Alk1 receptor and ligand antagonists and uses thereof

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 (ALK1) polypeptide may be used to inhibit angiogenesis in vivo, particularly in mammals suffering angiogenesis-related disorders. The disclosure also identifies ligands for ALK1 and demonstrates that such ligands have pro-angiogenic activity, and antibodies that inhibit receptor-ligand interaction.

We claim: 
     
         1 . An ALK1-Fc fusion protein comprising: a polypeptide having an amino acid sequence that is at least 97% identical to the sequence of amino acids 22-118 of SEQ ID NO:1, which polypeptide is fused to an Fc portion of an immunoglobulin, and wherein the ALK1-Fc fusion protein binds to GDF5, GDF7 and BMP9 with a K D  of less than 1×10 −7  M and binds to TGFβ-1 with a K D  of greater than 1×10 −6 . 
     
     
         2 . The ALK1-Fc fusion protein, wherein the Fc portion is an Fc portion of a human IgG 1. 
     
     
         3 . An ALK1-Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 3. 
     
     
         4 . An ALK1-Fc fusion protein that is produced by expression of the nucleic acid of SEQ ID NO:4 in a mammalian cell line. 
     
     
         5 . The ALK1-Fc fusion protein of  claim 4 , wherein the mammalian cell line is a Chinese Hamster Ovary (CHO) cell line. 
     
     
         6 . A pharmaceutical preparation that is substantially pyrogen free, comprising an ALK1-Fc fusion protein of  claim 5 . 
     
     
         7 . An antibody that binds to an ALK1 polypeptide consisting of amino acids 22-118 of SEQ ID NO:1 and inhibits the binding of at least one ALK1 ligand selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10. 
     
     
         8 . The antibody of  claim 7 , wherein the antibody binds to the ALK1 polypeptide with a K D  of less than 5×10 −8  M. 
     
     
         9 . The antibody of  claim 7 , wherein the antibody binds to the ALK1 polypeptide with a K D  of less than 1×10 −10  M. 
     
     
         10 . The antibody of  claim 8 , wherein the antibody inhibits angiogenesis stimulated by at least one ALK1 ligand selected from the group consisting of: GDF5, GDF6 and GDF7. 
     
     
         11 . The antibody of  claim 10 , wherein the antibody inhibits binding of BMP9 and BMP10 to ALK1. 
     
     
         12 . A pharmaceutical preparation that is substantially pyrogen free comprising the antibody of  claim 7 . 
     
     
         13 . A method of inhibiting angiogenesis in a mammal, the method comprising, administering to the mammal an effective amount of an ALK1 ECD protein. 
     
     
         14 . The method of  claim 13 , wherein the ALK-1 ECD protein is an ALK1-Fc fusion protein. 
     
     
         15 . The method of  claim 14 , wherein the ALK1-Fc fusion protein comprises a polypeptide having an amino acid sequence that is at least 90% identical to the sequence of amino acids 22-118 of SEQ ID NO:1, which polypeptide is fused to an Fc portion of an immunoglobulin, and wherein the ALK1-Fc fusion protein binds to TGFβ-1 with a K D  of greater than 1×10 −6 . 
     
     
         16 . The method of  claim 13 , wherein the ALK1 ECD protein binds to one or more ALK1 ligands selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10. 
     
     
         17 . The method of  claim 13 , wherein the ALK1 ECD polypeptide comprises an amino acid sequence that is at least 90% identical to the sequence of amino acids corresponding to amino acids 34-95 of SEQ ID NO:1. 
     
     
         18 . The method of  claim 13 , wherein the ALK1 ECD comprises an amino acid sequence encoded by a nucleic acid that hybridized under stringent hybridization conditions to nucleotides 100-285 of SEQ ID NO:2 or a variant of nucleotides 100-285 of SEQ ID NO:2 that has the same coding sequence. 
     
     
         19 . The method of  claim 14 , wherein the ALK1-Fc fusion protein has a sequence of SEQ ID NO:3. 
     
     
         20 . The method of  claim 13 , wherein the ALK1 ECD fusion protein is delivered intravenously or locally to the eye. 
     
     
         21 . The method of  claim 13 , wherein the method further comprises administering a second agent that inhibits angiogenesis. 
     
     
         22 . The method of  claim 13 , wherein the angiogenesis to be inhibited is angiogenesis occurring: in the eye of the mammal, in a tumor or in a bone or joint. 
     
     
         23 . A method of inhibiting angiogenesis in a mammal, the method comprising, administering to the mammal an effective amount of an antibody that binds to an ALK1 polypeptide consisting of amino acids 22-118 of SEQ ID NO:1 and inhibits the binding of at least one ALK1 ligand selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10. 
     
     
         24 . The method of  claim 23 , wherein the antibody binds to the ALK1 polypeptide with a K D  of less than 5×10 −8  M. 
     
     
         25 . The method of  claim 23 , wherein the antibody binds to the ALK1 polypeptide with a K D  of less than 1×10 −10  M. 
     
     
         26 . The method of  claim 23 , wherein the antibody inhibits angiogenesis stimulated by at least one ALK1 ligand selected from the group consisting of: GDF5, GDF6 and GDF7. 
     
     
         27 . The method of  claim 23 , wherein the antibody inhibits binding of ALK1 to an ALK1 ligand, wherein the ALK1 ligand is selected from the group consisting of: BMP9 and BMP10. 
     
     
         28 . The method of  claim 23 , wherein the antibody is delivered intravenously. 
     
     
         29 . The method of  claim 23 , wherein the method further comprises administering a second agent that inhibits angiogenesis. 
     
     
         30 . The method of  claim 23 , wherein the angiogenesis to be inhibited is angiogenesis occurring: in the eye of the mammal, in a tumor or in a bone or joint. 
     
     
         31 . An antibody that binds to an ALK1 ligand and inhibits the binding of the ALK1 ligand to ALK1, wherein the ALK1 ligand is selected from the group consisting of GDF5, GDF6 and GDF7. 
     
     
         32 . The antibody of  claim 31 , wherein the antibody binds to the ALK1 ligand with a K D  of less than 5×10 −8  M. 
     
     
         33 . The antibody of  claim 31 , wherein the antibody inhibits angiogenesis stimulated by the ALK1 ligand. 
     
     
         34 . A pharmaceutical preparation comprising the antibody of  claim 31 . 
     
     
         35 . An antibody that binds to an ALK1 ligand and inhibits the binding of the ALK1 ligand to ALK1, wherein the ALK1 ligand is selected from the group consisting of BMP9 and BMP10. 
     
     
         36 . The antibody of  claim 35 , wherein the antibody binds to the ALK1 ligand with a K D  of less than 1×10 −10  M. 
     
     
         37 . A pharmaceutical preparation comprising the antibody of  claim 35 . 
     
     
         38 . A method of inhibiting angiogenesis in a mammal, the method comprising, administering to the mammal an effective amount of an antibody that binds to an ALK1 ligand and inhibits the binding of the ALK1 ligand to ALK1, wherein the ALK1 ligand is selected from the group consisting of GDF5, GDF6, GDF7, BMP9 and BMP10. 
     
     
         39 . The method of  claim 38 , wherein the antibody inhibits angiogenesis stimulated by at least one ALK1 ligand selected from the group consisting of: GDF5, GDF6 and GDF7. 
     
     
         40 . The method of  claim 38 , wherein the antibody inhibits angiogenesis stimulated by at least one ALK1 ligand selected from the group consisting of: BMP9 and BMP10. 
     
     
         41 . The method  claim 38 , wherein the antibody is delivered intravenously. 
     
     
         42 . The method of  claim 38 , wherein the method further comprises administering a second agent that inhibits angiogenesis. 
     
     
         43 . The method of  claim 38 , wherein the angiogenesis to be inhibited is angiogenesis occurring: in the eye of the mammal, in a tumor or in a bone or joint. 
     
     
         44 . Use of an ALK1 ECD polypeptide or nucleic acids encoding the same in the manufacture of a medicament for the inhibition of angiogenesis in a mammal. 
     
     
         45 . The use of  claim 44 , wherein the ALK1 ECD polypeptide is an ALK-1 Fc fusion protein. 
     
     
         46 . The use of  claim 45 , wherein the ALK-1 Fc fusion protein comprises a polypeptide having an amino acid sequence that is at least 90% identical to the sequence of amino acids 22-118 of SEQ ID NO:1, which polypeptide is fused to an Fc portion of an immunoglobulin, and wherein the ALK1-Fc fusion protein binds to TGFβ-1 with a K D  of greater than 1×10 −6 . 
     
     
         47 . Use of an antibody that binds to ALK1 or nucleic acids encoding the same in the manufacture of a medicament for the inhibition of angiogenesis in a mammal, wherein the antibody binds to an ALK1 polypeptide consisting of amino acids 22-118 of SEQ ID NO:1 and inhibits the binding of at least one ALK1 ligand selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10. 
     
     
         48 . Use of an antibody that binds to an ALK1 ligand and inhibits the binding of the ALK1 ligand to ALK1, or nucleic acids encoding the same, in the manufacture of a medicament for the inhibition of angiogenesis in a mammal, wherein the antibody binds to an ALK1 ligand selected from the group consisting of GDF5, GDF6, GDF7, BMP9 and BMP10. 
     
     
         49 . A method of inhibiting angiogenesis in a mammal, the method comprising, administering to the mammal an effective amount of a DAN protein. 
     
     
         50 . The method of  claim 49 , wherein the DAN protein is a DAN-Fc fusion protein. 
     
     
         51 . The method of  claim 49 , wherein the DAN-Fc fusion protein comprises a polypeptide having an amino acid sequence that is at least 90% identical to the sequence of amino acids 21-125 of SEQ ID NO:10, which polypeptide is fused to an Fc portion of an immunoglobulin, and wherein the ALK1-Fc fusion protein binds to TGFβ-1 with a K D  of greater than 1×10 −6 . 
     
     
         52 . The method of  claim 49 , wherein the DAN protein binds to one or more ALK1 ligands selected from the group consisting of: GDF5, GDF6 and GDF7. 
     
     
         53 . The method of  claim 49 , wherein the DAN protein comprises an amino acid sequence that is at least 90% identical to the sequence of amino acids corresponding to amino acids 17-180 of SEQ ID NO:10. 
     
     
         54 . The method of  claim 49 , wherein the DAN protein comprises an amino acid sequence encoded by a nucleic acid that hybridizes under stringent hybridization conditions to nucleotides 153-467 of SEQ ID NO:11 or a variant of nucleotides 153-467 of SEQ ID NO:11 that has the same coding sequence. 
     
     
         55 . The method of  claim 49 , wherein the method further comprises administering a second agent that inhibits angiogenesis. 
     
     
         56 . The method of  claim 49 , wherein the angiogenesis to be inhibited is angiogenesis occurring: in the eye of the mammal, in a tumor or in a bone or joint. 
     
     
         57 . A method for treating rheumatoid arthritis in a mammal, the method comprising, administering to a mammal that has rheumatoid arthritis an effective amount of an agent selected from the group consisting of:
 (a) an ALK1 ECD protein;   (b) an antibody that binds to an ALK1 ligand and inhibits the binding of the ALK1 ligand to ALK1, wherein the ALK1 ligand is selected from the group consisting of GDF5, GDF6, GDF7, BMP9 and BMP10;   (c) an antibody that binds to an ALK1 polypeptide consisting of amino acids 22-118 of SEQ ID NO:1 and inhibits the binding of at least one ALK1 ligand selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10; and   (d) a DAN polypeptide.   
     
     
         58 . The method of  claim 57 , wherein the ALK-1 ECD protein is an ALK1-Fc fusion protein. 
     
     
         59 . The method of  claim 57 , wherein the ALK1-Fc fusion protein comprises a polypeptide having an amino acid sequence that is at least 90% identical to the sequence of amino acids 22-118 of SEQ ID NO:1, which polypeptide is fused to an Fc portion of an immunoglobulin, and wherein the ALK1-Fc fusion protein binds to TGFβ-1 with a K D  of greater than 1×10 −6 . 
     
     
         60 . The method of  claim 57 , wherein the ALK1 ECD protein binds to one or more ALK1 ligands selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10. 
     
     
         61 . The method of  claim 58 , wherein the ALK1-Fc fusion protein has a sequence of SEQ ID NO:3. 
     
     
         62 . The method of  claim 57 , wherein the ALK1 ECD polypeptide comprises an amino acid sequence that is at least 90% identical to the sequence of amino acids corresponding to amino acids 34-95 of SEQ ID NO:1. 
     
     
         63 . The method of  claim 57 , wherein the ALK1 ECD comprises an amino acid sequence encoded by a nucleic acid that hybridized under stringent hybridization conditions to nucleotides 100-285 of SEQ ID NO:2 or a variant of nucleotides 100-285 of SEQ ID NO:2 that has the same coding sequence. 
     
     
         64 . The method of  claim 57 , wherein the antibody of (b) binds to the ALK1 polypeptide with a K D  of less than 5×10 −8  M. 
     
     
         65 . The method of  claim 57 , wherein the antibody of (b) binds to the ALK1 polypeptide with a K D  of less than 1×10 −10  M. 
     
     
         66 . The method of  claim 57 , wherein the antibody of (b) inhibits angiogenesis stimulated by at least one ALK1 ligand selected from the group consisting of: GDF5, GDF6 and GDF7. 
     
     
         67 . The method of  claim 57 , wherein the antibody of (b) inhibits binding of ALK1 to an ALK1 ligand, wherein the ALK1 ligand is selected from the group consisting of: BMP9 and BMP10. 
     
     
         68 . The method of  claim 57 , wherein the antibody of (c) inhibits angiogenesis stimulated by at least one ALK1 ligand selected from the group consisting of: GDF5, GDF6 and GDF7. 
     
     
         69 . The method of  claim 57 , wherein the antibody of (c) inhibits angiogenesis stimulated by at least one ALK1 ligand selected from the group consisting of: BMP9 and BMP10. 
     
     
         70 . The method of  claim 57 , wherein the DAN protein is a DAN-Fc fusion protein. 
     
     
         71 . The method of  claim 70 , wherein the DAN-Fc fusion protein comprises a polypeptide having an amino acid sequence that is at least 90% identical to the sequence of amino acids 21-125 of SEQ ID NO:10, which polypeptide is fused to an Fc portion of an immunoglobulin, and wherein the ALK1-Fc fusion protein binds to TGFβ-1 with a K D  of greater than 1×10 −6 . 
     
     
         72 . The method of  claim 57 , wherein the DAN protein binds to one or more ALK1 ligands selected from the group consisting of: GDF5, GDF6 and GDF7. 
     
     
         73 . The method of  claim 57 , wherein the DAN protein comprises an amino acid sequence that is at least 90% identical to the sequence of amino acids corresponding to amino acids 17-180 of SEQ ID NO:10. 
     
     
         74 . The method of  claim 57 , wherein the DAN protein comprises an amino acid sequence encoded by a nucleic acid that hybridizes under stringent hybridization conditions to nucleotides 153-467 of SEQ ID NO:11 or a variant of nucleotides 153-467 of SEQ ID NO:11 that has the same coding sequence. 
     
     
         75 . The method of  claim 57 , wherein the agent is delivered intravenously. 
     
     
         76 . The method of  claim 57 , wherein the method further comprises administering a second agent that inhibits angiogenesis. 
     
     
         77 . A method for treating a tumor in a mammal, the method comprising, administering to a mammal that has a tumor an effective amount of an agent selected from the group consisting of:
 (a) an ALK1 ECD protein;   (b) an antibody that binds to an ALK1 ligand and inhibits the binding of the ALK1 ligand to ALK1, wherein the ALK1 ligand is selected from the group consisting of GDF5, GDF6, GDF7, BMP9 and BMP10;   (c) an antibody that binds to an ALK1 polypeptide consisting of amino acids 22-118 of SEQ ID NO:1 and inhibits the binding of at least one ALK1 ligand selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10; and   (d) a DAN polypeptide.   
     
     
         78 . The method of  claim 77 , wherein the ALK-1 ECD protein is an ALK1-Fc fusion protein. 
     
     
         79 . The method of  claim 78 , wherein the ALK1-Fc fusion protein comprises a polypeptide having an amino acid sequence that is at least 90% identical to the sequence of amino acids 22-118 of SEQ ID NO:1, which polypeptide is fused to an Fc portion of an immunoglobulin, and wherein the ALK1-Fc fusion protein binds to TGFβ-1 with a K D  of greater than 1×10 −6 . 
     
     
         80 . The method of  claim 77 , wherein the ALK1 ECD protein binds to one or more ALK1 ligands selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10. 
     
     
         81 . The method of  claim 78 , wherein the ALK1-Fc fusion protein has a sequence of SEQ ID NO:3. 
     
     
         82 . The method of  claim 77 , wherein the ALK1 ECD polypeptide comprises an amino acid sequence that is at least 90% identical to the sequence of amino acids corresponding to amino acids 34-95 of SEQ ID NO:1. 
     
     
         83 . The method of  claim 77 , wherein the ALK1 ECD comprises an amino acid sequence encoded by a nucleic acid that hybridized under stringent hybridization conditions to nucleotides 100-285 of SEQ ID NO:2 or a variant of nucleotides 100-285 of SEQ ID NO:2 that has the same coding sequence. 
     
     
         84 . The method of  claim 77 , wherein the antibody of (b) binds to the ALK1 polypeptide with a K D  of less than 5×10 −8  M. 
     
     
         85 . The method of  claim 77 , wherein the antibody of (b) binds to the ALK1 polypeptide with a K D  of less than 1×10 −10  M. 
     
     
         86 . The method of  claim 77 , wherein the antibody of (b) inhibits angiogenesis stimulated by at least one ALK1 ligand selected from the group consisting of: GDF5, GDF6 and GDF7. 
     
     
         87 . The method of  claim 77 , wherein the antibody of (b) inhibits binding of ALK1 to an ALK1 ligand, wherein the ALK1 ligand is selected from the group consisting of: BMP9 and BMP10. 
     
     
         88 . The method of  claim 77 , wherein the antibody of (c) inhibits angiogenesis stimulated by at least one ALK1 ligand selected from the group consisting of: GDF5, GDF6 and GDF7. 
     
     
         89 . The method of  claim 77 , wherein the antibody of (c) inhibits angiogenesis stimulated by at least one ALK1 ligand selected from the group consisting of: BMP9 and BMP10. 
     
     
         90 . The method of  claim 77 , wherein the DAN protein is a DAN-Fc fusion protein. 
     
     
         91 . The method of  claim 90 , wherein the DAN-Fc fusion protein comprises a polypeptide having an amino acid sequence that is at least 90% identical to the sequence of amino acids 21-125 of SEQ ID NO:10, which polypeptide is fused to an Fc portion of an immunoglobulin, and wherein the ALK1-Fc fusion protein binds to TGFβ-1 with a K D  of greater than 1×10 −6 . 
     
     
         92 . The method of  claim 77 , wherein the DAN protein binds to one or more ALK1 ligands selected from the group consisting of: GDF5, GDF6 and GDF7. 
     
     
         93 . The method of  claim 77 , wherein the DAN protein comprises an amino acid sequence that is at least 90% identical to the sequence of amino acids corresponding to amino acids 17-180 of SEQ ID NO:10. 
     
     
         94 . The method of  claim 77 , wherein the DAN protein comprises an amino acid sequence encoded by a nucleic acid that hybridizes under stringent hybridization conditions to nucleotides 153-467 of SEQ ID NO:11 or a variant of nucleotides 153-467 of SEQ ID NO:11 that has the same coding sequence. 
     
     
         95 . The method of  claim 77 , wherein the agent is delivered intravenously. 
     
     
         96 . The method of  claim 77 , wherein the method further comprises administering a second agent that inhibits angiogenesis. 
     
     
         97 . The method of  claim 77 , wherein the method further comprises administering a second agent that inhibits angiogenesis. 
     
     
         98 . The method of  claim 77 , wherein the tumor is associated with bone. 
     
     
         99 . The method of  claim 77 , wherein the tumor is a myeloma or a tumor that has metastasized to the bone. 
     
     
         100 . The method of  claim 77 , wherein the tumor is resistant to anti-VEGF therapy. 
     
     
         101 . An ophthalmic pharmaceutical formulation comprising an agent selected from the group consisting of:
 (a) an ALK1 ECD protein;   (b) an antibody that binds to an ALK1 ligand and inhibits the binding of the ALK1 ligand to ALK1, wherein the ALK1 ligand is selected from the group consisting of GDF5, GDF6, GDF7, BMP9 and BMP10;   (c) an antibody that binds to an ALK1 polypeptide consisting of amino acids 22-118 of SEQ ID NO:1 and inhibits the binding of at least one ALK1 ligand selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10; and   (d) a DAN polypeptide.   
     
     
         102 . A method of treating an angiogenesis related disease of the eye comprising administering systemically or to said eye a pharmaceutical formulation comprising: an effective amount of an agent selected from the group consisting of:
 (a) an ALK1 ECD protein;   (b) an antibody that binds to an ALK1 ligand and inhibits the binding of the ALK1 ligand to ALK1, wherein the ALK1 ligand is selected from the group consisting of GDF5, GDF6, GDF7, BMP9 and BMP10;   (c) an antibody that binds to an ALK1 polypeptide consisting of amino acids 22-118 of SEQ ID NO:1 and inhibits the binding of at least one ALK1 ligand selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10; and   (d) a DAN polypeptide.