AU2014200003A1 - ALK1 receptor and ligand antagonists and uses thereof - Google Patents

Abstract

H:\szp\Interwoven\NRPortbl\DCC\SZP\59 186931.DOC-24/12/2013 In certain aspects, the present disclosure relates to the insight that a polypeptide comprising a ligand-binding portion of the extracellular domain of activin-like kinase I 5 (ALKl) polypeptide may be used to inhibit angiogenesis in vivo, particularly in mammals suffering angiogenesis-related disorders. The disclosure also identifies ligands for ALK1 and demonstrates that such ligands have pro-angiogenic activity, and antibodies that inhibit receptor-ligand interaction.

Description

Australian Patents Act 1990 - Regulation 3.2A ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Invention Title "ALK1 receptor and ligand antagonists and uses thereof" The following statement is a full description of this invention, including the best method of performing it known to me/us: H:\szp\Interwoven\NRPortbl\DCC\SZP\5918560_1, DOC - 24/12/13 ALKI RECEPTOR AND LIGAND ANTAGONISTS AND USES THEREOF RELATED APPLICATION This application is a divisional of Australian Patent Application No. 5 2007317926, the entire content of which is incorporated herein by reference. This application claims the benefit of the filing hte of U.S. provisional application 60/856,592, filed November 2, 2006 and entitled ALKi Receptor and 10 Ligand Antagonists and Uses Thereof. The entire teachings of the referenced provisional application are expressly incorporated herein ty reference. BACKGROUND Angiogenesis, the process of forming new blood vessels, is critical in many 15 normal and abnormal physiological states. Under normal physiological conditions, humans and animals undergo angiogenesis in specific ami restricted situations. For example, angiogenesis is normally observed in wound tealiag, fetal and embryonic development and formation of the corpus luteum, endonetrium and placenta. Undesirable or inappropriately regulated angiopaesis occurs in many 20 disorders, in which abnormal endothelial growth may erseor participate in the pathological process. For example, angiogenesis partidpates in the growth of many tumors. Deregulated angiogenesis has been implicated im pathological processes such as rheumatoid arthritis, retinopathies, hemangiomas, and psoriasis. The diverse pathological disease states in which unregulated angiognesis is present have been 25 categorized as angiogenesis-associated diseases. Both controlled and uncontrolled angiogenesis are thought to proceed in a similar manner. Capillary blood vessels are composed primarily of endothelial cells and pericytes, surrounded by a basement membrane. Aagiogenesis begins with the erosion of the basement membrane by enzymes released b endothelial cells and leukocytes. The endothelial cells, which line the lumenof blood vessels, then protrude through the basement membrane; Angiogenic actors induce the endothelial cells to migrate through the eroded basement membrane. The migrating cells form a "sprout" protruding from the parent blood venel, where the endothelial cells undergo mitosis and proliferate. Endothelial sprouts merge with each other to form capillary loops, creating the new blood vessel. Agents that inhibit angiogenesis have proven to be effective in treating a variety of disorders. AvastinTM (bevacizumab), a monoclornal antibody that binds to 5 Vascular Endothelial Growth Factor (VEGF), has proven to be effective in the treatment of a variety of cancers. Macugen T M , an aptamer that binds to VEOF has proven to be effective in the treatment of neovascular (wet) age-related macular degeneration. Antagonists of the SDF/CXCR4 signaling pathway inhibit tumor neovascularization and are effective against cancer in mouse models (Guleng et al. 10 Cancer Res. 2005 Jul 1;65(13):5864-71). The isocoumarin 2-(8-hydroxy-6 methoxy-1 -oxo-1 H-2-benzopyran-3-yl) propionic acid (NM-3) has completed phase I clinical evaluation as an orally bioavailable angiogenesis inhibitor. NM-3 directly kills both endothelial and tumor cells in vitro and is effective in the treatment of diverse human tumor xenografts in mice (Agata et al. Cancer Chemother Pharmacol. 15 2005 Dec;56(6):610-4.). Thalidomide and related compounds have shown beneficial effects in the treatment of cancer, and although the molecular mechanism of action is not clear, the inhibition of angiogenesis appears to be an important component of the anti-tumor effect (see, e.g., Dredge etal Microvasc Res. 2005 Jan;69(1-2):56-63). The success of TNF-alpha antagonists in the treatment of 20 rheumatoid arthritis is partially attributed to anti-angiogenic effects on the inflamed joint tissue (Feldmann et al. Annu Rev Immunol. 2001;l9:163-96). Anti-angiogenic therapies are widely expected to have beneficial effects oa other inflammatory diseases, particularly psoriasis. Although many anti-angiogenic agents have an effect on angiogenesis regardless of the tissue that is affted, other angiogenic 25 agents may tend to have a tissue-selective effect. It is desirable to have additional compositions and methods for inhibiting angiogenesis. These include methods and compositions which can inhibit the unwanted growth of blood vessels, either generally or in certain tissues and/or disease states. 30 2 SUMMARY In part, the present disclosure presents a characterization of an activin-like kinase I (ALK )-mediated regulatory system and the role of this system in angiogenesis in vivo. In certain aspects, the disclosure provides antagonists of 5 ALK-1 ligands and the use of such antagonists as anti-angiogenic agents. Additionally, the disclosure provides antagonists of ALK- I itself, and the use of such antagonists as anti-angiogenic agents. As described herein, ALKi is a receptor for the GDF5 group of ligands, which includes GDF6 and GDF7, and also for the BMP9 group of ligands, which includes BMP 10. This disclosure demonstrates that 10 signaling mediated by ALKI and the ligands described above is involved in angiogenesis in vivo, and that inhibition of this regulatory system has a potent anti angiogenic effect. Thus, in certain aspects, the disclosue provides antagonists of the ALK I regulatory system, including antagonists of tie receptor or one or more of the ligands, for use in inhibiting angiogenesis. In certah aspects, the disclosure 15 provides antagonists of ALKI ligands for the treatmentof cancers, particularly multiple myeloma, rheumatoid arthritis, and disorders assoiated with pathological angiogenesis in the eye. In certain aspects, the disclosure provides polypeptides comprising a ligand binding portion of the extracellular domain of ALKI ("ALK1 ECD polypeptides") 20 for use in inhibiting angiogenesis. While not wishing to be bound to any particular mechanism of action, it is expected that such polypeptides act by binding to ligands of ALKI and inhibiting the ability of these ligands to interact with ALKI as well as other receptors. In certain embodiments, an ALKI ECD polypeptide comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 97%, 99% or 100% 25 identical to the sequence of amino acids 22-118 of the human ALKI sequence of SEQ ID NO;.L An ALK ECD-polypeptide may be used as a small monomeric protein or in a dimerized form (e.g., expressed as a fusion protein), particularly for local administration into tissues such as the eye. An ALKI ECD may also be fused to a second polypeptide portion to provide improved properties, such as an increased 30 half-life or greater ease of production or purification. Fusions to an Fc portion of an immunoglobulin or linkage to a polyoxyethylene moiety (e.g., polyethylene glycol) may be particularly useful to increase the serum half-life of the ALKI ECD 3 polypeptide in systemic administration (e.g., intravenous, intraarterial and intra peritoneal administration). As demonstrated herein, a :stcmically administered ALK I -Fc polypeptide has a potent anti-angiogenic effect in the eye and also provides positive effects in marine models of rheumatoid arthritis and multiple 5 myeloma. In certain embodiments, an ALKi-Fc fusion protein comprises a polypeptide having an amino acid sequence that is at least 70%, 80%, 90%, 95%, 97%, 99% or 100% identical to the sequence of amino cids 22-118 of SEQ ID NO: 1, which polypeptide is fused, either with or without am intervening linker, to an Fc portion of an immunoglobulin, and wherein the ALI 1-Pc fusion protein binds to 10 GDF5, GDF7 and BMP9 with a KD of less than I x 10' Weand binds to TGFp-1 with a KD of greater than 1 x 10.6. An Fe portion may le selected so as to be appropriate to the organism. Optionally, the Fe portionis an Fe portion of a human IgGl. In a preferred embodiment, the ALKI-Fc fusionprotein comprises amino acids 22-118 of SEQ ID NO:1. Optionally, the ALK1Fe fusion protein comprises 15 the amino acid sequence of SEQ ID NO: 3. Optionally, the ALKI-Fc fusion protein is the protein produced by expression of the nucleic acid of SEQ ID NO:4 in a ,mammalian cell line, particularly a Chinese Hamster Ovary (CHO) cell line. ALKI ECD polypeptides may be formulated as a pharmaceutial preparation that is substantially pyrogen free. The pharmaceutical prepantio may be prepared for 20 systemic delivery (e.g., intravenous, intraarterial or sulcutancous delivery) or local delivery (e.g., to the eye). In certain aspects, the disclosure provides methods for inhibiting angiogenesis in a mammal by administering any of the ALK 1 ECD polypeptides described generally or specifically herein. In one embodiment, a method comprises 25 administering to the mammal an effective amount of an ALK1-Fc fusion protein, wherein the ALK1 Fe fusion protein comprises a polypeptide having an amino acid sequence that is at least 90% identical to the sequence of amino acids 22-118 of SEQ ID NO: 1, which polypeptide is fused to an Fe portion of an immunoglobulin, and wherein the ALK I -Fe fusion protein binds to TGFD- I with a K 0 of greater than I x 30 10 6 . Optionally, the ALKl-Fc fusion protein binds toone or more ALKI ligands selected from the group consisting of: GDF5, GDF6, (DF7, BMP9 and BMP10. Optionally, the ALKl -Fc fusion protein has a sequence of SEQ ID NO:3. The 4 ALKI ECD polypeptide may be delivered locally (e.g.,to the eye) or systemically (e.g., intravenously, intraarterially or subcutaneously). [I a particular embodiment, the disclosure provides a method for inhibiting angiogenesis in the eye of a mammal by administering an ALKI-Fc protein to the mammal at a location distal to the eye, 5 e.g. by systemic administration. In certain aspects, the disclosure provides antibodies that bind to ALK1, particularly an epitope situated in the extracellular domirx, amino acids 22-118 of SEQ ID NO:l, and inhibit the binding of ALK1 to at lest one ALK1 ligand selected from the group consisting of: GDF5, GDF6, GDF7, BlF9 and BMP 10. Based on 10 the affinity of these ligands for ALKI, an antibody may biad with a KD of less than 5 x 10' 8 M, and optionally between 5 x 10-' and I x 1 10 . An antibody with affinity within this range would be expected to inhibit signaling by one or more of GDF5, 6 and 7 while having less effect on signaling by BMP9 and 10. Such an antibody preferably inhibits angiogenesis stimulated by at least mne ALK 1 ligand selected 15 from the group consisting of: GDF5, GDF6 and GDF7. While not wishing to be bound to a particular mechanism, it is expected that suchantibodies will act by inhibiting ALK activity directly, which should be contrasted to the activity of an ALK I -Fc fusion protein, which is expected to inhibit tle activity of ALKI ligands. An anti-ALKI antibody is not expected to interfere with the ability of GDF5, GDF6, 20 GDF7, BMP9 or BMP 10 to signal through alternative receptor systems, such as the BMPRla, BMPR1b and BMPRII complexes. However, aaanti-ALKl antibody is expected to interfere with the ability of low affinity ligands for ALKI (e.g., TGF-P, which is generally recognized as triggering significant signaling events through ALK-I even though binding is relatively weak) to signal through ALKI, even 25 though an ALK I ECD may not bind to or inhibit such low affinity ligands, An antibody may bind to the ALK 1 polypeptide with a K of less than 1 x 10~" M. An antibody with affinity within this range would be expected to inhibit signaling by BMP9 or 10. Such an antibody preferably inhibits binding of BMP9 and BMP1O to ALKI. Notably, based on the data disclosed herein, an antibody that binds 30 relatively poorly to ALKI may inhibit TGFP binding to ALKI while failing to inhibit the tighter binding ligands such as GDF5 or BNP9. The antibodies described herein are preferably recombinant antibodies, meaning an antibody expressed from a 5 nucleic acid that has been constructed using the techniques of molecular biology, such as a humanized antibody or a fully human antibody developed from a single chain antibody. Fv, Fab and single chain antibodies are also included within the term "recombinant antibody". Antibodies may also bepolyclonal or non 5 recombinant monoclonal antibodies (including human or murine forms, as well as human antibodies obtained from transgenic mice). Antibodies and ALKl-ECD polypeptides may be formulated as a pharmaceutical preparation that is substantially pyrogen free, The pharmaceutical preparation may be prepared for systemic delivery (e.g., intravenous, intraarterial or subcutaneous delivery) or local delivery 10 (e.g., to the eye). In certain aspects, the disclosure provides methods for inhibiting angiogenesis in a mammal by administering to the mammal an effective amount of an antibody that binds to an ALK 1 polypeptide; described herein either generally or specifically. An antibody useful for this purpose may bind to the extracellular 15 domain of ALKI (e.g., bind to a polypeptide consisting of amino acids 22-118 of SEQ ID NO: 1) or another portion of ALKI. The antibody may bind to a polypeptide consisting of amino acids 22-118 of SEQ ID NO: I and inhibits the binding of at least one ALK1 ligand selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10. The antibody may bind to the ALKI polypeptide 20 with a K of less than 5 x iO' M, and optionally between 5 x 10' and 1 x 1010. The antibody may inhibit angiogenesis stimulated by at least one ALK I ligand selected from the group consisting of GDF5, GDF6 and GDF. An antibody that selectively inhibits signaling mediated by GDF5, 6 or 7 relative to signaling by BMP9 or 10 may be used as a selective inhibitor of angiogenesis that occurs in tissues where 25 GDF5, 6 or 7 are localized: primarily bone or joints. The antibody may bind to the ALKI polypeptide with a KD of less than 1 x 1 0 -1 M. The antibody may inhibit the binding of ALKl to an ALKI ligand, wherein the ALKl ligand is selected from the group consisting of: BMP9 and BMP 10. The anti-ALKI antibody may be delivered locally (e.g., to the eye) or systemically (e.g., intravenously, intraarterially or 30 subcutaneously), In a particular embodiment, the disclosure provides a method for inhibiting angiogenesis in the eye of a mammal by administering an anti-ALKI antibody. In another particular embodiment, the disclosure provides a method for 6 treating patients with multiple myeloma. In a particular embodiment, the disclosure provides a method for inhibiting angiogenesis in disorders that are associated with pathological angiogenesis as a consequence of multiple pro-angiogenic factors, such as VEGF, PDGF and/or FGF. 5 In certain aspects, the disclosure provides antibodies that bind to an ALKI ligand disclosed herein and inhibit the binding of the ALKI ligand to ALKi. While not wishing to be bound to any particular mechanism, it is expected that antibodies that bind to ALKI ligands will have effects that are sinilar in nature to ALKI ECD polypeptides, because both types of agent bind to the ligmnds rather than the receptor 10 itself. In certain embodiments, the antibody binds to a ligand selected from the group consisting of GDF5, GDF6 and GDF7. The antibody may bind to the ALK 1 ligand with a Ko of less than 5 x 10' M. The antibody may be selected for inhibition of angiogenesis stimulated by the ALKI ligead. A CAM assay is an appropriate assay system for selection of desirable antibodies, Such antibodies are -15 preferably recombinant antibodies, and may be formulated as a pharmaceutical preparation that is substantially pyrogen free. The pharmaceutical preparation may be prepared for systemic delivery (e.g., intravenous, intraarterial or subcutaneous delivery) or local delivery (e.g., to the eye). In certain aspects, the disclosure provides antibodies that bind to an ALKI 20 ligand and inhibit the binding of the ALKI ligand to ALKI, wherein the ALKI ligand is selected from the group consisting of BMP9 and BMP 10. The antibody may bind to the ALKI ligand with a Ki of less than 1 x 10"o M. Such antibodies are preferably recombinant antibodies, and may be formulated as a pharmaceutical preparation that is substantially pyrogen free. The pharmaceutical preparation may 25 be prepared for systemic delivery (e.g., intravenous, intraarterial or subcutaneous delivery) or local delivery (e.g., to the eye). In certain aspects, the disclosure provides methods for inhibiting angiogenesis in a mammal, the method comprising, administering to the mammal an effective amount of an antibody that binds to an ALKI ligand and inhibits the 30 binding of the ALKI ligand to ALKI, wherein the ALKi ligand is selected from the group consisting of GDF5, GDF6, GDF7, BMP9 and BMP 10. The antibody may 7 inhibit angiogenesis stimulated by at least one ALK 1 ligand selected from the group consisting of: GDF5, GDF6 and GDF7. Members of the BMP/GDF family, including ByP9, BMP10, GDF5, GDF6 and GDF7 bind to a type I and a type Il receptor in order to forn a functional 5 signaling complex; The binding sites for these receptors are different. Accordingly, in certain embodiments, an antibody that binds to an ALL I ligand and inhibits the ligand to ALK 1 is an antibody that binds at or near the tpc I receptor binding site of the ligand, In certain aspects, the disclosure provides methods for inhibiting 10 angiogenesis in a mammal by administering other inhibtsof the ALKI signaling system disclosed herein. Such inhibitors may include noleic acids (e.g., antisense or RNAi constructs) that decrease the production of ALK1, GDF5, GDF6, GDF7, BMP9 or BMP 1Q). A variety of affinity binding reagents can also be used, such as aptamers, random peptides, protein scaffolds that can b modified to allow binding 15 to selected targets (examples of such scaffolds include mticalins and FNIII domains); in each case, an affinity binding reagent wotd le selected for the ability to disrupt the ALKI regulatory system disclosed herein, either by disrupting the ALK I -ligand interaction or by inhibiting the signaling hat occurs after binding. In a further embodiment, the disclosure describes the role of DAN as a 20 regulator of the ALK I regulatory system. As shown hercia, DAN binds to the GDF5 group of ligands but fails to bind to the BMP9 gioip of ligands. Thus, DAN is expected to inhibit angiogenesis mediated by GDF5, GDF6 or GDF7 but not angiogenesis mediated by BMP9 or BMP 10. DAN may therefore be used as a selective agent for inhibiting angiogenesis in the bone or joints, where the GDF5 25 group of proteins is primarily expressed. Thus, in certain embodiments the disclosure provides DAN proteins for use as anti-angiogenic agents in the context of bone or joint angiogenesis, including rheumatoid arthritis and cancers that involve the bone or joints (e.g., multiple myeloma and bone metastases). A DAN protein will generally bind to one or more ALK I ligands selected from the group consisting 30 of: GDF5, GDF6 and GDF7, while having relatively poor binding to BMP9-or BMP 10. A DAN protein may comprise an amino acid sequence that is at least 70%, 8 80%, 90%, 95%, 97%, 99% or 100% identical to the sequence of amino acids corresponding to amino acids 17-180 of SEQ ID NO:10(mature human DAN) or amino acids 21-125 of SEQ ID NO: 10 (conserved cysteine knot domain of DAN). A DAN protein may also be encoded by a nucleic acid what comprises a sequence the 5 complement of which hybridizes under stringent hybridiation conditions to nucleotides 153-467 of SEQ ID NO:1 1 or a variant of nacleotides 153-467 of SEQ ID NO: 11 that has the same coding sequence (a "silent"wariant, such as a variant containing one or more alterations at a wobble position in the triplet code), or to nucleotides 93-635 of SEQ ID NO: 1 or a silent variantthereof. In certain aspects, 10 the DAN protein is a fusion protein, such as an Fc fusicaprotein. While DAN is expected to be particularly useful for the inhibition of angiogenesis in bone and joints (including tumors located in the bone or joints, s4cl as multiple myeloma and bone metastases), it may also be useful in other contexts, such as in a tumor located elsewhere, or in the eye. 15 In certain aspects, the disclosure provides rpethods for treating rheumatoid arthritis in a mammal, the method comprising, administering to a mammal that has rheumatoid arthritis an effective amount of an agent sekcted from the group consisting of: an ALKI ECD protein; an antibody that biads to an ALKI ligand and inhibits the binding of the ALKI ligand to ALK1, wherein the ALKI ligand is 20 selected from the group consisting of GDF5, GDF6, GDFI, BMP9 and BMP10; an antibody that binds to an ALK1 polypeptide consistingof amino acids 22-118 of SEQ ID NO:l and inhibits the binding of at least one ALKI ligand selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMPI 0; and a DAN polypeptide. 25 In certain aspects the disclosure provides methods for treating a tumor in a mammal. Such a method may comprise administering to a mammal that has a tumor an effective amount of an agent selected from the group consisting of: an ALKl ECD protein; an antibody that binds to an ALKI ligand and inhibits the binding of the ALKI ligand to ALK1, wherein the ALKI ligand is selected from the group 30 consisting of GDF5, GDF6, GDF7, BMP9 and BMP10; an antibody that binds to an ALK I polypeptide consisting of amino acids 22-118 of SEQ ID NO: I and inhibits the binding of at least one ALK 1 ligand selected from the group consisting of: 9 GDF5, GDF6, GDF7, BMP9 and BMP 10; and a DAN polypeptide. A method may further comprise administering a second agent that inhibits angiogenesis. A tumor may be a tumor that is associated with bone, such as a lukemia, a bone marrow tumor, a multiple myeloma or bone metastases, such as those commonly associated 5 with breast or prostate cancer. A tumor may also be one that utilizes multiple pro angiogenic factors, such as a tumor that is resistant to anti-VEGF therapy. In certain aspects the disclosure provides ophthalnic formulations. Such formulations may comprise an agent selected from the grMup consisting of: an ALK I ECD protein; an antibody that binds to an ALKI ligand and inhibits the binding of 10 the ALK ligand to ALK1, wherein the ALKI ligand is selected from the group consisting of GDF5, GDF6, GDF7, BMP9 and BMP10; an antibody that binds to an ALK polypeptide consisting of amino acids 22-118 of SEQ ID NO:l and inhibits the binding of at least one ALK I ligand selected from the group consisting of; GDF5, GDF6, GDF7, BMP9 and BMP0; and a DAN polypeptide. 15 In certain aspects, the disclosure provides methods for treating an angiogenesis related disease of the eye. Such methods may comprise administering systemically or to said eye a pharmaceutical formulation comprising: an effective amount of an agent selected from the group consisting of: an ALK1 ECD protein; an antibody that binds to an ALKI ligand and inhibits the finding of the ALKI ligand 20 to ALKI, wherein the ALK1 ligand is selected from th group consisting of GDF5, GDF6, GDF7, BMP9 and BMP 10; an antibody that binds to an ALK1 polypeptide consisting of amino acids 22-118 of SEQ ID NO; I and inhibits the binding of at least one ALKI ligand selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP 10; and a DAN polypeptide. 25 In each instance, an agent described herein may be administered in conjunction with a second agent that inhibits angiogenesis. Where it is desirable to inhibit angiogenesis of a tumor, the agent may be administered in conjunction with a second agent that has an anti-cancer effect, such as a chemotherapeutic agent or a biologic anti-cancer agent. 30 The disclosure also provides an ophthalmic pharnmaceutical formulation comprising an ALK I-Fc fusion protein having an amino acid sequence that is at 10 least 97% identical to the sequence of amino acids 2 2 -11 of SEQ ID NO:1, which polypeptide is fused to an Fc portion of an immunoglobulin, and wherein the ALK 1 Fe fusion protein binds to ODF5, GDF7 and BMP9 with a KnD of less than I x 10-7 M and binds to TGFp- 1 with a KD of greater than 1 x 106, In one embodiment, the 5 fusion protein has the sequence of SEQ ID NO: 3. In one embodiment, the Fc portion is from human IgGl. In one embodiment, the fusion protein is produced by expression of the nucleic acid of SEQ ID NO:4 in a mammalian cell line. In one embodiment, the cell line is Chinese Hamster Ovary cell line. The formulation may further comprise one or more of the following medicanents: pegaptanib, 10 ranibizumab, or a glucocorticoid. In one embodiment, the formulation is substantially pyrogen free. The application also provides for an ophthalmic pharmaceutical formulation comprising an antibody that binds to an ALKi polypeopide consisting of amino acids 22-118 of SEQ ID NO: 1 and.inhibits the binding of at least one ALKI ligand 15 selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10. In one embodiment, the antibody inhibits angiogenesis stimulated by at least one ALKI ligand selected from the group consisting of: GDF5, GDF6 and GDF7. In one embodiment, the antibody binds to the ALK I polypeptde with a K 0 of less than 5 x 10-8 M. In another embodiment, the antibody binds to the ALKI polypeptide with a 20 KD of less than I x 10~ M. In one embodiment, the antibody inhibits angiogenesis stimulated by GDF5, GDF6, GDF7, BMP9, or BMP1O The formulation may further comprise one or more of the following medicaments: pegaptanib, ranibizumab, or a glucocorticoid. In one embodiment, the formulation is substantially pyrogen free. In certain aspects, the disclosure provides for an ophthalmic pharmaceutical 25 formulation comprising an antibody that binds to an ALKI ligand disclosed herein and inhibits the binding of the ALKI ligand to ALKI. Ta certain embodiments, the antibody binds to a ligand selected from the group consisting of GDF5, GDF6 and GDF7. The antibody may bind to the ALKl ligand with a KO of less than 5 x 10* M. The antibody may be selected for inhibition of angiogenesis stimulated by the 30 ALKI ligand. A CAM assay is an appropriate assay system for selection of desirable antibodies. Such antibodies are preferably recombinant antibodies. The formulation may further comprise one or more of the following medicaments: 11 pegaptanib, ranibizumab, or a glucocorticoid. In one embodiment, the formulation is substantially pyrogen free. The application also provides methods of treating an angiogenesis related disease of the eye comprising administering to said eye an ophthalmic 5 pharmaceutical formulation comprising an ALKI-Fc fusion protein comprising: a polypeptide having an amino acid sequence that is at least 97% identical to the sequence of amino acids 22-118 of SEQ ID NO:1, which polypeptide is fused to an Fe portion of an immunoglobulin, and wherein the ALIYl-Fc fusion protein binds to GDF5, -GDF7 and BMP9 with a KD of less than 1 x 1 0M and binds to TGFp-1 10 with a KD of greater than I x 10.6.. In one embodiment, the fusion protein has the sequence of SEQ ID NO: 3. In one embodiment, the Fc portion is from human IgG 1. In one embodiment, the fusion protein is produced by expression of the nucleic acid of SEQ ID NO:4 in a mammalian cell line. In one embodiment, the cell line is Chinese Hamster Ovary cell line. The formulation may further comprise one or more 15 of the following medicaments: pegaptanib, ranibizumab or a glucocorticoid. In one embodiment, the formulation is substantially pyrogen flee. The application also provides methods of treating an angiogenesis related disease of the eye comprising administering to said eye an ophthalmic pharmaceutical formulation comprising an antibody that binds to an ALKI 20 polypeptide consisting of amino acids 22-118 of SEQ ID NO: 1 and inhibits the binding of at least one ALKI ligand selected from the goup consisting of: GDF5, GDF6, GDF7, BMP9 and BMP 10. In one embodiment, the antibody inhibits angiogenesis stimulated by at least one ALK 1 ligand selected from the group consisting of: GDF5, GDF6 and GDF7. In one embodiment, the antibody binds to 25 the ALK I polypeptide with a KD of less than 5 x IO~ N. hi another embodiment, the antibody binds to the ALKI polypeptide with a KD of less than I x 10-10 M. In one embodiment, the antibody inhibits angiogenesis stimulated by GDF5, GDF6, GDF7, BMP9, or BMP10. The formulation may further comprise one or more of the following medicaments: pegaptanib, ranibizumab, or aglucocorticoid. In one 30 embodiment, the formulation is substantially pyrogen free. In one embodiment of the disclosed methods, the angiogenesis related disease of the eye is selected from the group consisting of a tumor, a tumor that is 12 resistant to anti-VEOF therapy, a multiple myeloma tuor, a tumor that has metastasized to the bone, joint or bone inflammation, rituMatoid arthritis, diabetic retinopathy, retinopathy of prematurity, macular degeneration, comeal graft rejection, neovascular glaucoma, and retrolental fibrophsias, 5 BRIEF DESCRIPTION OF THE DRAWINGS Figure I shows the amino acid sequence for the kunan Activin Like Kinase 1, ALKI (SEQ ID NO:). Single underlining shows the predicted extracellular domain. Double underlining shows the intracellular donain, The signal peptide and 10 the transmembrane domain are not underlined. Figure 2 shows the nucleic acid sequence of a hunan ALK1 cDNA (SEQ ID NO:2). The coding sequence is underlined. The portion encoding the extracellular domain is double underlined. Figure 3 shows an example of a fusion of the extracellular domain of human 15 ALKI to an Fe domain (SEQ ID NO:3). The hALKI-Fo protein includes amino acids 22-120 of the human ALKI protein, fused at the C-terminus to a linker (underlined) and an IgGi Fe region. Figure 4 shows the nucleic acid sequence for expression of the hALKI-Fc polypeptide of SEQ ID NO:3. The encoded amino acid sequence is also shown. 20 The leader sequence is cleaved such that Asp 22 is the N-terminal amino acid of the secreted protein. Figure 5 shows the anti-angiogenic effect of marine ALK-Fc CRAP") and human ALKI-Fe ("ACE") in an endothelial cell tube forming assay. All concentrations of RAP and ACE reduced the level of tube formation in response to 25 Endothelial Cell Growth Supplement (ECGF) to a greater degree than the positive control, Endostatin. Figure 6 shows the angiogenic effect of GDF7 it a chick chorioallantoic membrane (CAM) assay. The GDF7 effect is comparable to that of VEGF. 13 Figure 7 shows the anti-angiogenic effect of the mnan ALKI-Fc fusion in the CAM assay. hALK1-Fc inhibits angiogenesis stimulated by VEGF, FGF and GDF7. Figure 8 shows comparative anti-angiogenic effects of urine ALKI-Fe 5 (mALK I -Fe), hALK 1-Fe, a commercially available anti-AK1 monoclonal antibody (Anti-ALKI mAb) and a commercially available, neutralizing anti-VEGF monoclonal antibody. The anti-angiogenic effect of the ALKl-Fc constructs is comparable to the effects of the anti-VEGF antibody. Figure 9 shows the anti-angiogenic effects of hALK I -Fc and the anti-VEGF 10 antibody in vivo. hALK 1-Fc and anti-VEGF had compuable effects on angiogenesis in the eye as measured by the mouse corneal micropocket assay. Figure 10 shows the effects of mALKI -Fc in thu urine collagen-induced arthritis (CIA) model of rheumatoid arthritis, The graph slows mean group arthritic scores determined during the 42 day observation period ia. the collagen-induced 15 male DBA/l arthritic mice. RAP-041 is mALKI-Fe. AVastiJ is the anti-VEGF antibody bevacizumab. DETAILED DESCRIPTION 1. Overview 20 ALKI is a type I cell-surface receptor for the TGF- p superfamily of ligands and is also known as ACVRLI and ACVRLKI. ALKI has been implicated as a receptor for TGF-0j1, TGF- p3 and BMP-9 (Marchuk et aL, Hum Mol Genet. 2003; Brown et al., J Biol Chem. 2005 Jul 1;280(26):25111 -8). In mice, loss-of-function mutations in ALKI lead to a variety of 25 abnormalities in the developing vasculature (Oh et al., Proc. Natl Acad. Sci. USA 2000, 97, 2626-2631; Urness et al., Nat. Genet. 2000,26, 328-331). In humans, loss-of-function mutations in ALKl are associated with hereditary hemorrhagic telangiectasia (HHT, or Osler-Rendu-Weber syndrome), in which patients develop arteriovenous malformations tiat create direct flow 30 (communication) from an artery to a vein (arteriovenous shunt), without an 14 intervening capillary bed. Typical symptoms of patiens with HHT include recurrent epistaxis, gastrointestinal hemorrhage, cutaneous and mucocutaneous telangiectases, and arteriovenous malformations (AVM) in the pulmoary, cerebral, or hepatic vasculature. 5 Recent publications from David et al. (Blood. 27 Mar 1;109(5):1953-61.) and Scharpfenecker et al. (J Cell Sci. 2007 Mar 15; 120(Pt6):964-72) conclude that BMP9 and BMP 10 activate ALKI in endothelial cells, and that the consequence of this activation is to inhibit endothelial cell proliferation and migration. These effects are directly opposed to those of pro-angiogenic factors such as VEGF. Thus, these 10 publications conclude that BMP9 dind BMP 10 are thernselves anti-angiogenic factors, and further, that ALKI activation has an anti-angiogenic effect. By contrast, the present disclosure demonstrates that antagonists, rather than agonists, of BMP9 and BMP 10 have anti-angiogenic effects. The disclosure relates to the discovery that polypeptides comprising a 15 portion of the extracellular domain of ALK 1 ("ALK 1 BCD polypeptides") may be used to inhibit angiogenesis in vivo, including VEGF-independent angiogenesis and angiogenesis that is mediated by multiple angiogenic factors, including VEGF, FGF and PDGF. In part, the disclosure provides the identity of physiological, high affinity ligands for ALKI and demonstrates that ALK ECD polypeptides inhibit 20 angiogenesis. The data demonstrate that an ALK I ECD polypeptide can exert an anti-angiogenic effect even in the case where the ALKl ECD polypeptide does not exhibit meaningful binding to TGF-P 1. Moreover, ALMl ECD polypeptides inhibit angiogenesis that is stimulated by many different pro-angiogenic factors, including VEGF, FGF, and GDF7. Thus, the disclosure provides a description of an ALK I 25 regulatory system, in which ALKI is a receptor for the GDF5 group of ligands, which includes GDF6 and GDF7, and also for the BlvP9 group of ligands, which includes BMP 10, with different affinities for the two groups of ligands. Further, the disclosure demonstrates that signaling mediated by ALKI and the ligands described above is pro-angiogenic in vivo, and that inhibition of this regulatory system has a 30 potent anti-angiogenic effect in vivo. Thus, in certain aspects, the disclosure provides antagonists of the ALK I regulatory system, including antagonists of the receptor or one or more of the ligands, for use in inhibiting angiogenesis, including 15 both VEGF-dependent angiogenesis and VEGF-indeplent angiogenesis. However, it should be noted that antibodies directed to ALK1 I itself are expected to have different effects from an ALKI ECD polypeptide, A pan-neutralizing antibody against ALK1 (one that inhibits the binding of all strong and weak ligands) would be 5 expected to inhibit the signaling of such ligands through ALKI but would not be expected to inhibit the ability of such ligands to signal through other receptors (e.g., BMPR1a, BMPR1b, BMPRII in the case of GDF5-7 ad BMP9-10 and TBRI and TBRH in the case of TGFp). On the other hand, an ALK I ECD polypeptide would be expected to inhibit all of the ligands that it binds to igltly, including, for a 10 construct such as that shown in the Examples, GDF5-land BMP9-10, but would not affect ligands that it binds to weakly, such as TGF-p. So, while a pan-neutralizing antibody against ALKT would block BMP9 and TGF-p signaling through ALKI it would not block BMP9 and TGF-P signaling through mother receptor, and while an ALK I ECD polypeptide may inhibit BMP9 signaling though all receptors (even 15 receptors other than ALKI) it would not be expected to inhibit TGF-p signaling through any receptor, even ALK 1. Proteins described herein are the human forms,unless otherwise specified. Genbank references for the proteins are as follows: huwan GDF5, CAA56874; human GDF6, AAH43222; human GDF7, NP_878248; human BMP9, Q9UK05; 20 human BMP10, 095393; human DAN, BAA92265. ALK1 sequences are set forth in Figures 1-5. Human Dan amino acid sequence (SEQ ID NO:10) (Genbank BAA92265): MLRVLVGAVL PAMLLAAPPP INKLALFPDK SAWCEAKNIT QIVGHSGCEA KSIQNRACLG QCFSYSVPNT FPQSTESLVH .CDSCMPAQSM WEIVTLECPG HEEVPRVDKL VEKILHCSCQ 25 ACGKEPSHEG LSVYVQGEDG PGSQPGTHPH PHPHPHPGGQ TPEPEDPPGA.PHTEEEGAED The mature Dan protein is expected to correspond to amino acids 17-180. The conserved cysteine knot domain of Dan corresponds to amino acids 21-125 (underlined). Human Dan cDNA sequence (SEQ ID NOdl l)(Geabank BC012037): 30 gccgagcctc ctqgggcgcc ogggcccgcg acccccgcac ccagctccgc aggaccggcg ggcgcgcgCg ggctctggag gccacgggca tgatgcttcg ggtcctggtg ggggctgtcc 16 tccctgccat gctactggct gccccaccac ccatcaacaa gctggcaCtg ttcccagata agagtgcctg gtgcgaagcc aagaacatca occagatcgt gggccacag ggctgtgagg ccaagtccat ccagaacagg gcgtgcctag gacagtgctt cagctacagc gtccccaaca ccttcccaca gtccacagag tccctggttc actgtgactc ctgcatgcca gcccagtcca 5 tgtgggagat tgtgacgctg gagtgcccgg gccacgagga qggoccagg gtggacaagC tggtggagaa gatcctgcac tgtagctgcc aggcctgcgg Oaaggagcct agtcacgagg ggctgagcgt ctatgtgcag ggcgaggacg ggccgggatc ceagccggC acccaccctc acecccatcc ccacccccat cctggcgggc agacccctga qccCgaggac ccccctgggg ccccccacac agaggaagag ggggctgagg actgaggccc ccccaactct tCcteccctc 10 tcatccccct gtggaatgtt gggtotcact ctctggggaa qtcaggggag aagctgaagc ccccctttgg cactggatgg acttggcttc agacteggac ttgaatgctg cccggttgcc atggagatct gaaggggcgg ggttagagcc aagctgcaoa atttaatata ttcaagagtg gggggaggaa gcagaggtct tcagggctct ttttttgggg ggggggtggt ctcttcctgt ctggcttcta gagatgtgcc tgtgggaggg ggaggaagtt gectgagcca ttgagtgctg 15 ggggaggcCa tccaagatgg catgaatcgg gctaaggtcc ctgggggtgc agatggtact gctgaggtoc cgggcttagt gtgagcatct tgccagcctc aggcttgagg gagggctggg ctagaaagac cactggcaga aacaggaggc tccggcccca caggtttccC caaggcctct caccccactt cccatctcca gggaagcgtc gccccagtgg cactgaagtg gccctccctc agcggagggg tttgggagtc aggcctgggc aggaccctgc tgactcgtgg cgcgggagct 20 gggagccagg ctctccqggc ctttctctgg cttocttggc ttgcotggtg ggggaagggg aggaggggaa gaaggaaagg gaagagtctt ccaaggccag aeggaggggg acaacccccc aagaccatoc ctgaagacga gcatctccct cctctccctg ttagaaatgt tagtgccccg cactgtgccc caagttctag gccccccaga aagctgtcag aqccggccgC cttctcccct ctcccaggga tgotctttgt aaatatogga tgggtgtggg agtgaggggt tacctccetc 25 gccccaaggt tccagagjcc ctaggcggga tgggctagct gaacctcgag gaactccagg acgaggagga catgggactt gcgtggacag tcagggttca cttgggctct ctctagctcc ccaattctgc ctgcctcdtc octcccagot gcactttaac ectagaaggt ggggacctgg ggggagggac agggcagqgcg ggoccatgaa gaaagcocct cgttgcccag cactgtctgc gtctgctctt ctgtgcccag ggtggctgcc agcccactgc tcctgcctg gggtggcctg 30 gccctcctgg ctgttgcgac gcgggcttct ggagcttgtc accattggaC agtctccctg atggaccctc agtcttctca tgaataaatt ccttcaacgc caaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa The coding sequence for DAN precursor corresponds to nucleic acids 93 35 635, The coding sequence for the mature DAN protein corresponds to nucleic acids 141-632. The coding sequence for the conserved cysteine knot portion of DAN corresponds to nucleic acids 153-467, The terms used in this specification generally lave their ordinary meanings in the art, within the context of this disclosure and in the specific context where each 40 term is used. Certain terms are discussed in the specification, to provide additional guidance to the practitioner in describing the compositions and methods disclosed herein and how to make and use them. The scope or meaning of any use of a term will be apparent from the specific context in which the term is used. 45 17 2. Soluble ALKI Polypeptides Naturally occurring ALKI proteins are transmenbrane proteins, with a portion of the protein positioned outside the cell (the extricelluar portion) and a portion of the protein positioned inside the cell (the intacellular portion). Aspects of 5 the present disclosure encompass polypeptides comprising a portion of the extracellular domain of ALK . In certain embodiments, the disclosure provides"ALKI ECD polypeptides". The term "ALK 1 ECD polypeptide". is intended to refer to a polypeptide consisting of or comprising an amino acid sequence of an extracellular domain of a naturally 10 occurring ALK1 polypeptide, either including or excluding my signal sequence and sequence N-terminal to the signal sequence, or an amino acid sequence that is at least 33 percent identical to an extracellular domain of a naturally occurring ALKI polypeptide, and optionally at least 60%, at least 70%, at I east 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 1OVo identical to the sequence 15 of an extracellular domain of a naturally occurring AL( I polypeptide, as exemplified by the cysteine knot region of amino acids 3C-95 of SEQ ID No: 1 or the cysteine knot plus additional amino acids at the N- and C-termnini of the extracellular domain, such as amino acids 22-118 of SEQ ID No. 1. Likewise, an ALKI ECD polypeptide may comprise a polypeptide that is encoded by nucleotides 100-285 of 20 SEQ ID NO:2, or silent variants thereof or nucleic acids that hybridize to the complement thereof under stringent hybridization conditions (generally, such conditions are known in the art but may, for example, involve hybridization in 50% v/v formamide, 5x SSC, 2% w/v blocking agent, 0.1%N-lauroylsarcosin, 0.3% SDS at 65 C" overnight and washing in, for example, SaSSC at about 65 C 0 ). 25 Additionally, an ALKI ECD polypeptide may comprise a polypeptide that is encoded by nucleotides 64-3 84 of SEQ ID NO:2, or silent variants thereof or nucleic acids that hybridize to the complement thereof under stringent hybridization conditions (generally, such conditions are known in the art but may, for example, involve hybridization in 50% v/v formamide, 5x SSC,2% w/v blocking agent, 0.1% 30 N-lauroylsarcosine, 0.3% SDS at 65 C" overnight and washing in, for example, 5xSSC at about 65 C"). The term "ALK I BCD polypeptide" accordingly encompasses isolated extracellular portions of ALKI polypeptides, variants thereof 18 (including variants that comprise, for example, no more than 2, 3, 4, 5 or 10 amino acid substitutions, additions or deletions in the sequence corresponding to amino acids 22-118 of SEQ ID NO; 1 and including variants that comprise no more than 2, 3, 4, 5, or 10 amino acid substitutions, additions or deletions in the sequence 5 corresponding to amino acids 34-95 of SEQ ID NO: 1), fragments thereof and fusion proteins comprising any of the preceding, but in each case preferably any of the foregoing ALK I ECD polypeptides will retain substantial affinity for one or more of GDF5, GDF6, GDF7, BMP9 or BMP10. The term "ALKI ECD polypeptide" is explicitly intended to exclude any full-length, naturally occurring ALKI 10 polypeptide. Generally, an ALKI ECD polypeptide will be designed to be soluble in aqueous solutions at biologically relevant temperatures, pH levels and osmolarity. As described above, the disclosure provides ALI ECD polypeptides sharing a specified degree of sequence identity or similarity to a naturally occurring ALK I polypeptide, To determine the percent identity or two amino acid sequences, 15 -the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by 20 the same amino acid residue as the corresponding position in the second sequence, then the molecules are identical at that position (as usedherein amino acid "identity" is equivalent to amino acid "homology"). The percent identity between the two sequences is a function of the number of identical positbas shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be 25 introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm. (Computational Molecular Biology, Lesk, A M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. 30 W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; 19 and Sequence Analysis Primer, Gribskov, M. and Devereux, L, eds., M Stockton Press, New York, 1991). In one embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J Mol. Biol. (48);444-453 (1970)) 5 algorithm which has been incorporated into the GAP program in the GCO software package (available at http://www.gcg.com). In a specific embodiment, the following parameters are used in the GAP program: either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another embodiment, the percent identity between two nucleotide 10 sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res. 12(t):387 (1984)) (available at http://www.gg.com). Exemplary parameters include using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Unless otherwise specified, percent identity between two amino acid 15 sequences is to be determined using the GAP program using a Blosum 62 matrix, a GAP weight of 10 and a length weight of -3, and if such algorithm cannot compute the desired percent identity, a suitable alternative disclosed herein should be selected. In another embodiment, the percent identity between two amino acid 20 sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penity of 12 and a gap penalty of 4. Another embodiment for determining the best overall alignment between two 25 amino acid sequences can be determined using the FASTDB computer program based on the algorithm of Brutlag et al (Comp. App. Biosci., 6:237-245 (1990)). In a sequence alignment the query and subject sequences are both amino acid sequences. The result of said global sequence alignment is presented in terms of percent identity. In one embodiment, amino acid sequence identity is performed using the 30 FASTDB computer program based on the algorithm of 3rutlag et al (Comp. App. Biosci, 6:237-245 (1990)). In a specific embodiment, parameters employed to calculate percent identity and similarity of an amino acid alignment comprise: 20 Matrix=PAM 150, k-tuple=2, Mismatch Penalty=1, Joining Penalty20, Randomization Group Length=0, Cutoff Score=l, Gap penalty=5 and Gap Size Penalty=0.05. In certain embodiments, ALKJ ECD polypeptides comprise an extracellular 5 portion of a naturally occurring ALKI protein such as a sequence of SEQ ID NO:1, and preferably a ligand binding portion of the ALK1 extracellular domain. In certain embodiments, a soluble ALKI polypeptide comprises an amino acid sequence that is al least 60%, 70%, 80%, 85%, 90%, 95%, 97% or 99% identical to an amino acid sequence of amino acids 22-118 of the SEQ ID NO:l. In certain 10 embodiments, a truncated extracellular ALK1 polypeptide comprises at least 30, 40 or 50 consecutive amino acids of an amino acid sequence of an extracellular portion of SEQ ID NO:1. In preferred embodiments, an ALKI ECD polypeptide binds to one or more of GDF5, GDF6, GDF7, BMP9 and BMP1O. Optionally the ALK polypeptide 15 does not show substantial binding to TGF-pI or TGF-p3. Binding may be assessed using purified proteins in solution or in a surface plasmon resonance system, such as a BiacoreTm system, Preferred soluble ALKI polypeptides will exhibit an anti angiogenic activity. Bioassays for angiogenesis inhibitory activity include the chick chouioallantoic membrane (CAM) assay, the mouse coneal micropocket assay, an 20 assay for measuring the effect of administering isolated or synthesized proteins on implanted tumors. The CAM assay is described by O'Rcilly, et al. in "Angiogenic Regulation of Metastatic Growth" Cell, vol. 79 (2), Oct.1, 1994, pp. 315-328. Briefly, 3 day old chicken embryos with intact yolks are separated from the egg and placed in a petri dish. After 3 days of incubation, a metliylcellulose disc containing 25 the protein to be tested is applied to the CAM of individal embryos. After 48 hours of incubation, the embryos and CAMs are observed to determine whether endothelial growth has been inhibited. The mouse corneal micropocket assay involves implanting a growth factor-containing pellet, along with another pellet containing the suspected endothelial growth inhibitor, in the comea of a mouse and 30 observing the pattern of capillaries that are elaborated in the cornea. Other assays are described in the Examples. 21 ALK I ECD polypeptides may be produced by removing the cytoplasmic tail and the transmembrane region of an ALKI polypeptide, Alternatively, the transmembrane domain may be inactivated by deletion, or by substitution of the normally hydrophobic amino acid residues which comprise a transmembrane 5 domain with hydrophilic ones. In either case, a substantially hydrophilic hydropathy profile is created which will reduce lipid affinity and improve aqueous solubility. Deletion of the transmembrane domain is preferred over substitution with hydrophilic amino acid residues because it avoids introducing potentially immunogenic epitopes. 10 ALKi ECD polypeptides may additionally inchde any number of well known leader sequences at the N-terminus. Such a sequence would allow the peptides to be expressed and targeted to the secretion pathway in a eukaryotic system. See, e.g., Ernst et al., U.S. Pat. No. 5,082,783 (1992). Alternatively, a native ALKI signal sequence may be used to effect extrusion from the cell. Possible 15 leader sequences include native, tPa and honeybee mellitin leaders (SEQ ID Nos. 7 9, respectively). Processing of signal peptides may vary depending on the leader sequence chosen, the cell type used and culture'conditions, amqng other variables, and therefore actual N-terminal start sites for mature AIK ECD polypeptides, including that of SEQ ID NO:5, may shift by 1-5 amino acids in either the N 20 terminal or C-terminal direction. In certain embodiments, the present disclosure contemplates specific mutations of the ALK I polypeptides so as to alter the glycosylation of the polypeptide. Such mutations may be selected so as to introduce or eliminate one or more glycosylation sites, such as 0-linked or N-linked glycosylation sites. 25 Asparagine-linked glycosylation recognition sites generally comprise a tripeptide sequence, asparagine-X-threonine (or asparagines-X-serine) (where "X" is any amino acid) which is specifically recognized by appropriate cellular glycosylation enzymes. The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the wild-type ALKi 30 polypeptide (for O-linked glycosylation sites). A variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a glycosylation recognition site (and/or amino acid deletion at the second position) 22 results in non-glycosylation at the modified tripeptide sequence. Another means of increasing the number of carbohydrate moieties on an ALKI polypeptide is by chemical or enzymatic coupling of glycosides to the AIK 1 polypeptide. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and 5 histidine; (b) free carboxyl groups; (c) free sulthydryl groups such as those of cysteine; (d) free hydroxyl groups such as those of series, threonine, or hydroxyproline; (e) aromatic residues such as those of $henylalanine, tyrosine, or tryptophan; or (f) the aide group of glutamine. These methods are described in WO 87/05330 published Sep. 11, 1987, and in Aplin and Wriston (1981) CRC Crit. 10 Rev. Biochem., pp. 259-306, incorporated by referencelirein. Removal of one or more carbohydrate moieties present on an ALKi polypeptide may be accomplished chemically and/or enzymatically. Chemical deglycosylition may involve, for example, exposure of the ALKM polypeptide to the cornpaund trifluoromethanesulfonic acid, or an equivalent compomd.. This treatment results in 15 the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the amino acid sequence intact. Chemical deglycosylation is further described by Hakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and by Edge et al. (1981) Anal. Biochen. 118:131. Enzymatic cleavage of carbohydrate moieties on ALKI polypeptides can be achieved by the 20 use of a variety of endo- and exo-glycosidases as described by Thotakura et al. (1987) Meth. Enzymol. 138:350. The sequence of an ALKl polypeptide may be adjusted, as appropriate, depending on the type of exprssion system used, as mammalian, yeast, insect and plant cells may all introduce differing glycosylation patterns that can be affected by the amino acid sequence of the peptide. In general, 25 ALKI proteins for use in humans will be expressed in a mammalian cell line that provides proper glycosylation, such as HEK293 or CHO cell lines, although other mammalian expression cell lines, yeast cell lines with engineered glycosylation enzymes and insect cells are expected to be useful as vell This disclosure further contemplates a method of generating mutants, 30 particularly sets of combinatorial mutants of an ALKI polypeptide, as well as truncation mutants; pools of combinatorial mutants areespecially useful for identifying functional variant sequences. The purposeof screening such 23 combinatorial libraries may be to generate, for example, ALKI polypeptide variants which can act as either agonists or antagonist, or atternatively, which possess novel activities all together. A variety of screening assays are provided below, and such assays may be used to evaluate variants. For example, an ALKI polypeptide variant 5 may be screened for ability to bind to an ALKI ligand, to prevent binding of an ALKI ligand to an ALKI polypeptide or to interfere with signaling caused by an ALKI ligand. The activity of an ALK I polypeptide or its variants may also be tested in a cell-based or in vivo assay, particularly any of the assays disclosed in the Examples. 10 Combinatorially-derived variants can be generated which have a selective or generally increased potency relative to an ALKI ECD polypeptide comprising an extracellular domain of a naturally occurring ALKi polypeptide. Likewise, mutagenesis can give rise'to variants which have serum half-lives dramatically different than the corresponding a wild-type ALKI EC) polypeptide. For example, 15 the altered protein can be tendered either more stable od ess stable to proteolytic degradation or other processes which result in destruction of, or otherwise elimination or inactivation of a native ALKI ECD polypeptide. Such variants, and the genes which encode them, can be utilized to alter ALKl ECD polypeptide levels by modulating the half-life of the ALKI polypeptides. For instance, a short half-life 20 can give rise to more transient biological effects and can allow tighter control of recombinant ALKI ECD polypeptide levels within the patient. In an Fc fusion protein, mutations may be made in the linker (if any) ad/cr the Fc portion to alter the half-life of the protein. A combinatorial library may be produced by way of a degenerate library of 25 genes encoding a library of polypeptides which each include at least a portion of potential ALKI polypeptide sequences. For instance, araixture of synthetic oligonucleotides can be enzymatically ligated into genesequences such that the degenerate set of potential ALKI polypeptide nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., 30 for phage display). 24 There are many ways by which the library of potential ALKI ECD variants can be generated from a degenerate oligonucleotide sequenc. Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes then be ligated into an appropriate vector for expression. 5 The synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et a., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp 2 73

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28 9 ; Itakura et al., (1984) Annu, Rev. Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 11:477). Such 10 techniques have been employed in the directed evolution cf other proteins (see, for example, Scott et al., (1990) Science 249:386-390; Roberts et al., (1992) PNAS USA 89:2429-243 3; Devlin et al., (1990) Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; as well as U.S. Pateat Nos: 5,223,409, 5,198,346, and 5,096,815). 15 Alternatively, other forms of mutagenesis can be utilized to generate a combinatorial library. For example, ALKI polypeptideyvariants can be generated and isolated from a library by screening using, for example, alanine scanning mutagenesis and the like (Ruf et al., (1994) Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem. 269:3095-3099; Balint et al.,(1993) Gene 137:109-118; 20 Grodberg et al., (1993) Eur. J. Biochem. 218:597-601; gagashima et al., (1993) J. Biol. Chem. 268:2888-2892; Lowman et al., (1991) Bioclnemistry 30:10832-10838; and Cunningham et al., (1989) Science 244:1081-1085), by linker scanning mutagenesis (Gustin et al., (1993) Virology 193:653-660; Brown et al., (1992) Mol. Cell Biol. 12:2644-2652; McKnight et al., (1982) Science 232:316); by saturation 25 mutagenesis (Meyers et al., (1986) Science 232:613); by PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol 1:11-19); or by random mutagenesis, including chemical mutagenesis, etc. (Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY; and Greener et al., (1994) Strategies in Mol Biol 7:32-34). Linker scanning mutagenesis, particularly in a 30 combinatorial setting, is an attractive method for identifying truncated (bioactive) forms of ALKI polypeptides. 25 A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations and truncations, and, for that matter, for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries 5 generated by the combinatorial mutagenesis of ALKI polypeptides. The most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy 10 isolation of the vector encoding the gene whose product was detected. Preferred assays include ALKI ligand binding assays and ligand-nediated cell signaling assays. In certain embodiments, the ALKI ECD polypeptides of the disclosure may further comprise post-translational modifications in addition to any that are naturally 15 present in the ALKI polypeptides. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. As a result, the modified ALKI ECD polypeptides may contain non amino acid elements, such as polyethylene glycols, lipids, poly- or mono-saccharide, and phosphates. Effects of such non-amino acid elemerts on the functionality of an 20 ALKI ECD polypeptide may be tested as described hereim for other ALKI ECD polypeptide variants. When an ALK I ECD polypeptide is produced in cells by cleaving a nascent form of the ALK1 polypeptide, post-tanslational processing may also be important for correct folding and/or function of the protein. Different cells (such as CHO, HeLa, NO1DCK, 293, W138, NIH-3T3 or HEK293) have specific 25 cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the ALKI polypeptides. In certain aspects, functional variants or modified forms of the ALK1 ECD polypeptides include fusion proteins having at least a portion of the ALK I ECD 30 polypeptides and one or more fusion domains. Well lown examples of such fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, a immunoglobulin heavy 26 chain constant region (Fe), maltose binding protein (MP), or human serum albumin. A fusion domain may be selected so as to confer a desired property. For example, some fusion domains are particularly useful for isolation of the fusion proteins by affinity chromatography. For the purpose of affinity purification, 5 relevant matrices for affinity chromatography, such as glutathione-, amylase-, and nickel- or cobalt- conjugated resins are used. Many of such matrices are available in "kit" form, such as the Pharmacia GST purification system and the QlAexpressim system (Qiagen) useful with (HIS 6 ) fusion partners. As another example, a fusion domain may be selected so as to facilitate detection of the ALKI ECD polypeptides. 10 Examples of such detection domains include the various fluorescent proteins (e.g., GFP) as well as "epitope tags," which are usually shortpeptide sequences for which a specific antibody is available. Well known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus haemagglutinin (HA), and c-myc tags. In some cases, lhe fusion domains have a 15 protease cleavage site, such as for Factor Xa or Thrombia which allows the relevant protease to partially digest the fusion proteins and thereby liberate the recombinant proteins therefrom. The liberated proteins can then beisolated from the fusion domain by subsequent chromatographic separation. Incertain preferred embodiments, an ALKI ECD polypeptide is fused with & domain that stabilizes the 20 ALKI polypeptide in vivo (a "stabilizer" domain). By"stabilizing" is meant anything that increases serum half fife, regardless of whether this is because of decreased destruction, decreased clearance by the kidney, or other pharmacokinetic effect. Fusions with the Fr portion of an immunoglobulin are known to confer desirable pharmacokinetic properties on a wide range of proteins. Likewise, fusions 25 to human serum albumin can confer desirable properties. Other types of fusion domains that may be selected include multimerizing (e.g., dimerizing, tetramerizing) domains and functional domains. As a specific example, the present disclosure provides a fusion protein comprising a soluble extracellular domain of ALKI fused to an Fc domain (e.g., 30 SEQ ID NO: 6). THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVPCVVVD (A) VSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLRGKEYKCK (A) VSNKAL 27 PVP IEKT ISKAKGQ PRE PQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN (A) HYT QKSLSLSPGK* Optionally, the Fe domain has one or more mutations at residues such as 5 Asp-265, lysine 322, and Asn-434. In certain cases, the mutant Fe domain having one or more of these mutations (e.g., Asp-265 mutation) has reduced ability of binding to the Fey receptor relative to a wildtype Fc domain. In other cases, the mutant Fe domain having one-or more of these mutations (e.g., Asn-434 mutation) has increased ability of binding to the MHC class I-related Fc-receptor (FcRN) 10 relative to a wildtype Fc domain. It is understood that different elements of the fusion proteins may be arranged in any manner that is consistent with the desired functionality. For example, an ALKI ECD polypeptide may be placed C-terminal to a heterologous domain, or, alternatively, a heterologous domain may be placed C-terminal to an 15 ALK 1 ECD polypeptide. The ALK I ECD polypeptide domain and the heterologous domain need not be adjacent in a fusion protein, and additional domains or amino acid sequences may be included C- or N-terminal to either domain or between the domains. As used herein, the term, "immunoglobulin Fe rgiona" or simply "Fe" is 20 understood to mean the carboxyl-terminal portion of an immunoglobulin chain constant region, preferably an immunoglobulin heavy chain constant region, or a portion thereof. For example,'an immunoglobulin Fc region may comprise 1) a CH domain, a CH2 domain, and a CH3 domain, 2) a CHI domain and a CH2 domain, 3) a CH I domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, or 5) a 25 combination of two or more domains and an immunoglobulin hinge region. In a preferred embodiment the immunoglobulin Fc region comprises at least an immunoglobulin hinge region a CH2 domain and a CH3 domain, and preferably lacks the CH1 domain. In one embodiment, the class of immunoglobulin from which the heavy 30 chain constant region is derived is IgG (Igy) (7 subclasses 1, 2, 3, or 4). Other classes of immunoglobulin, IgA (Iga), IgD (IgS), IgE (ge) and IgM (Igp), may be 28 used. The choice of appropriate immunoglobulin heavy chain constant region is discussed in detail in U.S. Pat. Nos. 5,541,087, and 5,726,044. The choice of particular immunoglobulin heavy chain constant region sequences from certain immunoglobulin classes and subclasses to achieve a paiticular result is considered to 5 be within the level of skill in the art. The portion of the DNA construct encoding the immunoglobulin Fe region preferably comprises at leas a portion of a hinge domain, and preferably at least a portion of a CH 3 domain of Fc y or the homologous domains in any of IgA, lgD, IgE, or IgM. Furthermore, it is contemplated that substitution or deletion of amino acids 10 within the immunoglobulin heavy chain constant regions may be useful in the practice of the methods and compositions disclosed hereia. One example would be to introduce amino acid substitutions in the upper CH2 legion to create an Fc variant with reduced affinity for Fe receptors (Cole et at. (199') .Immunol. 159:3613). In certain embodiments, the present disclosure makes available isolated 15 and/or purified forms of the ALK I ECD polypeptides, vhich are isolated from, or otherwise substantially free of (e.g., at least 80%, 90%, 95 %, 97% or 99% free of, other proteins and/or other ALKl ECD polypeptide spies. ALKI polypeptides will generally be produced by expression from recombinant nucleic acids. In certain embodiments, the disclosure includesnucleic acids encoding 20 soluble ALK I polypeptides comprising the coding sequence for an extracellular portion of an ALKI proteins. In further embodiments, this disclosure also pertains to a host cell comprising such nucleic acids. The host tell may be any prokaryotic or eukaryotic cell. For example, a polypeptide of the present disclosure may be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus 25 expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art. Accordingly, some embodiments of the present disclosure further pertain to methods of producing the ALKI ECD polypeptides. It has been established that an ALK 1-Fc fusion protein set forth in SEQ ID NO:3 and expressed in CHO cells has potent anti-angiogenic activity. 29 DAN polypeptides, including variants of wild type DAN, and fusion proteins containing DAN proteins may be generated and characterized as described above with respect to ALK 1 ECD proteins. 5 3. Nucleic Acids Encoding ALKi Polypeptides In certain aspects, the disclosure provides isolated and/or recombinant nucleic acids encoding any of the ALKI polypeptides (e.g., ALKI BCD polypeptides), including fragments, functional variants wd fusion proteins disclosed herein. For example, SEQ ID NO: 2 encodes'the naturally occurring human ALKI 10 precursor polypeptide, while SEQ ID NO: 4 encodes the precursor of an ALKI extracellular domain fused to an IgGI Fe domain. Thesubject nucleic acids may be single-stranded or double stranded. Such nucleic acids may be DNA or RNA molecules. These nucleic acids may be used, for example, in methods for making ALK I polypeptides or as direct therapeutic agents (e~g., in an antisense, RNAi or 15 gene therapy approach). In certain aspects, the subject nucleic acids encoding ALKI polypeptides are further understood to include nucleic acids that are variants of SEQ ID NO: 2 or 4. Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants. 20 In certain embodiments, the disclosure provides isolated or recombinant nucleic acid sequences that are at least 80%, 85%, 90/, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2 or 4. One of ordinary skill in the art will appreciate that nucleic acid sequences complementary to SEQ ID NO: 2 or 4, and variants of SEQ ID NO: 2 or 4 are also within the scope of this disclosure. In further 25 embodiments, the nucleic acid sequences of the disclosure can be isolated, recombinant, and/or fused with a heterologous nucleotide sequence, or in a DNA library. In other embodiments, nucleic acids of the disclosure also include nucleotide sequences that hybridize under highly stringent conditions to the nucleotide 30 sequence designated in SEQ ID NO: 2 or 4, complement sequence of SEQ ID NO: 2 30 or 4, or fragments thereof. As discussed above, one of ordinary skill in the art will understand readily that appropriate stringency conditions which promote DNA hybridization can be varied. One of ordinary skill in tie art will understand readily that appropriate stringency conditions which promote DNA hybridization can be 5 varied. For example, one could perform the hybridization at 6.0 x sodium chloride/sodium citrate (SSC) at about 45 *C, followedby a wash of 2.0 x SSC at 50 *C. For example; the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50 .C to a high stringency of about 0.2 x SSC at 50 *C. In addition, the temperature in the wash step can be increased from low 10 stringency conditions at room temperature, about 22 C, to high stringency conditions at about 65 "C. Both temperature and salt nay be varied, or temperature or salt concentration may be held constant while the other variable is changed. In one embodiment, the disclosure provides nucleic acids which hybridize under low stringency conditions of 6 x SSC at room temperature foilwed by a wash at 2 x 15 SSC at room temperature. Isolated nucleic acids which differ from the nucleic acids as set forth in SEQ ID NOs: 2 or 4 due to degeneracy in the genetic code are also within the scope of the disclosure. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or syonyms (for example, CAU 20 and CAC are synonyms for histidine) may result in "silent" mutations which do not affect the amino acid sequence of the protein. However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject proteins will exist among mammalian cells. Cne skilled in the art will appreciate that these variations in one or more nucleolides (up to about 3-5% of the 25 nucleotides) of the nucleic acids encoding a particular protein may exist among individuals of a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this disclosure. In certain embodiments, the recombinant nucleic acids of the disclosure may 30 be operably linked -to one or more regulatory nucleotide sequences in an expression construct. Regulatory nucleotide sequences will generally be appropriate to the host 31 cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Typically, said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding 5 sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are contemplated by the disclosure. The promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter. An expression construct may be 10 present in a cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome. In a preferred embodimet, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used. 15 In certain aspects disclosed herein, the subject caleic acid is provided in an expression vector comprising a nucleotide sequence encoding an ALK1 polypeptide and operably linked to at least one regulatory sequence. Regulatory sequences are art-recognized and are selected to direct expression of he ALKi polypeptide. Accordingly, the term regulatory sequence includes promoters, enhancers, and other 20 expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enymology, Academic Press, San Diego, CA (1990). For instance, any of a wide varety of expression control sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding an ALKI 25 polypeptide. Such useful expression control sequences, include, for example, the early and late promoters of SV40, tet promoter, adenovinu or cytomegalovirus immediate early promoter, RSV promoters, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd 30 coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast a-mating factors, the polyhedron promoter of the bacubvirus system and other 32 sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. it should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. 5 Moreover, the vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered. A recombinant nucleic acid included in the disclosure can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in 10 either prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian), or both. Expression vehicles for production of a recombinant ALK I polypeptide include plasmids and other vectors, For instance, suitable vectors include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in 15 prokaryotic cells, such as K coli. Some mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dti, pTk2, pRSVneo, pMSG, 20 pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-1), or 25 Epstein-Barr virus (pHEBo, pREP-derived and p205) an be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found below in the description of gene therapy delivery systems. The various methods employed in the preparation of the plasmids and in transformation of host organisms are well known in the art. For other suitable 30 expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 3rd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 2001). 33 In some instances, it may be desirable to express the recombinant polypeptides by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL94 1), pAcUW-derived vectors (such as pAcUWl), and pBlueBac-derived 5 vectors (such as the B-gal containing pBlueBac III). In a preferred embodiment, a vector will be designed for production of the subject ALKI polypeptides in CHO cells, such as a Pcnv-Script vector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen, Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wisc.). As will be apparent, the subject gene constructs can be 10 used to cause expression of the subject ALKI polypeptides in cells propagated in culture, e.g., to produce proteins, including fusion proteins or variant proteins, for purification. This disclosure also pertains to a host cell transfected with a recombinant gene including a coding sequence (e.g., SEQ ID NO: 2or 4) for one or more of the 15 subject ALK I polypeptides. The host cell may be any prokaryotic or eukaryotic cell. For example, an ALK 1 polypeptide disclosed herein may be expressed in bacterial cells such as E coli, insect cells (e.g., using abaculovirus expression systemn, yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art. 20 Accordingly, the present disclosure further pertains to methods of producing the subject ALKI polypeptides, including ALKi ECDpolypeptides. For example, a host cell transfected with an expression vector encoding an ALKi polypeptide can be cultured under appropriate conditions to allow expression of the ALKi polypeptide to occur. The ALKI polypeptide may be secreted and isolated from a 25 mixture of cells and medium containing the ALKI polypeptide. Alternatively, the ALKI1 polypeptide may be retained cytoplasmically oz in a membrane fraction and the cells harvested, lysed and the protein isolated. A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. The subject ALKI polypeptides can be isolated from cell culture medium, host 30 cells, or both, using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, 34 electrophoresis, immunoaffinity purification with antibodies specific for particular epitopes of the ALK 1 polypeptides and affinity purification with an agent that binds to a domain fused to the ALKI polypeptide (e.g., a protein A column may be used to purify an ALKI -Fc fusion), In a preferred embodiment, the ALKI polypeptide is a 5 fusion protein containing a domain which facilitates its purification. In a preferred embodiment, purification is achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenysepharose chromatography, size exclusion chromatography, and cation exchange ciromatography. The 10 purification could be completed with viral filtration and buffer exchange. In another embodiment, a fusion gene coding for a purification leader sequence, such as a poly-(His)/enterokinase cleavage site sequence at the N terminus of the desired portion of the recombinant ALKl polypeptide, can allow purification of the expressed fusion protein by affinity chromatography using a Ni 2 " 15 metal resin. The purification leader sequence can then be subsequently removed by treatment with enterokinase to provide the purified ALKi polypeptide (e.g., see Hochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al., PNAS USA 88:8972). Techniques for making fusion genes are well known. Essentially, the joining 20 of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger ended termini for ligation, restriction enzyme digestionto provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In Mother embodiment, the 25 fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in 30 Molecular Biology, eds, Ausubel et al., John Wiley & Sons: 1992). 35 Examples of categories of nucleic acid compounds that are antagonists of ALK 1, BMP9, BMP 10, GDF5, GDF6 or GDF7 include antisense nucleic acids, RNAi constructs and catalytic nucleic acid constructs. A nucleic acid compound may be single or double stranded. A double stranded compound may also include 5 regions of overhang or non-complementarity, where one or the other of the strands is single stranded. A single stranded compound may include regions of self complementarity, meaning that the compound forms a so-called "hairpin" or "stem loop" structure, with a region of double helical structure. A nucleic acid compound may comprise a nucleotide sequence that is complementary to a region consisting of 10 no more than 1000, no more than 500, no more than 250, no more than 100 or no more than 50, 35, 30, 25, 22, 20 or 18 nucleotides of the Full-length ALKI nucleic acid sequence or ligand nucleic acid sequence. The region of complementarity will preferably be at least 8 nucleotides, and optionally at least 10 or at least 15 nucleotides, and optionally between 15 and 25 nucleotides. A region of 15 complementarity may fall within an intron, a coding sepience or a noncoding sequence of the target transcript, such as the coding sequence portion. Generally, a nucleic acid compound will have a length of about 8 toabaut 500 nucleotides or base pairs in length, and optionally the length will be abovt 14 to about 50 nucleotides. A nucleic acid may be a DNA (particularly for use as an antisense), 20 RNA or RNA:DNA hybrid. Any one strand may incliule a mixture of DNA and RNA, as well as modified forms that cannot readily beclassified as either DNA or RNA. Likewise, a double stranded compound may be DNA:DNA, DNA:RNA or RNA:RNA, and any one strand may also include a mixture of DNA and RNA, as well as modified forms that cannot readily be classified as either DNA or RNA. A 25 nucleic acid compound may include any of a variety of modifications, including one or modifications to the backbone (the sugar-phosphate portion in a natural nucleic acid, including intemucleotide linkages) or the base portion (the purine or pyrimidine portion of a natural nucleic acid). An antisense nucleic acid compound will preferably have a length of about 15 to about 30 nucleotides and will often 30 contain one or more modifications to improve characteristics such as stability in the serum, in a cell or in a place where the compound is likely to be delivered, such as the stomach in the case of orally delivered compounds and the lung for inhaled 36 compounds. In the case of an RNAi construct, the strand complenentary to the target transcript will generally be RNA or modifications thereof, The other strand may be RNA, DNA or any other variation. The duplex portion of double stranded or single stranded "hairpin" RNAi construct will preferably have a length of 18 to 40 5 nucleotides in length and optionally about 21 to 23 nuclectides in length, so long as it serves as a Dicer substrate. Catalytic or enzymatic nueleic acids may be ribozymes or DNA enzymes and may also contain modified forms. Nucleic acid compounds may inhibit expression of the target by aboit 50%, 75%, 90% or more when contacted with cells under physiological conditios and at a concentration 10 where a nonsense or sense control has little or no effect. Preferred concentrations for testing the effect of nucleic acid compounds are 1,5 and 10 micromolar. Nucleic acid compounds may also be tested for effects on, for example, angiogenesis. Nucleic acids encoding DAN polypeptides, including variants of wild type DAN, and those encoding fusion proteins containing DAN proteins may be 15 generated and characterized as described above with respect to nucleic acids encoding ALKI ECD proteins. 4. Antibodies Another aspect of the disclosure pertains to an antibody reactive with an 20 extracellular portion of an ALK 1 polypeptide, preferably antibodies that are specifically reactive with ALK I polypeptide. In a prefrred embodiment, such antibody may interfere with ALKi binding to a ligand such as GDF5, GDF6, GDF7 BMP-9 or BMP- 10 - it will be understood that an antibody against a ligand of ALKI should bind to -the mature, processed form of the relevant protein. The 25 disclosure also provides antibodies that bind to GDF5,GDF6, GDF7, BMP9 and/or BMP 10 and inhibit ALK 1 binding to such ligands. Preferred antibodies will exhibit an anti-angiogenic activity in a bioassay, such as a CAM assay or corneal micropocket assay (see above). The term "antibody" as used herein is intended to include whole antibodies, 30 e.g., of any isotype (Igo, IgA, IgM, IgE, etc), and includes fragments or domains of immunoglobulins which are reactive with a selected antigen, Antibodies can be 37 fragmented using conventional techniques and the fragments screened for utility and/or interaction with a specific epitope of interest, Tus, the term includes segments of proteolytically-cleaved or recombinantly-preparCd portions of an antibody molecule that are capable of selectively reacting with a certain protein. 5 Non-limiting examples of such proteolytic and/or recomabinant fragments include Fab, F(ab')2, Fab', Fv, and single chain antibodies (scFv) containing a V[L] and/or V[H] domain joined by a peptide linker. The scFvs may be covalently or non covalently linked to form antibodies having two or more binding sites. The term antibody also includes polyclonal, monoclonal, or other purified preparations of 10 antibodies and recombinant antibodies. The term "recombinant antibody", means an antibody, or antigen binding domain of an imrnunoglobalin, expressed from a nucleic acid that has been constructed using the techniques of molecular biology, such as a humanized antibody or a fully human antibody developed from a single chain antibody. Single domain and single chain antibodies are also included within 15 the term "recombinant antibody". Antibodies may be generated by any of the various methods known in the art, including administration of antigen to an animal, administration of antigen to an animal that carries human immunoglobulin genes, or screening with an antigen against a library of antibodies (often single chain antibodies or antibody domains). 20 Once antigen binding activity is detected, the relevant portions of the protein may be grafted into other antibody frameworks, including full-lngth IgG frameworks. For example, by using immunogens derived from an ALKI rolypeptide or an ALKI ligand, anti-protein/anti-peptide antisera or monoclonal ralibodies can be made by standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by 25 Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal, such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide (e.g., a ALKI polypeptide or an antigenic fragment wheh is capable of eliciting an antibody response, or a fusion protein). Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well 30 known in the art. An immunogenic portion -(preferably an extracellular portion) of an ALK I polypeptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or 38 serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies. Following immunization of an animal with an antigeuic preparation of an ALKI polypeptide, anti-ALKI antisera can be obtained and, if desired, polyclonal 5 anti-ALKI antibodies can be isolated from the serum. To produce monoclonal antibodies, antibody-producing cells lymphocytess) can be harvested from an immunized animal and fused by standard somatic cell vision procedures with immortalizing cells such as myeloma cells to yield hybridoma cells. Such techniques are well known in the art, and include, for example, the hybridoma 10 technique (originally developed by Kohler and Milsteim (1975) Nature, 256: 495 497), the human B cell hybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies snd Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridoma cells can be screenedinmunochemically for 15 production of antibodies specifically reactive with a manmalian ALKI polypeptide of the present disclosure and monoclonal antibodies isdated from a culture comprising such hybridoma cells. The term antibody as used herein is intended to include fragments thereof which are also specifically reactive with one of the subject ALK polypeptides. 20 Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab) 2 fragments can be generated by treating antibody with pepsin. The resulting F(ab)2 fragment can be treated to reduce disvlfide bridges to produce Fab fragments. The antibody of the present disclosure is father intended to include 25 bispecific, single-chain, and chimeric and humanized molecules having affinity for an ALK1 polypeptide conferred by at least one CDR region of the antibody. In preferred embodiments, the antibody further comprises a label attached thereto and is able to be detected, (e.g., the label can be a radioisotope, fluorescent compound, enzyme or enzyme co-factor). 39 In certain preferred embodiments, an antibody of the disclosure is a recombinant antibody, particularly a humanized monoclonal antibody or a fully human recombinant antibody. The adjective "specifically reactive with" as used in reference to an antibody 5 is intended to mean, as is generally understood in the art, that the antibody is sufficiently selective between the antigen of interest (e.g. an ALKI polypeptide or an ALKi ligand) and other antigens that are not of interest that the antibody is useful for, at minimum, detecting the presence of the antigen of irtorest in a particular type of biological sample. In certain methods employing the antibody, a higher degree of 10 specificity in binding may be desirable. For example, am antibody for use in detecting a low abundance protein of interest in the presence of one or more very high abundance protein that are not of interest may perfbrn better if it has a higher degree of selectivity between the antigen of interest and other cross-reactants. Monoclonal antibodies generally have a greater tendency (as compared to polyclonal 15 antibodies) to discriminate effectively between the desired antigens and cross reacting polypeptides. In addition, an antibody that is effective at selectively identifying an antigen of interest in one type of biological sample (e.g. a stool sample) may not be as effective for selectively identifying the same antigen in a different type of biological sample (e.g. a blood sample). Likewise, an antibody that 20 is effective at identifying an antigen of interest in a purified protein preparation that is devoid of other biological contaminants may not be as effective at identifying an antigen of interest in a crude biological sample, such as a blood or urine sample. Accordingly, in preferred embodiments, the application provides antibodies that have demonstrated specificity for an antigen of interest ii a sample type that is likely 25 to be the sample type of choice for use of the antibody. One characteristic that influences the specificity of an antibody:antigen interaction is the affinity of the antibody for the antigen. Although the desired specificity may be reached with a range of different affinities, generally preferred antibodies will have an affinity (a dissociation constant) of about 10, 10, 10 1 30 or less. Given the apparently low binding affinity of TGFj for ALKI, it is expected that many anti-ALK I antibodies will inhibit TGFP binding. However, the GDF5,6,7 group of ligands bind with a Kr of approximately 5x10~"M and the BMP9,10 40 ligands bind with a KD of approximately I XI 10 M. Thus, antibodies of appropriate affinity may be selected to interfere with the signaling activities of these ligands. In addition, the techniques used to screen antibodies in order to identify a desirable antibody may influence the properties of the antibody obtained. For 5 example, an antibody to be used for certain therapeutic purposes will preferably be able to target a particular cell type. Accordingly, to obtain antibodies of this type, it may be desirable to screen for antibodies that bind to cells that express the antigen of interest (e.g. by fluorescence activated cell sorting). Likewise, if an antibody is to be used for binding an antigen in solution, it may be desirable to test solution 10 binding. A variety of different techniques are available for testing antibody:antigen interactions to identify particularly desirable antibodies, Such techniques include ELISAs, surface plasmon resonance binding assays (e.g. the Biacore binding assay, Bia-core AB, Uppsala, Sweden), sandwich assays (e.g.the paramagnetic bead system of IGEN International, Inc., Gaithersburg, Marylad), western blots, 15 immunoprecipitation assays and immunohistochemisty. 5. Alterations in antibodies and Fc-fusion proteins The application further provides antibodies, ALK I .Fc fusion proteins and DAN-Fc fusion proteins with engineered or variant Fe regions. Such antibodies and 20 Fc fusion proteins may be useful, for example, in modulating effector functions, such as, antigen-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Additionally, the modifications may improve the stability of the antibodies and Fc fusion proteins. Amino acid sequence variants of the antibodies and Fc fusion proteins are prepared by introducing appropriate nucleotide 25 changes into the DNA, or by peptide synthesis. Such variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibodies and Fc fusion proteins disclosed herein, Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The 30 amino acid changes also may alter post-translational processes of the antibodies and Fc fusion proteins, such as changing the number or position of glycosylation sites. 41 Antibodies and Fc fusion proteins with reduced effector function may be produced by introducing changes in the amino acid sequence, including, but are not limited to, the Ala-Ala mutation described by Bluestone et al. (see WO 94/28027 and WO 98/47531; also see Xu et a. 2000 Cell Immuno 12 0 0 ; 16-26). Thus in 5 certain embodiments, antibodies and Fc fusion proteins of the disclosure with mutations within the constant region including the Ala-Ala mutation may be used to reduce or abolish effector function. According to these embodiments, antibodies and Fe fusion proteins may comprise a mutation to an alanine at position 234 or a mutation to an alanine at position 235, or a combination thereof. In one 10 embodiment, the antibody or Fc fusion protein comprises an IgG4 framework, wherein the Ala-Ala mutation would describe a mutations) from phenylalanine to alanine at position 234 and/or a mutation from leucine to alanine at position 235. In another embodiment, the antibody or Fe fusion protein comprises an IgGI framework, wherein the Ala-Ala mutation would describe a mutation(s) from 15 leucine to alanine at position 234 and/or a mutation from leucine to alanine at position 235. The antibody or Fc fusion protein may alternatively or additionally carry other mutations, including the point mutation K322A in the CR2 domain (Hezareh et al. 2001 J Virol. 75: 12161-8). In particular embodiments, the antibody or Fe fusion protein may be 20 modified to either enhance or inhibit complement dependent cytotoxicity (CDC). Modulated CDC activity may be achieved by introducing one or more amino acid substitutions, insertions, or deletions in an Fe region (see, e.g., U.S. Pat. No. 6,194,551). Alternatively or additionally, cysteine residte(s) may be introduced in the Fe region, thereby allowing interchain disulfide bond fonnation in this region. 25 The homodimeric antibody thus generated may have improved or reduced internalization capability and/or increased or decreased complement-mediated cell killing. See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992), W099/51642, Dunan & Winter Nature 322: 738 40 (1988); U.S. Pat, No. 5,648,260; U.S. Pat. No. 5,624,821; and W094/29351. 42 6. Methods and compositions for modulating anginlsis and certain disorders The disclosure provides methods of inhibiting angiogenesis in a mammal by administering to a subject an effective amount of a an ALKI ECD polypeptide, such as an ALKI-Fe fusion protein, a DAN protein, such as a DAN-Fc fusion protein, or 5 an antibody disclosed herein, such as an antibody against GDF5, GDF6, GDF7, BMP9, BMP 10, or the ECD of ALK1, or nucleic acid antagonists (e.g., antisense or siRNA) of any of the foregoing hereafter collectively referred to as "therapeutic agents". The data presented indicate specifically that the anti-angiogenic therapeutic agents disclosed herein may be used to inhibit angiogenesis in the eye of a mammal. 10 It is expected that these therapeutic agents will also be useful in inhibiting angiogenesis in bones and joints, and in tumors, particularly tumors associated with bones and joints. Angiogenesis associated diseases include, but are not limited to, angiogenesis-dependent cancer, including, for example, solid tumors, blood born 15 tumors such as leukemias, and tumor metastases; benig tumors, for example henangiomas, acoustic neuromas, neurofibromas, traclomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis; rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; and angiofibroma. 20 In particular, polypeptide therapeutic agents of the present disclosure are useful for treating or preventing a cancer (tumor), and particularly such cancers as are known to rely on angiogenic processes to support growth. Unlike most anti angiogenic agents, ALK 1 ECD polypeptides affect angogenesis that is stimulated by multiple factors. This is highly relevant in cancers,where a cancer will 25 frequently acquire multiple factors that support tumor angiogenesis. Thus, the therapeutic agents disclosed herein will be particularly effective in treating tumors that are resistant to treatment with a drug that targets a single angiogenic factor (e.g., bevacizumab, which targets VEGF). As demonstrated herein, an ALKl-Fe fusion protein is effective in reducing the pathological effectsof multiple myeloma. 30 Multiple myeloma is widely recognized as a cancer that includes a significant angiogenic component. Accordingly, it is expected that ALKI -Fo fusion proteins 43 and other therapeutic agents disclosed herein will be useful in treating multiple myeloma and other tumors associated with the bone. As demonstrated herein, therapeutic agents disclosed herein may be used to ameliorate the bone damage associated with multiple myeloma, and therefore may be used to ameliorate bone 5 damage associated with bone metastases of other tumors, such as breast or prostate tumors. As noted herein, the GDF5-7 ligands are highly expressed in bone, and, while not wishing to be limited to any particular mechanism, interference with these ligands may disrupt processes that are required for tumor development in bone. In certain embodiments of such methods, one or more polypeptide 10 therapeutic agents can be administered, together (simultleously) or at different times (sequentially). In addition, polypeptide therapeutic agents can be administered with another type of compounds for treating cancer or for inhibiting angiogenesis. In certain embodiments, the subject methods of the disclosure can be used alone. Alternatively, the subject methods may be used in combination with other 15 conventional anti-cancer therapeutic approaches directed to treatment or prevention of proliferative disorders (e.g., tumor). For example, suet methods can be used in prophylactic cancer prevention, prevention of cancer rearrence and metastases after surgery, and as an adjuvant of other conventional cancer therapy. The present disclosure recognizes that the effectiveness of conventional cancer therapies (e.g., 20 chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery) can be enhanced through the use of a subject polypeptide therapeutic agent. A wide array of conventional compounds have been shown to have anti neoplastic activities. These compounds have been used as pharmaceutical agents in chemotherapy to shrink solid tumors, prevent metastases and further growth, or 25 decrease the number of malignant cells in leukemic or bone marrow malignancies. Although chemotherapy has been effective in treating various types of malignancies, many anti-neoplastic compounds induce undesirable sile effects. It has been shown that when two or more different treatments are combined, the treatments may work synergistically and allow reduction of dosage of each of the treatments, thereby 30 reducing the detrimental side effects exerted by each compound at higher dosages. 44 In other instances, malignancies that are refractory to a treatment may respond to a combination therapy of two or more different treatments. When a polypeptide therapeutic agent disclosed herein is administered in combination with another conventional anti-neoplastic agent, either concomitantly 5 or sequentially, such therapeutic agent may enhance the therapeutic effect of the anti-neoplastic agent or overcome cellular resistance to such anti-neoplastic agent. This allows decrease of dosage of an anti-neoplastic agent, thereby reducing the undesirable side effects, or restores the effectiveness of an anti-neoplastic agent in resistant cells. 10 According to the present disclosure, the antiangiogenic agents described herein may be used in combination with other compositions and procedures for the treatment of diseases. For example, a tumor may be treated conventionally with surgery, radiation or chemotherapy combined with the ALKI or ALKI ligand antagonist and then the antagonist may be subsequently administered to the patient 15 to extend the dormancy of micrometastases and to stabilize any residual primary tumor. Angiogenesis-inhibiting agents can also be given prophylactically to individuals known to be at high risk for developing nev or re-current cancers. Accordingly, an aspect of the disclosure encompasses methods for prophylactic 20 prevention of cancer in a subject, comprising administnting to the subject an effective amount of an ALK 1 or ALK 1 ligand antagonism and/or a derivative thereof, or another angiogenesis-inhibiting agent of the present disclosure. As demonstrated herein, ALKI -Fc is effective for diminishing the phenotype of a murine model of rheumatoid arthritis. Accordingly, therapeutic agents 25 disclosed herein may be used for the treatment of rheumatoid arthritis and other type of bone or joint inflammation. Certain normal physiological processes are also associated with angiogenesis, for example, ovulation, menstruation, and placentation. The angiogenesis inhibiting proteins of the present disclosure are useful in the treatment 30 of disease of excessive or abnormal stimulation of endothelial cells. These diseases include, but are not limited to, intestinal adhesions, atherosclerosis, scleroderma, and 45 hypertrophic scars, i.e., keloids. They are also useful in the treatment of diseases that have angio genesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa) and ulcers (Helicobacter pylori). General angiogenesis inhibiting proteins can be ued as a birth control agent 5 by reducing or preventing uterine vascularization required for embryo implantation. Thus, the present disclosure provides an effective birth control method when an amount of the inhibitory protein sufficient to prevent embryo implantation is administered to a female. In one aspect of the birth control method, an amount of the inhibiting protein sufficient to block embryo implantation is administered before or 10 after intercourse and fertilization have occurred, thus providing an effective method of birth control, possibly a "morning after" method. While not wanting to be bound by this statement, it is believed that inhibition of vasculaization of the uterine endometrium interferes with implantation of the blastocyst. Similar inhibition of vascularization of the mucosa of the uterine tube interferes with implantation of the 15 blastocyst, preventing occurrence of a tubal pregnancy. Administration methods may include, but are not limited to, pills, injections (intravenmals, subcutaneous, intramuscular), suppositories, vaginal sponges, vaginal tampons, and intrauterine devices. It is also believed that administration of angiogenesis inhibiting agents of the present disclosure will interfere with normal enhanced vascularization of the 20 placenta, and also with the development of vessels within a successfully implanted blastocyst and developing embryo and fetus. In the eye, angiogenesis is associated with, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, and retrolental fibroplasias. The therapeutic agents disclosed 25 herein may be administered intra-ocularly or by other local administration to the eye. Furthermore, as shown in the Examples, ALKI-Fc may be administered systemically and yet have the desired effect on ocular angiogenesis. Other diseases associated with angiogenesis in the eye include, but are not liriited to, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens 30 overwear, atopic keratitis, superior limbic keratitis, pteiygium keratitis sicca, sjogrens, acne rosacea, phylectenulosis, syphilis, Mycotacteria infections, lipid 46 degeneration, chemical bums, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections, Kaposi sarcoma, Mooren ulcer, Terrien's marginal degeneration, mariginal keratolysis, rheumatoid arthritis, systemic lupus, polyarteritis, trauma, Wegeners sarcoidosis, Scieritis, Steven's 5 Johnson disease, periphigoid radial keratotomy, and comeal graph rejection. sickle cell anemia, sarcoid, pseudoxanthoma elasticum, Pagets disease, vein occlusion, artery occlusion, carotid obstructive disease, chronic uveitis/vitritis, mycobacterial infections, Lyme's disease, systemic lupus erythematosis, retinopathy of prematurity, Eales disease, Bechets disease, infections causing a retinitis or 10 choroiditis, presumed ocular histoplasmosis, Bests disease, myopia, optic pits, Stargarts disease, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma and post-laser complications. Other diseases include, but are not limited to, diseases associated with rubeosis (neovasculariation of the angle) and diseases caused by the abnormal proliferation of fibrovascular or 15 fibrous tissue including all forms of proliferative vitreoctinopathy. Conditions of the eye can be treated or prevented by, e.g., systemic, topical, intraocular injection of a therapeutic agent, or by insertioa of a sustained release device that releases a therapeutic agent. A therapeutic agent may be delivered in a pharmaceutically acceptable ophthalmic vehicle, such that the compound is 20 maintained in contact with the ocular surface for a sufficent time period to allow the compound to penetrate the corneal and internal regions of the eye, as for example the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris/ciliary, lens, choroid/retina and solea. The pharmaceutically acceptable ophthalmic vehicle may, for example, be an ointment, vegetable oil or an 25 encapsulating material. Alternatively, the therapeutic agents of the disclosure may be injected directly into the vitreous and aqueous humour. In a further alternative, the compounds may be administered systemically, such as by intravenous infusion or injection, for treatment of the eye. One or more therapeutic agents can be administered. The methods of the 30 disclosure also include co-administration with other medicaments that are used to treat conditions of the eye. When administering more than one agent or a combination of agents and medicaments, administration can occur simultaneously or 47 sequentially in time. The therapeutic agents and/or medicanients may be administered by different routes of administration or by the same route of administration. In one embodiment, a therapeutic agent and a medicament are administered together in an ophthalmic pharmaceutical formulation. 5 In one embodiment, a therapeutic agent is used to treat a disease associated with angiogenesis in the eye by concurrent administration with other medicaments that act to block angiogenesis by pharmacological mechanisms. Medicaments that can be concurrently administered with a therapeutic agent of the disclosure include, but are not limited to, pegapianib (Macugen

T

m), ranibizumab (LucentisT"), 10 squalamine lactate (Evizon

T

), heparinase, and glucocorticoids (e.g. Triamcinolone). In one embodiment, a method is provided to treat a disease associated with angio genesis is treated by administering an ophthalmic pharmaceutical formulation containing at least one therapeutic agent disclosed herein and at least one of the following medicaments: pegaptanib (Macugentm, 15 ranibizumab (Lucentis

T

"), squalamine lactate (Evizon"), heparinase, and glucocorticoids (e.g. Triamcinolone). 7. Formulations and Effective Doses The therapeutic agents described herein may be formulated into 20 pharmaceutical compositions. Pharmaceutical compositons for use in accordance with the present disclosure may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Such formulations will generally be substantially pyrogen free, in compliancewith most regulatory requirements. 25 In certain embodiments, the therapeutic method of the disclosure includes administering the composition systemically, or locally as an implant or device. When administered, the therapeutic composition for use in this disclosure is in a pyrogen-free, physiologically acceptable form, Therapeutically useful agents other than the ALKi signaling antagonists which may also optionally be included in the 30 composition as described above, may be administered simultaneously or 48 sequentially with the subject compounds (e.g., ALK ECD polypeptides or any of the antibodies disclosed herein) in the methods disclosed herein. Typically, protein therapeutic agents disclosed herein will be administered parentally, and particularly intravenously or subcutaneously. Pharmaceutical 5 compositions suitable for parenteral administration may comprise one or more ALKI ECD polypeptides or other antibodies in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or Ronaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which 10 may contain antioxddants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and 15 the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper thidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by he use of surfactants. In one embodiment, the antibodies and ALKi ECD proteins disclosed herein 20 are administered in an ophthalmic pharmaceutical foralation In some embodiments, the ophthalmic pharmaceutical formulation is a sterile aqueous solution, preferable of suitable concentration for injection, or a salve or ointment. Such salves or ointments typically comprise one or more antibodies or ALK I ECD proteins disclosed herein dissolved or suspended in a sterile pharmaceutically 25 acceptable salve or ointment base, such as a mineral oil-white petrolatum base. In salve or ointment compositions, anhydrous lanolin may also be included in the formulation. Thimerosal or chlorobutanol are also preferably added to such ointment compositions as antimicrobial agents. In one embodiment, the sterile aqueous solution is as described in U.S. Pat. No. 6,071,958. 30 The disclosure provides formulations that may be varied to include acids and bases to adjust the pH; and buffering agents to keep the pH within a narrow range. Additional medicaments may be added to the formulation. These include, but are not 49 limited to, pegaptanib, heparinase, ranibizumab, or glucocorticoids. The ophthalmic pharmaceutical formulation according to the disclosure is prepared by aseptic manipulation, or sterilization is performed at a suitable stage of preparation. The compositions and formulations may, if desired, be presented in a pack or 5 dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. 10 EXAMPLES: Example 1: Expression of ALKI -Fc fusion proteins Applicants constructed a soluble ALK I fusion protein that has the extracellular domain of human ALK I fused to a human Fe or mouse ALK 1 fused to a urine Fc domain with a minimal linker in between. the constructs are referred 15 to as hALKI-Fc and mALKI-Fc, respectively. hALK I -Fc is shown as purified from CHO cell lies in Figure 3 (SEQ ID NO: 3). Notably, while the conventional C-terminus ofthe extracellular domain of human ALK 1 protein is amino acid 118 of SEQ ID NO1, we have determined that it is desirable to avoid having a domain that ends at a gktamine residue. 20 Accordingly, the portion of SEQ ID NO:3 that derives from human ALKI incorporates two residues c-terminal to Q 118, a leucine and an alanine. The disclosure therefore provides ALK 1 ECD polypeptides(including Ec fusion proteins) having a c-terminus of the ALKI derived sequence that is anywhere from I to 5 amino acids upstream (113-117 relative to SEQ IDNO: 1) or downstream (119 25 123)ofQll8. The hALK) -Fc and mALKI -Fc proteins were expressed in CHO cell lines. Three different leader sequences were considered: (i) Honey bee mellitin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ ID NO: 7) 50 (ii) Tissue Plasminogen Activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 8) (iii) Native: MTLGSPRKGLLMLLMALVTQG (SEQ DNO: 9). The selected form employs the TPA leader and has the unprocessed amino 5 acid sequence shown in Figure 4 (SEQ ID NO:5). This polypeptide is encoded by the nucleic acid sequence shown in Figure 4 (SEQ ID NO:4). Purification can be achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A 10 chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography. The purification can be completed with viral filtration and buffer exchange. The hALKI-Fe protein was purified to a purity of >98% as determined by size exclusion chromatography and >95% as determined by -SDS PAGE 15 Example 2: Identification of ALKI-Fe Ligands ALKI is a type I receptors for members of the TOFp family. A variety of members of the TGFP family were tested for binding toa human ALKi -Fe fusion protein, using a BiacoreTm system. TGFp itself, GDF8,GDFI 1, BMP2 and BMP4 20 all failed to show substantial binding to the hALKl -Fc protein. BMP2 and BMP4 showed limited binding. GDF5, GDF7 and BMP9 shoved binding with K,, values of approximately 5 x 10 M, 5x 10 M and I x 100 N, respectively. Based on the similarity of GDF5 and GDF7 to GDF6, it is expected hit GDF6 will bind with similar affinity. BMP 10 is closely related to BMP9 and is also expected to bind 25 with similar affinity. Example 3: Characterization of ALK I -Fc and anti-ALK I Antibody Effects on Endothelial Cells 51 Using a luciferase reporter construct under the control of four sequential consensus SBE sites (SBE4-luc), which are responsive to Srnadl/5/8-mediated signaling, we measured BMP-9 mediated activity in the presence and absence of hALKI-Fc drug or neutralizing ALKI specific monoclonal antibody in HMVEC 5 cells. HMVEC cells were stimulated with rhBMP-9 (song/ml), which induced Smadl/5/8-mediated transcriptional activation, evidenced here by the increase in SBE4-luc modulated transcriptional upregulation. When added, the hALKl-Fc compound (1 Opg/ml) or antibody (I 0pg/ml) diminished this transcriptional response, each by nearly 60%, indicating that the presence of ALKI-Fc significantly 10 reduces BMP9 signaling, and moreover, that the BMP9 signaling is related to ALKI activity. Activation of SMAD phosphorylation is commonly used to assay activation of upstream activin receptors. ALK I is known to modulate phosphorylation of SMAD proteins 1,5 and 8 upon activation by its ligand. Here, we added rhBMP-9 15 (50ng/ml) to initiate SMAD phosphorylation in HUVEC cells, a human endothelial cell line which innately expresses ALKI receptor, overatinecourse of 30 minutes. Phosphorylation of SMAD 1/5/8 was seen 5 minutes after treatment of cells with ligand and phosphorylation was maintained for the entirety of the 30 minute period. In the presence of relatively low concentrations of hALKI-Fe (250ng/ml), SMAD 20 1/5/8 phosphorylation was reduced, confirming that this agent inhibits Smadl/5/8 activation in endothelial cells. In order to evaluate the angiogenic effect of AL -Fc in an in vitro system, we assayed the effectiveness of the compound in reducing tube formation of endothelial cells on a Matrigel substrate. This technique is commonly used to assess 25 neovascularization, giving both rapid and highly reproducible results. Endothelial Cell Growth Supplement (ECGS) is used to induce theformation of microvessels from endothelial cells on Matrigel, and the efficacy of anti-angiogenic compounds are then gauged as a reduction of cord formation in the presence of both the drug and ECGS over an 18 hour timecourse. As expected, addition of ECGS (200ng/ml) 30 induced significant cord formation, as compared to thenegative control (no treatment added), which indicates basal levels of endothelial cell cord formation produced on Matrigel substrate (Fig 5). Upon addition of either hALK1-Fc (100 52 ng/ml) or mALKI-Fe (100ng/ml), cord formation was visibly reduced. Final quantification of vessel length in all samples revealed that every concentration of hALKI-fe or mALKI-Fe reduced neovascularization to basal levels. Additionally, hALK 1-Fc and mALK -Fec in the presence of the strongly pro-angiogenic factor 5 ECGS maintained strong inhibition of neovascularization demonstrating even more potent anti-angiogenic activity than the negative control endostatin (100ng/mi). Example 4: CAM Assays VEGF and FGF are well-known to stimulate angiogenesis. A CAM (chick 10 chorioallantoic membrane) assay system was used to assess the angiogenic effects of GDF7. As shown in Figure 6, GDF7 stimulates angiogemesis with a potency that is similar to that of VEGF. Similar results were observed with GDF5 and GDF6. ALK I-Fe fusions were tested for anti-angiogenio activity in the CAM assay. These fusion proteins showed a potent anti-angiogenic efcct on angiogenesis 15 stimulated by VEGF, FGF and GDF7. See Figure 7. BMP9 and PDGF showed a relatively poor capability to induce angiogenesis in this assay, but such angiogenesic effect of these factors was nonetheless inhibited by ALKI . ALKI-Fc proteins and acommercially available, aati-angiogenic anti-VEGF monoclonal antibody were compared in the CAM assay. The ALKI-Fec proteins had 20 similar potency as compared to anti-VEGF. The anti-NEGF antibody bevacizumab is currently used in the treatment of cancer and macular degeneration in humans. See Figure 8. Interestingly, an anti-ALKI antibody (R&D Systems) failed to significantly inhibit angiogenesis in this assay system. We expect that this may reflect the 25 difference in the ALKI sequence in different species. Example 5: Mouse Corneal Micropocket Assay The mouse corneal micropocket assay was used to assess the effects of ALKl-Fc on angiogenesis in the mouse eye. hALKI-Fe,administered 30 intraperitoneally, significantly inhibited ocular angioguiesis. As shown in Figure 9, 53 hALK 1 -Fc inhibited ocular angiogenesis to the same degree as anti-VEGF. hALK1 Fe and anti-VEGF were used at identical weight/weight dosages. Similar data were obtained when a Matrigel plug impregnated with VEGF was implanted in a non ocular location. 5 These data demonstrate that high affinity ligands for ALK 1 promote angiogenesis and that an ALK I-Fc fusion protein has potent anti-angiogenic activity. The ligands for ALKI fall into two categories, with the GDF5,6,7 grouping having an intermediate affinity for ALKI and the BMP9,10 grouping having a high affinity for ALK 1. 10 GDF5, 6 and 7 are primarily localized to bone and joints, while BMP9 is circulated in the blood. Thus, there appears to be a pro.ugiogenic system of the bones and joints that includes ALK 1, GDF5, 6 and 7 anl a systemic angiogenic system that includes ALK 1 and BMP9 (and possibly BlvPi 0). 15 Example 6: Murine Model of Rheumatoid Arthritis The murine collagen-induced arthritis model is a well-accepted model of rheumatoid arthritis. In this study, groups of 10 mice vere treated with vehicle, anti VEOF (bevacizumab - as a negative control, because bevacizumab does not inhibit urine VEGF), or doses of mALK I -Fc ("RAP-041") at I mg/kg, 10 mg/kg or 25 20 mg/kg. Following the collagen boost on day 21 arthritic scores (see Figure 10) and paw swelling steadily increased in all groups, peaking around day 38. Mice treated with mALK 1-Fc C'RAP-041 ") showed reduced scores for both characteristics, particularly at the highest dose (25mg/kg), although the reduction did not achieve statistical significance. Nonetheless, a dose-related trend is apparent. 25 By study termination at day 42 the incidence of arthritis had reached 10/10 in the vehicle control treated mice, 9/10 in the bevacizumab treated mice, 8/10 in the mALK I-Fc at I mg/kg treated group and 9/1.0 in the mALK 1-Fc 10mg/kg treated group. In the mALK I-Fc 25mg/kg treated group disease incidence was lower at 6/10. 30 54 Example 7: Murine Model of Multiple Myeloma Multiple myeloma is a cancer primarily of the bone that is associated with substantial bone loss. The 5T2MM model of myeloma in mice is based on the use of tumor cells (5T2MM cells) from a type of spontaneous tumor that develops in 5 aged mice and causes effects in mice that are similar to those seen in human multiple myeloma patients. See, e.g., Vanderkerken et al,, Methods Mol Med. 2005;113:191 205. mALKl -Fc was tested for effects in this model. 5T2MM cells injected into C57BL/KaLwRij mice promotes an increase in osteoclast surface, the formation of osteolytic lesions nd caused a decrease in bone 10 area. Bone disease is associated with a decrease in osteoblast number, osteoblast surface and a reduction in mineralization. Mice bearing 5T2MM cells were treated with mALKI-FC (RAP-041) (10mg/kg, i.p, twice weekly), or a vehicle, from the time of 5T2MM injection, for a total of 12 weeks. MicroCT analysis of the proximal tibia and lumbar vertebrae 15 demonstrated a statistically significant reduction in cancellous bone volume and trabecular number in 5T2MM-bearing mice compared to naive mice (bone volume reduced by 40% relative to controls; trabecular numberreduced by 40%). RAP-041 completely prevented ST2MM-induced decreases in bone volume and trabecular number when compared to vehicle treated mice (treated nice had a bone volume of 20 120% relative to untutored controls and trabecular number of 115% relative to untumored controls). Additionally, the tumor treated nice developed lytic bone lesions that were detected by microCT. mALK1-Fc treatment reduced the number of lytic bone lesions by 50% relative to vehicle treated mice. Based on the anti-angiogenic effects of ALKI-Ec, we infer that the 25 protective effect for bone provided by this agent is a consequence of diminished tumor growth, Therefore, ALK 1 -Fc may be used to treat multiple myeloma and to decrease the effects of bone disease resulting from this tumor type, 30 Example 8: Ligand Binding Characteristics of DAN 55 DAN is a member of a family of secreted cystine knot proteins that inhibit BMP activity. DAN is known to bind to and antagonize ODF5. We determined that DAN also binds tightly to GDF7, but not to BMP9, Thus, we conclude that DAN inhibits the suite of bone and joint localized ligands for ALKI, and DAN is expected 5 to be a potent antagonist of bone and joint related angiogenesis. Thus DAN may be useful in treating cancers of the bone, e.g., multiple myeloma and bone metastases, as well as rheumatoid arthritis and osteoarthritis. Taken together, the findings disclosed in these Examples provide numerous reagents; described herein, for inhibiting angiogenesis in vivo, and particularly 10 ocular angiogenesis. These findings also indicate that agents targeted to GDF5, 6 and 7 can be used to selectively inhibit bone and joint angiogenesis. These findings further indicate that such agents can be used to treat cancers and rheumatoid arthritis. 15 INCORPORATION BY REFERENCE All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, In case of conflict, the present application, including any definitions herein, will control. 20 EQUIVALENTS While specific embodiments of the subject inventions are explicitly disclosed herein, the above specification is illustrative and not restictive, Many variations of the inventions will become apparent to those skilled in ihe art upon review of this 25 specification and the claims below. The full scope of the inventions should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations. 56 Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims (58)

1. An ALK 1 -Fc fusion protein comprising: a polypeptide having an amino acid sequence that is at least 97% identical to the sequence of amino acids 22-118 of SEQ ID NO:1, which polypeptide is fused to an Fc portion of an immunoglobulin, and wherein the ALK -Fe fusion protein binds to GDF5, GDF7 and BMP9 with a K 0 of less than I x 10- M and binds to TGFp- 1 with a K 0 of greater than 1 x 106.

2. The ALKI -Fe fusion protein, wherein the Fe portion is an Fe portion of a human IgGI,

3. An ALK I-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 3.

4. An ALKI-Fc fusion protein that is produced by expression of the nucleic acid of SEQ ID NO:4 in a mammalian cell line.

5. The ALKi-Fe fusion protein of claim 4, wherein the mammalian cell line is a Chinese Hamster Ovary (CHO) cell line.

6. A pharmaceutical preparation that is substantially pyrogen free, comprising an ALK I -Fc fusion protein of claim 5.

7. An antibody that binds to an ALK1 polypeptide consisting of amino acids

22-118 of SEQ ID NO:1 and inhibits the binding of at least one ALKI ligand selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10, 8. The antibody of claim 7, wherein the antibody binds to the ALKI polypeptide with a K 0 of less than 5 x 10 4 M. 57 9. The antibody of claim 7, wherein the antibody binds to the ALKI polypeptide with a KD of less than I x 1010 M. 10. The antibody of claim 8, wherein the antibody inhibits angiogenesis stimulated by at least one ALKI ligand selected from the group consisting of: GDF5, GDF6 and GDF7. 11. The antibody of claim 10, wherein the antibody inhibits binding of BMP9 and BMP10 to ALK1. 12. A pharmaceutical preparation that is substantially pyrogen free comprising the antibody of claim 7. 13. A method of inhibiting angiogenesis in a mammal, the method'comprising, administering to the mammal an effective amount of an ALKI ECD protein, 14. The method of claim 13, wherein the ALK-1 ECD protein is an ALKI-Fc fusion protein. 15. The method of claim 14, wherein the ALK1-Fc fusion protein comprises a polypeptide having an amino acid sequence that is at least 90% identical to the sequence of amino acids 22-118 of SEQ ID NO: 1, which polypeptide is fused to an Fc portion of an immunoglobulin, and wherein the ALK1-Fc fusion protein binds to TGFp- I with a KD of greater than I x 10 16, The method of claim 13, wherein the ALKI ECD protein binds to one or more ALKI ligands selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10. 17. The method of claim 13, wherein the ALKI ECD polypeptide comprises an amino acid sequence that is at least 90% identical to the sequence of amino acids corresponding to amino acids 34-95 of SEQ ID NO: 1. 58 18. The method of claim 13, wherein the ALKI ECD comprises an amino acid sequence encoded by a nucleic acid that hybridized under stringent hybridization conditions to nucleotides 100-285 of SEQ ID NO:2 or a variant of nucleotides 100-285 of SEQ ID NO:2 that has the same coding sequence. 19. The method of claim 14, wherein the ALK1-Fc fusion protein has a sequence of SEQ ID NO:3. 20. The method of claim 13, wherein the ALKI ECD fusion protein is delivered intravenously or locally to the eye. 21. The method of claim 13, wherein the method further comprises administering a second agent that inhibits angiogenesis. 22. The method of claim 13, wherein the angiogenesis to be inhibited is angiogenesis occurring: in the eye of the mammal, in a tumor or in a bone or joint.

23. A method of inhibiting angiogenesis in a mammal, the method comprising, administering to the mammal an effective amount of an antibody that binds to an ALK1 polypeptide consisting of amino acids 22-118 of SEQ ID NO:I and inhibits the binding of at least one ALKI ligand selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMPI0,

24. The method of claim 23, wherein the antibody binds to the ALKI polypeptide with a Ko of less than 5 x 10- M.

25. The method of claim 23, wherein the antibody binds to the ALK1 polypeptide with a KD of less than I x 0'1 0 M. 59

26. The method of claim 23, wherein the antibody inhibits angiogenesis stimulated by at least one ALKI ligand selected from the group consisting of: GDF5, GDF6 and GDF7.

27. The method of claim 23, wherein the antibody inhibits binding of ALKI to an ALKI ligand, wherein the ALKI ligand is selected from the group consisting of: BMP9 and BMP10.

28. The method of claim 23, wherein the antibody is delivered intravenously.

29. The method of claim 23, wherein the method further comprises administering a second agent that inhibits angiogenesis.

30. The method of claim 23, wherein the angiogenesis to be inhibited is angiogenesis occurring: in the eye of the mammal, in a tumor or in a bone or joint.

31. An antibody that binds to an ALKI ligand and inhibits the binding of the ALKI ligand to ALKI, wherein the ALKl ligand is selected from the group consisting of GDF5, GDF6 and GDF7.

32. The antibody of claim 31, wherein the antibody binds to the ALK1 ligand with a Ko of less than 5 x 10-' M.

33. The antibody of claim 31, wherein the antibody inhibits angiogenesis stimulated by the ALKI ligand.

34. A pharmaceutical preparation comprising the antibody of claim 31.

35. An antibody that binds to an ALKI ligand and inhibits the binding of the ALK1 ligand to ALKI, wherein the ALKI ligand is selected from the group consisting of BMP9 and BMP 10. 60

36. The antibody of claim 35, wherein the antibody binds to the ALKI ligand with a KD of less than 1 x 10-" M.

37. A pharmaceutical preparation comprising the antibody of claim 35.

38. A method of inhibiting angiogenesis in a mammal, the method comprising, administering to the mammal an effective amount of an antibody that binds to an ALKI ligand and inhibits the binding of the ALK1 ligand to ALKI, wherein the ALK ligand is selected from the group consisting of GDF5, GDF6, GDF7, BMP9 and BMP10.

39. The method of claim 38, wherein the antibody inhibits angiogenesis stimulated by at least one ALK1 ligand selected from the group consisting of: GDFS, GDF6 and GDF7.

40. The method of claim 38, wherein the antibody inhibits angiogenesis stimulated by at least one ALKi ligand selected from the group consisting of: BMP9 and BMP10.

41. The method claim 38, wherein the antibody is delivered intravenously.

42. The method of claim 38, wherein the method further comprises administering a second agent that inhibits angiogenesis.

43. The method of claim 38, wherein the angiogenesis to be inhibited is angiogenesis occurring: in the eye of the mammal, in a tumor or in a bone or joint.

44. Use of an ALK1 IECD polypeptide or nucleic acids encoding the same in the manufacture of a medicament for the inhibition of angiogenesis in a mammal. 61

45. The use of claim 44, wherein the ALKI ECD polypeptide is an ALK-1 Fe fusion protein.

46. The use of claim 45, wherein the ALK-l Fe fusion protein comprises a polypeptide having an amino acid sequence that is at least 90% identical to the sequence of amino acids 22-118 of SEQ ID NO:1, which polypeptide is fused to an Fe portion of an inununoglobulin, and wherein the ALKI-Fc fusion protein binds to TGFP-1 with a KD of greater than I x 10

47. Use of an antibody that binds to ALKI or nucleic acids encoding the same in the manufacture of a medicament for the inhibition of angiogenesis in a mammal, wherein the antibody binds to an ALK1 polypeptide consisting of amino acids 22-118 of SEQ ID NO:l and inhibits the binding of at. least one ALKI ligand selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10.

48. Use of an antibody that binds to an ALKI ligand and inhibits the binding of the ALK1 ligand to ALKI, or nucleic acids encoding the same, in the manufacture of a medicament for the inhibition of angiogenesis in a mammal, wherein the antibody binds to an ALK ligand selected from the group consisting of GDF5, GDF6, GDF7, BMP9 and BMPIO.

49. A method of inhibiting angiogenesis in a mammal, the method comprising, administering to the mammal an effective amount of a DAN protein.

50. The method of claim 49, wherein the DAN protein is a DAN-Fe fusion protein.

51. The method of claim 49, wherein the DAN-Fc fusion protein comprises a polypeptide having an amino acid sequence that is at least 90% identical to 62 the sequence of amino acids 21-125 of SEQ ID NO: 10, which polypeptide is fused to an Fc portion of an immunoglobulin, and wherein the ALKI-Fc fusion protein binds to TGFp- I with a KD of greater than 1 x 10~ ,

52. The method of claim 49, wherein the DAN protein binds to one or more ALKI ligands selected from the group consisting of: GDF5, GDF6 and GDF7.

53. The method of claim 49, wherein the DAN protein comprises an amino acid sequence that is at least 90% identical to the sequence of amino acids corresponding to amino acids 17-180 of SEQ ID NO: 10.

54. The method of claim 49, wherein the DAN protein comprises an amino acid sequence encoded by a nucleic acid that hybridizes under stringent hybridization conditions to nucleotides 153-467 of SEQ ID NO: 1 or a variant of nucleotides 153-467 of SEQ ID NO:I) that has the same coding sequence.

55. The method of claim 49, wherein the method further comprises administering a second agent that inhibits angiogenesis.

56. The method of claim 49, wherein the angiogenesis to be inhibited is angiogenesis occurring: in the eye of the mammal, in a tumor or in a bone or joint,

57. A method for treating rheumatoid arthritis in a mammal, the method comprising, administering to a mammal that has rheumatoid arthritis an effective amount of an agent selected from the group consisting of: (a) an ALKi ECD protein; 63 (b) an antibody that binds to an ALKi ligand and inhibits the binding of the ALKI ligand to ALKI, wherein the ALKI ligand is selected from the group consisting of GDFS, GDF6, GDF7, BMP9 and BMP10; (c) an antibody that binds to an ALKI polypeptide consisting of amino acids 22-118 of SEQ ID NO:1 and inhibits the binding of at least one ALKI ligand selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMPIO; and (d) a DAN polypeptide.

58. The method of claim 57, wherein the ALK-1 ECD protein is an ALKI-Fc fusion protein.

59. The method of claim 57, wherein the ALK1 -Fe fusion protein comprises a polypeptide having an amino acid sequence that is at least 90% identical to the sequence of amino acids 22-118 of SEQ ID NO: 1, which polypeptide is fused to an Fc portion of an immunoglobulin, and wherein the ALKl-Fc fusion protein binds to TGFp- I with a KD of greater than 1 x 10,6

60. The method of claim 57, wherein the ALKi ECD protein binds to one or more ALKI ligands selected from the group consisting of: GDFS, GDF6, GDF7, BMP9 and BMP10.

61. The method of claim 58, wherein the ALKl-Fc fusion protein has a sequence of SEQ ID NO:3. 64

62. The method of claim 57, wherein the ALKI ECD polypeptide comprises an amino acid sequence that is at least 90% identical to the sequence of amino acids corresponding to amino acids 34-95 of SEQ ID NO:1.

63. The method of claim 57, wherein the ALKI ECD comprises an amino acid sequence encoded by a nucleic acid that hybridized under stringent hybridization conditions to nucleotides 100-285 of SEQ ID NO:2 or a variant of nucleotides 100-285 of SEQ ID NO:2 that has the same coding sequence.

64. The method of claim 57, wherein the antibody of (b) binds to the ALK1 polypeptide with a KD of less than 5 x 10- 8 M. 65, The method of claim 57, wherein the antibody of (b) binds to the ALK1 polypeptide with a KD of less than 1 x 10.10 M. 66, The method of claim 57, wherein the antibody of (b) inhibits angiogenesis stimulated by at least one ALKi ligand selected from the group consisting of: GDF5, GDF6 and GDF7.

67. The method of claim 57, wherein the antibody of (b) inhibits binding of ALKI to an ALKI ligand, wherein the ALKi ligand is selected from the group consisting of: BMP9 and BMP 10. 68, The method of claim 57, wherein the antibody of (c) inhibits angiogenesis stimulated by at least one ALK1 ligand selected from the group consisting of: GDF5, GDF6 and GDF7.

69. The method of claim 57, wherein the antibody of (c) inhibits angiogenesis stimulated by at least one ALKI ligand selected from the group consisting of: BMP9 and BMP10. 65

70. The method of claim 57, wherein the DAN protein is a DAN-Fc fusion protein.

71. The method of claim 70, wherein the DAN-Fc fusion protein comprises a polypeptide having an amino acid sequence that is at least 90% identical to the sequence of amino acids 21-125 of SEQ ID NO:10, which polypeptide is fused to an Fc portion of an immunoglobulin, and wherein the ALKI-Fc fusion protein binds to TGFp- 1 with a KD of greater than l x 106,

72. The method of claim 57, wherein the DAN protein binds to one or more ALKI ligands selected from the group consisting of: GDF5, GDF6 and GDF7.

73. The method of claim 57, wherein the DAN protein comprises an amino acid sequence that is at least 90% identical to the sequence of amino acids corresponding to amino acids 17-180 of SEQ ID NO: 10.

74. The method of claim 57, wherein the DAN protein comprises an amino acid sequence encoded by a nucleic acid that hybridizes under stringent hybridization conditions to nucleotides

153-467 of SEQ ID NO: 11 or a variant of nucleotides 153-467 of SEQ ID NO:11 that has the same coding sequence. 75. The method of claim 57, wherein the agent is delivered intravenously. 76. The method of claim 57, wherein the method further comprises administering a second agent that inhibits angiogenesis. 77. A method for treating a tumor in a mammal, the method comprising, administering to a mammal that has a tumor an effective amount of an agent selected from the group consisting of: 66 (a) an ALKI ECD protein; (b) an antibody that binds to an ALKI ligand and inhibits the binding of the ALKI ligand to ALK1, wherein the ALKI ligand is selected from the group consisting of GDF5, GDF6, GDF7, BMP9 and BMP10; (c) an antibody that binds to an ALKI polypeptide consisting of amino acids 22-118 of SEQ ID NO: 1 and inhibits the binding of at least one ALKI ligand selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP 10; and (d) a DAN polypeptide. 78. The method of claim 77, wherein the ALK-1 ECD protein is an ALK1-Fc fusion protein. 79. The method of claim 78, wherein the ALKI-Fc fusion protein comprises a polypeptide having an amino acid sequence that is at least 90% identical to the sequence of amino acids 22-118 of SEQ ID NO: 1, which polypeptide is fused to an Fc portion of an immunoglobulin, and wherein the ALK I -Fc fusion protein binds to TGFp- I with a KD of greater than 1 x 106. 80. The method of claim 77, wherein the ALKI ECD protein binds to one or more ALKI ligands selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP 10. 81. The method of claim 78, wherein the ALK1-Fc fusion protein has a sequence of SEQ ID NO:3. 67 82. The method of claim 77, wherein the ALK1 ECD polypeptide comprises an amino acid sequence that is at least 90% identical to the sequence of amino acids corresponding to amino acids 34-95 of SEQ ID NO: 1. 83. The method of claim 77, wherein the ALKi ECD comprises an amino acid sequence encoded by a nucleic acid that hybridized under stringent hybridization conditions to nucleotides 100-285 of SEQ ID NO:2 or a variant of nucleotides 100-285 of SEQ ID NO:2 that has the same coding sequence. 84. The method of claim 77, wherein the antibody of (b) binds to the ALKi polypeptide with a K, of less than 5 x 10S M. 85. The method of claim 77, wherein the antibody of (b) binds to the ALK1 polypeptide with a Kr of less than I x 1010 M. 86. The method of claim 77, wherein the antibody of (b) inhibits angiogenesis stimulated by at least one ALKI ligand selected from the group consisting of: GDF5, GDF6 and GDF7. 87. The method of claim 77, wherein the antibody of (b) inhibits binding of ALKI to an ALK1 ligand, wherein the ALKI ligand is selected from the group consisting of: BMP9 and BMP 10. 88. The method of claim 77, wherein the antibody of (c) inhibits angiogenesis stimulated by at least one ALK1 ligand selected from the group consisting of: GDF5, GDF6 and GDF7. 89. The method of claim 77, wherein the antibody of (c) inhibits angiogenesis stimulated by at least one ALKI ligand selected from the group consisting of: BMP9 and BMP10. 68 90. The method of claim 77, wherein the DAN protein is a DAN-Fc fusion protein. 91. The method of claim 90, wherein the DAN-Fc fusion protein comprises a polypeptide having an amino acid sequence that is at least 90% identical to the sequence of amino acids 21-125 of SEQ ID NO:10, which polypeptide is fused to an Fc portion of an immunoglobulin, and wherein the ALKI-Fc fusion protein binds to TGFp- I with a KD of greater than 1 x 10. 92. The method of claim 77, wherein the DAN protein binds to one or more ALKi ligands selected from the group consisting of: GDF5, GDF6 and GDF7. 93. The method of claim 77, wherein the DAN protein comprises an amino acid sequence that is at least 90% identical to the sequence of amino acids corresponding to amino acids 17-180 of SEQ ID NO:10. 94. The method of claim 77, wherein the DAN protein comprises an amino acid sequence encoded by a nucleic acid that hybridizes under stringent hybridization conditions to nucleotides 153-467 of SEQ ID NO: 11 or a variant of nucleotides 153-467 of SEQ ID NO: 11 that has the same coding sequence. 95. The method of claim 77, wherein the agent is delivered intravenously. 96. The method of claim 77, wherein the method further comprises administering a second agent that inhibits angiogenesis. 97. The method of claim 77, wherein the method further comprises administering a second agent that inhibits angiogenesis. 98. The method of claim 77, wherein the tumor is associated with bone. 69 99, The method of claim 77, wherein the tumor is a myeloma or a tumor that has metastasized to the bone. 100. The method of claim 77, wherein the tumor is resistant to anti-VEGF therapy. 101. An ophthalmic pharmaceutical formulation comprising an agent selected from the group consisting of: (a) an ALKI ECD protein; (b) an antibody that binds to an ALKI ligand and inhibits the binding of the ALKI ligand to ALKI, wherein the ALKI ligand is selected from the group consisting of GDF5, GDF6, GDF7, BMP9 and BMP10; (c) an antibody that binds to an ALKI polypeptide consisting of amino acids 22-118 of SEQ ID NO: 1 and inhibits the binding of at least one ALKI ligand selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMPIO; and (d) a DAN polypeptide. 102. A method of treating an angiogenesis related disease of the eye comprising administering systemically or to said eye a pharmaceutical formulation comprising: an effective amount of an agent selected from the group consisting of: (a) an ALKi ECD protein; (b) an antibody that binds to an ALKI ligand and inhibits the binding of the ALKI ligand to ALKI, wherein the ALKI 70 ligand is selected from the group consisting of GDF5, GDF6, GDF7, BMP9 and BMP10; (c) an antibody that binds to an ALKI polypeptide consisting of amino acids 22-118 of SEQ ID NO: I and inhibits the binding of at least one ALK1 ligand selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10; and (d) a DAN polypeptide. 71