vol. 79; (1 994)). Antibodies that include intact molecules as well as fragments such as Fab and F (ab ') 2 fragments can be used to bind to and inhibit the polypeptides of the present invention by blocking the start of a signaling cascade. It is preferable that the antibodies are humanized, and more preferably that the antibodies are human. The antibodies of the present invention can be prepared by any of a variety of well-known methods. Specific classification methods are known in the art and many are extensively incorporated into high throughput test systems, so that large amounts of test compounds can be classified within a short time. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical classification assays, immunoassays, cell-based assays, etc. These assay formats are well known in the art. The classification assays of the present invention are receptive to the classification of chemical libraries and are suitable for the identification of drug candidates of small molecules, antibodies, peptides and other antagonists and agonists. One embodiment of a method for identifying molecules, which antagonize or inhibit the ligand-receptor interaction of TWEAK involves adding a candidate molecule to a medium, which contains cells expressing the polypeptides of the present invention; changing the conditions of said medium so that, but for the presence of the candidate molecule, the polypeptides would interact; and observe the binding and inhibition of angiogenesis. The binding of the ligand and TWEAK receptor can be determined according to the competitive binding assays outlined above, and well known in the art. The angiogenic effect of this binding can be determined via cell proliferation assays such as, for example, cell density assays, or other proliferation assays that are also well known in the art. The activity of the cells contacted with the candidate molecule can then be compared with the identical cells, which are not contacted and the agonists and antagonists of the TWEAK polypeptide interactions of the present invention can be identified. The measurement of biological activity can be performed by a variety of well-known methods, such as measuring the amount of protein present (e.g., an ELI SA) or protein activity. A decrease in biological stimulation or activation would indicate an antagonist. An increase would indicate an agonist. Classification assays can be further designed to find molecules that mimic the biological activity resulting from the TWEAK polypeptide interactions of the present invention. Molecules that mimic the biological activity of a polypeptide may be useful for enhancing the biological activity of the polypeptide. To identify compounds for therapeutically active agents that mimic the biological activity of a polypeptide, it must first be determined whether a candidate molecule binds to the polypeptide. A candidate molecule that binds is then added to a biological assay to determine its biological effects. The biological effects of the candidate molecule are then compared to those of the polypeptide. Additionally, the complex formation within the reaction mixtures containing the test compound and the normal TWEAK ligand or receptor protein protein can also be compared to complex formation within the reaction mixtures containing the test compound and a ligand gene protein or mutant TWEAK receptor. This comparison may be important in those cases where it is desirable to identify compounds that break the mutant interactions but not normal ligand or TWEAK receptor gene proteins. The assay for compounds that interfere with the interaction of the TWEAK ligand or receptor gene products and binding partners can be conducted in a heterogeneous or homogeneous format. The heterogeneous assays involve anchoring either the ligand or TWEAK receptor gene product or the binding partner on a solid phase and detecting complexes anchored in the solid phase at the end of the reaction. In homogeneous tests, the complete reaction is carried out in a liquid phase. In either approach, the order of adding reagents can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the TWEAK ligand or receptor gene products and the binding partners, for example, by competition, can be identified by conducting the reaction in the presence of the test substance.; that is, by adding the test substance to the reaction mixture before or simultaneously with the TWEAK ligand and receptor gene products. Alternatively, test compounds that break preformed complexes, e.g., compounds with higher binding constants that displace one of the components of the complex, can be tested by adding the test compound to the reaction mixture after the complexes they have formed. The various formats are briefly described below. In a heterogeneous assay system, either the ligand gene product or TWEAK receptor, it is anchored on a solid surface, while the non-anchored species is labeled, either directly or indirectly. In practice, microtitre plates are conveniently used. The anchored species can be immobilized by non-covalent or covalent bonds. Non-covalent binding can be achieved simply by coating the solid surface with a solution of the ligand gene product or TWEAK receptor and drying. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface. The surfaces can be prepared in advance and stored. In order to conduct the assay, the companion of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, the unreacted components are removed (eg, by washing) and any complex formed will remain immobilized on the solid surface. The detection of complexes anchored in the solid surface can be achieved in a variety of ways. Where the non-immobilized species is pre-labeled, detection of the tag immobilized on the surface indicates that the complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect tag can be used to detect complexes anchored on the surface; for example, using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Depending on the order of addition of the reaction components, test compounds which inhibit complex formation or which break preformed complexes can be detected. Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components and complexes detected; for example, using an immobilized antibody specific for one of the binding components to anchor any complex formed in solution, and a labeled antibody specific for the other partner for detecting anchored complexes. Again, depending on the order of addition of reagents to the liquid phase, test compounds can be identified that inhibit the complex or that break preformed complexes. In an alternate embodiment of the invention, a homogeneous assay can be used. In this approach, a preformed complex of the ligand gene product or TWEAK receptor is prepared in which either the ligand gene product or TWEAK receptor or its binding partner is labeled, but the signal generated by the label is extinguished due to complex formation (see, for example, US Patent No. 4, 1, 496 by Rubenstein, which uses this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species of the preformed complex will result in the generation of a signal above the bottom. In that manner, test substances that break the interaction of the TWEAK ligand or receptor gene product can be identified. In a particular embodiment, the ligand gene product or TWEAK receptor can be prepared for immobilization using recombinant DNA techniques. For example, the ligand encoding region or TWEAK receptor can be fused to a glutathione-S-transferase (GST) gene using a fusion vector, such as pG EX-5X-1, in such a way that its binding activity is maintained in the resulting fusion protein. The interactive binding partner can be purified and used to culture a monoclonal antibody, using methods routinely practiced in the art. This antibody can be labeled with the radioactive isotope <; 125 > l, for example, by methods routinely practiced in the art. In a heterogeneous assay, for example, the ligand fusion protein or TWEAK receptor can then be added in the presence or absence of the test compound in a manner that allows interaction and binding to occur. At the end of the reaction period, a non-bound material can be washed, and the labeled monoclonal antibody can be added to the system and allowed to bind the complex components. The interaction between the TWEAK ligand and receptor gene products can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. Successful inhibition of the interaction by the test compound will result in a decrease in the measured radioactivity. Alternatively, a TWEAK-GST receptor gene fusion protein and a TWEAK ligand gene product (or vice versa) can be mixed together in liquid in the absence of the solid glutathione-agarose beads. The test compound can be added either during or after the species allows interaction. This mixture can then be added to the glutathione-agarose beads and the unbonded material is washed. Again the degree of inhibition of the TWEAK igando-receptor gene product interaction can be detected by adding the labeled antibody and measuring the radioactivity associated with the beads. In another embodiment of the invention, these same techniques can be employed using peptide fragments corresponding to the binding domains of the ligand protein and / or TWEAK receptor, rather than one or both of the full-length proteins. Any variety of methods routinely practiced in the art can be used to identify and isolate the binding sites. These methods include, but are not limited to, mutagenesis of the gene encoding one of the proteins and classification for cleavage binding in a co-in-unoprecipitation assay. The compensation mechanisms in the gene that codes for the second species in the complex can then be selected. Sequence analysis of the genes encoding the respective proteins will reveal the mutations that correspond to the region of the protein involved in the interactive binding. Alternatively, a protein can be anchored to a solid surface using methods described in this previous section, and it is allowed to interact with and bind to its labeled binding partner, which has been brought with a proteolytic enzyme, such as trypsin. . After washing, a short labeled peptide, comprising the binding domain, can remain associated with the solid material, which can be isolated and identified by the sequencing of amino acids. In addition, once the gene encoding the segments can be designed to express peptide fragments of the protein, which can then be tested for binding activity and be purified or synthesized. For example, and not by way of limitation, a ligand gene product or TWEAK receptor can be anchored to a solid material as described above, in this Section, by making a ligand fusion protein or TWEAK-GST receptor. and allowing it to bind to glutathione agarose beads. The obtained interactive binding partner can be labeled with a radioactive isotope, such as < 35 > S, and cut with a proteolytic enzyme such as trypsin. The cleavage products can then be added to the TWEAK-GST receptor fusion protein or anchored TWEAK ligand fusion protein and allowed to bind. After washing off the unbound peptides, the tagged bound material, which represents the binding partner binding domain, can be levigated, purified and analyzed by amino acid sequence by well-known methods. The peptides thus identified can be produced synthetically or fused to appropriate facilitator proteins using recombinant DNA technology. The TWEAK ligand-receptor interactions of the invention, in vivo, initiate a cascade of events that either stimulate or suppress angiogenesis in a targeted cell or tissue group. Molecules, such as nucleic acid molecules, proteins or small molecules can, in turn, influence this cascade. Compounds that break the effects of TWEAK ligand-receptor interaction in this manner may be useful in regulating angiogenesis. The basic principle of the assay systems used to identify compounds that interfere with the angiogenic effect or anti-angiogenic interaction ligand-TWEAK receptor involves preparing a reaction mixture containing the receptor and ligand TWEAK under conditions and for a enough time to allow the two to interact and unite, thus forming a complex. In order to test a compound for inhibitory activity of the effect of this interaction, the reaction mixture is prepared in the presence and absence of the test compound. The precurve compound may be initially included in the reaction mixture, or may be added at a time subsequent to the addition of the TWEAK ligand-receptor complex. The control reaction mixtures are incubated without the test compound or with a placebo. The inhibition or potentiation of any effect of the TWEAK complex on vascularization is then detected. The normal angiogenic response in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the cascade of events initiated by the TWEAK ligand-receptor interaction. The enhanced angiogenesis in the culture containing the test compounds indicates a stimulator of the effect of the TWEAK ligand-receptor complex. The following examples are intended to illustrate particular embodiments and do not limit the scope of the invention.

EXAMPLE 1 This example presents the cloning and identification of the TWEAK receiver.

Cloning of expression of the TWEAK receptor cDNA To clone TWEAK receptor cDNA, an expression vector encoding a growth hormone leader, a leucine closure multimerization domain, and the C-terminal extracellular domain of human TWEAK ( see Chicheportiche et al., J. Biol. Chem. 272 (51): 32401, 1 997) was constructed. This expression vector, which was called pDC409-LZ-TWEAK, comprised the DNA sequence SEQ ID NO: 1 and encoded the polypeptide SEQ I D NO: 2. The supernatants conditioned with pDC409-LZ-TWEAK were produced by transient transfection in CV1-EBNA cells. These supernatants were incubated with magnetic beads coated with polyclonal goat anti-mouse antibody that had previously been incubated with a mouse monoclonal antibody against leucine closure. The control beads were produced by mixing the beads coated with supernatants from cells transfected with empty vector. A monolayer of COS cells cultured in a T175 flask was transfected with 1 5 μg of 1 000 000 complexity DNA deposits from a HUVEC cDNA expression library. After 2 days, these cells were lifted from the flask and incubated in 1.5 ml of binding medium plus 5% dehydrated skim milk for 3 hours at 4 degrees C on a rotating wheel. The cells were pre-cleared by adding control beads and turned at 4 degrees C for an additional 45 minutes, after which the cells attached to beads were removed with a magnet. The pre-rinse was repeated 2-3 times, then the beads coated with TWEAK were added to the cells and turned 30 minutes at 4 degrees C. The cells that were attached to the TWEAK beads were separated by the use of a magnet. and washed 4x in PBS. The plasmid DNA was extracted from these cells by lysate in 0.1% SDS and electroporation of the supernatants in DH 1 01 B cells. The colonies were culíivadas overnight in selective medium with ampicillin. Transformants were deposited and used as a source of plasmid DNA during an additional tamping round. After 2 rounds of batting, positive clones were collected from the resulting pool based on their ability to bind to TWEAK using a slide-binding protocol similar to that described in Part B, below. The human TWEAK receptor cDNA (also called TWEAKR) was determined to have the sequence SEQ I D NO: 3, which encodes a 129 residue polypeptide (S EQ I D NO: 4). The sequence screening predicts a polypeptide having an extracellular domain of about 78 amino acids (residues 1 -78 of SEQ ID NO: 4, including the signal peptide), a transmembrane domain of about 23 am inoperates (residues 79-1 01 of SEQ ID NO: 4), and an intracellular dominance of about 28 am inoperates (residues 1 02-129 of SEQ ID NO: 4). TWEAKR is the smallest known TN F receptor family member. It has a repeating region rich in simple cysteine in the extracellular domain, as compared to the 3-4 repeats of other TNF receptor family members. The TWEAKR polypeptide was previously described as a transmembrane protein encoded by a human liver cDNA clone (WO 98/55508, see also WO 99/61471), but had not been identified as the TWEAK receptor. A murine homologue, FNF-inducible Fn 14 (Meighan-Mantha et al., J. Biol. Chem. 274 (46): 331 66, 1999), is approximately 82% identical to human protein, as shown by aligning in Figure 1. The newly identified TWEAK receptor was tested side by side with DR3 (which had been identified as the TWEAK receptor by Marsters et al., Current Biology 8: 525, 1998) for the ability to bind to TWEAK.

B. The TWEAK receptor binds to TWEAK The COS cell slides were transfected with expression vectors containing TWEAKR, DR3 or vector without insertion (Control). After two days, the cells were incubated with concentrated supernatants of CV-1 cells transfected with a vector encoding the leucine-closing TWEAK extracellular domain fusion protein. One hour later, the cells were wd and probed with an antibody labeled with 1-125 against the leucine closing domain. The slides were wd, fixed and the autoradiography was performed using X-ray film. The transfected TWEAKR cells bound significant amounts of TWEAK. TWEAK did not bind to cells transfected with DR3 or control cells. This experiment confirmed that the TWEAKR polypeptide identified in part A above, instead of DR3, is the main receptor for TWEAK. After the discovery of the functional TWEAK receptor, other investigators also reported that DR3 is not the main receptor for TWEAK (Kaptein et al., FEBS Lett 485 (2-3): 1 35, 2000. The TWEAK-binding interaction TEAKR was further characterized by Scatchard analysis.VC-1 cells were transfected with full-length human TWEAK and mixed 1: 30 with Raji cells, which do not express TWEAK.The cells were incubated with serial dilutions of human TWEAK receptor -Fe labeled with 1 25-1 for 2 hours at 4 degrees Celsius.The free and bound probe was separated by micro-centrifugation of the samples through a mixture of phthalate oil in plastic tubes.The supernatants and pellets were gamma-counted. Scatchard analyzes of TWEAK ligand that binds to the TWEAK receptor showed a binding affinity constant (Ka) of approximately 4.5 x 1 08 M "1.

C. The TWEAK receptor is strongly expressed in cardiac tissue. To determine the expression pattern of the TWEAK receptor, Northern blot analysis was performed. Northern blots of human multiple tissue were purchased from Clontech (Palo Alto, CA) and probed with randomized initiated DNA labeled with P-32 from the TWEAK receptor coding region. The spots were washed and the autoradiography was performed using x-ray film. The results showed that in the adult TWEAKR was strongly expressed in samples of heart, placenta and some samples of skeletal muscle. The strong expression in heart tissue additionally supports the usefulness of TWEAKR in the diagnosis and treatment of heart disease. In contrast to the adult, fetal tissues expressed TWEAKR more ubiquitously; TWEAKR transcripts were seen in the lung and liver.

EXAMPLE 2 This example presents the recombinant production of soluble TWEAK Fc receptor fusion polypeptides (TWEAKR-Fc). To construct a nucleic acid encoding the extracellular domain of TWEAKR fused to Fc, a nucleic acid encoding the N-terminal 79 amino acids of TWEAKR, including the leader (signal peptide), was linked to a nucleic acid encoding an Fc portion of human IgG 1 The sequences for this construct are shown as SEQ ID NO: 6 (nucleic acid) and SEQ ID NO: 7 (amino acid). In SEQ ID NO: 7, residues 1-27 are the predicted signal peptide (predicted to be cut on cell secretion, the actual cut site was identified by N-terminal sequence analysis, see below), residues 28-79 are from the extracellular domain of TWEAKR rich in cysteine, residues 80-81 are from a BglII cloning site and the remainder is the Fc portion. Upon insertion into a mammalian expression vector and expression in and secretion of a mammalian host cell, this construct produced a polypeptide designated TWEAKR-Fc. N-terminal sequence analysis determined that the secreted polypeptide designated TWEAKR-Fc had an N-terminus corresponding to residue 28 (Glu) of SEQ I D NO: 7. The anti-angiogenic activity of TWEAKR-Fc was demonstrated using assays such as those described in the following examples. An analogous Fc fusion construct was prepared using the extracellular domain of murine TWEAKR.

EXAMPLE 3 This example presents a migration assay of flat endothelial cells (wound closure) to measure the activity of TWEAK receptor antagonists. In this assay, the migration of endothelial cells is measured as the closing rate of a circular wound in a monolayer of cultured cells. The rate of wound closure is linear and is regulated dynamically by agents that stimulate and inhibit angiogenesis in vivo.

The primary human renal mucosal endothelial cells, HRMEC, were isolated, cultured and used in the third passage after thawing, as described in Martin et al. , In vitro Cell Dev Biol 33: 261, 1 997. Replicable circular lesions, "wounds" (diameter 600-800 microns) were generated in confluent HRMEC monolayers using a silicon-tipped drill. At the time of wounding, the medium (DMEM + 1% BSA) was supplemented with 20 ng / ml of PMA (phorbol-1 2-myristate-13-acetate), EGF (4 ng / ml) and 0.1 50 to 5 μg / ml of TWEAKR-Fc, or a combination of 40 ng / ml of EGF and 0.1 50 to 5 μ / ml of TWEAKR-Fc. The residual wound area was measured as a function of time (0-12 hours) using a microscope and computer image analysis software (Bioquant, Nashville, TN). The relative migration speed was calculated for each agent and the combination of agents by linear regression of residual wound area was plotted against time. The results are shown in Figures 2-3. Compared with hulgG or medium + BSA, TWEAKR-Fc inhibited PMA-induced endothelial migration in a dose-responsive manner, reducing the migration rate from unstimulated levels to 5 μg / ml (Figure 2). Neither HulgG nor TWEAKR-Fc inhibited basal (non-induced) migration. When migration of HRM EC was induced by EGF, TWEAKR-Fc inhibited endothelial migration in a dose-dependent manner, reducing the migration rate to unstimulated levels at 5 μg / ml (Figure 3).

EXAMPLE 4 This example presents a mouse corneal pocket assay useful for measuring the activity of TWEAK receptor antagonists. In this assay, the agents to be tested by angiogenic or anti-angiogenic activity are immobilized in a slow release form in a hydron pellet, which is implanted in micro pockets created in the cornea epithelium of anesthetized mice. Vascularization is measured as the appearance, density and degree of congenital vessels of the vascularized corneal limbus in the normally avascular cornea. Hydron pellets, as described in Kenyon et al. , Invest Opthamol. & Visual Science 37: 1625, 1996, incorporated sucralphafo with bFGF (90 ng / pellet), bFGF and IgG (14 μg / pellet, control) or bFGF and TWEAKR-Fc (14 μg). The pellets were surgically implanted in stromal corneal micro-pockets created by micro-dissection of 1 mm medial to the lateral corneal limbus of 6-7 week old male C57BL mice. After five days, at the peak of neovascular response to bFGF, the corneas were photographed, using a Zeiss slit lamp, at an incipient angle of 35-50 ° of the polar axis in the meridian containing the pellet. The images were digitized and processed using subtractive color filters (Adobe Photoshop 4.0) to delineate microvessels established by hemoglobin content. The image analysis computation program (Bioquant, Nashville, TN) was used to calculate the fraction of the cornea image that was vascularized, the density of vessels within the vascularized area and the density of vessels within the total cornea. As shown in Table 1, TWEAKR-Fc (1000 pmol) inhibited corneal angiogenesis induced with bFGF (3 pmol), reducing vascular density to 50% of that induced by FGF alone or FGF + IgG.

Table 1 Effect of TWEAKR-Fc on angiogenesis induced by FGF in the mouse corneal pocket assay EXAMPLE 5 This example presents an endothelial cell poliferation assay useful for measuring the activity of a TWEAK receptor antagonist. In this assay, the proliferation of endothelial cells is measured after 4 days of cell growth in microtitre cavities using a cellular labeled molecule called calcein AM. The esterases expressed by the cells cut the calcein and cause it to fluoresce when cut at 485 nm. The uncut calcein does not fluoresce. The amount of fluorescence is directly related to the number of endothelial cells in the culture cavity. The proliferation of endothelial cells is frequently regulated by agents that stimulate and / or inhibit angiogenesis in vivo. The primary HUVEC (human umbilical vein endothelial cells) were obtained from a commercial source (Clonetics, Walkersivelle, MD), cultured and used in passage 2 to 7. The replica cultures were arranged by adding 3000 H UVEC to each microtidule cavity in basal medium of endoihelial cells (EBM, a basal medium of endoihelial cells that does not contain growth or serum favors and is based on medium formulations developed by Dr. Richard Ham at the University of Colorado, Clonetics) 0.05% FBS (fetal bovine serum). At the time of initiation of culture FGF-2 (fibroblast growth factor-2, 10 ng / ml) or human TWEAK (1000 ng / ml) was added to the cultures in the presence of human IgG (hulgG, control) or human TWEAKR-Fc at concentrations ranging from 0.08 μg / ml to 20 μg / ml for TWEAK-induced and 0.08 to 6.7 μg / ml for FGF-2-induced). The cultures containing HUVEC were incubated for 4 days at 37 degrees C, 5% CO2. On the fourth day of culture, 4 μM calcein-AM was added to the cultures and 2 hours later the cavities were evaluated by fluorescence. The results, expressed as the average fluorescence counts (485-530 nm) per replication cavities plus or minus SEM, are shown in Figures 4 and 5. TWEAKR-Fc specifically inhibited the proliferation of H UVEC TWEAK-induced in a manner dose dependent, when compared to hulgG, which did not effect TWEAK-induced proliferation (Figure 4). In addition, TWEAKR-Fc inhibited the basal proliferation of H UVBEC observed during culture in EBM plus 0.05% FBS, as compared to hulgG which did not. Interestingly, TWEAKR-Fc also inhibited the proliferation of HUVEC mediated with FGF-2 at concentrations of more than 2 μg / ml, as compared to hulgG, which did not effect the proliferative response of H UVEC induced with FGF-2. (Figure 5). These results show that TWEAKR-Fc inhibits the proliferation of HUVEC by the addition of exogenous recombinant human TWEAKR. That TWEAKRF-Fc partially inhibits the proliferation of serum-induced HIVEC, indicating that HUVEC produces endogenous TWEAK that promotes growth / survival of the EC (endothelial cell) via TWEAKR. The TWEAKR-Fc attenuation of FGF-2 induced proliferation indicates that at least part of the EC response to FGF-2 is dependent on the endogenous TWEAK / TWEAKR interaction.

EXAMPLE 6 This example presents a murine cardiac graft / ischemia model useful for measuring the activity of a TWEAK receptor antagonist. The survival of heterotopically transplanted cardiac tissue from a mouse donor to the ear skin of another genetically similar mouse requires adequate neovascularization by the transplanted heart and surrounding tissue to promote survival and energy for cardiac muscle function. Inadequate vasculature at the transplant site causes excessive ischemia, tissue damage, and tissue failure to graft. Agents that antagonize factors involved in endothelial cell migration and vessel formation can decrease angiogenesis at the transplant site, thereby limiting the function of graft tissue and ultimately grafting on its own. A murine heterotopic cardiac isograft model is used to demonstrate the effects of TWEAKR antagonists, including antibodies and TWEAKR-Fc, on neovascularization. Female BALB / c receptors ("1-2 weeks old") are given neonatal heart grafts from donor mice of the same species. The donor heart tissue is grafted into the pavilions of the left ear of the recipient on day 0 and the mice are divided into two groups. The control group receives human IgG (Hu IgG), while the other group receives the TWEAKR antagonist, both intraperitoneally. The treatments are continued for five consecutive days. The functionality of the grafts is determined by monitoring pulsatile activity visible on days 7 and 14 post-graft. The inhibition of functional graft, as a function of the dose of TWEAKR antagonist, is determined. The histology of the transplanted hearts is examined in order to visualize the effects of the TWEAKR antagonist on edema at the site of transplantation and host and donor tissue vasculature (using, for example, staining of Factor VI I I).

EXAMPLE 7 This example presents a method for treating tumors with a TWEAK receptor antagonist. TWEAKR antagonists are tested in animal models of solid tumors. The effect of TWEAKR antagonists is determined by measuring tumor frequency and tumor growth.

EXAMPLE 8 This example presents an ELISA-based assay useful for determining the binding properties of TWEAK binding molecules, eg, monomeric and multimeric TWEAKR-Fc oligomers. For this example, huTWEAKR was used. Fc (SEQ ID NO: 7), di-TWEAKR: Fc (SEQ ID NO: 1 1), tri-TWEAKR: Fc (SEQ ID NO: 13) and TWEAKR-DR5-Fc (SEQ ID NO: 9). IL-NBRO® / TITERTEK ™ R ninety-six cavity enzyme immunoassay plates (ICB Biochemicasl, Aurora, OH) were coated with TWEAK®: Fc (SEQ ID NO: 7) at a concentration of 1 mg / ml in PBS, 50 μl / cavity, applied plate sealer, and incubated overnight at 4 ° C. Each cavity was washed three times with PBST (PBS + 0.1% TWEEN 20; 200 μl / well, then incubated for one hour at 37 ° C in PBST + 3% dehydrated skim milk (NFDM).) In separate reactions, marked TWEAK with FLAG (TWEAKR-FLAG) was pre-incubated with each of the above TWEAKR polypeptides at room temperature for 30 min, in DMEM + 0.5% low Ig serum, in 96-well U-shaped bottom plates at a time. final TWEAK-FLAG concentration of 50 ng / ml and a final concentration of each TWEAKR polypeptide from 9,000 to 4 ng / ml, in serial 3-fold dilutions.The EIA plate was again washed three times with PBST + 3% NFDM. 50 μl / cavity of ligand / receptor mixture was added, incubated at room temperature for 30 min, then washed three times with PBST + 3% NFDM.

The FLAG-M2 biotinylated antibody, diluted 1: 500 in PBST + 3% NFDM, was added to 50 μl / well, incubated for 45 min at room temperature and washed three times with PBST + 3% NFDM. SA-HRP, diluted 1: 2000 in PBST + 3% NFDM, was added to 50 μl / well, then incubated for 45 min at room temperature. The plates were washed five times, 100 μl / 3.3 'cavity, 5,5'-tetramethylbenzidine (TMB) were added and the plates were incubated at room temperature for 5-20 minutes. The reaction was stopped with 50 μl / 1M H3PO4 cavity and the absorbances were read at A450 / 57 ° - As shown in Figure 6, TWEAKR: Fc showed the weakest binding, followed by TWEAKR: DR5: Fc, then di- TWEAKR: Fc (SEQ ID NO: 11), then tri-TWEAKR: Fc (SEQ ID NO: 13). In this way, the more soluble were the TWEAKR domains comprising the fusion protein, the stronger TWEAK bound. Moreover, the increase in binding was more than additive, as shown by the difference in binding between TWEAKR: fc and di-TWEAKR: Fc.

EXAMPLE 9 This example presents a competition binding test using TWEAKR.Fc labeled with Europium useful for determining the binding properties of TWEAK binding molecules, eg, monomeric and multimeric TWEAKR-Fc oligomers. For this example, huTWEAKR was used: Fc (SEQ ID NO: 7), di-TWEAKR: Fc (SEQ ID NO: 11), tri-TWEAKR: Fc (SEQ ID NO: 13) and TWEAKR: DR5: Fc (SEQ ID NO: 9).

An M2 anti-flag antibody was diluted to a concentration of 4 μg / ml in 0.1 M NaHCO3. 100μl / cavity was used to coat 96-well flat bottom plates. The plate sealer was applied and the plates were incubated at 4 ° C during the night. Each cavity was washed five times with PBST (PBS + 0. 1% TWEEN ), then 200 μl / well of PBST + 3% NFDM were added. The plates were incubated for one hour at 37 ° C, then washed five times with PBST. FLAG-TWEAK was diluted to 50 ng / ml in PBS and added to 1000 μl / well. Plates were incubated at room temperature, with shaking, for one hour. The plates were washed five more times in PBST. Europium-labeled and unlabeled receptors were pre-mixed in 96-well U-bottom plates, diluted in PBST + 3% NFDM to a final concentration of 35 ng / ml for TWEAKR labeled with europium, three-fold dilutions of unlabeled receptors, to final concentracoins of 9000-12 ng / ml. To each cavity, 1 00 μl of pre-mixed receptors were added. The plates were incubated for one hour at room temperature with shaking, then washed ten times as before. 1 μl / intensification solution cavity (Perkin Elmer, 1244-1 05) were added and the plates were incubated for five minutes with shaking. The absorbances were read to A6? It's in a Wallac plate reader. As shown in Figure 7, the monomeric TWEAKR showed the poorer capacity to compete with TWEAKR: Fc labeled with europium to bind to TWEAK, followed by TWEAKR dimeric and TWEAKR trimeric.

EXAMPLE 10 This example presents an ELISA-style assay useful for determining the binding properties of TWEAK binding molecules, for example, monomeric and multimeric TWEAKR-Fc oligomers. TWEAKR: Gly5: Fc (SEQ ID NO: 15) is a fusion protein comprising an N-terminal meyionin residue, residues 29 to 70 of the TWEAK receptor, five glycine residues, and a peptide derived from Fc fragment. TWEAKR: 1 KPEG: Fc (SEQ ID NO: 17) is a fusion protein comprising an N-terminal methionine residue, residues 29 to 70 of the TWEAK receptor, a linker and a peptide derived from Fc fragment. TWEAKR: 1 KPEG: TWEAKR: Gly5: Fc (SEQ ID NO: 18) is a fusion protein comprising an N-terminal methionine residue, residues 29 to 70 of the TWEAK receptor, a linker, residues 29 to 70 of the receptacle of TWEAK, five glycine residues and one peptide derived from Fc fragment. TWEAKR: Gly5: TWEAKR: Gly5: Fc (SEQ ID NO: 1 9) is a fusion protein comprising an N-terminal methionine residue, residues 29 to 70 of the TWEAK receptor, five glycine residues, residues 29 to 70 of the TWEAK receptor, five glycine residues and one peptide derived from Fc fragment. Using an ELISA style assay similar to that described in Example 8, it was determined that monomeric oligomers (TWEAKR: Gly5: Fc and TWEAKR: 1 KPEG: Fc) bind TWEAK approximately equally well, but much less than the dimeric oligomeric constructs (TWEAKR: 1KPEG: TWEAKR: Gly5: Fc and TWEAKR: Gly5: TWEAKR: Gly5: Fc) (Figure 8).

EXAMPLE 11 This example presents the results of an ELISA-based TWEAK binding assay using huTWEAKR: Fc (SEQ ID NO: 7), TWEAKR 40mono-3 (SEQ ID NO: 21), TWEAKR 40dimer-1 (SEQ ID NO. : 23), TWEAKR 40trimer-5 (SEQ ID NO: 25), TWEAKR 40quad-2 (SEQ ID NO: 27), TWEAKR 40quint-1 (SEQ ID NO: 29), TWEAKR 43mono-F4 (SEQ ID NO.31) ), TWEAKR 43dimer-F2 (SEQ ID NO: 33), TWEAKR 43trimer-1 (SEQ ID NO: 35), TWEAKR 43quad-2 (SEQ ID NO: 37), TWEAKR 43quint-3 (SEQ ID NO: 39), TWEAKR 43hex-1 (SEQ ID NO: 41) and TWEAKR 43sept-1 (SEQ ID NO: 43). The protocol described in Example 8 was used except that in the present example BSA was omitted from the wash buffers. The results are presented in Figures 9-12.

EXAMPLE 12 This example presents the results of a competition binding assay using TWEAKR: Fc labeled with Europium as described in Example 9. In the present example, huTWEAKR: Fc (SEQ ID NO: 7), TWEAKR 40mono-3 ( SEQ ID NO: 21), TWEAKR 40dimer-1 (SEQ ID NO: 23), TWEAKR 40trimer-5 (SEQ ID NO: 25), TWEAKR 40quad-2 (SEQ ID NO: 27), TWEAKR 40quint-1 (SEQ ID NO: 29), TWEAKR 43mono-F4 (SEQ ID NO: 31), TWEAKR 43dimer-F2 (SEQ ID NO: 33), TWEAKR 43trimer-1 (SEQ ID NO: 35), TWEAKR 43quad-2 (SEQ ID NO: 37), TWEAKR 43quint-3 (SEQ ID NO: 39), TWEAKR 443hex-1 (SEQ ID NO: 41) and TWEAKR 43sept-1 (SEQ ID NO: 43) were used. The results are presented in Figures 1 3 and 14. Relevant descriptions of publications cited herein are specifically incorporated by reference. The examples presented above are not intended to be exhaustive or to limit the scope of the invention. The skilled artisan will understand that variations and modifications are possible in light of the above teachings, and such modifications and variations are intended to be within the scope of the invention.

Claims (73)

1. REVIVALATION IS 1 . A polypeptide comprising a first soluble fragment. of a TWEAK receptor, a second soluble fragment of a TWEAK receptor and an oligomerization domain, wherein said polypeptide binds to TWEAK and said first soluble fragment consists of a sequence that is at least 90% identical to a sequence selected from the group consists of: a. residues 29 to 70 of the amino acid sequence of SEQ ID NO: 7; b. residues 28 to 79 of the amino acid sequence of SEQ ID NO: 7; c. residues 30 to 73 of the amino acid sequence of SEQ ID NO: 7; and d. residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7 2. The polypeptide of claim 1, wherein said first soluble fragment consists of a sequence that is at least 95% identical to a sequence selected from the group consisting of: a. residues 29 to 70 of the amino acid sequence of SEQ ID NO: 7; b. residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; c. residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and d. residues 30 to 70 of the amino acid sequence of SEQ ID NO: 7. 3. The polypeptide of claim 2, wherein said first soluble fragment consists of a sequence selected from the group consisting of: a. residues 29 to 70 of the amino acid sequence of SEQ I D NO: 7; b. residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; c. residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and d. residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. 4. The polypeptide of claim 1, wherein said first soluble fragment and said second soluble fragment each consist independently of a sequence that is at least 90% identical to a sequence selected from the group consisting of: a. residues 29 to 70 of the amino acid sequence of SEQ ID NO: 7; b. residues 28 to 79 of the amino acid sequence of SEQ ID NO: 7; c. residues 30 to 73 of the amino acid sequence of SEQ ID NO: 7; and d. residues 30 to 70 of the amino acid sequence of SEQ ID NO: 7. 5. The polypeptide of claim 4, wherein said first soluble fragment and said second soluble fragment each consist independently of a sequence that is at least 95% identical to a sequence selected from the group consisting of: a. residues 29 to 70 of the amino acid sequence of SEQ ID NO: 7; b. residues 28 to 79 of the amino acid sequence of SEQ ID NO: 7; c. residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and d. residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. 6. The polypeptide of claim 5, wherein said first soluble fragment and said second soluble fragment each consist independently of a sequence selected from the group consisting of: a. residues 29 to 70 of the amino acid sequence of SEQ I D NO: 7; b. residues 28 to 79 of the amino acid sequence of SEQ ID NO: 7; c. residues 30 to 73 of the amino acid sequence of SEQ ID NO: 7; and d. residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. 7. The polypeptide of claim 1, further comprising a third soluble fragment of a TWEAK receptor. The polypeptide of claim 1, wherein said first soluble fragment, second soluble fragment and third soluble fragment each independently consists of a sequence that is at least 90% identical to a sequence selected from the group consisting of: a. residues 29 to 70 of the amino acid sequence of SEQ ID NO: 7; b. residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; c. residues 30 to 73 of the amino acid sequence of SEQ ID NO: 7; and d. residues 30 to 70 of the amino acid sequence of SEQ ID NO: 7. 9. The polypeptide of claim 8, wherein said first soluble fragment, second soluble fragment and third soluble fragment each consists independently of a sequence which is at least 95% identical to a sequence selected from the group consisting of: a. residues 29 to 70 of the amino acid sequence of SEQ ID NO: 7; b. residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; c. residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and d. residues 30 to 70 of the amino acid sequence of SEQ ID NO: 7. The polypeptide of claim 9, wherein said first soluble fragment, second soluble fragment and third soluble fragment each independently of a sequence selected from the group consisting of: a. residues 29 to 70 of the amino acid sequence of SEQ I D NO: 7; b. residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; c. residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and d. residues 30 to 70 of the amino acid sequence of S EQ ID NO: 7. eleven . The polypeptide of claim 7, further comprising a fourth soluble fragment of a TWEAK receptor. The polypeptide of claim 1, wherein said first soluble fragment, second soluble fragment, third soluble fragment and fourth soluble fragment, each consisting independently of a sequence that is at least 90% identical to a sequence selected from the group which consists of: a. residues 29 to 70 of the amino acid sequence of SEQ I D NO: 7; b. residues 28 to 79 of the amino acid sequence of SEQ ID NO: 7; c. residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and d. residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. The polypeptide of claim 12, wherein said first soluble fragment, according to each soluble fragment, third soluble fragment and fourth soluble fragment, each independently of a sequence that is at least 95% identical to a sequence selected from the group consists of: a. residues 29 to 70 of the amino acid sequence of SEQ ID NO: 7; b. residues 28 to 79 of the amino acid sequence of SEQ ID NO: 7; c. residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and d. residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. The polypeptide of claim 1, wherein said first soluble fragment, second soluble fragment, third soluble fragment and fourth soluble fragment, each independently of a sequence selected from the group consisting of: a. residues 29 to 70 of the amino acid sequence of SEQ I D NO: 7; b. residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; c. residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and d. residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7 The polypeptide of claim 1, further comprising a fifth soluble fragment of a TWEAK receptor. The polypeptide of claim 1, wherein said first soluble fragment, second soluble fragment, soluble fraction fragment, fourth soluble fragment and fifth soluble fragment, each independently of a sequence that is at least 90% identical to one sequence selected from the group consisting of: a. residues 29 to 70 of the amino acid sequence of SEQ ID NO: 7; b. residues 28 to 79 of the amino acid sequence of SEQ ID NO: 7; c. residues 30 to 73 of the amino acid sequence of SEQ ID NO: 7; and d. residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. The polypeptide of claim 16, wherein said first soluble fragment, second soluble fragment, third soluble fragment, fourth soluble fragment and fifth soluble fragment, each independently of a sequence that is at least 95% identical to a sequence selected from the group consisting of: a. residues 29 to 70 of the amino acid sequence of SEQ I D NO: 7; b. residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; c. residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and d. residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. The polypeptide of claim 17, wherein said first soluble fragment, second soluble fragment, third soluble fragment, fourth soluble fragment and fifth soluble fragment, each independently of a sequence selected from the group consisting of: a. residues 29 to 70 of the amino acid sequence of SEQ ID NO: 7; b. residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; c. residues 30 to 73 of the amino acid sequence of SEQ ID NO: 7; and d. residues 30 to 70 of the amino acid sequence of S EQ I D NO: 7. 9. The polypeptide of claim 15, further comprising a sixth soluble fragment of a TWEAK receptor. The polypeptide of claim 19, wherein said first soluble fragment, second soluble fragment, third soluble fragment, fourth soluble fragment, fifth soluble fragment and sixth soluble fragment, each independently of a sequence that is at least 90% identical to a sequence selected from the group consisting of: a. residues 29 to 70 of the amino acid sequence of SEQ I D NO: 7; b. residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; c. residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and d. residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. twenty-one . The polypeptide of claim 20, wherein said first soluble fragment, second soluble fragment, third soluble fragment, fourth soluble fragment, fifth soluble fragment and sixth soluble fragment, each consists independently of a sequence that is at least 95% identical to one sequence selected from the group consisting of: a. residues 29 to 70 of the amino acid sequence of SEQ I D NO: 7; b. residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; c. residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and d. residues 30 to 70 of the amino acid sequence of SEQ ID NO: 7 22. The polypeptide of claim 21, wherein said first soluble fragment, second soluble fragment, third soluble fragment, fourth soluble fragment, fifth soluble fragment and sexuous soluble fragment, each independently of a sequence selected from the group consisting of: to. residues 29 to 70 of the amino acid sequence of SEQ ID NO: 7; b. residues 28 to 79 of the amino acid sequence of SEQ ID NO: 7; c. residues 30 to 73 of the amino acid sequence of SEQ ID NO: 7; and d. residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7 23. The polypeptide of claim 1, further comprising a seventh soluble fragment of a TWEAK receptor. The polypeptide of claim 23, wherein said first soluble fragment, second soluble fragment, third soluble fragment, fourth soluble fragment, fifth soluble fragment, sixth soluble fragment and seventh soluble fragment, each independently of a sequence that is minus 90% identical to a sequence selected from the group consisting of: a. residues 29 to 70 of the amino acid sequence of SEQ I D NO: 7; b. residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; c. residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and d. residues 30 to 70 of the amino acid sequence of SEQ ID NO: 7. 25. The polypeptide of claim 24, wherein said first soluble fragment, second soluble fragment, third soluble fragment, fourth soluble fragment, fifth soluble fragment, sixth fragment soluble and seventh soluble fragment, each consists independently of a sequence that is at least 95% identical to a sequence selected from the group consisting of: a. residues 29 to 70 of the amino acid sequence of SEQ ID NO: 7; b. residues 28 to 79 of the amino acid sequence of SEQ I D NO: 7; c. residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and d. residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7 The polypeptide of claim 25, wherein said first soluble fragment, second soluble fragment, third soluble fragment, fourth soluble fragment, fifth soluble fragment, sixth soluble fragment and seventh soluble fragment, each independently of a sequence selected from the group which consists of: a. residues 29 to 70 of the amino acid sequence of SEQ ID NO: 7; b. residues 28 to 79 of the amino acid sequence of SEQ ID NO: 7; c. residues 30 to 73 of the amino acid sequence of SEQ I D NO: 7; and d. residues 30 to 70 of the amino acid sequence of SEQ I D NO: 7. 27. The polypeptide of claim 1, further comprising a linker. 28. The polypeptide of claim 27, wherein said linker binds said first fragment of soluble TWEAKR and said second fragment of soluble TWEAKR. 29. The polypeptide of claim 27, wherein said linker binds said second fragment of soluble TWEAKR and said oligomerization domain. 30. The polypeptide of claim 1, further comprising a first linker and a second linker, wherein said first linker binds said first fragment of soluble TWEAKR and said second fragment of soluble TWEAKR and said oligomerization domain. 31 The polypeptide of claim 27, wherein said linker comprises the amino acid sequence GGGGG (SEQ I D NO: 45). 32. The polypeptide of claim 1, wherein said first soluble fragment and said second soluble fragment are linked together without a intervening polypeptide sequence. 33. The polypeptide of claim 1, wherein said first soluble fragment and said oligomerization domain are linked together without a intervening polypeptide sequence. 34. The polypeptide of claim 1, wherein said first soluble fragment, said second soluble fragment and said oligomerization domain are joined together, without an intervening polypeptide sequence, in a contiguous linear polypeptide. 35. The polypeptide of claim 1, wherein said oligomerization domain is N-terminal to said first soluble TWEAKR fragment and said second soluble TWEAKR fragment. 36. The polypeptide of claim 1, wherein said oligomerization domain is C-terminal to said first soluble TWEAKR fragment and said soluble TWEAKR fragment. 37. The polypeptide of claim 1, wherein said oligomerization domain comprises a leucine lock. 38. The polypeptide of claim 1, wherein said oligomerization domain comprises a fragment of an antibody. 39. The polypeptide of claim 38, wherein said fragment of an antibody comprises an Fc domain. 40. The polypeptide of claim 1, wherein said polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of: a. SEQ I D NO: 9; b. SEQ I D NO: 1 1; c. SEQ ID NO: 13; d. SEQ I D NO: 1 5; and. SEQ I D NO: 17; F. SEQ ID NO: 18; g. SEQ ID NO: 19; h. SEQ ID NO: 21; i. SEQ ID NO: 23; j. SEQ ID NO: 25; k. SEQ ID NO: 27; I. SEQ ID NO: 29; m. SEQ ID NO: 31; n. SEQ ID NO: 33; or. SEQ ID NO: 35; p. SEQ ID NO: 37; q. SEQ ID NO: 39; r. SEQ ID NO: 41; s. SEQ ID NO: 43; and t. SEQ ID NO: 44 41. The polypeptide of claim 40, wherein said polypeptide comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of: a. SEQ ID NO: 9; b. SEQ ID NO: 11 c. SEQ ID NO: 13 d. SEQ ID NO: 15 e. SEQ ID NO: 17 f. SEQ ID NO: 18 g- SEQ ID NO: 19 h. SEQ ID NO: 21; i. SEQ ID NO: 23; j- SEQ ID NO: 25; k. SEQ ID NO: 27; 1. SEQ ID NO: 29; m. SEQ ID NO: 31; n. SEQ ID NO: 33; or. SEQ ID NO: 35; P- SEQ ID NO: 37; q- SEQ ID NO: 39; r. SEQ ID NO: 41; s. SEQ ID NO: 43; and t. SEQ ID NO: 44 42. The polypeptide of claim 41, wherein said polypeptide comprises a sequence selected from the group consisting of: a. SEQ ID NO: 9; b. SEQ ID NO: 11; c. SEQ ID NO: 13; d. SEQ ID NO: 15; and. SEQ ID NO: 17; F. SEQ ID NO: 18; g- SEQ ID NO: 19; h. SEQ ID NO: 21; i. SEQ ID NO: 23; j- SEQ ID NO: 25; k. SEQ I D NO: 27; I. SEQ I D NO: 29; m. SEQ ID NO: 31; n. SEQ I D NO: 33; or. SEQ I D NO: 35; p. SEQ I D NO: 37; q. SEQ ID NO: 39; r. SEQ I D NO: 41; s. SEQ I D NO: 43; and t. SEQ I D NO: 44. 43. A protein comprising a first polypeptide of claim 1 and a second polypeptide of claim 1, wherein said first and second polypeptides are oligomerized to each other. 44. The protein of claim 43, wherein the amino acid sequence of said first polypeptide is identical to the amino acid sequence of said second polypeptide. 45. The protein of claim 43, wherein the amino acid sequence of said first polypeptide is not identical to the amino acid sequence of said second polypeptide. 46. A method for inhibiting a receptor in a subject comprising administering to said subject the polypeptide of claim 1. 47. A method for inhibiting angiogenesis in a subject comprising administering to said subject a therapeutically effective amount of a composition comprising the polypeptide of claim 1. 48. The method of claim 47, wherein said composition further comprises a pharmaceutically acceptable carrier. 49. The method of claim 47, wherein said fastener is a mammal. 50. The method of claim 49, wherein said mammal is a human. 51 The method of claim 47, wherein said subject has a disease or condition mediated or exacerbated by angiogenesis. 52. The method of claim 51, wherein said disease or condition is characterized by ocular neovascularization. 53. The method of claim 51, wherein said disease or condition is a solid tumor. 54. The method of claim 53, wherein said method further comprises treating said subject with radiation. 55. The method of claim 53, wherein said method further comprises bringing said subject with a second chemotherapeutic agent. 56. The method of claim 55, wherein said second chemotherapeutic agent is selected from the group consisting of: an alkylating agent, an antimetabolite, a vinca alkaloid, a plant-derived chemotherapeutic, a nitrosourea, an antitumor antibiotic, an enzyme antitumor, a topoisomerase inhibitor, a plaíin analog, an adrenocorticoid suppressant, a hormone, a hormone agonist, a hormone antagonism, an antibody, an immunotherapeutic, a blood cell factor, a radiotherapeutic and a biological response modifier. 57. The method of claim 55, wherein said second chemotherapeutic agent is selected from the group consisting of cisplatin, cyclophosphamide, mechlorethamine, melphalan, bleomycin, carboplatin, fluorouracil, 5-fluorodeoxyuridine, methotrexate, taxol, asparaginase, vincristine, vinblastine., a lymphokine, a cytokine, an interleukin, an interferon, alpha interferon, beta interferon, delta interferon, TNF, chlorambucil, busulfan, carmustine, lomustine, semustine, streptozocin, dacarbazine, cytarabine, mercaptopurine, thioguanine, vindesine, etoposide, teniposide, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, L-asparaginase, hydroxyurea, methylhydrazine, mitotane, tamoxifen, and fluoxymesterone. 58. The method of claim 51, wherein said disease or condition is an inflammatory disease or condition. 59. The method of claim 58, wherein said method further comprises treating said subject with a second therapeutic agent. 60. The method of claim 59, wherein said second therapeutic agent inhibits a cytokine or a cytokine receptor that promotes inflammation. 61 The method of claim 60, wherein said second therapeutic agent comprises a soluble fragment of said cytokine receptor, an antibody that binds said cytokine, or an antibody that binds said cytokine receptor. 62. The method of claim 59, wherein said second therapeutic agent activates a receptor that inhibits inflammation. 63. The method of claim 59, wherein said second therapeutic agent is selected from the group consisting of ligand Flt3, ligand CD40, interleukin-2, an antagonist of interleukin-4, an antagonist I L-13, interleukin-12, ligand 4-1BB, an anti-4-1BB antibody, a TNF antagonist, a TNF receptor antagonist, TRAIL, a CD148 agonist, a VEGF antagonist, a VEGF receptor antagonist, an IgE angiogonist and an antagonist of Tek. 64. A nucleic acid, or its complement, comprising a sequence encoding said polypeptide of claim 1. 65. The nucleic acid of claim 64, wherein said nucleic acid, or its complement, hybrid under moderately severe hybridization conditions. to a second nucleic acid, and said second nucleic acid comprises a sequence selected from the group consisting of: a. SEQ ID NO: 10; b. SEQ ID NO: 12; c. SEQ ID NO: 14; d. SEQ ID NO: 16; and. SEQ ID NO: 20; F. SEQ ID NO: 22; g. SEQ ID NO: 24; h. SEQ ID NO: 26; i. SEQ ID NO: 28; j. SEQ ID NO: 30; k. SEQ ID NO: 32; I. SEQ ID NO: 34; m. SEQ ID NO: 36; n. SEQ ID NO: 38; 0. SEQ ID NO: 40; And p. SEQ ID NO: 42 66. The nucleic acid of claim 64, wherein said nucleic acid, or its complement, comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of: a. SEQ ID NO: 10; b. SEQ ID NO: 12; c. SEQ ID NO: 14; d. SEQ ID NO: 16; and. SEQ ID NO: 20; F. SEQ ID NO: 22; g. SEQ ID NO: 24; h. SEQ ID NO: 26; i. SEQ ID NO: 28; j. SEQ ID NO: 30; k. SEQ ID NO: 32; 1. SEQ ID NO: 34; m. SEQ ID NO: 36; n. SEQ ID NO: 38; or. SEQ ID NO: 40; And p. SEQ ID NO: 42 67. The nucleic acid of claim 66, wherein said nucleic acid, or its complement, comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of: a. SEQ ID NO: 10; b. SEQ ID NO: 12; c. SEQ ID NO: 14; d. SEQ ID NO: 16; and. SEQ ID NO: 20; F. SEQ ID NO: 22; g. SEQ ID NO: 24; h. SEQ ID NO: 26; i. SEQ ID NO: 28; j. SEQ ID NO: 30; k. SEQ ID NO: 32; I. SEQ ID NO: 34; m. SEQ ID NO: 36; n. SEQ ID NO: 38; or. SEQ ID NO: 40; And p. SEQ ID NO: 42 68. The nucleic acid of claim 67, wherein said nucleic acid, or its complement, comprises a sequence selected from the group consisting of: a. SEQ ID NO: 10; b. SEQ ID NO: 12; c. SEQ ID NO: 14; d. SEQ ID NO: 16; and. SEQ ID NO: 20; F. SEQ ID NO: 22; g- SEQ ID NO: 24; h. SEQ ID NO: 26; i. SEQ ID NO: 28; j- SEQ ID NO: 30; k. SEQ ID NO: 32; I. SEQ ID NO: 34; m. SEQ ID NO: 36; n. SEQ ID NO: 38; or. SEQ ID NO: 40; and P- SEQ ID NO: 42. 69. The nucleic acid of claim 64, wherein said nucleic acid encodes a polypeptide sequence selected from the group consisting of: a. SEQ ID NO: 9; b. SEQ ID NO: 11; c. SEQ ID NO: 13; d. SEQ ID NO: 15; and. SEQ ID NO: 17; F. SEQ ID NO: 18; g- SEQ ID NO: 19; h. SEQ ID NO: 21; i. SEQ ID NO: 23; SEQ ID NO: 25; k. SEQ ID NO: 27; I. SEQ ID NO: 29; m. SEQ ID NO: 31; n. SEQ ID NO: 33; or. SEQ ID NO: 35; p. SEQ ID NO: 37; q. SEQ ID NO: 39; r. SEQ ID NO: 41; s. SEQ ID NO: 43; and t. SEQ ID NO: 44 70. A vector comprising said nucleic acid of claim 64. The vector of claim 70, wherein said vector is an expression vector. 72. A host cell comprising said nucleic acid of claim 64. 73. A method for producing a polypeptide comprising culturing the host cell of claim 72 under conditions that promote the expression of said polypeptide.