Anti-BMP9 Antibodies and Methods of Use Thereof

Abstract

Antibodies that specifically bind to bone morphogenetic protein-9 (BMP9), and antigen-binding fragments thereof, are provided. Embodiments include uses and related methods of using the antibodies and antigen-binding fragments thereof.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority and benefit to U.S. Provisional Application No. 63 / 347,543, filed May 31, 2022; U.S. Provisional Application No. 63 / 375,781, filed September 15, 2022; and U.S. Provisional Application No. 63 / 499,808, filed May 3, 2023, the contents of which are incorporated herein by reference.

[0002] Parties to the Joint Research Statement The presently claimed invention was made by or on behalf of the following listed parties to a joint research agreement. The joint research agreement was in effect on or before the date the claimed invention was made, and the claimed invention was made as a result of activities within the scope of the joint research agreement. The parties to the joint research agreement are PARTNERS HEALTHCARE and PFIZER INC.

Background Art

[0003] Pulmonary arterial hypertension (PAH) is a rare progressive disease characterized by high blood pressure (hypertension) in the pulmonary arteries of the lungs. PAH is defined as a mean pulmonary artery pressure (mPAP) greater than 20 mmHg in the setting of normal or decreased cardiac output and normal pulmonary capillary wedge pressure (Simonneau G, Hoeper MM, The revised definition of pulmonary hypertension: exploring the impact on patient management. European Heart Journal Supplements, 2019;21:K4 - K8). The initial symptom of PAH is shortness of breath after exercise. Additional symptoms include excessive fatigue, weakness, chest pain, dizziness, and fainting spells. In advanced stages of PAH, the body may have abnormal bluish discoloration of the skin (cyanosis) due to low levels of circulating oxygen and abnormal enlargement (hypertrophy) of the heart chambers (ventricles) that can cause heart failure.

[0004] Current methods for treating PAH include vasodilators, anti-proliferatives, calcium channel blockers, anticoagulants, and diuretics. These therapies focus on extending the patient's lifespan and improving the quality of life. However, these treatments have one or more drawbacks such as lack of efficacy, severe side effects, reduced patient compliance, and high costs. In fact, most patients still die from this disease or are unable to respond appropriately to medical therapy with a 5-year survival rate of 59% (Boucly A, Weatherald J, Savale L, et al. Risk assessment, prognosis, and guideline implementation in pulmonary arterial hypertension. Eur Respir J. 2017;50:1700889). Therefore, there is an important need in the art for new targeted therapies for the treatment and / or prevention of PAH, particularly to address the underlying pathophysiology.

Summary of the Invention

Means for Solving the Problems

[0005] The present disclosure provides an antibody that binds to bone morphogenetic protein-9 (BMP9) encoded by the growth differentiation factor 2 (GDF2) gene, as well as uses and related methods of the antibody. A polynucleotide encoding an antibody that binds to BMP9 is provided. An antibody heavy chain or light chain, or a polynucleotide encoding a therapy, is also provided. A host cell expressing the antibody is provided. The present disclosure also provides a process for making, preparing, and producing an antibody that binds to BMP9. The present disclosure further encompasses the expression of the antibody and the preparation and manufacture of a composition comprising the antibody of the present disclosure, for example, a pharmaceutical for use of the antibody.

[0006] The antibodies of the present disclosure are useful in one or more of the diagnosis, prevention, and / or treatment of disorders or conditions mediated by, or associated with, BMP9 activity, including, but not limited to, pulmonary arterial hypertension, anemia, liver disease, neuroinflammatory or neurodegenerative diseases such as multiple sclerosis, fibrotic disorders, and heart failure. Any one or more of the foregoing disorders or conditions may be excluded from the diagnosis, prevention, and / or treatment mediated by, or associated with, BMP9 activity.

[0007] In some aspects herein, isolated antibodies that bind to bone morphogenetic protein-9 (BMP9) and comprise a heavy chain variable region (VH) and a light chain variable region (VL) are disclosed. In some aspects, the VH complementarity determining region 1 (CDR1) comprises an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:24, SEQ ID NO:36, SEQ ID NO:45, and SEQ ID NO:53, the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:25, SEQ ID NO:37, SEQ ID NO:46, SEQ ID NO:54, SEQ ID NO:61, SEQ ID NO:70, and SEQ ID NO:76, the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:26, SEQ ID NO:38, SEQ ID NO:47, and SEQ ID NO:62, the VL CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:19, SEQ ID NO:30, SEQ ID NO:50, SEQ ID NO:57, SEQ ID NO:65, and SEQ ID NO:73, the VL CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:20, SEQ ID NO:31, SEQ ID NO:41, and SEQ ID NO:66, and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:32, SEQ ID NO:42, SEQ ID NO:58, and SEQ ID NO:67.

[0008] In this specification, in some embodiments, isolated antibodies are disclosed that bind to bone morphogenetic protein-9 (BMP9) and comprise a heavy chain variable region (VH) and a light chain variable region (VL), where (i) VH CDR1 comprises the amino acid sequence of SEQ ID NO: 80, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 81, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 26, and VL CDR1 comprises the amino acid sequence of SEQ ID NO: 82, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 83, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 84; (ii) VH CDR1 comprises the amino acid sequence of SEQ ID NO: 9, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 10, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 11, and VL CDR1 comprises the amino acid sequence of SEQ ID NO: 2, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 3, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 4; (iii) VH CDR1 comprises the amino acid sequence of SEQ ID NO: 24, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 25, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 26, and VL CDR1 comprises the amino acid sequence of SEQ ID NO: 19, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 20, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 21; (iv) VH CDR1 comprises the amino acid sequence of SEQ ID NO: 36, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 37, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 38, and VL CDR1 comprises the amino acid sequence of SEQ ID NO: 30, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 32; (v) VH CDR1 comprises the amino acid sequence of SEQ ID NO: 45, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 46, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 47, and VL CDR1 comprises the amino acid sequence of SEQ ID NO: 2, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 41, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 42;(vi) The VH CDR1 contains the amino acid sequence of SEQ ID NO: 53, the VH CDR2 contains the amino acid sequence of SEQ ID NO: 54, and the VH CDR3 contains the amino acid sequence of SEQ ID NO: 26, and the VL CDR1 contains the amino acid sequence of SEQ ID NO: 50, the VL CDR2 contains the amino acid sequence of SEQ ID NO: 20, and the VL CDR3 contains the amino acid sequence of SEQ ID NO: 21; (vii) The VH CDR1 contains the amino acid sequence of SEQ ID NO: 45, the VH CDR2 contains the amino acid sequence of SEQ ID NO: 61, and the VH CDR3 contains the amino acid sequence of SEQ ID NO: 62, and the VL CDR1 contains the amino acid sequence of SEQ ID NO: 57, the VL CDR2 contains the amino acid sequence of SEQ ID NO: 41, and the VL CDR3 contains the amino acid sequence of SEQ ID NO: 58; (viii) The VH CDR1 contains the amino acid sequence of SEQ ID NO: 53, the VH CDR2 contains the amino acid sequence of SEQ ID NO: 70, and the VH CDR3 contains the amino acid sequence of SEQ ID NO: 26, and the VL CDR1 contains the amino acid sequence of SEQ ID NO: 65, the VL CDR2 contains the amino acid sequence of SEQ ID NO: 66, and the VL CDR3 contains the amino acid sequence of SEQ ID NO: 67; or (ix) The VH CDR1 contains the amino acid sequence of SEQ ID NO: 53, the VH CDR2 contains the amino acid sequence of SEQ ID NO: 76, and the VH CDR3 contains the amino acid sequence of SEQ ID NO: 26, and the VL CDR1 contains the amino acid sequence of SEQ ID NO: 73, the VL CDR2 contains the amino acid sequence of SEQ ID NO: 20, and the VL CDR3 contains the amino acid sequence of SEQ ID NO: 67.;

[0009] In some embodiments of the isolated antibodies disclosed herein, (i) VH CDR1 comprises the amino acid sequence of SEQ ID NO: 80, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 81, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 26, and VL CDR1 comprises the amino acid sequence of SEQ ID NO: 82, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 83, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 84; (ii) VH CDR1 comprises the amino acid sequence of SEQ ID NO: 24, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 25, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 26, and VL CDR1 comprises the amino acid sequence of SEQ ID NO: 19, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 20, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 21; (iii) VH CDR1 comprises the amino acid sequence of SEQ ID NO: 53, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 54, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 26, and VL CDR1 comprises the amino acid sequence of SEQ ID NO: 50, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 20, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 21; (iv) VH CDR1 comprises the amino acid sequence of SEQ ID NO: 53, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 70, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 26, and VL CDR1 comprises the amino acid sequence of SEQ ID NO: 65, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 66, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 67; or (v) VH CDR1 comprises the amino acid sequence of SEQ ID NO: 53, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 76, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 26, and VL CDR1 comprises the amino acid sequence of SEQ ID NO: 73, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 20, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 67.

[0010] In some aspects herein, an isolated antibody that binds to bone morphogenetic protein-9 (BMP9) and includes a heavy chain variable region (VH) and a light chain variable region (VL) is disclosed, where VH CDR1 includes the amino acid sequence of SEQ ID NO: 53, VH CDR2 includes the amino acid sequence of SEQ ID NO: 76, and VH CDR3 includes the amino acid sequence of SEQ ID NO: 26, and wherein VL CDR1 includes the amino acid sequence of SEQ ID NO: 73, VL CDR2 includes the amino acid sequence of SEQ ID NO: 20, and VL CDR3 includes the amino acid sequence of SEQ ID NO: 67.

[0011] In some aspects, the VH of the isolated antibody disclosed herein includes the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 27, SEQ ID NO: 39, SEQ ID NO: 48, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 71, or SEQ ID NO: 77, or variants thereof that include 1 to 4 amino acid substitutions with residues that are not within the CDRs. In some aspects, the VL of the isolated antibody disclosed herein includes the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 33, SEQ ID NO: 44, SEQ ID NO: 51, SEQ ID NO: 59, SEQ ID NO: 68, or SEQ ID NO: 74, or variants thereof that include 1 to 4 amino acid substitutions with residues that are not within the CDRs. In some aspects, the VH of the isolated antibody disclosed herein includes the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 27, SEQ ID NO: 39, SEQ ID NO: 48, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 71, or SEQ ID NO: 77, or variants thereof that include 1 to 4 amino acid substitutions with residues that are not within the CDRs, and the VL of the isolated antibody disclosed herein includes the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 33, SEQ ID NO: 44, SEQ ID NO: 51, SEQ ID NO: 59, SEQ ID NO: 68, or SEQ ID NO: 74, or variants thereof that include 1 to 4 amino acid substitutions with residues that are not within the CDRs.

[0012] In some embodiments, the VH of the isolated antibodies disclosed herein comprises an amino acid sequence that is at least, at most, exactly, between any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical within any range thereof, to SEQ ID NO: 16, SEQ ID NO: 27, SEQ ID NO: 39, SEQ ID NO: 48, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 71, or SEQ ID NO: 77. In some embodiments, the VH of the isolated antibodies disclosed herein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 16, SEQ ID NO: 27, SEQ ID NO: 39, SEQ ID NO: 48, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 71, or SEQ ID NO: 77. In some embodiments, the VL of the isolated antibodies disclosed herein comprises an amino acid sequence that is at least, at most, exactly, between any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical within any range thereof, to SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 33, SEQ ID NO: 44, SEQ ID NO: 51, SEQ ID NO: 59, SEQ ID NO: 68, or SEQ ID NO: 74. In some embodiments, the VL of the isolated antibodies disclosed herein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 33, SEQ ID NO: 44, SEQ ID NO: 51, SEQ ID NO: 59, SEQ ID NO: 68, or SEQ ID NO: 74.In some embodiments, the VH of the isolated antibodies disclosed herein comprises an amino acid sequence that is at least, at most, exactly, between any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical within any range of these (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 16, SEQ ID NO: 27, SEQ ID NO: 39, SEQ ID NO: 48, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 71, or SEQ ID NO: 77. The VL of the isolated antibodies disclosed herein comprises an amino acid sequence that is at least, at most, exactly, between any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical within any range of these (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 33, SEQ ID NO: 44, SEQ ID NO: 51, SEQ ID NO: 59, SEQ ID NO: 68, or SEQ ID NO: 74. It is contemplated that any one or more of these aforementioned percentages may be excluded in embodiments.

[0013] In some embodiments, the isolated antibodies disclosed herein comprise: (i) a VH comprising the amino acid sequence of SEQ ID NO: 16 and a VL comprising the amino acid sequence of SEQ ID NO: 6; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 27 and a VL comprising the amino acid sequence of SEQ ID NO: 22; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 39 and a VL comprising the amino acid sequence of SEQ ID NO: 33; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 48 and a VL comprising the amino acid sequence of SEQ ID NO: 43; (v) a VH comprising the amino acid sequence of SEQ ID NO: 55 and a VL comprising the amino acid sequence of SEQ ID NO: 51; (vi) a VH comprising the amino acid sequence of SEQ ID NO: 63 and a VL comprising the amino acid sequence of SEQ ID NO: 59; (vii) a VH comprising the amino acid sequence of SEQ ID NO: 71 and a VL comprising the amino acid sequence of SEQ ID NO: 68; or (viii) a VH comprising the amino acid sequence of SEQ ID NO: 77 and a VL comprising the amino acid sequence of SEQ ID NO: 74.

[0014] In some embodiments, the isolated antibodies disclosed herein are: (i) VH comprising the amino acid sequence of SEQ ID NO: 27 and VL comprising the amino acid sequence of SEQ ID NO: 22; (ii) VH comprising the amino acid sequence of SEQ ID NO: 55 and VL comprising the amino acid sequence of SEQ ID NO: 51; (iii) VH comprising the amino acid sequence of SEQ ID NO: 71 and VL comprising the amino acid sequence of SEQ ID NO: 68; (iv) VH comprising the amino acid sequence of SEQ ID NO: 77 and VL comprising the amino acid sequence of SEQ ID NO: 74; (v) VH comprising an amino acid sequence that is at least, at most, exactly, between any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical within any range thereof, e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical, to SEQ ID NO: 27, and VL comprising an amino acid sequence that is at least, at most, exactly, between any of the following two (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical within any range thereof, e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical, to SEQ ID NO: 22;(vi) At least, at most, exactly, between any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical within any range thereof, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical VH containing an amino acid sequence, and at least, at most, exactly, between any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical within any range thereof, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical VL containing an amino acid sequence with respect to SEQ ID NO: 51; (vii) At least, at most, exactly, between any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical within any range thereof, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical VH containing an amino acid sequence, and at least, at most, exactly, between any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical within any range thereof, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical VL containing an amino acid sequence with respect to SEQ ID NO: 68;or (viii) a VH comprising an amino acid sequence that is at least, at most, exactly, between any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical within any range thereof, e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical, to SEQ ID NO: 77, and a VL comprising an amino acid sequence that is at least, at most, exactly, between any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical within any range thereof, e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical, to SEQ ID NO: 74. It is contemplated that any one or more of these aforementioned percentages may be excluded in embodiments. In some aspects, the isolated antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 77 and a VL comprising the amino acid sequence of SEQ ID NO: 74.;

[0015] In some aspects herein, a portion of an isolated antibody is disclosed, the isolated antibody binds to BMP9, and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:24, SEQ ID NO:36, SEQ ID NO:45, and SEQ ID NO:53, VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:25, SEQ ID NO:37, SEQ ID NO:46, SEQ ID NO:54, SEQ ID NO:61, SEQ ID NO:70, and SEQ ID NO:76, VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:26, SEQ ID NO:38, SEQ ID NO:47, and SEQ ID NO:62, VL CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:19, SEQ ID NO:30, SEQ ID NO:50, SEQ ID NO:57, SEQ ID NO:65, and SEQ ID NO:73, VL CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:20, SEQ ID NO:31, SEQ ID NO:41, and SEQ ID NO:66, and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:32, SEQ ID NO:42, SEQ ID NO:58, and SEQ ID NO:67. In some aspects, this portion binds to BMP9.

[0016] In some aspects, this portion comprises a VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:24, SEQ ID NO:36, SEQ ID NO:45, and SEQ ID NO:53, VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:25, SEQ ID NO:37, SEQ ID NO:46, SEQ ID NO:54, SEQ ID NO:61, SEQ ID NO:70, and SEQ ID NO:76, and VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:26, SEQ ID NO:38, SEQ ID NO:47, and SEQ ID NO:62.

[0017] In some aspects herein, an isolated antibody is disclosed that binds to BMP9 and comprises a VH encoded by the nucleic acid sequence of the insert of a plasmid deposited with the ATCC under accession number PTA-127292, a VL encoded by the nucleic acid sequence of the insert of a plasmid deposited with the ATCC under accession number PTA-127293, or both.

[0018] In some aspects herein, an isolated antibody is disclosed that comprises: (i) the VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NO: 16 and the VL CDR1, VL CDR2, and VL CDR3 sequences of SEQ ID NO: 6; (ii) the VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NO: 27 and the VL CDR1, VL CDR2, and VL CDR3 sequences of SEQ ID NO: 22; (iii) the VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NO: 39 and the VL CDR1, VL CDR2, and VL CDR3 sequences of SEQ ID NO: 33; (iv) the VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NO: 48 and the VL CDR1, VL CDR2, and VL CDR3 sequences of SEQ ID NO: 43; (v) the VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NO: 55 and the VL CDR1, VL CDR2, and VL CDR3 sequences of SEQ ID NO: 51; (vi) the VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NO: 63 and the VL CDR1, VL CDR2, and VL CDR3 sequences of SEQ ID NO: 59; (vii) the VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NO: 71 and the VL CDR1, VL CDR2, and VL CDR3 sequences of SEQ ID NO: 68; or (viii) the VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NO: 77 and the VL CDR1, VL CDR2, and VL CDR3 sequences of SEQ ID NO: 74.

[0019] In some aspects, the isolated antibody comprises a human V κ or V λ light chain constant domain. In some aspects, the antibody comprises a human V λIt includes a light chain constant domain. In some embodiments, the antibody includes a heavy chain constant domain. In some embodiments, the heavy chain constant domain includes IgA (e.g., IgA1 or IgA2), IgD, IgE, IgM, or IgG (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the heavy chain constant domain includes IgG. In some embodiments, IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, IgG is IgG1. In some embodiments, the antibody includes an Fc domain. In some embodiments, the Fc domain includes the IgG1 heavy chain CH2 domain and the IgG heavy chain CH3 domain.

[0020] As used herein, in some embodiments, there are disclosed isolated antibodies comprising a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:23, SEQ ID NO:35, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:60, SEQ ID NO:69, and SEQ ID NO:75. As used herein, in some embodiments, there are disclosed isolated antibodies comprising a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:18, SEQ ID NO:29, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:56, SEQ ID NO:64, and SEQ ID NO:72. As used herein, in some embodiments, there are disclosed isolated antibodies comprising (i) a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:23, SEQ ID NO:35, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:60, SEQ ID NO:69, and SEQ ID NO:75, and (ii) a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:18, SEQ ID NO:29, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:56, SEQ ID NO:64, and SEQ ID NO:72.

[0021] In this specification, in some embodiments, an isolated antibody comprising a heavy chain comprising an amino acid sequence that is at least, up to, exactly, between any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical within any range thereof, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8, SEQ ID NO: 23, SEQ ID NO: 35, SEQ ID NO: 44, SEQ ID NO: 52, SEQ ID NO: 60, SEQ ID NO: 69, or SEQ ID NO: 75 is disclosed. In this specification, in some embodiments, an isolated antibody comprising a light chain comprising an amino acid sequence that is at least, up to, exactly, between any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical within any range thereof, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1, SEQ ID NO: 18, SEQ ID NO: 29, SEQ ID NO: 40, SEQ ID NO: 49, SEQ ID NO: 56, SEQ ID NO: 64, or SEQ ID NO: 72 is disclosed.In some embodiments herein, (i) a heavy chain comprising an amino acid sequence that is at least, up to, exactly, between any two of the following (including or excluding the ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical within any range thereof, e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical, to SEQ ID NO: 8, SEQ ID NO: 23, SEQ ID NO: 35, SEQ ID NO: 44, SEQ ID NO: 52, SEQ ID NO: 60, SEQ ID NO: 69, or SEQ ID NO: 75; and (ii) a light chain comprising an amino acid sequence that is at least, up to, exactly, between any two of the following (including or excluding the ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical within any range thereof, e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical, to SEQ ID NO: 1, SEQ ID NO: 18, SEQ ID NO: 29, SEQ ID NO: 40, SEQ ID NO: 49, SEQ ID NO: 56, SEQ ID NO: 64, or SEQ ID NO: 72, are disclosed for an isolated antibody. It is contemplated that any one or more of these aforementioned percentages may be excluded in an embodiment.

[0022] In some embodiments herein, an isolated antibody is disclosed that comprises: (i) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 8 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 1; (ii) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 23 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 18; (iii) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 35 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 29; (iv) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 44 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 40; (v) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 52 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 49; (vi) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 60 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 56; (vii) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 69 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 64; or (viii) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 75 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 72.

[0023] In this specification, in some embodiments, (i) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 23 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 18; (ii) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 52 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 49; (iii) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 69 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 64; (iv) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 75 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 72; (v) an HC comprising or consisting of an amino acid sequence that is at least, at most, exactly, between any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical in any range thereof, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 23, and an LC comprising or consisting of an amino acid sequence that is at least, at most, exactly, between any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical in any range thereof, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 18.(vi) At least, up to, exactly, between any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical within any range thereof, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequences, including or consisting of, HC with respect to SEQ ID NO: 52, and at least, up to, exactly, between any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical within any range thereof, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequences, including or consisting of, LC with respect to SEQ ID NO: 49;(vii) At least, up to, exactly, between any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical within any range thereof, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequences, including or consisting of an HC with respect to SEQ ID NO: 69, and at least, up to, exactly, between any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical within any range thereof, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequences, including or consisting of an LC with respect to SEQ ID NO: 64;or (viii) an HC comprising, or consisting of, an amino acid sequence that is at least, at most, exactly, between any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical within any range of these, e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 75, and an LC comprising, or consisting of, an amino acid sequence that is at least, at most, exactly, between any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical within any range of these, e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 72. It is contemplated that one or more of these aforementioned percentages may be excluded in embodiments.;

[0024] In this specification, in some embodiments, (i) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 75 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 72, or (ii) an HC comprising or consisting of an amino acid sequence that is at least, at most, exactly, any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical in any range thereof, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 75, and an LC comprising or consisting of an amino acid sequence that is at least, at most, exactly, any two of the following (including or excluding both ends), or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or identical in any range thereof, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 72 are disclosed. It is contemplated that any one or more of these aforementioned percentages may be excluded in embodiments.

[0025] In this specification, in some embodiments, isolated antibodies that bind to BMP9 are disclosed, and the antibodies bind to an epitope on BMP9 that comprises one or more amino acid residues selected from the group consisting of Ala28, Pro29, Ser83, Leu85, Met90, Val92, Pro93, Leu95, and Tyr97 of SEQ ID NO: 79. In some embodiments, the antibody binds to Ala28, Pro29, Ser83, Leu85, Met90, Val92, Pro93, Leu95, and Tyr97 of SEQ ID NO: 79. In some embodiments, the antibody further binds to one or more amino acid residues selected from the group consisting of Pro81, Tyr86, Asp88, Asp89, Thr94, Lys96, His98, Tyr99, and Glu100 of SEQ ID NO: 79. In some embodiments, the antibody binds to Pro81, Tyr86, Asp88, Asp89, Thr94, Lys96, His98, Tyr99, and Glu100 of SEQ ID NO: 79. In some embodiments, the antibody further binds to one or more amino acid residues selected from the group consisting of Ile27, Lys30, and Glu31 of SEQ ID NO: 79. In some embodiments, the antibody binds to Ile27, Lys30, and Glu31 of SEQ ID NO: 79. It is contemplated that any one or more of these aforementioned amino acid residues may be excluded in an embodiment.

[0026] In this specification, in some embodiments, isolated antibodies that bind to BMP9 are disclosed, and the antibodies bind to an epitope on BMP9 that comprises one or more amino acid residues selected from the group consisting of Ile27, Ala28, Pro29, Lys30, Glu31, Tyr32, Ser80, Pro81, Ile82, Ser83, Val84, Leu85, Tyr86, Asp88, Asp89, Met90, Val92, Pro93, Thr94, Leu95, Lys96, Tyr97, His98, Tyr99, and Glu100 of SEQ ID NO: 79. It is contemplated that any one or more of these aforementioned amino acid residues may be excluded in an embodiment.

[0027] In some embodiments herein, isolated antibodies that bind to BMP9 are disclosed, wherein the antibodies bind to an epitope on BMP9 that includes Ile27, Ala28, Pro29, Lys30, Glu31, Tyr32, Ser80, Pro81, Ile82, Ser83, Val84, Leu85, Tyr86, Asp88, Asp89, Met90, Val92, Pro93, Thr94, Leu95, Lys96, Tyr97, His98, Tyr99, and Glu100 of SEQ ID NO: 79.

[0028] In some embodiments herein, isolated antibodies that bind to BMP9 are disclosed, wherein the antibodies bind to an epitope on BMP9 that comprises one or more amino acid residues selected from the group consisting of Ala28, Pro29, Pro81, Ser83, Leu85, Tyr86, Asp88, Asp89, Met90, Val92, Pro93, Thr94, Leu95, Lys96, Tyr97, His98, Tyr99, and Glu100 of SEQ ID NO: 79. It is contemplated that any one or more of these aforementioned amino acid residues may be excluded in embodiments.

[0029] In some embodiments herein, isolated antibodies that bind to BMP9 are disclosed, wherein the antibodies bind to an epitope on BMP9 that includes Ala28, Pro29, Pro81, Ser83, Leu85, Tyr86, Asp88, Asp89, Met90, Val92, Pro93, Thr94, Leu95, Lys96, Tyr97, His98, Tyr99, and Glu100 of SEQ ID NO: 79.

[0030] In this specification, in some embodiments, isolated antibodies that bind to BMP9 are disclosed, and the antibodies bind to an epitope on BMP9 that comprises one or more amino acid residues selected from the group consisting of Arg14, Ile27, Ala28, Pro29, Lys30, Glu31, Ser83, Leu85, Met90, Val92, Pro93, Leu95, and Tyr97 of SEQ ID NO: 79. It is contemplated that any one or more of these aforementioned amino acid residues may be excluded in an embodiment.

[0031] In this specification, in some embodiments, isolated antibodies that bind to BMP9 are disclosed, and the antibodies bind to an epitope on BMP9 that comprises Arg14, Ile27, Ala28, Pro29, Lys30, Glu31, Ser83, Leu85, Met90, Val92, Pro93, Leu95, and Tyr97 of SEQ ID NO: 79. In some embodiments, the antibody epitope on BMP9 does not include amino acid residues 21-25 and 43-60 of SEQ ID NO: 79.

[0032] In some embodiments of the isolated antibodies disclosed herein, when measured by surface plasmon resonance, the antibody binds to human BMP9 at 37°C with an affinity of at least, up to, exactly, between (including or excluding both ends) any two of the following, or about 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, 100 pM, 105 pM, 110 pM, 115 pM, 120 pM, 125 pM, 130 pM, 135 pM, 140 pM, 145 pM, or 150 pM, or within a range of any of these. In some embodiments of the isolated antibodies disclosed herein, when measured by surface plasmon resonance, the antibody binds to human BMP9 at 37°C with an affinity of about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 55 pM, about 60 pM, about 65 pM, about 70 pM, about 75 pM, about 80 pM, about 85 pM, about 90 pM, about 95 pM, about 100 pM, about 105 pM, about 110 pM, about 115 pM, about 120 pM, about 125 pM, about 130 pM, about 135 pM, about 140 pM, or about 150 pM. In some embodiments, when measured by surface plasmon resonance, the antibody binds to human BMP9 at 37°C with an affinity of about 75 pM to about 95 pM. In some embodiments, when measured by surface plasmon resonance, the antibody binds to human BMP9 at 37°C with an affinity of about 85 pM. In some embodiments, when measured by surface plasmon resonance, the antibody binds to human BMP9 at 37°C with an affinity of about 87 pM. It is contemplated that any one or more of these aforementioned binding affinities may be excluded in an embodiment.

[0033] In some embodiments of the isolated antibodies disclosed herein, the antibody binds to cynomolgus BMP9 with an affinity of at least, at most, exactly, between (including or excluding both ends) any two of the following, or about 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, 100 pM, 105 pM, 110 pM, 115 pM, 120 pM, 125 pM, 130 pM, 135 pM, 140 pM, 145 pM, or 150 pM, as measured by surface plasmon resonance at 37°C. In some embodiments of the isolated antibodies disclosed herein, the antibody binds to cynomolgus BMP9 with an affinity of about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 55 pM, about 60 pM, about 65 pM, about 70 pM, about 75 pM, about 80 pM, about 85 pM, about 90 pM, about 95 pM, about 100 pM, about 105 pM, about 110 pM, about 115 pM, about 120 pM, about 125 pM, about 130 pM, about 135 pM, about 140 pM, or about 150 pM, as measured by surface plasmon resonance at 37°C. In some embodiments, the antibody binds to cynomolgus BMP9 with an affinity of about 50 pM to about 100 pM at 37°C, as measured by surface plasmon resonance. In some embodiments, the antibody binds to cynomolgus BMP9 with an affinity of about 65 pM at 37°C, as measured by surface plasmon resonance. It is contemplated that any one or more of these aforementioned binding affinities may be excluded in an embodiment.

[0034] In some embodiments of the isolated antibodies disclosed herein, the antibody binds to rat BMP9 with an affinity of at least, up to, exactly, between (including or excluding both ends) any two of the following, or about 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, 100 pM, 105 pM, 110 pM, 115 pM, 120 pM, 125 pM, 130 pM, 135 pM, 140 pM, 145 pM, or 150 pM, or any range thereof, as measured by surface plasmon resonance at 37°C. In some embodiments of the isolated antibodies disclosed herein, the antibody binds to rat BMP9 with an affinity of about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 55 pM, about 60 pM, about 65 pM, about 70 pM, about 75 pM, about 80 pM, about 85 pM, about 90 pM, about 95 pM, about 100 pM, about 105 pM, about 110 pM, about 115 pM, about 120 pM, about 125 pM, about 130 pM, about 135 pM, about 140 pM, or about 150 pM as measured by surface plasmon resonance at 37°C. In some embodiments, the antibody binds to rat BMP9 with an affinity of about 75 pM to about 120 pM at 37°C as measured by surface plasmon resonance. In some embodiments, the antibody binds to rat BMP9 with an affinity of about 98 pM at 37°C as measured by surface plasmon resonance. It is contemplated that any one or more of these aforementioned binding affinities may be excluded in an embodiment.

[0035] In some embodiments of the isolated antibodies disclosed herein, the antibody comprises (i) the heavy chain variable region (VH) CDR3 of SEQ ID NO: 26 and the light chain variable region (VL) CDR3 of SEQ ID NO: 84, (ii) the heavy chain variable region (VH) CDR3 of SEQ ID NO: 26 and the light chain variable region (VL) CDR3 of SEQ ID NO: 21, or (iii) the heavy chain variable region (VH) CDR3 of SEQ ID NO: 26 and the light chain variable region (VL) CDR3 of SEQ ID NO: 67.

[0036] In some embodiments of the isolated antibodies disclosed herein, the antibody binds to human BMP9 and inhibits its binding to the BMP9 type II receptor of human BMP9 (e.g., BMPRII, ActRIIA, and / or ActRIIB), or (ii) binds to human BMP9, inhibits its binding to the BMP9 type II receptor of human BMP9 (e.g., BMPRII, ActRIIA, and / or ActRIIB), and weakly inhibits its binding to the BMP9 type I receptor of human BMP9 (e.g., ALK1). In some embodiments of the isolated antibodies disclosed herein, the antibody binds to human BMP9 and inhibits its binding to human endothelial cells. In some embodiments of the isolated antibodies disclosed herein, the antibody binds to human BMP9 but does not bind to other human transforming growth factor beta (TGFβ) superfamily ligands (e.g., GDF8, GDF11, activin A, TGFβ1, GDF9, and BMP10).

[0037] In some embodiments of the isolated antibodies disclosed herein, the antibody inhibits BMP9 activity as measured by phospho-SMAD1 / 5 / 9 nuclear translocation in endothelial cells.

[0038] In some embodiments, the isolated antibody inhibits human BMP9 activity with an IC of from about 0.01 nM to about 100 nM (e.g., at least, at most, exactly, between any two of the following (including or excluding both ends), or about 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, 100 pM, 105 pM, 110 pM, 115 pM, 120 pM, 125 pM, 130 pM, 135 pM, 140 pM, 145 pM, 150 pM, 160 pM, 170 pM, 180 pM, 190 pM, 200 pM, 225 pM, 250 pM, 275 pM, 300 pM, 325 pM, 350 pM, 375 pM, 400 pM, 425 pM, 450 pM, 475 pM, 500 pM, 550 pM, 600 pM, 650 pM, 700 pM, 750 pM, 800 pM, 850 pM, 900 pM, 950 pM, 1000 pM, 2000 pM, 3000 pM, 4000 pM, 5000 pM, 6000 pM, 7000 pM, 8000 pM, 9000 pM, 10000 pM, 20000 pM, 30000 pM, 40000 pM, 50000 pM, 60000 pM, 70000 pM, 80000 pM, 90000 pM, or 100000 pM, or any range thereof). 50 50 In some embodiments, the isolated antibody has an IC of about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 55 pM, about 60 pM, about 65 pM, about 70 pM, about 75 pM, about 80 pM, about 85 pM, about 90 pM, about 95 pM, about 100 pM, about 105 pM, about 110 pM, about 115 pM, about 120 pM, about 125 pM, about 130 pM, about 135 pM, about 140 pM, about 145 pM, about 150 pM, about 155 pM, about 160 pM, about 170 pM, about 180 pM, about 190 pM, about 200 pM, about 225 pM, about 250 pM, about 300 pM, about 350 pM, about 400 pM, about 450 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, or about 1000 pM to inhibit human BMP9 activity.Inhibits human BMP9 activity. In some embodiments, the isolated antibody has an IC 50 that inhibits human BMP9 activity. Any one or more of these aforementioned IC 50 values may be excluded in embodiments.

[0039] In some aspects, the isolated antibody has an IC 50 that inhibits cynomolgus monkey BMP9 activity in the range of about 15 pM to about 120 pM (e.g., at least, at most, exactly, between any two of the following (including or excluding both ends), or about 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, 100 pM, 105 pM, 110 pM, 115 pM, or 120 pM, or any range therebetween). 50 In some aspects, the isolated antibody has an IC 50 that inhibits cynomolgus monkey BMP9 activity in the range of about 17 pM to about 117 pM, e.g., but not limited to, about 17 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, about 100 pM, or about 117 pM. 50 In some aspects, the isolated antibody has an IC

[0040] In some embodiments of the isolated antibodies disclosed herein, the antibody inhibits BMP9 activity as measured by phospho-SMAD2 nuclear translocation in endothelial cells.

[0041] In some embodiments, the antibody inhibits human BMP9 activity with an IC of from about 0.01 nM to about 350 nM, (e.g., at least, at most, exactly, between any two of the following (including or excluding both ends), or about 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, 100 pM, 105 pM, 110 pM, 115 pM, 120 pM, 125 pM, 130 pM, 135 pM, 140 pM, 145 pM, 150 pM, 160 pM, 170 pM, 180 pM, 190 pM, 200 pM, 225 pM, 250 pM, 275 pM, 300 pM, 325 pM, or 350 pM, or a range of any of these). 50 In some embodiments, the antibody inhibits human BMP9 activity with an IC of about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 55 pM, about 60 pM, about 65 pM, about 70 pM, about 75 pM, about 80 pM, about 85 pM, about 90 pM, about 95 pM, about 100 pM, about 105 pM, about 110 pM, about 115 pM, about 120 pM, about 125 pM, about 130 pM, about 135 pM, about 140 pM, about 145 pM, about 150 pM, about 155 pM, about 160 pM, about 170 pM, about 180 pM, about 190 pM, about 200 pM, about 225 pM, about 250 pM, about 300 pM, about 350 pM, about 400 pM, about 450 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, or about 1000 pM. 50 In some embodiments, the isolated antibody inhibits human BMP9 activity with an IC of about 1 nM, about 2 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, or about 350 nM. 50 In some embodiments, the isolated antibody inhibits human BMP9 activity with an IC of about 1 nM, about 2 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, or about 350 nM. It is contemplated that any one or more of these aforementioned IC 50 values may be excluded in an embodiment.

[0042] In some embodiments, the isolated antibody inhibits cynomolgus BMP9 activity with an IC of from about 15 pM to about 75 pM (e.g., at least, at most, exactly, between any two of the following (including or excluding both ends), or about 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, or 75 pM, or a range of any of these). 50 In some embodiments, the isolated antibody inhibits cynomolgus BMP9 activity with an IC of from about 27 pM to about 42 pM, such as, but not limited to, about 27 pM, about 30 pM, about 35 pM, about 40 pM, or about 42 pM. 50 In some embodiments, the isolated antibody inhibits rat BMP9 activity with an IC of from about 450 pM to about 1 nM, such as, but not limited to, about 650 pM, about 700 pM, about 750 pM, about 800 pM, about 850 pM, about 900 pM, about 950 pM, or about 1000 pM. 50 Any one or more of these aforementioned IC 50 values may be excluded in an embodiment, as is contemplated.

[0043] In some embodiments of the isolated antibodies disclosed herein, the antibody reduces the right ventricular systolic pressure in a subject by at least, up to, exactly, between any two of the following (including or excluding both ends), or by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, or any range between them, for example, by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% as compared to before antibody administration. In some embodiments of the isolated antibodies disclosed herein, the antibody reduces the right ventricular systolic pressure in a subject by at least, up to, exactly, between any two of the following (including or excluding both ends), or by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, or any range between them, for example, by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% when compared to the percentage (%) difference observed between the isotype control antibody and normoxia. In some embodiments of the isolated antibodies disclosed herein, the antibody reduces right ventricular hypertrophy in a subject by at least, up to, exactly, between any two of the following (including or excluding both ends), or by about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, or any range between them, for example, by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% as compared to before antibody administration.In some embodiments of the isolated antibodies disclosed herein, the antibody, when compared to the percentage (%) difference observed between the isotype control antibody and normoxia, is precisely between any two of the following (including or excluding both ends), or by about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, or any range thereof, for example, by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% reduces right ventricular hypertrophy in a subject. In some embodiments, the subject is a SuGen-Hypoxia animal model of pulmonary arterial hypertension (PAH). It is contemplated that any one or more of these aforementioned reduction percentages may be excluded in an embodiment.

[0044] In some embodiments herein, an isolated polynucleotide encoding any one of the isolated antibodies disclosed herein is disclosed. In some embodiments herein, an isolated nucleic acid encoding the VH, VL, or both of an antibody that binds BMP9 is disclosed. In some embodiments, the isolated nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 85, the nucleic acid sequence of SEQ ID NO: 86, or both. In some embodiments herein, an isolated nucleic acid encoding the heavy chain, light chain, or both of an antibody that binds BMP9 is disclosed. In some embodiments, the isolated nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 87, the nucleic acid sequence of SEQ ID NO: 88, or both. In some embodiments herein, an isolated nucleic acid encoding the VH, VL, or both of an antibody that binds BMP9 is disclosed. In some embodiments, the isolated nucleic acid comprises the nucleic acid sequence of the insert of a plasmid deposited by the ATCC and having accession number PTA-127292, the nucleic acid sequence of the insert of a plasmid deposited by the ATCC and having accession number PTA-127293, or both.

[0045] In some embodiments herein, vectors are disclosed that contain an isolated polynucleotide disclosed herein that encodes any one of the isolated antibodies disclosed herein. In some embodiments herein, vectors are disclosed that contain an isolated nucleic acid disclosed herein that encodes the VH, VL, or both of an antibody that binds BMP9. In some embodiments herein, vectors are disclosed that contain an isolated nucleic acid disclosed herein that encodes the heavy chain, light chain, or both of an antibody that binds BMP9.

[0046] In some embodiments herein, isolated host cells are disclosed that contain an isolated polynucleotide disclosed herein that encodes any one of the isolated antibodies disclosed herein. In some embodiments herein, isolated host cells are disclosed that contain an isolated nucleic acid disclosed herein that encodes the VH, VL, or both of an antibody that binds BMP9. In some embodiments herein, isolated host cells are disclosed that contain an isolated nucleic acid disclosed herein that encodes the heavy chain, light chain, or both of an antibody that binds BMP9. In some embodiments, the host cell is a mammalian cell or an insect cell. In some embodiments, the host cell is a mammalian cell selected from the group consisting of CHO cells, HEK-293 cells, NS0 cells, PER.C6® cells, or Sp2.0 cells.

[0047] In some embodiments herein, methods for producing an antibody are disclosed that include culturing a host cell disclosed herein under conditions that result in the production of the antibody. In some embodiments, the method further includes recovering the antibody.

[0048] In some embodiments herein, a pharmaceutical composition is disclosed that comprises a therapeutically effective amount of an antibody disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the composition comprises 1.12 mg / mL of L-histidine, 2.67 mg / mL of L-histidine hydrochloride monohydrate, 85 mg / mL of sucrose, 0.05 mg / mL of disodium EDTA dihydrate, 0.2 mg / mL of polysorbate 80 (at pH 5.8). In some embodiments, the composition comprises 20 mM of histidine, 8.5% of sucrose, and 0.02% of polysorbate 80, 0.005% of EDTA (at pH 5.8). In some embodiments, the composition comprises an antibody at about 2 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 50 mg / mL, 75 mg / mL, 100 mg / mL, 125 mg / mL, 150 mg / mL, 175 mg / mL, or 200 mg / mL. In some embodiments, the composition is suitable for subcutaneous (SC) and / or intravenous (IV) administration. In some embodiments, the composition is suitable for intravenous (IV) administration. In some embodiments, the composition is suitable for subcutaneous (SC) administration.

[0049] In some embodiments herein, a method for reducing the expression or activity of BMP9 in a subject is disclosed, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition disclosed herein or an antibody disclosed herein.

[0050] In some embodiments herein, a method for treating hypertension in a subject is disclosed, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition disclosed herein or an antibody disclosed herein. In some embodiments, the hypertension is pulmonary hypertension.

[0051] In some aspects herein, a method of treating pulmonary arterial hypertension (PAH) in a subject is disclosed, the method comprising administering to the subject in need thereof a therapeutically effective amount of a pharmaceutical composition disclosed herein or an antibody disclosed herein. In some aspects, the antibody is administered in combination with a therapeutically effective amount of one or more additional therapeutically active compounds or therapies effective in treating at least one symptom and / or sign of hypertension, optionally pulmonary arterial hypertension.

[0052] In some aspects herein, an isolated antibody disclosed herein, or a pharmaceutical composition disclosed herein, for use as a medicament is disclosed.

[0053] In some aspects herein, an isolated antibody disclosed herein, or a pharmaceutical composition disclosed herein, for use in the treatment of at least one symptom and / or sign of hypertension, optionally pulmonary arterial hypertension, is disclosed.

[0054] In some aspects herein, use of an isolated antibody disclosed herein, or a pharmaceutical composition disclosed herein, in the manufacture of a medicament for treating at least one symptom and / or sign of hypertension, optionally pulmonary arterial hypertension, is disclosed.

[0055] It is contemplated that any aspect discussed herein may be practiced with respect to any method or composition of the disclosure and vice versa. Further, the compositions of the disclosure may be used to achieve the methods of the disclosure.

[0056] Any method for therapeutic, diagnostic, or physiological purposes or effects may be described in "use" claim language such as "use" of any compound, composition, or agent discussed herein to achieve or effect the described therapeutic, diagnostic, or physiological purposes or effects. The use of one or more compositions may be based on any of the methods described herein.

[0057] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific aspects of the present disclosure, are provided by way of illustration only, and various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description.

Brief Description of the Drawings

[0058]

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BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The present invention can be more readily understood by reference to the following detailed description of the embodiments of the present invention and the examples included herein. It should be understood that the present invention is, of course, not limited to the particular fabrication methods that may vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments and are not intended to be limiting.

[0060] Exemplary embodiments (E) of the present invention provided herein include: E1. An isolated antibody that binds to bone morphogenetic protein-9 (BMP9) and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH complementarity determining region 1 (CDR1) comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 24, SEQ ID NO: 36, SEQ ID NO: 45, and SEQ ID NO: 53, the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 25, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 54, SEQ ID NO: 61, SEQ ID NO: 70, and SEQ ID NO: 76, the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 26, SEQ ID NO: 38, SEQ ID NO: 47, and SEQ ID NO: 62, the VL CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 19, SEQ ID NO: 30, SEQ ID NO: 50, SEQ ID NO: 57, SEQ ID NO: 65, and SEQ ID NO: 73, The VL CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 20, SEQ ID NO: 31, SEQ ID NO: 41, and SEQ ID NO: 66, and The VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 21, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 58, and SEQ ID NO: 67, an isolated antibody. An isolated antibody that binds to E2. Bone morphogenetic protein-9 (BMP9) and comprises a heavy chain variable region (VH) and a light chain variable region (VL), (i) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 80, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 81, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 26, and the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 82, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 83, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 84; (ii) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 9, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 10, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 11, and the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 2, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 3, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 4; (iii) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 24, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 25, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 26, and the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 19, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 20, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 21; (iv) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 36, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 37, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 38, and the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 30, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 32; (v) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 45, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 46, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 47, and the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 2, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 41, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 42; (vi) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 53, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 26, and the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 50, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 20, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 21; (vii) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 45, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 61, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 62, and the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 57, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 41, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 58; (viii) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 53, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 70, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 26, and the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 65, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 66, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 67; or (ix) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 53, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 76, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 26, and the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 73, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 20, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 67, an isolated antibody. E3. (i) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 80, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 81, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 26, and the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 82, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 83, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 84; (ii) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 24, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 25, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 26, and the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 19, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 20, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 21; (iii) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 53, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 26, and the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 50, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 20, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 21; (iv) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 53, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 70, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 26, and the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 65, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 66, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 67; or (v) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 53, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 76, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 26, and the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 73, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 20, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 67, the isolated antibody according to E2. An isolated antibody that binds to bone morphogenetic protein-9 (BMP9) and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 53, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 76, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 26, and wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 73, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 20, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 67. E5. The isolated antibody according to any one of E1 to E4, wherein VH comprises the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 27, SEQ ID NO: 39, SEQ ID NO: 48, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 71, or SEQ ID NO: 77, or variants thereof comprising 1 to 4 amino acid substitutions with residues not within the CDRs, and VL comprises the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 33, SEQ ID NO: 44, SEQ ID NO: 51, SEQ ID NO: 59, SEQ ID NO: 68, or SEQ ID NO: 74, or variants thereof comprising 1 to 4 amino acid substitutions with residues not within the CDRs. E6. The isolated antibody according to any one of E1 to E4, wherein VH comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 16, SEQ ID NO: 27, SEQ ID NO: 39, SEQ ID NO: 48, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 71, or SEQ ID NO: 77, and VL comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 33, SEQ ID NO: 44, SEQ ID NO: 51, SEQ ID NO: 59, SEQ ID NO: 68, or SEQ ID NO: 74. E7. The antibody is (i) VH comprising the amino acid sequence of SEQ ID NO: 16 and VL comprising the amino acid sequence of SEQ ID NO: 6; (ii) VH containing the amino acid sequence of SEQ ID NO: 27 and VL containing the amino acid sequence of SEQ ID NO: 22; (iii) VH containing the amino acid sequence of SEQ ID NO: 39 and VL containing the amino acid sequence of SEQ ID NO: 33; (iv) VH containing the amino acid sequence of SEQ ID NO: 48 and VL containing the amino acid sequence of SEQ ID NO: 43; (v) VH containing the amino acid sequence of SEQ ID NO: 55 and VL containing the amino acid sequence of SEQ ID NO: 51; (vi) VH containing the amino acid sequence of SEQ ID NO: 63 and VL containing the amino acid sequence of SEQ ID NO: 59; (vii) VH containing the amino acid sequence of SEQ ID NO: 71 and VL containing the amino acid sequence of SEQ ID NO: 68; or (viii) An isolated antibody according to any one of E5 to E6, comprising VH containing the amino acid sequence of SEQ ID NO: 77 and VL containing the amino acid sequence of SEQ ID NO: 74. E8. The antibody is (i) VH containing the amino acid sequence of SEQ ID NO: 27 and VL containing the amino acid sequence of SEQ ID NO: 22; (ii) VH containing the amino acid sequence of SEQ ID NO: 55 and VL containing the amino acid sequence of SEQ ID NO: 51; (iii) VH containing the amino acid sequence of SEQ ID NO: 71 and VL containing the amino acid sequence of SEQ ID NO: 68; (iv) VH containing the amino acid sequence of SEQ ID NO: 77 and VL containing the amino acid sequence of SEQ ID NO: 74; (v) VH containing an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 27, and VL containing an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 22; (vi) a VH comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 55, and a VL comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 51; (vii) a VH comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 71, and a VL comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 68; or (viii) an isolated antibody according to E7, comprising a VH comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 77, and a VL comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 74. E9. An isolated antibody according to E8, wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 77 and a VL comprising the amino acid sequence of SEQ ID NO: 74. E10. A portion of an isolated antibody, wherein the isolated antibody binds to BMP9 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), The VH CDR1 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 24, SEQ ID NO: 36, SEQ ID NO: 45, and SEQ ID NO: 53; The VH CDR2 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 25, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 54, SEQ ID NO: 61, SEQ ID NO: 70, and SEQ ID NO: 76; The VH CDR3 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 26, SEQ ID NO: 38, SEQ ID NO: 47, and SEQ ID NO: 62; The VL CDR1 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 19, SEQ ID NO: 30, SEQ ID NO: 50, SEQ ID NO: 57, SEQ ID NO: 65, and SEQ ID NO: 73; The VL CDR2 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 20, SEQ ID NO: 31, SEQ ID NO: 41, and SEQ ID NO: 66; and The VL CDR3 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 21, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 58, and SEQ ID NO: 67, A portion of an isolated antibody, wherein this portion binds to BMP9. The E11. portion contains a VH CDR1 that contains an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 24, SEQ ID NO: 36, SEQ ID NO: 45, and SEQ ID NO: 53; The VH CDR2 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 25, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 54, SEQ ID NO: 61, SEQ ID NO: 70, and SEQ ID NO: 76; and The VH CDR3 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 26, SEQ ID NO: 38, SEQ ID NO: 47, and SEQ ID NO: 62, the portion according to E10. An isolated antibody that binds to BMP9 and contains the VH encoded by the nucleic acid sequence of the insert of the plasmid deposited with the ATCC and having the deposit number PTA - 127292, the VL encoded by the nucleic acid sequence of the insert of the plasmid deposited with the ATCC and having the deposit number PTA - 127293, or both of them. An isolated antibody comprising: (i) the VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NO: 16, and the VHL CDR1, VL CDR2, and VL CDR3 sequences of SEQ ID NO: 6; (ii) the VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NO: 27, and the VHL CDR1, VL CDR2, and VL CDR3 sequences of SEQ ID NO: 22; (iii) the VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NO: 39, and the VHL CDR1, VL CDR2, and VL CDR3 sequences of SEQ ID NO: 33; (iv) the VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NO: 48, and the VHL CDR1, VL CDR2, and VL CDR3 sequences of SEQ ID NO: 43; (v) the VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NO: 55, and the VHL CDR1, VL CDR2, and VL CDR3 sequences of SEQ ID NO: 51; (vi) the VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NO: 63, and the VHL CDR1, VL CDR2, and VL CDR3 sequences of SEQ ID NO: 59; (vii) the VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NO: 71, and the VHL CDR1, VL CDR2, and VL CDR3 sequences of SEQ ID NO: 68; or (viii) an isolated antibody comprising the VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NO: 77, and the VHL CDR1, VL CDR2, and VL CDR3 sequences of SEQ ID NO: 74. E14. The isolated antibody according to any one of E1 to E13, wherein the antibody comprises a human Vκ or V λ light chain constant domain. E15. The isolated antibody according to E14, wherein the antibody comprises a human V λ light chain constant domain. E16. The isolated antibody according to any one of E1 to E15, wherein the antibody comprises a heavy chain constant domain. The isolated antibody according to E16, wherein the heavy chain constant domain comprises IgA (e.g., IgA1 or IgA2), IgD, IgE, IgM, or IgG (e.g., IgG1, IgG2, IgG3, or IgG4). The isolated antibody according to E17, wherein the heavy chain constant domain comprises IgG. The isolated antibody according to E18, wherein IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. The isolated antibody according to E19, wherein IgG is IgG1. The isolated antibody according to any one of E1 to E20, wherein the antibody comprises an Fc domain. The isolated antibody according to E21, wherein the Fc domain comprises an IgG1 heavy chain CH2 domain and an IgG1 heavy chain CH3 domain. An isolated antibody, (i) a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 23, SEQ ID NO: 35, SEQ ID NO: 44, SEQ ID NO: 52, SEQ ID NO: 60, SEQ ID NO: 69, and SEQ ID NO: 75; and (ii) a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 18, SEQ ID NO: 29, SEQ ID NO: 40, SEQ ID NO: 49, SEQ ID NO: 56, SEQ ID NO: 64, and SEQ ID NO: 72. An isolated antibody, (i) a heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8, SEQ ID NO: 23, SEQ ID NO: 35, SEQ ID NO: 44, SEQ ID NO: 52, SEQ ID NO: 60, SEQ ID NO: 69, or SEQ ID NO: 75; and (ii) An isolated antibody comprising a light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 18, SEQ ID NO: 29, SEQ ID NO: 40, SEQ ID NO: 49, SEQ ID NO: 56, SEQ ID NO: 64, or SEQ ID NO: 72. E25. An isolated antibody, (i) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 8 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 1; (ii) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 23 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 18; (iii) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 35 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 29; (iv) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 44 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 40; (v) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 52 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 49; (vi) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 60 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 56; (vii) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 69 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 64; or (viii) an isolated antibody comprising an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 75 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 72. E26. An isolated antibody, (i) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 23 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 18; (ii) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 52 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 49; (iii) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 69 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 64; (iv) an HC comprising or consisting of the amino acid sequence of SEQ ID NO: 75 and an LC comprising or consisting of the amino acid sequence of SEQ ID NO: 72; (v) an HC comprising or consisting of an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 23, and an LC comprising or consisting of an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 18; (vi) an HC comprising or consisting of an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 52, and an LC comprising or consisting of an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 49; (vii) an HC comprising, or consisting of, an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 69, and an LC comprising, or consisting of, an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 64; or (viii) an isolated antibody comprising an HC comprising, or consisting of, an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 75, and an LC comprising, or consisting of, an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 72. E27. An isolated antibody, (i) an HC comprising, or consisting of, the amino acid sequence of SEQ ID NO: 75 and an LC comprising, or consisting of, the amino acid sequence of SEQ ID NO: 72; or (ii) an isolated antibody comprising an HC comprising, or consisting of, an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 75, and an LC comprising, or consisting of, an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 72. An isolated antibody that binds to E28.BMP9, wherein the antibody binds to an epitope on BMP9 that comprises one or more amino acid residues selected from the group consisting of Ala28, Pro29, Ser83, Leu85, Met90, Val92, Pro93, Leu95, and Tyr97 of SEQ ID NO: 79. The isolated antibody according to E28, wherein the antibody binds to Ala28, Pro29, Ser83, Leu85, Met90, Val92, Pro93, Leu95, and Tyr97 of SEQ ID NO: 79. The isolated antibody according to any one of E28 to E29, wherein the antibody further binds to one or more amino acid residues selected from the group consisting of Pro81, Tyr86, Asp88, Asp89, Thr94, Lys96, His98, Tyr99, and Glu100 of SEQ ID NO: 79. The isolated antibody according to E30, wherein the antibody binds to Pro81, Tyr86, Asp88, Asp89, Thr94, Lys96, His98, Tyr99, and Glu100 of SEQ ID NO: 79. The isolated antibody according to any one of E28 to E31, wherein the antibody further binds to one or more amino acid residues selected from the group consisting of Ile27, Lys30, and Glu31 of SEQ ID NO: 79. The isolated antibody according to E32, wherein the antibody binds to Ile27, Lys30, and Glu31 of SEQ ID NO: 79. An isolated antibody that binds to BMP9, wherein the antibody binds to an epitope on BMP9 that comprises one or more amino acid residues selected from the group consisting of Ile27, Ala28, Pro29, Lys30, Glu31, Tyr32, Ser80, Pro81, Ile82, Ser83, Val84, Leu85, Tyr86, Asp88, Asp89, Met90, Val92, Pro93, Thr94, Leu95, Lys96, Tyr97, His98, Tyr99, and Glu100 of SEQ ID NO: 79. An isolated antibody that binds to E35.BMP9, wherein the antibody binds to an epitope on BMP9 that comprises Ile27, Ala28, Pro29, Lys30, Glu31, Tyr32, Ser80, Pro81, Ile82, Ser83, Val84, Leu85, Tyr86, Asp88, Asp89, Met90, Val92, Pro93, Thr94, Leu95, Lys96, Tyr97, His98, Tyr99, and Glu100 of SEQ ID NO:79. An isolated antibody that binds to E36.BMP9, wherein the antibody binds to an epitope on BMP9 that comprises one or more amino acid residues selected from the group consisting of Ala28, Pro29, Pro81, Ser83, Leu85, Tyr86, Asp88, Asp89, Met90, Val92, Pro93, Thr94, Leu95, Lys96, Tyr97, His98, Tyr99, and Glu100 of SEQ ID NO:79. An isolated antibody that binds to E37.BMP9, wherein the antibody binds to an epitope on BMP9 that comprises Ala28, Pro29, Pro81, Ser83, Leu85, Tyr86, Asp88, Asp89, Met90, Val92, Pro93, Thr94, Leu95, Lys96, Tyr97, His98, Tyr99, and Glu100 of SEQ ID NO:79. An isolated antibody that binds to E38.BMP9, wherein the antibody binds to an epitope on BMP9 that comprises one or more amino acid residues selected from the group consisting of Arg14, Ile27, Ala28, Pro29, Lys30, Glu31, Ser83, Leu85, Met90, Val92, Pro93, Leu95, and Tyr97 of SEQ ID NO:79. An isolated antibody that binds to E39.BMP9, wherein the antibody binds to an epitope on BMP9 that comprises Arg14, Ile27, Ala28, Pro29, Lys30, Glu31, Ser83, Leu85, Met90, Val92, Pro93, Leu95, and Tyr97 of SEQ ID NO:79. An isolated antibody according to any one of E28 - E39, wherein the epitope on E40.BMP9 does not include amino acid residues 21 - 25 and 43 - 60 of SEQ ID NO: 79. An isolated antibody according to any one of E1 - E40, wherein when measured by surface plasmon resonance, the antibody binds to human BMP9 with an affinity of about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 55 pM, about 60 pM, about 65 pM, about 70 pM, about 75 pM, about 80 pM, about 85 pM, about 90 pM, about 95 pM, about 100 pM, about 105 pM, about 110 pM, about 115 pM, about 120 pM, about 125 pM, about 130 pM, about 135 pM, about 140 pM, or about 150 pM at 37°C. An isolated antibody according to E41, wherein when measured by surface plasmon resonance, the antibody binds to human BMP9 with an affinity of about 30.48 pM or about 87.41 pM at 37°C. An isolated antibody according to any one of E1 - E42, wherein when measured by surface plasmon resonance, the antibody binds to cynomolgus monkey BMP9 with an affinity of about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 55 pM, about 60 pM, about 65 pM, about 70 pM, about 75 pM, about 80 pM, about 85 pM, about 90 pM, about 95 pM, about 100 pM, about 105 pM, about 110 pM, about 115 pM, about 120 pM, about 125 pM, about 130 pM, about 135 pM, about 140 pM, or about 150 pM at 37°C. An isolated antibody according to E43, wherein when measured by surface plasmon resonance, the antibody binds to cynomolgus monkey BMP9 with an affinity of about 20.23 pM or about 65.82 pM at 37°C. The isolated antibody according to any one of E1 to E44, wherein when the antibody is measured by surface plasmon resonance, it binds to rat BMP9 with an affinity of about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 55 pM, about 60 pM, about 65 pM, about 70 pM, about 75 pM, about 80 pM, about 85 pM, about 90 pM, about 95 pM, about 100 pM, about 105 pM, about 110 pM, about 115 pM, about 120 pM, about 125 pM, about 130 pM, about 135 pM, about 140 pM, or about 150 pM at 37°C. The isolated antibody according to E45, wherein when the antibody is measured by surface plasmon resonance, it binds to rat BMP9 with an affinity of about 41.75 pM or about 98.63 pM at 37°C. E47. The antibody, wherein (i) it comprises the heavy chain variable region (VH) CDR3 of SEQ ID NO: 26 and the light chain variable region (VL) CDR3 of SEQ ID NO: 84; (ii) it comprises the heavy chain variable region (VH) CDR3 of SEQ ID NO: 26 and the light chain variable region (VL) CDR3 of SEQ ID NO: 21; or (iii) it comprises the heavy chain variable region (VH) CDR3 of SEQ ID NO: 26 and the light chain variable region (VL) CDR3 of SEQ ID NO: 67, and is the antibody according to any one of E28 to E31, E34 to E37, E40 to E46. E48. The isolated antibody according to any one of E1 to E47, wherein the antibody (i) binds to human BMP9 and inhibits the binding of human BMP9 to the BMP9 type II receptor (e.g., BMPRII, ActRIIA or ActRIIB), or (ii) binds to human BMP9, inhibits the binding of human BMP9 to the BMP9 type II receptor (e.g., BMPRII, ActRIIA or ActRIIB), and weakly inhibits the binding of human BMP9 to the BMP9 type I receptor (e.g., ALK1). E49. The isolated antibody according to any one of E1 to E48, wherein the antibody binds to human BMP9 and inhibits the binding of human BMP9 to human endothelial cells. An isolated antibody according to any one of E1-E49, wherein the antibody binds to human BMP9 but does not bind to other human transforming growth factor beta (TGFβ) superfamily ligands (e.g., GDF8, GDF11, activin A, GDF9, TGFβ1, and BMP10). An isolated antibody according to any one of E1-E50, wherein the antibody inhibits BMP9 activity as measured by phospho-SMAD1 / 5 / 9 nuclear translocation in endothelial cells. E52. The antibody inhibits human BMP9 activity with an IC of about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 55 pM, about 60 pM, about 65 pM, about 70 pM, about 75 pM, about 80 pM, about 85 pM, about 90 pM, about 95 pM, about 100 pM, about 105 pM, about 110 pM, about 115 pM, about 120 pM, about 125 pM, about 130 pM, about 135 pM, about 140 pM, about 145 pM, about 150 pM, about 155 pM, about 160 pM, about 170 pM, about 180 pM, about 190 pM, about 200 pM, about 225 pM, about 250 pM, about 300 pM, about 350 pM, about 400 pM, about 450 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, about 1000 pM, about 2 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, or about 100 nM. 50 The isolated antibody according to E51, wherein the antibody inhibits human BMP9 activity with an IC of about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 55 pM, about 60 pM, about 65 pM, about 70 pM, about 75 pM, about 80 pM, about 85 pM, about 90 pM, about 95 pM, about 100 pM, about 105 pM, about 110 pM, about 115 pM, about 120 pM, about 125 pM, about 130 pM, about 135 pM, about 140 pM, about 145 pM, about 150 pM, about 155 pM, about 160 pM, about 170 pM, about 180 pM, about 190 pM, about 200 pM, about 225 pM, about 250 pM, about 300 pM, about 350 pM, about 400 pM, about 450 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, about 1000 pM, about 2 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, or about 100 nM. E53. The antibody inhibits rat BMP9 activity with an IC of about 110 pM, about 115 pM, about 120 pM, about 125 pM, about 130 pM, about 135 pM, about 140 pM, about 150 pM, about 175 pM, about 200 pM, about 225 pM, about 250 pM, about 275 pM, or about 300 pM. 50 The isolated antibody according to E51, wherein the antibody inhibits rat BMP9 activity with an IC of about 110 pM, about 115 pM, about 120 pM, about 125 pM, about 130 pM, about 135 pM, about 140 pM, about 150 pM, about 175 pM, about 200 pM, about 225 pM, about 250 pM, about 275 pM, or about 300 pM. E54. The antibody inhibits human BMP9 activity with an IC of about 17 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, about 100 pM, about 110 pM, about 115 pM, about 120 pM, about 125 pM, about 130 pM, about 135 pM, or about 140 pM. 50The isolated antibody described in E51 that inhibits cynomolgus monkey BMP9 activity. E55. The isolated antibody described in any one of E1 - E54, wherein the antibody inhibits BMP9 activity as measured by phospho - SMAD2 nuclear translocation in endothelial cells. E56. The isolated antibody described in E55, wherein the antibody inhibits BMP9 activity with an IC 50 at about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 50 pM, about 100 pM, about 250 pM, about 500 pM, about 750 pM, about 1000 pM, about 10 nM, about 100 nM, about 200 nM, about 300 nM, or about 350 nM. E57. The isolated antibody described in any one of E1 - E56, wherein the antibody reduces the right ventricular systolic pressure in a subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% compared to before antibody administration. E58. The isolated antibody described in any one of E1 - E57, wherein the antibody reduces right ventricular hypertrophy in a subject by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% compared to before antibody administration. E59. The isolated antibody described in any one of E57 - E58, wherein the subject is a SuGen - Hypoxia animal model of pulmonary arterial hypertension (PAH). E60. An isolated polynucleotide encoding the antibody described in any one of E1 - E59, E91, or E92. E61. The polynucleotide described in E60, wherein the polynucleotide is RNA. E62. The polynucleotide described in E61, wherein the polynucleotide contains at least one chemical modification. E63. The polynucleotide according to E62, wherein the chemical modification is selected from pseudouridine, 1-methylpseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine. E64. The polynucleotide according to E61, wherein the polynucleotide does not contain a chemical modification. E65. An isolated nucleic acid encoding the VH, VL, or both of an antibody that binds to BMP9, wherein the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 85, the nucleic acid sequence of SEQ ID NO: 86, or both. E66. An isolated nucleic acid encoding the heavy chain, light chain, or both of an antibody that binds to BMP9, wherein the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 87, the nucleic acid sequence of SEQ ID NO: 88, or both. E67. An isolated nucleic acid encoding the VH, VL, or both of an antibody that binds to BMP9, wherein the nucleic acid comprises the nucleic acid sequence of the insert of the plasmid deposited with ATCC under the accession number PTA-127292, the nucleic acid sequence of the insert of the plasmid deposited with ATCC under the accession number PTA-127293, or both. E68. A vector comprising the polynucleotide according to any one of E60 to E64 or the nucleic acid according to any one of E65 to E67. E69. An isolated host cell comprising the polynucleotide according to any one of E60 to E64 or the nucleic acid according to any one of E65 to E67. E70. The host cell according to E69, wherein the cell is a mammalian cell or an insect cell. The host cell according to E70, which is a mammalian cell selected from the group consisting of CHO cells, HEK-293 cells, NS0 cells, PER.C6 (registered trademark) cells, or Sp2.0 cells. A method for producing an antibody, comprising culturing the host cell according to any one of E69 to E71 under conditions that result in the production of the antibody, and recovering the antibody. A pharmaceutical composition comprising a therapeutically effective amount of the antibody according to any one of E1 to E59, E90, or E91 and a pharmaceutically acceptable carrier. The pharmaceutical composition according to E73, wherein the composition comprises 1.12 mg / mL of L-histidine, 2.67 mg / mL of L-histidine hydrochloride monohydrate, 85 mg / mL of sucrose, 0.05 mg / mL of disodium EDTA dihydrate, and 0.2 mg / mL of polysorbate 80 (at pH 5.8). The pharmaceutical composition according to any one of E73 to E74, wherein the composition comprises 20 mM of histidine, 8.5% of sucrose, 0.02% of polysorbate 80, and 0.005% of EDTA (at pH 5.8). The pharmaceutical composition according to any one of E72 to E74, wherein the composition comprises an antibody at about 2 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 50 mg / mL, 75 mg / mL, 100 mg / mL, 125 mg / mL, 150 mg / mL, 175 mg / mL, or 200 mg / mL. The pharmaceutical composition according to any one of E73 to E76, wherein the composition comprises an antibody at about 50 mg / mL, about 100 mg / mL, about 150 mg / ml, or about 200 mg / mL. The pharmaceutical composition according to any one of E73 to E77, wherein the dose is a 1 mL dose. The pharmaceutical composition according to any one of E73 to E78, wherein the composition is suitable for subcutaneous (SC) and / or intravenous (IV) administration. E80. A method for reducing the expression or activity of BMP9 in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition described in any one of E73 - E79 or an antibody described in any one of E1 - E59, E90, or E91. E81. A method for treating hypertension in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition described in any one of E73 - E79 or an antibody described in any one of E1 - E59, E90, or E91. E82. The method according to E81, wherein the hypertension is pulmonary hypertension. E83. A method for treating pulmonary arterial hypertension (PAH) in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition described in any one of E73 - E79 or an antibody described in any one of E1 - E59, E90, or E91. E84. The method according to any one of E80 - E83, wherein the antibody is administered in combination with one or more additional therapeutic active compounds or therapies effective in the treatment of at least one symptom and / or sign of hypertension, optionally pulmonary arterial hypertension. E85. The method according to E84, wherein the combination therapy is administered according to the same dosing regimen (e.g., both therapies are administered daily) or according to different dosing regimens (e.g., one therapy is administered daily and the other is administered weekly). E86. An isolated antibody described in any one of E1 - E59, E90, or E91, or a pharmaceutical composition described in any one of E73 - E79, for use as a medicament. E87. An isolated antibody described in any one of E1 - E59, E90, or E91, or a pharmaceutical composition described in any one of E73 - E79, for use in the treatment of at least one symptom and / or sign of hypertension, optionally pulmonary arterial hypertension. Use of an isolated antibody as described in any one of E1 to E59, E90, or E91, or a pharmaceutical composition as described in any one of E73 to E79, in the manufacture of a medicament for treating hypertension and optionally at least one sign and / or symptom of pulmonary arterial hypertension. E89. A pharmaceutical composition as described in any one of E73 to E79, or an isolated antibody as described in any one of E1 to E59, E90, or E91, for treating hypertension and optionally at least one sign and / or symptom of pulmonary arterial hypertension. E90. Use of an isolated antibody as described in any one of E1 to E59, E91, or E92, or a pharmaceutical composition as described in any one of E73 to E79, for treating hypertension and optionally at least one sign and / or symptom of pulmonary arterial hypertension. E91. An isolated antibody as described in any one of E1 to E59, wherein the isolated antibody inhibits one or more proteins associated with hypertension and optionally pulmonary hypertension. E92. An isolated antibody as described in any one of E1 to E59 or E91, wherein the isolated antibody inhibits CXCL12, IGFBP4, INHBA, MALL, FRZB, CPE, CCL2, PDGFBB, endothelin-1, or a combination thereof. E93. A method as described in any one of E80 to E85, wherein a pharmaceutical composition as described in any one of E73 to E79 or an antibody as described in any one of E1 to E59, E90, or E91 inhibits one or more proteins associated with hypertension and optionally pulmonary hypertension. E94. A method as described in any one of E80 to E85 or E93, wherein a pharmaceutical composition as described in any one of E73 to E79 or an antibody as described in any one of E1 to E59, E91, or E92 inhibits CXCL12, IGFBP4, INHBA, MALL, FRZB, CPE, CCL2, PDGFBB, endothelin-1, or a combination thereof. A method for monitoring the effectiveness of a pulmonary arterial hypertension (PAH) therapy in a subject in need thereof, comprising: (i) administering the PAH therapy to the subject; (ii) determining the level of one or more biomarkers in a biological sample obtained from the subject; and (iii) comparing the level of the one or more biomarkers from step (ii) with the level of the one or more same biomarkers before administering the PAH therapy, wherein a change (e.g., a decrease) in the level of the one or more biomarkers after administering the PAH therapy indicates that the therapy is effective, and the PAH therapy comprises an isolated antibody described in any one of E1 - E59, E91, or E92. The method according to E100, wherein the biological sample comprises urine, blood, serum, plasma, saliva, sputum, exhaled condensate, bronchoalveolar lavage fluid, cerebrospinal fluid, tears, tissue, or mucus, or a combination thereof. The method according to any one of E100 - E101, wherein determining the level of one or more biomarkers comprises determining the mRNA level. The method according to any one of E100 - E101, wherein determining the level of one or more biomarkers comprises determining the protein level.

[0061] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0062] All references cited herein, including literature references, patent applications, patent publications, UniProtKB accession numbers, and GenBank accession numbers, are specifically and expressly incorporated herein by reference in their entirety as if each individual reference were specifically and individually indicated to be incorporated by reference. Where definitions of terms in a document incorporated herein by reference conflict with those used herein, the definitions used herein apply.

[0063] The techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art using conventional methods, such as the widely used methodologies described below: Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (3rd ed.) (2001) (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F.M. Ausubel, et al. eds.) (2003); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M.J. MacPherson, B.D. Hames and G.R. Taylor eds.) (1995), ANTIBODIES, A LABORATORY MANUAL (Harlow and Lane, eds.) (1988), and ANIMAL CELL CULTURE (R.I. Freshney, ed.) (1987); OLIGONUCLEOTIDE SYNTHESIS (M.J. Gait, ed.) (1984); METHODS IN MOLECULAR BIOLOGY (Humana Press); CELL BIOLOGY: A LABORATORY NOTEBOOK (J.E. Cellis, ed.) (1998) (Academic Press); INTRODUCTION TO CELL AND TISSUE CULTURE (J.P. Mather and P.E. Roberts) (1998) (Plenum Press); CELL AND TISSUE CULTURE LABORATORY PROCEDURES (A. Doyle, J.B. Griffiths, and D.G. Newell, eds.) (1993-8) (J.Wiley and Sons); HANDBOOK OF EXPERIMENTAL IMMUNOLOGY (D.M. Weir and C.C.Blackwell, eds); GENE TRANSFER VECTORS FOR MAMMALIAN CELLS (J.M. Miller and M.P. Calos, eds.) (1987); PCR: THE POLYMERASE CHAIN REACTION (Mullis et al., eds.) (1994); CURRENT PROTOCOLS IN IMMUNOLOGY (J.E. Coligan et al., eds.) (1991); SHORT PROTOCOLS IN MOLECULAR BIOLOGY (1999) (Wiley and Sons); IMMUNOBIOLOGY (C.A. Janeway and P. Travers) (1997); ANTIBODIES (P. Finch) (1997); ANTIBODIES: A PRACTICAL APPROACH (D. Catty., ed.) (1988-1989) (IRL Press); MONOCLONAL ANTIBODIES: A PRACTICAL APPROACH (P. Shepherd and C. Dean, eds.) (2000) (Oxford University Press); USING ANTIBODIES: A LABORATORY MANUAL (E. Harlow and D. Lane (1999) (Cold Spring Harbor Laboratory Press); THE ANTIBODIES (M. Zanetti and J.D. Capra, eds.) (1995) (Harwood Academic Publishers); and their updated versions.

[0064] Definitions Unless otherwise defined herein, scientific and technical terms used in connection with the present invention have the meanings commonly understood by one of ordinary skill in the art.

[0065] As used herein, the terms "comprising," "including," "having," or "containing," or the use of the words "a" or "an" or "the" used in conjunction with variations of these terms may mean "one," but it also is consistent with the meaning of "one or more," "at least one," and "one or more than one." For example, "an" antibody includes one or more antibodies.

[0066] When aspects or embodiments of the present invention are described in terms of Markush groups or other alternative groups, the present invention encompasses not only the entire recited group as a whole, but also each member of the group individually, and all possible subgroups of the main group, as well as the main group lacking one or more of the members of the group. The present invention also anticipates any one or more express exclusions of group members in the claimed invention.

[0067] The phrase "and / or" means "and" or "or." By way of illustration, A, B, and / or C includes A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, "and / or" functions as an inclusive disjunction.

[0068] Any example following the term "e.g." or "for example" is not meant to be exhaustive or limiting.

[0069] As used herein, when used to modify a numerically used parameter (e.g., the dosage of "X"), the terms "about", "approximately", and "substantially" mean that the parameter may vary by up to about 10% below or above the numerically specified value for that parameter. Accordingly, in any disclosed embodiment, it can be replaced within the " [%] range" of what is specified. In a non-limiting embodiment, the percentages include 0.1, 0.5, 1, 5, and 10 percent. For example, a dosage of about 5 mg means 5 mg ± 10%, i.e., it may vary from 4.5 mg to 5.5 mg.

[0070] The description of a range of values herein is intended to serve simply as a shorthand notation for individually referring to each separate value within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually recited herein.

[0071] The phrase "substantially all" is defined as "at least 95%", and when substantially all members of a group have a particular trait, at least 95% of the members of that group have that trait. In some embodiments, substantially all is equal to any one of 95, 96, 97, 98, 99, or 100% of the members of the group having that trait, or at least any one of them, or between any two of the following (including or excluding both ends).

[0072] The compositions and methods for using them can “comprise,” “consist essentially of,” or “consist of” any of the components or steps disclosed throughout the specification. Throughout the specification, unless the context requires otherwise, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or non-limiting and are understood to imply including the recited step or element or group of steps or elements but not excluding any other step or element or group of steps or elements. As a result, the compositions and methods of the invention that “comprise,” “have,” “include,” or “contain” one or more elements have those one or more elements but are not limited to having only those one or more elements. Similarly, the elements of the compositions or methods of the present disclosure that “comprise,” “have,” “include,” or “contain” one or more features have those one or more features but are not limited to having only those one or more features.

[0073] As used herein, aspects described in terms of the term "comprising" are also contemplated to be implemented in terms of the terms "consisting of" or "consisting essentially of". Compositions and methods "consisting essentially of" any of the disclosed components or steps limit the claims to those specified materials or steps that do not materially affect the basic and novel characteristics of the claimed disclosure. The term "consisting of" (and any form of consisting of, e.g., "consist of" and "consists of") means, without limitation, that what follows the phrase "consisting of" includes nothing else. Thus, the phrase "consisting of" indicates that the recited elements are necessary or essential and that no other elements may be present.

[0074] Throughout this specification, references to "one aspect", "an aspect", "certain aspects", "related aspects", "certain aspects", "additional aspects", "further aspects", "one embodiment", "an embodiment", "certain embodiments", "related embodiments", "certain embodiments", "additional embodiments", "further embodiments", or combinations thereof mean that the particular features, structures, or characteristics described in connection with the aspect are included in at least one aspect or embodiment of the present disclosure. Thus, appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same aspect or embodiment. Further, the particular features, structures, and / or characteristics may be combined in any suitable manner in one or more aspects or embodiments.

[0075] "Antibody" refers to an immunoglobulin molecule that can specifically bind to a target, such as a polypeptide, carbohydrate, polynucleotide, lipid, etc., through at least one antigen-binding site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" can encompass any type of antibody (e.g., monospecific, bispecific), a portion of an intact antibody that retains the ability to bind to a given antigen (e.g., "antigen-binding fragment"), and any other modified configuration of an immunoglobulin molecule that contains an antigen-binding site.

[0076] Antibodies include antibodies of any class, such as IgG, IgA, or IgM (or their subclasses), and the antibody need not be of any particular class. Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the antibody's constant region of the heavy chain (HC). There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant regions corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structure and three-dimensional arrangement of the different classes of immunoglobulins are well known. It is contemplated that any one or more of the foregoing immunoglobulins or their subclasses may be excluded from the embodiments.

[0077] Examples of antibodies, antigen-binding fragments, and modified structures include: (i) Fab fragments (monovalent fragments consisting of the VL, VH, CL, and CH1 domains); (ii) F(ab’)2 fragments (bivalent fragments containing two Fab fragments linked by disulfide bridges in the hinge region); and (iii) Fv fragments consisting of the VL and VH domains of a single arm of an antibody. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that enables them to pair and be made as a single protein chain (known as a single-chain Fv (scFv)) that forms a monovalent molecule. See, for example, Bird et al., Science 1988; 242:423-426 and Huston et al., Proc. Natl. Acad. Sci. 1988 USA 85:5879-5883. Other forms of single-chain antibodies such as diabodies are also included. It is contemplated that any one of the aforementioned antibodies, antigen-binding fragments, or modified structures thereof may be excluded from a particular embodiment.

[0078] Furthermore, antibodies are further included in which the C-terminal lysine (K) amino acid residue on the heavy chain polypeptide (e.g., a human IgG1 heavy chain includes a terminal lysine) is absent. As is known in the art, the C-terminal lysine may be cleaved during antibody production, resulting in an antibody having a heavy chain lacking the C-terminal lysine. Alternatively, the antibody heavy chain may be produced using a nucleic acid that does not include the C-terminal lysine. Accordingly, the present disclosure includes compositions comprising anti-BMP9 antibodies having a heavy chain lacking the C-terminal lysine residue. In some embodiments, the present disclosure includes compositions comprising anti-BMP9 antibodies having a heavy chain with a C-terminal lysine residue and anti-BMP9 antibodies having a heavy chain lacking the C-terminal lysine residue. In some embodiments, the present disclosure includes compositions comprising anti-BMP9 antibodies lacking the C-terminal lysine residue.

[0079] The "variable region" of an antibody refers to either an antibody light chain variable region or an antibody heavy chain variable region, alone or in combination. As is known in the art, the variable regions of the heavy and light chains, each consisting of four framework regions (FRs) connected by three complementarity determining regions (CDRs), are also known as hypervariable regions and contribute to the formation of the antigen binding site of the antibody. In particular, when variants of the variable region of the subject having substitutions at amino acid residues outside the CDR regions (i.e., within the framework regions) are desired, appropriate amino acid substitutions, preferably conservative amino acid substitutions, can be identified by comparing the variable region of the subject with the variable regions of other antibodies containing CDR1 and CDR2 arrangements in the same canonical class as the variable region of the subject (Chothia and Lesk, J. Mol. Biol. 196(4):901-917, 1987).

[0080] In certain embodiments, the definitive delineation of the CDRs and the identification of the residues comprising the binding site of the antibody are achieved by elucidating the structure of the antibody or the structure of the antibody-ligand complex. In certain embodiments, it can be achieved by any of a variety of descriptions known to those of skill in the art, such as X-ray crystallographic analysis. In certain embodiments, the CDR regions can be defined or approximated using a variety of analytical methods. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, the contact definition, the extended definition, and the conformational definition.

[0081] The Kabat definition is a standard for numbering residues in an antibody and is typically used to identify CDR regions. See, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8. The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account the positions of specific structural loop regions. See, for example, Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83. The extended definition is a combination of the Kabat definition and the Chothia definition. The AbM definition uses an integrated suite of computer programs created by the Oxford Molecular Group for modeling antibody structures. See, for example, Martin et al., 1989, Proc. Natl. Acad. Sci. (USA), 86:9268-9272; “AbM (trademark), A Computer Program for Modeling Variable Regions of Antibodies,” Oxford, UK; Oxford Molecular, Ltd. The AbM definition uses a combination of a knowledge database such as that described by Samudrala et al., 1999, “Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach,” in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198 and an ab initio method to model the tertiary structure of an antibody from its primary sequence. The contact definition is based on the analysis of available complex crystal structures. See, for example, MacCallum et al., 1996, J. Mol. Biol., 5:732-45. In this specification, in another approach called the “conformational definition” of CDRs, the positions of CDRs can be defined as residues that contribute enthalpically to antigen binding. See, for example, Makabe et al., 2008, J. Biol. Chem., 283:1156-1166.Still other CDR boundaries may not strictly follow one of the above approaches, but overlap at least in part with the Kabat CDR and yet may be too short or too long in view of predictions or experimental results that certain residues or groups of residues do not significantly affect antigen binding. As used herein, CDR may refer to CDRs defined by any approach known in the art, including combinations of approaches. The methods used herein can utilize CDRs defined according to any of these approaches. For any given embodiment containing two or more CDRs, the CDRs may be defined according to any one or more of the Kabat, Chothia, extended, AbM, contact, and / or conformation definitions. It is contemplated that any one or more of these definitions may be excluded in a particular embodiment.

[0082] The "constant region" of an antibody refers to the constant region of any antibody light chain or the constant region of an antibody heavy chain, either alone or in combination. The IgG heavy chain constant region contains three sets of immunoglobulin domains (CH1, CH2, and CH3), with a hinge region between the CH1 and CH2 domains. The IgG light chain constant region contains a single immunoglobulin domain (CL).

[0083] The "Fc domain" refers to the part of an immunoglobulin (Ig) molecule associated with the crystallizable fragment obtained by papain digestion of the Ig molecule. As used herein, the term refers to the two-chain constant region of an antibody in which each chain excludes the first constant region immunoglobulin domain. Within the Fc domain, there are two "Fc chains" (e.g., "first Fc chain" and "second Fc chain"). An "Fc chain" generally refers to the C-terminal portion of an antibody heavy chain. Thus, an Fc chain refers to the last two constant region immunoglobulin domains (CH2 and CH3) of IgA, IgD, and IgG heavy chains, and the last three constant region immunoglobulin domains of IgE and IgM heavy chains, and optionally the flexible hinge N-terminus to these domains.

[0084] The boundary of the Fc chain may vary, but the Fc chain of the human IgG heavy chain is typically defined to include residue C226 or P230 at its carboxyl terminus, and this numbering follows the EU index of Edelman et al., Proc. Natl. Acad. Sci. USA 1969;63(1):78-85, as described in Kabat et al., 1991. Typically, the Fc chain includes amino acid residues from about 236 to about 447 of the human IgG1 heavy chain constant region. The "Fc chain" may refer to this polypeptide separately or in association with a larger molecule (e.g., an antibody heavy chain or an Fc fusion protein).

[0085] A "functional" Fc domain refers to an Fc domain that possesses at least one effector function of the native sequence Fc domain. Exemplary "effector functions" include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Such effector functions generally require an Fc domain that is combined with a binding domain (e.g., an antibody variable region) and can be evaluated using various assays known in the art for evaluating such antibody effector functions. A "native sequence" Fc chain refers to an Fc chain that contains an amino acid sequence identical to that of the Fc chain found in nature. A "variant" Fc chain contains an amino acid sequence that differs from that of the native sequence Fc chain by at least one amino acid modification. It is contemplated that any one of the foregoing Fc chains or domains may be excluded from a particular embodiment.

[0086] A "monoclonal antibody" (mAb) refers to an antibody derived from a single copy or clone, including, for example, any eukaryotic clone, prokaryotic clone, or phage clone. Monoclonal antibodies are highly specific and target a single antigenic site. Further, typically in contrast to polyclonal antibody preparations that include antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. The modifier "monoclonal" indicates the characteristic of the antibody when obtained from a substantially homogeneous group of antibodies, but it is not to be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present disclosure may be produced by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or by recombinant DNA methods as described in U.S. Patent No. 4,816,567. In another example, monoclonal antibodies may be isolated from phage libraries such as those generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554.

[0087] A "human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, or an antibody produced using any technique for making a fully human antibody. For example, fully human antibodies can be obtained by using commercially available mice engineered to express specific human immunoglobulin proteins, or by phage, yeast, or ribosome display techniques for preparing fully human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0088] A "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, for example, an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0089] A "humanized" antibody refers to a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin, which is a non-human (e.g., mouse) antibody. Preferably, a humanized antibody is a human immunoglobulin (recipient antibody) in which the residues derived from the CDRs of the recipient are replaced by the residues derived from the CDRs of a non-human species (donor antibody) such as a mouse, rat, or rabbit that has the desired specificity, affinity, and ability. A humanized antibody may contain residues not found in the CDRs imported into the recipient antibody or in the framework sequences, but which are included for further improvement or optimization of antibody performance.

[0090] It is contemplated that any one or more of the antibody types described herein may be excluded from a particular embodiment.

[0091] "Antigen" refers to a molecular entity used in the immunization of an immunocompetent vertebrate to produce an antibody that recognizes the antigen, or for the screening of an expression library (e.g., especially a phage, yeast, or ribosome display library) for antibody selection. As used herein, antigen is more broadly defined and is intended to generally include a target molecule specifically recognized by an antibody, and thus includes fragments or mimetics of molecules used in an immunization process to induce an antibody or in a library screening to select an antibody.

[0092] "Epitope" refers to the region or domain of an antigen to which an antibody specifically binds, e.g., a region or domain that contains residues that interact with an antibody as determined by any method well known in the art. There are many methods known in the art for mapping and characterizing the location of epitopes on a protein, including, for example, elucidation of the crystal structure of an antibody-antigen complex as described in Chapter 11 of Harlow and Lane, USING ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999, competitive assays, gene fragment expression assays, epitope mapping, and synthetic peptide-based assays. Additionally, or alternatively, during the discovery process, information regarding the desired epitope may be elucidated by the generation and characterization of antibodies. From this information, it is then possible to competitively screen antibodies for binding to the same epitope.

[0093] Additionally, the epitope to which an antibody binds can be determined in a systematic screening by using overlapping peptides derived from the antigen and determining binding by the antibody. According to a gene fragment expression assay, the open reading frame encoding the antigen can be fragmented randomly or by a specific gene structure, and the reactivity of the expressed fragments of the antigen with the antibody being tested is determined. The gene fragments can be generated, for example, by PCR and then transcribed in vitro in the presence of radiolabeled amino acids and translated into protein. The binding of the antibody to the radiolabeled antigen fragment is then determined by immunoprecipitation and gel electrophoresis.

[0094] Specific epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries) or yeast (yeast display). Alternatively, defined libraries of overlapping peptide fragments can be tested for binding to a test antibody in a simple binding assay. In additional examples, mutagenesis of the antigen, domain swapping experiments, and alanine scanning mutant analysis can be performed to identify residues that are sufficient or essential for epitope binding.

[0095] At its most detailed level, an epitope for the interaction between an antigen and an antibody can be identified by the spatial coordinates that define the atomic contacts present in the antigen-antibody interaction, as well as information regarding the relative contributions to the binding thermodynamics. At a less detailed level, an epitope can be characterized by the spatial coordinates that define the atomic contacts between the antigen and the antibody. At an even less detailed level, an epitope can be characterized by the amino acid residues it contains, such that it is identified by specific criteria, e.g., distances between atoms (e.g., heavy atoms, i.e., non-hydrogen atoms) in the antibody and the antigen. At an even less detailed level, an epitope can be characterized through function, e.g., by competitive binding with other antibodies. An epitope can also be more generally defined as including amino acid residues where substitution by another amino acid alters the nature of the interaction between the antibody and the antigen (e.g., using alanine scanning).

[0096] Given the fact that descriptions and definitions of epitopes are obtained at various levels of detail depending on the epitope mapping method used, the comparison of epitopes of different antibodies to the same antigen can likewise be performed at different levels of detail.

[0097] For example, epitopes that are described at the amino acid level determined from X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, hydrogen / deuterium exchange mass spectrometry (H / D-MS) are said to be identical if they contain the same set of amino acid residues. Epitopes are said to overlap if at least one amino acid is shared by the epitopes. Epitopes are said to be distinct (unique) if no amino acid residues are shared by the epitopes.

[0098] Yet another method that can be used to characterize an antibody is to use a competition assay with other antibodies known to bind to the same antigen to determine whether the antibody of interest binds to the same epitope as the other antibodies. Competition assays are well known to those skilled in the art. Epitopes characterized by competitive binding are said to overlap if the binding of the corresponding antibodies is mutually exclusive, i.e., the binding of one antibody precludes the simultaneous or sequential binding of the other antibody. Epitopes are said to be distinct (unique) if the antigen can accommodate the binding of both corresponding antibodies simultaneously.

[0099] Epitopes can be linear or conformational. In a linear epitope, all of the points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. A "non-linear epitope" or "conformational epitope" includes non-contiguous polypeptides (or amino acids) within the antigen protein to which an antibody specific for the epitope binds.

[0100] The term "binding affinity" refers to the overall strength of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y is generally the dissociation constant (K D) can be represented by. Affinity can be measured by conventional methods known in the art. Low-affinity antibodies generally bind to antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind to antigens more quickly and tend to remain bound for a longer time. In particular, the term "binding affinity" is intended to refer to the dissociation rate of a particular antigen-antibody interaction. K D is the ratio of the dissociation rate and is the "off-rate (k off )" or "k d " with respect to the association rate, or the "on-rate (k on )" or "k a ". Thus, KD is equal to k off / k on (or k d / k a ) and is expressed as a molecular concentration (M). The smaller the K D , the stronger the binding affinity. Thus, a K D of 1 μM exhibits a weaker binding affinity compared to a K D of 1 nM. The K D value for an antibody can be determined using methods well established in the art. One exemplary method for determining the K D of an antibody typically involves using surface plasmon resonance (SPR) using a biosensor system such as a BIACORE™ system. BIACORE™ kinetic analysis involves analyzing the binding and dissociation of an antigen from a chip having immobilized molecules (e.g., molecules containing an epitope-binding domain) on their surfaces. Another method for determining the K D of an antibody typically involves using biolayer interferometry using an OCTET® technology (OCTET® QK e system, ForteBio, now Sartorius). Alternatively, or additionally, a KINEXA® (binding equilibrium exclusion method) assay available from Sapidyne Instruments (Boise, ID) can also be used.

[0101] A "monospecific antibody" refers to an antibody that contains one or more antigen-binding sites per molecule such that all binding sites of the antibody specifically recognize the same epitope on the antigen. Thus, if a monospecific antibody has two or more antigen-binding sites, the binding sites compete with each other for binding to one antigen molecule.

[0102] A "bispecific antibody" refers to a molecule having binding specificities for at least two different epitopes. In some embodiments, a bispecific antibody can bind two different antigens simultaneously. In other embodiments, the two different epitopes may be present on the same antigen.

[0103] "Half maximal effective concentration (EC 50 )" refers to the concentration of a therapeutic agent that elicits a response halfway between the baseline and the maximum value after a specified exposure time. EC 90 refers to the concentration of a therapeutic agent corresponding to 90% of the maximum possible response after a specified exposure time. The therapeutic agent can cause inhibition or stimulation. EC 50 is commonly used and is used herein as a measure of potency.

[0104] "Half maximal inhibitory concentration" (IC 50 ) refers to the concentration of a therapeutic agent required to inhibit 50% of the maximum response obtained.

[0105] An "agonist" refers to a substance that promotes (i.e., induces, causes, enhances, and / or increases) the biological activity or effect of another molecule. The term agonist encompasses substances (such as antibodies) that bind to a molecule and promote its activity.

[0106] An "antagonist" refers to a molecule, e.g., a receptor, that prevents, blocks, inhibits, neutralizes, and / or reduces the biological activity or effect of another molecule. The term antagonist encompasses substances (such as antibodies) that bind to a molecule and prevent or reduce its activity.

[0107] As used herein with respect to antibodies, the term "competing" means that a first antibody binds to an epitope in a manner sufficiently similar to the binding of a second antibody such that the result of the binding of the second antibody to its cognate epitope is detectably decreased in the presence of the first antibody as compared to the binding of the second antibody in the absence of the first antibody. It is possible, but not necessary, that the binding of the first antibody to the epitope is also detectably decreased in the presence of the second antibody. That is, a first antibody can inhibit the binding of a second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its epitope. However, antibodies are said to "cross-compete" with each other for binding to their respective epitopes if each antibody detectably inhibits the binding of the other antibody to its cognate epitope or ligand, whether to the same, greater, or lesser extent. Both competing and cross-competing antibodies are encompassed by the present disclosure. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational changes, and / or binding to a common epitope or a portion thereof), one of ordinary skill in the art will understand, based on the teachings provided herein, that such competing or cross-competing antibodies are encompassed and may be useful in the methods disclosed herein.

[0108] "Fc receptor" (FcR) refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is an FcR (gamma receptor) that binds to IgG antibodies and includes receptors of the FcgRI, FcgRII, and FcgRIII subclasses, including allelic variants and alternative splice forms of these receptors. The FcgRII receptor includes FcgRIIA ("activating receptor") and FcgRIIB ("inhibitory receptor"), which have similar amino acid sequences but differ mainly in their cytoplasmic domains. The activating receptor FcgRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcgRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 1997;15:203-234). FcRs are outlined, for example, in Ravetch and Kinet, Annu. Rev. Immunol. 1991;9:457-92; Capel et al., Immunomethods 1994;4:25-34; and de Haas et al., J. Lab. Clin. Med. 1995;126:330-41. Other FcRs, including those to be identified in the future, are encompassed by the term "Fc receptor" herein. The term "Fc receptor" also includes the neonatal receptor FcRn, which contributes to the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 1976;117:587 and Kim et al., J. Immunol. 1994;24:249) and contributes to the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today 1997;18(12):592-598; Ghetie et al., Nature Biotechnology, 1997;15(7):637-640; Hinton et al., J. Biol. Chem. 2004;279(8):6213-6216; WO 2004 / 92219 pamphlet).

[0109] "Effector cell" refers to a leukocyte that expresses one or more FcRs and performs an effector function. In certain embodiments, the effector cell expresses at least one FcgRIII and performs an ADCC effector function. Examples of leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, macrophages, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources, such as blood.

[0110] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which Ig secreted binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), thereby enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. NK cells, which are the major cells mediating ADCC, express only FcgRIII, while monocytes express FcgRI, FcgRII, and FcgRIII. To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay such as those described in U.S. Patent Nos. 5,500,362, 5,821,337, or 6,737,056 may be performed. Effector cells useful in such assays include PBMCs and NK cells. Alternatively, or additionally, the ADCC activity of a molecule of interest can be evaluated in vivo in an animal model such as those disclosed in, e.g., Clynes et al., Proc. Natl. Acad. Sci. (USA) 1998;95:652-656. Additional antibodies having an altered amino acid sequence in the Fc region and increased or decreased ADCC activity are described, e.g., in U.S. Patent Nos. 7,923,538, and 7,994,290.

[0111] The term "enhanced ADCC activity" refers to an antibody that is more effective in mediating ADCC in vitro or in vivo compared to a parental antibody, where the antibody and the parental antibody differ in at least one structural feature, and the amounts of such antibody and parental antibody used in the assay are essentially the same. In some embodiments, the antibody and the parental antibody have the same amino acid sequence, but the antibody is defucosylated while the parental antibody is fucosylated. In some embodiments, the ADCC activity is determined using an in vitro ADCC assay, but other assays or methods for determining ADCC activity, such as those in animal models, are contemplated. In some embodiments, an antibody having enhanced ADCC activity has enhanced affinity for FcgRIIIA.

[0112] The term "altered" FcR binding affinity or ADCC activity refers to an antibody having either enhanced or decreased activity for one or more of FcR binding activity or ADCC activity compared to a parental antibody, where the antibody and the parental antibody differ in at least one structural feature. An antibody that "shows increased binding" to an FcR binds to at least one FcR with better affinity than the parental antibody. An antibody that "shows decreased binding" to an FcR binds to at least one FcR with lower affinity than the parental antibody. Such an antibody that shows decreased binding to an FcR may not have little or no appreciable binding to the FcR, for example, 0 to 20% binding to the FcR compared to the native sequence IgG Fc region.

[0113] The term "complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass) that are bound to their cognate antigens. To assess complement activation, for example, a CDC assay such as that described in Gazzano-Santoro et al., J. Immunol. Methods 1996;202:163 may be performed. Antibodies having an altered amino acid sequence in the Fc region and an increased or decreased Clq binding ability are described, for example, in U.S. Patent No. 6,194,551, U.S. Patent No. 7,923,538, U.S. Patent No. 7,994,290, and International Publication No. 1999 / 51642 pamphlet.

[0114] A "host cell" may be a recipient for a vector for the incorporation of a polypeptide insert, or refers to an individual cell or cell culture that is the recipient. Host cells include the progeny of a single host cell, which progeny may not necessarily be identical to the original parent cell in morphology or in the complement of genomic DNA due to natural, accidental, and / or intentional mutations. Host cells include cells transfected in vivo with the polynucleotides disclosed herein.

[0115] "Vector" refers to a construct that can deliver and preferably express one or more genes or sequences of interest (e.g., antibody-encoding genes) in a host cell. Examples of vectors include, but are not limited to, plasmids and viral vectors, which may contain naked nucleic acids or nucleic acids associated with delivery aids (e.g., cationic condensing agents, liposomes, etc.). Vectors can contain DNA or RNA. As used herein, "expression vector" refers to a vector containing at least one polypeptide-encoding gene and at least one regulatory element (e.g., promoter sequence, poly(A) sequence) associated with the transcription or translation of the gene. Typically, the vectors used herein contain at least one antibody-encoding gene and one or more of regulatory elements or selectable markers. Vector components may include, for example, one or more of the following: signal sequences; origins of replication; one or more marker genes; suitable transcriptional control elements (such as promoters, enhancers, and terminators). For translation, one or more translation control elements such as ribosome binding sites, translation initiation sites, and stop codons may also be included. It is contemplated that any one or more of these aforementioned components or elements may be excluded in certain embodiments.

[0116] An "isolated" molecule (e.g., an antibody) is, by its origin or source, (1) a molecule not associated with its naturally associated components, (2) a molecule substantially free of other molecules from the same source, e.g., species, the cells in which it is expressed, libraries, etc., (3) a molecule expressed by cells from different species, or (4) a molecule that does not occur in nature. Thus, a molecule that is chemically synthesized or expressed in a cell line different from the system of its natural origin is "isolated" from its naturally associated components. Molecules can also be made substantially free of naturally associated components by isolation using purification techniques well known in the art.

[0117] "Polypeptide" or "protein" (used interchangeably herein) refers to a chain of amino acids of any length. The chain may be linear or branched. The chain may contain one or more of the modified amino acids. The term also encompasses naturally modified amino acid chains, or amino acids modified by the following interventions: for example, formation of disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation, and / or any other manipulation or modification, such as binding to a labeling component. This definition further includes polypeptides containing, for example, one or more amino acid analogs (including, for example, non-natural amino acids), as well as other modifications known in the art. A polypeptide is understood to be capable of occurring as a single chain or associated chains.

[0118] "Polynucleotide" or "nucleic acid" (used interchangeably herein) refers to a chain of nucleotides of any length and includes DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, or their analogs, or any substrate that can be incorporated into the chain by DNA polymerase or RNA polymerase. Polynucleotides may include, for example, modified nucleotides such as methylated nucleotides and their analogs. When present, modifications to the nucleotide structure are made either before or after chain assembly. The nucleotide sequence may be interrupted by non-nucleotide components. Polynucleotides may be further modified after multimerization, for example, by conjugation to a labeling component. Other types of modifications include, for example, "cap" substitution of one or more of the naturally occurring nucleotides with analogs, modifications between nucleotides using, for example, uncharged linkages (e.g., methyl phosphonate, phosphotriester, phosphoramidate, carbamate, etc.) and charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.), those containing pendant moieties of proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those using intercalating agents (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, those using modified linkages (e.g., alpha-anomeric nucleic acids, etc.), as well as the unmodified form of the polynucleotide. Further, any of the hydroxyl groups normally present on the sugar may be substituted, for example, with a phosphonate group, a phosphate group, etc., protected by a standard protecting group, or activated to prepare additional linkages to additional nucleotides, or attached to a solid support. The 5' end OH and 3' end OH may be phosphorylated or substituted with an amine or an organic capping group moiety of 1 to 20 carbon atoms. Other hydroxyls may be derivatized with standard protecting groups.The polynucleotide may also contain analog forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. It is contemplated that any one or more of these aforementioned modifications may be excluded in certain embodiments.

[0119] "Conservative substitution" refers to the substitution of one amino acid by a biologically, chemically, and / or structurally similar residue. Biologically similar means that the substitution does not destroy biological activity. Structurally similar means that the amino acids have side chains of similar length, such as alanine, glycine and serine, or have similar size. Chemical similarity means that the residues have the same charge, or both are hydrophilic or hydrophobic. Specific examples include substitution of one hydrophobic residue by another, such as isoleucine, valine, leucine or methionine, or substitution of one polar residue by another, such as substitution of arginine by lysine, substitution of glutamic acid by aspartic acid or substitution of glutamine by asparagine, or substitution of serine by threonine. Specific examples of conservative substitutions include substitution of hydrophobic residues for each other, such as isoleucine, valine, leucine or methionine, or substitution of one polar residue by another, such as substitution of arginine by lysine, substitution of glutamic acid by aspartic acid, or substitution of glutamine by asparagine. Conservative amino acid substitutions typically include substitutions within the following groups, for example: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0120] The term "identity" or "identical to" refers to the overall relatedness between polymer molecules, for example, between nucleic acid molecules (e.g., DNA molecules or RNA molecules), or between polypeptide molecules. "Identity" is determined by the percentage of exact matches between two or more sequences using gap alignment, which is addressed by a specific mathematical model (e.g., an algorithm) of a computer program well-known in the art.

[0121] The terms "increasing", "improving", "decreasing", or "reducing" refer to a value relative to a baseline measurement, such as a measurement in the same individual prior to the disclosure of the treatment described herein, or a value relative to a measurement in a control individual or subject (or multiple control individuals or subjects) in the absence of the treatment described herein. In some embodiments, the "control individual" is an individual suffering from the same form of disease or injury as the individual being treated. In some embodiments, the "control individual" is an individual not suffering from the same form of disease or injury as the individual being treated.

[0122] The term "excipient" refers to any substance that, when combined with a target active ingredient (e.g., an antibody), enables the active ingredient to retain its biological activity. The choice of excipient will depend largely on factors such as the method of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, carriers, diluents, etc. that are physiologically compatible. Examples of excipients include water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc., and one or more of combinations thereof, and isotonic agents such as sugars, sodium chloride, and / or polyhydric alcohols such as mannitol or sorbitol may be included in the composition. It is contemplated that any one or more of these aforementioned excipients may be excluded in certain embodiments.

[0123] The terms "treating," "treat," or "treatment" refer to any type of treatment, for example, to alleviate, reduce, improve, and / or delay the progression of a patient's disease, disorder, or condition, or damage to any tissue associated with the disease. In some embodiments, the disease, disorder, or condition is hypertension, optionally pulmonary arterial hypertension.

[0124] The terms "prevent" or "prevention" refer to at least one of delaying the onset, reducing the frequency, and / or reducing the severity of at least one symptom of a particular disease, disorder, or condition (e.g., pulmonary arterial hypertension), such as for pulmonary arterial hypertension, shortness of breath, fatigue, dizziness, chest pressure or pain, swelling (edema), cyanosis, palpitations, ventricular hypertrophy, and / or reduced function of the right side of the heart (leading to heart failure). In some embodiments, prevention is evaluated on a population basis such that a statistically significant decrease in the occurrence, frequency, and / or intensity of one or more symptoms of a disease, disorder, or condition is observed in a population susceptible to the disease, disorder, or condition, the agent is considered to "prevent" the particular disease, disorder, or condition. Prevention can be considered complete if the onset of the disease, disorder, or condition is delayed over a predetermined period.

[0125] The terms "subject," "individual," or "patient" (used interchangeably herein) refer to any animal, including mammals. Mammals according to embodiments include dogs, cats, cows, goats, horses, sheep, pigs, rodents, rabbits, primates, humans, etc., including mammalian fetuses. In certain embodiments, humans are suitable subjects. Human subjects can be of either sex and at any stage of development. In some embodiments, the subject is a patient having hypertension, optionally pulmonary arterial hypertension.

[0126] The term "therapeutically effective amount" refers to the amount of an active ingredient that elicits a biological or medical response in a tissue, system, animal, individual, and / or human being as required by a researcher, veterinarian, physician, and / or other clinician, and the requirements may include one or more of the following: (1) Preventing a disease; for example, preventing a disease, condition, or disorder in an individual who is susceptible to but has not yet experienced a disease, condition, or disorder and / or who may exhibit the pathology and / or symptoms of the disease; (2) Inhibiting a disease; for example, inhibiting a disease, condition, or disorder in an individual who is experiencing and / or exhibiting the pathology and / or symptoms of the disease, condition, or disorder (i.e., preventing and / or slowing further development of the pathology and / or symptoms); and (3) Improving a disease; for example, improving a disease, condition, or disorder in an individual who is experiencing and / or exhibiting the pathology and / or symptoms of the disease, condition, or disorder (i.e., reversing the pathology and / or symptoms).

[0127] Antibodies against BMP9 The present disclosure provides an antibody that binds to bone morphogenetic protein-9 (BMP9). BMP9, also known as growth differentiation factor 2 (GDF2), is encoded in humans by the GDF2 gene. It belongs to the transforming growth factor beta (TGFβ) superfamily. BMPs are synthesized as large precursors of approximately 400-500 amino acids consisting of an N-terminal signal peptide that directs secretion, a prodomain for proper folding and the C-terminal mature peptide (Bragdon B, Moseychuk O, Saldanha S, King D, Julian J, Nohe A (2011) Bone morphogenetic proteins: a critical review. Cell Signal 23:609-620). Upon dimerization, the carboxy-terminal mature protein is cleaved from the prodomain (Nelsen SM, Christian JL (2009) Site-specific cleavage of BMP4 by furin, PC6, and PC7. J. Biol. Chem. 284:27157-27166). After cleavage, the prodomain remains non-covalently associated with the mature active BMP dimer. Three type II receptors [bone morphogenetic protein receptor type II (BMPRII), activin receptor type 2A (ActRIIA), and type 2B (ActRIIB)], and four type I receptors [activin receptor-like kinase (ALK1, ALK2, ALK3, and ALK6)] are involved in BMP signaling. After ligand-induced receptor hetero-oligomerization, the type I receptor is phosphorylated and activated by the type II receptor. The complex of these activated receptors phosphorylates receptor-regulated Smads (R-Smads: Smad1, Smad5, and Smad8), enabling the formation of a complex with co-Smad Smad4.The R-Smad / Smad4 complex translocates into the nucleus, binds to regulatory elements, and recruits transcriptional corepressors and / or activation complexes onto their promoters to regulate target gene expression (Bidart, M., Ricard, N., Levet, S. et al. BMP9 is produced by hepatocytes and circulates mainly in an active mature form complexed to its prodomain. Cell. Mol. Life Sci. 69, 313-324 (2012)).

[0128] As used herein, the term bone morphogenetic protein-9 or bone morphogenetic protein 9 (BMP9), which is also known as growth differentiation factor 2 (GDF2), includes variants, isoforms, homologs, orthologs, and paralogs of BMP9. In some embodiments, the antibodies disclosed herein cross-react with BMP9 from non-human species such as cynomolgus monkey BMP9 and / or rat BMP9, as well as different forms of BMP9. In some embodiments, the antibody can be completely specific for human BMP9 and not exhibit species cross-reactivity (e.g., does not bind to mouse BMP9). In some embodiments, the antibody can be completely specific for human BMP9 but does not exhibit cross-reactivity with other transforming growth factor beta superfamily members (e.g., does not bind to TGFβ1, GDF8, GDF9, GDF11, activin A, and BMP10). As used herein, the term BMP9 refers to naturally occurring human BMP9 unless the context indicates otherwise. Thus, "BMP9 antibody", "anti-BMP9 antibody", or other similar designations mean any antibody (as defined herein) that binds to or reacts with BMP9, or an isoform, fragment, and / or derivative thereof. The full-length human BMP9 represented by UniProtKB / Swiss-Prot accession number Q9UK05 is provided herein as SEQ ID NO: 78. The full-length mature form (monomer) of human BMP9 is provided herein as SEQ ID NO: 79.

[0129] While not wishing to be bound by any particular theory, perturbation of the BMP9 signaling pathway has been shown to be highly important for endothelial function (dysfunction) and vascular remodeling, particularly for pulmonary arterial hypertension. Inhibition of BMP9 in different complementary and well-established animal models of PAH has been shown to provide partial protection against experimental pulmonary hypertension (Tu L., Desroches-Castan A., Mallet C., et al. Selective BMP-9 inhibition partially protects against experimental pulmonary hypertension. Circulation Research. 2019;124:846-855; Bouvard C., Tu L., Rossi M., et al. Different cardiovascular and pulmonary phenotypes are observed in single- and double-knock-out mice deficient in BMP9 and / or BMP10. Cardiovasc Res. 2021; Jun 4: cvab187).

[0130] A neutralizing or “blocking” antibody refers to an antibody whose binding to BMP9 either (i) interferes with, limits, and / or inhibits the interaction between BMP9 and a type I receptor or a type II receptor or both a type I receptor and a type II receptor, such as (ALK1, ALK2, ALK3, ALK6, BMPRII, ActRIIA, and ActRIIB); and / or (ii) results in the inhibition of at least one biological function of BMP9 binding. Assays for determining neutralization by the antibodies of the present disclosure are well known in the art.

[0131] A "biological function" or "biological activity" of BMP9 is meant to include binding of BMP9 to endothelial cells, induction of phospho-SMAD1 / 5 / 9 nuclear translocation in endothelial cells, and phospho-SMAD2 nuclear translocation in endothelial cells. A biological function or biological activity of BMP9 may, but need not, be mediated by an interaction between BMP9 and its receptor.

[0132] In some embodiments, the anti-BMP9 antibodies of the present disclosure include one or both of an antibody having the amino acid sequence of a heavy chain variable region set forth in SEQ ID NO: 77 and the amino acid sequence of a light chain variable region set forth in SEQ ID NO: 74 that i) competes for binding to human BMP9 and / or ii) binds to the same epitope as that antibody.

[0133] The anti-BMP9 antibodies of the present disclosure may include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chain (ScFv), variants thereof, fusion proteins comprising antibody fragments (e.g., domain antibodies), humanized antibodies, and any other modified structure of an immunoglobulin molecule comprising an antigen-binding site of the required specificity, including glycosylation variants of antibodies, amino acid variants of antibodies, and covalently modified antibodies. The antibodies may be of mouse, rat, human, or any other origin (including chimeric or humanized antibodies). In some embodiments, the anti-BMP9 antibody is a monoclonal antibody. In some embodiments, the anti-BMP9 antibody is a human or humanized antibody. In some embodiments, the anti-BMP9 antibody is a chimeric antibody. It is contemplated that any one or more of these aforementioned antibody types may be excluded in certain embodiments.

[0134] In some embodiments, antibodies having light chain variable region (VL) and heavy chain variable region (VH) sequences as seen in Table 3 / Table 4, or variants thereof, are disclosed. In Table 4, the underlined sequences are CDR sequences (Kabat definition) and the italicized sequences are CDR sequences (Chothia definition). In Table 1, KD shows the affinity for human BMP9 when measured using surface plasmon resonance at 37°C, unless otherwise indicated.

[0135] Tables 3 and 4 provide the VH and VL sequences for the mAbs of certain embodiments of the present disclosure.

[0136] Embodiments also provide the CDR portions of the antibodies against BMP9. Determination of the CDR regions is well within the skill of those in the art. In some embodiments, the CDR can be a combination of Kabat and Chothia CDRs (also referred to as "combined CDRs" or "extended CDRs"). It is understood that in another approach herein referred to as "conformation definition" of the CDR, the positions of the CDRs can be defined as residues that contribute enthalpically to antigen binding. See, for example, Makabe et al., 2008, J. Biol. Chem., 283:1156-1166. Generally, "conformation CDRs" include the residue positions within the Kabat CDRs and the Vernier zones that are constrained to maintain the appropriate loop structure for the antibody to bind to the specific antigen. Determination of the conformation CDRs is well within the skill of those in the art. In some embodiments, the CDR is a Kabat CDR. In other embodiments, the CDR is a Chothia CDR. In other embodiments, the CDR is an extended, AbM, conformation, and / or contact CDR. In other words, in embodiments having two or more CDRs, the CDRs can be any of Kabat, Chothia, extended, AbM, conformation, contact CDRs, or combinations thereof. It is contemplated that any one or more of these aforementioned CDRs may be excluded in certain embodiments.

[0137] Table 4 provides examples of CDR sequences of the anti-BMP9 antibodies provided herein. In some embodiments, the antibody comprises any one of the three CDRs of the heavy chain variable region shown in Table 4. In some embodiments, the antibody comprises any one of the three CDRs of the light chain variable region shown in Table 4. In some embodiments, the antibody comprises any one of the three CDRs of the heavy chain variable region shown in Table 4 and any one of the three CDRs of the light chain variable region shown in Table 4. In some embodiments, the antibody comprises three light chain CDRs and three heavy chain CDRs from Table 4.

[0138] In some embodiments, the antibody comprises i) a full-length heavy chain, with or without the C-terminal lysine of an anti-BMP9 antibody (e.g., Ab1076, Ab93, Ab352, Ab804, Ab101, Ab732, Ab100, and Ab89), and / or ii) one or both of the full-length light chains. The amino acid sequences of the full-length heavy and light chains for antibody BMP9 are shown in Table 4 below.

[0139] In certain embodiments, the antibodies described herein comprise an Fc domain. The Fc domain can be derived from IgA (e.g., IgA1 or IgA2), IgD, IgE, IgM, or IgG (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the anti-BMP9 antibody is an IgG antibody.

[0140] The embodiments encompass modifications to the variable regions, CDR regions, and / or heavy and light chain sequences shown in Tables 4 and 5. For example, the embodiments include antibodies comprising functionally equivalent variable regions and CDRs that do not significantly affect their properties, as well as variants with enhanced or decreased activity or affinity. For example, the amino acid sequence may be mutated to obtain an antibody with a desired binding affinity to BMP9. Modification of polypeptides is a routine practice in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, polypeptides with one or more deletions or additions of amino acids that do not significantly adversely alter the functional activity or mature (enhance) the affinity of the polypeptide for its ligand, or polypeptides that use chemical analogs. It is contemplated that any one or more of these aforementioned modifications may be omitted in certain embodiments.

[0141] Modifications or mutations may be made in the framework or constant regions to increase the half-life of the antibodies provided herein, see, for example, PCT Publication WO 00 / 09560. Mutations in the framework or constant regions may be made to alter the immunogenicity of the antibody, to provide sites for covalent or non-covalent attachment to another molecule, and / or to alter such properties as complement fixation, FcR binding, and antibody-dependent cell-mediated cytotoxicity. In some embodiments, no more than 1 to 5 (e.g., at least, at most, or exactly 1, 2, 3, 4, or 5) conservative amino acid substitutions are made in the framework or constant regions. In other embodiments, no more than 1 to 3 (e.g., at least, at most, or exactly 1, 2, or 3) conservative amino acid substitutions are made in the framework or constant regions. According to certain embodiments, a single antibody may have mutations in any one or more of the CDRs or framework regions of the variable domain, or the constant region.

[0142] In some embodiments, the antibody has an increased or decreased binding affinity for human Fc gamma receptors and is immunologically inert or partially inert, e.g., does not cause complement-mediated lysis, does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC), does not activate microglia, and / or has a modified constant region that has reduced activity (compared to an unmodified antibody) in one or more of the following: induction of complement-mediated lysis, stimulation of ADCC, and / or activation of microglia. It is contemplated that any one or more of these aforementioned properties due to the modified constant region may be excluded in certain embodiments. Different modifications of the constant region may be used to achieve an optimal level or combination of effector functions. See, e.g., Morgan et al., Immunology 86:319-324, 1995; Lund et al., J. Immunology 157:4963-9 157:4963-4969, 1996; Idusogie et al., J. Immunology 164:4178-4184, 2000; Tao et al., J. Immunology 143:2595-2601, 1989; and Jefferis et al., Immunological Reviews 163:59-76, 1998. In some embodiments, the constant region is modified as described in Eur. J. Immunol., 1999, 29:2613-2624; PCT Publication No. WO 99 / 058572 pamphlet.

[0143] Modifications also include glycosylated and non-glycosylated polypeptides, and polypeptides containing other post-translational modifications such as, for example, glycosylation with different sugars, acetylation, and phosphorylation. Antibodies are glycosylated at conserved positions in their constant regions (Jefferis and Lund, 1997, Chem. Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32). The oligosaccharide side chains of immunoglobulins affect the function of the protein (Boyd et al., 1996, Mol. Immunol. 32:1311-1318; Wittwe and Howard, 1990, Biochem. 29:4175-4180), affect the intramolecular interactions between moieties of the glycoprotein, which can affect the conformation and presented three-dimensional surface of the glycoprotein (Jefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech. 7:409-416). Oligosaccharides can also function to target a given glycoprotein to a particular molecule based on specific recognition structures. The glycosylation of antibodies has also been reported to affect antibody-dependent cell-mediated cytotoxicity (ADCC). In particular, antibodies produced by CHO cells using tetracycline-regulated expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glucosyltransferase that catalyzes the formation of bisecting GlcNAc, have been reported to have improved ADCC activity (Umana et al., 1999, Nature Biotech. 17:176-180).

[0144] In some embodiments, the disclosure provides anti-BMP9 antibodies containing variations of the variable regions, CDRs, or heavy and light chain sequences set forth in Tables 4 and 5, where such variant polypeptides share an amino acid sequence that is at least, up to, exactly, between any two of the following (inclusive or excluding the extremes), or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences disclosed in Tables 4 or 5, or any range therebetween. For example, in some embodiments, the present disclosure provides anti-BMP9 antibodies containing the variable region CDRs or heavy and light chain sequence variations shown in Tables 4 and 5, such variant polypeptides share at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical amino acid sequences to any of the amino acid sequences disclosed in Tables 4 or 5. These amounts are not meant to be limiting, and increments between the recited percentages are specifically contemplated as part of the disclosure. It is contemplated that any one or more of these aforementioned percentages may be excluded in embodiments.

[0145] Regions or fragments of the polypeptides of the present disclosure have at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, contiguous amino acid additions, and / or contiguous amino acid deletions relative to any of SEQ ID NOs: 1-77, 80-84, or 89. In some embodiments, the anti-BMP9 antibody, or antigen-binding fragment thereof, comprises or consists of an amino acid sequence that is at least or at most 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% (or within any range thereof) identical to any of SEQ ID NOs: 1-77, 80-84, or 89. It is contemplated that any one or more of these aforementioned percentages may be excluded in an embodiment.

[0146] Furthermore, in some embodiments, the region, or fragment, is at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263 of any of SEQ ID NOs: 1-77, 80-84, or 89.264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 positions (1 position is at the N-terminus of the sequence number), starting at least, at most, exactly,or between any two of the following (including or excluding both ends) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,It contains 500 or more consecutive amino acids (or any range thereof).

[0147] The anti-BMP9 antibody of the present disclosure, or an antigen-binding fragment thereof, may include at least, at most, exactly, or one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine, forty, forty-one, forty-two, forty-three, forty-four, forty-five, forty-six, forty-seven, forty-eight, forty-nine, or fifty or more (or any range thereof) variant amino acids or amino acid substitutions between any two of the following (including or excluding both ends). In some embodiments, the variant or amino acid substitution is at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、at positions 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, and / or 500 (where position 1 is at the N-terminus of the SEQ ID NO.), the anti-BMP9 antibody, or an antigen-binding fragment thereof, comprises at least, or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 600 or more consecutive amino acids, or any range thereof, and at least, at most, exactly, or between any two of the following (including or excluding both ends) 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical or may be homologous. It is contemplated that any one or more of these aforementioned percentages may be excluded in embodiments.,

[0148] In some embodiments, the present disclosure provides anti-BMP9 antibodies containing variations of the variable regions shown in Tables 4 and 5. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 27, SEQ ID NO: 39, SEQ ID NO: 48, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 71, or SEQ ID NO: 77, or a variant thereof comprising 1 to 4 amino acid substitutions with residues not within the CDRs. In some embodiments, the 1 to 4 amino acid substitutions (i.e., 1, 2, 3, or 4 amino acid substitutions) can be selected from the group consisting of K13Q, D73N, S74A, T77S, K83R, and T84A of SEQ ID NO: 27. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 33, SEQ ID NO: 44, SEQ ID NO: 51, SEQ ID NO: 59, SEQ ID NO: 68, or SEQ ID NO: 74, or a variant thereof comprising 1 to 4 (i.e., 1, 2, 3, or 4) amino acid substitutions with residues not within the CDRs. In some embodiments, the 1 to 4 amino acid substitutions can be selected from the group consisting of M43T, V76S, Q79R, G84A, and Y85D of SEQ ID NO: 22. In some embodiments, an anti-BMP9 antibody comprising VH and VL is provided herein, wherein the VH of the antibody has the amino acid sequence encoded by the nucleic acid sequence of the insert of a plasmid deposited by the ATCC and having the ATCC deposit number PTA-127292, and / or the VL of the antibody has the amino acid sequence encoded by the nucleic acid sequence of the insert of a plasmid deposited by the ATCC and having the ATCC deposit number PTA-127293.

[0149] The embodiments also encompass fusion proteins comprising one or more components of the antibodies disclosed herein. In some embodiments, fusion proteins can be made that comprise all or part of an anti-BMP9 antibody disclosed herein linked to another polypeptide. In another embodiment, only the variable domains of the anti-BMP9 antibody are linked to the polypeptide. In another embodiment, the VH domain of the anti-BMP9 antibody is linked to a first polypeptide, while the VL domain of the anti-BMP9 antibody is linked to a second polypeptide associated with the first polypeptide in such a way that the VL domains interact with each other to form an antigen-binding site. In another embodiment, the VH domain is separated from the VL domain by a linker such that the VH and VL domains can interact with each other. The VH-linker-VL antibody is then linked to the polypeptide of interest. In addition, fusion antibodies can be formed in which two (or more) single-chain antibodies are linked to each other. This is useful when one wishes to create a bivalent or multivalent antibody on a single-chain polypeptide or when one wishes to create a bispecific antibody.

[0150] Biological activity of the anti-BMP9 antibody In addition to binding to an epitope on BMP9, the antibodies of the present disclosure are capable of mediating a biological activity. That is, the present disclosure includes isolated antibodies that specifically bind to BMP9 and mediate at least one detectable activity selected from: (i) specifically binds to human BMP9; (ii) specifically binds to cynomolgus (cyno) BMP9; (iii) specifically binds to rat BMP9; (iv) reduces, inhibits, and / or neutralizes binding to type II receptors of BMP9 (BMPRII, ActRIIA, and / or ActRIIB) (e.g., Ab89, Ab93, Ab100, Ab101); (v)Reducing, inhibiting, and / or neutralizing one or more of the binding of BMP9 to type II receptors (BMPRII, ActRIIA, and / or ActRIIB), and weakly inhibiting the binding of BMP9 to type I receptor (ALK1) (e.g., Ab352, Ab804, and Ab1076); (vi)Reducing, inhibiting, and / or neutralizing one or more of the binding of BMP9 to endothelial cells; (vii)Reducing, inhibiting, and / or neutralizing one or more of SMAD1 / 5 / 9 phosphorylation and / or phospho-SMAD1 / 5 / 9 nuclear translocation in endothelial cells; (viii)Reducing, inhibiting, and / or neutralizing one or more of SMAD2 phosphorylation or phospho-SMAD2 nuclear translocation in endothelial cells; (ix)Reducing right ventricular systolic pressure; (x)Reducing right ventricular hypertrophy; (xi)Reducing pulmonary artery pressure (e.g., at least, up to, precisely, between any two of the following (including or excluding both ends), or about 1 mmHg, 2 mmHg, 3 mmHg, 4 mmHg, 5 mmHg, 6 mmHg, 7 mmHg, 8 mmHg, 9 mmHg, 10 mmHg, 11 mmHg, 12 mmHg, 13 mmHg, 14 mmHg, 15 mmHg, 16 mmHg, 17 mmHg, 18 mmHg, 19 mmHg, 20 mmHg, 21 mmHg, 22 mmHg, 23 mmHg, 24 mmHg, or 25 mmHg, or any range thereof) (e.g., at least 3 mmHg, at least 5 mmHg, at least 7 mmHg, at least 10 mmHg, at least 12 mmHg, at least 15 mmHg, at least 20 mmHg, or at least 25 mmHg); (xii)Reducing pulmonary vascular resistance; (xiii)Increasing cardiac output or cardiac index; (xiv)Increasing pulmonary capillary wedge pressure; (xv)Increasing left ventricular end-diastolic pressure; (xvi)Increasing the exercise capacity of the subject; (xvii) The 6-minute walk distance of the subject with maximum effort (e.g., at least, at most, exactly, between any two of the following (including or excluding both ends), or about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 meters, or any range thereof) only (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 meters or more); and (xviii) Reduce the Borg Dyspnea Index (BDI) of the subject (e.g., Clini Enrico et al. Multidiscip. Respir. Med. 2010;5(3):202 - 210). It is contemplated that any one or more of these aforementioned activities may be excluded in certain embodiments.

[0151] Assays for measuring the functional activities described above are well known in the art. For example, binding activity can be measured using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), Western blot assay, BIACORE™ assay, and / or by surface plasmon resonance. Each of these assays generally detects the presence of a protein-antibody complex of a specific target by utilizing a labeled reagent (e.g., an antibody) specific to the target complex.

[0152] The ability to block or compete with a reference molecule (e.g., a receptor or an antibody) may be determined, for example, by a competitive binding assay. By using a competitive binding assay, the antibody under test can be examined for its ability to inhibit specific binding to a common antigen such as the BMP9 polypeptide of the reference molecule. The test antibody competes with the reference molecule for specific binding to the antigen if an excess of the test antibody substantially inhibits the binding of the reference molecule. Substantially inhibit means that the test antibody usually reduces the specific binding of the reference molecule by at least, at most, exactly, any two of the following (including or excluding both ends), or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, or any range between them (e.g., at least 10%, 25%, 50%, 75%, or 90%). There are many known competitive binding assays that can be used to evaluate the competition between an antibody and a reference antibody for binding to a specific protein, in this case, BMP9. These include, for example, solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assay (see Stahli et al., Methods in Enzymology 9:242-253, 1983); solid-phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619, 1986); solid-phase direct labeled assay, solid-phase direct labeled sandwich assay (Harlow & Lane, supra); solid-phase direct labeled RIA using 1-125 label (see Morel et al., Molec. Immunol. 25:7-15, 1988); solid-phase direct biotin-avidin EIA (Cheung et al., Virology 176:546-552, 1990); and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol. 32:77-82, 1990).

[0153] The BMP9-binding antibodies and antigen-binding fragments thereof of the present disclosure can be further tested for reactivity with BMP9 polypeptides or antigen fragments by Western blotting. Briefly, purified BMP9 polypeptides or fusion proteins, and / or cell extracts from cells expressing BMP9 can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to a nitrocellulose membrane, blocked with 10% fetal bovine serum, and probed with the monoclonal antibody to be tested. Human IgG binding is detected using anti-human IgG alkaline phosphatase and developed using a BCIP / NBT substrate tablet (Sigma Chem. Co., St. Louis, Mo.).

[0154] Examples of functional assays are also described in the Examples section below. It is contemplated that one or more of any of these aforementioned functional assays may be excluded in certain embodiments.

[0155] In some embodiments, the antibody that mediates at least one of the functional activities described herein is selected from the group consisting of Ab89, Ab93, Ab100, Ab101, Ab352, Ab732, Ab804, and Ab1076. In some embodiments, the antibody is Ab93, Ab352, Ab804, or Ab1076. In some embodiments, the antibody is Ab1076. In some embodiments, the antibody is Ab101. In some embodiments, the antibody is Ab732. In some embodiments, the antibody is Ab101 or Ab732. It is contemplated that one or more of any of these aforementioned antibodies may be excluded in certain embodiments.

[0156] In some embodiments, the antibody binds to the human BMP9 antibody with an affinity of about 15 pM to about 80 pM at 37°C when measured by surface plasmon resonance. In some embodiments, the antibody binds to human BMP9 with an affinity of about 15 pM to about 150 pM at 37°C when measured by surface plasmon resonance. In some embodiments, the antibody, when measured by surface plasmon resonance, at 37°C, has an affinity of at least, at most, exactly, between any two of the following (including or excluding both ends), or about 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, 100 pM, 105 pM, 110 pM, 115 pM, 120 pM, 125 pM, 130 pM, 135 pM, 140 pM, 145 pM, or 150 pM, or any range thereof (e.g., about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 55 pM, about 60 pM, about 65 pM, about 70 pM, about 75 pM, about 80 pM, about 85 pM, about 90 pM, about 95 pM, about 100 pM, about 105 pM, about 110 pM, about 115 pM, about 120 pM, about 125 pM, about 130 pM, about 135 pM, about 140 pM, or about 150 pM) and binds to human BMP9. In some embodiments, the antibody binds to human BMP9 with an affinity of about 87 pM at 37°C when measured by surface plasmon resonance. In some embodiments, the antibody binds to human BMP9 with an affinity of about 87.41 pM or about 90 pM at 37°C when measured by surface plasmon resonance. In some embodiments, the antibody is Ab1076.

[0157] Polynucleotide encoding anti-BMP9 antibody, and method of manufacture The present disclosure also provides a polynucleotide encoding any of the antibodies of the present disclosure, including the antibody portions and modified antibodies described herein. Embodiments also provide a method of making any of the antibodies and polynucleotides described herein. The polynucleotide is made and the protein is expressed therefrom by procedures known in the art.

[0158] If desired, the anti-BMP9 antibody of interest can be sequenced, and then the polynucleotide sequence can be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in a vector in a host cell, and then the host cell can be expanded and frozen for future use. Production of recombinant monoclonal antibodies in cell culture can be carried out by known means in the art through cloning of antibody genes from B cells. See, for example, Tiller et al., 2008, J. Immunol. Methods 329, 112; U.S. Patent No. 7,314,622.

[0159] In some embodiments, provided herein are polynucleotides comprising a sequence encoding one or both of the heavy chain and / or light chain variable regions of the anti-BMP9 antibodies provided herein. The sequence encoding the antibody of interest can be maintained in a vector in a host cell, and then the host cell can be expanded and frozen for future use. Vectors (including expression vectors) and host cells are further described herein.

[0160] In some embodiments, the disclosure provides polynucleotides encoding the amino acid sequence of any of the following anti-BMP9 antibodies: Ab89, Ab93, Ab100, Ab101, Ab352, Ab732, Ab804, and Ab1076. In one embodiment, the disclosure provides a polynucleotide encoding the amino acid sequence of Ab93, Ab1076, Ab101, or Ab732 of the anti-BMP9 antibody. In one embodiment, the disclosure provides a polynucleotide encoding the amino acid sequence of Ab1076 of the anti-BMP9 antibody.

[0161] In some embodiments, the disclosure provides polynucleotides encoding the heavy chain polypeptides of one or more anti-BMP9 antibodies comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 23, 35, 44, 52, 60, 69, and 75.

[0162] In some embodiments, the present disclosure provides a polynucleotide encoding a light chain polypeptide of one or more anti-BMP9 antibodies comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 18, 29, 40, 49, 56, 64, and 72.

[0163] In some embodiments, the present disclosure provides a polynucleotide comprising SEQ ID NO: 87. In some embodiments, the present disclosure provides a polynucleotide comprising SEQ ID NO: 88.

[0164] In some embodiments, the present disclosure provides a polynucleotide encoding a VH polypeptide of one or more anti-BMP9 antibodies comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 27, 39, 48, 55, 63, 71, and 77.

[0165] In some embodiments, the present disclosure provides a polynucleotide encoding a VL polypeptide of one or more anti-BMP9 antibodies comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 33, 43, 51, 59, 68, and 74.

[0166] In some embodiments, the present disclosure provides a polynucleotide comprising SEQ ID NO: 85. In some embodiments, the present disclosure provides a polynucleotide comprising SEQ ID NO: 86.

[0167] The present disclosure provides a polypeptide comprising an amino acid sequence encoded by a DNA insert of a plasmid deposited with the ATCC and having the deposit number PTA-127292, which encodes the VH domain of antibody Ab1076. The present disclosure further provides a polypeptide comprising an amino acid sequence encoded by an insert of a plasmid deposited with the ATCC and having the deposit number PTA-127293, which encodes the VL domain of antibody Ab1076.

[0168] As a result of the degeneracy of the genetic code, those skilled in the art will appreciate that there are many nucleotide sequences encoding the polypeptides described herein. Some of these polynucleotides possess minimal homology to the nucleotide sequences of any native gene. Nevertheless, different polypeptides are specifically contemplated by the present disclosure due to differences in codon usage frequency. Furthermore, alleles of the genes containing the polynucleotides disclosed herein are within the scope of the present disclosure. Alleles are endogenous genes that vary as a result of one or more mutations such as nucleotide deletions, additions, and / or substitutions. The resulting mRNA and proteins may have an altered structure or function, although this is not necessarily required. Alleles can be identified using standard techniques such as hybridization, amplification, and / or database sequence comparison.

[0169] In one embodiment, the VH and / or VL domains, and / or the full-length HC and / or LC are encoded by separate polynucleotides. Alternatively, both the VH and / or VL, and / or the HC and / or LC are encoded by a single polynucleotide.

[0170] Polynucleotides complementary to any such sequences are also encompassed by the present disclosure. The polynucleotide may be single-stranded (coding or antisense) or double-stranded, and may be a DNA (genomic, cDNA, or synthetic) or RNA molecule. The RNA molecule may contain introns, including HnRNA molecules that correspond 1:1 to the DNA molecule and mRNA molecules that do not contain introns. Additional coding or non-coding sequences are not necessarily required, but may be present within the polynucleotides of the present disclosure, and the polynucleotide may optionally be linked to other molecules or support materials.

[0171] The polynucleotides of the present disclosure can be obtained using chemical synthesis, recombinant methods, or PCR. Chemical synthesis methods of polynucleotides are well known in the art and need not be described in detail herein. One of ordinary skill in the art can produce the desired DNA sequence using the sequences provided herein and a commercially available DNA synthesizer.

[0172] For the preparation of polynucleotides using recombinant methods, a polynucleotide containing the desired sequence can be inserted into a suitable vector, and then the vector can be introduced into a suitable host cell for replication and amplification, as further discussed herein. The polynucleotide can be inserted into the host cell by any means known in the art. The cell is transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, F - mating, or electroporation. Once introduced, the exogenous polynucleotide can be maintained intracellularly as a non - integrating vector (such as a plasmid) or can be integrated into the host cell genome.

[0173] Suitable cloning vectors may be constructed according to standard techniques or selected from among the numerous cloning vectors available in the art. The cloning vector selected may vary depending on the host cell intended for use, and useful cloning vectors will generally have one or more characteristics such as i) the ability to self-replicate, ii) a single target for a particular restriction endonuclease, or iii) the ability to carry a gene for a marker that can be used in the selection of clones containing the vector. Suitable examples include plasmids and bacteriophages such as pUC18, pUC19, Bluescript (e.g., pBS SK+ ) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as, for example, pSA3 and pAT28. These vectors and many other cloning vectors are available from commercial sources such as, for example, BioRad, Strategene, and Invitrogen.

[0174] An expression vector is further provided. An expression vector is generally a replicable polynucleotide construct containing a polypeptide according to the present disclosure. It is suggested that the expression vector must be replicable in a host cell, either as an episome or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, adenoviruses, adeno-associated viruses, viral vectors including retroviruses, cosmids, and the expression vectors disclosed in PCT Publication WO 87 / 04462. Vector components generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; and suitable transcriptional control elements (such as promoters, enhancers, and terminators). For expression (i.e., translation), usually one or more translational control elements are also required, such as a ribosome binding site, a translation initiation site, and a stop codon. It is contemplated that any one or more of these aforementioned vector components or elements may be excluded in certain embodiments.

[0175] A vector containing a target polynucleotide can be introduced into a host cell by any of a number of suitable means, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (e.g., if the vector is an infectious entity such as vaccinia virus). The choice of means for introducing the vector or polynucleotide often depends on the characteristics of the host cell.

[0176] Embodiments also provide host cells comprising any of the polynucleotides described herein. Any host cell having the ability to overexpress heterologous DNA can be used for the purpose of isolating a gene encoding a target antibody, polypeptide, or protein. Non-limiting examples of mammalian host cells include, but are not limited to, COS cells, HeLa cells, and CHO cells. See also PCT Publication No. WO 87 / 04462 pamphlet. Suitable non-mammalian host cells include prokaryotes (such as E. coli or B. subtilis) and yeast (such as S. cerevisae, S. pombe; or K. lactis).

[0177] Additionally, commercially available cell lines and non-commercially available cell lines that express a polypeptide or protein may be utilized in accordance with the present disclosure. One of ordinary skill in the art will understand that different cell lines may have different nutritional requirements or may require different culture conditions for optimal growth and expression of the polypeptide or protein, and can modify the conditions as needed.

[0178] In some embodiments, the heavy and light chain antibodies described herein are produced using a single peptide (e.g., SEQ ID NO: 89). As used herein, "leader peptide" or "leader sequence" or "leader signal sequence" or "signal sequence" or "signal peptide" (used interchangeably herein) means any nucleic acid sequence, or the amino acid sequence encoded thereby, which can be present at the 5' end of a nucleic acid molecule and / or at or near the N-terminus of a polypeptide, and which, when present, can mediate the transport of the polypeptide to cellular organelles including, but not limited to, the secretion of the polypeptide from the cell. Such leader sequences include, but are not limited to, the nucleic acid sequence encoding SEQ ID NO: 89. Embodiments include this leader signal and any leader signal (nucleic acid sequence and amino acid sequence) known or identified in the art that can effect the transport of a polypeptide to a desired cellular organelle, e.g., the endoplasmic reticulum, and / or the secretion of the polypeptide from the cell. In preferred embodiments, the signal peptide is removed from the mature polypeptide and / or is not present in the mature polypeptide.

[0179] Pharmaceutical composition In another embodiment, the disclosure includes a pharmaceutical composition. "Pharmaceutical composition" refers to a mixture of an antibody and one or more excipients.

[0180] The pharmaceutical compositions of the disclosure can be in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as, for example, liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, and lyophilized powders. The form depends on the intended method of administration and therapeutic use. It is contemplated that one or more of any of the foregoing forms may be excluded in certain embodiments.

[0181] Other excipients and administration methods known in the pharmaceutical field may be used. The pharmaceutical compositions of the present disclosure can be prepared by any of the well-known techniques of pharmacy, such as effective formulation and administration procedures. The above considerations regarding effective formulation and administration procedures are well-known in the art and are described in standard texts. The formulation of drugs is discussed, for example, in Hoover, John E., REMINGTON’S PHARMACEUTICAL SCIENCES, Mack Publishing Co., Easton, Pennsylvania, 1975; Liberman et al., Eds., PHARMACEUTICAL DOSAGE FORMS, Marcel Decker, New York, N.Y., 1980; and Kibbe et al., Eds., HANDBOOK OF PHARMACEUTICAL EXCIPIENTS (3rd Ed.), American Pharmaceutical Association, Washington, 1999.

[0182] Excipients that are acceptable are non-toxic to the recipient at the dosages and concentrations employed, buffers such as phosphoric acid, citric acid, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzetonium chloride; de-nol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, and / or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, and / or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, and / or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). It is contemplated that any one or more of these aforementioned excipients may be excluded in certain embodiments.

[0183] In some embodiments, the pharmaceutical composition comprises at least, at most, exactly, between any two of the following (including or excluding both ends), or about 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, 105 mg / mL, 110 mg / mL, 115 mg / mL, 120 mg / mL, 125 mg / mL, 130 mg / mL, 135 mg / mL, 140 mg / mL, 145 mg / mL, or 150 mg / mL of an antibody (or antigen-binding fragment thereof), or any range thereof (e.g., about 25 mg / mL, 50 mg / mL, 75 mg / mL, 100 mg / mL, 125 mg / mL, 150 mg / mL of an antibody). In some embodiments, the pharmaceutical composition comprises about 100 mg / mL of an antibody (or antigen-binding fragment thereof). In some embodiments, the pharmaceutical composition suitable for SC administration and / or IV administration comprises about 100 mg / mL of an anti-BMP9 antibody (or antigen-binding fragment thereof) (e.g., Ab1076).

[0184] In some embodiments, the anti-BMP9 antibody, or antigen-binding fragment thereof, is administered as an intravenous formulation or subcutaneous formulation as a sterile aqueous solution comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 75. In some embodiments, the anti-BMP9 antibody, or antigen-binding fragment thereof, is administered as an intravenous formulation or subcutaneous formulation as a sterile aqueous solution comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-BMP9 antibody, or antigen-binding fragment thereof, is administered as an intravenous formulation or subcutaneous formulation as a sterile aqueous solution comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 75 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 72.

[0185] In some embodiments, the anti-BMP9 antibody (or antigen-binding fragment thereof) (e.g., Ab1076) is administered as an intravenous or subcutaneous formulation that is a sterile aqueous solution containing at least, up to, exactly, between any two of the following (including or excluding both ends), or about 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, 105 mg / mL, 110 mg / mL, 115 mg / mL, 120 mg / mL, 125 mg / mL, 130 mg / mL, 135 mg / mL, 140 mg / mL, 145 mg / mL, or 150 mg / mL of the antibody, or any range thereof (e.g., about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 75 mg / mL, about 100 mg / mL, about 125 mg / mL, or about 150 mg / mL of the antibody).

[0186] In some embodiments, the intravenous or subcutaneous formulation is a sterile aqueous solution containing sodium acetate, polysorbate 80, and sodium chloride at a pH in the range of about 5 to 6. In some embodiments, the intravenous or subcutaneous formulation is a sterile aqueous solution containing at least, at most, exactly, between any two of the following (including or excluding both ends), or about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, or 40 mM of sodium acetate, or a range of sodium acetate between any of them. In some embodiments, the intravenous or subcutaneous formulation is a sterile aqueous solution containing 20 mM of sodium acetate. In some embodiments, the intravenous or subcutaneous formulation is a sterile aqueous solution containing at least, at most, exactly, between any two of the following (including or excluding both ends), or about 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, or 0.4 mg / mL of polysorbate 80, or a range of polysorbate 80 between any of them. In some embodiments, the intravenous or subcutaneous formulation is a sterile aqueous solution containing 0.2 mg / mL of polysorbate 80. In some embodiments, the intravenous or subcutaneous formulation is a sterile aqueous solution containing at least, at most, exactly, between any two of the following (including or excluding both ends), or about 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, or 200 mM of sodium chloride, or a range of sodium chloride between any of them. In some embodiments, the intravenous or subcutaneous formulation is a sterile aqueous solution containing 140 mM of sodium chloride. In some embodiments, the sodium chloride is at least, at most, exactly, or about pH 5 (e.g., pH 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6, or a range of pH between any of them).In some embodiments, the intravenous formulation is a sterile aqueous solution containing 5 or 10 mg / mL of an antibody or antigen-binding fragment thereof, together with 20 mM sodium acetate, 0.2 mg / mL polysorbate 80, and 140 mM sodium chloride (at pH 5.5), with a nominal fill volume of 1.0 mL. It is contemplated that one or more of any of the foregoing components may be excluded in certain embodiments.

[0187] Furthermore, a solution containing an antibody, or antigen-binding fragment thereof, may include, among many other compounds, glutamic acid, histidine, mannitol, sucrose, trehalose, glycine, poly(ethylene) glycol, EDTA, methionine, polysorbate 80, or any combination thereof, and many other compounds known in the relevant art. It is contemplated that one or more of any of the foregoing components may be excluded in certain embodiments.

[0188] In some embodiments, the pharmaceutical composition comprises at least, at most, exactly, between any two of the following (including or excluding both ends), or about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, or 40 mM of histidine, or histidine in any range thereof. In some embodiments, the pharmaceutical composition comprises 20 mM of histidine. In some embodiments, the pharmaceutical composition comprises 10 mM of histidine. In some embodiments, the pharmaceutical composition comprises at least, at most, exactly, between any two of the following (including or excluding both ends), or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of sucrose, or sucrose in any range thereof. In some embodiments, the pharmaceutical composition comprises 8.5% of sucrose. In some embodiments, the pharmaceutical composition comprises 5% of sucrose. In some embodiments, the pharmaceutical composition comprises at least, at most, exactly, between any two of the following (including or excluding both ends), or about 0.001%, 0.005%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04% of polysorbate 80, or polysorbate 80 in any range thereof. In some embodiments, the pharmaceutical composition comprises 0.02% of polysorbate 80. In some embodiments, the pharmaceutical composition comprises 0.01% of polysorbate 80. In some aspects, the polysorbate 80 is at least, at most, exactly, or about pH 5.8 (e.g., pH 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, or 6.5, or pH in any range thereof). In some embodiments, the pharmaceutical composition comprises at least, at most, exactly, between any two of the following (including or excluding both ends), or about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, or 0.01% of EDTA, or EDTA in any range thereof.In some embodiments, the pharmaceutical composition comprises 0.005% EDTA. In some aspects, the EDTA is at least, at most, exactly, or about pH 5.8 (e.g., pH 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, or 6.5, or a pH in the range of any of these). In one embodiment, the pharmaceutical composition of the present disclosure comprises the following components: 50 mg / mL or 100 mg / mL of the anti-BMP9 antibody of the present disclosure or an antigen-binding fragment thereof (e.g., Ab1076), 20 mM histidine, 8.5% sucrose, and 0.02% polysorbate 80, 0.005% EDTA (at pH 5.8). In one embodiment, the pharmaceutical composition comprises the following components: 100 mg / mL of the anti-BMP9 antibody of the present disclosure or an antigen-binding fragment thereof (e.g., Ab1076) or antigen-binding fragment, 10 mM histidine, 5% sucrose, and 0.01% polysorbate 80 (at pH 5.8). It is contemplated that any one or more of these aforementioned components may be excluded in certain embodiments.

[0189] In some embodiments, the pharmaceutical composition comprises at least, at most, exactly, between any two of the following (including or excluding both ends), or about 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.6 mg / mL, 1.8 mg / mL, or 2 mg / mL of L-histidine, or L-histidine in any range thereof. In some embodiments, the pharmaceutical composition comprises 1.12 mg / mL of L-histidine. In some embodiments, the pharmaceutical composition comprises at least, at most, exactly, between any two of the following (including or excluding both ends), or about 1.5 mg / mL, 1.7 mg / mL, 1.9 mg / mL, 2.1 mg / mL, 2.3 mg / mL, 2.5 mg / mL, 2.7 mg / mL, 2.9 mg / mL, 3.1 mg / mL, 3.3 mg / mL, or 3.5 mg / mL of L-histidine hydrochloride monohydrate, or L-histidine hydrochloride monohydrate in any range thereof. In some embodiments, the pharmaceutical composition comprises 2.67 mg / mL of L-histidine hydrochloride monohydrate. In some embodiments, the pharmaceutical composition comprises at least, at most, exactly, between any two of the following (including or excluding both ends), or about 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, 105 mg / mL, 110 mg / mL, 115 mg / mL, 120 mg / mL, 125 mg / mL, 130 mg / mL, 135 mg / mL, 140 mg / mL, 145 mg / mL, or 150 mg / mL of sucrose, or sucrose in any range thereof. In some embodiments, the pharmaceutical composition comprises 85 mg / mL of sucrose. In some embodiments, the pharmaceutical composition comprises at least, at most, exactly, between any two of the following (including or excluding both ends), or about 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, or 0.1 mg / mL of EDTA, or EDTA in any range thereof.In some embodiments, the pharmaceutical composition comprises 0.05 mg / mL of EDTA. In some aspects, the EDTA is at least, at most, exactly, or about pH 5.8 (e.g., pH 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, or 6.5, or a pH in the range of any of these). In some embodiments, the pharmaceutical composition comprises at least, at most, exactly, between any two of the following (including or excluding both ends), or about 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, or 0.4 mg / mL of polysorbate 80, or polysorbate 80 in the range of any of these. In some embodiments, the pharmaceutical composition comprises 0.2 mg / mL of polysorbate 80. In some aspects, the polysorbate 80 is at least, at most, exactly, or about pH 5.8 (e.g., pH 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, or 6.5, or a pH in the range of any of these). In some embodiments, the pharmaceutical composition comprises an anti-BMP9 antibody, or an antigen-binding fragment thereof (e.g., Ab1076), at a concentration of 105 mg / mL (or about 99.6 mg / mL to about 118.2 mg / mL) or 100 mg / mL, 1.12 mg / mL of L-histidine, 2.67 mg / mL of L-histidine hydrochloride monohydrate, 85 mg / mL of sucrose, 0.05 mg / mL of disodium edetate dihydrate, and 0.2 mg / mL of polysorbate 80 (at pH 5.8) in a nominal fill volume of 1.0 mL. Such a pharmaceutical composition is suitable for SC or IV administration. It is contemplated that one or more of any of the foregoing components may be excluded in certain embodiments.

[0190] Treatment methods, diagnostic methods, and other methods The antibodies and antibody conjugates of the present disclosure are useful in a variety of applications including, but not limited to, methods of therapeutic treatment and / or diagnostic treatment.

[0191] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein can inhibit, antagonize, and / or modulate the activity of BMP9 and its receptors and can be useful in the treatment, prevention, suppression, and / or amelioration of diseases, disorders, or conditions mediated by increased BMP9 activity, including, but not limited to, hypertension (e.g., pulmonary arterial hypertension), anemia, liver diseases (e.g., liver fibrosis, cirrhosis, portal hypertension, end-stage liver disease such as varices, jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, spontaneous bacterial peritonitis, non-alcoholic fatty liver disease, portopulmonary hypertension, and hepatopulmonary syndrome), neuroinflammatory or neurodegenerative diseases such as multiple sclerosis, fibrotic disorders, and / or heart failure. In some embodiments, the disease, disorder, or condition is selected from the group consisting of hereditary hemorrhagic telangiectasia syndrome; cardiovascular malformations; cardiac structural malformations; progressive osseous heteroplasia; juvenile familial polyposis syndrome; parathyroid diseases; cancer (e.g., breast cancer, prostate cancer, renal cell carcinoma, bone metastatic cancer, lung metastatic cancer, osteosarcoma, and multiple myeloma); vascular calcification; vasculitis; atherosclerosis; acquired or congenital hypercholesterolemia or hyperlipoproteinemia; diseases, disorders, or syndromes associated with defects in lipid absorption or metabolism; diseases, disorders, or syndromes caused by hyperlipidemia; valvular calcification; renal osteodystrophy; inflammatory disorders (e.g., ankylosing spondylitis); and infectious diseases caused by viruses, bacteria, fungi, mycobacteria, and parasites.

[0192] In one aspect, the present disclosure provides a method for treating hypertension (e.g., pulmonary arterial hypertension). In some embodiments, a method of treating hypertension (e.g., pulmonary arterial hypertension) in a subject comprises administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of either a BMP9 antibody or an antigen-binding fragment thereof described herein. In some embodiments, a method of reducing or inhibiting BMP9 activity in a subject comprises administering to a subject in need thereof a composition comprising an effective amount of an antibody or an antigen-binding fragment thereof provided herein.

[0193] In another aspect, the present disclosure further provides an antibody, an antigen-binding fragment thereof, or a pharmaceutical composition thereof described herein for use in the described method of treating hypertension (e.g., pulmonary arterial hypertension). The present disclosure also provides the use of an antibody, an antigen-binding fragment thereof described herein in the manufacture of a medicament for treating hypertension (e.g., pulmonary arterial hypertension).

[0194] In another aspect, without limitation, diseases, disorders, or conditions mediated by increased and / or dysregulated BMP9 activity include, but are not limited to, hypertension (e.g., pulmonary arterial hypertension), anemia, liver diseases (e.g., liver fibrosis, cirrhosis, portal hypertension, end-stage liver disease, such as varices, jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, spontaneous bacterial peritonitis, non-alcoholic fatty liver disease, portopulmonary hypertension, and hepatopulmonary syndrome), neuroinflammatory or neurodegenerative diseases, such as multiple sclerosis, fibrotic disorders, and / or heart failure. One or more methods are provided for detecting, prognosticating, diagnosing, and / or monitoring diseases, disorders, or conditions mediated by increased and / or dysregulated BMP9 activity. In some embodiments, the methods include determining, measuring, detecting, or assaying the level of BMP9 in a sample obtained from a subject using a BMP9 antibody or antigen-binding fragment thereof described herein. For example, the anti-BMP9 or antigen-binding fragment thereof described herein can be labeled with a detectable moiety such as a contrast agent, enzyme-substrate label, or other compound or element that can be detected due to their specific functional properties and / or chemical properties, the use of which enables the detection of the antibody and / or enables further quantification as needed. Examples of detectable labels include, but are not limited to, radioisotopes, fluorescers, semiconductor nanocrystals, chemiluminescers, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, metal sols, ligands (e.g., biotin, streptavidin or hapten). Specific examples of labels include, but are not limited to, horseradish peroxidase (HRP), fluorescein, FITC, rhodamine, dansyl, umbelliferone, dimethylacridinium ester (DMAE), Texas red, luminol, NADPH, and α- or β-galactosidase. Different diagnoses and / or prognoses can be made for different subjects based on the level of BMP9 determined, measured, detected, or assayed. The antibodies or antigen-binding fragments thereof described herein can also be used in in vivo diagnostic assays, such as in vivo imaging (e.g., PET or SPECT), or as a staining reagent.

[0195] In another aspect, a method of monitoring the effectiveness of a therapy (e.g., a BMP9 antibody or an antigen-binding fragment thereof described herein) in a subject having a disease, disorder, or condition is provided. The disease, disorder, or condition may be mediated by an increase and / or dysregulation of BMP9 activity, including but not limited to hypertension (e.g., pulmonary arterial hypertension), anemia, liver diseases (e.g., liver fibrosis, cirrhosis, portal hypertension, end-stage liver disease such as varices, jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, spontaneous bacterial peritonitis, non-alcoholic fatty liver disease, portopulmonary hypertension, and hepatopulmonary syndrome), neuroinflammatory or neurodegenerative diseases such as multiple sclerosis, fibrotic disorders, and / or heart failure. In a specific aspect, the disease, disorder, or condition is pulmonary arterial hypertension. In some aspects, the method includes administering a therapy (e.g., a BMP9 antibody or an antigen-binding fragment thereof described herein) to a subject in need thereof.

[0196] In some embodiments, the method further includes determining, measuring, detecting, or assaying the level of expression or activity of one or more biomarkers in a biological sample obtained from a subject. In some embodiments, the one or more biomarkers include CXCL12, IGFBP4, INHBA, MALL, FRZB, CPE, CCL2, PDGFBB, endothelin-1, or combinations thereof. The level of a biomarker can be determined, measured, detected, or assayed in a biological sample obtained from a subject by measuring the mRNA and / or protein level. The protein level can be determined, measured, detected, or assayed, for example, using an antibody that specifically binds to the biomarker or an antigen-binding fragment thereof. For example, an antibody to a biomarker, or an antigen-binding fragment thereof, can be labeled with a detectable moiety such as a contrast agent, an enzyme-substrate label, or other compound or element that can be detected due to its specific functional and / or chemical properties, and by its use, the antibody can be detected, and / or, if desired, further quantified. Examples of detectable labels include, but are not limited to, radioisotopes, fluorescers, semiconductor nanocrystals, chemiluminescers, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, metal sols, ligands (e.g., biotin, streptavidin or hapten), and the like. Specific examples of labels include, but are not limited to, horseradish peroxidase (HRP), fluorescein, FITC, rhodamine, dansyl, umbelliferone, dimethylacridinium ester (DMAE), Texas red, luminol, NADPH, and α- or β-galactosidase.

[0197] In some embodiments, the method further comprises comparing the level of one or more biomarkers measured in a biological sample of a subject to the level of one or more biomarkers measured in a biological sample obtained from the subject prior to administration of a therapy (e.g., a BMP9 antibody or antigen-binding fragment thereof described herein). In some embodiments, the method further comprises determining the efficacy of a therapy (e.g., a BMP9 antibody or antigen-binding fragment thereof described herein) based on the level at which one or more biomarkers are determined, measured, detected, or assayed. Different treatments may be prescribed or recommended for different subjects and / or a treatment may be modified for a subject based on the profile of the expression or activity level of a biomarker.

[0198] For example, in some embodiments, a biomarker being measured, detected, or assayed is determined to be effective for a therapy (e.g., a BMP9 antibody or an antigen-binding fragment thereof described herein) if: i) it is not measured, detected, or assayed in a biological sample from a subject; ii) there is no significant difference from a control, where the control represents the level of the biomarker in a biological sample from a subject without a disease, disorder, or condition (e.g., PAH); iii) there are few controls, where the control represents the level of the biomarker in a biological sample from a subject with a disease, disorder, or condition (e.g., PAH); or iv) it is decreased as compared to the level of the biomarker before treatment of the subject with a therapy (e.g., a BMP9 antibody or an antigen-binding fragment thereof described herein). In some embodiments, a biomarker is determined to be ineffective for a therapy (e.g., a BMP9 antibody or an antigen-binding fragment thereof described herein) if: i) it is measured, detected, or assayed in a biological sample from a subject; ii) it is increased as compared to a control, where the control represents the level of the biomarker in a biological sample from a subject without a disease, disorder, or condition (e.g., PAH); iii) there is no significant difference from or it exceeds the control, where the control represents the level of the biomarker in a biological sample from a subject with a disease, disorder, or condition (e.g., PAH); or iv) there is no significant difference from or it is increased as compared to the level of the biomarker before treatment of the subject with a therapy (e.g., a BMP9 antibody or an antigen-binding fragment thereof described herein).

[0199] In some embodiments, the method further comprises measuring, detecting, or assaying the level of a biomarker in a biological sample from a subject obtained prior to treatment. In some embodiments, the method further comprises measuring, detecting, or assaying the level of a biomarker in a biological sample from a subject obtained after one or more treatments with a therapy (e.g., a BMP9 antibody or an antigen-binding fragment thereof described herein). For example, the method further comprises determining, measuring, detecting, or assaying biomarker levels after 1, 2, 3, 4, 5, 6, or more administrations of a therapy (e.g., a BMP9 antibody or an antigen-binding fragment thereof described herein). Therapeutic efficacy can be determined based on the measured, detected, or assayed levels of one or more biomarkers.

[0200] In some embodiments, the determined, measured, detected, or assayed levels of BMP9 and / or a biomarker are normalized. In some embodiments, the levels of BMP9 and / or a biomarker are compared to a control. In some embodiments, the measured, detected, or assayed levels of BMP9 and / or a biomarker are determined to be above the control. In some embodiments, the measured, detected, or assayed levels of BMP9 and / or a biomarker are determined to be below the control. In some embodiments, a subject is determined to have, or have a measured, detected, or assayed level of BMP9 and / or a biomarker in a sample that is above the level of BMP9 and / or a biomarker in a control sample. In some embodiments, a subject is determined to have, or have a measured, detected, or assayed level of BMP9 and / or a biomarker in a sample that is below the level of BMP9 and / or a biomarker in a control sample. In some embodiments, a subject is determined to have, or have a measured, detected, or assayed level of BMP9 and / or a biomarker in a sample that is not significantly different from the level of BMP9 and / or a biomarker in a control sample. The control can include levels of BMP9 and / or a biomarker representative of the levels of BMP9 and / or a biomarker in samples from subjects with or without a disease, disorder, or condition (e.g., PAH). In some cases, the control may include levels of BMP9 and / or a biomarker representative of the levels of BMP9 and / or a biomarker in samples from subjects with a disease, disorder, or condition (e.g., PAH). In some embodiments, the control may include levels of BMP9 and / or a biomarker representative of the levels of BMP9 and / or a biomarker in samples from subjects without a disease, disorder, or condition (e.g., PAH).

[0201] The subject sample for use in the methods described herein can be obtained using any method known in the art that can provide a sample suitable for the analytical methods described herein. Methods for obtaining a sample from a subject provided herein can include, for example, obtaining a tissue, cell, or biological material by scraping, swabbing, performing a biopsy, or collecting in another manner. Samples can include, but are not limited to, a subject's tissue, cells, or biological material from or derived from cells. General methods for obtaining a biological sample are also known in the art. For example, publications such as Ramzy, Ibrahim, Clinical Cytopathology and Aspiration Biopsy (2001), which is hereby incorporated by reference in its entirety, also describe general methods for biopsies and cytological methods. A biological sample can be a heterogeneous or homogeneous population of cells or tissues. In certain embodiments, the sample is obtained from a source including, but not limited to, urine, blood, serum, plasma, sputum, mucus, saliva, breath condensate, bronchoalveolar lavage fluid, cerebrospinal fluid, sweat, tissue, hair follicles, oral tissue, tears, menstrual secretions, or feces. In a specific embodiment, the sample is obtained from serum. In certain embodiments of the method, any medical professional, such as a physician, nurse, or medical technician, can obtain a biological sample for testing. Additionally, a biological sample can be obtained without the assistance of a medical professional. In some embodiments of the method, the biological sample is obtained directly from the subject, from a medical professional, from a third party, or from a kit provided by a molecular profiling business or a third party. In some cases, the subject, medical professional, or third party can obtain a biological sample and send it to a third party before the biological sample is obtained. In some cases, a third party can provide a container and excipient suitable for storage and transport the biological sample to the third party.

[0202] Although not wishing to be bound by theory, in an affected subject (e.g., in a patient having PAH), reduction of BMPRII by either mutation or downregulation is hypothesized to shift BMP9 from a vascular quiescence factor to a driver of vascular remodeling via modulation of EC proliferation and survival (i.e., BMPRII dysregulation leads to BMP9 pathogenic gain of function). Thus, the methods herein treat a subject with a BMP9 antibody, an antigen-binding fragment thereof, or a pharmaceutical composition thereof to block the binding of BMP9 to type II receptors including BMPRII, ActRIIAm, and ActRIIB, thereby neutralizing their downstream (pathogenic) signaling in endothelial cells (ECs) and smooth muscle cells (SMCs) dysregulated in PAH. In some embodiments, the BMP9 antibody, an antigen-binding fragment thereof, or a pharmaceutical composition thereof described herein binds to BMP9 and blocks the binding of BMP9 to type II receptors by at least, up to, exactly, any two of the following (including or excluding both ends), or by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% less than, compared to prior to administration, or by any range between them (e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% less).

[0203] For all methods described herein, reference to an anti-BMP9 antibody also includes its antigen-binding fragments and pharmaceutical compositions comprising the anti-BMP9 antibody or its antigen-binding fragment and one or more additional agents.

[0204] Administration and Dosage Typically, the antibodies of the disclosure, or antigen-binding fragments thereof, are administered in an amount effective to treat a condition as described herein. The antibodies of the disclosure can be administered as the antibody itself or as a pharmaceutical composition containing the antibody.

[0205] In certain embodiments, the antibodies, antigen-binding fragments thereof, or pharmaceutical compositions thereof of the present disclosure are administered by any suitable route in a form of a pharmaceutical composition adapted to such route and in a therapeutically effective dose for the intended treatment.

[0206] In some embodiments, the antibody, antigen-binding fragment thereof, or pharmaceutical composition thereof can be administered parenterally, for example, directly into the bloodstream, into muscle, or into internal organs. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intracerebroventricular, intraurethral, intrasternal, intracranial, intramuscular, and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) syringes, needleless syringes, and infusion techniques. In some embodiments, the antibody, antigen-binding fragment thereof, or pharmaceutical composition thereof can be administered subcutaneously. In some embodiments, the antibody, antigen-binding fragment thereof, or pharmaceutical composition thereof can be administered intravenously. It is contemplated that any one or more of these aforementioned routes of administration may be excluded in certain embodiments.

[0207] In another embodiment, the compounds disclosed herein may be administered topically to the skin or mucosa, i.e., by cutaneous, transdermal, or sublingual administration. In another embodiment, the compounds disclosed herein can be administered by intranasal administration or instillation. In another embodiment, the compounds disclosed herein may be administered rectally or vaginally. In another embodiment, the compounds disclosed herein may also be administered directly to the eye or ear. It is contemplated that any one or more of these aforementioned routes of administration may be excluded in certain embodiments.

[0208] The dosing regimen for the antibodies of the present disclosure, or antigen-binding fragments thereof, or compositions containing such antibodies, is based on various factors including the type, age, weight, sex, and / or medical condition of the subject; the severity of the condition; the route of administration; and the activity of the particular antibody being used. Thus, the dosing regimen can vary widely. In one embodiment, the total daily dose of the antibody of the present disclosure is from about 0.01 to 1000 mg / kg (i.e., mg of the antibody of the present disclosure per kg of body weight) (e.g., at least, at most, exactly, between any two of the following (including or excluding both ends), or about 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 200 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg, 600 mg / kg, 700 mg / kg, 800 mg / kg, 900 mg / kg, or 1000 mg / kg, or any range thereof) for the treatment of the indicated conditions discussed herein. In another embodiment, the total daily dose of the antibody of the present disclosure is from about 0.1 to about 50 mg / kg, and in another embodiment, from about 0.5 to about 30 mg / kg. In some embodiments, the total daily dose is about 0.5 mg / kg (i.e., about 35 mg per day in a human of typical dimensions).

[0209] In some embodiments, the method or use comprises administering a dose of from about 1 mg to about 1000 mg. In some embodiments, the method or use comprises administering a dose of from about 1 mg to 1000 mg (e.g., at least, at most, exactly, between any two of the following (including or excluding both ends), or about 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 200 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg, 600 mg / kg, 700 mg / kg, 800 mg / kg, 900 mg / kg, or 1000 mg / kg, or a range of any of them) as an initial fixed dose. In some embodiments, the method or use comprises optionally administering as an initial fixed dose a dose of from about 1 mg to about 2 mg, from about 2 mg to about 5 mg, from about 5 mg to about 10 mg, from about 10 mg to about 20 mg, from about 20 mg to about 30 mg, from about 30 mg to about 40 mg, from about 40 mg to about 50 mg, from about 50 mg to about 60 mg, from about 60 mg to about 70 mg, from about 70 mg to about 80 mg, from about 80 mg to about 90 mg, from about 90 mg to about 100 mg, from about 100 mg to about 150 mg, from about 150 mg to about 200 mg, from about 200 mg to about 300 mg, from about 300 mg to about 400 mg, from about 400 mg to about 500 mg, from about 500 mg to about 600 mg, from about 600 mg to about 700 mg, from about 700 mg to about 800 mg, from about 800 mg to about 900 mg, or from about 900 mg to about 1000 mg. In some embodiments, the method or use comprises administering an anti-BMP9 antibody or a pharmaceutical composition thereof at a dose of about 15 mg, 30 mg, 40 mg, 50 mg, 75 mg, 100 mg, 150 mg, 300 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg. In some embodiments, the dose is an initial fixed dose.

[0210] In some embodiments, the method or use comprises subcutaneous administration of a dose of from about 12 mg to 500 mg (e.g., at least, at most, exactly, between any two of the following (including or excluding both ends), or about 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 200 mg / kg, 300 mg / kg, 400 mg / kg, or 500 mg / kg, or a range of any of them) every two weeks, every three weeks or every four weeks. In some embodiments, the method of use comprises subcutaneous administration of a dose of about 12 mg, about 15 mg, about 20 mg, about 24 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 175 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, or about 500 mg every two weeks, every three weeks or every four weeks. In some embodiments, the method or use comprises subcutaneous administration of a dose of about 110 mg every three weeks. In some embodiments, the method or use comprises administration of a dose of about 30 mg, about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 165 mg, about 180 mg, or about 200 mg every two weeks. In some, the method or use comprises administration of a dose of about 165 mg every two weeks. In some embodiments, the method or use comprises administration of a dose of about 50 mg, about 120 mg, about 230 mg, about 240 mg, about 250 mg, about 255 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, or about 300 mg every three weeks. In some aspects, the method or use comprises administration of a dose of about 255 mg every three weeks.In some embodiments, the method or use comprises administering at a dosage of about 65 mg, about 160 mg, about 200 mg, about 250 mg, about 300 mg, about 325 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg every four weeks. In some aspects, the method or use comprises administering at a dosage of about 355 mg every four weeks. In some embodiments, the antibody is Ab1076.

[0211] The dosage administered provides an average suppression of free BMP9 in the steady state of about 90% to about 99% (e.g., at least, at most, exactly, between any two of the following (including or excluding both ends), or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range thereof) in a standard antagonist antibody model. In some embodiments, the antibody, or an antigen-binding fragment thereof, or a pharmaceutical composition thereof is administered subcutaneously every two weeks at about 30 mg, 35 mg, about 50 mg, about 75 mg, about 85 mg, about 100 mg, about 125 mg, about 150 mg, or about 165 mg, providing an average suppression of 95%, 98%, or 99% of free BMP9 in the steady state. In some embodiments, the antibody, or an antigen-binding fragment thereof, or a pharmaceutical composition thereof is administered subcutaneously every three weeks at about 50 mg, about 55 mg, about 120 mg, about 130 mg, about 150 mg, about 200 mg, about 230 mg, about 250 mg, or about 255 mg, providing an average suppression of 95%, 98%, or 99% of free BMP9 in the steady state. In some embodiments, the antibody, or an antigen-binding fragment thereof, or a pharmaceutical composition thereof is administered subcutaneously every four weeks at about 65 mg, about 75 mg, about 100 mg, about 160 mg, about 180 mg, about 200 mg, about 250 mg, about 300 mg, about 325 mg, about 350 mg, or about 355 mg, providing an average suppression of 95%, 98%, or 99% of free BMP9 in the steady state. In some embodiments, the antibody is Ab1076.

[0212] In some embodiments, the method or use comprises administering an anti-BMP9 antibody of the disclosure, or an antigen-binding fragment thereof, or a pharmaceutical composition thereof, in a dosage of about 2 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 75 mg, 100 mg, 110 mg, 125 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, or 500 mg. In some embodiments, the method or use comprises administering an anti-BMP9 antibody of the disclosure, or an antigen-binding fragment thereof, or a pharmaceutical composition thereof, in a dosage of about 2 mg, 10 mg, 30 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, or 500 mg on a weekly basis. In some embodiments, the method or use comprises subcutaneously administering an anti-BMP9 antibody (e.g., Ab1076), or an antigen-binding fragment thereof, or a pharmaceutical composition thereof, in a dosage of about 2 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, or 500 mg on a weekly basis. In some embodiments, the method or use comprises subcutaneously administering an anti-BMP9 antibody (e.g., Ab1076), or an antigen-binding fragment thereof, or a pharmaceutical composition thereof, in a dosage of about 2 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, or 500 mg on a monthly basis. The required dosage may be administered subcutaneously as a single injection or delivered by more than one injection at one or more injection sites. For example, the required dosage may be administered subcutaneously once a month as two injections.

[0213] In some embodiments, the method or use comprises administering, optionally as a first dose, an antibody, or an antigen-binding fragment thereof, or a pharmaceutical composition, at a dose of from about 0.01 mg / kg to about 300 mg / kg, from about 1 mg / kg to about 250 mg / kg, from about 10 mg / kg to about 200 mg / kg, from about 50 mg / kg to about 150 mg / kg, or from about 75 mg / kg to about 100 mg / kg. The first dose may be followed by one or more subsequent doses. In some embodiments, one or more subsequent doses may be administered at least any of every week, every other week, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, every ten weeks, every eleven weeks, or every twelve weeks. In some embodiments, the dose is administered every three weeks.

[0214] One or more subsequent doses may follow the first dose. In some embodiments, the subsequent dose is the same dose, a lower dose, or a higher dose of the anti-BMP9 antibody as compared to the first dose. In some embodiments, one or more subsequent doses may be administered at least any of every week, every other week, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, every ten weeks, every eleven weeks, or every twelve weeks. The specific dosing protocol is involved in the maximum dose or dosing frequency to avoid significant undesirable side effects.

[0215] It is contemplated that any one or more of these aforementioned doses and / or dosing regimens may be excluded in certain embodiments.

[0216] Concurrent administration The antibodies or antigen-binding fragments thereof of the present disclosure can be used alone or in combination with one or more other therapeutic agents. The present disclosure provides any of the uses, methods, or compositions as defined herein in which the antibodies or antigen-binding fragments thereof of the present disclosure are used in combination with one or more other therapeutic agents. For example, in the treatment of pulmonary arterial hypertension, the BMP9 antibodies or antigen-binding fragments thereof described herein may be used in combination with one or more of the current therapies for pulmonary arterial hypertension. Such therapies include vasodilators such as prostacyclin, epoprostenol, and sildenafil; endothelin receptor antagonists such as bosentan; calcium channel blockers such as amlodipine, diltiazem, and nifedipine; anticoagulants such as warfarin; and diuretics, but are not limited thereto. The treatment of pulmonary hypertension may also be performed in combination with oxygen therapy, atrial septostomy, pulmonary thromboendarterectomy, and / or lung and / or heart transplantation. It is contemplated that one or more of these aforementioned therapies may be excluded in certain embodiments.

[0217] Administering two or more agents "in combination" means that all of the agents are administered in close proximity within a time sufficient to affect the treatment of the subject. The two or more agents may be administered simultaneously or sequentially. Additionally, simultaneous administration may be effected by mixing the agents prior to administration or by administering the agents at the same time but as separate dosage forms at the same or different sites of administration.

[0218] Kit Another aspect of the present disclosure provides a kit comprising the antibodies or antigen-binding fragments thereof of the present disclosure (e.g., Ab1076) or a pharmaceutical composition comprising the antibodies or antigen-binding fragments thereof (e.g., Ab1076). The kit may comprise a diagnostic or therapeutic agent in addition to the antibodies or antigen-binding fragments thereof or the pharmaceutical composition of the present disclosure. The kit may also comprise instructions for use in a diagnostic or therapeutic method. In some embodiments, the kit comprises the antibodies or antigen-binding fragments thereof or the pharmaceutical composition thereof and a diagnostic agent.

[0219] In another embodiment, the kit comprises an antibody or an antigen-binding fragment thereof or a pharmaceutical composition thereof, and one or more therapeutic agents such as prostacyclin, epoprostenol, and sildenafil; endothelin receptor antagonists such as bosentan; calcium channel blockers such as amlodipine, diltiazem, and nifedipine; anticoagulants such as warfarin; and / or diuretics. It is contemplated that any one or more of these aforementioned therapeutic agents may be excluded in certain embodiments.

[0220] In yet another embodiment, the disclosure includes a kit suitable for use in practicing the treatment methods described herein. In one embodiment, the kit contains a first dosage form comprising one or more of the antibodies or antigen-binding fragments thereof of the disclosure in an amount sufficient to carry out the methods disclosed herein. In another embodiment, the kit comprises one or more antibodies or antigen-binding fragments thereof of the disclosure in an amount sufficient to carry out the methods disclosed herein, at least a first container for the first dosage form, and a second container for the second dosage form.

[0221] Deposit of Biological Materials Representative materials of the disclosure were deposited with the American Type Culture Collection (10801 University Boulevard, Manassas, VA 20110-2209, USA) on April 19, 2022. The vector "Ab1076-VH" with ATCC Deposit No. PTA-127292 contains a DNA insert encoding the anti-BMP9 heavy chain variable region, and the vector "Ab1076-VL" with ATCC Deposit No. PTA-127293 contains a DNA insert encoding the anti-BMP9 light chain variable region.

[0222]

Table A

[0223] This deposit was made in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the Regulations thereunder (Budapest Treaty). This guarantees that viable cultures of the deposit will be maintained for 30 years from the date of the deposit. The deposited material can be obtained from the ATCC in accordance with the terms of the Budapest Treaty and the agreement between Genentech and the ATCC, which guarantees that, whichever comes first, progeny of the deposited culture will be made permanently and unrestrictedly available upon the issuance of the relevant U.S. patent or the publication of any U.S. or foreign patent application, and that progeny will be made available to those determined by the Commissioner of Patents and Trademarks to have the right under 35 U.S.C. § 122 of the U.S. Patent Act and the Commissioner's rules thereunder (including, in particular, 37 C.F.R. § 1.14 of MPEP § 886 OG 638).

[0224] The assignee of this application agrees that if the deposited material dies, is lost or is destroyed when cultured under appropriate conditions, the material will be promptly replaced with another of the same at the time of notice. The availability of the deposited material is not considered to be a license to practice the invention contrary to any rights recognized under the authority of any government in accordance with patent laws.

Examples

[0225] The following are examples of specific embodiments for carrying out the present disclosure. The following examples are included to demonstrate aspects of the present disclosure. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. It will be understood by those skilled in the art that the techniques disclosed in the following examples should be followed in accordance with representative techniques discovered by the inventors in order to function well in the practice of the present disclosure. However, it will be understood by those skilled in the art that, in light of the present disclosure, many changes can be made in the specific embodiments disclosed without departing from the spirit and scope of the disclosure and that the same or similar results can be obtained.

[0226] Example 1: Selection of anti-BMP9 antibodies by phage display. Hits of anti-BMP9 single-chain Fv (scFv) were selected from the human phage display antibody library WyHN5 by panning against mature human BMP9 as follows.

[0227] Human mature BMP9 protein was biotinylated using sulfo-NHS-LC-biotin (Thermo) according to the manufacturer's protocol. Four rounds of selection were performed in solution while decreasing the concentration of biotinylated BMP9 as follows: 200 nM biotinylated BMP9 (first round), 50 nM biotinylated BMP9 (second round), 1 nM biotinylated BMP9, then 1 μM non-biotinylated BMP9 was added and incubated overnight (third round), and performed with 1 nM biotinylated BMP9 (fourth round). In each round, phages that bound to biotinylated BMP9 were captured with magnetic DYNABEADS® M-280 streptavidin (Invitrogen), followed by washing to remove non-specifically bound phages. BMP9-specific binders were eluted with 100 mM triethylamine and then amplified in Escherichia coli (E. coli) for the next round of selection and / or screening ELISA.

[0228] The scFv can be expressed either on the surface of phage particles or in solution in the periplasmic space of bacteria, depending on the growth conditions used. To induce the release of scFv into the periplasm, 96-well plates containing 2×YT medium supplemented with 0.1% glucose and 100 μg / ml ampicillin were inoculated from 2×YT agar plates (one clone per well) and grown at 37 °C for approximately 4 h with shaking at 850 rpm. Cultures were induced with IPTG at a final concentration of 0.02 mM and grown overnight at 30 °C with shaking at 850 rpm. The contents of the bacterial periplasms were released by osmotic shock. Briefly, the plates were centrifuged, the pellets were resuspended in 150 ml of TES periplasm buffer (50 mM Tris, 1 mM EDTA, 20% sucrose, pH 7.4), followed by the addition of 150 ml of 1:5 TES:water and incubation on ice for 30 min. The plates were centrifuged at 4000 rpm for 20 min and the scFv-containing supernatant was collected.

[0229] For the binding assay with scFv peripreps, high-binding 96-well ELISA plates (Costar 3590) were coated overnight at 4°C with 0.1 mg of human BMP9 (100 μl at 1 mg / ml) in PBS buffer per well. After discarding the coating solution, the plates were blocked with PBS + 3% BSA for 2 hours at room temperature. After discarding the blocking solution, 100 μl of peripreps was added. After a 1-hour incubation at room temperature with gentle shaking, the plates were washed 5 times with 300 μl per well of wash buffer (Perkin Elmer 1244-114) and incubated for 1 hour with 100 μl of secondary HRP-labeled anti-His tag Ab. After washing 5 times with 300 μl of wash buffer, the plates were developed with TMB substrate solution for 10 minutes, and then 0.18 M H2SO4 was added to stop the reaction. Absorbance at 450 nM was measured, the data were plotted, and analyzed with Microsoft Excel. From this screening, 100 scFv hits were identified as binding to human BMP9 by periprep ELISA, and 15 unique clones including lead clones BMP9-0093 (also referred to herein as Ab93) and BMP9-0101 (also referred to herein as Ab101) were converted to human IgG1 / lambda using standard molecular biology methods.

[0230] Example 2: Binding of anti-BMP9 antibody to human BMP9 protein. This example illustrates the binding of the identified anti-BMP9 antibody to human BMP9 protein in an enzyme-linked immunosorbent assay (ELISA).

[0231] Purified human mature BMP9 protein was coated at 1 μg / ml in 100 μl of PBS (calcium- and magnesium-free) on a NUNC™-MAXISORP™ 96-well ELISA plate at 4°C. The plate was washed three times with PBS + 0.05% TWEEN™ 20 and blocked with PBS + 1% BSA with shaking for 1 hour at RT. The Ab to be tested was serially diluted 5-fold starting from 100 nM in PBS + 1% BSA. The blocking solution was removed, the plate was washed as before, and then the serially diluted Ab was added and incubated with shaking for 1 hour at RT. The plate was washed as before, a secondary antibody (goat anti-human IgG Fc-HRP, manufactured by Invitrogen, catalog number 31413, 1:10000) was added, and then incubated for 1 hour at RT. The plate was washed as before, the signal was developed using a TMB substrate, and the reaction was stopped with 0.18 M H2SO4. The absorbance was read at 450 nm using an ENVISION® plate reader (Perkin Elmer).

[0232] The results are shown in Figure 1. All the identified antibodies (Ab89, Ab93, Ab100, and Ab101) bound to human mature BMP9 with high potency. BM01 (referred to as AM0100 in WO 2016 / 193872 pamphlet) and BM02 (referred to as AM4405 in WO 2016 / 193872 pamphlet) were used as reference antibodies. In contrast, the isotype control antibody was unable to bind to human BMP9.

[0233] Example 3: Competitive ELISA for measuring the neutralizing activity of anti-BMP9 antibodies This example illustrates in a blocking (competitive) ELISA the ability of the identified anti-BMP9 antibodies to inhibit the interaction between BMP9 and its receptor.

[0234] Using type I receptor (ALK1) and type II receptors (BMPRII, ActRIIA, or ActRIIB), it was confirmed whether anti-BMP9 Ab could compete with the binding of these receptors to BMP9. Each receptor was coated on an ELISA plate at 1 - 15 μg / ml, incubated with serially diluted biotinylated BMP9 to determine the optimal receptor coating concentration, and the EC 90 of biotinylated BMP9 binding to each receptor was determined (data not shown).

[0235] For competitive ELISA, each receptor protein was coated on a 384-well ELISA plate at 4°C overnight at its optimal coating concentration, 20 μl / well in PBS-CMF. The next day, the plate was washed 3 times with PBS + 0.05% TWEEN™ 20 to determine the optimal receptor coating concentration and blocked for 1 hour at RT with shaking in PBS + 1% BSA. Anti-BMP9 Ab was serially diluted 3-fold starting from 100 nM in PBS + 1% BSA and then mixed with the same volume of optimally concentrated biotinylated BMP9. The final concentration of biotinylated BMP9 corresponded to the EC 90 for each receptor as determined in the preliminary experiment described above.

[0236] The serially diluted Ab was incubated with biotinylated BMP9 at room temperature for at least 15 minutes. The plate was washed as before, and then the mixture of serially diluted Ab and biotinylated BMP9 was added. After incubation for 1 hour at RT, the plate was washed 3 times with PBS + 0.05% TWEEN™ 20. The secondary antibody (HRP-streptavidin from BioLegend, catalog number 405210, 1:2000) was added and then incubated for 1 hour at RT. The plate was washed as before, developed using the TMB substrate, and the reaction was stopped with 0.18 M H2SO4. Absorbance was read at 450 nm using an ENVISION® plate reader (Perkin Elmer).

[0237] The results of these competitive ELISAs are shown in Figure 2. Antibodies Ab89, Ab93, Ab100, and Ab101 inhibit the binding of BMP9 to type II receptors (BMPRII, ActRIIA, or ActRIIB), but do not inhibit the binding of BMP9 to the type I receptor ALK1 (Figures 2A-2D). Antibodies Ab352, Ab804, and Ab1076, which are derived from the parental antibody Ab93, strongly inhibit the binding of BMP9 to the type II receptor (BMPRII) and weakly inhibit the binding of BMP9 to the type I receptor ALK1 (Figures 2E-2F). In contrast, the reference antibody BM02 is a type I receptor blocker, which strongly inhibited the binding of BMP9 to ALK1 (Figure 2F) and did not inhibit the binding to the type II receptor (not shown).

[0238] Example 4: Effect of anti-BMP9 antibodies on the binding of BMP9 to endothelial cells. This example illustrates the ability of the identified anti-BMP9 antibodies to dose-dependently inhibit the binding of human BMP9 to endothelial cells.

[0239] Human telomerase immortalized microvascular endothelial (TIME) cells were obtained from ATCC and cultured according to the provider's instructions. Recombinant human BMP9 was conjugated to NHS-ALEXA FLUOR 647 (registered trademark) dye. On the day of the experiment, the cells were harvested by enzymatic detachment and resuspended in pre-cooled culture medium. Subsequently, equal aliquots of the cell suspension were dispensed into 96-well plates and placed on ice.

[0240] The BMP9 Ab was serially diluted in pre-cooled cell growth medium to four final concentrations of 0.2 nM, 1 nM, 5 nM, 25 nM, and 125 nM. The BMP9-ALEXA FLUOR 647 (registered trademark) protein was diluted in pre-cooled cell growth medium to four final concentrations of 1 nM and combined with the serially diluted BMP9 Ab at a 1:1 (v / v) ratio to obtain two final concentrations of BPM9 and Ab, respectively. Additionally, aliquots of the four BMP9 solutions were combined with pre-cooled medium without Ab at a 1:1 (v / v) ratio.

[0241] Next, the above samples were incubated refrigerated and shielded from light for 40 minutes, after which aliquots from each sample were dispensed at a 1:1 (v / v) ratio into wells containing the TIME cell suspension in a 96-well plate. Cell samples mixed with medium without BMP9 or Ab were regarded as untreated controls. The plates were shielded from light and further incubated on ice for 60 minutes, after which the samples in the wells were washed with an excess of pre-cooled buffer containing fetal bovine serum protein and sodium azide to prevent active uptake of materials from the cell environment.

[0242] Next, the samples were analyzed using a BD FACSYMPHONY® A5 flow cytometer equipped with an appropriate optical setup for analysis of the median fluorescence intensity of the ALEXA FLUOR 647® dye per cell using BD FACSDIVA™ software. The raw data files were exported for analysis using FlowJo software and the numerical data were plotted using GraphPad Prism.

[0243] The results are shown in Figure 3. Anti-BMP9 antibodies Ab89, Ab93, and Ab100 demonstrated dose-dependent inhibition of BMP9 binding to TIME cells.

[0244] Example 5: Determination of the affinity of Ab1076 for human, cynomolgus monkey, and rat BMP9. This example confirms the binding affinity of anti-BMP9 antibody Ab1076 (analyzed as Fab) for human, cynomolgus monkey, and rat BMP9.

[0245] The binding affinities of Ab1076 (as a monovalent Fab fragment) for mature human, cynomolgus monkey, and rat BMP9 were determined by surface plasmon resonance (SPR) at 37 °C at a collection rate of 10 Hz using a BIACORE™ T200 (Cytiva). The running buffer and sample buffer were 10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% P-20 (HBS-EP+). Biotinylated mature human, cynomolgus monkey, and rat BMP9 proteins were reversibly captured onto Sensor Chip CAP according to the manufacturer's instructions using Biotin CAPture reagent (Cytiva, 28920234). The final capture levels were 100 resonance units (RU) for human BMP9, 50 RU for cynomolgus monkey BMP9, and 60 RU for rat BMP9. Flow cell 1 without BMP9 was used as the reference flow cell. Two-fold serial dilutions of Ab1076 Fab in the range of 100 nM to 6.2 nM were injected for 60 s at a flow rate of 100 μl per minute, and dissociation was monitored for 1800 s. Rate constants and affinities were determined by fitting the double-reference sensorgram data to a 1:1 model of BIACORE® Insight Evaluation software version 3.0.1.12.15655 (Cytiva).

[0246] As summarized in Table 1, the equilibrium dissociation constant (K D ) values for human, cynomolgus monkey, and rat BMP9 were 87.41 pM, 65.82 pM, and 98.63 pM, respectively. The K D values for cynomolgus monkey and rat BMP9 were within two-fold of that of human BMP9. Representative sensorgrams are shown in Figure 4.

[0247] Table 1. Affinities of Ab1076 Fab fragment for human, cynomolgus monkey, and rat BMP9 determined by SPR at 37 °C. Sensorgram data from three parallel measurements from three independent dilutions of the Ab1076 Fab analyte were used to calculate k a , k d , and K D ± SD values.

[0248]

Table 1

[0249] Example 6: Evaluation of Ab1076 for off-target binding to a panel of TGFβ family members. In this example, the off-target binding of Ab1076 (tested as Fab) to a panel of TGFβ family members was evaluated.

[0250] Using biolayer interferometry (BLI) on an OCTET® Red 384 instrument, the off-target binding of Ab1076 Fab to a panel of TGFβ family members including human TGFβ1, GDF8, GDF9, GDF11, activin A, and BMP10 was evaluated at 37°C. Human BMP9 was used as a positive control. The TGFβ family members were amine-coupled to AR2G biosensors (Sartorius ForteBio, 18-5092) at 20 μg / ml for 300 seconds. The immobilization levels were as follows: GDF8, 0.72 nm; GDF11, 0.28 nm; BMP9, 0.58 nm; BMP10, 1.0 nm; GDF9, 0.35 nm; TGFb1, 0.62 nm and activin A, 0.6 nm. Biosensors with immobilized TGFβ family members were immersed in 100 nM Ab1076 Fab diluted in Kinetics buffer (Sartorius ForteBio 18-5032) for 300 seconds. Dissociation was continued for 180 seconds. Data were collected using OCTET® Acquisition software version 11.0.0.64 and double-referenced in OCTET® Data Analysis software version 11.0.

[0251] At 100 nM, Ab1076 Fab did not demonstrate observable off-target binding to the TGFβ family members evaluated but bound to BMP9 as expected (Figure 5). All of the TGFβ family members were active as demonstrated by their binding to their respective positive controls (not shown).

[0252] Example 7: Anti-BMP9 antibody inhibits BMP9-induced phospho-SMAD1 / 5 / 9 and phospho-SMAD2 signaling in endothelial cells. In this example, the ability of an anti-BMP9 antibody to inhibit BMP9-induced phospho-SMAD1 / 5 / 9 and phospho-SMAD2 nuclear translocation as a measure of activation of SMAD1 / 5 / 9 or SMAD2 signaling in human, cynomolgus monkey, and rat endothelial cells was tested. Briefly, TIME cells (immortalized human microvascular endothelial cell line), primary human pulmonary artery endothelial cells (HPAEC), primary cynomolgus monkey pulmonary artery endothelial cells (PAEC), and primary rat liver sinusoidal endothelial cells (LSEC) were treated with human BMP9 or species-specific BMP9 at 10 pM or 1000 pM for 30 minutes in the presence of the anti-BMP9 antibody. Cells were fixed, blocked, permeabilized, and stained overnight with an antibody to pSMAD1 / 5 / 9 or pSMAD2. The next day, cells were washed and stained with a secondary antibody labeled with ALEXA FLUOR® 488 to detect pSMAD1 / 5 / 9 or pSMAD2, the nuclei were detected with Hoechst 33342, and the outline of the whole cell was shown with CellMask Deep Red staining. After the final wash, cells were imaged with a high-contrast imager. To analyze pSMAD1 / 5 / 9 or pSMAD2 nuclear translocation, the mean nuclear fluorescence in the ALEXA FLUOR® 488 channel was calculated for each well of cells. IC50 values were calculated using GraphPad Prism version 9.0.0.

[0253] Dose-dependent inhibition of BMP9-induced pSMAD1 / 5 / 9 and pSMAD2 nuclear translocation was observed in all endothelial cells upon treatment with the anti-BMP9 antibody.

[0254] In some experiments to be conducted, the IC of the anti-BMP9 antibody against human BMP9 in the pSMAD1 / 5 / 9 assay 50 values were in the range of 0.02 nM to 86 nM. In the pSMAD1 / 5 / 9 assay, the IC of the anti-BMP9 antibody against cynomolgus BMP9 50 values were in the range of 17 pM to 117 pM, and for rat BMP9 they were in the range of 120 pM to 300 pM. In some experiments to be conducted, the IC of the anti-BMP9 antibody against human BMP9 in the pSMAD2 assay 50 values were in the range of 0.01 nM to 330 nM. In the pSMAD2 assay, the IC of Ab1076 against cynomolgus BMP9 50 values were in the range of 27 pM to 42 pM, and for rat BMP9 they were in the range of 465 pM to 1 nM. Representative images are presented in Figure 6.

[0255] Example 8: Ab732 and Ab1076 of the anti-BMP9 antibody are effective in a rat Sugen-Hypoxia model of pulmonary hypertension. An efficacy study to evaluate the sclerosis of Ab732 and Ab1076 against pulmonary hypertension was conducted in a preclinical Sugen-Hypoxia (SuHx) model of pulmonary arterial hypertension (PAH) against isotype control and activin receptor type IIA-Fc (ActRIIA-Fc). In this vascular proliferative model, rodents develop progressive obstructive and complex lesions that closely resemble severe human diseases (Abe, K., Toba, M., Alzoubi, A., Ito, M., Fagan, K.A., Cool, C.D., Voelkel, N.F., McMurtry, I.F., and Oka, M. (2010) Formation of plexiform lesions in experimental severe pulmonary arterial hypertension. Circulation 121, 2747-2754).

[0256] Male Sprague-Dawley rats (150 - 200 g) received a single subcutaneous injection of the vascular endothelial growth factor receptor 1 / 2 antagonist, Sugen 5416 (20 mg / kg), and were placed in a normobaric hypoxic condition (FiO2 = 0.1) to induce moderate disease. An untreated (normoxic) control group of the same age was also included. After 4 weeks, the SuHx-treated rats were returned to normoxia and randomized into different treatment groups of equal body weight.

[0257] Treatments were administered intraperitoneally twice a week for 3 weeks. Ab732 and Ab1076 were tested at 0.3 mg / kg and 3 mg / kg. Human ActRIIA-Fc protein was administered at 2.1 mg / kg, and the isotype control human IgG1 was administered at 3 mg / kg. At the end of the study, cardiovascular hemodynamic evaluation was performed. The rats were anesthetized, and the right ventricular systolic pressure (RVSP) was measured by catheterization. To evaluate the degree of right ventricular hypertrophy (RVH), the right ventricular free wall was excised from the left ventricle and septum (LV + S) of the excised heart and weighed. RVH was expressed as the ratio of the weight of the RV to the weight of the (LV + S). The results are shown in Figure 7.

[0258] The mean RVSP and RVH in the untreated (normoxic) group were 29.2 ± 1.25 mmHg and 0.197 ± 0.00669, respectively. When SuHx was administered after 4 weeks, Ab1076, at 3 mg / kg, significantly improved RVSP to 63.7 ± 3.82 mmHg compared to 94.9 ± 7.27 mmHg in the isotype control - this was a reduction of 31.2 mmHg or 47.4% of the difference [subtracting normoxia from the isotype control] (p < 0.05). Ab1076 also improved RVH (0.331 ± 0.0240) compared to the isotype control (0.410 ± 0.0236) at 3 mg / kg, corresponding to a 37.1% decrease in the difference [subtracting normoxia from the isotype control]. Ab732 also induced significant improvements in hemodynamic parameters: at 3 mg / kg, Ab732 significantly improved RVSP to 55.4 ± 6.81 mmHg - a reduction of 39.5 mmHg or 60.0% of the difference [subtracting normoxia from the isotype control] (p < 0.01). At 3 mg / kg, Ab732 improved RVH (0.329 ± 0.0216) compared to the isotype control, corresponding to a 37.9% decrease in the difference [subtracting normoxia from the isotype control].

[0259] Treatment with ActRIIA-Fc also significantly improved RVSP (62.9 ± 4.60 mmHg, a reduction of 31.9 mmHg compared to the isotype control; p < 0.05) and also improved RVH compared to the isotype control (0.325 ± 0.0171).

[0260] In summary, administration of Ab732, Ab1076, and ActRIIA-Fc attenuated the magnitude of PAH developed in this study.

[0261] Example 9: Evaluation of total serum BMP9 and BMP10 concentrations in the SuGen-Hypoxia (SuHx) model. The effect of anti-BMP9 treatment on the total soluble BMP9 and BMP10 concentrations in rat serum samples collected at the end point of the SuHx efficacy dose study of Example 8 was evaluated by immunoaffinity LCMS analysis. This functional readout was used for the quantitative determination of target:antibody complexes in serum as a measure of target engagement. The results are shown in Figure 8.

[0262] Circulating BMP9 and BMP10 concentrations that were similar or consistent were measured in the sera of rats maintained in normoxia and SuHx rats treated with isotype controls. No significant changes were observed in BMP9 and BMP10 circulating concentrations after treatment with ActRIIA-Fc.

[0263] When treated with 3 mg / kg of Ab732, a 1.7-fold increase in circulating BMP9 concentration was observed compared to the hypoxia and isotype control groups, and no change was seen at 0.3 mg / kg. Treatment with Ab1076 resulted in a dose-dependent accumulation of circulating BMP9, with 1.6-fold and 7.9-fold increases measured at 0.3 mg / kg and 3 mg / kg, respectively, compared to the normoxia and isotype control groups (p≤0.0001) (Figure 8A). This is consistent with a higher affinity for Ab1076 compared to Ab732 for BMP9. The magnitude of the change in circulating BMP10 was reduced compared to BMP9 (Figure 8B).

[0264] Example 10: Molecular interactions of Ab93, Ab101, and Ab1076 with human BMP9 analyzed by X-ray crystallography. Crystallization and structure determination of mature human BMP9 complexed with Ab1076 Fab For the crystallization study, a complex between Ab1076 Fab and mature human BMP9 was formed at a 1:1.5 molar ratio and concentrated to 9.2 mg / ml in a protein solution containing TBS at pH 7.5.

[0265] Crystals were obtained by the hanging-drop vapor diffusion method under conditions containing 25% PEG 1500 and 100 mM SPG buffer (pH 5). The crystals had symmetry consistent with the orthorhombic space group C222 with unit cell parameters a = 141.98 Å; b = 271.05 Å; c = 62.59 Å and contained two copies of the BMP9+Ab1076 Fab complex in the crystallographic asymmetric unit. The crystals were flash-frozen in liquid nitrogen using 15% ethylene glycol (EG) as the cryoprotectant. Data were collected from a single frozen crystal at the IMCA beamline 17-ID at Argonne National Laboratory at a resolution of 2.55 Å. The data were processed, normalized using AutoPROC, and the final data set had a completeness of 62.7%.

[0266] The structure was solved by molecular replacement with PHASER. Several rounds of manual adjustment and model rebuilding using COOT and crystal refinement using AutoBUSTER resulted in a final model of BMP9+Ab1076 Fab with R work of 25.6% and R free of 27.7%, where R work =||F obs |-|F calc || / |F obs | and R free is equivalent to R work but with a randomly selected 5% that reflects what was omitted in the refinement assumptions calculated.

[0267] The BMP9 epitopes of antibodies Ab1076, Ab93, and Ab101, and the BMP9 epitope of type II receptor ActRIIB are defined by a descriptor method based on the underlined structures detailed below. The structure-based descriptor method utilizes the crystal structure complex and defines epitope residues as residues that are: a) significantly buried by complex formation (≥20 Å 2(a) buried surface area), b) residues participating in hydrogen bonds, c) residues participating in water-mediated hydrogen bonds, d) residues participating in salt bridges, and e) residues in close contact (<3.8 Å). This combination of interacting and proximal residues is then combined as a list of all potentially important epitope residues. This list is limited by the fact that sites not in close contact with the ligand can still be important for binding and sites that are proximal may not be important. For the calculations, the H, L, and G chains from the Ab1076 structure, the H, L, and C chains from the Ab93 structure, the H, L, and I chains from the Ab101 structure, and the A and E chains from Protein Data Bank entry 4fao were used to generate descriptors for the interface residues.

[0268] The BMP9 epitopes of antibodies Ab1076, Ab93, and Ab101, and the BMP9 epitope of type II receptor ActRIIB are summarized in Table 2. From this analysis, there is significant overlap in the epitopes for antibodies Ab1076, Ab93, and Ab101, and the epitope for type II receptor ActRIIB (Figure 9), and it is also clear that there is significant overlap in other type II receptors such as BMPRII, ActRIIA, and endoglin (data not shown). The overlap of the BMP9 epitopes of antibody Ab1076 and ActRIIB is shown in Figure 9.

[0269] Table 2: BMP9 epitopes of antibodies Ab1076, Ab93, and Ab101, and type II receptor ActRIIB defined by the structure-based descriptor method.

[0270]

Table 2

[0271] Structure-based epitope residue descriptors Here, descriptor of interface residues was generated using the H, L, and G chains from the Ab1076:BMP9 complex structure, the H, L, and C chains from the Ab93:BMP9 complex structure, and the H, L, and I chains from the Ab101:BMP9 complex structure. Residues of the antigen and antibody are said to be hydrogen-bonded to a hydrogen atom if they contain a hydrogen-bond donor atom (bonded to a hydrogen with a positive electrical charge) within 3.2 Å of a hydrogen-bond acceptor atom having a lone pair of electrons in another molecule. Further, a given residue was considered to be part of a water-mediated hydrogen bond if it participated in a hydrogen bond with a water molecule that was also hydrogen-bonded to the antibody. Residues of the antibody and antigen are said to form a salt bridge if they contain a positively charged atom in one molecule within 4 Å of a negatively charged atom in another molecule. Residues are in close contact if one atom in the antigen is within 3.8 Å of one atom in the antibody.

[0272] The buried surface area for each residue was determined by calculating the solvent-accessible surface area of each residue of the antibody and antigen in the complex and subtracting this from the sum of the solvent-accessible surface areas of the two components considered individually. The solvent-accessible surface area was calculated according to the method of Strake and Rupley (J. Mol. Biol. 79(2):351 - 71, 1973). Using the buried surface areas paired in two, the individual contributions to the overall effect of the buried surface area on the binding energy of residue pairs from the antibody and antigen were estimated. Since the buried surface area cannot be decomposed for each pair, the buried surface area of each residue within the epitope was calculated in the presence of the individual antigen residue with the remaining antigen absent. These individual contributions were then normalized such that the sum of all individual contributions of all antigen residues to the buried surface area of a particular epitope residue is equal to the sum of the buried surface area of that epitope residue for the binding of the entire antigen. This process was repeated in reverse for the individual contributions of epitope residues to the buried surface area of antibody residues. Antigen residues were considered to be part of the epitope if the buried surface area was ≧20 Å 2 and was so regarded.

[0273] Using the A chain and E difference from the Protein Data Bank structure 4fao, the same calculations were also performed on the interaction of the binding epitope of BMP9 to ActRIIB.

[0274] The binding interaction of Ab1076 Fab in the crystal structure with human BMP9 is shown in Figure 10.

[0275] Example 11: Anti-BMP9 antibodies inhibit serum-induced phospho-SMAD1 / 5 / 9 and phospho-SMAD2 signaling in human endothelial cells. Using rat serum samples collected at the end point of the SuHx efficacy dose study of Example 8, the effects of anti-BMP9 and ActRIIA-Fc treatments on pSMADF1 / 5 / 9 and pSMAD2 nuclear translocation in immortalized human microvascular endothelial cells were evaluated. Briefly, serum-starved TIME cells were treated with 10% rat serum for 20 minutes. The cells were fixed, blocked, permeabilized, and stained overnight with an antibody against pSMAD1 / 5 / 9 or pSMAD2. The next day, the cells were washed and stained with a secondary antibody labeled with ALEXA FLUOR® 488 to detect pSMAD1 / 5 / 9 or pSMAD2, the nuclei were detected with Hoechst 33342, and the outline of the whole cell was shown with CellMask Deep Red staining. After the final wash, the cells were imaged with a high-contrast imager. To analyze pSMAD1 / 5 / 9 or pSMAD2 nuclear translocation, the average nuclear fluorescence in the ALEXA FLUOR® 488 channel was calculated for each well of the cells. IC 50 values were calculated using GraphPad Prism version 9.0.0.

[0276] Serum from rats treated with 0.3 mg / kg of Ab1076 induced a significantly lower mean nuclear fluorescence intensity of pSMAD1 / 5 / 9: 2270 ± 155 compared to 2810 ± 160 for Iso Ctrl (isoform control), which was a 19.3% reduction (p < 0.0001). An 18.7% reduction in the mean nuclear intensity of pSMAD1 / 5 / 9 compared to Iso Ctrl was observed upon treatment with 3 mg / kg of Ab1076 (2290 ± 325) (p < 0.0001). Treatment with serum from Ab732-treated rats at 0.3 mg / kg (2550 ± 140) and treatment with serum from Ab732-treated rats at 3 mg / kg (2530 ± 74.6; p < 0.05) inhibited the mean nuclear fluorescence of pSMAD1 / 5 / 9 by 9.2% and 10%, respectively, compared to serum from rats treated with Iso Ctrl (Figure 11A).

[0277] The mean average pSMAD2 nuclear intensity was reduced by only 14.5% upon treatment with serum from the 0.3 mg / kg Ab1076 group (2400 ± 79.1; p < 0.0001) and by 11.6% upon treatment with serum from the 3 mg / kg Ab1076 group (2481 ± 171; p = 0.0002) compared to Iso Ctrl (2810 ± 189). Treatment with serum from Ab732-treated rats at 0.3 mg / kg (2720 ± 127) and treatment with serum from Ab732-treated rats at 3 mg / kg (2580 ± 75.7; p < 0.05) inhibited the mean nuclear fluorescence of pSMAD2 compared to serum from rats treated with Iso Ctrl (Figure 11B).

[0278] Similar levels of nuclear translocation of pSMAD1 / 5 / 9 (2150 ± 169) and nuclear translocation of pSMAD2 (2320 ± 42.4) were observed in untreated TIME endothelial cells and cells treated with empty serum from the 0.3 mg / kg or 3 mg / kg Ab1076 groups.

[0279] No significant changes in the nuclear translocation of pSMAD1 / 5 / 9 (2780 ± 104) and pSMAD2 (2790 ± 144) were observed upon treatment with ActRIIA-Fc compared to Iso Ctrl.

[0280] Example 12: Evaluation of the off-target effects of anti-BMP9 antibodies on GDF8 and GDF11-induced signaling of TGFβ-family members. The ability of Ab1076 and Ab732 to inhibit the activity of GDF8 and GDF11, BMP9 family members, was evaluated using C2C12 mouse myoblasts stably expressing the CAGA promoter inserted in a firefly luciferase reporter construct. Briefly, C2C12-CagaLuc cells were treated with 3 nM GDF8 or GDF11 for 24 hours in the presence of a series of doses of Ab1076, Ab732, or an isotype control antibody. Cells were lysed, and luciferase activity was evaluated by adding the firefly-specific substrate D-luciferein and then measuring luminescence with a plate reader.

[0281] Ab1076 had no effect on GDF8-induced luciferase activity (Figure 12A) or GDF11-induced luciferase activity (Figure 12B) compared to an unbound isotype control antibody. Ab732 inhibited GDF8-induced luciferase activity (Figure 12A) but did not inhibit GDF11-induced luciferase activity (Figure 12B). ActRIIA-Fc was used as a positive control and inhibited GDF8 and GDF11 at IC 50 values in the range of 0.2 nM to 1.5 nM (data not shown).

[0282] Example 13: Evaluation of the effect of anti-BMP9 antibody Ab1076 on the endothelial secretion of endothelin-1. In this example, the ability of Ab1076, an anti-BMP9 antibody that inhibits the endothelial cell secretion of endothelin-1 (ET-1), was tested. Briefly, human pulmonary artery endothelial cells (HPAECs) were treated with 100 pM of BMMP9 for 24 hours in the presence of a series of doses of Ab1076 or an isotype control. At the end of the treatment, the medium was collected from each well of the cells and subjected to ET-1 ELISA. A standard curve was generated using 4-parameter logistic curve fitting, and this was used to determine the amount of ET-1 in each of the sample wells. IC 50 values were calculated using GraphPad Prism version 9.0.0.

[0283] BMP9 stimulates an increase in ET-1 secretion (data not shown), and Ab1076 induced an increase in ET-1 secretion at IC 50 values in the range of 0.021 nM to 0.065 nM in several experiments performed (Figure 13).

[0284] Example 14: Identification of gene and protein targets for the treatment of pulmonary hypertension by anti-BMP9 antibodies. In this example, potential target genes of BMP9 in pulmonary hypertension were identified.

[0285] Bulk RNAseq data from rat lungs treated with normoxia (Nx), SU5416+Hypoxia+isotype control (SuHx_Iso), and SU5416+Hypoxia+Ab93 (SuHx_Ab93) were analyzed. The induction of gene expression and the secretion of gene products that are correlated with pulmonary hypertension in response to BMP9 expression were also tested in HPMVECs.

[0286] The number of paired-end raw data reads in FASTQ format was aligned to the Rattus norvegicus rn7 reference genome using the Rsubread R package (2.8.2). FeatureCounts was used to count the number of reads mapped to each gene. Differential expression analysis was performed using the DESeq2 R package (1.34.0). The relative mRNA expression levels (fold change) for the following target genes were increased in the lungs of SU5416+Hypoxia-treated (SuHx) rats compared to the lungs of normoxic (Nx) rats, decreased in the lungs of SuHx rats treated with Ab93, and mapped to specific cell types as shown below in human lung scRNAseq data (Saygin et al., Pulm Circ, 2020) (Figure 14).

[0287] a. Cxcl12, C-X-C motif chemokine ligand 12 (also known as stromal cell-derived factor 1 (SDF1)) (Figure 14A). The expression of Cxck12 was increased in human IPAH lung endothelial cells and fibroblasts. The protein encoded by this gene is a secreted protein. In some aspects, neutralization of CXCL12 can attenuate established pulmonary hypertension in rats.

[0288] b. Igfbp4, insulin-like growth factor-binding protein 4 (Figure 14B). The expression of Igfbp4 was increased in human IPAH lung endothelial cells and smooth muscle cells / pericytes. The protein encoded by this gene is a secreted protein. In some aspects, IGFBP4 inhibits angiogenesis.

[0289] c. Inhba, inhibin, beta A (also known as activin A, activin AB alpha polypeptide, or erythroid differentiation inducing protein) (Figure 14C). The protein encoded by this gene is a member of the TGF-beta superfamily of proteins. Increased expression of activin A was mapped to SPP1 macrophages in human IPAH lung, but it was decreased in FABP4 macrophages and proliferating cells (macrophages and EC).

[0290] d. Mall, or Mal, T cell differentiation protein-like (Figure 14D). Increased expression of MALL was mapped to endothelial cells in human IPAH lung.

[0291] e. Frzb, secreted frizzled related protein 3 (Figure 14E). The protein encoded by this gene is an osteosecretory protein involved in the regulation of bone development. Increased expression of FRZB was mapped to SMC / pericytes in human IPAH lung.

[0292] f. Cpe, carboxypeptidase E (Figure 14F). Cpe encodes a member of the M14 family of metallocarboxypeptidases. The encoded protein is a secreted protein. In some embodiments, CPE upregulates the anti-apoptotic protein Bcl-2 and inhibits the activation of caspase 3. Increased expression of CPE was mapped to SMC / pericytes in human IPAH lung.

[0293] Therefore, among the genes that were upregulated in the lungs of SuHx_Iso rats compared to the lungs of Nx rats but downregulated in the lungs of SuHx-Ab93 rats compared to the lungs of SuHx_Iso rats, Cxcl12, Igfbp4, Inhba, Mall, Frzb, and Cpe were selected as potential target genes of BMP9. The expression of these genes was also shown to be upregulated in certain cell types of the lungs of IPAH patients when tested by scRNAseq (Saygin et al., Pulm. Circ., 2020). Among the six genes, the expression of Cxcl12, Inhba, Mall, Frzb, and Cpe was shown to be upregulated in the lungs of IPAH patients when tested by microarray assay (Stearman et al., Am J Respir Cell Mol Biol, 2019).

[0294] Human pulmonary microvascular endothelial cells (HPMVEC, Lonza) with a low passage numb...

Claims

1. It binds to bone morphogenetic protein-9 (BMP9), (a)(i) Heavy chain variable region complementarity determination region 1 (VH CDR1), containing amino acid sequence NAWMS (SEQ ID NO: 90), VH CDR2 containing the amino acid sequence of SEQ ID NO: 76, VH CDR3 containing the amino acid sequence of SEQ ID NO: 26, Light chain variable region complementarity determination region 1 (VL CDR1), containing the amino acid sequence of SEQ ID NO: 73, VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 67; (ii) VH CDR1 containing the amino acid sequence GFTFSNA (SEQ ID NO: 91), VH CDR2 containing the amino acid sequence KSKTEGGD (SEQ ID NO: 92), VH CDR3 containing the amino acid sequence of SEQ ID NO: 26, VL CDR1 containing the amino acid sequence of SEQ ID NO: 73, VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 67; (iii) VH CDR1 containing the amino acid sequence of SEQ ID NO: 53, VH CDR2 containing the amino acid sequence of SEQ ID NO: 76, VH CDR3 containing the amino acid sequence of SEQ ID NO: 26, VL CDR1 containing the amino acid sequence of SEQ ID NO: 73, VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 67; (b) VH CDR1 containing the amino acid sequence of SEQ ID NO: 53, VH CDR2 containing the amino acid sequence of SEQ ID NO: 54, VH CDR3 containing the amino acid sequence of SEQ ID NO: 26, VL CDR1 containing the amino acid sequence of SEQ ID NO: 50, VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 21; or (c) VH CDR1 containing the amino acid sequence of SEQ ID NO: 53, VH CDR2 containing the amino acid sequence of SEQ ID NO: 70, VH CDR3 containing the amino acid sequence of SEQ ID NO: 26, VL CDR1 containing the amino acid sequence of SEQ ID NO: 65, VL CDR2 containing the amino acid sequence of SEQ ID NO: 66, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 67 Isolated antibodies containing [the specified substance].

2. The aforementioned antibody (i) VH containing the amino acid sequence of SEQ ID NO: 55 and VL containing the amino acid sequence of SEQ ID NO: 51; (ii) VH containing the amino acid sequence of SEQ ID NO: 71 and VL containing the amino acid sequence of SEQ ID NO: 68; (iii) VH containing the amino acid sequence of SEQ ID NO: 77 and VL containing the amino acid sequence of SEQ ID NO: 74; or (iv) VH encoded by the nucleic acid sequence of a plasmid insert deposited at ATCC with deposit number PTA-127292, VL encoded by the nucleic acid sequence of a plasmid insert deposited at ATCC with deposit number PTA-127293, or both. The isolated antibody according to claim 1, comprising:

3. The aforementioned antibody (i) Human Vκ or V λ Light chain constant domain; and / or (ii) Heavy chain constant domain The isolated antibody according to claim 1, comprising:

4. The heavy chain constant domain is IgA, IgA １ IgA ２ , IgD, IgE, IgM, IgG, IgG １ , IgG ２ , IgG ３ , or IgG ４ The isolated antibody according to claim 3, comprising

5. The aforementioned antibody (i) FC domain; or (ii) Fc domains containing the IgG1 heavy chain CH2 domain and the IgG heavy chain CH3 domain The isolated antibody according to claim 1, comprising:

6. An isolated antibody according to Claim 1, (i) HCs containing at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% the same amino acid sequence as SEQ ID NO: 52, and LCs containing at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% the same amino acid sequence as SEQ ID NO: 49; (ii) an HC containing at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% the same amino acid sequence as SEQ ID NO: 69, or an LC containing at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% the same amino acid sequence as SEQ ID NO: 64, or an LC consisting of the same; or (iii) an isolated antibody according to claim 1, comprising an HC containing or consisting of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% the same amino acid sequence as SEQ ID NO: 75, and an LC containing or consisting of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% the same amino acid sequence as SEQ ID NO:

72.

7. An isolated nucleic acid encoding the VH, VL, or both of the antibody according to any one of claims 1 to 6.

8. The isolated nucleic acid according to claim 7, (i) the nucleic acid sequence of SEQ ID NO: 85, the nucleic acid sequence of SEQ ID NO: 86, or both; (ii) the nucleic acid sequence of sequence number 87, the nucleic acid sequence of sequence number 88, or both; or (iii) The nucleic acid sequence of an insert in a plasmid deposited by ATCC with deposit number PTA-127292, the nucleic acid sequence of an insert in a plasmid deposited by ATCC with deposit number PTA-127293, or both. Isolated nucleic acids containing [the specified substance].

9. A vector comprising the isolated nucleic acid described in claim 7.

10. An isolated host cell comprising the isolated nucleic acid described in claim 7.

11. The host cell according to claim 10, wherein the cell is a mammalian cell or an insect cell.

12. The host cell according to claim 11, wherein the host cell is a CHO cell, a HEK-293 cell, an NS0 cell, a PER. C6 cell, or an Sp2.0 cell.

13. A method for producing antibodies, The host cells described in claim 10 are cultured under conditions that result in the production of the antibody, A method comprising recovering the aforementioned antibody.

14. A pharmaceutical composition comprising a therapeutically effective amount of an antibody according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition according to claim 14, (a) comprising 1.12 mg / mL of L-histidine, 2.67 mg / mL of L-histidine hydrochloride monohydrate, 85 mg / mL of sucrose, 0.05 mg / mL of EDTA disodium dihydrate, and 0.2 mg / mL of polysorbate 80 at pH 5.8; (b) comprising 20 mM histidine, 8.5% sucrose, and 0.02% polysorbate 80, and 0.005% EDTA at pH 5.8; (c) Containing antibodies in concentrations of approximately 2 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 50 mg / mL, 75 mg / mL, 100 mg / mL, 125 mg / mL, 150 mg / mL, 175 mg / mL, or 200 mg / mL; and / or (d) Suitable for subcutaneous (SC) and / or intravenous (IV) administration, The aforementioned pharmaceutical composition.

16. A pharmaceutical composition comprising an isolated antibody according to any one of claims 1 to 6, for use in the treatment of hypertension or at least one sign and / or symptom of hypertension.

17. The pharmaceutical composition according to claim 16, wherein the hypertension is pulmonary arterial hypertension.

18. The pharmaceutical composition according to claim 17, wherein the pharmaceutical composition is administered in combination with one or more additional therapeutic active compounds or treatments effective in treating at least one sign and / or symptom of hypertension in a therapeutically effective amount.

19. A pharmaceutical composition comprising an isolated antibody according to any one of claims 1 to 6 for monitoring the efficacy of the pharmaceutical composition in a subject having pulmonary arterial hypertension (PAH) requiring the same, wherein a change (e.g., a decrease) in the level of one or more biomarkers after administration of the pharmaceutical composition indicates that the pharmaceutical composition is effective.

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