Methods and compositions for modulating angiogenesis and pericyte composition

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. Additionally, the disclosure demonstrates that inhibitors of ALK1 may be used to increase pericyte coverage in vascularized tissues, including tumors and the retina. The disclosure also identifies ligands for ALK1 and demonstrates that such ligands have pro-angiogenic activity, and describes antibodies that inhibit receptor-ligand interaction.

We claim: 
     
         1 . A pharmaceutical preparation comprising 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 .and wherein at least 90% of the ALK1-Fc fusion protein is present in a dimeric form. 
     
     
         2 . The pharmaceutical preparation of  claim 1 , wherein the Fc portion of the ALK1-Fc fusion protein is an Fc portion of a human IgG1. 
     
     
         3 . The pharmaceutical preparation of  claim 1 , wherein the ALK1-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 3. 
     
     
         4 . The pharmaceutical preparation of  claim 1 , wherein the ALK1-Fc fusion protein is produced by expression of the nucleic acid of SEQ ID NO:4 in a mammalian cell line. 
     
     
         5 . The pharmaceutical preparation of  claim 4 , wherein the ALK1-Fc fusion protein is produced by expression of the nucleic acid of SEQ ID NO:4 in a Chinese Hamster Ovary (CHO) cell line. 
     
     
         6 . The pharmaceutical preparation of  claim 1 , wherein the pharmaceutical preparation is a pharmaceutical preparation that is suitable for administration to the eye. 
     
     
         7 . The pharmaceutical preparation of  claim 1 , wherein at least 95% of the ALK1-Fc fusion protein is present in a dimeric form. 
     
     
         8 . The pharmaceutical preparation of  claim 1 , wherein at least 99% of the ALK1-Fc fusion protein is present in a dimeric form. 
     
     
         9 . A method for inhibiting angiogenesis in a mammal in need thereof, the method comprising administering to the mammal an effective amount of a pharmaceutical preparation of  claim 1 . 
     
     
         10 . A method for treating a tumor in a mammal in need thereof, the method comprising administering to the mammal an effective amount of a pharmaceutical preparation of  claim 1 . 
     
     
         11 . A method of treating an ocular disorder associated with angiogenesis in a mammal in need thereof, the method comprising administering to the mammal an effective amount of a pharmaceutical preparation of  claim 1 . 
     
     
         12 . A method for treating rheumatoid arthritis in a mammal in need thereof, the method comprising, administering to the mammal an effective amount of a pharmaceutical preparation of  claim 1 . 
     
     
         13 . The method of  claim 9 , wherein the method further comprises administering a second agent that inhibits angiogenesis. 
     
     
         14 . A method of promoting an increase in pericyte coverage in a vascularized tissue of a mammal in need thereof, the method comprising administering to the mammal an effective amount of an ALK1 ECD protein. 
     
     
         15 . The method of  claim 14 , wherein the ALK1 ECD protein is an ALK1-Fc fusion protein. 
     
     
         16 . The method of  claim 15 , 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 . 
     
     
         17 . The method of  claim 14 , wherein the ALK1 ECD protein binds to one or more ALK1 ligands selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10. 
     
     
         18 . The method of  claim 14 , 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. 
     
     
         19 . The method of  claim 14 , wherein the ALK1 ECD comprises an amino acid sequence encoded by a nucleic acid that hybridizes 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 encodes the same amino acid sequence. 
     
     
         20 . The method of  claim 15 , wherein the ALK1-Fc fusion protein has a sequence of SEQ ID NO:3. 
     
     
         21 . The method of  claim 15 , wherein the ALK1-Fc fusion protein is administered in a pharmaceutical preparation wherein at least 90% of the ALK1-Fc fusion protein is in a dimeric form. 
     
     
         22 . The method of  claim 14 , wherein the ALK1 ECD fusion protein is delivered intravenously or locally to the eye. 
     
     
         23 . The method of  claim 14 , wherein the method further comprises administering a second agent that inhibits angiogenesis. 
     
     
         24 . The method of  claim 14 , wherein the vascularized tissue is selected from the group consisting of: the eye of the mammal, a tumor, a bone and a joint. 
     
     
         25 . The method of  claim 14 , wherein the mammal has diabetic retinopathy or other retinal disorder characterized by a loss of pericyte coverage in the retinal vasculature. 
     
     
         26 . The method of  claim 14 , wherein the mammal has a tumor selected from the group consisting of: melanoma, a lung tumor, multiple myeloma and breast cancer. 
     
     
         27 . The method of  claim 14 , wherein the mammal has a pancreatic tumor. 
     
     
         28 . The method of  claim 27 , wherein the mammal has a tumor of the pancreatic endocrine tissue. 
     
     
         29 . A method of promoting an increase in pericyte coverage in a vascularized tissue of a mammal in need thereof, 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. 
     
     
         30 . The method of  claim 29 , wherein the antibody binds to the ALK1 polypeptide with a K D  of less than 5×10 −8  M. 
     
     
         31 . The method of  claim 29 , wherein the antibody binds to the ALK1 polypeptide with a K D  of less than 1×10 −10  M. 
     
     
         32 . The method of  claim 29 , 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. 
     
     
         33 . The method of  claim 29 , wherein the antibody is delivered intravenously. 
     
     
         34 . The method of  claim 29 , wherein the method further comprises administering a second agent that inhibits angiogenesis. 
     
     
         35 . The method of  claim 29 , wherein the vascularized tissue is selected from the group consisting of: the eye of the mammal, a tumor, a bone and a joint. 
     
     
         36 . The method of  claim 29 , wherein the mammal has diabetic retinopathy or other retinal disorder characterized by a loss of pericyte coverage in the retinal vasculature. 
     
     
         37 . The method of  claim 29 , wherein the mammal has a tumor selected from the group consisting of: melanoma, a lung tumor, multiple myeloma, and breast cancer. 
     
     
         38 . The method of  claim 29 , wherein the mammal has a pancreatic tumor. 
     
     
         39 . The method of  claim 38 , wherein the mammal has a tumor of the pancreatic endocrine tissue. 
     
     
         40 . 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,   
       wherein the tumor is selected from the group consisting of: melanoma, multiple myeloma, a tumor associated with bone, a tumor that has metastasized to bone, a lung tumor, a pancreatic tumor and breast cancer. 
     
     
         41 . The method of  claim 40 , wherein the ALK-1 ECD protein is an ALK1-Fc fusion protein. 
     
     
         42 . The method of  claim 41 , 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 . 
     
     
         43 . The method of  claim 40 , wherein the ALK1 ECD protein binds to one or more ALK1 ligands selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10. 
     
     
         44 . The method of  claim 41 , wherein the ALK1-Fc fusion protein has a sequence of SEQ ID NO:3. 
     
     
         45 . The method of  claim 40 , 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. 
     
     
         46 . The method of  claim 40 , 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. 
     
     
         47 . The method of  claim 40 , wherein the antibody of (b) binds to the ALK1 polypeptide with a K D  of less than 5×10 −8  M. 
     
     
         48 . The method of  claim 40 , wherein the antibody of (b) binds to the ALK1 polypeptide with a K D  of less than 1×10 −10  M. 
     
     
         49 . The method of  claim 40 , wherein the antibody of (b) inhibits angiogenesis stimulated by at least one ALK1 ligand selected from the group consisting of: GDF5, GDF6 and GDF7. 
     
     
         50 . The method of  claim 40 , 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. 
     
     
         51 . The method of  claim 40 , wherein the antibody of (c) inhibits angiogenesis stimulated by at least one ALK1 ligand selected from the group consisting of: GDF5, GDF6 and GDF7. 
     
     
         52 . The method of  claim 40 , wherein the antibody of (c) inhibits angiogenesis stimulated by at least one ALK1 ligand selected from the group consisting of: BMP9 and BMP10. 
     
     
         53 . The method of  claim 40 , wherein the DAN protein is a DAN-Fc fusion protein. 
     
     
         54 . The method of  claim 53 , 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 . 
     
     
         55 . The method of  claim 40 , wherein the DAN protein binds to one or more ALK1 ligands selected from the group consisting of: GDF5, GDF6 and GDF7. 
     
     
         56 . The method of  claim 40 , 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. 
     
     
         57 . The method of  claim 40 , 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. 
     
     
         58 . The method of  claim 40 , wherein the agent is delivered intravenously. 
     
     
         59 . The method of  claim 40 , wherein the method further comprises administering a second agent that inhibits angiogenesis. 
     
     
         60 . The method of  claim 10 , wherein the tumor is selected from the group consisting of: melanoma, a lung tumor, multiple myeloma, pancreatic tumor, and breast cancer.