Anti-BMP9 antibodies and methods of use thereof

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 347,543, filed on May 31, 2022, U.S. Provisional Application No. 63 / 375,781, filed on September 15, 2022, and U.S. Provisional Application No. 63 / 499,808, filed on May 3, 2023, the contents of which are incorporated herein by reference.

[0003] Parties to the Joint Research Statement

[0004] The presently claimed invention was made by or on behalf of the parties to the joint research agreement listed below. 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 performed within the scope of the joint research agreement. The parties to the joint research agreement are PARTNERS HEALTHCARE and PFIZER INC. Background Art

[0005] Pulmonary arterial hypertension (PAH) is a rare, progressive disorder characterized by high blood pressure (hypertension) in the pulmonary arteries. PAH is defined as a mean pulmonary artery pressure (mPAP) greater than 20 mmHg in the presence of normal or reduced 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). Initial symptoms of PAH include shortness of breath after exertion. Additional symptoms include excessive fatigue, weakness, chest pain, dizziness, and fainting episodes. In the late stages of PAH, individuals may have an abnormal bluish discoloration of the skin (cyanosis) due to low levels of circulating oxygen, and the right chamber of the heart (ventricle) may be abnormally enlarged (hypertrophy), potentially leading to heart failure.

[0006] Current treatments for PAH include vasodilators, antiproliferative agents, calcium channel blockers, blood thinners, and diuretics. These therapies focus on extending the patient's lifespan and improving the quality of life. However, these treatments have one or more disadvantages, such as lack of effectiveness, severe side effects, low patient compliance, and high costs. In fact, most patients still die from the disease or do not respond adequately 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 a significant need in the art for novel targeted therapies for the treatment and / or prevention of PAH, particularly addressing the underlying pathophysiology. Summary of the invention

[0007] The present disclosure provides antibodies that bind to bone morphogenetic protein-9 (BMP9) encoded by the growth differentiation factor 2 (GDF2) gene, as well as the uses of these antibodies and related methods. Polynucleotides encoding antibodies that bind to BMP9 are provided. Polynucleotides encoding antibody heavy chains or light chains or both are also provided. Host cells expressing these antibodies are provided. The present disclosure also provides methods for manufacturing, preparing and producing antibodies that bind to BMP9. The present disclosure further encompasses the expression of antibodies, and the preparation and manufacture of compositions (such as agents for using these antibodies) containing the antibodies of the present disclosure.

[0008] The antibodies disclosed herein can be used for the diagnosis, prevention and / or treatment of one or more of disorders or conditions mediated by or associated with BMP9 activity (including but not limited to pulmonary hypertension, anemia, liver disease, neuritis or neurodegenerative diseases such as multiple sclerosis, fibrotic disorders and heart failure). Any one or more of the aforementioned disorders or conditions can be excluded from the diagnosis, prevention and / or treatment mediated by or associated with BMP9 activity.

[0009] In some aspects, disclosed herein is 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). In some aspects, the VH complementarity determining region one (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 VLCDR3 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.

[0010] In some aspects, disclosed herein is 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 (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. 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, 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) 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; (vii) VH CDR1 comprises the amino acid sequence of SEQ ID NO:45, VH CDR2 comprises the amino acid sequence of SEQ ID NO:61, and VH CDR3 comprises the amino acid sequence of SEQ ID NO:62, and VL CDR1 comprises the amino acid sequence of SEQ ID NO:57, VL CDR2 comprises the amino acid sequence of SEQ ID NO:41, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:58; (viii) 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:71. (ix) 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.;

[0011] 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, 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: 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.

[0012] In some aspects, disclosed herein is 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 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.

[0013] In some aspects, the VH of the isolated antibodies disclosed herein 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 containing one to four amino acid substitutions at residues not within the CDRs. In some aspects, the VL of the isolated antibodies disclosed herein 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 containing one to four amino acid substitutions at residues not within the CDRs. In some aspects, the VH of the isolated antibody disclosed herein 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 containing one to four amino acid substitutions at residues not within the CDRs, and the VL of the isolated antibody disclosed herein 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 containing one to four amino acid substitutions at residues not within the CDRs.

[0014] In some aspects, the VH of the isolated antibodies disclosed herein comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 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, at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 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, at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: In some aspects, 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 aspects, the VL of the isolated antibody disclosed herein comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 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 an amount that is at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 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 an amount that is at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: In some aspects, the VL of the isolated antibody 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 aspects, the VH of the isolated antibodies disclosed herein comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 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 the VL of the isolated antibodies disclosed herein comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 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 the VL of the isolated antibodies disclosed herein comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 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: 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 are at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amount or any range thereof (including or excluding the end values), or about the following amount or 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 amino acid sequences: 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. It is contemplated that any one or more of these aforementioned percentages may be excluded in embodiments.

[0015] In some aspects, 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: 72 and a VL comprising the amino acid sequence of SEQ ID NO: 73. VH comprising the amino acid sequence of SEQ ID NO:77 and VL comprising the amino acid sequence of SEQ ID NO:74.

[0016] In some aspects, the isolated antibodies disclosed herein comprise: (i) 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; (ii) 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; (iii) 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; (iv) 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; (v) a VH comprising the amino acid sequence of SEQ ID NO: 79 and a VL comprising the amino acid sequence of SEQ ID NO: 80; NO:27 is at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount or 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 an amino acid sequence: 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and a VL that comprises the same amino acid sequence as SEQ ID NO:22 is at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount or 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 an amino acid sequence: 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%;(vi) a VH comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:55, and a VL comprising an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:55. NO:51 is at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount or 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 an amino acid sequence: 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%; (vii) a VH comprising the same amino acid sequence as SEQ ID NO:71 is at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount or 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 an amino acid sequence: 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and a VL comprising the same amino acid sequence as SEQ ID NO:68 is at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount or 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 an amino acid sequence: 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%;or (viii) a VH comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 77, and a VL comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 77, and a VL comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 77. NO: 74 at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amount or any range thereof (including or excluding the end value) or about the following amount or 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 amino acid sequence: 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. 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. ;

[0017] In some aspects, disclosed herein is an isolated antibody portion, wherein 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, VHCDR2 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, and 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: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 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, the portion binds to BMP9.

[0018] In some aspects, the 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, a VH CDR2 comprising 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 a VH CDR3 comprising 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.

[0019] In some aspects, disclosed herein is an isolated antibody that binds to BMP9 and comprises a VH encoded by a nucleic acid sequence of an insert of a plasmid deposited with ATCC and having accession number PTA-127292, a VL encoded by a nucleic acid sequence of an insert of a plasmid deposited with ATCC and having accession number PTA-127293, or both.

[0020] In some aspects, disclosed herein is an isolated antibody comprising: (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, VLCDR2, 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 VHCDR3 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: 49, and the VL CDR1, VL CDR2, and VL CDR3 sequences of SEQ ID NO: 50; NO:55, and the VL CDR1, VL CDR2, and VL CDR3 sequence of SEQ ID NO:51; (vi) the VH CDR1, VH CDR2, and VH CDR3 sequence of SEQ ID NO:63, and the VL CDR1, VL CDR2, and VL CDR3 sequence of SEQ ID NO:59; (vii) the VH CDR1, VH CDR2, and VH CDR3 sequence of SEQ ID NO:71, and the VL CDR1, VL CDR2, and VL CDR3 sequence of SEQ ID NO:68; or (viii) the VH CDR1, VH CDR2, and VH CDR3 sequence of SEQ ID NO:77, and the VL CDR1, VL CDR2, and VL CDR3 sequence of SEQ ID NO:74.

[0021] In some aspects, the isolated antibody comprises a human Vκ or V λ In some aspects, the antibody comprises a human V λLight chain constant domain. In some aspects, the antibody comprises a heavy chain constant domain. In some aspects, the heavy chain constant domain comprises IgA (e.g., IgA1 or IgA2), IgD, IgE, IgM or IgG (e.g., IgG1, IgG2, IgG3 or IgG4). In some aspects, the heavy chain constant domain comprises IgG. In some aspects, the IgG is selected from the group consisting of: IgG1, IgG2, IgG3 and IgG4. In some aspects, the IgG is IgG1. In some aspects, the antibody comprises an Fc domain. In some aspects, the Fc domain comprises an IgG1 heavy chain CH2 domain and an IgG heavy chain CH3 domain.

[0022] In some aspects, disclosed herein is an isolated antibody 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. In some aspects, disclosed herein is an isolated antibody 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. In some aspects, disclosed herein is an isolated antibody 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.

[0023] In some aspects, disclosed herein is an isolated antibody comprising a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 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, or any range thereof. In some aspects, disclosed herein is an isolated antibody comprising a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 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, or any range thereof.In some aspects, disclosed herein is an isolated antibody comprising: (i) a heavy chain comprising a residue corresponding 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 at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount or 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 an amino acid sequence: 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and (ii) a light chain comprising an amino acid sequence 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: 65. NO:72 at least following amount or its any scope, at most following amount or its any scope, exactly following amount or its any scope, between any two in following amount or its any scope (comprising end value or not containing end value) or approximately following amount or its any scope (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 sequence: 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.Expectation can exclude any one or more of these aforementioned percentages in an embodiment.

[0024] In some aspects, disclosed herein is an isolated antibody comprising: (i) a HC comprising or consisting of an amino acid sequence of SEQ ID NO: 8, and a LC comprising or consisting of an amino acid sequence of SEQ ID NO: 1; (ii) a HC comprising or consisting of an amino acid sequence of SEQ ID NO: 23, and a LC comprising or consisting of an amino acid sequence of SEQ ID NO: 18; (iii) a HC comprising or consisting of an amino acid sequence of SEQ ID NO: 35, and a LC comprising or consisting of an amino acid sequence of SEQ ID NO: 29; (iv) a HC comprising or consisting of an amino acid sequence of SEQ ID NO: 44, and a LC comprising or consisting of an amino acid sequence of SEQ ID NO: 40; (v) a HC comprising or consisting of an amino acid sequence of SEQ ID NO: 52, and a LC comprising or consisting of an amino acid sequence of SEQ ID NO: 49; (vi) a HC comprising or consisting of an amino acid sequence of SEQ ID NO: 60, and a LC comprising or consisting of an amino acid sequence of SEQ ID NO: 56; (vii) a HC comprising or consisting of an amino acid sequence of SEQ ID NO: 61 NO:69 or a HC consisting of the amino acid sequence thereof, and a LC consisting of the amino acid sequence of SEQ ID NO:64 or a LC consisting of the amino acid sequence thereof; or (viii) a HC consisting of the amino acid sequence of SEQ ID NO:75 or a HC consisting of the amino acid sequence thereof, and a LC consisting of the amino acid sequence of SEQ ID NO:72 or a LC consisting of the amino acid sequence thereof.

[0025] In some aspects, disclosed herein is an isolated antibody comprising: (i) a HC comprising or consisting of an amino acid sequence of SEQ ID NO: 23, and a LC comprising or consisting of an amino acid sequence of SEQ ID NO: 18; (ii) a HC comprising or consisting of an amino acid sequence of SEQ ID NO: 52, and a LC comprising or consisting of an amino acid sequence of SEQ ID NO: 49; (iii) a HC comprising or consisting of an amino acid sequence of SEQ ID NO: 69, and a LC comprising or consisting of an amino acid sequence of SEQ ID NO: 64; (iv) a HC comprising or consisting of an amino acid sequence of SEQ ID NO: 75, and a LC comprising or consisting of an amino acid sequence of SEQ ID NO: 72; (v) a HC comprising or consisting of an amino acid sequence of SEQ ID NO: 76, and a LC comprising or consisting of an amino acid sequence of SEQ ID NO: 77; ID NO: 23 at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount or 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 amino acid sequence or consisting of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and such LC, the LC comprises the same amino acid sequence as SEQ ID NO: 23 at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount or 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%). NO:18 is at least or consists of an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the following amount or any range thereof, at most, exactly, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount or 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%);(vi) a HC comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:52 in an amount that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% or more, or a LC comprising an amino acid sequence that is ... identical to SEQ ID NO:52 in an amount that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:52 NO:49 is at least or consists of an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:49; (vii) an HC that comprises or consists of an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:49; (vii) an HC that comprises or consists of an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:49; NO:69 is at least or consists of an amino acid sequence identical to SEQ ID NO:69 in an amount of at least or any range thereof, at most in an amount of at least or any range thereof, in an amount of at least or any range thereof, in an amount of at least or any range thereof, in an amount of at least or any range thereof, in an amount of at least or any range thereof, in an amount of at least or any range thereof, in an amount of at least or any range thereof, in an amount of at least or any range thereof, in an amount of at least or any range thereof, in an amount of at least or any range thereof, in an amount of at least or any range thereof, in an amount of at least or any range thereof, in an amount of at least or any range thereof, in an amount of at least or any range thereof, in an amount of at least or any range thereof, in an amount of at least or any range thereof, in an amount of at least or any range thereof, in an amount of at least or any range thereof, in an amount of at least or any range thereof, in an amount of at least or any range thereof, in an amount of at least or any range thereof, in an amount of at least or any range thereof, in NO:64 is at least or consists of an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the following amount or any range thereof, at most, exactly, between or about any two of the following amounts or any range thereof (including or excluding the end values), or 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%);or (viii) a HC comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:75, and a LC comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:75, and a LC comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:75 NO:72 is at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amount or any range thereof (including or excluding the end value) or about the following amount or 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 amino acid sequence or consisting of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. It is contemplated that any one or more of these aforementioned percentages may be excluded in embodiments. ;

[0026] In some aspects, disclosed herein is an isolated antibody comprising: (i) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 75 and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 72; or (ii) a HC comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 75, and a LC comprising or consisting of an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 75, and a LC comprising or consisting of an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: NO:72 at least following amount or its any scope, at most following amount or its any scope, exactly following amount or its any scope, between any two in following amount or its any scope (comprising end value or not containing end value) or approximately following amount or its any scope (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 sequence or consisting of: 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.Expectation can exclude any one or more of these aforementioned percentages in an embodiment.

[0027] In some aspects, disclosed herein is 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: Ala28, Pro29, Ser83, Leu85, Met90, Val92, Pro93, Leu95, and Tyr97 of SEQ ID NO: 79. In some aspects, the antibody binds to Ala28, Pro29, Ser83, Leu85, Met90, Val92, Pro93, Leu95, and Tyr97 of SEQ ID NO: 79. In some aspects, 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 aspects, the antibody binds Pro81, Tyr86, Asp88, Asp89, Thr94, Lys96, His98, Tyr99, and Glu100 of SEQ ID NO:79. In some aspects, the antibody further binds one or more amino acid residues selected from the group consisting of: Ile27, Lys30, and Glu31 of SEQ ID NO:79. In some aspects, the antibody binds 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 embodiments.

[0028] In some aspects, disclosed herein is 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. It is contemplated that any one or more of these aforementioned amino acid residues may be excluded in an embodiment.

[0029] In some aspects, disclosed herein is an isolated antibody that binds to 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.

[0030] In some aspects, disclosed herein is 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: 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.

[0031] In some aspects, disclosed herein is an isolated antibody that binds to 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.

[0032] In some aspects, disclosed herein is 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 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 embodiments.

[0033] In some aspects, disclosed herein is an isolated antibody that binds to 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. In some aspects, the antibody epitope on BMP9 does not comprise amino acid residues 21-25, 43-60 of SEQ ID NO: 79.

[0034] In some aspects of the isolated antibodies disclosed herein, the antibody binds to human BMP9 with an affinity of at least 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 at 37°C, as measured by surface plasmon resonance. In some aspects of the isolated antibodies disclosed herein, 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, as measured by surface plasmon resonance. In some embodiments, the antibody binds to human BMP9 with an affinity of about 75 pM to about 95 pM at 37°C, as measured by surface plasmon resonance. In some embodiments, the antibody binds to human BMP9 with an affinity of about 85 pM at 37° C., as measured by surface plasmon resonance. In some embodiments, the antibody binds to human BMP9 with an affinity of about 87 pM at 37° C., as measured by surface plasmon resonance. It is contemplated that any one or more of these foregoing binding affinities may be excluded in embodiments.

[0035] In some aspects of the isolated antibodies disclosed herein, the antibody binds to cynomolgus monkey BMP9 with an affinity of at least 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 at 37°C as measured by surface plasmon resonance. In some aspects of the isolated antibodies disclosed herein, the antibody binds to cynomolgus monkey 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 as measured by surface plasmon resonance. In some embodiments, the antibody binds to cynomolgus monkey 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 monkey 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 foregoing binding affinities may be excluded in embodiments.

[0036] In some aspects of the isolated antibodies disclosed herein, the antibody binds to rat BMP9 with an affinity of at least 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 at 37°C as measured by surface plasmon resonance. In some aspects of the isolated antibodies disclosed herein, the antibody binds to rat 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, as measured by surface plasmon resonance. In some embodiments, the antibody binds to rat BMP9 at 37° C. with an affinity of about 75 pM to about 120 pM, as measured by surface plasmon resonance. In some embodiments, the antibody binds 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 foregoing binding affinities may be excluded in embodiments.

[0037] In some aspects of the isolated antibodies disclosed herein, the antibody comprises: (i) a heavy chain variable region (VH) CDR3 of SEQ ID NO:26 and a light chain variable region (VL) CDR3 of SEQ ID NO:84, (ii) a heavy chain variable region (VH) CDR3 of SEQ ID NO:26 and a light chain variable region (VL) CDR3 of SEQ ID NO:21, or (iii) a heavy chain variable region (VH) CDR3 of SEQ ID NO:26 and a light chain variable region (VL) CDR3 of SEQ ID NO:67.

[0038] In some aspects of the separated antibodies disclosed herein, the antibody (i) binds to human BMP9 and inhibits the binding of human BMP9 to BMP9 type II receptors (e.g., BMPRII, ActRIIA and / or ActRIIB); or (ii) binds to human BMP9 and inhibits the binding of human BMP9 to BMP9 type II receptors (e.g., BMPRII, ActRIIA and / or ActRIIB), and weakly inhibits the binding of human BMP9 to BMP9 type 1 receptors (e.g., ALK1). In some aspects of the separated antibodies disclosed herein, the antibody binds to human BMP9 and inhibits the binding of human BMP9 to human endothelial cells. In some aspects of the separated antibodies disclosed herein, the antibody binds to human BMP9, but does not bind to other human transforming growth factor β (TGFβ) superfamily ligands (e.g., GDF8, GDF11, activin A, TGFβ1, GDF9 and BMP10).

[0039] In some aspects of the isolated antibodies disclosed herein, the antibody inhibits BMP9 activity measured by phospho-SMAD1 / 5 / 9 nuclear translocation in endothelial cells.

[0040] In some aspects, the isolated antibody is present in an amount of about 0.01 nM to about 100 nM (e.g., at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount or any range thereof: 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, M, 45pM, 50pM, 55pM, 60pM, 65pM, 70pM, 75pM, 80pM, 85pM, 90pM, 95pM, 100pM, 105pM, 11 0pM, 115pM, 120pM, 125pM, 130pM, 135pM, 140pM, 145pM, 150pM, 160pM, 170pM, 180pM, 1 90pM, 200pM, 225pM, 250pM, 275pM, 300pM, 325pM, 350pM, 375pM, 400pM, 425pM, 450pM, 475pM, 500pM, 550pM, 600pM, 650pM, 700pM, 750pM, 800pM, 850pM, 900pM, 950pM, 1000p pM, 2000pM, 3000pM, 4000pM, 5000pM, 6000pM, 7000pM, 8000pM, 9000pM, 10000pM, 20000pM, 30000pM, 40000pM, 50000pM, 60000pM, 70000pM, 80000pM, 90000pM or 100000pM)50 Inhibit human BMP9 activity. In some embodiments, the isolated antibody is about 15pM, about 20pM, about 25pM, about 30pM, about 35pM, about 40pM, about 45pM, about 50pM, about 55pM, about 60pM, about 65pM, about 70pM, about 75pM, about 80pM, about 85pM, about 90pM, about 95pM, about 100pM, about 105pM, about 110pM, about 115pM, about 120pM, about 125pM, about 13 The IC values ​​of about 100 pM, about 150 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 are preferably selected from the group consisting of: 50 Inhibit human BMP9 activity. In some embodiments, the isolated antibody has 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, or about 100 nM. 50 Inhibit human BMP9 activity. It is expected that these aforementioned IC 50 Any one or more of the values.

[0041] In some aspects, the isolated antibody has an IC of about 15 pM to about 120 pM (e.g., at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount or any range thereof: 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). 50 Inhibits cynomolgus monkey BMP9 activity. In some aspects, the isolated antibody has an IC of about 17 pM to about 117 pM (for example, but not limited to 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 or about 117 pM). 50Inhibits cynomolgus monkey BMP9 activity. In some aspects, the isolated antibody has an IC of about 120 pM to about 300 pM (for example, but not limited to about 120 pM, about 150 pM, about 175 pM, about 200 pM, about 225 pM, about 250 pM, about 275 pM or about 300 pM). 50 Inhibits rat BMP9 activity. It is expected that these aforementioned IC 50 Any one or more of the values.

[0042] In some aspects of the isolated antibodies disclosed herein, the antibody inhibits BMP9 activity measured by phospho-SMAD2 nuclear translocation in endothelial cells.

[0043] In some aspects, the antibody is expressed in an amount of about 0.01 nM to about 350 nM (e.g., at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount or any range thereof: 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 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) 50 In some embodiments, the antibody is at 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 The IC values ​​of about 100 pM, about 110 pM, about 120 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 are preferably selected from the group consisting of: 50Inhibit human BMP9 activity. In some embodiments, the isolated antibody has 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 Inhibit human BMP9 activity. It is expected that these aforementioned IC 50 Any one or more of the values.

[0044] In some aspects, the isolated antibody has an IC of about 15 pM to about 75 pM (e.g., at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount or any range thereof: 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). 50 Inhibits cynomolgus monkey BMP9 activity. In some aspects, the isolated antibody has an IC of about 27 pM to about 42 pM (for example, but not limited to about 27 pM, about 30 pM, about 35 pM, about 40 pM or about 42 pM). 50 Inhibits cynomolgus monkey BMP9 activity. In some aspects, the isolated antibody has an IC of about 450 pM to about 1 nM (for example, 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 Inhibits rat BMP9 activity. It is expected that these aforementioned IC 50 Any one or more of the values.

[0045] In some aspects of the isolated antibodies disclosed herein, the antibody reduces right ventricular systolic pressure in the subject by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, compared to before administration of the antibody. In some aspects of the isolated antibodies disclosed herein, the antibody reduces right ventricular systolic pressure in the subject by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, compared to the percent difference (%) observed between an isotype control antibody and normoxic conditions, at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, exactly 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, between any two of the following amounts or any ranges thereof, inclusive or exclusive, or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%. In some aspects of the isolated antibodies disclosed herein, the antibody reduces right ventricular hypertrophy in the subject by at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount or any range thereof (e.g., 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 administration of the antibody: 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%.In some aspects of the isolated antibodies disclosed herein, the antibody reduces right ventricular hypertrophy in the subject by at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, compared to the percent difference (%) observed between an isotype control antibody and normoxic conditions, at most 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, exactly 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, between any two of the following amounts or any ranges thereof (including or excluding the end values), or about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%. In some aspects, the subject is a Sugen-hypoxia animal model of pulmonary arterial hypertension (PAH).It is contemplated that any one or more of these aforementioned percentage reductions may be excluded in embodiments.

[0046] In some aspects, a separated polynucleotide is disclosed herein, which encodes any of the separated antibodies disclosed herein. In some aspects, a separated nucleic acid is disclosed herein, which encodes VH, VL or both of the antibody binding BMP9. In some aspects, the separated nucleic acid comprises SEQ ID NO:85 nucleic acid sequence, SEQ ID NO:86 nucleic acid sequence or both. In some aspects, a separated nucleic acid is disclosed herein, which encodes the heavy chain, light chain or both of the antibody binding BMP9. In some aspects, the separated nucleic acid comprises SEQ ID NO:87 nucleic acid sequence, SEQ ID NO:88 nucleic acid sequence or both. In some aspects, a separated nucleic acid is disclosed herein, which encodes VH, VL or both of the antibody bound to BMP9. In some aspects, the isolated nucleic acid comprises the nucleic acid sequence of the insert of the plasmid deposited with the ATCC and having accession number PTA-127292, the nucleic acid sequence of the insert of the plasmid deposited with the ATCC and having accession number PTA-127293, or both.

[0047] In some aspects, a vector is disclosed herein, comprising a polynucleotide disclosed herein encoding any of the isolated antibodies disclosed herein. In some aspects, a vector is disclosed herein, comprising a nucleic acid disclosed herein encoding the VH, VL, or both of an antibody that binds BMP9. In some aspects, a vector is disclosed herein, comprising a nucleic acid disclosed herein encoding the heavy chain, light chain, or both of an antibody that binds BMP9.

[0048] In some aspects, disclosed herein is an isolated host cell comprising an isolated polynucleotide disclosed herein encoding any of the isolated antibodies disclosed herein. In some aspects, disclosed herein is an isolated host cell comprising an isolated nucleic acid disclosed herein encoding VH, VL, or both of an antibody that binds BMP9. In some aspects, disclosed herein is an isolated host cell comprising an isolated nucleic acid disclosed herein encoding a heavy chain, a light chain, or both of an antibody that binds BMP9. In some aspects, the host cell is a mammalian cell or an insect cell. In some aspects, the host cell is a mammalian cell selected from the group consisting of: CHO cells, HEK-293 cells, NS0 cells, cells or Sp2.0 cells.

[0049] In some aspects, disclosed herein is a method of producing an antibody, the method comprising culturing a host cell disclosed herein under conditions leading to the production of the antibody. In some aspects, the method further comprises recovering the antibody.

[0050] In some aspects, a pharmaceutical composition is disclosed herein, comprising a therapeutically effective amount of an antibody disclosed herein and a pharmaceutically acceptable carrier. In some aspects, the composition comprises 1.12 mg / mL L-histidine, 2.67 mg / mL L-histidine hydrochloride monohydrate, 85 mg / mL sucrose, 0.05 mg / mL EDTA disodium dihydrate, 0.2 mg / mL polysorbate 80, and a pH of 5.8. In some aspects, the composition comprises 20 mM histidine, 8.5% sucrose and 0.02% polysorbate 80, 0.005% EDTA, and a pH of 5.8. In some aspects, the composition comprises about 2mg / mL, 5mg / mL, 10mg / mL, 15mg / mL, 20mg / mL, 25mg / mL, 50mg / mL, 75mg / mL, 100mg / mL, 125mg / mL, 150mg / mL, 175mg / mL or 200mg / mL antibody. In some aspects, the composition is suitable for subcutaneous (SC) and / or intravenous (IV) administration. In some aspects, the composition is suitable for intravenous (IV) administration. In some aspects, the composition is suitable for subcutaneous (SC) administration.

[0051] In some aspects, disclosed herein is 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 a pharmaceutical composition disclosed herein or an antibody disclosed herein.

[0052] In some aspects, disclosed herein is a method of treating hypertension in a subject, 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 aspects, the hypertension is pulmonary hypertension.

[0053] In some aspects, disclosed herein is 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 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 treatment modalities that are effective in treating hypertension, optionally at least one sign and / or symptom of pulmonary arterial hypertension.

[0054] In some aspects, disclosed herein is an isolated antibody disclosed herein or a pharmaceutical composition disclosed herein for use as a medicament.

[0055] In some aspects, disclosed herein is an isolated antibody disclosed herein or a pharmaceutical composition disclosed herein for use in treating at least one sign and / or symptom of hypertension, optionally pulmonary hypertension.

[0056] In some aspects, disclosed herein is the use of an isolated antibody disclosed herein or a pharmaceutical composition disclosed herein in the manufacture of a medicament for treating at least one sign and / or symptom of hypertension, optionally pulmonary hypertension.

[0057] It is contemplated that any aspect discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. In addition, the compositions of the present disclosure can be used to implement the methods of the present disclosure.

[0058] Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect can also be described in "use" claim language (e.g., "use" of any compound, composition, or medicament discussed herein for achieving or carrying out the described therapeutic, diagnostic, or physiological purpose or effect). The use of one or more compositions can be employed based on any method described herein.

[0059] According to the following specific embodiments, other objects, features and advantages of the present disclosure will become clear. However, it should be understood that the specific embodiments and specific examples, although indicating specific aspects of the present disclosure, are given only in an illustrative manner, because according to the specific embodiments, various changes and modifications within the spirit and scope of the present disclosure will become clear to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1An exemplary graph showing the binding of antibodies to human BMP9 in an enzyme-linked immunosorbent assay (ELISA) is depicted.

[0061] Figure 2A-2F Depicted is the use of BMP9 antibodies and the receptor protein ActRIIA ( Figure 2A )、BMPRII( Figure 2B , Figure 2E )、ActRIIB( Figure 2C ) and ALK1( Figure 2D , Figure 2F ) obtained by competitive ELISA. Antibodies Ab89, Ab93, Abl00 and Abl01 are parent anti-BMP9 antibodies identified from human antibody phage library selection activities. Ab352, Ab804 and Ab1076 are optimized variants of Ab93. BM01 (referred to as AM0100 in WO 2016 / 193872) and BM02 (referred to as AM4405 in WO2016 / 193872) were used as reference antibodies.

[0062] Figure 3 Depicted is an exemplary graph showing the effect of anti-BMP9 antibodies on the binding of BMP9 to human telomerase-immortalized microvascular endothelial (TIME) cells.

[0063] Figure 4A-4C Depicts Ab1076 Fab with human ( Figure 4A )、Cynomolgus monkey( Figure 4B ) and rats ( Figure 4C ) Representative BIACORE binding to BMP9 TM Sensor diagram.

[0064] Figure 5 Depicted are exemplary Octet sensorgrams of Ab1076 Fab binding to human Activin A, BMP10, GDF8, GDF9, GDF11, TGFβ1, and BMP9 proteins at 37°C.

[0065] Figures 6A-6K Depicted are exemplary data showing that anti-BMP9 antibodies inhibit BMP9-induced phosphorylation of SMAD1 / 5 / 9 ( Fig. 6A , Figure 6C , Fig. 6E , Figure 6G , Fig.6I and Figure 6K ) and phosphorylated-SMAD2( Figure 6B , Fig.6D , Fig. 6F , Figure 6H and Figure 6J )Inhibition of nuclear translocation.

[0066] Figure 7A-7B Depicted are exemplary graphs showing right ventricular systolic pressure (RVSP; Fig. 7A ) and right ventricular hypertrophy (RV / (LV+S); Figure 7B ). Data represent mean ± SEM. One-way ANOVA and Tukey's multiple comparison test were performed for both RVSP and RV / (LV+S) relative to isotype control. * = p ≤ 0.05; ** = p ≤ 0.01; and **** = p ≤ 0.0001.

[0067] Figure 8A-8B Depicted are exemplary data showing total circulating BMP9 ( Fig. 8A ) and BMP10( Figure 8B ) concentrations. Data represent mean ± SD. For all treatment comparisons, one-way ANOVA with Tukey's multiple comparison test was performed on both BMP9 and BMP10 circulating concentrations. * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001.

[0068] Figure 9A-9B Depicted are exemplary data showing the BMP9 binding epitope of the type II receptor ActRIIB ( Fig. 9A ) and the overlapping epitope of Ab1076 ( Fig. 9B Binding epitopes were calculated using the structure-based descriptor method described in the Examples section below. Here, the structure of BMP9 is shown in white, and the ActRIIB epitope ( Fig. 9A ) is shown in black, and the Ab1076 epitope ( Fig. 9B ) is shown in grey, overlaid on the non-binding ActRIIB epitope shown in black. Calculations for the ActRIIB epitope were performed on the BMP9:ActRIIB complex in Protein Data Bank entry 4fao chains A(BMP9) and E(ActRIIB). Calculations for the Ab1076 epitope were performed using the Ab1076 structure chains H+L(Fab) and G(BMP9).

[0069] Figures 10A-10D Depicted are exemplary data showing the binding interface of BMP9 and Ab1076. Fig. 10A Depicted is a ribbon diagram of the interaction of BMP9 dimer (black) with the Fab of Abl076 (VH: dark grey, VL: light grey) as seen in the crystal structure of their complex. A 2-Fab:2-BMP9 ratio was observed in the interaction. Fig. 10B , Fig. 10C and Fig. 10D The important mutations shown in contributed to the approximately 20-fold increase in binding affinity from Ab93 to Ab1076. These include the D_H53_E mutation, predicted to introduce a more optimal salt bridge geometry with BMP9_K96; the D_L31_F mutation, predicted to increase packing with BMP9_P29 and BMP9_Y32; and the V_L93_E mutation, predicted to add additional hydrogen bonds to BMP9_S80.

[0070] Figure 11A-11B Depicted are exemplary data showing that serum from rats treated with isotype control (isotype control (Iso Ctrl)), ActRIIA-Fc, Ab732, or Ab1076 induced phosphorylated-SMAD1 / 5 / 9 ( Fig.11A ) and phosphorylated-SMAD2( Fig. 11B ) Serum-mediated regulation of nuclear translocation. Data represent mean ± SD. One-way ANOVA and Tukey's multiple comparison test were performed for both phospho-SMAD1 / 5 / 9 and phospho-SMAD2 relative to isotype control. * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001, and **** = p ≤ 0.0001.

[0071] Figure 12A-12B Exemplary graphs showing the effects of anti-BMP9 antibodies Ab732 and Abl076 on GDF8-induced luciferase activity and GDF11-induced luciferase activity are depicted.

[0072] Fig.13 An exemplary graph showing inhibition of endothelial secretion of endothelin-1 by anti-BMP9 antibody Ab1076 in an enzyme-linked immunosorbent assay (ELISA) is depicted.

[0073] Figure 14A-14F Depicted is a differential expression analysis to determine the expression of Cxcl12 ( Fig.14A )、Igfbp4( Fig. 14B )、Inhba( Fig. 14C )、Mall( Fig.14D )、Frzb( Fig.14E ) and Cpe( Fig.14F ) relative mRNA expression levels (fold change) of Nx. Isotype: 10 mg / kg, ip. Ab93: 10 mg / kg, ip. Data analysis was performed using GraphPad Prism 9 (GraphPad Software). Results are shown as mean and SD. Statistical significance was analyzed by t-test (nonparametric test). Values ​​of P<0.05 were considered significant. For Nx, N=8, for SuHx_isotype and SuHx_Ab93, N=6.

[0074] Figures 15A-15D Figure 3 shows the expression of CXCL12 in HPMVEC culture medium in response to BMP9 treatment. Fig.15A )、CCL2( Fig. 15B )、PDGFBB( Fig. 15C ) and endothelin-1( Fig.15D ) protein concentration. Data analysis was performed using GraphPad Prism 9 (GraphPad Software). Results are shown as mean and SD. Statistical significance was analyzed by t-test (non-parametric test). *P<0.05. DETAILED DESCRIPTION

[0075] The present invention can be more easily understood by reference to the following specific embodiments and the examples included herein. It should be understood that the present invention is not limited to specific manufacturing methods, which can certainly vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be restrictive.

[0076] Exemplary embodiments of the present invention provided herein (E) include:

[0077] 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:

[0078] the VH complementarity determining region one (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,

[0079] 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,

[0080] 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,

[0081] 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,

[0082] 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

[0083] 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.

[0084] 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:

[0085] (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;

[0086] (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;

[0087] (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;

[0088] (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;

[0089] (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;

[0090] (vi) 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;

[0091] (vii) VH CDR1 comprises the amino acid sequence of SEQ ID NO:45, VH CDR2 comprises the amino acid sequence of SEQ ID NO:61, and VH CDR3 comprises the amino acid sequence of SEQ ID NO:62, and VL CDR1 comprises the amino acid sequence of SEQ ID NO:57, VL CDR2 comprises the amino acid sequence of SEQ ID NO:41, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:58;

[0092] (viii) 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

[0093] (ix) 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.

[0094] E3. The isolated antibody of E2, wherein:

[0095] (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;

[0096] (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;

[0097] (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;

[0098] (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

[0099] (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.

[0100] E4. 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 VLCDR1 comprises the amino acid sequence of SEQ ID NO: 73, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 20, and VLCDR3 comprises the amino acid sequence of SEQ ID NO: 67.

[0101] E5. An isolated antibody as described in any one of E1-E4, wherein the 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 containing one to four amino acid substitutions at residues not within the CDRs, and the 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 containing one to four amino acid substitutions at residues not within the CDRs.

[0102] E6. An isolated antibody as described in any one of E1-E4, wherein the 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 the 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: 79. NO:74 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 the amino acid sequence.

[0103] E7. The isolated antibody of any one of E5-E6, wherein the antibody comprises:

[0104] (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;

[0105] (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;

[0106] (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;

[0107] (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;

[0108] (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;

[0109] (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;

[0110] (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

[0111] (viii) VH comprising the amino acid sequence of SEQ ID NO: 77 and VL comprising the amino acid sequence of SEQ ID NO: 74.

[0112] E8. The isolated antibody of E7, wherein the antibody comprises:

[0113] (i) 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;

[0114] (ii) 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;

[0115] (iii) 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;

[0116] (iv) 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;

[0117] (v) 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:27 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:22;

[0118] (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;

[0119] (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

[0120] (viii) 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.

[0121] E9. The isolated antibody of 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.

[0122] E10. An isolated antibody portion, wherein the isolated antibody binds to BMP9 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0123] 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;

[0124] 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;

[0125] 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;

[0126] 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;

[0127] 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

[0128] 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, and

[0129] This part binds to BMP9.

[0130] E11. The portion as described in E10, wherein the portion comprises:

[0131] 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;

[0132] a VH CDR2 comprising 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

[0133] A VH CDR3 comprising 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.

[0134] E12. An isolated antibody that binds to BMP9 and comprises VH encoded by the nucleic acid sequence of an insert of a plasmid deposited with ATCC and having accession number PTA-127292, VL encoded by the nucleic acid sequence of an insert of a plasmid deposited with ATCC and having accession number PTA-127293, or both.

[0135] E13. An isolated antibody comprising:

[0136] (i) the VH CDR1, VH CDR2 and VH CDR3 sequences of SEQ ID NO: 16, and the VLCDR1, VL CDR2 and VL CDR3 sequences of SEQ ID NO: 6;

[0137] (ii) the VH CDR1, VH CDR2 and VH CDR3 sequences of SEQ ID NO:27, and the VLCDR1, VL CDR2 and VL CDR3 sequences of SEQ ID NO:22;

[0138] (iii) the VH CDR1, VH CDR2 and VH CDR3 sequences of SEQ ID NO:39, and the VLCDR1, VL CDR2 and VL CDR3 sequences of SEQ ID NO:33;

[0139] (iv) the VH CDR1, VH CDR2 and VH CDR3 sequences of SEQ ID NO:48, and the VLCDR1, VL CDR2 and VL CDR3 sequences of SEQ ID NO:43;

[0140] (v) the VH CDR1, VH CDR2 and VH CDR3 sequences of SEQ ID NO:55, and the VLCDR1, VL CDR2 and VL CDR3 sequences of SEQ ID NO:51;

[0141] (vi) the VH CDR1, VH CDR2 and VH CDR3 sequences of SEQ ID NO:63, and the VLCDR1, VL CDR2 and VL CDR3 sequences of SEQ ID NO:59;

[0142] (vii) the VH CDR1, VH CDR2 and VH CDR3 sequences of SEQ ID NO:71, and the VLCDR1, VL CDR2 and VL CDR3 sequences of SEQ ID NO:68; or

[0143] (viii) the VH CDR1, VH CDR2 and VH CDR3 sequences of SEQ ID NO:77, and the VLCDR1, VL CDR2 and VL CDR3 sequences of SEQ ID NO:74.

[0144] E14. An isolated antibody as described in any one of E1-E13, wherein the antibody comprises human Vκ or V λ Light chain constant domain.

[0145] E15. The isolated antibody of E14, wherein the antibody comprises human V λ Light chain constant domain.

[0146] E16. The isolated antibody of any one of E1-E15, wherein the antibody comprises a heavy chain constant domain.

[0147] E17. The isolated antibody of E16, wherein the heavy chain constant domain comprises IgA (eg, IgA1 or IgA2), IgD, IgE, IgM, or IgG (eg, IgG1, IgG2, IgG3, or IgG4).

[0148] E18. The isolated antibody of E17, wherein the heavy chain constant domain comprises IgG.

[0149] E19. The isolated antibody of E18, wherein the IgG is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

[0150] E20. The isolated antibody of E19, wherein the IgG is IgG1.

[0151] E21. The isolated antibody of any one of E1-E20, wherein the antibody comprises an Fc domain.

[0152] E22. The isolated antibody of E21, wherein the Fc domain comprises an IgG1 heavy chain CH2 domain and an IgG1 heavy chain CH3 domain.

[0153] E23. An isolated antibody comprising:

[0154] (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

[0155] (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.

[0156] E24. An isolated antibody, comprising:

[0157] (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

[0158] (ii) a light 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: 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.

[0159] E25. An isolated antibody, comprising:

[0160] (i) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 8, and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 1;

[0161] (ii) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 23, and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 18;

[0162] (iii) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 35, and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 29;

[0163] (iv) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 44, and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 40;

[0164] (v) HC comprising or consisting of the amino acid sequence of SEQ ID NO:52, and LC comprising or consisting of the amino acid sequence of SEQ ID NO:49;

[0165] (vi) HC comprising or consisting of the amino acid sequence of SEQ ID NO: 60, and LC comprising or consisting of the amino acid sequence of SEQ ID NO: 56;

[0166] (vii) HC comprising or consisting of the amino acid sequence of SEQ ID NO: 69, and LC comprising or consisting of the amino acid sequence of SEQ ID NO: 64; or

[0167] (viii) HC comprising or consisting of the amino acid sequence of SEQ ID NO: 75, and LC comprising or consisting of the amino acid sequence of SEQ ID NO: 72.

[0168] E26. An isolated antibody, comprising:

[0169] (i) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 23, and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 18;

[0170] (ii) a HC comprising or consisting of the amino acid sequence of SEQ ID NO:52, and a LC comprising or consisting of the amino acid sequence of SEQ ID NO:49;

[0171] (iii) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 69, and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 64;

[0172] (iv) HC comprising or consisting of the amino acid sequence of SEQ ID NO: 75, and LC comprising or consisting of the amino acid sequence of SEQ ID NO: 72;

[0173] (v) a 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 a 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;

[0174] (vi) a 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 a 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;

[0175] (vii) a 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 a 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

[0176] (viii) a 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 a 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.

[0177] E27. An isolated antibody, comprising:

[0178] (i) HC comprising or consisting of the amino acid sequence of SEQ ID NO: 75, and LC comprising or consisting of the amino acid sequence of SEQ ID NO: 72; or

[0179] (ii) a 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 a 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.

[0180] E28. 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 Ala28, Pro29, Ser83, Leu85, Met90, Val92, Pro93, Leu95, and Tyr97 of SEQ ID NO:79.

[0181] E29. The isolated antibody of E28, wherein the antibody binds to Ala28, Pro29, Ser83, Leu85, Met90, Val92, Pro93, Leu95 and Tyr97 of SEQ ID NO:79.

[0182] E30. The isolated antibody of any one of E28-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.

[0183] E31. The isolated antibody of E30, wherein the antibody binds to Pro81, Tyr86, Asp88, Asp89, Thr94, Lys96, His98, Tyr99 and Glu100 of SEQ ID NO:79.

[0184] E32. The isolated antibody of any one of E28-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.

[0185] E33. The isolated antibody of E32, wherein the antibody binds to Ile27, Lys30 and Glu31 of SEQ ID NO:79.

[0186] E34. 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.

[0187] E35. An isolated antibody that binds to 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.

[0188] E36. 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 Ala28, Pro29, Pro81, Ser83, Leu85, Tyr86, Asp88, Asp89, Met90, Val92, Pro93, Thr94, Leu95, Lys96, Tyr97, His98, Tyr99 and Glu100 of SEQ ID NO:79.

[0189] E37. An isolated antibody that binds to 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.

[0190] E38. 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 Arg14, Ile27, Ala28, Pro29, Lys30, Glu31, Ser83, Leu85, Met90, Val92, Pro93, Leu95 and Tyr97 of SEQ ID NO:79.

[0191] E39. An isolated antibody that binds to 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.

[0192] E40. The isolated antibody of any one of E28-E39, wherein the epitope on BMP9 does not comprise amino acid residues 21-25, 43-60 of SEQ ID NO:79.

[0193] E41. An isolated antibody as described in any of E1-E40, wherein 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, as measured by surface plasmon resonance.

[0194] E42. The isolated antibody of E41, wherein the antibody binds to human BMP9 with an affinity of about 30.48 pM or about 87.41 pM at 37°C as measured by surface plasmon resonance.

[0195] E43. An isolated antibody as described in any of E1-E42, wherein 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, as measured by surface plasmon resonance.

[0196] E44. The isolated antibody of E43, wherein the antibody binds cynomolgus monkey BMP9 with an affinity of about 20.23 pM or about 65.82 pM at 37°C as measured by surface plasmon resonance.

[0197] E45. An isolated antibody as described in any of E1-E44, wherein the antibody 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, as measured by surface plasmon resonance.

[0198] E46. The isolated antibody of E45, wherein the antibody binds rat BMP9 with an affinity of about 41.75 pM or about 98.63 pM at 37°C as measured by surface plasmon resonance.

[0199] E47. The antibody of any one of E28-E31, E34-E37, E40-E46, wherein the antibody comprises:

[0200] (i) a heavy chain variable region (VH) CDR3 of SEQ ID NO: 26 and a light chain variable region (VL) CDR3 of SEQ ID NO: 84;

[0201] (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

[0202] (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.

[0203] E48. The isolated antibody of any one of E1-E47, wherein the antibody:

[0204] (i) binds to human BMP9 and inhibits the binding of human BMP9 to BMP9 type II receptor (e.g., BMPRII, ActRIIA or ActRIIB); or

[0205] (ii) binds to human BMP9 and inhibits the binding of human BMP9 to BMP9 type II receptor (e.g., BMPRII, ActRIIA or ActRIIB), and weakly inhibits the binding of human BMP9 to BMP9 type 1 receptor (e.g., ALK1).

[0206] E49. The isolated antibody of any one of E1-48, wherein the antibody binds to human BMP9 and inhibits the binding of human BMP9 to human endothelial cells.

[0207] E50. The isolated antibody of any one of E1-E49, wherein the antibody binds to human BMP9, but does not bind to other human transforming growth factor β (TGFβ) superfamily ligands (eg, GDF8, GDF11, activin A, GDF9, TGFβ1, and BMP10).

[0208] E51. The isolated antibody of any one of E1-E50, wherein the antibody inhibits BMP9 activity measured by phospho-SMAD1 / 5 / 9 nuclear translocation in endothelial cells.

[0209] E52. The isolated antibody of E51, wherein the antibody is expressed at 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 15 0 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 Inhibits human BMP9 activity.

[0210] E53. The isolated antibody of E51, wherein the antibody has 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 Inhibits rat BMP9 activity.

[0211] E54. The isolated antibody of E51, wherein the antibody has 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. 50 Inhibits BMP9 activity in cynomolgus monkeys.

[0212] E55. The isolated antibody of any one of E1-E54, wherein the antibody inhibits BMP9 activity measured by phospho-SMAD2 nuclear translocation in endothelial cells.

[0213] E56. The isolated antibody of E55, wherein the antibody has 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 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. 50 Inhibits BMP9 activity.

[0214] E57. An isolated antibody as described in any of E1-E56, wherein the antibody reduces the subject's right ventricular systolic pressure 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 administration of the antibody.

[0215] E58. An isolated antibody as described in any of E1-E57, wherein the antibody reduces right ventricular hypertrophy in the 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 administration of the antibody.

[0216] E59. The isolated antibody of any one of E57-E58, wherein the subject is a Sugen-hypoxia animal model of pulmonary arterial hypertension (PAH).

[0217] E60. An isolated polynucleotide encoding the antibody of any one of E1-E59, E91 or E92.

[0218] E61. The polynucleotide of E60, wherein the polynucleotide is RNA.

[0219] E62. The polynucleotide of E61, wherein said polynucleotide comprises at least one chemical modification.

[0220] E63. A polynucleotide as described in 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.

[0221] E64. The polynucleotide of E61, wherein the polynucleotide does not comprise a chemical modification.

[0222] 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.

[0223] E66. An isolated nucleic acid encoding the heavy chain, the 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.

[0224] 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 an insert of a plasmid deposited with ATCC and having accession number PTA-127292, the nucleic acid sequence of an insert of a plasmid deposited with ATCC and having accession number PTA-127293, or both.

[0225] E68. A vector comprising the polynucleotide of any one of E60-E64 or the nucleic acid of any one of E65-E67.

[0226] E69. An isolated host cell comprising the polynucleotide of any one of E60-E64 or the nucleic acid of any one of E65-E67.

[0227] E70. The host cell of E69, wherein the cell is a mammalian cell or an insect cell.

[0228] E71. The host cell of E70, wherein the host cell is a mammalian cell selected from the group consisting of: CHO cells, HEK-293 cells, NS0 cells, cells or Sp2.0 cells.

[0229] E72. A method for producing an antibody, the method comprising:

[0230] Cultivating the host cell of any one of E69-E71 under conditions resulting in production of the antibody; and recovering the antibody.

[0231] E73. A pharmaceutical composition comprising a therapeutically effective amount of an antibody as described in any one of E1-E59, E90 or E91 and a pharmaceutically acceptable carrier.

[0232] E74. A pharmaceutical composition as described in E73, wherein the composition comprises 1.12 mg / mL L-histidine, 2.67 mg / mL L-histidine hydrochloride monohydrate, 85 mg / mL sucrose, 0.05 mg / mL EDTA disodium dihydrate and 0.2 mg / mL polysorbate 80, and the pH is 5.8.

[0233] E75. The pharmaceutical composition of any one of E73-E74, wherein the composition comprises 20 mM histidine, 8.5% sucrose and 0.02% polysorbate 80, 0.005% EDTA, and has a pH of 5.8.

[0234] E76. A pharmaceutical composition as described in any of E72-E74, wherein the composition comprises 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 of the antibody.

[0235] E77. The pharmaceutical composition of any one of E73-E76, wherein the composition comprises about 50 mg / mL, about 100 mg / mL, about 150 mg / ml or about 200 mg / mL of the antibody.

[0236] E78. The pharmaceutical composition of any one of E73-E77, wherein the dose is a 1 mL dose.

[0237] E79. The pharmaceutical composition of E73-E78, wherein the composition is suitable for subcutaneous (SC) and / or intravenous (IV) administration.

[0238] 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 a pharmaceutical composition as described in any one of E73-E79 or an antibody as described in any one of E1-E59, E90 or E91.

[0239] E81. A method for treating hypertension in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition as described in any one of E73-E79 or an antibody as described in E1-E59, E90 or E91.

[0240] E82. The method of E81, wherein the hypertension is pulmonary hypertension.

[0241] E83. A method of treating pulmonary arterial hypertension (PAH) in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition as described in any one of E73-E79 or an antibody as described in E1-E59, E90 or E91.

[0242] E84. The method of any one of E80-E83, wherein the antibody is administered in combination with a therapeutically effective amount of one or more additional therapeutically active compounds or treatment modalities effective to treat at least one sign and / or symptom of hypertension, optionally pulmonary hypertension.

[0243] E85. The method of E84, wherein the combination therapies are administered according to the same dosing schedule (eg, both therapies are administered daily) or according to different dosing schedules (eg, one therapy is administered daily and the other therapy is administered weekly).

[0244] E86. The isolated antibody of any one of E1-E59, E90 or E91 or the pharmaceutical composition of any one of E73-E79 for use as a medicament.

[0245] E87. The isolated antibody of any one of E1-E59, E90 or E91 or the pharmaceutical composition of any one of E73-E79 for use in treating at least one sign and / or symptom of hypertension, optionally pulmonary hypertension.

[0246] E88. Use of the isolated antibody of any one of E1-E59, E90 or E91 or the pharmaceutical composition of any one of E73-E79 in the manufacture of a medicament for treating at least one sign and / or symptom of hypertension, optionally pulmonary hypertension.

[0247] E89. The pharmaceutical composition of any one of E73-E79 or the isolated antibody of any one of E1-E59, E90 or E91, for use in treating at least one sign and / or symptom of hypertension, optionally pulmonary hypertension.

[0248] E90. Use of the isolated antibody of any one of E1-E59, E91 or E92 or the pharmaceutical composition of any one of E73-E79 for treating at least one sign and / or symptom of hypertension, optionally pulmonary hypertension.

[0249] E91. The isolated antibody of any one of E1-E59, wherein the isolated antibody inhibits one or more proteins associated with hypertension, optionally pulmonary hypertension.

[0250] E92. The isolated antibody of any one of E1-E59 or E91, wherein the isolated antibody inhibits CXCL12, IGFBP4, INHBA, MALL, FRZB, CPE, CCL2, PDGFBB, endothelin-1, or a combination thereof.

[0251] E93. A method as described in any of E80-E85, wherein the pharmaceutical composition as described in any of E73-E79 or the antibody as described in any of E1-E59, E90 or E91 inhibits one or more proteins associated with hypertension, optionally pulmonary hypertension.

[0252] E94. The method of any one of E80-E85 or E93, wherein the pharmaceutical composition of any one of E73-E79 or the antibody of any one of E1-E59, E91 or E92 inhibits CXCL12, IGFBP4, INHBA, MALL, FRZB, CPE, CCL2, PDGFBB, endothelin-1 or a combination thereof.

[0253] E100. A method for monitoring the effectiveness of a pulmonary arterial hypertension (PAH) therapy in a subject in need thereof, the method comprising: (i) administering a 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 same one or more biomarkers before administration of the PAH therapy, wherein a change (e.g., a decrease) in the level of the one or more biomarkers after administration of the PAH therapy indicates that the therapy is effective, and wherein the PAH therapy comprises an isolated antibody as described in any one of E1-E59, E91 or E92.

[0254] E101. The method as described in E100, wherein the biological sample comprises urine, blood, serum, plasma, saliva, sputum, exhaled breath condensate, bronchoalveolar lavage fluid, cerebrospinal fluid, tears, tissue or mucus or a combination thereof.

[0255] E102. The method of any one of E100-E101, wherein determining the level of one or more biomarkers comprises determining mRNA levels.

[0256] E103. The method of any one of E100-E101, wherein determining the level of one or more biomarkers comprises determining protein levels.

[0257] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0258] All references cited herein (including literature references, patent applications, patent publications, UniProtKB accession numbers, and GenBank accession numbers) are hereby specifically and expressly incorporated by reference, as if each individual reference was specifically and individually indicated to be incorporated by reference in its entirety. In the event of a definition of a term in a document incorporated by reference that conflicts with the definition used herein, the definition used herein shall prevail.

[0259] The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are commonly employed using conventional methods, such as, for example, the widely used methods described in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (3rd ed.) (2001) (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., eds.) (2003); METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor, eds.) (1995); ANTIBODIES, A LABORATORY TECHNOLOGY (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); ANTIBODIES: A LABORATORY MANUAL (Harlow and Lane, eds.) (1988), and ANIMAL CELL CULTURE (RI Freshney, ed.) (1987); OLIGONUCLEOTIDE SYNTHESIS (MJ Gait, ed.) (1984); METHODS IN MOLECULAR BIOLOGY (Humana Press); CELL BIOLOGY: A LABORATORY NOTE BOOK (JE Cellis, ed.) (1998) (Academic Press); INTRODUCTION TO CELL AND TISSUE CULTURE (JP Mather and PE Roberts, ed.) (1998) (Plenum Press); CELL AND TISSUE CULTURE LABORATORY PROCEDURES [Cell and Tissue Culture Laboratory Procedures] (A. Doyle, JB Griffiths and DG Newell, eds.) (1993-8) (J. Wiley and Sons); HANDBOOK OF EXPERIMENTAL IMMUNOLOGY [Handbook of Experimental Immunology] (D.GENE TRANSFER VECTORS FOR MAMMALIAN CELLS (JM Miller and MP Calos, eds.) (1987); PCR: THE POLYMERASE CHAIN ​​REACTION (Mullis et al., eds.) (1994); CURRENT PROTOCOLS IN IMMUNOLOGY (JE Coligan et al., eds.) (1991); SHORT PROTOCOLS IN MOLECULAR BIOLOGY (1999) (Wiley and Sons); IMMUNOBIOLOGY (CA Janeway and P. Travers, 1997); ANTIBODIES (P. Finch, 1997); ANTIBODIES: APRACTICAL APPROACH [Antibodies: A Practical Approach] (D. Catty., ed.) (1988-1989) (IRL Press); MONOCLONAL ANTIBODIES: A PRACTICAL APPROACH [Monoclonal Antibodies: A Practical Approach] (P. Shepherd and C. Dean, eds.) (2000) (Oxford University Press); USING ANTIBODIES: A LABORATORY MANUAL [Using Antibodies: A Laboratory Manual] (E. Harlow and D. Lane (1999) (Cold Spring Harbor Laboratory Press); THE ANTIBODIES [Antibodies] (M. Zanetti and JD Capra, eds.) (1995) (Harwood Academic Publishers); and updated versions thereof.

[0260] definition

[0261] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention have the meanings that are commonly understood by one of ordinary skill in the art.

[0262] As used herein, the use of the words "a" or "an" or "the" when used in conjunction with the terms "comprising," "including," "having," or "containing," or variations of these terms, can mean "one," but it is also 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.

[0263] Where aspects or embodiments of the invention are described according to Markush groups or other alternative groupings, the invention encompasses not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, and also encompasses the main group without one or more of the group members present. The invention also contemplates the explicit exclusion of one or more of any group members in the claimed invention.

[0264] The phrase "and / or" means "and" or "or." For 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" operates as an inclusive or.

[0265] Any one or more examples following the term "eg" or "for example" are not intended to be exhaustive or limiting.

[0266] As used herein, the terms "about" and "approximately" and "substantially" when used to modify a numerically defined parameter (e.g., an "X" dose) mean that the parameter can vary up to 10% lower or higher than the stated value of the parameter. Thus, in any disclosed aspect, these terms can be replaced with a specified "within [percentage] of ...". In one non-limiting aspect, percentages include 0.1%, 0.5%, 1%, 5%, and 10%. For example, a dose of about 5 mg means 5 mg ± 10%, i.e., it can vary between 4.5 mg and 5.5 mg.

[0267] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein.

[0268] The phrase "substantially all" is defined as "at least 95%"; if substantially all members of a group have a certain property, then at least 95% of the members of the group have that property. In some aspects, substantially all means that equal to any one of 95%, 96%, 97%, 98%, 99%, or 100% of the members of the group, at least any one of them, or between any two of them (including or excluding the end values) have that property.

[0269] Compositions and methods of use thereof may "comprise," "consist essentially of," or "consist of" any ingredient or step disclosed throughout this specification. Unless the context requires otherwise, throughout this specification, 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 open-ended, and will be understood to imply the inclusion of a stated step or element or groups of steps or elements, but not the exclusion of any other step or element or groups of steps or elements. Thus, compositions and methods of the present disclosure that "comprise," "have," "include," or "contain" one or more elements possess those one or more elements, but are not limited to possessing only those one or more elements. Similarly, elements of compositions or methods of the present disclosure that "comprise," "have," "include," or "contain" one or more features possess those one or more features, but are not limited to possessing only those one or more features.

[0270] It is contemplated that aspects described herein in the context of the term "comprising" may also be implemented in the context of the term "consisting of" or "consisting essentially of." Compositions and methods that "consist essentially of any of the disclosed ingredients or steps" limit the scope of the claim to the specified materials or steps that do not materially affect the basic and novel characteristics of the claimed disclosure. The phrase "consisting of" (and any forms of consisting of, such as "consisting of" and "consists of") is meant to include and be limited to any words that follow the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0271] Reference throughout this specification to "an aspect," "aspect," "particular aspects," "related aspects," "an aspect," "another aspect," "other aspects," "one embodiment," "an embodiment," "particular embodiments," "related embodiments," "an embodiment," "another embodiment," "other embodiments," or combinations thereof means that a particular characteristic, structure, or feature described in conjunction with an aspect is included in at least one aspect or embodiment of the present disclosure. Therefore, the appearances of the aforementioned phrases in various places throughout this specification do not necessarily all refer to the same aspect or embodiment. In addition, particular characteristics, structures, and / or features may be combined in any suitable manner in one or more aspects or embodiments.

[0272] "Antibody" refers to an immunoglobulin molecule that is capable of specifically binding 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 antibody, bispecific antibody), and includes intact antibody portions (e.g., "antigen-binding fragments") that retain the ability to bind to a given antigen and any other modified configurations of immunoglobulin molecules that contain an antigen binding site.

[0273] Antibody includes any classification such as IgG, IgA or IgM (or its subclass), and antibody need not be any particular class. According to the antibody amino acid sequence of the constant region of the heavy chain (HC) of immunoglobulin, immunoglobulin can be assigned to different classes. There are following five main immunoglobulin classes: IgA, IgD, IgE, IgG and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant region corresponding to different immunoglobulin classes is respectively referred to as α, δ, ε, γ and μ. The subunit structure and the three-dimensional configuration of different immunoglobulin classes are well known. It is expected that any one or more of the aforementioned immunoglobulin or its subclass can be excluded from the embodiments.

[0274] Examples of antibody antigen-binding fragments and modified configurations include (i) Fab fragments (monovalent fragments consisting of VL, VH, CL and CH1 domains); (ii) F(ab')2 fragments (bivalent fragments comprising two Fab fragments connected by a disulfide bridge at the hinge region); and (iii) Fv fragments consisting of the VL and VH domains of a single antibody arm. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be connected by synthetic linkers using recombinant methods, enabling them to be made into a single protein chain in which the VL and VH regions are paired to form monovalent molecules (called single-chain Fv (scFv)); see, for example, Bird et al., Science [Science] 1988; 242: 423-426 and Huston et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 1988 85: 5879-5883. Other forms of single-chain antibodies, such as diabodies, are also contemplated. It is contemplated that any one or more of the foregoing antibody antigen-binding fragments or modified configurations thereof may be excluded from the Examples.

[0275] In addition, antibodies (e.g., human IgG1 heavy chains contain terminal lysine) that lack C-terminal lysine (K) amino acid residues on the heavy chain polypeptide are further contemplated. As known in the art, the C-terminal lysine is sometimes cut off during antibody production to produce antibodies with heavy chains lacking C-terminal lysine. Alternatively, nucleic acids can be used to produce antibody heavy chains that do not contain C-terminal lysine. Therefore, the present disclosure includes such a composition, which includes an anti-BMP9 antibody with a heavy chain lacking a C-terminal lysine residue. In certain embodiments, the present disclosure includes such a composition, which includes an anti-BMP9 antibody with a heavy chain containing a C-terminal lysine residue and an anti-BMP9 antibody with a heavy chain lacking a C-terminal lysine residue. In certain embodiments, the present disclosure includes such a composition, which includes an anti-BMP9 antibody lacking a C-terminal lysine residue.

[0276] The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. As known in the art, the variable regions of the heavy and light chains are each composed of four framework regions (FR) connected by three complementary determining regions (CDRs) (also referred to as hypervariable regions), and contribute to the formation of the antigen binding site of the antibody. If a variant of the subject variable region is desired, particularly one with substitutions in amino acid residues outside the CDR region (i.e., in the framework region), then the subject variable region can be compared with the variable regions of other antibodies containing CDR1 and CDR2 sequences in the same classical class as the subject variable region to identify appropriate amino acid substitutions, preferably conservative amino acid substitutions (Chothia and Lesk, J. Mol. Biol. [Journal of Molecular Biology] 196 (4): 901-917, 1987).

[0277] In certain embodiments, the clear description of CDR and the identification of residues comprising antibody binding sites are completed by analyzing the structure of the antibody or analyzing the structure of the antibody-ligand complex. In certain embodiments, this can be completed by any of a variety of techniques known to those skilled in the art (such as X-ray crystallography). In certain embodiments, various analytical methods can be used to identify or simulate CDR regions. In certain embodiments, various analytical methods can be used to identify or simulate CDR regions. Examples of such methods include but are not limited to Kabat definition, Chothia definition, AbM definition, contact definition, extended definition and conformational definition.

[0278] The Kabat definition is a standard for numbering residues in antibodies and is typically used to identify CDR regions. See, e.g., Johnson and 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 position of certain structural loop regions. See, e.g., 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 and Chothia definitions. The AbM definition uses an integrated suite of computer programs produced by the Oxford Molecular Group that model antibody structure. See, e.g., Martin et al., 1989, Proc. Natl. Acad. Sci. (USA), 86:9268-9272; "AbM TM,A Computer Program for Modeling Variable Regions of Antibodies[AbM TM [A computer program for modeling antibody variable regions]," Oxford, United Kingdom; Oxford Molecular, Ltd. [Oxford Molecular Ltd.]. The AbM definition uses a combination of knowledge databases and de novo methods (e.g., Samudrala et al., 1999, "AbInitio Protein Structure Prediction Using a Combined Hierarchical Approach," PROTEINS, Structure, Function and The tertiary structure of the antibody is modeled from the primary sequence (see, e.g., those described in Proteins, Structure, Function and Genetics Suppl., 3:194-198). The contact definitions are based on analysis of available complex crystal structures. See, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45. In another approach, referred to herein as "conformational definition" of the CDRs, the positions of the CDRs can be identified as residues that make enthalpic contributions to antigen binding. See, e.g., Makabe et al., 2008, J. Biol. Chem., 283:1156-1166. Still other CDR boundary definitions may not strictly follow one of the above approaches, but will still be consistent with Kabat's method. At least a portion of the CDR overlaps, although they can be shortened or extended based on predictions or experimental findings that a specific residue or residue group does not significantly affect antigen binding. As used herein, CDR can refer to a CDR defined by any method known in the art (including a combination of methods). The method used herein can utilize CDRs defined according to any of these methods. For any given embodiment containing more than one CDR, CDR can be defined according to any one or more of the following: Kabat definition, Chothia definition, extended definition, AbM definition, contact definition and / or conformational definition. It is expected that any one or more of these definitions can be excluded in the embodiments.

[0279] The "constant region" of an antibody refers to the constant region of an antibody light chain or the constant region of an antibody heavy chain, alone or in combination. The IgG heavy chain constant region contains three consecutive 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).

[0280] "Fc domain" refers to the portion of an immunoglobulin (Ig) molecule associated with a crystallizable fragment obtained by papain digestion of the Ig molecule. As used herein, the term relates to a two-chain constant region of an antibody, each chain excluding the first constant region immunoglobulin domain. Within the Fc domain, there are two "Fc chains" (e.g., "the first Fc chain" and "the second Fc chain"). "Fc chain" generally refers to the C-terminal portion of an antibody heavy chain. Therefore, the 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 at the N-terminus of these domains.

[0281] Although the boundaries of the Fc chain can vary, a human IgG heavy chain Fc chain is generally defined as comprising residues C226 or P230 at its carboxyl terminus, where numbering is according to the EU index as described in Edelman et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 1969; 63(1):78-85 and as described in Kabat et al., 1991. Typically, an Fc chain comprises from about amino acid residues 236 to about 447 of the constant region of a human IgG1 heavy chain. "Fc chain" may refer to this polypeptide in isolation or in the context of a larger molecule (e.g., in an antibody heavy chain or Fc fusion protein).

[0282] A "functional" Fc domain refers to an Fc domain having at least one effector function of a 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, etc. Such effector functions generally require an Fc domain in combination with a binding domain (e.g., an antibody variable region), and can be assessed using various assays known in the art for evaluating such antibody effector functions. A "native sequence" Fc chain refers to an Fc chain comprising an amino acid sequence identical to that of an Fc chain found in nature. A "variant" Fc chain comprises an amino acid sequence that is different from the amino acid sequence of a native sequence Fc chain due to at least one amino acid modification. It is contemplated that any one or more of the aforementioned Fc chains or domains may be excluded from the embodiments.

[0283] "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 directly antagonize a single antigenic site. In addition, in contrast to polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the characteristics of an antibody obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring antibodies to be produced by any particular method. For example, the monoclonal antibody to be used according to the present disclosure can be prepared by the hybridoma method first described by Kohler and Milstein, 1975, Nature [nature] 256: 495, or can be prepared by a recombinant DNA method (as described in U.S. Patent No. 4,816,567). In another example, monoclonal antibodies can be separated from phage libraries (such as those produced using the technology described in McCafferty et al., 1990, Nature [nature] 348: 552-554).

[0284] "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 has been prepared using any technique for preparing a fully human antibody. For example, a fully human antibody can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulins or by display techniques for libraries (e.g., phage, yeast or ribosomes) for preparing fully human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues.

[0285] "Chimeric antibody" refers to an antibody whose variable region sequence is derived from one species and whose constant region sequence is derived from another species, such as an antibody whose variable region sequence is derived from a mouse antibody and whose constant region sequence is derived from a human antibody.

[0286] "Humanized" antibodies refer to non-human (e.g., mouse) antibodies, which are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. Preferably, humanized antibodies are such human immunoglobulins (receptor antibodies) in which residues from the CDRs of receptors are replaced by residues from the CDRs of non-human species (donor antibodies) (such as mice, rats or rabbits) with desired specificity, affinity and ability. Humanized antibodies may include residues that are neither found in the receptor antibody nor in the CDR or framework sequences introduced, but are included to further refine and optimize antibody performance.

[0287] It is contemplated that any one or more of the antibody types described herein may be excluded from the Examples.

[0288] "Antigen" refers to a molecular entity that is used to immunize an immunocompetent vertebrate to produce antibodies that recognize the antigen or to screen expression libraries (e.g., phage, yeast or ribosome display libraries, etc.) for antibody selection. In this article, antigens are more broadly defined and are generally intended to include target molecules that are specifically recognized by antibodies, thus including fragments or mimetics of molecules used in the immunization process to produce antibodies or used in library screening to select antibodies.

[0289] "Epitope" refers to an antigen region or region that specifically binds to an antibody, such as a region or region 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 position of an epitope on a protein, including crystal structures, competitive assays, gene fragment expression assays, epitope mapping, and synthetic peptide-based assays of parsing antibody-antigen complexes, such as described in Harlow and Lane, USING ANTIBODIES, A LABORATORY MANUAL [Using Antibodies: Laboratory Manual], Cold Spring Harbor Laboratory Press [Cold Spring Harbor Laboratory Press], Cold Spring Harbor, New York, Chapter 11 of 1999. Additionally or alternatively, during the discovery process, the generation and characterization of antibodies can illustrate information about a desired epitope. Based on this information, it is then possible to screen antibodies for binding to the same epitope competitively.

[0290] In addition, the epitope combined with the antibody can be determined in the system screening by using overlapping peptides derived from the antigen and determining the combination of the antibody. According to the gene fragment expression assay, the open reading frame encoding the antigen can be randomly or fragmented by specific genetic construction, and the reactivity of the expressed fragment of the antigen and the antibody to be tested is determined. The gene fragment can be produced, for example, by PCR, and then transcribed in vitro and translated into protein in the presence of radioactive amino acids. The combination of the antibody and the radiolabeled antigen fragment is then determined by immunoprecipitation and gel electrophoresis.

[0291] Certain 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, a defined library of overlapping peptide fragments can be tested for binding to a test antibody in a simple binding assay. In other examples, antigenic mutagenesis, domain exchange experiments, and alanine scanning mutagenesis can be performed to identify residues required, sufficient, or essential for epitope binding.

[0292] In its most detailed level, the epitope that interacts between antigen and antibody can be defined by the spatial coordinates of the atomic contacts present in the antigen-antibody interaction and the information about their relative contribution to binding thermodynamics. In a less detailed level, the epitope can be characterized by the spatial coordinates of the atomic contacts between the antigen and antibody. In a further less detailed level, the epitope can be characterized by the amino acid residues (as defined by a specific standard) contained therein, such as by the distance between the atom (for example, heavy atoms, i.e. non-hydrogen atoms) in the antibody and the antigen. In a further less detailed level, the epitope can be characterized by function (for example, by combining with competition of other antibodies). The epitope can also be more generally defined as comprising such an amino acid residue, which is replaced by another amino acid and will change the interactive feature (for example, using alanine scanning) between the antibody and the antigen.

[0293] From the fact that descriptions and definitions of epitopes (depending on the epitope mapping method used) are obtained at different levels of detail, it follows that comparisons of epitopes of different antibodies on the same antigen can similarly be made at different levels of detail.

[0294] Epitopes described at the amino acid level (e.g., as 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 they share at least one amino acid. Epitopes are said to be separate (unique) if they do not share amino acid residues.

[0295] Another method that can be used to characterize antibodies is to use competition assays with other antibodies known to bind to the same antigen to determine whether the target antibody binds to the same epitope as the other antibodies. Competition assays are well known to those skilled in the art. If the binding of the corresponding antibodies is mutually exclusive, i.e., the binding of one antibody excludes the simultaneous or continuous binding of another antibody, then it is said that the epitopes characterized by competitive binding are overlapping. If the antigen can accommodate the binding of two corresponding antibodies at the same time, then the epitopes are said to be separate (unique).

[0296] Epitopes can be linear or conformational. In a linear epitope, all interaction points between a protein and an interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. A "non-linear epitope" or "conformational epitope" comprises non-contiguous polypeptides (or amino acids) within an antigenic protein that bind to an antibody specific for the epitope.

[0297] The term "binding affinity" refers to the strength of the sum of 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 indicated, as used herein, "binding affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D ). Affinity can be measured by common methods known in the art. Low-affinity antibodies generally bind antigen slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigen faster and tend to remain bound longer. Specifically, the term "binding affinity" is intended to refer to the dissociation rate of a specific antigen-antibody interaction. D is the rate of dissociation (also called the off-rate (k off )" or "k d ”) and the association rate or “on-rate” (k on )" or "k a ". Therefore, K D Equal to k off / k on (or k d / k a ) and is expressed as a molar concentration (M). It can be seen that K D The smaller the value, the stronger the binding affinity. D Compared with 1 μM K D Indicates a weaker binding affinity. The K of an antibody D The K value can be determined using methods established in the art. D An exemplary method is by using surface plasmon resonance (SPR), typically using a biosensor system such as BIACORE TM system). BIACORE TM Kinetic analysis involves analyzing the binding and dissociation of an antigen to a chip having immobilized molecules (e.g., molecules comprising an epitope binding domain) on its surface. D Another approach is through the use of biolayer interferometry, typically using technology( QK e System, ForteBio, now Sartorius). Alternatively or additionally, the ELISA system available from Sapidyne Instruments (Boise, Idaho) may also be used. (Kinetic Exclusion Assay) determination.

[0298] "Monospecific antibody" refers to an antibody that contains one or more antigen binding sites per molecule, such that any and all binding sites of the antibody specifically recognize the same epitope on the antigen. Thus, where a monospecific antibody has more than one antigen binding site, the binding sites compete with each other for binding to one antigen molecule.

[0299] "Bispecific antibody" refers to a molecule that has binding specificity for at least two different epitopes. In some embodiments, a bispecific antibody can bind to two different antigens simultaneously. In other embodiments, the two different epitopes can be located on the same antigen.

[0300] The term “half maximal effective concentration (EC 50 )” refers to the concentration of the therapeutic agent that elicits a response halfway between baseline and maximum after a specified exposure time. EC 90 The concentration of a therapeutic agent that corresponds to 90% of the maximum possible response after a specified exposure time. Therapeutic agents can cause either inhibition or stimulation. EC 50 value and is used herein as a measure of efficacy.

[0301] The term "half maximal inhibitory concentration (IC 50 )" refers to the concentration of the therapeutic agent required to inhibit 50% of the maximal response achieved.

[0302] "Agonist" refers to a substance that promotes (ie, induces, causes, enhances and / or increases) the biological activity or action of another molecule. The term agonist encompasses substances (such as antibodies) that bind to a molecule to promote the activity of that molecule.

[0303] "Antagonist" refers to a substance that prevents, blocks, inhibits, neutralizes and / or reduces the biological activity or action of another molecule (such as a receptor). The term antagonist encompasses substances (such as antibodies) that bind to a molecule to prevent or reduce the activity of that molecule.

[0304] As used herein with respect to antibodies, the term "competition" means that the first antibody binds to the epitope in a sufficiently similar manner to the binding of the second antibody, so that the binding of the second antibody in the absence of the first antibody is compared to the binding of the second antibody in the presence of the first antibody, and the result of the binding of the second antibody to its cognate epitope can be detectably reduced. The alternative scheme that the binding of the first antibody to its epitope can also be detectably reduced in the presence of the second antibody can but need not be the case. That is, the first antibody can inhibit the binding of the second antibody to its epitope, and the second antibody does not inhibit the binding of the first antibody to its corresponding epitope. However, in the case where each antibody can detectably inhibit the binding of another antibody to its cognate epitope or ligand (whether in the same, greater or lesser degree), these antibodies are said to "cross-compete" with each other to bind to one or more epitopes of each of them. Both competitive antibodies and cross-competitive antibodies are covered by this disclosure. Regardless of the mechanism (e.g., steric hindrance, conformational changes and / or binding to a common epitope or part thereof) of such competition or cross-competition, the technical staff will understand that based on the teachings provided herein, such competitive or cross-competitive antibodies are covered and can be used for the methods disclosed herein.

[0305] "Fc receptor" (FcR) refers to a receptor that binds to the Fc region of an antibody. In some embodiments, FcR is a natural human FcR. In some embodiments, FcR is a receptor that binds to an IgG antibody (gamma receptor), and includes receptors of the FcgRI, FcgRII, and FcgRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcgRII receptors include FcgRIIA ("activating receptor") and FcgRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ primarily 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 inhibition motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. [Annual Review of Immunology] 1997; 15: 203-234). FcRs are reviewed in, e.g., 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 is responsible for 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 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).

[0306] "Effector cells" refer to leukocytes that express one or more FcRs and perform effector functions. In certain embodiments, effector cells express at least FcgRIII and perform one or more ADCC effector functions. 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 (e.g., from blood).

[0307] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to target cells carrying antigens and then kill target cells with cytotoxins. Primary cells NK cells used to mediate ADCC express only FcgRIII, while monocytes express FcgRI, FcgRII, and FcgRIII. In order to assess the ADCC activity of target molecules, in vitro ADCC assays can be performed, such as those described in U.S. Patent Nos. 5,500,362, 5,821,337, or 6,737,056. Useful effector cells for such assays include PBMC and NK cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo (e.g., in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 1998; 95: 652-656). Additional antibodies with altered Fc region amino acid sequences and increased or decreased ADCC activity are described, for example, in U.S. Pat. No. 7,923,538 and U.S. Pat. No. 7,994,290.

[0308] The term "enhanced ADCC activity" refers to an antibody that mediates ADCC more effectively in vitro or in vivo than a parent antibody, wherein the antibody and the parent antibody differ in at least one structural aspect and when the amounts of such antibody and the parent antibody used in the assay are substantially the same. In some embodiments, the antibody and the parent antibody have the same amino acid sequence, but the antibody is defucosylated and the parent antibody is fucosylated. In some embodiments, an in vitro ADCC assay will be used to determine ADCC activity, but other assays or methods for determining ADCC activity are contemplated, such as in an animal model, etc. In some embodiments, an antibody with enhanced ADCC activity has an enhanced affinity for FcgRIIIA.

[0309] The term "altered" FcR binding affinity or ADCC activity refers to an antibody with enhanced or decreased activity for one or more of FcR binding activity or ADCC activity compared to a parent antibody, wherein the antibody and the parent antibody differ in at least one structural aspect. An antibody that "exhibits increased binding to an FcR" binds to at least one FcR with better affinity than the parent antibody. An antibody that "exhibits decreased binding to an FcR" binds to at least one FcR with lower affinity than the parent antibody. Such antibodies that exhibit decreased binding to an FcR may have little or no appreciable binding to an FcR, e.g., 0-20% binding to an FcR compared to a native sequence IgG Fc region.

[0310] 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 (Clq) of the complement system to antibodies (of the appropriate subclass) that bind to their cognate antigens. To assess complement activation, a CDC assay may be performed, for example as described in Gazzano-Santoro et al., J. Immunol. Methods 1996; 202: 163. Antibodies with altered Fc region amino acid sequences and increased or decreased Clq binding capacity are described, for example, in U.S. Pat. No. 6,194,551, U.S. Pat. No. 7,923,538, U.S. Pat. No. 7,994,290, and WO 1999 / 51642.

[0311] "Host cell" refers to a single cell or cell culture that can be or has been a recipient of one or more vectors for incorporating a polynucleotide insert. Host cells include the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental and / or deliberate mutation. Host cells include cells transfected in vivo with one or more polynucleotides disclosed herein.

[0312] "Vector" refers to a construct capable of delivering and preferably expressing one or more target genes or sequences (e.g., antibody encoding genes) in a host cell. Examples of vectors include, but are not limited to, plasmids and viral vectors, and may include naked nucleic acids, or may include nucleic acids associated with delivery auxiliary materials (e.g., cationic condensing agents, liposomes, etc.). The vector may include DNA or RNA. "Expression vector" as used herein refers to a vector comprising at least one polypeptide encoding gene, at least one regulatory element (e.g., promoter sequence, poly (A) sequence) associated with the transcription or translation of the gene. Typically, the vector used herein contains at least one antibody encoding gene, and one or more of a regulatory element or a selective marker. The vector components may include, for example, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcription control elements (e.g., promoters, enhancers, and terminators). In order to translate, one or more translation control elements may also be included, such as ribosome binding sites, translation start sites, and stop codons. It is contemplated that any one or more of these aforementioned components or elements may be excluded in an embodiment.

[0313] An "isolated" molecule (e.g., an antibody) is one that, by virtue of its source or source of derivation, is (1) not associated with naturally associated components that accompany it in its native state, (2) is substantially free of other molecules from the same source (e.g., species, cell expressing it, library, etc.), (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized or expressed in a cellular system that is different from the system from which it originates will be "isolated" from its naturally associated components. A molecule may also be rendered substantially free of naturally associated components by isolation using purification techniques well known in the art.

[0314] "Polypeptide" or "protein" (used interchangeably herein) refers to an amino acid chain of any length. The chain may be linear or branched. The chain may contain one or more of the modified amino acids. These terms also encompass amino acid chains that have been modified naturally or by intervention (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, and / or any other manipulation or modification, such as conjugation with a labeling component). Also included within the definition are, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. It should be understood that a polypeptide may exist as a single chain or an associated chain.

[0315] "Polynucleotide" or "nucleic acid" (used interchangeably herein) refers to a chain of nucleotides of any length, and includes DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, or analogs thereof, or any substance that can be incorporated into the chain by DNA or RNA polymerase. The polynucleotide may contain modified nucleotides (such as methylated nucleotides) and their analogs. If present, modifications to the nucleotide structure may be imparted before or after chain assembly. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example by conjugation with a labeling component. Other types of modifications include, for example, "caps", substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing overhangs such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), and unmodified forms of the one or more polynucleotides. Further, any hydroxyl group typically present in a sugar may be replaced, for example, by a phosphonate group, a phosphate group, protected by a standard protecting group, or activated to prepare additional linkages with additional nucleotides, or may be conjugated to a solid support. 5' and 3' terminal OH can be phosphorylated, or partially replaced by amine or organic capping groups of 1 to 20 carbon atoms. Other hydroxyls can also be derived as standard protecting groups. Polynucleotides can also contain similar forms of ribose or deoxyribose commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α- or β-anomeric sugars, epimeric sugars (such as arabinose, xylose or lyxose, pyranose, furanose, sedoheptulose), acyclic analogs and abasic nucleoside analogs (such as methyl ribonucleoside). It is expected that any one or more of these aforementioned modifications can be excluded in the embodiments.

[0316] "Conservative substitution" refers to an amino acid replaced 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 lengths, such as alanine, glycine and serine or similar sizes. Chemical similarity means that the residues have the same charge or both are hydrophilic or hydrophobic. Specific examples include a hydrophobic residue (such as isoleucine, valine, leucine or methionine) replaced by another hydrophobic residue, or a polar residue replaced by another polar residue, such as arginine replaced lysine, glutamic acid replaced aspartic acid or glutamine replaced asparagine, serine replaced threonine, etc. Specific examples of conservative substitutions include a hydrophobic residue (such as isoleucine, valine, leucine or methionine) replaced by another hydrophobic residue, a polar residue replaced by another polar residue, such as arginine replaced lysine, glutamic acid replaced aspartic acid or glutamine replaced asparagine, etc. Conservative amino acid substitutions typically include, for example, substitutions within the following group: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0317] The term "identity" or "same as..." refers to the overall relatedness between polymer molecules (e.g., between nucleic acid molecules (e.g., DNA molecules or RNA molecules) or between polypeptide molecules). "Identity" measures the percentage of identical matches between two or more sequences, and gapped alignments are solved by a specific mathematical model of a computer program (e.g., an algorithm), which is well known in the art.

[0318] The terms "increase," "improve," "decrease," or "reduction" refer to values ​​relative to a baseline measurement, such as a measurement in the same individual prior to the start of a treatment as described herein, or a measurement in a control individual or subject (or multiple control individuals or subjects) in the absence of a treatment as described herein. In some embodiments, a "control individual" is an individual suffering from the same form of disease or injury as the individual being treated. In some embodiments, a "control individual" is an individual who does not suffer from the same form of disease or injury as the individual being treated.

[0319] The term "excipient" refers to any material that allows the active ingredient to retain biological activity in combination with the target active ingredient (e.g., antibody). The selection of excipient will depend to a great extent on factors such as the following: mode of administration, the influence of excipient on solubility and stability and the properties of the dosage form. As used herein, "excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, carriers, diluents, etc. that are compatible physiologically. The example of excipient includes one or more of the following: water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc. and combinations thereof, and isotonic agents, such as sugar, sodium chloride and / or polyols (such as mannitol or sorbitol) can be included in the composition. It is expected that any one or more of these aforementioned excipients can be excluded in an embodiment.

[0320] The terms "treating", "treat" or "treatment" refer to any type of treatment, such as, for example, alleviating, relieving, ameliorating and / or slowing the progression of a patient's disease, disorder or condition or any tissue damage associated with the disease. In some embodiments, the disease, disorder or condition is hypertension, optionally pulmonary hypertension.

[0321] The terms "prevent" or "prevention" refer to one or more of the following: a delay in the onset, reduction in frequency, and / or reduction in severity of at least one sign or symptom (e.g., shortness of breath, fatigue, dizziness, chest tightness or pain, swelling (edema), cyanosis, palpitations, ventricular hypertrophy, and / or decreased function of the right portion of the heart (leading to heart failure for pulmonary hypertension)) of a particular disease, disorder, or condition (e.g., pulmonary hypertension). In some embodiments, prevention is assessed on a population basis such that an agent is considered to "prevent" a disease, disorder, or condition if a statistically significant reduction in the development, frequency, and / or intensity of one or more symptoms of the disease, disorder, or condition is observed in a population susceptible to the particular disease, disorder, or condition. Prevention can be considered complete when the onset of the disease, disorder, or condition has been delayed for a predefined period of time.

[0322] The terms "subject", "individual" or "patient" (used interchangeably herein) refer to any animal, including mammals. Mammals according to embodiments include canines, felines, bovines, goats, equines, sheep, porcines, rodents, lagomorphs, primates, humans, etc., and encompass mammals in utero. In embodiments, humans are suitable subjects. Human subjects may be of any sex and at any stage of development. In some embodiments, the subject is a patient suffering from the disease hypertension, optionally pulmonary hypertension.

[0323] 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 that is being sought by a researcher, veterinarian, physician and / or other clinician, which biological or medical response may include one or more of the following:

[0324] (1) preventing a disease; for example, preventing a disease, condition or disorder in an individual who is susceptible to the disease, condition or disorder but does not yet experience and / or exhibit the pathology and / or symptomology of the disease;

[0325] (2) inhibiting a disease; e.g., inhibiting a disease, condition, or disorder in an individual who is experiencing and / or exhibiting pathology and / or symptomology of the disease, condition, or disorder (i.e., arresting and / or slowing the further development of the pathology and / or symptomology); and

[0326] (3) ameliorating a disease; e.g., ameliorating a disease, condition, or disorder in an individual who is experiencing and / or exhibiting the pathology and / or symptomology of the disease, condition, or disorder (i.e., reversing the pathology and / or symptomology).

[0327] Antibodies against BMP9

[0328] The present disclosure provides antibodies that bind to bone morphogenetic protein-9 (BMP9). BMP9 (also known as growth differentiation factor 2 (GDF2)) is encoded by the GDF2 gene in humans. It belongs to the transforming growth factor β (TGFβ) superfamily. BMP is synthesized as a large precursor of about 400-500 amino acids, which consists of an N-terminal signal peptide that guides secretion, a prodomain for correct folding, and a C-terminal mature peptide (Bragdon B, Moseychuk O, Saldanha S, King D, Julian J, Nohe A (2011) Bone morphogenetic proteins: a critical review [bone morphogenetic protein: a critical review]. Cell Signal [Cell Signal] 23: 609-620). After dimerization, the carboxyl-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 activin receptor type 2B (ActRIIB)] and four type I receptors [activin receptor-like kinases (ALK1, ALK2, ALK3 and ALK6)] are involved in BMP signaling. After ligand-induced receptor hetero-oligomerization, type I receptors are trans-phosphorylated and activated by type II receptors. These activated receptor complexes phosphorylate receptor-regulated Smads (R-Smad: Smad1, Smad5, and Smad8), enabling the formation of complexes with co-Smad (Smad4). The R-Smad / Smad4 complex translocates into the nucleus and regulates target gene expression by binding to regulatory elements and recruiting transcription co-repressors and / or activation complexes on their promoters (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 [BMP9 is produced by hepatocytes and circulates mainly in an active mature form complexed to its prodomain. Cell. Mol. Life Sci. [Cell and Molecular Life Sciences] 69, 313-324 (2012)).

[0329] As used herein, the term bone morphogenetic protein-9 or bone morphogenetic protein 9 (BMP9) (also referred to as growth differentiation factor 2 (GDF2)) includes variants, subtypes, homologs, orthologs and paralogs of BMP9. In some embodiments, the antibodies disclosed herein cross-react with BMP9 (such as BMP9 of cynomolgus monkeys and / or rats) and different forms of BMP9 from species other than humans. In some embodiments, the antibody can have complete specificity to human BMP9 and may not exhibit species cross-reactivity (e.g., not in conjunction with mouse BMP9). In some embodiments, the antibody can have complete specificity to human BMP9, but does not exhibit cross-reactivity to other transforming growth factor β superfamily members (e.g., not in conjunction with TGFβ1, GDF8, GDF9, GDF11, activin A and BMP10). Unless the context otherwise provides, as used herein, the term BMP9 refers to naturally occurring human BMP9. Therefore, "BMP9 antibody", "anti-BMP9 antibody" or other similar names refer to any antibody that binds or reacts with BMP9 or its isoforms, fragments and / or derivatives (as defined herein). 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 of human BMP9 (monomer) is provided herein as SEQ ID NO: 79.

[0330] Without wishing to be bound by any particular theory, perturbations in the BMP9 signaling pathway have appeared to be essential in endothelial dysfunction and vascular remodeling, particularly in pulmonary hypertension. It has been shown that inhibition of BMP9 can partially prevent experimental pulmonary hypertension in different complementary and established animal models of PAH (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).

[0331] Neutralizing antibodies or "blocking" antibodies refer to antibodies that bind to BMP9 and result in one or both of the following: (i) interfering with, limiting and / or inhibiting the interaction between BMP9 and type I receptors or type II receptors or both type I and type II receptors such as (ALK1, ALK2, ALK3, ALK6, BMPRII, ActRIIA and ActRIIB); and / or (ii) resulting in inhibition of at least one biological function of BMP9 binding. Assays for determining neutralization of antibodies of the present disclosure are well known in the art.

[0332] The "biological function" or "biological activity" of BMP9 is intended to include the binding of BMP9 to endothelial cells, the induction of nuclear translocation of phosphorylated-SMAD1 / 5 / 9 in endothelial cells, and the induction of nuclear translocation of phosphorylated-SMAD2 in endothelial cells. The biological function or biological activity of BMP9 can be, but not necessarily, mediated by the interaction between BMP9 and its receptor.

[0333] In some embodiments, the anti-BMP9 antibodies of the present disclosure encompass antibodies that have one or both of the following: i) compete for binding to human BMP9 with an antibody having an amino acid sequence of a heavy chain variable region as shown in SEQ ID NO: 77 and an amino acid sequence of a light chain variable region as shown in SEQ ID NO: 74, and / or ii) bind to the same epitope as an antibody having an amino acid sequence of a heavy chain variable region as shown in SEQ ID NO: 77 and an amino acid sequence of a light chain variable region as shown in SEQ ID NO: 74.

[0334] The anti-BMP9 antibodies disclosed herein may include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heterologous conjugated antibodies, single chains (ScFv), mutants thereof, fusion proteins comprising antibody fragments (e.g., domain antibodies), humanized antibodies, and any other modified configurations of immunoglobulin molecules comprising antigen-binding sites of desired specificity (including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies). The antibody may be of murine, rat, human, or any other source (including chimeric antibodies or humanized antibodies). In some embodiments, the anti-BMP9 antibody is a monoclonal antibody. In some embodiments, the anti-BMP9 antibody is a human antibody or a 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 the embodiments.

[0335] In some embodiments, antibodies or variants thereof having light chain variable region (VL) sequences and heavy chain variable region (VH) sequences as found in Table 3 / Table 4 are disclosed. In Table 4, the underlined sequences are CDR sequences (Kabat definition), and the italicized sequences are CDR sequences (Chothia definition). Unless otherwise indicated, in Table 1, K D Indicated is the affinity for human BMP9 as measured using surface plasmon resonance at 37°C.

[0336] Tables 3 and 4 provide the VH and VL sequences of mAbs of specific embodiments of the disclosure.

[0337] Embodiments also provide CDR portions of antibodies for BMP9. The determination of CDR regions is completely within the technical scope of this art. It should be understood that in some embodiments, CDRs can be a combination of Kabat and Chothia CDRs (also referred to as "combined CDRs" or "extended CDRs"). In another method referred to herein as "conformation definition" of CDRs, the positions of CDRs can be identified as residues that contribute enthalpy to antigen binding. See, for example, Makabe et al., 2008, J.Biol.Chem. [Journal of Biological Chemistry], 283: 1156-1166. Typically, "conformation CDRs" include such residue positions in Kabat CDRs and Vernier regions that are constrained to maintain the correct loop structure of antibody-specific antigen binding. The determination of conformation CDRs is completely within the technical scope of this art. In some embodiments, CDRs are Kabat CDRs. In other embodiments, CDRs are Chothia CDRs. In other embodiments, CDRs are extended CDRs, AbM CDRs, conformation CDRs and / or contact CDRs. In other words, in embodiments with more than one CDR, the CDR can be any of the following: Kabat CDR, Chothia CDR, extended CDR, AbM CDR, conformational CDR, contact CDR, or a combination thereof. It is contemplated that any one or more of these foregoing CDRs may be excluded in embodiments.

[0338] Table 4 provides examples of CDR sequences of anti-BMP9 antibodies provided herein. In some embodiments, the antibody comprises three CDRs of any one of the heavy chain variable regions shown in Table 4. In some embodiments, the antibody comprises three CDRs of any one of the light chain variable regions shown in Table 4. In some embodiments, the antibody comprises three CDRs of any one of the heavy chain variable regions shown in Table 4 and three CDRs of any one of the light chain variable regions shown in Table 4. In some embodiments, the antibody comprises three light chain CDRs and three heavy chain CDRs from Table 4.

[0339] In some embodiments, the antibody comprises one or both of the following of anti-BMP9 antibodies (e.g., Ab1076, Ab93, Ab352, Ab804, Ab101, Ab732, Ab100, and Ab89): i) full-length heavy chains with or without C-terminal lysine, and / or ii) full-length light chains. The amino acid sequences of the full-length heavy and light chains of antibody BMP9 are shown in Table 4 below.

[0340] 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 certain embodiments, the anti-BMP9 antibody is an IgG antibody.

[0341] Embodiments encompass modifications of the variable regions, CDR regions and / or heavy and light chain sequences shown in Tables 4 and 5. For example, embodiments include antibodies comprising functionally equivalent variable regions and CDRs that do not significantly affect their characteristics, and variants with enhanced or reduced activity or affinity. For example, the amino acid sequence can be mutated to obtain an antibody with a desired binding affinity to BMP9. The modification of polypeptides is a routine practice in the art and does not need to be described in detail herein. Examples of modified polypeptides include polypeptides having conservative substitutions of amino acid residues, one or more deletions or additions of amino acids (which do not significantly and harmfully change functional activity or mature the affinity of polypeptides to their ligands (enhancement)), or the use of chemical analogs. It is contemplated that any one or more of these aforementioned modifications may be excluded in an embodiment.

[0342] Modification or mutation can also be carried out in framework region or constant region to increase the half-life of antibody provided herein.See, for example, PCT Publication No. WO 00 / 09560. Mutation can also be carried out in framework region or constant region to change the immunogenicity of antibody, provide for the site of covalent or non-covalent binding with another molecule, and / or change the characteristic of cytotoxicity such as complement fixation, FcR combination and antibody-dependent cell-mediated. In certain embodiments, no more than one to five (for example, at least, at most or accurately 1,2,3,4 or 5) conservative amino acid substitutions are carried out in framework region or constant region. In other embodiments, no more than one to three (for example, at least, at most or accurately 1,2 or 3) conservative amino acid substitutions are carried out in framework region or constant region. According to certain embodiments, single antibody can have mutation in any one or more of CDR or framework region of variable domain or in constant region.

[0343] In certain embodiments, the antibody comprises a modified constant region that has an increased or reduced binding affinity to human Fcγ receptors, is immunologically inert or partially inert, such as not triggering complement-mediated lysis, not stimulating antibody-dependent cell-mediated cytotoxicity (ADCC), not activating microglia, and / or having reduced activity (compared to unmodified antibodies) in any one or more of the following: triggering complement-mediated lysis, stimulating ADCC, and / or activating microglia. It is contemplated that any one or more of these aforementioned features caused by a modified constant region can be excluded in an embodiment. Different modifications of the constant region can 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; 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.

[0344] Modifications also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications such as glycosylation with different sugars, acetylation, and phosphorylation. Antibodies are glycosylated at conserved positions in their constant regions (Jefferis and Lund, 1997, Chem. Immunol. [Chemical Immunology] 65: 111-128; Wright and Morrison, 1997, TibTECH [Biotechnology Trends] 15: 26-32). The oligosaccharide side chains of immunoglobulins affect the function of the protein (Boyd et al., 1996, Mol. Immunol. [Molecular Immunology] 32: 1311-1318; Wittwe and Howard, 1990, Biochem. [Biochemistry] 29: 4175-4180) and the intramolecular interactions between the parts of the glycoprotein, which can affect the conformation of the glycoprotein and the three-dimensional surface presented (Jefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech. [Biotechnology New View] 7: 409-416). Oligosaccharides can also be used to target a given glycoprotein to certain molecules based on specific recognition structures. It has also been reported that glycosylation of antibodies affects antibody-dependent cellular cytotoxicity (ADCC). Specifically, antibodies produced by CHO cells with tetracycline-regulated expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase that catalyzes the formation of bisecting GlcNAc, were reported to have improved ADCC activity (Umana et al., 1999, Nature Biotech. 17: 176-180).

[0345] In some embodiments, the present disclosure provides anti-BMP9 antibodies containing variants of the variable regions, CDRs, or heavy and light chain sequences shown in Tables 4 and 5, wherein such variant polypeptides have an amino acid sequence identity of at least 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% with any of the amino acid sequences disclosed in Tables 4 or 5. For example, in some embodiments, the disclosure provides anti-BMP9 antibodies containing variants of the variable regions, CDRs, or heavy and light chain sequences shown in Tables 4 and 5, wherein such variant polypeptides have 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% amino acid sequence identity with any of the amino acid sequences disclosed in Tables 4 or 5. These amounts are not intended to be limiting, and the 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 the embodiments.

[0346] It is contemplated that a region or fragment of a polypeptide of the present disclosure may have an amino acid sequence having, having at least or having at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, consecutive amino acid additions and / or consecutive amino acid deletions relative to any one of SEQ ID NOs: 1-77, 80-84 or 89. In some embodiments, an anti-BMP9 antibody or antigen-binding fragment thereof comprises or consists of an amino acid sequence having, having at least or having at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, consecutive amino acid additions and / or consecutive amino acid deletions relative to any one of SEQ ID NOs: 1-77, 80-84 or 89. any of NOs: 1-77, 80-84 or 89 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 any range thereof) identical. It is contemplated that embodiments may exclude any one or more of these foregoing percentages.

[0347] Furthermore, in some embodiments, a region or fragment comprises an amino acid region in SEQ ID NO: 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 5, 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 22, 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 2, 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 (wherein position 1 is in SEQ ID NO) has at least the following amounts (or any range thereof),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, 7, 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, 1 69, 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 0, 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 2, 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, 3 82、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 or more.

[0348] The anti-BMP9 antibodies or antigen-binding fragments thereof disclosed herein may comprise at least the following amounts (or any ranges thereof), at most the following amounts (or any ranges thereof), exactly the following amounts (or any ranges thereof), or between any two of the following amounts (or any ranges thereof), including or excluding the end values: 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, or 50 or more of the variant amino acids or amino acid substitutions. In some embodiments, the variant or amino acid substitution is in SEQ ID NO: 1-77, 80-84 or 89, 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 6, 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 2, 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、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 (wherein position 1 is at the N-terminus of SEQ ID NO), and the anti-BMP9 antibody or antigen-binding fragment thereof may be fused to SEQ ID NO. at least 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 6, 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, 300, 400, 500, 550, 600 or more consecutive amino acids, or any range thereof, of at least the following amount, at most The following amounts, exactly the following amounts, or between any two of the following amounts (inclusive or exclusive) are identical or homologous: 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%. It is contemplated that any one or more of these aforementioned percentages may be excluded in embodiments.

[0349] In some embodiments, the present disclosure provides anti-BMP9 antibodies containing variants of the variable regions shown in Tables 4 and 5. In some embodiments, VH comprises an 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 containing one to four amino acid substitutions at residues not within a CDR. In some embodiments, one to four (i.e., 1, 2, 3 or 4) amino acid substitutions may be selected from the group consisting of: K13Q, D73N, S74A, T77S, K83R and T84A of SEQ ID NO: 27. In some embodiments, VL comprises an 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 containing one to four (i.e., 1, 2, 3 or 4) amino acid substitutions at residues not within a CDR. In some embodiments, one to four amino acid substitutions may be selected from the group consisting of: M43T, V76S, Q79R, G84A and Y85D of SEQ ID NO:22. In some embodiments, provided herein is an anti-BMP9 antibody comprising VH and VL, wherein the antibody VH has an amino acid sequence encoded by a nucleic acid sequence of an insert of a plasmid deposited at ATCC and having ATCC accession number PTA-127292, and / or the antibody VL has an amino acid sequence encoded by a nucleic acid sequence of an insert of a plasmid deposited at ATCC and having ATCC accession number PTA-127293.

[0350] Embodiments also encompass fusion proteins comprising one or more components of antibodies disclosed herein. In some embodiments, such fusion proteins can be manufactured, and the fusion proteins include all or part of the anti-BMP9 antibodies disclosed herein connected to another polypeptide. In another embodiment, only the variable domains of the anti-BMP9 antibodies are connected to the polypeptide. In another embodiment, the VH domain of the anti-BMP9 antibody is connected to the first polypeptide, and the VL domain of the anti-BMP9 antibody is connected to the second polypeptide, and the second polypeptide associates with the first polypeptide in a manner such that the VH and VL domains can interact with each other to form an antigen binding site. In another embodiment, the VH domain is separated from the VL domain by a joint so that the VH and VL domains can interact with each other. The VH-joint-VL antibody is then connected to the polypeptide of interest. In addition, such fusion antibodies can be produced in which two (or more) single-chain antibodies are connected to each other. If you want to produce a divalent or multivalent antibody on a single polypeptide chain, or if you want to produce a bispecific antibody, this is useful.

[0351] Biological activity of anti-BMP9 antibodies

[0352] In addition to binding to an epitope on BMP9, the antibodies disclosed herein can also mediate biological activity. That is, the disclosure includes such isolated antibodies that specifically bind to BMP9 and mediate at least one detectable activity selected from the following:

[0353] (i) specifically binds to human BMP9;

[0354] (ii) specifically binds to cynomolgus monkey (cynomolgus monkey (cyno)) BMP9;

[0355] (iii) specifically binds to rat BMP9;

[0356] (iv) one or more of the following: reducing, inhibiting and / or neutralizing the binding of BMP9 to type II receptors (BMPRII, ActRIIA and / or ActRIIB) (e.g., Ab89, Ab93, AblOO, AblOl);

[0357] (v) one or more of the following: reducing, inhibiting and / or neutralizing the binding of BMP9 to the type II receptor (BMPRII, ActRIIA and / or ActRIIB), and weakly inhibiting the binding of BMP9 to the type I receptor (ALK1) (e.g., Ab352, Ab804 and Ab1076);

[0358] (vi) one or more of the following: reducing, inhibiting and / or neutralizing the binding of BMP9 to endothelial cells;

[0359] (vii) one or more of the following: reducing, inhibiting and / or neutralizing SMAD1 / 5 / 9 phosphorylation and / or phosphorylated-SMAD1 / 5 / 9 nuclear translocation in endothelial cells;

[0360] (viii) one or more of the following: reducing, inhibiting and / or neutralizing SMAD2 phosphorylation or phosphorylated-SMAD2 nuclear translocation in endothelial cells;

[0361] (ix) decreases right ventricular systolic pressure;

[0362] (x) Reduce right ventricular hypertrophy;

[0363] (xi) reducing pulmonary artery pressure (e.g., at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof, inclusive or exclusive, or about the following amount or any range thereof: 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) (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);

[0364] (xii) reduce pulmonary vascular resistance;

[0365] (xiii) increase cardiac output or cardiac index;

[0366] (xiv) Increased pulmonary capillary wedge pressure;

[0367] (xv) Increase left ventricular end-diastolic pressure;

[0368] (xvi) increasing the subject's motor ability;

[0369] (xvii) increases a subject's 6-minute walk distance (e.g., by at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof, inclusive or exclusive, or about the following amount or any range thereof: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 meters) (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more meters); and

[0370] (xviii) reducing the Borg dyspnea index (BDI) of a subject (eg, Clini Enrico et al. Multidiscip. Respir. Med. 2010; 5(3): 202-210).

[0371] It is contemplated that embodiments may exclude any one or more of these foregoing activities.

[0372] Assays for measuring the above functional activities are well known in the art. For example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, bioassay (e.g., growth inhibition), Western blot assay, BIACORE TM Binding activity is measured by surface plasmon resonance and / or by assays. Each of these assays typically detects the presence of a protein-antibody complex of particular interest by employing a labeled reagent (eg, an antibody) specific for the complex of interest.

[0373] The ability to block a reference molecule (e.g., receptor or antibody) or compete with a reference molecule (e.g., receptor or antibody) can be determined by, for example, competitive binding assays. The ability of the tested antibody to inhibit the specific binding of a reference molecule to a common antigen (such as a BMP9 polypeptide) is checked by competitive binding assays. If an excess of test antibody greatly inhibits the binding of a reference molecule, the test antibody competes with the reference molecule for antigen-specific binding. Greatly inhibiting means that the test antibody generally reduces the specific binding of a reference molecule by at least the following amount or any range thereof, at most the following amount or any range thereof, accurately the following amount or any range thereof, between any two of the following amount or any range thereof (including end values ​​or without end values) or about the following amount or any range thereof: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% (e.g., at least 10%, 25%, 50%, 75% or 90%). There are many known competitive binding assays that can be used to assess the competition of an antibody with a reference antibody for binding to a particular protein (in this case, BMP9). These include, for example, solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see Stahli et al., Methods in solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619, 1986); solid phase direct label assay, solid phase direct label sandwich assay (see Harlow and Lane, supra); solid phase direct label 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 label RIA (Moldenhauer et al., Scand. J. Immunol. 32:77-82, 1990).

[0374] The reactivity of the BMP9 binding antibodies and antigen-binding fragments thereof disclosed herein and BMP9 polypeptides or antigen fragments can be further tested by Western blotting. In short, 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 nitrocellulose membranes, blocked with 10% fetal bovine serum, and detected with the monoclonal antibody to be tested. Human IgG binding can be detected using anti-human IgG alkaline phosphatase, and developed with BCIP / NBT substrate tablets (Sigma Chem. Co., St. Louis, Missouri).

[0375] Examples of functional assays are also described in the Examples section below. It is contemplated that any one or more of these foregoing functional assays may be excluded in embodiments.

[0376] 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, Abl00, Abl01, 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 Abl01. In some embodiments, the antibody is Ab732. In some embodiments, the antibody is Abl01 or Ab732. It is contemplated that any one or more of these aforementioned antibodies may be excluded in embodiments.

[0377] In some embodiments, the antibody binds to human BMP9 with an affinity of about 15 pM to about 80 pM at 37° C., as 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., as measured by surface plasmon resonance. In some embodiments, the antibody is expressed at least in an amount or any range thereof, at most in an amount or any range thereof, exactly in an amount or any range thereof, between any two of the amounts or any range thereof (including or excluding the end values), or about in an amount 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 In some embodiments, 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), as measured by surface plasmon resonance. In some embodiments, the antibody binds to human BMP9 with an affinity of about 87 pM at 37°C, as measured by surface plasmon resonance. In some embodiments, the antibody binds human BMP9 with an affinity of about 87.41 pM or about 90 pM at 37° C. as measured by surface plasmon resonance. In some embodiments, the antibody is Abl076.

[0378] Polynucleotides encoding anti-BMP9 antibodies and methods for making them

[0379] The disclosure also provides polynucleotides encoding any of the antibodies disclosed herein (including antibody portions and modified antibodies described herein). The embodiments also provide methods for making any of the antibodies and polynucleotides described herein. Polynucleotides can be made by procedures known in the art, and proteins expressed therefrom.

[0380] If desired, the target anti-BMP9 antibody can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequence encoding the target antibody can be maintained in a vector in a host cell, which can then be amplified and frozen for future use. The production of recombinant monoclonal antibodies in cell culture can be performed by cloning antibody genes from B cells by means known in the art. See, for example, Tiller et al., 2008, J. Immunol. Methods [Journal of Immunology Methods] 329,112; U.S. Patent No. 7,314,622.

[0381] In some embodiments, polynucleotides are provided herein, comprising sequences 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 amplified and frozen for future use. Vectors (including expression vectors) and host cells are further described herein.

[0382] In some embodiments, the disclosure provides polynucleotides encoding the amino acid sequence of any of the following anti-BMP9 antibodies: Ab89, Ab93, Abl00, Abl01, Ab352, Ab732, Ab804, and Ab1076. In one embodiment, the disclosure provides polynucleotides encoding the amino acid sequence of anti-BMP9 antibodies Ab93, Ab1076, Abl01, or Ab732. In one embodiment, the disclosure provides polynucleotides encoding the amino acid sequence of anti-BMP9 antibody Ab1076.

[0383] In some embodiments, the present disclosure provides polynucleotides encoding one or more anti-BMP9 antibody heavy chain polypeptides comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 8, 23, 35, 44, 52, 60, 69 and 75.

[0384] In some embodiments, the present disclosure provides polynucleotides encoding one or more anti-BMP9 antibody light chain polypeptides comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, 18, 29, 40, 49, 56, 64, and 72.

[0385] In some embodiments, the disclosure provides a polynucleotide comprising SEQ ID NO: 87. In some embodiments, the disclosure provides a polynucleotide comprising SEQ ID NO: 88.

[0386] In some embodiments, the present disclosure provides polynucleotides encoding one or more anti-BMP9 antibody VH polypeptides comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: 16, 27, 39, 48, 55, 63, 71, and 77.

[0387] In some embodiments, the present disclosure provides polynucleotides encoding one or more anti-BMP9 antibody VL polypeptides comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 6, 22, 33, 43, 51, 59, 68, and 74.

[0388] In some embodiments, the disclosure provides a polynucleotide comprising SEQ ID NO: 85. In some embodiments, the disclosure provides a polynucleotide comprising SEQ ID NO: 86.

[0389] The present disclosure provides a polypeptide comprising an amino acid sequence encoded by a DNA insert of a plasmid deposited at ATCC with accession number PTA-127292, encoding the VH domain of antibody Ab1076. The present disclosure provides a polypeptide comprising an amino acid sequence encoded by an insert of a plasmid deposited at ATCC with accession number PTA-127293, encoding the VL domain of antibody Ab1076.

[0390] Those of ordinary skill in the art will appreciate that, due to the degeneracy of the genetic code, there are many nucleotide sequences encoding polypeptides as described herein. Some of these polynucleotides have minimal homology with the nucleotide sequence of any natural gene. Nevertheless, the present disclosure specifically contemplates polynucleotides that vary due to differences in codon usage. Further, the alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present disclosure. Alleles are endogenous genes that change due to one or more mutations (such as deletions, additions and / or substitutions of nucleotides). The resulting mRNA and protein may but need not have a changed structure or function. Standard techniques (such as hybridization, amplification and / or database sequence comparisons) can be used to identify alleles.

[0391] In one embodiment, the VH and / or VL domains and / or full-length HC and / or LC are encoded by separate polynucleotides. Alternatively, the VH and / or VL and / or HC and / or LC are all encoded by a single polynucleotide.

[0392] Polynucleotides complementary to any such sequences are also encompassed in the present disclosure. Polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules that contain introns and correspond to DNA molecules in a one-to-one manner, and mRNA molecules that do not contain introns. Additional coding or non-coding sequences may, but need not, be present in the polynucleotides of the present disclosure, and polynucleotides may, but need not, be connected to other molecules or support materials.

[0393] The polynucleotides disclosed herein can be obtained using chemical synthesis, recombinant methods or PCR. The method of chemical polynucleotide synthesis is well known in the art and does not need to be described in detail herein. Those skilled in the art can use the sequence provided herein and a commercial DNA synthesizer to produce the desired DNA sequence.

[0394] In order to prepare polynucleotides using recombinant methods, the polynucleotides comprising 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 polynucleotides can be inserted into a host cell by any means known in the art. Cells are transformed by introducing exogenous polynucleotides via direct uptake, endocytosis, transfection, F-conjugation or electroporation. After introduction, the exogenous polynucleotides can be maintained in the cell or integrated into the host cell genome as a non-integrated vector (such as a plasmid).

[0395] Suitable cloning vectors can be constructed according to standard techniques, or can be selected from a large number of cloning vectors available in the art. Although the selected cloning vector can be changed according to the host cell used in the expectation, useful cloning vectors will generally have one or more characteristics such as the following: i) the ability to self-replicate, ii) the single target of a specific restriction endonuclease, or iii) the gene that can be used to select the marker of the clone containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (for example, pBS SK+) and derivatives thereof, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA and shuttle vectors (such as pSA3 and pAT28). These and many other cloning vectors can be obtained from commercial suppliers (such as BioRad, Strategene and Invitrogen).

[0396] An expression vector is further provided. An expression vector is typically a reproducible polynucleotide construct containing a polynucleotide according to the present disclosure. This implies that the expression vector must be reproducible as an episome or as a component of chromosomal DNA in a host cell. Suitable expression vectors include, but are not limited to, plasmids, viral vectors (including adenovirus, adeno-associated virus, retrovirus), cosmids, and one or more expression vectors disclosed in PCT Publication No. WO 87 / 04462. Vector components may typically 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 transcription control elements (such as promoters, enhancers, and terminators). In order to express (i.e., translate), one or more translation control elements, such as ribosome binding sites, translation start sites, and stop codons, are typically also required. It is expected that any one or more of these aforementioned vector components or elements may be excluded in an embodiment.

[0397] The vector containing the polynucleotide of interest can be introduced into the host cell by any of a variety of appropriate means, including electroporation, transfection, using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent, such as vaccinia virus). The choice of the vector or polynucleotide to be introduced will generally depend on the characteristics of the host cell.

[0398] Embodiments also provide host cells comprising any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA can be used for the purpose of isolating genes encoding target antibodies, polypeptides or proteins. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa and CHO cells. Also refer to PCT Publication No. WO 87 / 04462. Suitable non-mammalian host cells include prokaryotes (such as Escherichia coli or Bacillus subtilis) and yeast (such as Saccharomyces cerevisiae, Schizosaccharomyces pombe or Kluyveromyces lactis).

[0399] Additionally, any number of commercially available and non-commercially available cell lines expressing polypeptides or proteins can be utilized according to the present disclosure. Those skilled in the art will appreciate that different cell lines may have different nutritional requirements or may require different culture conditions to achieve optimal growth and polypeptide or protein expression, and will be able to modify the conditions as needed.

[0400] In some embodiments, heavy and light chain antibodies described herein with signal peptides (e.g., SEQ ID NO: 89) are produced. As used herein, the term "leader peptide" or "leader sequence" or "leader signal sequence" or "signal sequence" or "signal peptide" (used interchangeably herein) means any nucleic acid sequence or amino acid sequence encoded thereby, which may be present at or near the 5' end of a nucleic acid molecule and / or the N-terminus of a polypeptide, and when present, it may mediate the transport of a polypeptide to a target organelle, including but not limited to the secretion of a polypeptide from a cell. Such leader sequences include but are not limited to the nucleic acid sequence encoding SEQ ID NO: 89. Embodiments encompass such and any other leader signals (nucleic acids and amino acid sequences) known in the art or to be identified, which may result in transport of a polypeptide to a desired organelle (e.g., endoplasmic reticulum) and / or secretion from a cell. In a preferred embodiment, the signal peptide is removed from the mature polypeptide and / or is not present in the mature polypeptide.

[0401] Pharmaceutical composition

[0402] In another embodiment, the disclosure includes a pharmaceutical composition. A "pharmaceutical composition" refers to a mixture of an antibody and one or more excipients.

[0403] The pharmaceutical compositions of the present disclosure can be in various forms. These include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions and lyophilized powders. The form depends on the intended mode of administration and therapeutic application. It is expected that any one or more of these aforementioned forms can be excluded in the embodiments.

[0404] Other excipients and modes of administration known in the pharmaceutical field can also be used. The pharmaceutical compositions disclosed herein can be prepared by any of the well-known pharmaceutical techniques (such as effective formulations and administration procedures). The above considerations on effective formulations and administration procedures are well known in the art and are described in standard textbooks. The formulation of drugs is discussed in, for example, Hoover, John E., REMINGTON'S PHARMACEUTICAL SCIENCES [Remington Pharmaceutical Sciences], Mack Publishing Co. [Mark Publishing Company], Easton, Pennsylvania, 1975; Liberman et al., ed., PHARMACEUTICAL DOSAGE FORMS [drug dosage forms], Marcel Decker [Marcel Decker Company], New York, New York, 1980; and Kibbe et al., ed., HANDBOOK OF PHARMACEUTICAL EXCIPIENTS [drug excipients handbook] (3rd edition), American Pharmaceutical Association [American Pharmaceutical Association], Washington, 1999.

[0405] Acceptable excipients are nontoxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphate, citrate and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, 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 1 0 residues) polypeptides; proteins, such as serum albumin, gelatin and / or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine and / or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose and / or dextrins; 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®. TM 、PLURONICS TM Or polyethylene glycol (PEG). It is contemplated that any one or more of these foregoing excipients may be excluded in the embodiments.

[0406] In some embodiments, the pharmaceutical composition comprises at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount 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 antibody): 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL In some embodiments, the pharmaceutical composition comprises about 100 mg / mL antibody (or its Fab). In some embodiments, the pharmaceutical composition suitable for subcutaneous and / or intravenous administration comprises about 100 mg / mL anti-BMP9 antibody (or its Fab) (e.g., Ab1076).

[0407] In some embodiments, the anti-BMP9 antibody or antigen-binding fragment thereof is administered as a sterile aqueous solution comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 75 in an intravenous or subcutaneous formulation. In some embodiments, the anti-BMP9 antibody or antigen-binding fragment thereof is administered as a sterile aqueous solution comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 72 in an intravenous or subcutaneous formulation. In some embodiments, the anti-BMP9 antibody or antigen-binding fragment thereof is administered 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 in an intravenous or subcutaneous formulation.

[0408] In some embodiments, an anti-BMP9 antibody (or antigen-binding fragment thereof) (e.g., Ab1076) is administered as an intravenous or subcutaneous formulation containing at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount 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): 5 mg / mL, 10mg / mL, 15mg / mL, 20mg / mL, 25mg / mL, 30mg / mL, 35mg / mL, 40mg / mL, 45mg / mL, 50mg / mL, 55mg / mL, 60mg / mL, 65mg / mL, 70mg / mL, 75mg / mL, 80mg / mL , 85mg / mL, 90mg / mL, 95mg / mL, 100mg / mL, 105mg / mL, 110mg / mL, 115mg / mL, 120mg / mL, 125mg / mL, 130mg / mL, 135mg / mL, 140mg / mL, 145mg / mL or 150mg / mL.

[0409] In some embodiments, the intravenous or subcutaneous formulation is a sterile aqueous solution comprising sodium acetate, polysorbate 80 and sodium chloride, with a pH ranging from about 5 to 6. In some embodiments, the intravenous or subcutaneous formulation is a sterile aqueous solution comprising sodium acetate in an amount of at least 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM or 40 mM, or any range thereof, at most 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM or 40 mM, or any range thereof. In some embodiments, the intravenous or subcutaneous formulation is a sterile aqueous solution comprising 20 mM sodium acetate. In some embodiments, the intravenous or subcutaneous formulation is a sterile aqueous solution comprising at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount or any range thereof: 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. In some embodiments, the intravenous or subcutaneous formulation is a sterile aqueous solution comprising 0.2 mg / mL polysorbate 80. In some embodiments, the intravenous or subcutaneous formulation is a sterile aqueous solution comprising sodium chloride in an amount of at least 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, or any range thereof. In some embodiments, the intravenous or subcutaneous formulation is a sterile aqueous solution comprising 140 mM sodium chloride. In some aspects, 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 any range thereof). In some embodiments, the intravenous formulation is a sterile aqueous solution optionally containing 5 or 10 mg / mL of the antibody or antigen-binding fragment thereof, and 20 mM sodium acetate, 0.2 mg / mL polysorbate 80, and 140 mM sodium chloride in a nominal fill volume of 1.0 mL, at a pH of 5.5.It is contemplated that embodiments may exclude any one or more of these foregoing ingredients.

[0410] Further, the solution containing the antibody or antigen-binding fragment thereof may also contain, among many other compounds, glutamic acid, histidine, mannitol, sucrose, trehalose, glycine, polyethylene glycol, EDTA, methionine, polysorbate 80, or any combination thereof, and many other compounds known in the relevant art. It is contemplated that any one or more of these aforementioned ingredients may be excluded in embodiments.

[0411] In some embodiments, the pharmaceutical composition comprises at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount or any range thereof: 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, or 40 mM of histidine. In some embodiments, the pharmaceutical composition comprises 20 mM histidine. In some embodiments, the pharmaceutical composition comprises 10 mM histidine. In some embodiments, the pharmaceutical composition comprises sucrose in at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amount or any range thereof (including or excluding the end values), or about the following amount or any range thereof: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%. In some embodiments, the pharmaceutical composition comprises 8.5% sucrose. In some embodiments, the pharmaceutical composition comprises 5% sucrose. In some embodiments, the pharmaceutical composition comprises at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount or any range thereof: 0.001%, 0.005%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04% polysorbate 80. In some embodiments, the pharmaceutical composition comprises 0.02% polysorbate 80. In some embodiments, the pharmaceutical composition comprises 0.01% polysorbate 80. In some aspects, 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 any range thereof). In some embodiments, the pharmaceutical composition comprises at least 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, or 0.01% EDTA, at most, at most, exactly, between any two of the following amounts or any range thereof (including or excluding the end values), or about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, or 0.01%. In some embodiments, the pharmaceutical composition comprises 0.005% EDTA.In some aspects, EDTA is at least, at most, precisely 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 any range thereof). In one embodiment, the pharmaceutical composition of the present disclosure comprises the following components: 50 mg / mL or 100 mg / mL of an anti-BMP9 antibody or antigen-binding fragment thereof (e.g., Ab1076) of the present disclosure, 20 mM histidine, 8.5% sucrose and 0.02% polysorbate 80, 0.005% EDTA, pH 5.8. In one embodiment, the pharmaceutical composition comprises the following components: 100 mg / mL of the anti-BMP9 antibody or antigen-binding fragment thereof (e.g., Ab1076) disclosed herein, or antigen-binding fragment thereof, 10 mM histidine, 5% sucrose, and 0.01% polysorbate 80, at a pH of 5.8. It is contemplated that any one or more of these aforementioned ingredients may be excluded in the embodiments.

[0412] In some embodiments, the pharmaceutical composition comprises at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amount or any range thereof (including or excluding the end values), or about the following amount or any range thereof of L-histidine: 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. In some embodiments, the pharmaceutical composition comprises 1.12 mg / mL L-histidine. In some embodiments, the pharmaceutical composition comprises at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amount or any range thereof (including or excluding the end values) or about the following amount or any range thereof of L-histidine hydrochloride monohydrate: 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. In some embodiments, the pharmaceutical composition comprises 2.67 mg / mL L-histidine hydrochloride monohydrate. In some embodiments, the pharmaceutical composition comprises at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amount or any range thereof (including or excluding the end values) or about the following amount or any range thereof of sucrose: 50mg / mL, 55mg / mL, 60mg / mL, 65mg / mL, 70mg / mL, 75mg / mL, 80mg / mL, 85mg / mL, 90mg / mL, 95mg / mL, 100mg / mL, 105mg / mL, 110mg / mL, 115mg / mL, 120mg / mL, 125mg / mL, 130mg / mL, 135mg / mL, 140mg / mL, 145mg / mL or 150mg / mL. In some embodiments, the pharmaceutical composition comprises 85mg / mL sucrose. In some embodiments, the pharmaceutical composition comprises at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amount or any range thereof (including or excluding the end values), or about the following amount or any range thereof EDTA: 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. In some embodiments, the pharmaceutical composition comprises 0.05 mg / mL 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 any range thereof). In some embodiments, the pharmaceutical composition comprises at least 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 any range thereof. In some embodiments, the pharmaceutical composition comprises 0.2 mg / mL polysorbate 80. In some aspects, 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 any range thereof). In some embodiments, the pharmaceutical composition comprises an anti-BMP9 antibody or its antigen-binding fragment (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 in a nominal fill volume of 1.0 mL, 1.12 mg / mL L-histidine, 2.67 mg / mL L-histidine hydrochloride monohydrate, 85 mg / mL sucrose, 0.05 mg / mL disodium ethylenediaminetetraacetic acid dihydrate and 0.2 mg / mL polysorbate 80, and a pH of 5.8. Such a pharmaceutical composition is suitable for subcutaneous or intravenous administration. It is expected that any one or more of these aforementioned ingredients can be excluded in the embodiments.

[0413] Treatment, diagnosis and other methods

[0414] The antibodies and antibody conjugates of the present disclosure can be used in a variety of applications, including but not limited to therapeutic treatment methods and / or diagnostic treatment methods.

[0415] 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 used to treat, prevent, inhibit and / or improve diseases, disorders or conditions mediated by increased BMP9 activity, including but not limited to hypertension (e.g., pulmonary hypertension), anemia, liver disease (e.g., liver fibrosis, cirrhosis, portal hypertension, end-stage liver disease such as varicose veins, jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, spontaneous bacterial peritonitis, non-alcoholic fatty liver disease, portopulmonary hypertension and hepatopulmonary syndrome), neuroinflammatory or neurodegenerative diseases (e.g., 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; heart valve malformations; structural heart malformations; fibrodysplasia ossificans progressiva; familial juvenile polyposis syndrome; parathyroid disease; cancer (e.g., breast cancer, prostate cancer, renal cell carcinoma, bone metastasis, lung metastasis, osteosarcoma, and multiple myeloma); vascular calcification; vascular inflammation; 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); viral infections; bacterial infections; fungal infections; tuberculosis; and parasitic infections.

[0416] On the one hand, the present disclosure provides a method for treating hypertension (e.g., pulmonary hypertension). In some embodiments, the method for treating hypertension (e.g., pulmonary hypertension) of a subject includes administering an effective amount of a pharmaceutical composition to a subject in need, the pharmaceutical composition comprising any BMP9 antibody or its antigen-binding fragment as described herein. In some embodiments, there is provided a method for reducing or inhibiting the BMP9 activity of a subject, the method comprising administering an effective amount of a composition to a subject in need, the composition comprising an antibody or its antigen-binding fragment provided herein.

[0417] On the other hand, the disclosure further provides an antibody, an antigen-binding fragment thereof, or a pharmaceutical composition thereof as described herein, which is used in the described method for treating hypertension (e.g., pulmonary hypertension). The disclosure also provides the use of an antibody, an antigen-binding fragment thereof, as described herein, in the manufacture of a medicament for treating hypertension (e.g., pulmonary hypertension).

[0418] On the other hand, there is provided a method for detecting, prognosing, diagnosing and / or monitoring one or more of a disease, disorder or condition mediated by increased and / or dysregulated BMP9 activity, including but not limited to hypertension (e.g., pulmonary hypertension), anemia, liver disease (e.g., liver fibrosis, cirrhosis, portal hypertension, end-stage liver disease, such as varicose veins, jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, spontaneous bacterial peritonitis, non-alcoholic fatty liver disease, portopulmonary hypertension and hepatopulmonary syndrome), neuroinflammatory or neurodegenerative diseases (e.g., multiple sclerosis), fibrotic disorders and / or heart failure. In some embodiments, these methods include determining, measuring, detecting or determining the level of BMP9 in a sample obtained from a subject using a BMP9 antibody or antigen-binding fragment thereof as described herein. For example, an anti-BMP9 antibody or its antigen-binding fragment as described herein can be labeled with a detectable portion, such as an imaging agent, an enzyme-substrate label or other compounds or elements that can be detected due to its specific functional properties and / or chemical characteristics, using the detectable portion allows the antibody to be detected and / or further quantified (if necessary). Examples of detectable labels include, but are not limited to, radioisotopes, fluorescent agents, semiconductor nanocrystals, chemiluminescent agents, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, metal sols, ligands (e.g., biotin, streptavidin or haptens), etc. Specific examples of labels are, but are not limited to, horseradish peroxidase (HRP), fluorescein, FITC, rhodamine, dansyl, umbelliferone, dimethyl acridinium ester (DMAE), Texas Red, luminol, NADPH and α- or β-galactosidase. Based on the determination, measurement, detection or determination level of BMP9, different diagnoses and / or prognoses can be performed on different subjects. The antibodies or antigen-binding fragments thereof as described herein can also be used in in vivo diagnostic assays, such as in vivo imaging (eg, PET or SPECT), or as staining reagents.

[0419] On the other hand, there is provided a method for monitoring the effectiveness of therapy (e.g., BMP9 antibody or its Fab as described herein) in a subject suffering from a disease, disorder or condition.Disease, disorder or condition can be mediated by increased and / or disordered BMP9 activity, and may include but is not limited to hypertension (e.g., pulmonary hypertension), anemia, liver disease (e.g., liver fibrosis, cirrhosis, portal hypertension, end-stage liver disease, such as varicose veins, jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, spontaneous bacterial peritonitis, nonalcoholic fatty liver disease, portopulmonary hypertension and hepatopulmonary syndrome), neuritis or neurodegenerative diseases (e.g., multiple sclerosis), fibrotic disorders and / or heart failure.In particular aspects, disease, disorder or condition is pulmonary hypertension.In some aspects, the method includes administering therapy (e.g., BMP9 antibody or its Fab as described herein) to a subject in need.

[0420] In some aspects, the method further includes determining, measuring, detecting or determining the expression or activity level of one or more biomarkers in the biological sample obtained from the subject. In some aspects, one or more biomarkers include CXCL12, IGFBP4, INHBA, MALL, FRZB, CPE, CCL2, PDGFBB, endothelin-1 or a combination thereof. Biomarker levels can be determined, measured, detected or determined in the biological sample obtained from the subject by measuring mRNA and / or protein levels. For example, antibodies or their antigen-binding fragments that specifically bind biomarkers can be used to determine, measure, detect or determine protein levels. For example, biomarker antibodies or their antigen-binding fragments can be labeled with a detectable portion, which can be detected as developer, enzyme-substrate labeling or other compounds or elements that can be detected due to its specific functional characteristics and / or chemical characteristics, using the detectable portion allows the antibody to be detected and / or further quantified (if necessary). Examples of detectable labels include, but are not limited to, radioisotopes, fluorescent agents, semiconductor nanocrystals, chemiluminescent agents, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, metal sols, ligands (e.g., biotin, streptavidin, or haptens), etc. Specific examples of labels are, but are not limited to, horseradish peroxidase (HRP), fluorescein, FITC, rhodamine, dansyl, umbelliferone, dimethyl acridinium ester (DMAE), Texas Red, luminol, NADPH, and α- or β-galactosidase.

[0421] In some aspects, the method further includes comparing the level of one or more biomarkers measured in the biological sample of the subject with the level of one or more biomarkers measured in the biological sample obtained from the subject before administering therapy (e.g., BMP9 antibody or its antigen-binding fragment described herein). In some aspects, the method further includes determining, measuring, detecting or determining the efficacy of therapy (e.g., BMP9 antibody or its antigen-binding fragment described herein) based on the determination, measurement, detection or determination level of one or more biomarkers. Based on the profile of biomarker expression or activity level, different treatments can be prescribed or recommended for different subjects and / or treatment can be modified for subjects.

[0422] For example, in some aspects, a therapy (e.g., a BMP9 antibody or antigen-binding fragment thereof described herein) is determined to be effective when one or more biomarkers that are measured, detected or determined are: i) not measured, detected or determined in a biological sample from a subject; ii) not significantly different from a control, wherein the control includes levels of the one or more biomarkers that represent the level of the one or more biomarkers in a biological sample from a subject that does not suffer from a disease, disorder or condition (e.g., PAH); iii) less than a control, wherein the control includes levels of the one or more biomarkers that represent the level of the one or more biomarkers in a biological sample from a subject that suffers from a disease, disorder or condition (e.g., PAH); or iv) decreased compared to the level of the one or more biomarkers before the subject was treated with the therapy (e.g., a BMP9 antibody or antigen-binding fragment thereof described herein). In some aspects, a therapy (e.g., a BMP9 antibody or antigen-binding fragment thereof described herein) is determined to be ineffective when one or more biomarkers are: i) measured, detected, or determined in a biological sample from a subject; ii) increased compared to a control, wherein the control includes levels of the one or more biomarkers that represent the levels of the one or more biomarkers in a biological sample from a subject not suffering from a disease, disorder, or condition (e.g., PAH); iii) not significantly different from or greater than the control, wherein the control includes levels of the one or more biomarkers that represent the levels of the one or more biomarkers in a biological sample from a subject suffering from a disease, disorder, or condition (e.g., PAH); or iv) not significantly different from or increased compared to the levels of the one or more biomarkers before the subject was treated with the therapy (e.g., a BMP9 antibody or antigen-binding fragment thereof described herein).

[0423] In some aspects, the method further includes measuring, detecting or determining the level of one or more biomarkers in the biological sample obtained from the subject before treatment. In some aspects, the method further includes measuring, detecting or determining the level of one or more biomarkers in the biological sample obtained from the subject after one or more treatments with therapy (e.g., BMP9 antibody or its Fab as described herein). For example, the method may include or further include after the therapy (e.g., BMP9 antibody or its Fab as described herein) of a dosage, after the therapy of two dosages, after the therapy of three dosages, after the therapy of four dosages, after the therapy of five dosages, after the therapy of six dosages, or more after the therapy of more dosages, determine, measure, detect or determine biomarker levels. Therapeutic efficacy can be determined based on the measurement, detection or determination level of one or more biomarkers.

[0424] In some aspects, the determination, measurement, detection or determination level of BMP9 and / or biomarker is normalized. In some aspects, the level of BMP9 and / or biomarker is compared with a control. In some aspects, it is determined that the measurement, detection or determination level of BMP9 and / or biomarker is greater than the control. In some aspects, it is determined that the measurement, detection or determination level of BMP9 and / or biomarker is less than the control. In some aspects, the subject has or has been determined to have greater than the BMP9 and / or biomarker level in the control sample, the measurement, detection or determination level of biomarker in the sample. In some aspects, the subject has or has been determined to have less than the BMP9 and / or biomarker level in the control sample, the measurement, detection or determination level of biomarker in the sample. In some aspects, the subject has or has been determined to have less than the BMP9 and / or biomarker level in the control sample, the measurement, detection or determination level of biomarker in the sample. In some aspects, the subject has or has been determined to have no significant difference in the level of BMP9 and / or biomarker in the control sample, the measurement, detection or determination level of biomarker in the sample. The control may include such a level of BMP9 and / or a biomarker, which represents the level of BMP9 and / or a biomarker in a sample from a subject with or without a disease, disorder or condition (e.g., PAH). In some cases, the control may include such a level of BMP9 and / or a biomarker, which represents the level of BMP9 and / or a biomarker in a sample from a subject with a disease, disorder or condition (e.g., PAH). In some aspects, the control may include such a level of BMP9 and / or a biomarker, which represents the level of BMP9 and / or a biomarker in a sample from a subject without a disease, disorder or condition (e.g., PAH).

[0425] The subject sample used in the method described herein can be obtained using any method known in the art that can provide a sample suitable for the analytical method described herein. The method for obtaining a sample from a subject provided herein can include, for example, obtaining tissue, cell or biomaterial by scraping, wiping, biopsy or otherwise collecting tissue, cell or biomaterial. The sample can include, but is not limited to, tissue, cell or biomaterial derived from the cell of the subject. The general method for obtaining biological samples is also known in the art. For example, publications such as Ramzy, Ibrahim, Clinical Cytopathology and Aspiration Biopsy [clinical cell pathology and aspiration biopsy] (2001) (which is incorporated herein by reference in its entirety) describe the general method for biopsy and cytological methods. The biological sample can be a heterogeneous or homogeneous cell or tissue colony. In some aspects, the sample is obtained from, including but not limited to, the following sources: urine, blood, serum, plasma, sputum, mucus, saliva, exhaled breath condensate, bronchoalveolar lavage fluid, cerebrospinal fluid, sweat, tissue, hair follicle, cheek tissue, tears, menstruation or feces. In certain aspects, sample is obtained from serum. In some aspects of the inventive method, any medical professional (such as doctor, nurse or medical technician) can obtain the biological sample for testing. Further, biological sample can be obtained without the assistance of medical professional. In some aspects of the inventive method, biological sample is obtained from experimenter (directly), medical professional, third party or the test kit provided by molecular analysis enterprise or third party. In some cases, biological sample can be obtained after experimenter, medical professional or third party obtain and send biological sample to third party. In some cases, third party can provide suitable container and excipient for storing biological sample and transporting to third party.

[0426] Without wishing to be bound by theory, it is hypothesized that in diseased subjects (such as in patients with PAH), reduction in BMPRII due to mutation or downregulation converts BMP9 from a vasoquiescent factor to a driver of vascular remodeling via regulation of EC proliferation and survival (i.e., BMPRII dysregulation results in BMP9 pathogenic gain of function). Accordingly, these methods include treating a subject with a BMP9 antibody, antigen-binding fragment thereof, or pharmaceutical composition thereof as described herein to block binding of BMP9 to type II receptors (including BMPRII, ActRIIA, and ActRIIB), thereby neutralizing their downstream (pathological) signaling in endothelial cells (ECs) and smooth muscle cells (SMCs) that are dysregulated in PAH. In some embodiments, the BMP9 antibody, antigen-binding fragment thereof, or pharmaceutical composition thereof described herein binds to BMP9 and blocks the binding of BMP9 to the type II receptor by at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount or any range thereof (e.g., 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%): 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to before administration.

[0427] With respect to all methods described herein, reference to an anti-BMP9 antibody also includes antigen-binding fragments thereof and pharmaceutical compositions comprising an anti-BMP9 antibody or antigen-binding fragment thereof and one or more additional agents.

[0428] Administration and dosing

[0429] Typically, the antibodies or antigen-binding fragments thereof of the disclosure are administered in an amount effective to treat a condition as described herein.The antibodies of the disclosure may be administered as the antibody itself, or alternatively, as a pharmaceutical composition containing the antibody.

[0430] In certain embodiments, an antibody or antigen-binding fragment thereof of the present disclosure, or a pharmaceutical composition thereof, is administered by any suitable route, in the form of a pharmaceutical composition suitable for such route, and at a dose effective for the intended treatment.

[0431] In certain embodiments, antibody or its Fab or its pharmaceutical composition can be administered parenterally, for example, directly administered to bloodstream, muscle or internal organs. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) syringe, needle-free syringe and infusion technology. In certain embodiments, antibody or its Fab or its pharmaceutical composition can be administered subcutaneously. In certain embodiments, antibody or its Fab or its pharmaceutical composition can be administered intravenously. It is expected that any one or more of these aforementioned routes of administration can be excluded in the embodiments.

[0432] In another embodiment, the compounds disclosed herein can also be topically applied to the skin or mucous membrane, i.e., cutaneous, transdermal or sublingual administration. In another embodiment, the compounds disclosed herein can also be administered intranasally or by inhalation. In another embodiment, the compounds disclosed herein can be administered rectally or vaginally. In another embodiment, the compounds disclosed herein can also be directly administered to the eyes or ears. It is contemplated that any one or more of these aforementioned routes of administration can be excluded in the embodiments.

[0433] The dosage regimen for the antibodies or antigen-binding fragments thereof or compositions containing the antibodies disclosed herein is based on a variety of 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 specific antibody employed. Therefore, the dosage regimen can vary widely. In one embodiment, the total daily dose of an antibody of the disclosure for treating a given disorder discussed herein is typically from about 0.01 to about 1000 mg / kg (i.e., mg antibody of the disclosure / kg body weight) (e.g., at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount or any range thereof: 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, / kg, 0.5mg / kg, 0.6mg / kg, 0.7mg / kg, 0.8mg / kg, 0.9mg / kg, 1mg / kg, 2mg / kg, 3mg / kg, 4mg / kg, 5mg / kg, 6mg / kg, 7mg / kg, 8mg / kg, 9mg / kg, 10mg / kg, 20mg / kg, 30mg / kg, 40mg / kg, 50mg / kg, 60mg / kg, 70mg / kg, 80mg / kg, 90mg / kg, 100mg / kg, 200mg / kg, 300mg / kg, 400mg / kg, 500mg / kg, 600mg / kg, 700mg / kg, 800mg / kg, 900mg / kg or 1000mg / kg). 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 daily dose is about 0.5 mg / kg (i.e., about 35 mg / day in a person of typical size).

[0434] In some embodiments, the method or use comprises administering a dose of about 1 mg to about 1000 mg. In some embodiments, the methods or uses include administering a dose of about 1 mg to 1000 mg (e.g., at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount or any range thereof: 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) as an initial fixed dose. In some embodiments, the methods or uses comprise administering a dose of about 1 mg to about 2 mg, about 2 mg to about 5 mg, about 5 mg to about 10 mg, about 10 mg to about 20 mg, about 20 mg to about 30 mg, about 30 mg to about 40 mg, about 40 mg to about 50 mg, about 50 mg to about 60 mg, about 60 mg to about 70 mg, about 70 mg to about 80 mg, about 80 mg to about 90 mg, about 90 mg to about 100 mg, about 100 mg to about 150 mg, about 150 mg to about 200 mg, about 200 mg to about 300 mg, about 300 mg to about 400 mg, about 400 mg to about 500 mg, about 500 mg to about 600 mg, about 600 mg to about 700 mg, about 700 mg to about 800 mg, about 800 mg to about 900 mg, or about 900 mg to about 1000 mg, optionally as an initial fixed dose. In some embodiments, the method or use comprises administering an anti-BMP9 antibody or a pharmaceutical composition thereof at a dosage 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 dosage is an initial fixed dose.

[0435] In some embodiments, the methods or uses comprise subcutaneous administration of a dose of about 12 mg to 500 mg (e.g., at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount or any range thereof: 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) every 2 weeks, every 3 weeks, or every 4 weeks. In some embodiments, the method of use comprises subcutaneously administering 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 every 2 weeks, every 3 weeks, or every 4 weeks. , about 110mg, about 115mg, about 120mg, about 125mg, about 130mg, about 135mg, about 140mg, about 145mg, about 150mg, about 175mg, about 200mg, about 210mg, about 220mg, about 230mg, about 240mg, about 250mg, about 300mg, about 350mg, about 400mg, about 450mg or about 500mg. In some embodiments, the method or use comprises a dose of about 110mg subcutaneously every 3 weeks. In some embodiments, the method or use comprises a dose of about 30mg, about 50mg, about 75mg, about 100mg, about 150mg, about 165mg, about 180mg or about 200mg every 2 weeks. In some embodiments, the method or use comprises a dose of about 165mg every 2 weeks. In some embodiments, the method or use includes a dosage 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 3 weeks. In some aspects, the method or use includes a dosage of about 255 mg every 3 weeks. In some embodiments, the method or use includes 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 4 weeks. In some aspects, the method or use includes a dosage of about 355 mg every 4 weeks. In some embodiments, the antibody is Ab1076.

[0436] In a standard antagonist antibody model at steady state, the administered dose provides an average inhibition of free BMP9 of about 90% to about 99% (e.g., at least the following amount or any range thereof, at most the following amount or any range thereof, exactly the following amount or any range thereof, between any two of the following amounts or any range thereof (including or excluding the end values), or about the following amount or any range thereof: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%). In some embodiments, the antibody or its antigen-binding fragment or its pharmaceutical composition is administered subcutaneously 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 every 2 weeks, and the antibody or its antigen-binding fragment or its pharmaceutical composition provides 95%, 98% or 99% average inhibition of free BMP9 at steady state. In some embodiments, the antibody or its antigen-binding fragment or its pharmaceutical composition is administered subcutaneously 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 every 3 weeks, and the antibody or its antigen-binding fragment or its pharmaceutical composition provides 95%, 98% or 99% average inhibition of free BMP9 at steady state. In some embodiments, the antibody or its antigen binding fragment or its pharmaceutical composition is administered subcutaneously 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 every 4 weeks, and the antibody or its antigen binding fragment or its pharmaceutical composition provides 95%, 98% or 99% average inhibition of free BMP9 at steady state. In some embodiments, the antibody is Ab1076.

[0437] In some embodiments, the method or use includes administering 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 of the present disclosure of the anti-BMP9 antibody or its antigen-binding fragment or its pharmaceutical composition. In some embodiments, the method or use includes administering 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 of the present disclosure of the anti-BMP9 antibody or its antigen-binding fragment or its pharmaceutical composition on a weekly basis. In some embodiments, the method or use includes subcutaneous administration of about 2mg, 10mg, 20mg, 30mg, 40mg, 50mg, 75mg, 100mg, 125mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg or 500mg of an anti-BMP9 antibody (e.g., Ab1076) or its antigen-binding fragment or its pharmaceutical composition on a weekly basis. In some embodiments, the method or use includes subcutaneous administration of about 2mg, 10mg, 20mg, 30mg, 40mg, 50mg, 75mg, 100mg, 125mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg or 500mg of an anti-BMP9 antibody (e.g., Ab1076) or its antigen-binding fragment or its pharmaceutical composition on a monthly basis. The desired dose may be given subcutaneously as a single injection, or may be delivered in more than one injection in one or more injection sites. For example, the desired dose may be administered subcutaneously as two bolus injections once a month.

[0438] In some embodiments, the method or use comprises administering an antibody or its antigen-binding fragment or pharmaceutical composition of a dosage of about 0.01 mg / kg to about 300 mg / kg, about 1 mg / kg to about 250 mg / kg, about 10 mg / kg to about 200 mg / kg, about 50 mg / kg to about 150 mg / kg, or about 75 mg / kg to about 100 mg / kg, optionally as an initial dose. The initial dose may be followed by one or more subsequent doses. In some embodiments, one or more subsequent doses may be administered at least weekly, 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.

[0439] The initial dose may be followed by one or more subsequent doses. In certain embodiments, the subsequent dose is the same dose, a lower dose, or a higher dose of anti-BMP9 antibody compared to the initial dose. In certain embodiments, one or more subsequent doses may be administered at least weekly, 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. A specific dosage regimen is a regimen involving the maximum dose or dosage frequency that avoids significant undesirable side effects.

[0440] It is contemplated that any one or more of these foregoing dosages and / or dosing regimens may be excluded in the embodiments.

[0441] Co-administration

[0442] The antibodies or antigen-binding fragments thereof disclosed herein can be used alone or in combination with one or more other therapeutic agents. The present disclosure provides any use, method or composition as defined herein, wherein the antibodies or antigen-binding fragments thereof disclosed herein are used in combination with one or more other therapeutic agents. For example, in the treatment of pulmonary hypertension, the BMP9 antibodies or antigen-binding fragments thereof described herein can be used in combination with one or more current pulmonary hypertension therapies. Examples of such therapies include, but are not limited to, 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. The treatment of pulmonary hypertension can also be combined with oxygen therapy, atrial septostomy, pulmonary endarterial thrombectomy and / or lung and / or heart transplantation. It is expected that any one or more of these aforementioned therapies can be excluded in the embodiments.

[0443] Administration of two or more agents "in combination" means that all agents are administered close enough in time to affect treatment of the subject. Two or more agents may be administered simultaneously or sequentially. Additionally, simultaneous administration may be performed by mixing the agents prior to administration or by administering the agents as separate dosage forms at the same time point but at the same or different administration sites.

[0444] Reagent test kit

[0445] Another aspect of the disclosure provides kits, which include antibodies or antigen-binding fragments thereof (e.g., Ab1076) disclosed herein or pharmaceutical compositions containing antibodies or antigen-binding fragments thereof (e.g., Ab1076). In addition to antibodies or antigen-binding fragments thereof or pharmaceutical compositions disclosed herein, the kits may also include diagnostic agents or therapeutic agents. The kits may also include instructions for use in diagnostic or therapeutic methods. In some embodiments, the kits include antibodies or antigen-binding fragments thereof or pharmaceutical compositions thereof and diagnostic agents.

[0446] In other embodiments, the kit comprises an antibody or 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 embodiments.

[0447] In yet another embodiment, the disclosure includes a kit suitable for use in performing the methods of treatment described herein. In one embodiment, the kit contains a first dosage form comprising one or more antibodies or antigen-binding fragments thereof of the disclosure in an amount sufficient to perform 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 perform the methods disclosed herein, and at least a first container for the first dose and a second container for the second dose.

[0448] Biological Deposit

[0449] Representative materials of the present disclosure were deposited at the American Type Culture Collection, 10801 University Avenue, Manassas, Virginia, USA (20110-2209) on April 19, 2022. The vector "Ab1076-VH" with ATCC accession number PTA-127292 contains a DNA insert encoding the anti-BMP9 heavy chain variable region, and the vector "Ab1076-VL" with ATCC accession number PTA-127293 contains a DNA insert encoding the anti-BMP9 light chain variable region.

[0450]

[0451] The deposit is made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and its Regulations (Budapest Treaty). This ensures that living cultures of the deposit will be maintained for 30 years from the date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty and is subject to an agreement between Pfizer and ATCC that ensures that progeny of the cultures of the deposit will be available to the public in perpetuity and without restriction upon the issuance of a related U.S. patent or the public availability of any U.S. or foreign patent application, whichever comes first, and that the progeny will be available for use by a party determined by the Commissioner of the United States Patent and Trademark to have such rights pursuant to 35 U.S.C. Chapter 122 and the Commissioner's Rules thereunder (including 37 C.FR Chapter 1.14, specifically referring to 886 OG 638).

[0452] The assignee of this application has agreed that if a culture of the deposited material dies or is lost or destroyed when grown under suitable conditions, the material will be replaced with another identical material immediately upon notification. The availability of the deposited material shall not be construed as a license to practice the invention in violation of the rights granted by any government under its patent laws.

[0453] Examples

[0454] The following are examples for carrying out the specific aspects of the present disclosure. The following examples are included to show the aspects of the present disclosure. These 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 appreciated by those skilled in the art that the technology disclosed in the following examples represents the technology found by the inventors to function well in the practice of the present disclosure. However, in view of the present disclosure, it will be appreciated by those skilled in the art that many changes can be made to the disclosed specific aspects without departing from the spirit and scope of the present disclosure and similar or similar results are still obtained. Efforts have been made to ensure the accuracy of the numerals (e.g., amount, temperature, etc.) used, but of course some experimental errors and deviations should still be allowed.

[0455] Example 1: Selection of anti-BMP9 antibodies by phage display.

[0456] Anti-BMP9 single chain Fv (scFv) hits were selected from the human phage display antibody library WyHN5 by panning on mature human BMP9 as follows.

[0457] Human mature BMP9 protein was biotinylated with sulfo-NHS-LC-biotin (Thermo) according to the manufacturer's protocol. Four rounds of selection were performed in solutions with decreasing concentrations of biotinylated human BMP9 as follows: 200 nM biotinylated BMP9 (round 1); 50 nM biotinylated BMP9 (round 2); 1 nM biotinylated BMP9, followed by addition of 1 μM non-biotinylated BMP9 and incubation overnight (round 3); and 1 nM biotinylated BMP9 (round 4). In each round, the cells were stained with a magnetic column. Phages bound to biotinylated BMP9 were captured with M-280 streptavidin (Invitrogen), followed by washing to remove non-specifically bound phages. BMP9-specific binders were eluted with 100 mM triethylamine and subsequently amplified in E. coli for subsequent selection rounds and / or screening ELISA.

[0458] scFv can be expressed on the surface of phage particles or in solution in the bacterial periplasmic space, depending on the growth conditions used. In order to induce scFv to be released into the periplasm, 96-well plates containing 2X YT culture medium (it has 0.1% glucose and 100 μg / ml ampicillin) are inoculated from 2X YT agar plates (one clone per hole), and it is grown with 850rpm shaking for about 4h at 37°C. The culture is induced with the IPTG that final concentration is 0.02mM, and it is grown with 850rpm shaking overnight at 30°C. The content (periplasmic preparation (periprep)) of the bacterial periplasm is released by osmotic pressure shock. In brief, the plate is centrifuged, and the precipitate is resuspended in 150ml TES periplasmic buffer (50mM Tris, 1mMEDTA, 20% sucrose, pH 7.4), 150 μl 1:5TES: water is added subsequently, and incubated on ice for 30min. The plate was centrifuged at 4000 rpm for 20 min, and the supernatant containing the scFv was harvested.

[0459] For the binding assay performed with scFv periplasmic preparations, high binding 96-well ELISA plates (Costar 3590) were coated overnight with 0.1 μg / well (100 μl, 1 μg / ml) human BMP9 in PBS buffer at 4°C. After discarding the coating solution, the plate was blocked with PBS+3% BSA for 2h at room temperature. After discarding the blocking solution, 100 μl periplasmic preparations were added. After incubation for 1h at room temperature with slow shaking, the plate was washed 5 times with 300 μl / well washing buffer (Perkin Elmer 1244-114) and incubated for 1h with 100 μl HRP-labeled anti-His tag secondary antibodies. After washing 5 times with 300 μl / well washing buffer, the plate was developed for 10min with TMB substrate solution, and then 0.18M H2SO4 was added to terminate the reaction. The absorbance at 450 nM was measured and the data were plotted and analyzed using Microsoft Excel. From this screening, 100 scFv hits were identified as binding to human BMP9 by periplasmic preparation 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 subsequently converted to human IgG1 / λ using standard molecular biology methods.

[0460] Example 2: Binding of anti-BMP9 antibodies to human BMP9 protein.

[0461] This example demonstrates the binding of the identified anti-BMP9 antibodies to human BMP9 protein in an enzyme-linked immunosorbent assay (ELISA).

[0462] Purified human mature BMP9 protein was coated on NUNC at 1 μg / ml at 4°C. TM -MAXISORP TM 100 μl PBS (without calcium and magnesium) on a 96-well ELISA plate. TM The plates were washed 3 times at 20 °C and blocked with PBS + 1% BSA for 1 h at room temperature with shaking. The antibodies tested were serially diluted 5-fold starting from 100 nM in PBS + 1% BSA. The blocking solution was removed and the plates were washed as described above, followed by the addition of serially diluted antibodies and incubation for 1 h at room temperature with shaking. The plates were washed as described above, and the secondary antibody (goat anti-human IgG Fc-HRP from Invitrogen, catalog number 31413, 1:10000) was added, followed by incubation for 1 h at room temperature. The plates were washed as described above, the signals were developed using TMB substrate, and the reaction was stopped with 0.18 M H2SO4. The absorbance was read at 450 nm on a plate reader (PerkinElmer).

[0463] The results are shown in Figure 1 All identified antibodies (Ab89, Ab93, Abl00 and Abl01) bind to human mature BMP9 with high potency. BM01 (referred to as AM0100 in WO 2016 / 193872) and BM02 (referred to as AM4405 in WO 2016 / 193872) were used as reference antibodies. In contrast, isotype control antibodies were unable to bind to human BMP9.

[0464] Example 3: Competitive ELISA to measure the neutralizing activity of anti-BMP9 antibodies.

[0465] This example demonstrates the ability of the identified anti-BMP9 antibodies to inhibit the interaction between BMP9 and its receptor in a blocking (competition) ELISA.

[0466] Type I receptor (ALK1) and type II receptor (BMPRII, ActRIIA, or ActRIIB) were used to see if anti-BMP9 antibodies could compete with these receptors for binding to BMP9. Each receptor was coated on an ELISA plate at 1-15 μg / ml and then incubated with serially diluted biotinylated BMP9 to determine the optimal receptor coating concentration and determine the EC of biotinylated BMP9 binding to each receptor. 90 (Data not shown).

[0467] For competitive ELISA, each receptor protein was coated in PBS-CMF at its optimal coating concentration (20 μl / well) on a 384-well ELISA plate overnight at 4°C. The next day, the plate was washed with PBS + 0.05% TWEEN TM The cells were washed 3 times at 20 °C and blocked with PBS + 1% BSA for 1 h at room temperature with shaking. Anti-BMP9 antibody was serially diluted 3-fold starting from 100 nM in PBS + 1% BSA and then mixed with the same volume of the optimal concentration of biotinylated BMP9. The final concentration of biotinylated BMP9 corresponded to the EC of each receptor. 90 , as determined in the preliminary experiments described above.

[0468] The serially diluted antibodies were incubated with biotinylated BMP9 for at least 15 min at room temperature. The plates were washed as before, followed by the addition of the serially diluted antibody and biotinylated BMP9 mixture. After incubation at room temperature for 1 h, the plates were washed with PBS + 0.05% TWEEN TM20 washes 3 times. Secondary antibody (HRP-streptavidin from BioLegend, catalog number 405210, 1:2000) was added and then incubated for 1 h at room temperature. The plates were washed as before, the signals were developed using TMB substrate, and the reaction was stopped with 0.18 M H2SO4. The absorbance was read at 450 nm on a plate reader (PerkinElmer).

[0469] The results of these competitive ELISAs are shown in Figure 2. Antibodies Ab89, Ab93, Abl00, and Abl01 inhibited the binding of BMP9 to type II receptors (BMPRII, ActRIIA, or ActRIIB), but did not inhibit the binding of BMP9 to type I receptor ALK1 ( Figure 2A-2D ). Antibodies Ab352, Ab804, and Ab1076 derived from parent antibody Ab93 strongly inhibited the binding of BMP9 to the type II receptor (BMPRII), and weakly inhibited the binding of BMP9 to the type I receptor ALK1 ( Figure 2E-2F ). In contrast, the reference antibody BM02 is a type I receptor blocker as it strongly inhibits the binding of BMP9 to ALK1 ( Figure 2F ), and did not inhibit type II receptor binding (not shown).

[0470] Example 4: Effect of anti-BMP9 antibodies on the binding of BMP9 to endothelial cells.

[0471] This example demonstrates the ability of the identified anti-BMP9 antibodies to inhibit the binding of human BMP9 to endothelial cells in a dose-dependent manner.

[0472] Human telomerase-immortalized microvascular endothelial (TIME) cells were obtained from ATCC and cultured according to the supplier's instructions. Recombinant human BMP9 was incubated with NHS-ALEXA FLUOR Dye conjugation. On the day of the experiment, cells were collected by enzymatic dissociation and resuspended in pre-chilled growth medium. Equal aliquots of the cell suspension were then distributed into the wells of a 96-well plate and placed on ice.

[0473] The BMP9 antibody was serially diluted 4-fold in pre-chilled cell growth medium to a final concentration of 0.2 nM, 1 nM, 5 nM, 25 nM, and 125 nM. The protein was diluted 4-fold in pre-cooled cell growth medium to a final concentration of 1 nM to be combined with serially diluted BMP9 antibodies at a 1:1 (v / v) ratio to obtain 2-fold final concentrations of BMP9 and antibodies, respectively. Additionally, aliquots of the 4X BMP9 solution were combined 1:1 (v / v) with pre-cooled medium without antibodies.

[0474] The above samples were then incubated under refrigeration and in dark for 40 min, then the aliquots from each sample were distributed to the wells containing TIME cell suspensions in 96-well plates at a ratio of 1:1 (v / v). The cell samples mixed with the culture medium without BMP9 or antibody were considered as untreated controls. The plate was further incubated in dark for 60 min on ice, then the samples in the wells were washed with an excessive amount of precooled buffer containing fetal bovine serum albumin and sodium azide, to prevent the active uptake of the material from the cell environment.

[0475] Then use a BD FACSYMPHONY equipped with appropriate optical settings. TM A5 flow cytometer for analyzing samples using BD FACSDIVA TM Software analyzes ALEXA FLUOR of each cell Median dye intensity. Raw data files were exported for analysis with FlowJo software and numerical data were plotted using GraphPad Prism.

[0476] The results are shown in Figure 3 Anti-BMP9 antibodies Ab89, Ab93, and AblOO exhibited dose-dependent inhibition of BMP9 binding to TIME cells.

[0477] Example 5: Determination of the affinity of Ab1076 for human, cynomolgus monkey and rat BMP9.

[0478] This example confirms the binding affinity of anti-BMP9 antibody Ab1076 (analyzed as a Fab) to human, cynomolgus monkey, and rat BMP9.

[0479] The BIACORE was used at 10 Hz collection rate at 37 °C. TMT200 (Cytiva) determines the binding affinity of Ab1076 (as monovalent Fab fragment) to mature human, cynomolgus monkey and rat BMP9 by surface plasmon resonance (SPR). Running buffer and sample buffer are 10mM HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.05% P-20 (HBS-EP+). Biotinylated mature human, cynomolgus monkey and rat BMP9 proteins are reversibly captured on sensor chip CAP using biotin capture reagent (Cytiva, 28920234) according to the manufacturer's instructions. The final capture level of human BMP9 is 100 resonance units (RU), the final capture level of cynomolgus monkey BMP9 is 50RU, and the final capture level of rat BMP9 is 60RU. Use flow cell 1 without BMP9 as reference flow cell. A two-fold dilution series of Ab1076 Fab ranging from 100 nM to 6.2 nM was injected at a flow rate of 100 μl / min for 60 sec, and dissociation was monitored for 1800 sec. The dual reference sensorgram data were fitted to the BIACORE TM The rate constants and affinities were determined using a 1:1 model in Insight Evaluation Software Version 3.0.1.12.15655 (Science & Technology Corporation).

[0480] As summarized in Table 1, the equilibrium dissociation constants (K) of human, cynomolgus monkey, and rat BMP9 are D ) values ​​were 87.41pM, 65.82pM and 98.63pM, respectively. D The values ​​are within 2-fold of human BMP9. Representative sensorgrams are shown in FIG4 .

[0481] Table 1. Affinity of Ab1076 Fab fragments for human, cynomolgus monkey and rat BMP9 determined by SPR at 37°C. Sensorgram data from 3 replicate measurements of 3 independent dilutions of Ab1076 Fab analyte were used to calculate k a , k d and K D ±SD values.

[0482]

[0483] Example 6: Evaluation of off-target binding of Abl076 to a panel of TGFβ family members.

[0484] In this example, Abl076 (tested as a Fab) was evaluated for off-target binding to a panel of TGFβ family members.

[0485] Use at 37°C The biolayer interferometry (BLI) of the Red 384 instrument was used to evaluate the off-target binding of Ab1076 Fab to a panel of TGFβ family members, including human TGFβ1, GDF8, GDF9, GDF11, activin A, and BMP10. Human BMP9 was used as a positive control. TGFβ family members were amine coupled to AR2G biosensors (Sartorius ForteBio, 18-5092) at 20 μg / ml for 300 seconds. The fixed levels were as follows: GDF8, 0.72 nm; GDF11, 0.28 nm; BMP9, 0.58 nm; BMP10, 1.0 nm; GDF9, 0.35 nm; TGFβ1, 0.62 nm; and activin A, 0.6 nm. The biosensor with immobilized TGFβ family members was immersed in Ab1076 Fab diluted to 100 nM in Kinetics buffer (Sartorius Fortebio 18-5032) for 300 seconds. After that, the dissociation was continued for 180 seconds. The data were collected using acquisition software version 11.0.0.64 and Double referencing was performed in the data analysis software version 11.0.

[0486] At 100 nM, the Abl076 Fab exhibited no observable off-target binding to the TGFβ family members evaluated, but did bind BMP9 ( Figure 5 ). All TGFβ family members were active as demonstrated by their binding to their respective positive controls (not shown).

[0487] Example 7: Anti-BMP9 antibodies inhibit BMP9-induced phosphorylation of SMAD1 / 5 / 9 and phosphorylation of SMAD2 signaling.

[0488] In this example, the ability of anti-BMP9 antibodies to inhibit phosphorylation-SMAD1 / 5 / 9 and phosphorylation-SMAD2 nuclear translocation induced by BMP9 (as a measure of activation of SMAD1 / 5 / 9 or SMAD2 signaling in people, cynomolgus monkeys and rat endothelial cells) was tested. In brief, in the presence of anti-BMP9 antibodies, 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 10pM or 1000pM people or species-specific BMP9 for 30 minutes. Cells were fixed, closed and permeabilized, and stained overnight with antibodies for pSMAD1 / 5 / 9 or pSMAD2. The next day, cells were washed and stained with ALEXA The cells were stained with 488-labeled secondary antibodies to detect pSMAD1 / 5 / 9 or pSMAD2, Hoechst 33342 to detect the nucleus, and cell mask deep red stain to depict the entire cell. After a final wash, the cells were imaged on a high-content imager. To analyze pSMAD1 / 5 / 9 or pSMAD2 nuclear translocation, ALEXA was calculated for each well of the cells. Average nuclear fluorescence in channel 488. IC50 values ​​were calculated using GraphPad Prism version 9.0.0.

[0489] Dose-dependent inhibition of BMP9-induced nuclear translocation of pSMAD1 / 5 / 9 and pSMAD2 was observed in all endothelial cells following treatment with anti-BMP9 antibodies.

[0490] In several experiments performed, the IC values ​​of anti-BMP9 antibodies against human BMP9 in the pSMAD1 / 5 / 9 assay were 50 The values ​​ranged from 0.02 nM to 86 nM. IC values ​​of anti-BMP9 antibodies against cynomolgus monkey BMP9 in the pSMAD1 / 5 / 9 assay 50 The values ​​ranged from 17pM to 117pM, and the IC values ​​for rat BMP9 50 The values ​​ranged from 120 pM to 300 pM. In several experiments performed, the IC values ​​of anti-BMP9 antibodies against human BMP9 in the pSMAD2 assay were 50 The values ​​ranged from 0.01 nM to 330 nM. IC of Ab1076 against cynomolgus monkey BMP9 in the pSMAD2 assay 50 The values ​​ranged from 27pM to 42pM, and the IC 50 The values ​​ranged from 465 pM to 1 nM. Representative images are shown in FIG6 .

[0491] Example 8: Anti-BMP9 antibodies Ab732 and Abl076 are effective in the rat Sugen-hypoxia model of pulmonary hypertension.

[0492] Efficacy studies were performed in the preclinical Sugen-hypoxia (SuHx) model of pulmonary arterial hypertension (PAH) to evaluate the effects of Ab732 and Ab1076 relative to isotype control and activin ligand trap (ActRIIA-Fc). In this model of vascular proliferation, rodents develop progressive occlusive and complex lesions reminiscent of severe human disease (Abe, K., Toba, M., Alzoubi, A., Ito, M., Fagan, KA, Cool, CD, Voelkel, NF, McMurtry, IF, and Oka, M. (2010) Formation of plexiform lesions in experimental severe pulmonary arterial hypertension. Circulation 121, 2747-2754).

[0493] 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 normobaric hypoxia (FiO2=0.1) for moderate disease induction. An age-matched untreated (normoxic) control group was also included. Four weeks later, the SuHx-treated rats were returned to normoxia and randomly assigned to different treatment groups with equal body weights.

[0494] Treatment was administered intraperitoneally twice a week for a period of 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 isotype control human IgG1 was administered at 3 mg / kg. At the end of the study, cardiovascular hemodynamic assessment was performed. Rats were anesthetized and right ventricular systolic pressure (RVSP) was measured by catheterization. In order to assess the extent of right ventricular hypertrophy (RVH), the right ventricular free wall was dissected from the left ventricle plus the septum (LV+S) in the explanted heart, and weighed; RVH is expressed as the ratio of the weight of the RV to the weight of (LV+S). The results are shown in Figure 7.

[0495] The mean RVSP and RVH in the untreated (normoxia) group were 29.2±1.25 mmHg and 0.197±0.00669, respectively. When administered after 4 weeks of SuHx, 3 mg / kg of Ab1076 significantly improved RVSP: 63.7±3.82 mmHg, compared to 94.9±7.27 mmHg for isotype control—a 31.2 mmHg or 47.4% decrease in the difference [isotype control minus normoxia] (p<0.05). 3 mg / kg of Ab1076 also improved RVH (0.331±0.0240) relative to isotype control (0.410±0.0236), representing a 37.1% decrease in the difference [isotype control minus normoxia]. Ab732 also induced significant improvements in hemodynamic parameters: 3 mg / kg of Ab732 significantly improved RVSP: 55.4 ± 6.81 mmHg - [isotype control minus normoxia] difference decreased by 39.5 mmHg or 60.0% (p < 0.01). 3 mg / kg of Ab732 improved RVH (0.329 ± 0.0216) relative to isotype control, [isotype control minus normoxia] difference decreased by 37.9%.

[0496] ActRIIA-Fc treatment also significantly improved RVSP (62.9±4.60 mmHg, a decrease of 31.9 mmHg compared to isotype control; p<0.05) and improved RVH relative to isotype control (0.325±0.0171).

[0497] In conclusion, administration of Ab732, Abl076, and ActRIIA-Fc attenuated the magnitude of PAH seen in this study.

[0498] Example 9: Evaluation of total serum BMP9 and BMP10 concentrations in the Sugen-hypoxia (SuHx) model.

[0499] The effect of anti-BMP9 treatment on total soluble BMP9 and BMP10 concentrations in rat serum samples collected at the end of the SuHx efficacy dose study of Example 8 was evaluated by immunoaffinity LCMS analysis. This functional readout was used to quantitatively determine target:antibody complexes in serum as a measure of target engagement. The results are shown in FIG8 .

[0500] Similar and consistent circulating BMP9 and BMP10 concentrations were measured in sera from rats maintained under normoxic conditions and SuHx rats treated with isotype controls. No significant changes in circulating concentrations of BMP9 and BMP10 were observed following treatment with ActRIIA-Fc.

[0501] A 1.7-fold increase in circulating BMP9 concentrations was observed following treatment with 3 mg / kg Ab732 relative to normoxic and isotype controls, and no changes were noted at 0.3 mg / kg. A dose-dependent accumulation of circulating BMP9 was observed with Ab1076 treatment: a 1.6-fold and 7.9-fold increase was measured at 0.3 mg / kg and 3 mg / kg, respectively, relative to normoxic and isotype controls (p≤0.0001)( Fig. 8A This is consistent with the higher affinity of BMP9 for Ab1076 compared to Ab732. The magnitude of change in circulating BMP10 was reduced relative to BMP9 ( Figure 8B ).

[0502] Example 10: Analysis of the molecular interaction of Ab93, AblOl and Abl076 with human BMP9 by X-ray crystallography.

[0503] Crystallization and structure determination of mature human BMP9 in complex with Ab1076 Fab

[0504] For crystallization experiments, the complex between Ab1076 Fab and mature human BMP9 was formed at a molar ratio of 1:1.5 and concentrated to 9.2 mg / ml in a protein solution containing TBS (pH 7.5).

[0505] Crystals were obtained by hanging drop vapor diffusion from 25% PEG 1500, 100 mM SPG buffer, pH 5. The crystals have symmetry consistent with orthorhombic space group C222, with unit cell parameters The crystals were rapidly frozen in liquid nitrogen using 15% ethylene glycol (EG) as a cryoprotectant solution. The crystals were collected from a single frozen crystal at the IMCA beamline 17-ID at Argonne National Laboratory. The data were processed and scaled using autoPROC and the final dataset was 62.7% complete.

[0506] The structure was solved by molecular replacement with PHASER. Several rounds of manual adjustment and model rebuilding using COOT and crystallographic refinement using autoBUSTER produced the final model of BMP9+Ab1076 Fab. work is 25.6% and R free is 27.7%, of which R work =||F 实测值 |-|F 计算值 || / |F 实测值 | and R free Equal to R work, but is calculated for a randomly selected 5% of reflections that are omitted from the refinement process.

[0507] The BMP9 epitopes for antibodies Abl076, Ab93 and Abl01 and the type II receptor ActRIIB were defined by a structure-based descriptor approach, as outlined in detail below. The structure-based descriptor approach utilized the crystal structure complex and defined the epitope residues as those residues that are: a) significantly buried by complex formation residues involved in hydrogen bonds, residues involved in water-mediated hydrogen bonds, residues involved in salt bridges, and residues involved in close contact with The residues of the interaction residues and the combination of the residues close to each other are then merged into a list of all potentially important epitope residues. This list is limited by the fact that sites that are not in close contact with the ligand may still be critical for binding, and sites close to each other may not be critical. The calculations use 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 chains A and E from the Protein Data Bank entry 4fao to generate descriptors for interface residues.

[0508] The BMP9 epitopes of antibodies Ab1076, Ab93 and Ab101 and type II receptor ActRIIB are summarized in Table 2. From this analysis, it is clear that there is a significant overlap between the epitopes of antibodies Ab1076, Ab93 and Ab101 and the epitopes of type II receptor ActRIIB (Figure 9) and other type II receptors (such as BMPRII, ActRIIA and endothelin) (data not shown). The overlap of the BMP9 epitopes of antibodies Ab1076 and ActRIIB is shown in Figure 9.

[0509] Table 2: BMP9 epitopes of antibodies Abl076, Ab93 and AblOl and type II receptor ActRIIB defined by structure-based descriptor approach.

[0510]

[0511]

[0512] Structure-based epitope residue descriptors

[0513] Here, 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 were used to generate descriptors for the interface residues. Residues are said to be hydrogen bonded if they contain hydrogen bond donor atoms (bonded to positively charged hydrogen) in the antibody. Alternatively, if a given residue participates in a hydrogen bond with a water molecule (which also hydrogen bonds to the antibody), the residue is considered to be part of a water-mediated hydrogen bond. If an atom in the antigen residue is in the These residues are in close contact.

[0514] The buried surface area per residue is determined by calculating the solvent accessible surface area for each residue of the antibody and antigen in the complex and subtracting it from the sum of the solvent accessible surface areas of the two components considered individually. Solvent accessible surface areas are calculated according to the method of Strake and Rupley (J. Mol. Biol. 79(2):351-71, 1973). Pairwise buried surface areas are used to estimate the individual contributions of residue pairs from the antibody and antigen to the overall effect of the buried surface area on the binding energy. Since the buried surface area is not pairwise resolvable, the buried surface area of ​​each residue in the epitope is calculated in the presence of each individual antigen residue in the absence of the rest of the antigen. These individual contributions are then normalized so that the sum of all individual contributions of all antigen residues to the buried surface area of ​​a given epitope residue will equal the total buried surface area of ​​the epitope residues due to the binding of the entire antigen. This process is repeated in reverse for the individual contributions of the epitope residues to the buried surface area of ​​the antibody residues. If The antigenic residues are considered to be part of the epitope.

[0515] The same calculations were performed for the interaction of the BMP9 binding epitope with ActRIIB using chains A and E from the Protein Data Bank structure 4fao.

[0516] The binding interaction of Ab1076 Fab with human BMP9 in the crystal structure is shown in FIG10 .

[0517] Example 11: Anti-BMP9 antibodies inhibit serum-induced phosphorylation of SMAD1 / 5 / 9 and phospho- SMAD2 signaling.

[0518] Rat serum samples collected at the end of the SuHx efficacy dose study of Example 8 were used to evaluate the effects of anti-BMP9 and ActRIIA-Fc treatment on pSMAD1 / 5 / 9 and pSMAD2 nuclear translocation in immortalized human microvascular endothelial cells. Briefly, serum-starved TIME cells were treated with 10% rat serum for 20 minutes. The cells were fixed, blocked and permeabilized, and stained overnight with antibodies against pSMAD1 / 5 / 9 or pSMAD2. The next day, the cells were washed and stained with ALEXA The cells were stained with 488-labeled secondary antibodies to detect pSMAD1 / 5 / 9 or pSMAD2, Hoechst 33342 to detect the nucleus, and cell mask deep red stain to depict the entire cell. After a final wash, the cells were imaged on a high-content imager. To analyze pSMAD1 / 5 / 9 or pSMAD2 nuclear translocation, ALEXA was calculated for each well of the cells. Average nuclear fluorescence in channel 488. IC was calculated using GraphPad Prism version 9.0.0 50 value.

[0519] Serum from rats treated with 0.3 mg / kg Ab1076 induced significantly less pSMAD1 / 5 / 9 mean nuclear fluorescence intensity: 2270±155, compared to 2810±160 for isotype control, a decrease of 19.3% (p≤0.0001). Relative to isotype control, an 18.7% decrease in pSMAD1 / 5 / 9 nuclear intensity was observed after treatment with 3 mg / kg Ab1076 (2290±325) (p≤0.0001). Treatment with serum from rats treated with 0.3 mg / kg (2550±140) and 3 mg / kg (2530±74.6; p≤0.05) Ab732 inhibited pSMAD1 / 5 / 9 mean nuclear fluorescence by 9.2% and 10%, respectively, relative to serum from rats treated with isotype control. Fig.11A ).

[0520] Compared to isotype control (2810±189), mean pSMAD2 nuclear intensity decreased by 14.5% after treatment with serum from the 0.3 mg / kg Ab1076 group (2400±79.1; p≤0.0001) and by 11.6% after treatment with serum from the 3 mg / kg Ab1076 group (2481±171; p=0.0002). Treatment with serum from rats treated with 0.3 mg / kg (2720±127) and 3 mg / kg (2580±75.7; p≤0.05) Ab732 inhibited pSMAD2 mean nuclear fluorescence ( Fig. 11B ).

[0521] Similar levels of pSMAD1 / 5 / 9 (2150±169) and pSMAD2 (2320±42.4) nuclear translocation were observed in untreated TIME endothelial cells and cells treated with sera from the 0.3 mg / kg or 3 mg / kg Ab1076 groups.

[0522] No significant changes in nuclear translocation of pSMAD1 / 5 / 9 (2780±104) and pSMAD2 (2790±144) were observed following treatment with ActRIIA-Fc relative to isotype controls.

[0523] Example 12: Effect of anti-BMP9 antibodies on signal transduction induced by TGFβ family members GDF8 and GDF11 Evaluation of off-target effects of induced leukemia.

[0524] The ability of Ab1076 and Ab732 to inhibit the activity of BMP9 family members GDF8 and GDF11 was evaluated using C2C12 mouse myoblasts stably expressing the CAGA promoter inserted into the firefly luciferase reporter construct. In brief, in the presence of a certain range of doses of Ab1076, Ab732 or isotype control antibodies, C2C12-CagaLuc cells were treated with 3nM GDF8 or GDF11 for 24 hours. Luciferase activity was evaluated by lysing cells and adding firefly-specific substrate D-luciferin, then measuring luminescence on a plate reader.

[0525] Ab1076 inhibited GDF8-induced luciferase activity relative to a non-binding isotype control antibody ( Fig. 12A ) or GDF11-induced luciferase activity ( Fig. 12B ) had no effect. Ab732 inhibited GDF8-induced luciferase activity ( Fig. 12A ), but did not inhibit GDF11-induced luciferase activity ( Fig. 12B ActRIIA-Fc was used as a positive control and inhibited GDF8 and GDF11, IC 50 Values ​​ranged from 0.2 nM to 1.5 nM (data not shown).

[0526] Example 13: Evaluation of the effect of anti-BMP9 antibody Ab1076 on the inhibition of endothelial secretion of endothelin-1.

[0527] In this example, the ability of anti-BMP9 antibody Ab1076 to inhibit endothelial cell secretion of endothelin-1 (ET-1) was tested. In brief, human pulmonary artery endothelial cells (HPAEC) were treated with 100pM BMP9 for 24 hours in the presence of a range of doses of Ab1076 or isotype controls. At the end of the treatment period, culture medium was collected from each well of the cell and subjected to ET-1 ELISA. A standard curve was generated using four-parameter logistic curve fitting and used to de...

Claims

1. 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 one (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.

2. 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: (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) 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; (vii) VH CDR1 comprises the amino acid sequence of SEQ ID NO:45, VH CDR2 comprises the amino acid sequence of SEQ ID NO:61, and VH CDR3 comprises the amino acid sequence of SEQ ID NO:62, and VL CDR1 comprises the amino acid sequence of SEQ ID NO:57, VL CDR2 comprises the amino acid sequence of SEQ ID NO:41, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:58; (viii) 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 (ix) 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.

3. The isolated antibody of claim 2, wherein: (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.

4. 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 VLCDR1 comprises the amino acid sequence of SEQ ID NO: 73, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 20, and VLCDR3 comprises the amino acid sequence of SEQ ID NO:

67.

5. The isolated antibody of any one of claims 1 to 4, wherein the 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 containing one to four amino acid substitutions at residues not within a CDR, and the 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 containing one to four amino acid substitutions at residues not within a CDR.

6. The isolated antibody of any one of claims 1-4, wherein the 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 the 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: NO:74 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 the amino acid sequence.

7. The isolated antibody of any one of claims 1-6, wherein the antibody comprises: (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) VH comprising the amino acid sequence of SEQ ID NO: 77 and VL comprising the amino acid sequence of SEQ ID NO:

74.

8. The isolated antibody of claim 7, wherein the antibody comprises: (i) 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; (ii) 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; (iii) 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; (iv) 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; (v) 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:27 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: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) 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.

9. The isolated antibody of claim 8, 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.

10. An isolated antibody portion, wherein 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, and wherein the portion binds to BMP9.

11. The portion of claim 10, wherein the 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; a VH CDR2 comprising 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 A VH CDR3 comprising 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.

12. An isolated antibody that binds to BMP9 and comprises a VH encoded by a nucleic acid sequence of an insert of a plasmid deposited with ATCC and having accession number PTA-127292, a VL encoded by a nucleic acid sequence of an insert of a plasmid deposited with ATCC and having accession number PTA-127293, or both.

13. An isolated antibody, comprising: (i) the VH CDR1, VH CDR2 and VH CDR3 sequences of SEQ ID NO: 16, and the VL CDR1, VLCDR2 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 VLCDR1, 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 VLCDR1, 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 VLCDR1, VL CDR2 and VL CDR3 sequences of SEQ ID NO:

74.

14. The isolated antibody of any one of claims 1-13, wherein the antibody comprises human Vκ or V λ Light chain constant domain.

15. The isolated antibody of claim 14, wherein the antibody comprises a human V λ Light chain constant domain.

16. The isolated antibody of any one of claims 1-15, wherein the antibody comprises a heavy chain constant domain.

17. The isolated antibody of claim 16, 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).

18. The isolated antibody of claim 17, wherein the heavy chain constant domain comprises IgG.

19. The isolated antibody of claim 18, wherein the IgG is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

20. The isolated antibody of claim 19, wherein the IgG is IgG1.

21. The isolated antibody of any one of claims 1-20, wherein the antibody comprises an Fc domain.

22. The isolated antibody of claim 21, wherein the Fc domain comprises an IgG1 heavy chain CH2 domain and an IgG heavy chain CH3 domain.

23. An isolated antibody, 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.

24. An isolated antibody, comprising: (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) a light 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: 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.

25. An isolated antibody, comprising: (i) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 8, and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 1; (ii) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 23, and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 18; (iii) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 35, and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 29; (iv) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 44, and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 40; (v) HC comprising or consisting of the amino acid sequence of SEQ ID NO:52, and LC comprising or consisting of the amino acid sequence of SEQ ID NO:49; (vi) HC comprising or consisting of the amino acid sequence of SEQ ID NO: 60, and LC comprising or consisting of the amino acid sequence of SEQ ID NO: 56; (vii) HC comprising or consisting of the amino acid sequence of SEQ ID NO: 69, and LC comprising or consisting of the amino acid sequence of SEQ ID NO: 64; or (viii) HC comprising or consisting of the amino acid sequence of SEQ ID NO: 75, and LC comprising or consisting of the amino acid sequence of SEQ ID NO:

72.

26. An isolated antibody, comprising: (i) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 23, and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 18; (ii) a HC comprising or consisting of the amino acid sequence of SEQ ID NO:52, and a LC comprising or consisting of the amino acid sequence of SEQ ID NO:49; (iii) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 69, and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 64; (iv) HC comprising or consisting of the amino acid sequence of SEQ ID NO: 75, and LC comprising or consisting of the amino acid sequence of SEQ ID NO: 72; (v) a 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 a 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) a 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 a 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) a 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 a 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) a 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 a 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.

27. An isolated antibody, comprising: (i) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 75, and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 72; or (ii) a 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 a 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.

28. 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 Ala28, Pro29, Ser83, Leu85, Met90, Val92, Pro93, Leu95, and Tyr97 of SEQ ID NO:

79.

29. The isolated antibody of claim 28, wherein the antibody binds Ala28, Pro29, Ser83, Leu85, Met90, Val92, Pro93, Leu95, and Tyr97 of SEQ ID NO:

79.

30. The isolated antibody of any one of claims 28-29, 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.

31. The isolated antibody of claim 30, wherein the antibody binds Pro81, Tyr86, Asp88, Asp89, Thr94, Lys96, His98, Tyr99, and Glu100 of SEQ ID NO:

79.

32. The isolated antibody of any one of claims 28-31, 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.

33. The isolated antibody of claim 32, wherein the antibody binds to Ile27, Lys30, and Glu31 of SEQ ID NO:

79.

34. An isolated antibody that binds to BMP9, wherein the antibody binds to an epitope on BMP9, the epitope comprising 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.

35. An isolated antibody that binds to 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.

36. An isolated antibody that binds to BMP9, wherein the antibody binds to an epitope on BMP9, the epitope comprising 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.

37. An isolated antibody that binds to 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.

38. 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 Arg14, Ile27, Ala28, Pro29, Lys30, Glu31, Ser83, Leu85, Met90, Val92, Pro93, Leu95, and Tyr97 of SEQ ID NO:

79.

39. An isolated antibody that binds to 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.

40. The isolated antibody of any one of claims 28-39, wherein the antibody epitope on BMP9 does not comprise amino acid residues 21-25, 43-60 of SEQ ID NO:

79.

41. The isolated antibody of any one of claims 1-40, wherein 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 as measured by surface plasmon resonance.

42. The antibody of any one of claims 28-31, 34-37, 40-41, wherein the antibody comprises: (i) a heavy chain variable region (VH) CDR3 of SEQ ID NO: 26 and a 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.

43. The isolated antibody of any one of claims 1-42, wherein the antibody (i) binds to human BMP9 and inhibits the binding of human BMP9 to BMP9 type II receptor (e.g., BMPRII, ActRIIA or ActRIIB); or (ii) binds to human BMP9 and inhibits the binding of human BMP9 to BMP9 type II receptor (e.g., BMPRII, ActRIIA or ActRIIB), and weakly inhibits the binding of human BMP9 to BMP9 type 1 receptor (e.g., ALK1).

44. The isolated antibody of any one of claims 1-43, wherein the antibody binds to human BMP9 and inhibits the binding of human BMP9 to human endothelial cells.

45. The isolated antibody of any one of claims 1-44, wherein the antibody binds to human BMP9, but does not bind to other human transforming growth factor β (TGFβ) superfamily ligands (e.g., GDF8, GDF11, activin A, TGFβ1, GDF9, and BMP10).

46. ​​The isolated antibody of any one of claims 1-45, wherein the antibody inhibits BMP9 activity measured by phospho-SMAD1 / 5 / 9 nuclear translocation in endothelial cells.

47. The isolated antibody of claim 46, wherein the antibody is expressed at 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 156 pM, about 157 pM, about 158 ​​pM, about 161 pM, about 162 pM, about 164 pM, about 165 pM, about 166 pM, about 167 pM, about 168 pM, about 169 pM, about 170 pM, about 171 pM, about 172 pM, about 173 pM, about 174 pM, about 175 pM, about 176 pM, about 177 pM, about 178 pM, about 179 pM, about 180 pM, about 181 pM, about 182 pM, about 183 pM, about 184 pM, about 185 pM, about 186 pM, about 187 pM, about 188 pM, about 189 pM, about 190 pM The IC values ​​of about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1000 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, or about 100 nM are preferably selected from the group consisting of: 50 Inhibits human BMP9 activity.

48. The isolated antibody of any one of claims 1-47, wherein the antibody inhibits BMP9 activity measured by phosphorylated-SMAD2 nuclear translocation in endothelial cells.

49. The isolated antibody of claim 48, wherein the antibody has an IC of about 10 pM, about 15 pM, about 17 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, about 100 pM, or about 117 pM. 50 Inhibits BMP9 activity.

50. The isolated antibody of any one of claims 1-49, wherein the antibody reduces right ventricular systolic pressure in the 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 administration of the antibody.

51. The isolated antibody of any one of claims 1-50, wherein the antibody reduces right ventricular hypertrophy in the 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 administration of the antibody.

52. The isolated antibody of any one of claims 50-51, wherein the subject is a Sugen-hypoxia animal model of pulmonary arterial hypertension (PAH).

53. An isolated polynucleotide encoding the antibody of any one of claims 1-52.

54. 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.

55. 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.

56. An isolated nucleic acid encoding VH, VL or both of an antibody that binds to BMP9, wherein the nucleic acid comprises the nucleic acid sequence of an insert of a plasmid deposited with ATCC and having accession number PTA-127292, the nucleic acid sequence of an insert of a plasmid deposited with ATCC and having accession number PTA-127293, or both.

57. A vector comprising the polynucleotide of claim 53 or the nucleic acid of any one of claims 54-56.

58. An isolated host cell comprising the polynucleotide of claim 53 or the nucleic acid of any one of claims 54-56.

59. The host cell of claim 58, wherein the cell is a mammalian cell or an insect cell.

60. The host cell of claim 59, wherein the host cell is a mammalian cell selected from the group consisting of: CHO cells, HEK-293 cells, NS0 cells, PER. cells or Sp2.0 cells.

61. A method for producing an antibody, the method comprising: Cultivating the host cell of any one of claims 58-60 under conditions that result in production of the antibody; as well as The antibody is recovered.

62. A pharmaceutical composition comprising a therapeutically effective amount of the antibody of any one of claims 1-52 and a pharmaceutically acceptable carrier.

63. The pharmaceutical composition of claim 62, wherein the composition comprises 1.12 mg / mL L-histidine, 2.67 mg / mL L-histidine hydrochloride monohydrate, 85 mg / mL sucrose, 0.05 mg / mL EDTA disodium dihydrate, and 0.2 mg / mL polysorbate 80, and has a pH of 5.

8.

64. A pharmaceutical composition as described in any one of claims 62-63, wherein the composition comprises 20 mM histidine, 8.5% sucrose and 0.02% polysorbate 80, 0.005% EDTA, and has a pH of 5.

8.

65. The pharmaceutical composition of any one of claims 62-64, wherein the composition comprises 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 of the antibody.

66. A pharmaceutical composition as described in any one of claims 62-65, wherein the composition is suitable for subcutaneous (SC) and / or intravenous (IV) administration.

67. 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 a pharmaceutical composition as described in any one of claims 62-66 or an antibody as described in any one of claims 1-52.

68. A method of treating hypertension in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of any one of claims 62-66 or the antibody of any one of claims 1-52.

69. The method of claim 68, wherein the hypertension is pulmonary hypertension.

70. A method of 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 of any one of claims 62-66 or the antibody of any one of claims 1-52.

71. The method of any one of claims 68-70, wherein the antibody is administered in combination with a therapeutically effective amount of one or more additional therapeutically active compounds or treatment modalities effective to treat at least one sign and / or symptom of hypertension, optionally pulmonary hypertension.

72. An isolated antibody as described in any one of claims 1-52 or a pharmaceutical composition as described in any one of claims 62-66 for use as a medicament.

73. An isolated antibody as described in any one of claims 1-52 or a pharmaceutical composition as described in any one of claims 62-66, for use in treating at least one sign and / or symptom of hypertension, optionally pulmonary hypertension.

74. Use of the isolated antibody of any one of claims 1-52 or the pharmaceutical composition of any one of claims 62-66 in the manufacture of a medicament for treating at least one sign and / or symptom of hypertension, optionally pulmonary hypertension.

75. A method for monitoring the effectiveness of a pulmonary arterial hypertension (PAH) therapy in a subject in need thereof, the method comprising: (i) administering a 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) to the level of the same one or more biomarkers before administration of the PAH therapy, wherein a change (e.g., a decrease) in the level of the one or more biomarkers after administration of the PAH therapy indicates that the therapy is effective, and wherein the PAH therapy comprises the isolated antibody of any one of claims 1-52.

Images