AU2018279010B2 - ALK1 receptor and ligand antagonists and uses thereof - Google Patents

Abstract

H:\szp\Interwoven\NRPortbl\DCC\SZP\59186931.DOC-24/12/2013 ABSTRACT 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 (ALK) polypeptide may be used to inhibit angiogenesis in vivo, particularly in mammals suffering angiogenesis-related disorders. The disclosure also identifies ligands for ALK and demonstrates that such ligands have pro-angiogenic activity, and antibodies that inhibit receptor-ligand interaction.

Description

This application claims the benefit of the filing hteof U.S. provisional application 60/856,592, filed November 2, 2006 andentitled ALKi Receptor and of the referenced Ligand Antagonists and Uses Thereof. The entire teachigs provisional application are expressly incorporated hereintyreference.

BACKGROUND

Angiogenesis, the process of forming new blood vessels, is critical in many normal and abnormal physiological states. Under nomal pysiological conditions, humans and animals undergo agiogenesis in specific mirestricted situations.For example, angiogenesis is normally observed in woundtealiag, fetal and embryonic development and formation of the corpus luteum, endonetrium and placenta.

Undesirable or inappropriately regulated angiopwesis occurs in many disorders, in which abnormal endothelial growth may eseor participate in the pathological process. For example, angiogenesis partidpates in the growth of many tumors. Deregulated'angiogenesis has been implicatedi pathological processes such as rheumatoid arthritis, retinopathies, hemangiomas, and psoriasis. The diverse pathological disease states in which unregulated angionesis is present have been categorized as angiogenesis-associated diseases.

Both controlled and uncontrolled angiogenesis are thought to proceed in a similar manner. Capillary blood-vessels are composed primarily ofendothelial cells and pericytes, surrounded by a basement membrane. Agiogenesis begins with the erosion of the basement membrane by enzymes releasedb endothelial cells and leukocytes. The endothelial cells, which line the lumenofblood vessels, then protrude through the basement membrane; Angiogenicactors induce the endothelial cells to migrate through the eroded basemet membrane. The migrating cells form a "sprout" protruding from the parent bloodvNel, 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 Vascular Endothelial Growth Factor (VEGF), has proven to be effective in the treatment of a variety of cancers. MacugenT , 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. Cancer Res. 2005 Jul 1;65(13):5864-71). The isocoumrin 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. Cncer Chemother Pharmacol. 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 appars to be an important component'of the anti-tumor effect (see, e.g., Dredge eta Microvasc Res. 2005 Jan;69(1-2):56-63). The success of TNF-alpha antagonists in the treatment of rheumatoid arthritis is partially attributed to anti-angiognic effects on the inflamed joint tissue (Feldmann et al. Annu Rev Immunol. 2001;9:163-96). Anti-angiogenic therapies are widely expected to have beneficial effectson other inflammatory diseases, particularly psoriasis. Although many anti-angiogenic agents have an effect on angiogenesis regardless of the tissue that is affected, other angiogenic agents may tend to have a tissue-selective effect.

It is desirable to have additional compositions ad 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.

SUMMARY

In part, the present disclosure presents a characterization of an activin-like kinase I (ALK 1)-mediated regulatory system and the role of this system in of angiogenesis in vivo. In certain aspects, the disclosure provides antagonists ALK-1 ligands and the use of such antagonists as anti-angiogenic agents. Additionally, the disclosure provides antagonists of ALK-Iitself, 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 BMP10. This disclosure demonstrates that signaling mediated by ALKi and the ligands describedaove is involved in a potent anti angiogenesis in vivo, and that inhibition of this regulatory system has of angiogenic effect. Thus, in certain aspects, the disclosue provides antagonists the ALK Iregulatory system, including antagonists of tie receptor or one or more of the ligands, for use in inhibiting angiogenesis. In certa aspects, the disclosure provides antagonists of ALKI ligands for the treatmentof cancers, particularly multiple myeloma, rheumatoid arthritis, and disordersassoiated with pathological angiogenesis in the eye.

In certain aspects, the disclosure provides polypptides comprising a ligand binding portion of the extracellular domain of ALK ("ALK1 ECD polypeptides") 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 an other receptors. In certain embodiments, an ALK ECD polypeptide comprises amino acid sequence that is at least 70%, 80%, 90%, 95%, 97%, 99% or 100% identical to the sequence of amino acids 22-118 of the human ALK Isequence of monomeric SEQ ID NO;l An ALK ECD-polypeptide may be used as a small 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 as an increased to a second polypeptide portion to provide improved properties, such of an 30 half-life or greater ease of production or purification. Fusions to an Fe portion immunoglobulin or linkage to a polyoxyethylene moiety (e.g., polyethylene glycol) ECD may be particularly useful to increase the serum half-life of the ALKI polypeptide in systemic administration (e.g., intravenous, intraarterial and intra peritoneal administration). As demonstrated herein, a:stcmically administered ALKI-Fc polypeptide has a potent anti-angiogenic effect in the eye and also provides positive effects in marine models of rheumatoid arthritis and multiple myeloma. In certain embodiments, an ALKi-Fc fusionprotein comprises a polypeptide having an amino acid sequence that is at least70%, 80%, 90%, 95%, 97%, 99% or 100% identical to the sequence of amino cids 22-118 of SEQ ID an NO:1, which polypeptide is fused, either with or without am intervening linker, to to Fc portion of an immunoglobulin, and wherein the ALI-Pc fusion protein binds GDF5, GDF7 and BMP9 with a KD of less than I x 1'Wand binds to TGFp-1 with a KD of greater than 1 x 106.An Fe portion may le selected so as to be human appropriate to the organism. Optionally, the Fe portions an Fe portion of a IgGl. In a preferred embodiment, the ALKI-Fc fusionprotein comprises amino acids 22-118 of SEQ ID NO:1. Optionally, the ALKIe fusion protein comprises 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 that is ECD polypeptides may be formulated as a pharmaceutial preparation substantially pyrogen free. The pharmaceutical prepantiom ay be prepared for or local systemic delivery (e.g., intravenous, intraarterial or sulcutancous delivery) delivery (e.g., to the eye).

In certain aspects, the disclosure provides methods for inhibiting polypeptides angiogenesis in a mammal by administering any of theALK ECD described generally or specifically herein. In one embodiment, a method comprises administering to the mammal an effective amount ofan ALKl-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 ALKI-Fe fusion protein binds to TGFD-I with a Ko 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. The Optionally, the ALK-Fc fusion protein has a sequenceof SEQ ID NO:3.

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, e.g. by systemic administration.

In certain aspects, the disclosure provides antibodies that bind to ALK1, acids 22-118 of particularly an epitope situated in the extracellular domir, amino ligand selected SEQ ID NO1, and inhibit the binding of ALK to at lest one ALK1 on from the group consisting of: GDF5, GDF6, GDF7, Bl9 and BMP10. Based the affinity of these ligands for ALKI, an antibody may biad with a KD of less than 10 . An antibody with affinity 5 x 10' M, and optionally between 5 x 10- andI x within this range would be expected to inhibit signalingby one or more of GDF5, 6 and 7 while having less effect on signaling by BMP9 and 10. Such an antibody selected preferably inhibits angiogenesis stimulated by at least me ALK ligand to be from the group consisting of: GDF5, GDF6 and GDF7. While not wishing 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 ligands. ALKI-Fc fusion protein, which is expected to inhibittle activity of ALKI An anti-ALKI antibody is not expected to interfere with the ability of GDF5, GDF6, as the GDF7, BMP9 or BMP10 to signal through alternativereceptor systems, such BMPRla, BMPR1b and BMPRII complexes. However,aaanti-ALKl antibody is expected to interfere with the ability of low affinity ligands for ALK (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 though an ALKI ECD may not bind to or inhibit suchlow affinity ligands, An antibody may bind to the ALK1 polypeptide with a KDof less than 1 x 10~" M. An antibody with affinity within this range would be expeted to inhibit signaling by to BMP9 or 10. Such an antibody preferably inhibits binding of BMP9 and BMP1O ALKI. Notably, based on the data disclosed herein,an antibody that binds 30 relatively poorly to ALKI may inhibit TGFP bindingto 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 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 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 prepartion 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 methods for inhibiting amount of angiogenesis in a mammal by administering to the mamal an effective an antibody that binds to an ALK1 polypeptide; described herein either generally or to the extracellular specifically. An antibody useful for this purpose may bind domain of ALKI (e.g., bind to a polypeptide consisting of amino acids 22-118 of a SEQ ID NO:1) or another portion of ALKI. The antibody may bind to and inhibits the polypeptide consisting of amino acids 22-118 of SEQ ID NO:I binding of at least one ALK1 ligand selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10. The antibody maybind to the ALK polypeptide The with a Kj of less than 5 x iO' M, and optionally between 5 x 10' and 1 x1010. antibody may inhibit angiogenesis stimulated by at least one ALKIligand selected from the group consisting of GDF5, GDF6 and GDF An antibody that selectively 10 inhibits signaling mediated by GDF5, 6 or 7 relative to signaling by BMP9 or in tissues where may be used as a selective inhibitor of angiogenesis that occurs GDF5, 6 or 7 are localized: primarily bone or joints. The antibody may bind to the 0 the ALKI polypeptide with a K of less than 1 x 1 -1 M. The antibody may inhibit the binding of ALKl to an ALK ligand, wherein the ALK ligand is selected from may be delivered group consisting of: BMP9 and BMP10. The anti-ALKI antibody 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 treating patients with multiple myeloma. In a particul embodiment, the disclosure with provides a method for inhibiting angiogenesis in disorder that are associated pathological angiogenesis as a consequence of multiple pro-angiogenic factors, such as VEGF, PDGF and/or FGF.

In certain aspects, the disclosure provides antibodies that bind to an ALKI ALKi. While ligand disclosed herein and inhibit the binding of the ALK ligand to not wishing to be bound to any particular mechanism, it is expected that antibodies that bind to ALKI ligands will have effects that are sinila in nature to ALKI ECD polypeptides, because both types of agent bind to the lignds rather than the receptor itself. In certain embodiments, the antibody binds to a ligand selected from the the ALK1 group consisting of GDF5, GDF6 and GDF7. The antibody may bind to for ligand with a Ko of less than 5 x 10 M. The antibody may be selected inhibition of angiogenesis stimulated by the ALKI ligead. A CAM assay is an are appropriate assay system for selection of desirable antibodies, Such antibodies pharmaceutical -15 preferably recombinant antibodies, and may be formulated as a preparation that is substantially pyrogen free. The pharmaceutical preparation may or subcutaneous be prepared for systemic delivery (e.g., intravenous, intraarterial delivery) or local delivery (e.g., to the eye).

In certain aspects, the disclosure provides antibodies that bind to an ALKI ligand and inhibit the binding of the ALKIligand to ALK, wherein the ALKI ligand is selected from the group consisting of BMP9and BMP10. The antibody Such antibodies may bind to the ALKIligand with a K of less than I x10° M. pharmaceutical are preferably recombinant antibodies, and may be formulated as a 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 methods for inhibiting to the mammal an angiogenesis in a mammal, the method comprising, administering effective amount of an antibody that binds to an ALK ligand and inhibits the the 30 binding of the ALKI ligand to ALKI, wherein the ALKi ligand is selected from group consisting of GDF5, GDF6, GDF7, BMP9 and BMP10. The antibody may inhibit angiogenesis stimulated by at least one ALK Iligand selected from the group consisting of: GDF5, GDF6 and GDF7.

Members of the BMP/GDF family, including ByP9, BMP10, GDF5, GDF6 and GDF7bind to a type I and a type Il receptor in order to forn a functional signaling complex; The binding sites for these receptorsae different. Accordingly, in certain embodiments, an antibody that binds to an AL Iligand and inhibits the ligand to ALK1 is an antibody that binds at ornear the tp I receptor binding site of the ligand,

In certain aspects, the disclosure provides methods for inhibiting angiogenesis in a mammal by administering other inhibtsofthe ALKI signaling system disclosed herein. Such inhibitors may include mleic acids (e.g., antisense or RNAi constructs) that decrease the production of ALK1, GDF5, GDF6, GDF7, BMP9 or BMP1Q). A variety of affinity binding reagents can also be used, such as aptamers, random peptides, protein scaffolds that can modified to allow binding to selected targets (examples of such scaffolds includemticalins 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 ALKI-ligand interaction or by inhibiting the signaling hatoccurs after binding.

In a further embodiment, the disclosure describes the role of DAN as a regulator of the ALK Iregulatory system. As shown heria, DAN binds to the GDF5 group of ligands but fails to bind to the BMP9 gpof ligands. Thus, DAN is expected to inhibit angiogenesis mediated by GDF5,GF6 or GDF7 but not angiogenesis mediated by BMP9 or BMP10. DAN may therefore be used as a selective agent for inhibiting angiogenesis in the boneor joints, where the GDF5 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 ALKIligands selected from the group consisting of: GDF5, GDF6 and GDF7, while having relatively poor binding to BMP9-or BMP10. A DAN protein may comprise an amino acidsequence that is at least 70%,

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 at comprises a sequence the complement of which hybridizes under stringent hybridiation conditions to nucleotides 153-467 of SEQ ID NO:11 or a variant of nacleotides 153-467 of SEQ ID NO:1I that has the same coding sequence (a "silentwariant, such as a variant containing one or more alterations at a wobble positioning the triplet code), or to nucleotides 93-635 of SEQ ID NO: 1 or a silent varianttereof. In certain aspects, is the DAN protein is a fusion protein, such as an Fc fusicaprotein. While DAN in bone and expected to be particularly useful for the inhibition of angiogenesis myeloma and joints (including tumors located in the bone or joints, s4 as multiple bone metastases), it may also be useful in other contexts,such as in a tumor located elsewhere, or in the eye.

In certain aspects, the disclosure providesmethods for treating rheumatoid has arthritis in a mammal, the method comprising, administering to a mammal that rheumatoid arthritis an effective amount of an agent sekted from the group and consisting of: an ALK IECD protein; an antibody that biads to an ALK ligand is inhibits the binding of the ALKI ligand to ALK1, wherein the ALK Iligand selected from the group consisting of GDF5, GDF6, GDFI, BMP9 and BMP10; an of antibody that binds to an ALK polypeptide consistingof amino acids 22-118 selected from the SEQ ID NO:l and inhibits the binding of at least one ALKIligand a DAN group consisting of: GDF5, GDF6, GDF7, BMP9 and BMPI0; and polypeptide.

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 of ECD protein; an antibody that binds to an ALK ligandand inhibits the binding the ALK Iligand to ALK1, wherein the ALK ligand is selected from the group 30 consisting of GDF5, GDF6, GDF7, BMP9 and BMP10; an antibody that binds to an ALK Ipolypeptide consisting of amino acids 22-118 of SEQ ID NO:I and inhibits the binding of at least one ALK Iligand selected from te group consisting of: may GDF5, GDF6, GDF7, BMP9 and BMP10; and a DAN polypeptide. A method A tumor further comprise administering a second agent that inhibits angiogenesis. a bone marrow may be a tumor that is associated with bone, such as a leukemia, tumor, a multiple myeloma or bone metastases, such as those commonly associated 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 of: an ALKI formulations may comprise an agent selected from thegrMup consisting the binding of ECD protein; an antibody that binds to an ALKI ligand and inhibits the ALK ligand to ALK1, wherein the ALK ligand is selected from the group that binds to an consisting of GDF5, GDF6, GDF7, BMP9 and BMP10; an antibody NO:l and inhibits ALK polypeptide consisting of amino acids 22-118 ofSEQ ID the binding of at least one ALKI ligand selected from the group consisting of GDF5, GDF6, GDF7, BMP9 and BMPO0; and a DAN polypeptide.

In certain aspects, the disclosure provides methods for treating an administering angiogenesis related disease of the eye. Such methods may comprise comprising: an effective systemically or to said eye a pharmaceutical formulation an amount of an agent selected from the group consisting of: an ALK1 ECD protein; antibody that binds to an ALKIligand and inhibits thefinding of the ALKI ligand of GDF5, to ALKI, wherein the ALK1 ligand is selected from th group consisting GDF6, GDF7, BMP9 and BMP10; an antibody that binds to an ALK1 polypeptide the binding of at consisting of amino acids 22-118 of SEQ ID NO; Iand inhibits least one ALK Iligand selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10; and a DAN polypeptide.

In each instance, an agent described herein may be administered in Where it is desirable to conjunction with a second agent that inhibits angiogenesis. conjunction with a inhibit angiogenesis of a tumor, the agent may be administered in agent or a second agent that has an anti-cancer effect, such as a hemotherapeutic biologic anti-cancer agent.

30 The disclosure also provides an ophthalmic pharnmaceutical formulation acid sequence that is at comprising an ALKI-Fc fusion protein having an amino least 97% identical to the sequence of amino acids 2 2 -119 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 ODF5, GDF7 and BMP9 with a Kn of less than I x 10-7 M and binds to TGFP-1 with a KD of greater than1 x 106, In one embodiment, the fusion protein has the sequence of SEQ ID NO: 3. In one embodiment, the c portion is from human IgGl. In one embodiment, the fsion 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 cellline. The formulation may further comprise one or more of the following medicanents: pegaptanib, 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 ALK polyppide consisting of amino acids 22-118 of SEQ ID NO:1 and.inhibits the bindingof at least one ALKI ligand 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:- DF5, GDF6 and GDF7. In one embodiment, the antibody binds to the ALK Ipolypptde with a KD of lessthan 5 x 10-8 M. In another embodiment, the antibody binds to the ALKI polypeptide with a KDof less than I x 100M. In one embodiment, the antibody inhibits angiogenesis stimulated by GDF5, GDF6, GDF7, BMP9, or BMP1OThe formulation may further comprise one or more of the following medicaments: pegaptanib, ranibizumab, or a glucocorticoid. In one embodiment, the formulation issubstantially pyrogen free. In certain aspects, the disclosure provides for an ophthalmic pharmaceutical formulation comprising an antibody that binds to an ALK ligand disclosed herein and inhibits the binding of the ALKI ligand to ALKI. a 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 KOof less than 5 x 10 M. The antibody may be selected for inhibition of angiogenesis stimulated by the 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: is pegaptanib, ranibizumab, or a glucocorticoid. In one embodiment, the formulation substantially pyrogen free. 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 ALKI-Fc fusion protein comprising: a polypeptide having an amino acid sequence that is at least 97% identical to the to an sequence of amino acids 22-118 of SEQ ID NO:1, which polypeptide is fused binds to Fe portion of an immunoglobulin, and wherein the ALIYl-Fc fusion protein GDF5,-GDF7 and BMP9 with a KD of less than1x1I04M and binds to TGFp-1 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 IgG1. 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 medicaments: pegaptanib, ranibizumab or a glucocorticoid. In one embodiment, the formulation is substantially pyrogen fee. The application also provides methods of treating an angiogenesis related disease of the eye comprising administering to said eyean ophthalmic pharmaceutical formulation comprising an antibody that binds to an ALKI inhibits the polypeptide consisting of amino acids 22-118 of SEQ ID NO:1 and binding of at least one ALK ligand selected from thegroup consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10. In one embodimentthe antibody inhibits angiogenesis stimulated by at least one ALK1 ligand selected from the group to consisting of: GDF5, GDF6 and GDF7. In one embodiment, the antibody binds the ALK Ipolypeptide with a KD of less than 5 xOMN. 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, the BMP9, or BMP10. The formulation may further comprise one or more of following medicaments: pegaptanib, ranibizumab, or aglucocorticoid. In one 30 embodiment, the formulation is substantially pyrogenfree. 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 resistant to anti-VEOF therapy, a multiple myeloma tuor, a tumor that has metastasized to the bone, joint or bone inflammation, ruMatoid arthritis,diabetic retinopathy, retinopathy of prematurity, macular degeneration, comeal graft rejection, neovascular glaucoma, and retrolental fibropsias,

BRIEF DESCRIPTION OF THE DRAWINGS

Figure I shows the amino acid sequence for thekunan 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 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 ALK Ito an Fe domain (SEQ ID NO:3). The hALK-F 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 shown. polypeptide of SEQ ID NO:3. The encoded amino acid sequence is also 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 ofmrine 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 Endothelial Cell Growth Supplement (ECGF) to a greater degree than the positive control, Endostatin.

Figure 6 shows the angiogenic effect of GDF7it a chick chorioallantoic membrane (CAM) assay. The GDF7 effect is comparable to that of VEGF.

Figure 7 shows the anti-angiogenic effect of the mnan ALKI-Fc fusion in the CAM assay. hALK1-Fc inhibits angiogenesis stimuated by VEGF, FGF and GDF7.

Figure 8 shows comparative anti-angiogenic effects of urine ALKI-Fe (mALKI-Fe), hALK1-Fe, a commercially available anti-ALK1 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 hALKI-Fc and the anti-VEGF antibody in vivo. hALK1-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 mALK-Fc in thunurine collagen-induced arthritis (CIA) model of rheumatoid arthritis, The graphsows mean group arthritic scores determined during the 42 day observation periodic the collagen-induced male DBA/l arthritic mice. RAP-041 is mALKI-F. AVastiJ is the anti-VEGF

antibody bevacizumab.

DETAILED DESCRIPTION

1. Overview

ALKI is a type I cell-surface receptor for the TGF- superfamily of ligands and is also known as ACVRLI and ACVRLKI. ALKI has been implicated as a receptor for TGF-01, TGF- 3 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 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, orOsler-Rendu-Weber syndrome), in which patients develop arteriovenous malformations tlat create direct flow 30 (communication) from an artery to a vein (arteriovenous shunt), without an intervening capillary bed. Typical symptoms of patiens with HHT include recurrent epistaxis, gastrointestinal hemorrhage, cutaneous and ncocutaneous telangiectases, and arteriovenous malformations (AVM) in the pulmoary, cerebral, or hepatic vasculature.

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(Pt:6):964-72) conclude that BMP9 and BMP10 activate ALK 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 factorssuch as VEGF. Thus, these publications conclude that BMP9 aind BMP10 are thernselves anti-angiogenic factors, and further, that ALKI activation has an anti-angiogenic effect. By contrast, of BMP9 the present disclosure demonstrates that antagonists, rather than agonists, and BMP10 have anti-angiogenic effects.

Thedisclosure relates to the discovery that polypeptides comprising a portion of the extracellular domain of ALK1 ("ALK1 BCD polypeptides") may be and used to inhibit angiogenesis in vivo, including VEGF-independent angiogenesis VEGF, FGF angiogenesis that is mediated by multiple angiogenic factors, including and PDGF. In part, the disclosure provides the identity of physiological, high affinity ligands for ALKI and demonstrates that ALK ECD polypeptides inhibit exert an angiogenesis. The data demonstrate that an ALKI ECD polypeptide can not anti-angiogenic effect even in the case where the ALKl ECD polypeptide does inhibit exhibit meaningful binding to TGF- 1. Moreover, ALM ECD polypeptides angiogenesis that is stimulated by many different pro-angiogenic factors, including of an ALKI VEGF, FGF, and GDF7. Thus, the disclosure provides a description regulatory system, in which ALKI is a receptor for theGDF5 group of ligands, which includes GDF6 and GDF7, and also for the BlP9 group of ligands, which includes BMP10, with different affinities for the two groups of ligands. Further, the disclosure demonstrates that signaling mediated by ALKI and the ligands described has a above is pro-angiogenic in vivo, and that inhibition of this regulatory system the disclosure 30 potent anti-angiogenic effect in vivo. Thus, in certain aspects, of the provides antagonists of the ALKIregulatory system,including antagonists receptor or one or more of the ligands, for use in inhibiting angiogenesis, including both VEGF-dependent angiogenesis and VEGF-indeplent angiogenesis. However, it should be noted that antibodies directed to AL1 Iitself are expected to have different effects from an ALK ECD polypeptide, A pan-neutralizing antibody against ALK1 (one that inhibits the binding of all strong and weak ligands) would be expected to inhibit the signaling of such ligands through ALKI but would not be expected to inhibit the ability of such ligands to signaltrough 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 ALKEICD polypeptide would be expected to inhibit all of the ligands that it binds to igtly, including, for a 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 ALKI would block BMP9 and TGF-signaling through ALKI it would not block BMP9 and TGF-P signaling through moher receptor, and while an ALKI ECD polypeptide may inhibit BMP9 signaling tough all receptors (even receptors other than ALKI).it would not be expected to inhibit TGF-p signaling through any receptor, even ALK1.

Proteins described herein are the human forms,uness otherwise specified. Genbank references for the proteins are as follows: huan GDF5, CAA56874; human GDF6, AAH43222; human GDF7, NP_878248; human BMP9, Q9UK05; 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 WEIVTLECPGHEEVPRVDKL VEKILHCSCQ 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 correspods to amino acids 21-125 (underlined).

Human Dan cDNA sequence (SEQ ID NO l)(Gebank BC012037):

gccgagcctc ctqgggcgcc ogggcccgcg acccccgcac cagtcg aggaccggcg ggcgcgcgg ggctctggag gccacgggca tgatgcttcg ggtcctggtg ggggctgtcc ttcccagata tccctgccat gctactggct gccccaccac ccatcaacaa gtggcaCtg g gccacag ggctgtgagg agagtgcctg gtgcgaagcc aagaacatca occagatcg g gacagtgctt cagctacag gtccccaaca ccaagtccat ccagaacagg gcgtgcctag ccttcccaca gtccacagag tccctggttc actgtgact ctgcatgcca gcccagtcca gagtgcccgg gccacgagga qgggoccagg gtggacaagC tgtgggagat tgtgacgctg Oaaggagcct agtcacgagg tggtggagaa gatcctgcac tgtagctgcc aggcctgcgg ggcgggate ceagccgg acccaccctc ggctgagcgt ctatgtgcag ggcgaggacg acecccatcc ccacccccat cctggcgggc agacccctgaqccCgaggac ccccctgggg ccccccacac agaggaagag ggggctgaggactgaggcc c ccaaCtct tCctccctc aagtgaagc tcatccccct gtggaatgtt gggtotcact ctctggggaaqtcaggggag agactggac ttgaatgctg cccggttgcc ccccctttgg cactggatgg acttggcttc aagctgeaca atttaatata ttcaagagtg atggagatct gaaggggcgg ggttagagcc tcagggctct ttttttgggg ggggggtggt ctcttcctgt gggggaggaa gcagaggtct gectgagcca ttgagtgctg ctggcttcta gagatgtgcc tgtgggaggg ggaggaagtt ciggggtgc agatggtact ggggaggcca tccaagatgg catgaatcgg gctaaggtoc tgccagcctc aggttgagg gagggctggg gctgaggtocc gggcttagt gtgagcatct caggtttacc caaggcctct cactggcaga aacaggaggc tccggcccca ctagaaagac cactgaagtg gccctccctc caccccactt cccatctcca gggaaggtc gccccagtgg aggaccctgc tgactcgtgg cgcgggagct agcggagggg tttgggagtc aggcctgggc tttctctgg cttcttggc ttgcotggtg ggggaagggg gggagccagg ctctccggcc gaaggaaagg gaagagtctt ccaaggcag aeggaggggg acaacccccc aggaggggaa ttagaaatgt tagtgccccg aagaccatcc tgaagacga gcatcccct cctctccctg gcccccaga aagctgtcag accggccgC cttctcccct cactgtgccc caagttctag agtgaggggt tacctccetc ctcccaggga tgotctttgt aaatatogga tgggtgtggg gaacctcgag gaactccagg gccccaaggt tccagagjec ctaggcggga tgggctagct tcagggttca cttgggtct ctctagctcc acgaggagga catgggactt gcgtggacag ccaattctgc ctgcctcdtc octcccagot gcatttaac ectagaaggt ggggacctgg ggoccatgaa gaaagcocct cgttgcccag cactgtctgc ggggagggac agggcaggcg gggtggcctg etcctgcctg gtctgctctt ctgtgcccag ggtggctgcc agcccactgc agtctccctg ggagcttgte accattgga gccctctgg ctgttgcgagcgggcttct ccttcaacgccaaaaaaaaa aaaaaaaaaa atggaccctc agtcttctca tgaataaatt aaaaaaaaaa aaaaaaaaaa aaa acids 93 The coding sequence for DAN precursor corresponds to nucleic to nucleic acids 635, The coding sequence for the mature DAN protein corresponds portion of DAN 141-632. The coding sequence for the conserved cysteine knot corresponds to nucleic acids 153-467,

The terms used in this specification generally lave their ordinary meanings context where each in the art, within the context of this disclosure and in the specific provide additional 40 term is used. Certain terms are discussed in the specification, to and methods disclosed guidance to the practitioner in describing the compositions any use of a term herein and how to make and use them. The scope ormeaning of used. will be apparent from the specific context in which the term is

45

2. Soluble ALKI Polypeptides

Naturally occurring ALKI proteins are transmenbrane proteins, with a portion) and a portion of the protein positioned outside the cell (the extricelluar portion). Aspects of portion of the protein positioned inside the cell (the intacellular the present disclosure encompass polypeptides comprising a portion of the extracellular domain of ALK1.

In certain embodiments, the disclosure provides"ALK ECD polypeptides". The term "ALK1 ECD polypeptide".is intended to refer o apolypeptide consisting of or comprising an amino acid sequence of an extracellular domain of a naturally sequence and occurring ALK1 polypeptide, either including or excluding my signal acid sequence that is at sequence N-terminal to the signal sequence, or an amino least 33 percent identical to an extracellular domain of a naturally occurring ALKI 80%, at least 85%, at polypeptide, and optionally at least 60%, at least 70%,atIeast to the sequence least 90%, at least 95%, at least97%,atleast 99% or 10Vo identical of an extracellular domain of a natrally occurring AL( Ipolypeptide, as 3C-95 of SEQ ID No:1 or the exemplified by the cysteine knot region of amino acids and C-termini of the extracellular cysteine knot plus additional amino acids at the N- an ALKI ECD domain, such as amino acids 22-118 of SEQ ID No. 1. Likewise, encoded by nucleotides 100-285 of polypeptide may comprise a polypeptide that is that hybridize to the SEQ ID NO:2, or silent variants thereof or nucleic acids (generally, such complement thereof under stringent hybridization conditions in 50% conditions are known in the art but may, for example, involve hybridization v/v formamide, 5x SSC, 2% w/v blocking agent, 0.1%N-lauroylsarcosine, 0.3% SDS at 65 C° overnight and washing in, for example, KSSC at about 65 C). that is Additionally, an ALKI ECD polypeptide may comprise a polypeptide thereof or nucleic encoded by nucleotides 64-384 of SEQ ID NO:2, or silent variants 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 CO overnight and washing in, for example, 5xSSC at about 65 C°). The term "ALK BCD polypptide" accordingly thereof encompasses isolated extracellular portions of ALK polypeptides, variants

(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 corresponding to amino acids 34-95 of SEQ ID NO:1), fragments thereof and fusion the proteins comprising any of the preceding, but in each case preferably any of or more of foregoing ALKI ECD polypeptides will retain substantial affinity for one is GDF5, GDF6, GDF7, BMP9 or BMP10. The term "ALK ECD polypeptide" explicitly intended to exclude any full-length, naturally occurring ALKI to be soluble polypeptide. Generally, an ALK ECD polypeptide will be designed 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 Ipolypeptide, To determine the percent identity otwo amino acid sequences, -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 amino acid comparisonpurposes). The amino acid residues at corresponding positions are then compared. When a position in the first sequence is occupied by 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" the two is equivalent to amino acid "homology"). The percent identity between sequences is a function of the number of identical positbas shared by the sequences, be taking into account the number of gaps, and the length of each gap, which need to introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplishedusing 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., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; 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)) the GCO software algorithm which has been incorporated into the GAP program in the following package (available at http://www.gcg.com). In a specific embodiment, 62 matrix or a PAM250 parameters are used in the GAP program: either a Blosm matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, between two nucleotide 4, 5, or 6. In yet another embodiment, the percent identity sequences is determined using the GAP program in the GCG software package at (Devereux, J., et al., Nucleic Acids Res. 12(t):387 (1984))(available using a NWSgapdna.CMP http://www.gcg.com). Exemplary parameters include 5, matrix and a gap weight of 40, 50, 60, 70, or 80 and alength weight of 1, 2, 3, 4, acid or 6. Unless otherwise specified, percent identity between two amino 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 should be the desired percent identity, a suitable-alternative disclosed herein selected. acid In another embodiment, the percent identity between two amino sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 2.0), 4:11-17 (1989)) which has been incorporated into the ALIGN program(version and a gap penalty using a PAM120 weight residue table, a gap length peity of 12 of 4. between two Another embodiment for determining the best overall alignment 25 amino acid sequences can be determined using the FASTDB computer program (1990)). In a based on the algorithm of Brutlag et al (Comp. App. Biosci., 6:237-245 are both amino acid sequences. sequence alignment the query and subject sequences of percent The result of said global sequence alignment is presented in terms identity. In one embodiment, amino acid sequence identity is performed using the 30 FASTDB computer program based on the algorithm of3rutlag et al (Comp. App. employed to Biosci, 6:237-245 (1990)). In a specific embodiment, parameters comprise: calculate percent identity and similarity of an amino acid alignment

Matrix=PAM 150, k-tuple=2, Mismatch Penalty=1, Joining Penalty20, Randomization Group Length=0, Cutoff Score=l, Gappenalty=5 and Gap Size Penalty=0.05. In certain embodiments, ALK ECD polypeptides comprise an extracellular 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 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 does not show substantial binding to TGF-p Ior TGF-p3. Binding may be assessed using purified proteins in solution or in a surface plasmn 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 choioallantoic membrane (CAM) assay, the mouse coneal micropocket assay, an assay for measuring the effect of administering isolatedor synthesized proteins on implanted tumors. The CAM assay is described byO'Rilly, 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 metlylcellulose disc containing the protein to be tested is applied to the CAM of individual 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 observing the pattern of capillaries that are elaborated in the cornea. Other assays are described in the Examples.

ALKI ECD polypeptides may be produced by roving the cytoplasmic tail and the transmembrane region of an ALK polypeptide, Alternatively, the transmembrane domain may be inactivated by deletion,or by substitution of the normally hydrophobic amino acid residues which comprise a transmembrane domain with hydrophilic ones. In either case, a substanially hydrophilichydropathy 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.

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 Possible native ALKI signal sequence may be used to effect extrusion from the cell. Nos. 7 leader sequences include native, tPa and honeybee mellitin leaders (SEQ ID on the leader 9, respectively). Processing of signal peptides may vary depending variables, sequence chosen, the cell type used and culture'onditions, amng other 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 terminal or C-terminal direction.

In certain embodiments, the present disclosure ontemplates specific mutations of the ALK Ipolypeptides so as to alter the glycsylation of the or eliminate one or polypeptide. Such mutations may be selected so as to introduce more glycosylation sites, such as0-linked or N-linked glycosylation sites. 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 amino acid substitutions 30 polypeptide (for O-linked glycosylation sites). A variety of 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) results in non-glycosylation at the modified tripeptide sequence. Another means of increasing the-number of carbohydrate moieties on an ALK Ipolypeptide is by chemical or enzymatic coupling of glycosides to the AIK Ipolypeptide. Depending and on the coupling mode used, the sugar(s) may be attached to (a) arginine of histidine; (b) free carboxyl groups; (c) free sulthydryl groups such as those cysteine; (d) free hydroxyl groups such as those of series, threonine, or tyrosine, or hydroxyproline; (e) aromatic residues such as those of henylalanine, are described in tryptophan; or (f) the amide group of glutamine. These methods CRC Crit. WO 87/05330 published Sep. 11, 1987, and in Aplin andWriston (1981) of one or Rev. Biochem., pp. 259-306, incorporated by referenceliorein. Removal accomplished more carbohydrate moieties present on an ALK polypeptide may be chemically and/or enzymatically. Chemical deglycosylition may involve, for example, exposure of the AL polypeptide to the cornpaund treatment results in trifluoromethanesulfonic acid, or an equivalent compomd.. This or the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine Chemical N-acetylgalactosamine), while leaving the amino acid sequence intact. et l. (1987) Arch. Biochem. deglycosylation is further described by Hakimuddin 18:131. Enzymatic Biophys. 259:52 and by Edge et al. (1981) Anal. Biochen cleavage of carbohydrate moieties on ALKI polypeptides can be achieved by the et al. use of a variety of endo- and exo-glycosidases as descrbed by Thotakura may be (1987) Meth. Enzymol. 138:350. The sequence of an ALK polypeptide used, as adjusted, as appropriate, depending on the type of exprssion system glycosylation mammalian, yeast, insect and plant cells may all introduce differing the peptide. In general, patterns that can be affected by the amino acid sequence of cell line that 25 ALK Iproteins for use in humans will be expressed in a mammalian cell lines, although other provides proper glycosylation, such as HEK293 or CHO mammalian expression cell lines, yeast cell lines with engineered glycosylation as ell enzymes and insect cells are expected to be useful This disclosure further contemplates a method of generating mutants, as well as 30 particularly sets of combinatorial mutants of an ALKpoypeptide, for truncation mutants; pools of combinatorial mutants areespecially useful identifying functional variant sequences. The purposeof screening such combinatorial libraries may be to generate, for example, ALK 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 ALK polypeptide variant may be screened for ability to bind to an ALK ligand, to prevent binding of an ALKI ligand to an ALKIpolypeptide or to interfere with signaling caused by an ALKI ligand. The activity of an ALKIpolypeptide or its variants may also be tested in a cell-based or in vivo assay, particularly any ofthe assays disclosed in the Examples.

Combinatorially-derived variants can be generated which have a selective or generally increased potency relative to an ALK ECD polypeptide comprising an extracellular domain ofa naturally occurring ALKi polypeptide. ikewise, mutagenesis can give riseto variants which haveserum half-lives dramatically different than the corresponding a wild-type ALKI EC)polypeptide. For example, the altered protein can betendered either more stable oess 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 levels the genes which encode them, can be utilized to alter ALK1 CD polypeptide by modulating the half-life of the ALK polypeptides. For instance, a short half-life 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 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 ALKIpolypeptide nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g., for phage display).

There are many ways by which the library of potetial 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. The synthesis of degenerate oligonucleotides is well known in the art (seefor example, Narang, SA (1983) Tetrahedron 39:3; Itakuraet a., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp2 73 -28 9 ; Itakura et al., (1984) Annu, Rev. Bichem. 53:323; Itakura et Such al., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 11:477). techniques have been employed in the directed evolutioaf other proteins (see, for example, Scott et al., (1990) Science 249:386-390; Roberts et al., (1992) PNAS USA 89:2429-2433; 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).

Alternatively, other forms of mutagenesis can be tilized to generate a combinatorial library. For example, ALK polypeptideyariants can be generated and isolated from a library by screening using, for exaple, 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; Grodberg et al., (1993) Eur. J. Biochem. 218:597-601; gagashima et al., (1993) J. Biol. Chem. 268:2888-2892; Lowman et al.,.(1991) Biolnemistry 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 mutagenesis (Meyers et al., (1986) Science 232:613); byPCR mutagenesis (Leung et al., (1989) Method Cell Mo 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; andGreener 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.

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 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 isolation of the vector encoding the gene whose productwas 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 limited present in the ALKI polypeptides. Such modifications include, but are not to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and non acylation. As a result, the modified ALKI ECD polypeptides may contain amino acid elements, suchas polyethylene glycols, lipids, poly- or mono-saccharide, of an and phosphates. Effects of such non-amino acid element on the functionality ALKi ECD polypeptide may be tested as described hereim for other ALK ECD polypeptide variants. When an ALK 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 ofthe protein. Different cells (such as CHO, HeLa, NDCK, 293, W138, NIH-3T3 or HEK293) have specific cellular machinery and characteristic mechanisms forsuch 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 ALK ECD polypeptides include fusion proteins having at least a portion of the ALKI 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 chain constant region (Fe), maltose binding protein (MW),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, 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 QAexpressim fusion system (Qiagen) useful with (HIS6 ) fusion partners. As another example, a domain may be selected so as to facilitate detection of the ALK ECD polypeptides. Examples of such detection domains include the various fluorescent proteins (e.g., for which GFP) as well as "epitope tags," which are usually shortpeptide sequences 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,he fusion domains have a the relevant protease cleavage site, such as for Factor Xa orThrombi which allows protease to partially digest the fusion proteins and therby liberate the recombinant the fusion proteins therefrom. The liberated proteins can then beisolated from domain by subsequent chromatographic separation. Incertain preferred embodiments, an ALKI ECD polypeptide is fused with &domain that stabilizes the ALKI polypeptide in vivo (a "stabilizer" domain). By"stabilizing" is meant of anything that increases serum half fife, regardless ofwhether this is because decreased destruction, decreased clearance by the kidney, or other pharmacokinetic effect. Fusions with the Fr portion of an immunoglobuin are known to confer desirable pharmacokinetic properties on a wide range of proteins. Likewise, fusions 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 Fe domain (e.g., 30 SEQ ID NO: 6).

THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVCVVVD(A)VSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLRGKEYKCK(A)VSNKAL

PVP IEKT ISKAKGQ PRE PQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN(A)HYT QKSLSLSPGK* Optionally, the Fe domain has one or more mutations at residues such as

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) 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 ALK1 ECD polypeptide. The ALKI ECD polypeptide domain and the heterologous domain need not be adjacent in a fusion protein, and aditional domains or amino the acid sequences may be included C- or N-terminal to either domain or between domains.

As used herein, the term, "immunoglobulin Fergiona" or simply "Fe"is understood to mean the carboxyl-terminal portion of an immunoglobulin chain constant region, preferably an immunoglobulin heavy chain constant region, or a 1) a CH portion thereof. For example,'an immunoglobulin Fc region may comprise 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 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 immunoglobuli 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 gs) and IgM (Igp), may be is used. The choice of appropriate immunoglobulin heavy chain constant region discussed in detail in U.S. Pat. Nos. 5,541,087, and 5,726,044. The choice of from certain particular immunoglobulin heavy chain constant region sequences is considered to immunoglobulin classes and subclasses to achieve a paiticular result 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 domain of Fc y or the homologous domains in any of IgA, lgD, IgE, or IgM. acids Furthermore, it is contemplated that substitutionor deletion of amino in the within the immunoglobulin heavy chain constant regions may be useful One example would be practice of the methods and compositions disclosed hereiL. an Fe variant to introduce amino acid substitutions in the upper CH2egion to create with reduced affinity for Fe receptors (Cole et at. (199) L Immunol. 159:3613).

In certain embodiments, the present disclosure makes available isolated and/or purified forms ofthe ALKIECD polypeptides, vhich are isolated from, or otherwise substantially free of (e.g., at least 80%, 90%,95%, 97% or 99% free of, polypeptides other proteins and/or other ALK ECD polypeptide spies. ALK will generally be produced by expression from recombinant nucleic acids.

In certain embodiments, the disclosure includesnucleic acids encoding an extracellular soluble ALK Ipolypeptides comprising the coding sequence for disclosure also pertains portion of an ALKIproteins. In further embodiments,this 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 cells(e.g., using a baculovirus expressed in bacterial cells such as E. coli, insect cells are known 25 expression system), yeast, or mammalian cells. Other suitable host present disclosure to those skilled in the art. Accordingly, some embodiments of the It has been further pertain to methods of producing the ALK ECD polypeptides. and expressed established that an ALK1-Fc fusion protein set orth in SEQ ID NO:3 in CHO cells has potent anti-angiogenic activity.

and fusion proteins DAN polypeptides, including variants of wild type DAN, as described above containing DAN proteins may be generated and characterized with respect to ALK1 ECD proteins.

3. Nucleic Acids Encoding ALKi Polypeptides

In certain aspects, the disclosure provides isolated and/or recombinant (e.g.,ALKI BCD nucleic acids encoding any of the ALK polypeptides variantswd fusion proteins disclosed polypeptides), including fragments, functional ocurring human ALKI herein. For example, SEQ ID NO: 2 encodesthe naturally the precursor of an ALK precursor polypeptide, while SEQ ID NO: 4 encodes acids may be extracellular domain fused to an IgG Fe domain. Thesubject nucleic acids may be DNA or RNA single-stranded or double stranded. Such nucleic for making molecules. These nucleic acids may be used, for example,in methods in an antisense, RNAi or ALK Ipolypeptides or as direct therapeutic agents (eg., gene therapy approach). polypeptides are In certain aspects, the subject nucleic acids encoding ALK variants of SEQ ID NO: 2 or 4. further understood to include nucleic acids that are differ by one or more Variant nucleotide sequences include sequences that asallelic variants. nucleotide substitutions, additions or deletions, such or recombinant 20 In certain embodiments, the disclosure provides isolated 95%, 97%, 98%, 99% or nucleic acid sequences that are at least80%, 85%, 90, skill in the art will 100% identical to SEQ ID NO: 2 or 4. One of ordinary to SEQ ID NO:2 or 4, and appreciate that nucleic acid sequences complementary scope of this disclosure. In further variants of SEQ ID NO: 2 or 4 are also within the can be isolated, 25 embodiments, the nucleic acid sequences of the disclosure sequence, or in a DNA recombinant, and/or fused with a heterologous nucleotide library. also include nucleotide In other embodiments, nucleic acids of the disclosure conditions to the nucleotide sequences that hybridize under highly stringent sequence of SEQ ID NO: 2 30 sequence designated in SEQ ID NO: 2 or 4, complement in the art will or 4, or fragments thereof. As discussed above, one ofordinary skill understand readily that appropriate stringency conditions which promote DNA readily hybridization can be varied. One of ordinary skill in te art will understand be that appropriate stringency conditions which promote DNA hybridization can varied. For example, one could perform the hybridization at 6.0 x sodium 2.0 x SSC at 50 chloride/sodium citrate (SSC) at about 45 C, followedby a wash of from a low °C. For example; the salt concentration in the wash step can be selected x SSC at 50 stringency of about 2.0 x SSC at 50C to a high stringency of about 0.2 from low °C. In addition, the temperature in the wash step canbe increased stringency conditions at room temperature, about 22 C, to high stringency or temperature conditions at about 65 °. Both temperature and salt nay be varied, changed. In or salt concentration may be held constant while the other variable is under low one embodiment, the disclosure provides nucleic acidswhich hybridize a wash at 2 x stringency conditions of 6 x SSC at room temperature foilwed by SSC at room temperature.

Isolated nucleic acids which differ from the nucleic acids as set forth in SEQ the scope of the ID NOs: 2 or 4 due to degeneracy in the genetic code re also within by more than one disclosure. For example, a number of amino acids are designated (for example, CAU triplet. Codons that specify the same amino acid, or sonyms which do not and CAC are synonyms for histidine) may result in "silent"mutations that DNA affect the amino acid sequence of the protein. However, it is expected in the amino acid sequences of the sequence polymorphisms that do lead to changes ne skilled in the art will subject proteins will exist among mammalian cells.C (up to about 3-5% of the appreciate that these variations in one or more nucleoides 25 nucleotides) of the nucleic acids encoding a particular protein may exist among and all such individuals of a given species due to natural allelic variation. Any 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 in an expression 30 be operably linked -to one or more regulatory nucleotide sequences to the host construct. Regulatory nucleotide sequences will generally be appropriate cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art forvariety 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 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 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.

In certain aspects disclosed herein, the subject ucleic 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 ofhe ALK polypeptide. Accordingly, the term regulatory sequence includes promoters, enhancers, and other 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 vaety 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 polypeptide. Such useful expression control sequencesinclude, for example, the early and late promoters of SV40, tet promoter, adenoinu 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 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 oreukaryotic sequences known to control the expression of genes of prokaryotic cells or their viruses, and various combinations thereof. it should be understood that of the the design of the expression vector may depend on such factors as the choice host cell to be transformed and/or the type of protein desired to be expressed. and the Moreover, the vector's copy number, the ability to control that copy number markers, expression of any other protein encoded by the vector, such as antibiotic should also be considered.

A recombinant nucleic acid included in the disclosure can be produced by suitable for expression in ligating the cloned gene, or a portion thereof, into a vector either prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian), or both. Expression vehicles for production of a recombinant ALKIpolypeptide include plasmids include plasmids and other vectors, For instance, suitable vectors of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived for expression in plasmids, pBTac-derived plasmids and pUC-derived plasmids prokaryotic cells, such as K coli. to Some mammalian expression vectors contain both prokaryotic sequences facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pMSG, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dti, pTk2, pRSVneo, mammalian expression pSVT7, pko-neo and pHyg derived vectors are examplesof vectors are vectors suitable for transfection of eukaryotic cells. Some of these to facilitate modified with sequences from bacterial plasmids, suchas pBR322, andeukaryotic cells. replication and drug resistance selection in both prokaryotic Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-1), or an be used for transient 25 Epstein-Barr virus (pHEBo, pREP-derived and p205) (including expression of proteins in eukaryotic cells. Examples of other viral retroviral) expression systems can be found below inthe description of gene therapy of the plasmids delivery systems. The various methods employed in the preparation For other suitable and in transformation of host organisms are well known in the art. well as general 30 expression systems for both prokaryotic and eukaryoticcells, as recombinant procedures, see Molecular Cloning A LaboratoryManual, 3rd Ed., ed. Press, 2001). by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory

In some instances, it may be desirable to express the recombinant polypeptides by the use of a baculovirus expression system. Examples of such baculovirus pVL1393 and expression systems include pVL-derived vectors (such as pVL1392, pVL941), pAcUW-derived vectors (such as pAcUW),and pBlueBac-derived vectors (such as the -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 Ponv-Script vector (Stratagene, vectors La Jolla, Calif.), pcDNA4 vectors (Invitrogen, Carlsbad, Calif.) and pCI-neo gene constructs can be (Promega, Madison, Wisc.). As will be apparent, the subject in used to cause expression of the subject ALKI polypeptides in cells propagated 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 4) for one or more of the gene including a coding sequence (e.g., SEQ ID NO: 2or subject ALK Ipolypeptides. The host cell may be any prokaryotic or eukaryotic in cell. For example, an ALK polypeptide disclosed herein may be expressed expression bacterial cells such as E coli, insect cells (e.g., using abaculovirus are known to those systemn, yeast, or mammalian cells. Other suitable host cells skilled in the art.

Accordingly, the present disclosure further pertains to methods of producing the subject ALKI polypeptides, including ALKi ECDpolypeptides. For example, a can host cell transfected with an expression vector encoding an ALKi polypeptide be cultured under appropriate conditions to allow expression of the ALKi isolated from a polypeptide to occur. The ALKI polypeptide may be secreted and 25 mixture of cells and medium containing the ALK polypeptide. Alternatively, the and ALK Ipolypeptide may be retained cytoplasmically oz in a membrane fraction the cells harvested, lysed and the protein isolated. A cell culture includes host cells, in the media and other byproducts. Suitable media for cell culture are well known art. The subject ALKIpolypeptides 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, for particular electrophoresis, immunoaffinity purification with antibodies specific with an agent that binds epitopes of the ALK1 polypeptides and affinity purification may be used to to a domain fused to the ALK polypeptide (e.g., a protin A column the ALKI polypeptide is a purify an ALKI-Fc fusion), In a preferred embodiment, In a preferred fusion protein containing a domain which facilitates its purification. embodiment, purification is achieved by a series of column chromatography steps, order: protein A including, for example, three or more of the following, in any chromatography, Q sepharose chromatography, phenysepharose chromatography, The size exclusion chromatography, and cation exchange ciromatography. purification could be completed with viral filtration and buffer exchange. In another embodiment, a fusion gene coding for a purification leader site sequence at the N sequence, such as a poly-(His)/enterokinase cleavage can allow terminus of the desired portion of the recombinant ALK1 polypeptide, affinity chromatography using a Ni" purification of the expressed fusion protein by by metal resin. The purification leader sequence can then be subsequently removed treatment with enterokinase to provide the purified AL1 polypeptide (e.g., see et al., PNAS USA Hochuli et al., (1987) J. Chromatography411:177; andJanknecht 88:8972).

Techniques for making fusion genes are wellknown. Essentially, the joining sequences is performed of various DNA fragments coding for different polypeptide in accordance with conventional techniques, employing blunt-ended or stagger provide for appropriate ended termini for ligation, restriction enzyme digestionto phosphatase treatment to termini, filling-in of cohesive ends as appropriate, alkaline the avoid undesirable joining, and enzymatic ligation. In Mother embodiment, automated 25 fusion gene can be synthesized by conventional techniques including fragments can be DNA synthesizers. Alternatively, PCR amplification of gene carried out using anchor primers which give rise to complementary overhangs be annealed to between two consecutive gene fragments which can subsequently CurrentProtocols in generate a chimeric gene sequence (see, for example, 30 Molecular Biology, eds, Ausubel et al.,John Wiley &Sons: 1992).

Examples of categories of nucleic acid compounds that are antagonists of ALK1, BMP9, BMP10, GDF5, GDF6 or GDF7 include antisense nucleic acids, RNAi constructs and catalytic nucleic acid constructs. A nuleic acid compound may also include may be single or double stranded. A double stranded compound other of the strands is regions of overhang or non-complementarity, where one or the of self single stranded. A single stranded compound may include regions "hairpin" or "stem complementarity, meaning that the compound forms a so-called loop" structure, with a region of double helical structure. A nucleic acid compound of may comprise a nucleotide sequence that is complementary to a region consisting no no more than 1000, no more than 500, no more than 250, no more than 100 or more than 50, 35, 30, 25, 22, 20 or 18 nucleotides of the Full-length ALK nucleic acid sequence or ligand nucleic acid sequence. The region of complementarity will 10 or at least 15 preferably be at least 8 nucleotides, and optionally at least nucleotides, and optionally between 15 and 25 nucleotides. A region of complementarity may fall within an intron, a coding sepience or a noncoding a sequence of the target transcript, such as the coding sequence portion. Generally, or nucleic acid compound will have a length of about 8 toabut 500 nucleotides 50 base pairs in length, and optionally the length will be abovt 14 to about nucleotides. A nucleic acid may be a DNA (particularly for use as an antisense), and RNA or RNA:DNA hybrid. Any one strand may include a mixture of DNA as either DNA or RNA, as well as modified forms that cannot readily beclassified or RNA. Likewise, a double stranded compound may beDNA:DNA, DNA:RNA RNA:RNA, and any one strand may also include a mixture of DNA and RNA, as A well as modified forms that cannotreadily be classified as either DNA or RNA. nucleic acid compound may include any of a variety of modifications, including one nucleic or modifications to the backbone (the sugar-phosphate portion in a natural (the purine or acid, including intemucleotide linkages) or the base portion pyrimidine portion of a natural nucleic acid). An antisense nucleic acid compound will preferably have a length of about 15 to about 30 ucleotides and will often as stability in the 30 contain one or more modifications to improve characteristics such such as serum, in a cell or in a place where the compound is likely to be delivered, for inhaled the stomach in the case oforally delivered compounds and the lung the compounds. In the case of an RNAi construct, the strand complenentary to other strand target transcript will generally be RNA or modifications thereof, The double stranded or may be RNA, DNA or any other variation. The duplex portionof a length of 18 to 40 single stranded "hairpin" RNAi construct will preferably have nucleotides in length and optionally about 21 to 23 nuclectides in length, so long as it serves as a Dicer substrate. Catalytic or enzymatic nuleic acids may be acid ribozymes or DNA enzymes and may also contain modified forms. Nucleic 75%, 90% or more compounds may inhibit expression of the target by about 50%, when contacted with cells under physiological conditios and at a concentration where a nonsense or sense control has little or no effect. Preferred concentrations Nucleic for testing the effect of nucleic acid compounds are 1,5and 10 micromolar. 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 DA proteins may be to nucleic acids generated and characterized as described above with respect encoding ALKI ECD proteins.

4. Antibodies an Another aspect of the disclosure pertains to anantibody reactive with are extracellular portion of an ALKl polypeptide, preferably antibodies that embodiment, such specifically reactive with ALK Ipolypeptide. In a prefrred antibody may interfere with ALKi binding to a ligand such as GDF5, GDF6, GDF7 of BMP-9 or BMP-10 - it will be understood that an antibody against a ligand The ALKI should bind to -the mature, processed form of the relevant protein. BMP9 and/or disclosure also provides antibodies that bind to GDF5,GDF6, GDF7, will exhibit BMP10 and inhibit ALK Ibinding to such ligands. Preferred antibodies or corneal an anti-angiogenic activity in a bioassay, such as a CAM assay micropocket assay (see above).

The term "antibody" as used herein is intended to include whole antibodies, or domains of 30 e.g., of any isotype (Igo,IgA, IgM, IgE, etc), and includes fragments can be immunoglobulins which are reactive with a selected antigen, Antibodies utility fragmented using conventional techniques and the fragments screened for and/or interaction with a specific epitope of interest, Tus, the term includes of an segments of proteolytically-cleaved or recombinantly-preparCd portions protein. antibody molecule that are capable of selectively reacting with a certain include Non-limiting examples of such proteolytic and/or recomabinant fragments containing a V[L] and/or Fab, F(ab')2, Fab', Fv, and single chain antibodies (scFv) may be covalently or non V[H] domain joined by a peptide linker. The scFvs The term covalently linked to form antibodies having two or more binding sites. of antibody also includes polyclonal, monoclonal, or other purified preparations antibody", means an antibodies and recombinant antibodies. The term "recombinant from a antibody, or antigen binding domain of an imrnunoglobalin, expressed 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 also included within chain antibody. Single domain and single chain antibodies are the term "recombinant antibody". in the Antibodies may be generated by any of the various methods known administration of antigen to an art, including administration of antigen to an animal, animal that carries human immunoglobulin genes, or screening with an antigen antibody domains). against a library of antibodies (often single chain antibodies or Once antigen binding activity is detected, the relevant portions of the protein may be full-bngth IgG frameworks. For grafted into other antibody frameworks, including or an ALKI example, by using immunogens derived from an ALKIrolypeptide can be made by ligand, anti-protein/anti-peptide antisera or monoclonal ralibodies standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by mouse, 25 Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal, such as a a hamster or rabbit can be immunized with an immunogenic form of the peptide of eliciting an (e.g., a ALKI polypeptide or an antigenic fragment whe is capable antibody response, or a fusion protein). Techniques for conferring immunogenicity techniques well on a protein or peptide include conjugation to carriersor other 30 known in the art. An immunogenic portion -(preferably an extracellular portion) of an ALK Ipolypeptide can be administered in the presece of adjuvant. The progress in plasma or of immunization can be monitored by detection of antibody titers the immunogen as serum. Standard ELISA or other immunoassays can beused with antigen to assess the levels of antibodies.

Following immunization of an animal with an antigeic preparation of an polyclonal ALK polypeptide, anti-ALK Iantisera can be obtainedand, if desired, monoclonal anti-ALKI antibodies can be isolated from the serum. To produce from an antibodies, antibody-producing cells lymphocytess) can be harvested with immunized animal and fused by standard somatic cell fsion procedures Such immortalizing cells such as myeloma cells to yield hybridoma cells. techniques are well known in the art, and include, for example, the hybridoma 256: 495 technique (originally developed by Kohler and Milstei (1975)Nature, 497), the human B cell hybridoma technique (Kozbar et al., (1983) Immunology produce human monoclonal Today, 4:72), and the EBV-hybridoma technique to Therapy, Alan R. antibodies (Cole et al., (1985) Monoclonal Antibodies nd Cancer for Liss, Inc. pp. 77-96). Hybridoma cells can be screenedinmunochemically polypeptide production of antibodies specifically reactive with a manmalian ALKI a culture of the present disclosure and monoclonal antibodies isdated from 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. and the fragments Antibodies can be fragmented using conventional techniques antibodies. For screened for utility in the same manner as describedabove for whole antibody with pepsin. The example, F(ab) 2 fragments can begenerated by treating to produce Fab resulting F(ab)2 fragment can be treated to reduce disfide bridges is father intended to include fragments. The antibody of the present disclosure 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 a label attached thereto and preferred embodiments, the antibody further comprises compound, is able to be detected, (e.g., the label can be a radioisotope, fluorescent enzyme or enzyme co-factor).

disclosure is a In certain preferred embodiments, an antibody ofthe antibody or a fully recombinant antibody, particularly a humanized monoclonal human recombinant antibody. antibody The adjective "specifically reactive with" as used in reference to an antibody is is intended to mean, as is generally understood in the art, that the (e.g.an ALKIpolypeptide or sufficiently selective between the antigen of interest interest that the antibody is useful an ALK ligand) and other antigens that are not of in a particular type for, at minimum, detecting the presence of the antigen ofirtorest the antibody, a higher degree of of biological sample. In certain methods employing For example, a antibody for use in specificity in binding may be desirable. of one or more very detecting a low abundance protein of interest in the presence perfbrn better if it has a higher high abundance protein that are not of interest may of interest and other cross-reactants. degree of selectivity between the antigen tendency (as compared to polyclonal Monoclonal antibodies generally have a greater the desired antigens and cross 15 antibodies) to discriminate effectively between at selectively reacting polypeptides. In addition, an antibody that iseffective sample (e.g. a stool identifying an antigen of interest in one type of biological identifying the same antigen in a sample) may not be as effective for selectively an antibody that different type of biological sample (e.g. a blood sample). Likewise, in a purified protein preparation that 20 is effective at identifying an antigen of interest at identifying an is devoid of other biological contaminants may not be as effective as a blood or urine sample. antigen of interest in a crude biological sample, such antibodies that Accordingly, in preferred embodiments, the applicatiomprovides sample type that is likely have demonstrated specificity for an antigen of interest ii a 25 to be the sample type of choice for use of the antibody.

One characteristic that influences the specificity of an antibody:antigen Although the desired interaction is the affinity of the antibody for the antigen. different affinities, generally preferred specificity may be reached with a range of of about 10, 10-, 104 antibodies will have an affinity (a dissociation constant) of TGFj for ALKI, it is expected 30 or less. Given the apparently low binding affinity theGDF5,6,7 that many anti-ALK I antibodies will inhibit TGF§ binding. However, 5x10~"M and the BMP9,10 group of ligands bind with a Kof approximately

1 0 M. Thus, antibodies of appropriate ligands bind with a KD of approximately IX ligands. affinity may be selected to interfere with the signaling activities of these

In addition, the techniques used to screen antibodies in order to identify a For desirable antibody may influence the properties of the antibody obtained. example, an antibody to be used for certain therapeuticpurposes will preferably be it able to target a particular cell type. Accordingly, to obain antibodies of this type, to calls that express the antigen of may be desirable to screen for antibodies that bind if an antibody is to interest (e.g. by fluorescence activated cell sorting). Likewise, test solution be used for binding an antigen in solution, it may be desirable to binding. A variety of different techniques are available for testing antibody:antigen techniques include interactions to identify particularly desirable antibodies, Such ELISAs, surface plasmon resonance binding assays (e.g.the Biacore binding assay, bead Bia-core AB, Uppsala, Sweden), sandwich assays (e.g.the paramagnetic western blots, system of IGEN International, Inc., Gaithersburg, Marylad), immunoprecipitation assays and immunohistochemisty.

5. Alterations in antibodies and Fc-fusion proteins proteins and The application further provides antibodies, ALK1.-Fc fusion Such antibodies and DAN-Fc fusion proteins with engineered or variant Fe regions. functions, Fc fusion proteins may be useful, for example, in modulating effector such as, antigen-dependent cytotoxicity (ADCC) and complement-dependent improve the stability of cytotoxicity (CDC). Additionally, the modifications may variants of the the antibodies and Fe fusion proteins. Amino acid sequence appropriate nucleotide antibodies and Fc fusion proteins are prepared by introducing 25 changes into the DNA, or by peptide synthesis. Such variants include, for example, residues within the deletions from, and/or insertions into and/or substitutions of, Any amino acid sequences of the antibodies and Fo fusion proteins disclosed herein, arrive at the final combination of deletion, insertion, and substitution ismade to characteristics. The construct, provided that the final construct possesses the desired of the antibodies and 30 amino acid changes also may alter post-translational processes glycosylation sites. Fc fusion proteins, such as changing the number or position of function may be Antibodies and Fc fusion proteins with reducedeffector acid sequence, including, but are not produced by introducing changes in the amino et al. (see WO 94/28027 limited to, the Ala-Ala mutation described by Bluestone 2 0 0 ; 16-26). Thus in and WO 98/47531; also see Xu et a. 2000Cell Immuno the disclosure with certain embodiments, antibodies and Fc fusion proteinsof mutation may be used to mutations within the constant region including the Ala-Ala theseembodiments, antibodies reduce or abolish effector function. According to to an alpine at position 234 or a and Fe fusion proteins may comprise a mutation thereof. Inone mutation to an alanine at position 235, or a combination IgG4 framework, embodiment, the antibody or Fc fusion protein compriss an mutation(s)from phenyalanine to wherein the Ala-Ala mutation would describe a to alanine at position 235. In alanine at position 234 and/or a mutation from leucine another embodiment, the antibody or Fe fusion proteincomprises an IgG from framework, wherein the Ala-Ala mutation would describe a mutation(s) from leucine to alanine at leucine to alanine at position 234 and/or a mutation may alternatively or additionally position 235. The antibody or Fc fusion protein mutation K322A in the C2 domain carry other mutations, including the point (Hezareh et al. 2001 J Virol. 75: 12161-8). protein may be In particular embodiments, the antibody or Fefusion cytotoxicity (CDC). modified to either enhance or inhibitcomplement dependent one or more amino acid Modulated CDC activity may be achieved by introducing region (see, e.g., U.S. Pat. No. substitutions, insertions, or deletions in an Fe residte(s) may be introduced in 6,194,551). Alternatively or additionally, cysteine region. the Fe region, thereby allowing interchain disulfide bond fonnation in this have improved or reduced 25 The homodimeric antibody thus-generated may complement-mediated cell internalization capability and/or increased or decreased (1992) and Shopes, B. J. killing. See Caron et al., J. Exp Med. 176:1191-1195 & Winter Nature 322: 738 Immunol. 148:2918-2922 (1992), W099/51642, Dunan No. 5,624,821; and W094/29351. 40 (1988); U.S. Pat, No. 5,648,260; U.S. Pat.

6. Methods and compositions for modulating anginlsisand 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 polypptide, such as an ALKI-Fe fusion protein, a DAN protein, such as aDAN-Fc fusion protein, or an antibody disclosed herein, such as an antibody against GDF5, GDF6, GDF7, (e.g., antisense or BMP9, BMP10, or the ECD of ALK1, or nucleic acid antagonists siRNA) of any of the foregoing hereafter collectively referred to as "therapeutic the anti-angiogenic therapeutic agents". The data presented indicate specifically that in the eye of a mammal. agents disclosed herein may be used to inhibit angiogenesis It is expected that these therapeutic agents will also be useful in inhibiting tumors associated with angiogenesis in bones and joints, and in tumors, particularly bones and joints.

Angiogenesis associated diseases include, but arenot limited to, tumors, blood born angiogenesis-dependent cancer, including, for examplesolid tumors such as leukemias, and tumor metastases; benig tumors, for example henangiomas, acoustic neuromas, neurofibromas, traclomas, and pyogenic Syndrome; granulomas; rheumatoid arthritis; psoriasis; rubeosis; Osler-Webber telangiectasia; hemophiliac myocardial angiogenesis; plaque neovascularization; joints; and angiofibroma. are In particular, polypeptide therapeutic agents of the present disclosure such cancers as useful for treating or preventing a cancer (tumor), and particularly most anti are known to rely on angiogenic processes to support growth. Unlike that is stimulated angiogenic agents, ALK1 ECD polypeptides affect anogenesis a cancer will by multiple factors. This is highly relevant in cancers,where 25 frequently acquire multiple factors that support tumoragiogenesis. Thus, the tumors therapeutic agents disclosed herein will be particularlyeffective in treating that are resistant to treatment with a drug that targets asingle angiogenic factor (e.g., fusion bevacizumab, which targets VEGF). As demonstratedherein, an ALKl-Fe multiple myeloma. protein is effective in reducing the pathological effectsof 30 Multiple myeloma is widely recognized as a cancer that includes a significant fusion proteins angiogenic component. Accordingly, it is expected that.ALKI-Fo and other therapeutic agents disclosed herein will be useful in treating multiple herein, myeloma and other tumors associated with the bone. As demonstrated therapeutic agents disclosed herein may be used to ameliorate the bone damage ameliorate bone associated with multiple myeloma, and therefore may be used to damage associated with bone metastases of other tumors, such as breast orprostate tumors. As noted herein, the GDF5-7 ligands are highly expressed in bone, and, interference with these while not wishing to be limited to any particular mechanism, for tumor development in bone. ligands may disrupt processes that are required In certain embodiments of such methods, one or more polypeptide or at different therapeutic agents can be administered, together (simultleously) can be administered times (sequentially). In addition, polypeptide therapeutic agents angiogenesis. with another type of compounds for treating cancer or for inhibiting can be used In certain embodiments, the subject methods ofthe disclosure with other alone. Alternatively, the subjectmethods may be used in combination or prevention conventional anti-cancer therapeutic approaches directed to treatment suet methods can be used in of proliferative disorders (e.g., tumor). For example, cancer rearrence and metastases after prophylactic cancer prevention, prevention of cancer therapy. The present surgery, and as an adjuvant of other conventional (e.g., disclosure recognizes that the effectiveness of conventioal cancer therapies and surgery) can be chemotherapy, radiation therapy, phototherapy, immunotherapy, enhanced through the use of a subject polypeptide theraputic agent. have anti A wide array of conventional compounds have been shown to usedas pharmaceutical agents in neoplastic activities. These compounds have been chemotherapy to shrink solid tumors, prevent metastases and further growth, or malignancies. 25 decrease the number of malignant cells in leukemic or bone marrow Although chemotherapy has been effective in treatingvarious types of malignancies, sile effects. It has been shown many anti-neoplastic compounds induce undesirable that when two or more different treatments are combined, the treatments may work treatments, thereby synergistically and allow reduction of dosage of each of the 30 reducing the detrimental side effects exerted by each compound at higher dosages.

respond to a In other instances, malignancies that are refractory to a treatment may combination therapy of two or more different treatments. in When a polypeptide therapeutic agent disclosed herein is administered combination with another conventional anti-neoplastic agent, either concomitantly effect of the or sequentially, such therapeutic agent may enhance the therapeutic 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 agent in undesirable side effects, or restores the effectiveness of an anti-neoplastic resistant cells.

According to the present disclosure, the antiangiogenic agents described procedures for the herein may bemused in combination with other compositions and treatment of diseases. For example, a tumor may be treated conventionally with ALKI ligand surgery, radiation or chemotherapy combined with theALKI or to the patient antagonist and then the antagonist may be subsequentlyeadministered to extend the dormancy of micrometastases and to stabilize any residual primary tumor. to Angiogenesis-inhibiting agents can also be given prophylactically cancers. individuals known to be at high risk for developing nev or re-current prophylactic Accordingly, an aspect of the disclosure encompasses methods for to the subject an prevention of cancer in a subject, comprising administnting thereof, effective amount of an ALK1 or ALK ligand antagonis and/or a derivative or another angiogenesis-inhibiting agent of the presentdisclosure.

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 and placentation. The angiogenesis, for example, ovulation, menstruation, are useful in the treatment angiogenesis inhibiting proteins of the present disclosure These diseases 30 of disease of excessive or abnormal stimulation of endthelial cells. include, but are not limited to, intestinal adhesions, atherosclerosis, scleroderma, and of diseases that hypertrophic scars, i.e., keloids. They are also useful in the treatment have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa) and ulcers (Helicobacter pylori).

General angiogenesis inhibiting proteins can beued as a birth control agent embryo implantation. by reducing or preventing uterine vascularization required for when an Thus, the present disclosure provides an effective birth control method is amount of the inhibitory protein sufficient to pevent embryo implantation of the administered to a female. In one aspect of the birth control method, an amount before or inhibiting protein sufficient to block embryo implantation is administered method after intercourse and fertilization have occurred, thus providing an effective to be bound of birth control, possibly a "morning after" method. While not wanting of the uterine by this statement, it is believed that inhibition of vasculization of endometrium interferes with implantation of the blastocyst. Similar inhibition of the vascularization of the mucosa of the uterine tube interferes with implantation blastocyst, preventing occurrence of a tubal pregnancy. Administration methods may include, but are not limited to, pills, injections (intraveas, subcutaneous, intrauterine intramuscular), suppositories, vaginal sponges, vaginaltampons, and agents of devices. It is also believed that administration of angiogeesis inhibiting vascularization of the the present disclosure will interfere with normal enhanced a successfully implanted placenta, and also with the development of vessels within 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, disclosed neovascular glaucoma, and retrolental fibroplasias. The therapeutic agents 25 herein may be administered intra-ocularly or by otherlocal administration to the eye. Furthermore, as shown in the Examples, ALKI-Fc may be administered systemically and yet have the desired effect on ocular agiogenesis. not Other diseases associated with angiogenesis inthe eye include, but are liriited to, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens 30 overwear, atopic keratitis, superior limbic keratitis, pteygium keratitis sicca, lipid sjogrens, acne rosacea, phylectenulosis, syphilis, Mycotacteria infections, 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, Sceritis, Steven's Johnson disease, periphigoid radial keratotomy, and coeal 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 or of prematurity, Eales disease, Bechets disease, infections causing a retinitis choroiditis, presumed ocular histoplasmosis, Bests disease, myopia, optic pits, Stargarts disease, pars planitis, chronic retinal detachment, hyperviscosity diseases syndromes, toxoplasmosis, trauma and post-laser complications. Other include, but are not limited to, diseases associated withrubeosis (neovasculariation of the angle) and diseases caused by the abnormal proliferation of fibrovascular or fibrous tissue including all forms of proliferative vitreotinopathy.

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 is pharmaceutically acceptable ophthalmic vehicle, such that the compound maintained in contact with the ocular surface for a sufficent time period to allow the compound to penetrate the corneal and internal regionsof the eye, as for example the anterior chamber, posterior chamber, vitreous bodyaqueous humor, vitreous humor, cornea, iris/ciliary, lens, choroid/retina and solea. The pharmaceutically oil or an acceptable ophthalmic vehicle may, for example, be anointment, vegetable 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 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 agentand a medicament are administered together in an ophthalmic pharmaceutical formulation.

In one embodiment, a therapeutic agent is used to treat a disease associated with angiogenesis in the eye by concurrent administrationwith other medicaments that act to block angiogenesis by pharmacological mechanisms. Medicaments that can be concurrently administered with a therapeutic agent of the disclosure include, T but are not limited to, pegapianib (Macugen m), ranibizumab (LucentisT"), squalamine lactate (EvizonT ), heparinase, and glucocorticoids (e.g. Triamcinolone). In one embodiment, a method is provided to treat a disease associated with angiogenesis is treated by administering an ophthalmic agent disclosed pharmaceutical formulation containing at least one therpeutic herein and at least one of the following medicaments: pegaptanib (Macugentm, ranibizumab (Lucentis T"), squalaine lactate (Evizon), heparinase, and glucocorticoids (e.g. Triamcinolone).

7. Formulations and Effective Doses

The therapeutic agents described herein may be formulated into for use in accordance pharmaceutical compositions. Pharmaceutical compositons one or with the present disclosure may be formulated in conventional manner using will more physiologically acceptable carriers or excipients. Such formulations generally be substantially pyrogen free, in complianceith most regulatory requirements. 25 In certain embodiments, the therapeutic method of the disclosure includes device. administering the composition systemically, or locally as an implant or in a When administered, the therapeutic composition for use in this disclosure is useful agents other pyrogen-free, physiologically acceptable form, Therapeutically in the than the ALKi signaling antagonists which may also optionally be included 30 composition as described above, may be administered simultaneously or or any of sequentially with the subject compounds (e.g., ALK ECD polypeptides the antibodies disclosed herein) in the methods disclosed herein.

Typically, protein therapeutic agents disclosed herein will be administered Pharmaceutical parentally, and particularly intravenously or subcutaneously. compositions suitable for parenteral administration may comprise one or more more ALKI ECD polypeptides or other antibodies in combination with one or solutions, pharmaceutically acceptable sterile isotonic aqueous or onaqueous be dispersions, suspensions or emulsions, or sterile powders which may to use, which reconstituted into sterile injectable solutions or dispersionsjust prior the may contain antioddants, buffers, bacteriostats, solutes which render formulation isotonic with the blood of the intended recipient or suspending or which thickening agents. Examples of suitable aqueous and nonaqueous carriers disclosure include may be employed in the pharmaceutical compositions ofthe water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and be maintained, for injectable organic esters, such as ethyl oleate. Proper hidity can by the maintenance of the example, by the use of coating materials, such as lecithin, he use of surfactants. required particle size in the case of dispersions, and by herein In one embodiment, the antibodies and ALK ECD proteins disclosed are administered in an ophthalmic pharmaceutical foralation.In some aqueous embodiments, the ophthalmic pharmaceutical formulation is a sterile or ointment. solution, preferable of suitable concentration for injection, or a salve or ALKI ECD Such salves or ointments typically comprise one or more antibodies proteins disclosed herein dissolved or suspended in a sterile pharmaceutically base. In 25 acceptable salve or ointment base, such as a mineral oil-white petrolatum in the salve or ointment compositions, anhydrous lanolin may also be included to such ointment formulation. Thimerosal or chlorobutanol are also preferably added the sterile aqueous compositions as antimicrobial agents. In one embodiment, solution is as described in U.S. Pat. No. 6,071,958. acids and 30 The disclosure provides formulations that may be varied to include bases to adjust the pH; and buffering agents to keep the pH within a narrow range. include, but are not Additional medicaments may be added to the formulaion. These limited to, pegaptanib, heparinase, ranibizumab, orglucocrticoids. The ophthalmic pharmaceutical formulation according to the disclosure is prepared byaseptic of preparation. manipulation, or sterilization is performed at a suitable stage The compositions and formulations may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the such as active ingredient. The pack may for example comprise metal or plastic foil, for a blister pack. The pack or dispenser device may be accompanied by instructions administration.

EXAMPLES:

Example 1: Expression of ALKI-Fc fusion proteins

Applicants constructed a soluble ALKI fusion protein that has the fused to extracellular domain of human ALK Ifused to a humanFe or mouse ALK1 are referred a urine Fc domain with a minimal linker in between. the constructs to as hALKI-Fc andmALKI-Fc, respectively.

hALKI-Fc is shown as purified from CHO cell lies in Figure 3 (SEQ ID domain of NO: 3). Notably, while the conventional C-terminus ofthe extracellular that human ALK1 protein is amino acid 118 of SEQ ID NO1, we have determined it is desirable to avoid having a domain that ends at a gtamine residue. Accordingly, the portion of SEQ ID NO:3 that derives fom human ALKI The incorporates two residues c-terminal to Q118, a leucineand an alanine. disclosure therefore provides ALK1 ECD polypeptides(including Fc fusion that is anywhere from I proteins) having a c-terminus of the ALKI derived sequence to 5 amino acids upstream (113-117 relative to SEQ IDNO:1) or downstream (119 25 123)ofQll8.

The hALK)-F and mALKI-Fc proteins were expressed in CHO cell lines. Three different leader sequences were considered: (SEQ ID NO: (i) Honey bee mellitin (HBML): MKFLVNVALVFMVVYISYIYA 7)

(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 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, A including, for example, three or more of the following, in any order: protein chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chomatography. The buffer exchange. The purification can be completed with viral filtration and exclusion hALKI-Fe protein was purified to a purity of>98% as determined by size chromatography and >95% as determined by-SDS PAGE

Example 2: Identification of ALKI-Fe Ligands

ALKI is a type I receptors for members of the TFp family. A variety of ALKI-Fe fusion members of the TGFP family were tested for binding toahuman BMP2 and BMP4 protein, using a BiacoreTm system. TGFP itself, GDF8,GDFI1, BMP4 all failed to show substantial binding to the hALK-Fc potein. BMP2 and K values showed limited binding. GDF5, GDF7 and BMP9 shoved binding with Based on the of approximately 5 x 10 M, 5x 10 M and I x 10W ,respectively. similarity of GDF5 and GDF7 to GDF6, it is expected hit GDF6 will bind with bind similar affinity. BMP10 is closely related to BMP9 and is also expected to with similar affinity.

Antibody Effects on Example 3: Characterization of ALKI-Fc and anti-ALKI Endothelial Cells

Using a luciferase reporter construct under the control of four sequential consensus SBE sites (SBE4-luc), which are responsive toSrnad/5/8-mediated and absence of signaling, we measured BMP-9 mediated activity in thepresence hALKI-Fc drug or neutralizing ALKI specific monoclonal antibody in HMVEC 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 (1Opg/ml) or antibody (I0pg/ml) diminished this transcriptional response, each by nearly 60%, indicating that the presence of ALK-F significantly reduces BMP9 signaling, and moreover, that the BMP9signaling is related to ALKI activity.

Activation of SMAD phosphorylation is commonly used to assay activation of of upstream activin receptors. ALK Iis known to modulate phosphorylation SMAD proteins 1,5 and 8 upon activation by its ligand. Here, we added rhBMP-9 a human endothelial (50ng/ml) to initiate SMAD phosphorylation in HUVEC cells, cell line which innately expresses ALK receptor, overtinecourse of 30 minutes. of cells with Phosphorylation of SMAD 1/5/8 was seen 5 minutes after treatment 30 minute period. ligand and phosphorylation was maintained for the entirety of the In the presence of relatively low concentrations of hALK-F (250ng/ml), SMAD 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 ALM-Fc in an in vitro system, we assayed the effectiveness of the compound in reducing tube formation of assess endothelial cells on a Matrigel substrate. This technique is commonly used to 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 ofanti-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 endotheial cell cord formation produced on Matrigel substrate (Fig 5). Upon addition of either hALK-Fc (100 visibly reduced. Final ng/ml) or mALKI-Fe (100ng/ml), cord formation was that every concentration of quantification of vessel length in all samples revealed hALKI-fe or mALKI-F reduced neovascularization to basal levels. Additionally, pro-angiogenic factor hALK1-Fc and mALK-Fec in the presence of the strongly even more ECGS maintained strong inhibition of neovascularization demonstrating endostatin (100ng/mi). potent anti-angiogenic activity than the negative control

Example 4: CAM Assays (chick VEGF and FGF are well-known to stimulate angiogenesis. A CAM the angiogenic effects of chorioallantoic membrane) assay system was used to assess with a potency that is GDF7. As shown in Figure 6, GDF7 stimulates angiogemesis GDF5 and GDF6. similar to that of VEGF. Similar results were observedwith

ALKI-Fe fusions were tested for anti-angiogeni activity in the CAM assay. angiogenesis These fusion proteins showed a potent anti-angiogenic efcct on and PDGF showed a stimulated by VEGF, FGF and GDF7. See Figure 7. BMP9 but such angiogenesic relatively poor capability to induce angiogenesis in this assay, effect of these factors was nonetheless inhibited by ALK .

anti-VEGF ALKI-Fc proteins and acommercially available, aati-angiogenic The ALK-Fec proteins had monoclonal antibody were compared in the CAM assay. antibody bevacizumab similar potency as compared to anti-VEGF. The anti-NEGF degeneration in humans. See is currently used in the treatment of cancer and macular Figure 8.

Interestingly, an anti-ALKIantibody (R&D Systems) failed to significantly that this may reflect the inhibit angiogenesis in this assay system. We expect difference in the ALKI sequence in different species.

Example 5: Mouse Corneal Micropocket Assay the effects of The mouse corneal micropocket assay was used to assess ALKl-Fc on angiogenesis in the mouse eye. hALK-Fe,administered shown in Figure 9, 30 intraperitoneally, significantly inhibited ocular angioguiesis. As hALK hALK1-Fc inhibited ocular angiogenesis to the same degreeas anti-VEGF. dosages. Similar data were Fe and anti-VEGF were used at identical weight/weight VEGFwas implanted in a non obtained when a Matrigel plug impregnated with ocular location. for ALK1 promote These data demonstrate that high affinity ligands has potent anti-angiogenic angiogenesis and that an ALK-F fusion protein the GDF5,6,7 grouping activity. The ligands for ALK fall into two categories,with grouping having a high having an intermediate affinity for ALKI and the BMP9,10 affinity for ALK1. and joints, while BMP9 is GDF5, 6 and 7 are primarily localized to bone to be a pro.ugiogenic system of the circulated in the blood. Thus, there appears systemic angiogenic bones and joints that includes ALK1, GDF5, 6 and 7 an a possibly BlvPi0) system that includes ALK1 and BMP9 (and

Arthritis Example 6: Murine Model of Rheumatoid is a well-accepted model of The murine collagen-induced arthritis model of 10 mice vere treated with vehicle, anti rheumatoid arthritis. In this study, groups becausebevacizumab does not inhibit VEOF (bevacizumab - as a negative control, at I mg/kg, 10 mg/kg or 25 urine VEGF), or doses of mALKI-Fc ("RAP-041") arthritic scores (see Figure 10) and 20 mg/kg. Following the collagen boost on day 21 treated groups, peakingaround day 38. Mice paw swelling steadily increased in all scores for both characteristics, with mALK1-Fc C'RAP-041") showed reduced although the reduction did not achieve particularly at the highest dose (25mg/kg), trend is apparent. statistical significance. Nonetheless, a dose-related of arthritis had reached 10/10 in 25 By study termination at day 42 the incidence bevacizumab treated mice, 8/10 in the the vehicle control treated mice, 9/10 in the in the mALK1-Fc 10mg/kg treated mALKI-Fc at Img/kg treated group and 9/1.0 group disease incidence was lower at group. In themALKI-Fc 25mg/kg treated 6/10.

30

Example 7: Murine Model of Multiple Myeloma is associated with Multiple myeloma is a cancer primarily of the bone that in mice is based on the use substantial bone loss. The 5T2MM model of myeloma develops in of tumor cells (5T2MM cells) from a type of spontaneous tumor that to those seen in human multiple aged mice and causes effects in mice that are similar etal, Methods MolMed. 2005;113:191 myeloma patients. See, e.g., Vanderkerken 205. mALKl-Fc was tested for effects in this model. an increase in 5T2MM cells injected into C57BL/KaLwRij mice promotes nd caused a decrease in bone osteoclast surface, the formation of osteolyti lesions osteoblast number, osteoblast area. Bone disease is associated with a decrease in surface and a reduction in mineralization.

Mice bearing 5T2MM cells were treated with mALKI-FC (RAP-041) from the time of5T2MM injection, for a (10mg/kg, i.p, twice weekly), or a vehicle, tibia and lumbar vertebrae total of 12 weeks. MicroCT analysis of the proximal in cancellous bone volume and demonstrated a statistically significant reduction to naive mice (bone volume trabecular number in 5T2MM-bearing mice compared numberreduced by 40%). RAP-041 reduced by 40% relative to controls; trabecular decreases in bone volume and trabecular completely prevented ST2MM-induced (treated nice had a bone volume of number when compared to vehicle treated mice number of 115% relative to 20 120% relative to untutored controls and trabecular nice developed lytic bone untumored controls). Additionally, the tumor treated treatment reduced the number lesions that were detected by microCT. mALK-Fc to vehicle treatedmice. of lytic bone lesions by 50% relative we infer that the Based on the anti-angiogenic effects of ALKI-c, agent is a consequence of diminished 25 protective effect for bone provided by this tumor growth, myeloma and to decrease Therefore, ALK1-Fc may be used to treat multiple the effects of bone disease resulting from this tumortype,

DAN 30 Example 8: Ligand Binding Characteristics of

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 localizedI igands for ALKI, and DAN is expected 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 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.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein arehereby 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, willcontrol.

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 inihe art upon review of this specification and the claims below. The full scope of the inventions should be determined by reference to the claims, along with theirful scope of equivalents, and the specification, along with such variations.

unless the context Throughout this specification and the claims which follow, variations such as "comprises" and requires otherwise, the word "comprise", and a stated integer or step or "comprising", will be understood to imply the inclusion of any other integer or step or group group of integers or steps but not the exclusion of of integers or steps.

(or information The reference in this specification to any prior publication and should not be taken as derived from it), or to any matter which is known, is not, that prior an acknowledgment or admission or any form of suggestion that it) or known matter forms part of the publication (or information derived from to which this specification common general knowledge in the field of endeavour relates.

Claims (21)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A pharmaceutical composition comprising a polypeptide comprising a ligand binding portion of the extracellular domain of ALKI (ALKI ECD polypeptide), wherein the ALKI ECD polypeptide binds to TGF -1 with a KD of greater than 1 x 10-6 M, wherein the C-terminal amino acid residue of the ALKI-ECD polypeptide is not glutamine 118 (Q118) of SEQ ID NO: 1, wherein the composition is formulated for human administration, wherein the pharmaceutical composition is pyrogen free and wherein the ALKI ECD polypeptide is not a full-length ALKI polypeptide.

2. A pharmaceutical composition comprising a polypeptide comprising a ligand binding portion of the extracellular domain of ALKI (ALKI ECD polypeptide), wherein the C-terminal amino acid residue of the ALKI-ECD polypeptide is not glutamine 118 (Q118) of SEQ ID NO: 1, wherein the ALK ECD polypeptide binds to TGF -1 with a KD of greater than 1 x 10-6 M and wherein the ALKI ECD polypeptide is not a full length ALKI polypeptide.

3. The pharmaceutical composition of claim 1 or claim 2, wherein the ALKI ECD polypeptide comprises an amino acid sequence that is at least 70% identical to the sequence of amino acids 22-118 of the human ALKI sequence of SEQ ID NO: 1.

4. The pharmaceutical composition of claim 3, wherein the ALKI ECD polypeptide comprises an amino acid sequence that is at least 80% identical to the sequence of amino acids 22-118 of the human ALKI sequence of SEQ ID NO: 1.

5. The pharmaceutical composition of claim 3, wherein the ALKI ECD polypeptide comprises an amino acid sequence that is at least 90% identical to the sequence of amino acids 22-118 of the human ALKI sequence of SEQ ID NO: 1.

6. The pharmaceutical composition of claim 3, wherein the ALKI ECD polypeptide comprises an amino acid sequence that is at least 95% identical to the sequence of amino acids 22-118 of the human ALKI sequence of SEQ ID NO: 1.

7. The pharmaceutical composition of claim 3, wherein the ALKI ECD polypeptide comprises an amino acid sequence that is at least 97% identical to the sequence of amino acids 22-118 of the human ALKI sequence of SEQ ID NO: 1.

8. The pharmaceutical composition of claim 3, wherein the ALKI ECD polypeptide comprises an amino acid sequence that is at least 99% identical to the sequence of amino acids 22-118 of the human ALKI sequence of SEQ ID NO: 1.

9. The pharmaceutical composition of claim 3, wherein the ALKI ECD polypeptide comprises an amino acid sequence that is at least 100% identical to the sequence of amino acids 22-118 of the human ALKI sequence of SEQ ID NO: 1.

10. The pharmaceutical composition of any one of claims 1-9, wherein the ALKI ECD polypeptide binds to ligands of ALKI thereby inhibiting their ability to interact with ALKI and other receptors.

11. The pharmaceutical composition of claim 10, wherein the ALKI ECD polypeptide binds to one or more of GDF5, GDF6, GDF7, BMP9 and BMP10.

12. The pharmaceutical composition of any one of the preceding claims, wherein the ALKI ECD is fused to a second polypeptide portion.

13. The pharmaceutical composition of claim 12, wherein the ALKI ECD is fused to an Fc portion of an immunoglobulin, being an ALKI-Fc fusion protein.

14. The pharmaceutical composition of claim 13, wherein the ALK1-Fc fusion protein binds to one or more ALKI ligands selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10.

15. The pharmaceutical composition of claim 13 or claim 14, wherein the ALK1-Fc fusion protein comprises a polypeptide having an amino acid sequence that is at least %, 80%, 90%, 95%, 97%, 99% or 100% identical to the sequence of amino acids 22 118 of SEQ ID NO:1, wherein the ALK1-Fc fusion protein binds to GDF5, GDF7 and BMP9 with a KD of less than 1 x 10-7 M and binds to TGF -1 with a KD of greater than 1 x 10-6 M.

16. The pharmaceutical composition of any one of claims 13-15, wherein the Fc portion is an Fc portion of a human IgG1.

17. The pharmaceutical composition of any one of the preceding claims, which is an ophthalmic formulation.

18. The pharmaceutical composition of any one of claims 1-17, wherein the ALKI ECD polypeptide comprises an amino acid sequence that is of amino acids 22-120 of the human ALKI sequence of SEQ ID NO: 1.

19. The pharmaceutical composition of any one of claims 1-17, wherein the C terminal amino acid residue of the polypeptide is proline 113 (P113), glycine 114 (G114), threonine 115 (T115), aspartic acid 116 (D116), glycine 117 (G117), leucine 119 (L119), alanine 120 (A120), leucine 121 (L121), isoleucine 122 (1122), or leucine 123 (L123) of SEQ ID NO: 1.

20. The pharmaceutical composition of any one of claims 1-17, wherein the ALKI ECD polypeptide: (a) begins at amino acid 22 of SEQ ID NO: 1, and

(b) ends at any one of amino acids proline 113 (P113), glycine 114 (G114), threonine 115 (T115), aspartic acid 116 (D116), or glycine 117 (G117).

21. The pharmaceutical composition of any one of claims 1-17, wherein the ALKI ECD polypeptide: (a) begins at amino acid 22 of SEQ ID NO: 1, and (b) ends at any one of amino acids leucine 119 (L119), alanine 120 (A120), leucine 121 (L121), isoleucine 122 (1122), or leucine 123 (L123) of SEQ ID NO: 1.