Selective regulation of transforming growth factor beta superfamily signaling via multispecific antibodies

Abstract

Compositions and methods for selective targeting of BMP / TGFβ signaling using heteromeric complexes, preferably heteromeric complexes comprising single-chain variable domain (scFv) antibodies (Abs) that target the extracellular domains of BMP type I and type II receptors.

Description

[Technical Field]

[0001] Claim of priority This application claims the benefits of U.S. Provisional Patent Application No. 62 / 983,374, filed on 28 February 2020. The entire foregoing is incorporated herein by reference.

[0002] Research and development funded by the federal government This invention was made with government support under grant numbers AR057374 and HL131910, granted by the National Institutes of Health. The government has certain rights in this invention.

[0003] Compositions and methods for selective targeting of bone morphogenetic protein (BMP) / transforming growth factor-beta (TGF-β) signaling, using heteromer complexes of single-chain variable domain (scFv) antibodies (Ab) that optionally target the extracellular domains of BMPI-type and type II receptors, are described herein. [Background technology]

[0004] The BMP / TGF-beta / activin / growth-differentiation-factor (GDF) signaling pathway broadly regulates developmental patterning and postnatal tissue remodeling (Waite and Eng, Nat Rev Genet. 2003 Oct;4(10):763-73). Thirty-three known ligands of this pathway interact with five constitutively activated type II receptors and seven conditionally activated type I receptors on the cell surface to initiate downstream signaling. This two-receptor signaling system confers combinatorial diversity (at least 5 × 7 possible heterotetramer complexes) in receptor pairs, each responding to a limited number of ligands. The physiological consequences of this signaling are highly tissue- and spatiotemporal-dependent and may include regulation of osteoplasia or fibrosis, more generally, control of cellular plasticity, cell hypertrophy, cell proliferation, and apoptosis. Recombinant BMP / TGF-beta / activin / GDF ligands can be used therapeutically for the treatment of various pathological conditions or for tissue manipulation applications. However, many of these ligands may have a short half-life in circulation or be sequestered in the extracellular matrix via their heparin-binding domain, which can make delivery to target tissues difficult.

[0005] TGF-beta signaling requires cross-linking of type I and type II receptors in a multimeric complex to initiate signaling. Dimeric ligand molecules facilitate the assembly of heteromeric complexes of type II and type I receptors, and the constitutively active kinase domain of the type II receptor transphosphorylates and activates the kinase domain of the type I receptor. The type I receptor can then mimic signaling through multiple signaling cascades, including SMADs. SMADs are a family of proteins analogous to the gene products of the Drosophila gene "Mother's Against Decapentaplesic" (Mad) and the C. elegans gene Sma, and include effector proteins that mediate the functions of BMP, activin, GDF, and TGF-ligands. These SMADs include SMAD2 and 3 downstream of TGF-beta, as well as many activin and GDF ligands, and SMAD1, 5, and 9 downstream of most BMP ligands and some GDF ligands. They are phosphorylated at their C-terminus by type I receptor kinases and then selectively retained in the cell nucleus to regulate gene transcriptional activity. Other signaling pathways targeted by the BMP / TGF-beta / activin / GDF receptor complex include MAP kinase, PI3K / Akt, protein kinase G, and other signal transduction cascades. Generally, these ligands, receptors, and their downstream effector signals function to coordinate cell growth, apoptosis, differentiation, and plasticity. These ligands, their receptors, co-receptors, and antagonists work to encode spatiotemporal gradients essential for developmental patterning and tissue remodeling. These ligands and receptors also function to regulate physiology in mature organisms, including iron homeostasis. These ligands and receptors are considered cytokines, which are multifunctional growth factors. [Overview of the project]

[0006] The BMP / TGFβ combinatorial system, comprising two receptors, enables functional specificity and spatiotemporal control of cellular signaling. Function is determined by the specific pairing of type II and type I receptors, which determines the activation of SMAD (SMAD1 / 5 / 9 vs. SMAD2 / 3) and non-SMAD (i.e., mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and / or protein kinase G (PKG)) effector pathways. The expression of type II and type I receptors is tissue-specific and dynamically regulated to enable context-dependent function. Compositions and methods for selective targeting of BMP / TGFβ signaling using antibodies or their antigen-binding fragments, preferably heteromer complexes of single-chain fragment-variable (scFv) antibodies, that target the extracellular domains of type I and type II receptors of the BMP / TGF-beta / activin / GDF family are described herein.

[0007] Accordingly, multiple or bispecific antibody molecules comprising at least a first antigen-binding domain that binds to osteomorphic protein receptor (BMPR) type I (BMPRI) and a second antigen-binding domain that binds to osteomorphic protein receptor (BMPR) type II (BMPRII) are provided herein, where BMPRI or BMPRII can be interpreted as referring to either type I or type II receptors of the BMP / TGF-beta / activin / GDF signaling pathway, respectively, where the first and second antigen-binding domains are linked to each other by a mobile linker, and each antigen-binding domain can act as an agonist against the BMPR to which it binds, preferably each antigen-binding domain is scFv. The first and second antigen-binding domains can be present in the molecule in any order, for example, the molecule may consist of N-terminus-first domain-linker-second domain-C-terminus or N-terminus-second domain-linker-first domain-C-terminus (see, for example, Figures 10A-D). In some embodiments, the BMPRII antigen-binding domain is N-terminus relative to the BMPRI antigen-binding domain. Also provided are tetrameric or more (e.g., hexamers, octamers, decamers, or more) multimer arrays consisting of a first and second antigen-binding domain separated by a mobile linker domain, in which the antigen-binding domains are present in any order within the molecule. Generally, the domains exist in pairs, for example, one BMPRI-binding domain for each BMPRI-binding domain, but the ratio can also vary (in either direction), for example, from 1:1 to 2:1, or from 3:1 or more.

[0008] In some embodiments, the binding of antibody molecules to cells initiates BMP / TGF-beta / activin / GDF signaling.

[0009] In some embodiments, the first antigen-binding domain binds to BMPRI, selected from the group consisting of, for example, ALK1 (also known as activin A receptor-like type 1 or ACVRL1); ALK2 (ACVR1A); ALK3 (BMPR1A); ALK4 (ACVR1B); ALK5 (TGFBR1); ALK6 (BMPR1B); or ALK7 (ACVR1C).

[0010] In some embodiments, the first antigen-binding domain binds to ALK1 and optionally includes a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; includes VH CDR1, 2, 3 and VL CDR1, 2, 3 which are identical to the complementarity-determining regions in SEQ ID NOs. 146, 148, 150, or 152; includes VH and / or VL sequences that are at least 95% identical to the sequence in Figure 7; or includes sequences that are at least 95% identical to SEQ ID NOs. 146, 148, 150, or 152.

[0011] In some embodiments, the first antigen-binding domain binds to ALK2 and optionally includes a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; includes VH CDR1, 2, 3 and VL CDR1, 2, 3 which are identical to the complementarity-determining regions in SEQ ID NOs. 160, 162, 164, 166, or 168; includes VH and / or VL sequences that are at least 95% identical to the sequence in Figure 7; or includes sequences that are at least 95% identical to SEQ ID NOs. 160, 162, 164, 166, or 168.

[0012] In some embodiments, the first antigen-binding domain binds to ALK3 and optionally includes a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; VH CDR1, 2, 3 and VL CDR1, 2, 3 that are identical to the complementarity-determining region in SEQ ID NO: 156 or 158; VH and / or VL sequences that are at least 95% identical to the sequence in Figure 7; or sequences that are at least 95% identical to the sequence in SEQ ID NO: 156 or 158.

[0013] In some embodiments, the first antigen-binding domain binds to ALK4 and optionally includes a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; VH CDR1, 2, 3 and VL CDR1, 2, 3 that are identical to the complementarity-determining region in SEQ ID NO: 178; VH and / or VL sequences that are at least 95% identical to the sequence in Figure 7; or sequences that are at least 95% identical to SEQ ID NO: 178.

[0014] In some embodiments, the first antigen-binding domain binds to ALK6 and optionally includes a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; VH CDR1, 2, 3 and VL CDR1, 2, 3 that are identical to the complementarity-determining region in SEQ ID NO: 180; VH and / or VL sequences that are at least 95% identical to the sequence in Figure 7; or sequences that are at least 95% identical to SEQ ID NO: 180.

[0015] In some embodiments, the first antigen-binding domain binds to ALK7 and optionally includes a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; includes VH CDR1, 2, 3 and VL CDR1, 2, 3 which are identical to the complementarity-determining regions in SEQ ID NOs. 182, 184, 186, or 188; includes VH and / or VL sequences that are at least 95% identical to the sequence in Figure 7; or includes sequences that are at least 95% identical to SEQ ID NOs. 182, 184, 186, or 188.

[0016] In some embodiments, the second antigen-binding domain binds to BMPRII selected from the group consisting of ACTRIIA (ACVR2A); ACTRIIB (ACVR2B); BMPRII (BMPR2); TGFBRII (TGFBR2); or AMHRII (AMHR2).

[0017] In some embodiments, the second antigen-binding domain binds to BMPR2 and includes a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; includes VH CDR1, 2, 3 and VL CDR1, 2, 3 which are identical to the complementarity-determining region in SEQ ID NO: 154 or 212; includes VH and / or VL sequences that are at least 95% identical to the sequence in Figure 7; or includes sequences that are at least 95% identical to SEQ ID NO: 154 or 212.

[0018] In some embodiments, the second antigen-binding domain binds to ACTRIIA(ACVR2A) and includes a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; includes VH CDR1, 2, 3 and VL CDR1, 2, 3 which are identical to the complementarity-determining regions in SEQ ID NOs. 170 and 172; includes VH and / or VL sequences that are at least 95% identical to the sequence in Figure 7; or includes a sequence that is at least 95% identical to SEQ ID NOs. 170 or 172.

[0019] In some embodiments, the second antigen-binding domain binds to ACTRIIB(ACVR2B) and includes a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; includes VH CDR1, 2, 3 and VL CDR1, 2, 3 which are identical to the complementarity-determining region in SEQ ID NO: 174 or 176; includes a VH and / or VL sequence that is at least 95% identical to the sequence in Figure 7; or includes a sequence that is at least 95% identical to the sequence in SEQ ID NO: 174 or 176.

[0020] In some embodiments, the first and second antigen-binding domains include any of the 5 × 7 = 35 possible combinations, for example, BMPR2 with ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, or ALK7; ACVR2A with ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, or ALK7; ACVR2B with ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, or ALK7; TGFBR2 with ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, or ALK7; or AMHR2 with any of the ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, or ALK7.

[0021] In some embodiments, (i) The first antigen-binding domain binds to BMPR2 and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7, and / or the second antigen-binding domain binds to ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7 and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; (ii) The first antigen-binding domain binds to ACVR2A and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7, and / or the second antigen-binding domain binds to ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7 and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; (iii) The first antigen-binding domain binds to ACVR2B and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7, and / or the second antigen-binding domain binds to ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7 and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; (iv) The first antigen-binding domain binds to TGFBR2 and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or FIG. 7, and / or the second antigen-binding domain binds to ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7 and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or FIG. 7; (v) The first antigen-binding domain binds to AMHR2 and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or FIG. 7, and / or the second antigen-binding domain binds to ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7 and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or FIG. 7.

[0022] An antibody or an antigen-binding portion thereof that binds to ALK1 and optionally contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or FIG. 7; contains VH CDR1, 2, 3 and VL CDR1, 2, 3 that are identical to the complementarity determining regions in SEQ ID NO: 146, 148, 150, or 152; contains a VH and / or VL sequence that is at least 95% identical to the sequence in FIG. 7; or contains a sequence that is at least 95% identical to SEQ ID NO: 146, 148, 150, or 152 is also provided herein.

[0023] An antibody or an antigen-binding portion thereof that binds to ALK2 and optionally contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or FIG. 7; contains VH CDR1, 2, 3 and VL CDR1, 2, 3 that are identical to the complementarity determining regions in SEQ ID NO: 160, 162, 164, 166, or 168; contains a VH and / or VL sequence that is at least 95% identical to the sequence in FIG. 7; or contains a sequence that is at least 95% identical to SEQ ID NO: 160, 162, 164, 166, or 168 is also provided herein.

[0024] An antibody or antigen-binding portion thereof that binds to ALK3 and optionally comprises a CDR sequence that is at least 95% identical to the CDR sequences shown in Table 1 or FIG. 7; comprises VH CDR1, 2, 3 and VL CDR1, 2, 3 that are identical to the complementarity-determining regions in SEQ ID NO: 156 or 158; comprises VH and / or VL sequences that are at least 95% identical to the sequences shown in FIG. 7; or comprises a sequence that is at least 95% identical to SEQ ID NO: 156 or 158 is also provided herein.

[0025] An antibody or antigen-binding portion thereof that binds to ALK4 and optionally comprises a CDR sequence that is at least 95% identical to the CDR sequences shown in Table 1 or FIG. 7; comprises VH CDR1, 2, 3 and VL CDR1, 2, 3 that are identical to the complementarity-determining regions in SEQ ID NO: 178; comprises VH and / or VL sequences that are at least 95% identical to the sequences shown in FIG. 7; or comprises a sequence that is at least 95% identical to SEQ ID NO: 178 is also provided herein.

[0026] An antibody or antigen-binding portion thereof that binds to ALK6 and optionally comprises a CDR sequence that is at least 95% identical to the CDR sequences shown in Table 1 or FIG. 7; comprises VH CDR1, 2, 3 and VL CDR1, 2, 3 that are identical to the complementarity-determining regions in SEQ ID NO: 180; comprises VH and / or VL sequences that are at least 95% identical to the sequences shown in FIG. 7; or comprises a sequence that is at least 95% identical to SEQ ID NO: 180 is also provided herein.

[0027] An antibody or antigen-binding portion thereof that binds to ALK7 and optionally comprises a CDR sequence that is at least 95% identical to the CDR sequences shown in Table 1 or FIG. 7; comprises VH CDR1, 2, 3 and VL CDR1, 2, 3 that are identical to the complementarity-determining regions in SEQ ID NO: 182, 184, 186, or 188; comprises VH and / or VL sequences that are at least 95% identical to the sequences shown in FIG. 7; or comprises a sequence that is at least 95% identical to SEQ ID NO: 182, 184, 186, or 188 is also provided herein.

[0028] Antibodies or antigen-binding moieties thereof that bind to BMPR2 and contain a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; contain VH CDR1, 2, 3 and VL CDR1, 2, 3 which are identical to the complementarity-determining region in SEQ ID NO: 154 or 212; contain VH and / or VL sequences that are at least 95% identical to the sequence in Figure 7; or contain a sequence that is at least 95% identical to the sequence in SEQ ID NO: 154 or 212 are also provided herein.

[0029] Antibodies or antigen-binding moieties thereof that bind to ACTRIIA(ACVR2A) and contain a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; contain VH CDR1, 2, 3 and VL CDR1, 2, 3 which are identical to the complementarity-determining region in SEQ ID NO: 170 or 172; contain VH and / or VL sequences that are at least 95% identical to the sequence in Figure 7; or contain sequences that are at least 95% identical to the sequences in SEQ ID NOs: 170 and 172 are also provided herein.

[0030] Antibodies or antigen-binding moieties conjugated to ACTRIIB(ACVR2B) and containing a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; containing VH CDR1, 2, 3 and VL CDR1, 2, 3 which are identical to the complementarity-determining region in SEQ ID NO: 174 or 176; containing a VH and / or VL sequence that is at least 95% identical to the sequence in Figure 7; or containing a sequence that is at least 95% identical to the sequence in SEQ ID NO: 174 or 176 are also provided herein.

[0031] In addition, pharmaceutical compositions are provided that optionally include an antibody molecule and an antibody or its antigen-binding moiety together with a pharmaceutically acceptable carrier, as described herein.

[0032] Methods using the antibody molecules and antibodies or their antigen-binding moieties (or compositions) described herein for treating a vascular condition in a subject, optionally the vascular condition being pulmonary arterial hypertension (PAH) or hereditary hemorrhagic telangiectasia (HHT) syndrome; more generally, for treating pulmonary hypertension (PH); for treating pulmonary vascular leakage syndrome, optionally the pulmonary vascular leakage syndrome being acute respiratory distress syndrome (ARDS) or acute lung injury (ALI); and / or for treating hepatic fibrosis in subjects having intrahepatic BMP9 signaling deficiency are also provided herein. In some embodiments, the first antigen-binding domain binds to ALK1 and the second antigen-binding domain binds to BMPR2. The method may include administering a therapeutically effective amount of the antibody molecule described herein to a subject in need.

[0033] Furthermore, nucleic acids encoding the antibody molecules described herein, and optionally host cells containing nucleic acids that express the antibody molecules are also provided herein.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Methods and materials for use in this invention are described herein, and other suitable methods and materials known in the art may also be used. Materials, methods, and examples are for illustrative purposes only and are not intended to limit the scope of this invention. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, this specification, including definitions, shall prevail.

[0035] Other features and advantages of the present invention will be apparent from the following detailed description and drawings, as well as from the claims. [Brief explanation of the drawing]

[0036] [Figure 1-1]Binding of scFv proteins to ALK1 and BMPR2. (A-B) Octet Red biolayer interferometry (BLI) shows that scFv clones ALK1.3 and ALK1.8 bind to immobilized ALK1. (C-D) BLI shows that scFv clones BMPR2.12 and BMPR2.23 bind to immobilized BMPR2. [Figure 1-2] This is a continuation of Figure 1-1. [Figure 2-1] Specific binding of scFv protein to ALK1 and BMPR2. (A) Octet Red biolayer interferometry (BLI) shows that clones ALK1.3 and ALK1.8 bind to ALK1 but not to clones BMPR2.12 or BMPR2.23. (B) Octet Red biolayer interferometry (BLI) shows that clones BMPR2.12 and BMPR2.23 bind to BMPR2 but not to clones ALK1.3 or ALK1.8; (C) None of clones ALK1.3, ALK1.8, BMPR2.12, or BMPR2.23 bind to immobilized ACVR2A. [Figure 2-2] This is a continuation of Figure 2-1. [Figure 3] ALK1 and BMPR2-specific scFv inhibit BMP9 binding in endothelial cells. Various scFv showed dose-dependent inhibition of BMP9 (1 ng / mL, 18 hours)-mediated transcriptional activity in TIME (human telomerase-immortalized capillary endothelial cells) as measured by BMP response element (BRE-luciferase) reporter activity, compared to BMP9 ligand trap ALK1-Fc. [Figure 4-1]Biotinylated scFv clones retain selective binding to ALK1 and BMPR2. (A) Immunoblotting of various scFv proteins using streptavidin-HRP confirms biotinylation of 25Kd species. (B) Using BLI, it is shown that both unmodified and biotinylated scFv ALK1.3 bind to immobilized ALK1, but unmodified and biotinylated scFv BMPR2.12 do not. (C) Using BLI, it is shown that both unmodified and biotinylated scFv BMPR2.12 bind to immobilized BMPR2, but unmodified and biotinylated scFv ALK1.3 do not. (D) Using BLI, it is shown that both unmodified and biotinylated scFv ALK1.8 bind to immobilized ALK1, but unmodified and biotinylated scFv BMPR2.23 do not. [Figure 4-2] This is a continuation of Figure 4-1. [Figure 5] Assembled streptavidin conjugates of biotinylated scFV clones ALK1.8 and BMPR2.12 induce BMP signaling in capillary endothelial cells. Immunoblotting shows activation of SMAD1 and SMAD3, and to a lower degree of SMAD2, after stimulation of human pulmonary capillary endothelial cells (PMVECs) with BMP9 (1 ng / mL, 30 min, 37°C), along with inhibition by concurrent treatment with monoclonal anti-BMP9 neutralizing antibody (mAb3209, 10 ng / mL). Treatment of cells with an equimolar mixture of biotinylated scFv clones ALK1.8 and BMPR2.12 (1 nm, 30 min, 37°C, respectively) induces activation of SMAD1 and SMAD3 in the presence of streptavidin (1 μg / mL), but not in the absence of streptavidin. Activation of SMAD1 / 3 by a biotinylated ALK1.8:BMPR2.12 complex assembled with streptavidin was not inhibited by simultaneous treatment with an anti-BMP9 neutralizing antibody, suggesting ligand-independent signaling activity. [Figure 6]A complex assembled with streptavidin of biotinylated scFv clones ALK1.8 and BMPR2.12 induces SMAD1 / 3 signaling in pulmonary capillary endothelial cells in a manner that requires ALK1 rather than BMP9 ligand. (A) Treatment of wild-type (WT) mouse pulmonary capillary endothelial cells (PMVECs) cultured with streptavidin-assembled (1 μg / mL) biotinylated ALK1.8:BMPR2.12 clones (1 nM each) resulted in activation of SMAD1 / 3 (30 min, 37°C), as assayed by immunoblotting, which is consistent with the requirement for ALK1 expression. Recombinant human BMP9 (rhBMP9, 1 ng / mL, 30 min, 37°C) induced SMAD1 / 3 activation in wild-type (WT) and Acvrl1 KO cells in a manner inhibited by mAb3209. In contrast, the activity of the streptavidin-assembled biotinylated ALK1.8:BMPR2.12 complex was not inhibited by mAb3209 (10 ng / mL), which again is consistent with ligand-independent signaling. (B) Quantitative measurement of phosphorylated SMAD1 / 5 using an in-cell Western assay in cultured human pulmonary capillary endothelial cells (PMVECs) revealed SMAD1 / 5 activation by recombinant human BMP9 (rhBMP9) and the streptavidin-assembled (1 μg / mL) biotinylated ALK1.8:BMPR2.12 (1 nM each) complex. Treatment of cells with the BMP9 / BMP10 ligand trap ALK1-Fc inhibited BMP9-mediated SMAD1 / 5 activation, but did not inhibit streptavidin-assembled ALK1.8 / BMPR2.12 complex-mediated SMAD1 / 5 activation. [Figure 7-1] CDR sequence. The heavy and light chain sequences for each of the exemplary antibodies described herein, having a CDR sequence identified using the different definitions described herein, are shown. [Figure 7-2] This is a continuation of Figure 7-1. [Figure 7-3] This is a continuation of Figure 7-1. [Figure 7-4] This is a continuation of Figure 7-1. [Figure 7-5] This is a continuation of Figure 7-1. [Figure 7-6] This is a continuation of Figure 7-1. [Figure 7-7] This is a continuation of Figure 7-1. [Figure 7-8] This is a continuation of Figure 7-1. [Figure 7-9] This is a continuation of Figure 7-1. [Figure 7-10] This is a continuation of Figure 7-1. [Figure 7-11] This is a continuation of Figure 7-1. [Figure 7-12] This is a continuation of Figure 7-1. [Figure 7-13] This is a continuation of Figure 7-1. [Figure 7-14] This is a continuation of Figure 7-1. [Figure 7-15] This is a continuation of Figure 7-1. [Figure 7-16] This is a continuation of Figure 7-1. [Figure 7-17] This is a continuation of Figure 7-1. [Figure 7-18] This is a continuation of Figure 7-1. [Figure 7-19] This is a continuation of Figure 7-1. [Figure 7-20] This is a continuation of Figure 7-1. [Figure 7-21] This is a continuation of Figure 7-1. [Figure 7-22] This is a continuation of Figure 7-1. [Figure 7-23] This is a continuation of Figure 7-1. [Figure 8] BMP transcriptional activity in TIME cells. In vitro activity assay data for the bispecific and quadruspecific constructs shown. The His-tagged bispecific constructs did not produce activity when they did not crosslink and form a tetramer. The IgG Fc-fused bispecific constructs induced signal transduction just as they did when expressed as disulfide-linked homodimers and formed a tetramer of the antigen-binding domain. [Figure 9]Intravenous recombinant human BMP9 and a tetravalent Fc fusion molecule induce in vivo BMP transcriptional activity in mice. BMP signaling activity based on Id1 gene transcriptional activity was assayed in whole lung tissue from 8-week-old mice 24 hours after tail vein injection of physiological saline (50 μL), recombinant human BMP9 (rhBMP9, 150 μg / kg in 50 μL), or recombinant tetrameric Fc fusion protein ACVRL1.8-L-BMPR2.12-Short-Fc (150 μg / kg in 50 μL). Injection of BMP9 or ACVRL1.8-L-BMPR2.12-Short-Fc induced increased BMP transcriptional activity in whole lung tissue compared to physiological saline. [Figure 10-1] Schematic diagram of an exemplary construct. (A) A tetravalent or polyvalent signaling molecule may contain an anti-type I BMP receptor antigen-binding region and an anti-type II BMP receptor antibody-binding region, delimited by a mobile linker region and expressed as a fusion molecule with an immunoglobulin G constant domain (IgG Fc). The antigen-binding regions are alternating and form a functional heterotetramer by a combination of homodimers of two identical IgG Fc fusion molecules via disulfide bonds. Alternatively, the heterotetramer may contain a combination of heterodimers of two dissimilar IgG Fc fusion molecules, each expressing a unique combination of type I and type II antigen-binding regions (B). Larger polyvalent molecules may contain two or more antigen-binding regions, occurring in different numbers in various cyclic or acyclic sequences, along with an even number of antibody-binding regions (C). These molecules may include combinations of homodimers of the same IgG Fc fusion molecule, or combinations of heterodimers of two dissimilar IgG Fc fusion molecules (D). [Figure 10-2] This is a continuation of Figure 10-1. [Figure 10-3] This is a continuation of Figure 10-1. [Figure 10-4] This is a continuation of Figure 10-1. [Figure 10-5] This is a continuation of Figure 10-1. [Figure 10-6] This is a continuation of Figure 10-1. [Modes for carrying out the invention]

[0037] The BMP / TGFβ signaling pathway, which includes ligands for activin and the growth factor / differentiation factor (GDF) family, acts as a critical regulator of organogenesis, embryonic patterning, and postnatal tissue remodeling and regeneration. The endogenous ligands of the BMP / TGFβ / activin / GDF signaling pathway exhibit a chaotic state, often binding to several type II and type I receptors, forming diverse ligand-receptor signaling complexes with distinct functional consequences. These ligands are often sequestered in the extracellular matrix thanks to their heparin-binding domains, and therefore exhibit poor pharmacokinetics as therapeutic molecules. Simplified gain-of-function and loss-of-function studies targeting individual type I receptors have confirmed the general roles of individual ligands and receptors in chondrogenic differentiation, osteogenic differentiation and bone mass regulation, adipogenic differentiation, fibrosis, and tendinogenic differentiation. 4、5 However, these studies yielded contradictory results, and there was a lack of clear data on which signaling complexes were involved in which tissues to produce these outcomes. The combinatorial diversity of ligand-receptor pairing within this family has complicated attempts to comprehensively map BMP / TGFβ family signaling inputs to cellular function.

[0038] Engineered bispecific antibodies that recognize the extracellular domain of the surface BMP / TGF-β / activin / GDF receptor to promote heterodimerization and initiate signal transduction are described herein. In some embodiments, such antibodies may be expressed with or without a C-terminal IgG Fc domain to have a more restricted or extended half-life in circulation, and may be delivered to target tissues through circulation, or bind to homing peptides or other homing molecules to target specific tissues, or adsorb or bind to solid substrates to promote the engraftment or growth of specific tissues.

[0039] Also described are tetravalent or polyvalent signaling molecules that may contain an anti-type I BMP receptor antigen-binding domain and an anti-type II BMP receptor antibody-binding domain, expressed as a fusion molecule with a mobile linker region and an immunoglobulin G constant domain (IgG Fc, the Fc domain may be derived from any of the wild-type IgG1, IgG2, IgG3, or IgG4 sequences, or from a sequence modified to alter binding to the Fcγ receptor, alter complement binding, alter antibody-dependent cytotoxicity, alter dimerization, or alter other functional properties). Methods for manipulating the Fc domain are known in the art; see, for example, Yang et al., Front Immunol. 2018 Jan 8;8:1860. The antigen-binding domains are alternating and can form a functional heterotetramer by a combination of homodimers of two identical IgG Fc fusion molecules via disulfide bonds (Figure 10A). Alternatively, heterotetramers may contain a combination of heterodimers of two dissimilar IgG Fc fusion molecules, each expressing a unique combination of type I and type II antigen-binding regions (Figure 10B). Larger polyvalent molecules may contain two or more antigen-binding regions, occurring in varying numbers in various cyclic or acyclic sequences, along with an even number of antibody-binding regions (Figure 10C). These molecules may contain a combination of homodimers of the same IgG Fc fusion molecule, or a combination of heterodimers of two dissimilar IgG Fc fusion molecules (Figure 10D). Single-chain multimeric signaling molecules are divalent or polyvalent molecules, but more typically, tetravalent molecules, containing anti-type I and anti-type II antigen-binding regions in alternating, sequential, or palindromic configurations, and in cyclic or acyclic sequences (Figure 10E). Polyvalent single-chain signaling molecules may contain two or more signaling molecules in alternating, sequential, palindromic, or acyclic sequences. These molecules will likely, but not necessarily, contain an even number of antibody-binding regions. These single-chain multimeric signaling molecules can also be expressed as monomeric IgG Fc fusion proteins (Figure 10F).

[0040] Therefore, these molecules described herein may include an Fc region. IgG Fc CH3 is necessary for homodimerization, but can be modified to prevent it for specific clinical applications. Methods for manipulating the Fc domain are known in the art; see, for example, Yang et al., Front Immunol. 2018 Jan 8;8:1860. Alternatively, or in addition, the molecules herein may include other domains to facilitate their targeting to a functional context, such as retaining specific cells, tissues, or molecules in tissues exhibiting changes due to inflammation, proliferation, or neoplastic changes, or other disease-related changes in protein expression, pH, extracellular fluid composition with respect to any solute or iron, extracellular matrix expression, glycosylation, or polysaccharide coat structure or composition.

[0041] Therefore, the tetramer may be a single molecule without an Fc region, for example, a molecule with a short half-life, preferably. Homodimeric Fc fusions in which each Fc chain has a double specificity pair of scFv are also described. The Fc region can also be attached to any tetramer, hexamer, octamer, etc. Tetramers with disulfide bonds to Fc are also described.

[0042] Methods for selective agonism, signal activation, or signal modulation of specific members of the bone morphogenetic protein (BMP), activin, growth and differentiation factor (GDF), and transforming growth factor (TGF)-beta signaling superfamily, using engineered bispecific or multispecific antibodies that recognize receptors in this pathway, are also described herein.

[0043] I. Definition The term "a" or "an" entity should be understood to refer to one or more such entities; for example, "an antibody" is understood to represent one or more antibodies. Therefore, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0044] As used herein, the term “polypeptide” is intended to encompass both the singular and plural forms “polypeptide” and refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or more of two or more amino acids and does not refer to a product of a specific length. Thus, peptide, dipeptide, tripeptide, oligopeptide, “protein,” “amino acid chain,” or any other term used to refer to a chain or more of two or more amino acids are included within the definition of “polypeptide,” and the term “polypeptide” may be used in place of or interchangeably with any of these terms.

[0045] The term “polypeptide” is also intended to refer, non-limitingly, to the products of post-expression modifications of polypeptides, including glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, protein cleavage, or modification with non-spontaneous amino acids. Polypeptides may originate from natural biological sources or be produced by recombinant technologies, but are not necessarily translated from the given nucleic acid sequence. They may be produced in any manner, including by chemical synthesis.

[0046] Polypeptides described herein may have a size of approximately 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, but they do not necessarily have such a structure. Polypeptides having a defined three-dimensional structure are referred to as folded, while polypeptides not having a defined three-dimensional structure can rather conform to a number of different stereochemistrys and are referred to as unfolded. As used herein, the term glycoprotein refers to a protein bound to at least one carbohydrate moiety, which is bound to the protein via an oxygen-containing or nitrogen-containing side chain of an amino acid residue, such as a serine residue or an asparagine residue.

[0047] The intended polypeptides are those not found in their natural environment, represented by "isolated" polypeptides, fragments, variants, or derivatives thereof. No specific level of purification is required. For example, isolated polypeptides can be extracted from their natural or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are natural or recombinant polypeptides that are considered for isolation for the purposes described herein and are delimited, partitioned, or partially or substantially purified by any appropriate technique.

[0048] Polypeptides also include fragments, derivatives, analogs, or variants of the polypeptides described herein, and any combination thereof. When referring to antibodies or antibody polypeptides described herein, the terms “fragment,” “variant,” “derivative,” and “analog” include any polypeptide that retains at least some of the antigen-binding properties of the corresponding native binding molecule, antibody, or polypeptide. Polypeptide fragments described herein include proteolytic fragments and deletion fragments, in addition to certain antibody fragments considered elsewhere herein. Antibody and antibody polypeptide variants described herein also include the fragments described above, as well as polypeptides having altered amino acid sequences resulting from amino acid substitutions (e.g., conserved or non-conserved amino acid substitutions), deletions, or insertions. Variants may occur naturally or not. Non-native variants may be produced using mutagenesis techniques known in the art. Variant polypeptides may include conserved or non-conserved amino acid substitutions (e.g., conserved or non-conserved amino acid substitutions), deletions, or additions. BMPRI / BMPRII-specific binding molecules, such as the antibodies and antibody polypeptide derivatives described herein, are polypeptides that have been modified to exhibit further properties not found on the natural polypeptide. Examples include fusion proteins. Variant polypeptides may also be referred to herein as “polypeptide analogs.” As used herein, a “derivative” of a binding molecule or its fragment, antibody, or antibody polypeptide refers to a target polypeptide having one or more residues chemically derivatized by a functional side chain reaction. Peptides containing one or more naturally occurring amino acid derivatives of 20 standard amino acids are also included as “derivatives.” For example, 4-hydroxyproline may be substituted with proline, 5-hydroxylysine may be substituted with lysine, 3-methylhistidine may be substituted with histidine, homoserine may be substituted with serine, and ornithine may be substituted with lysine.

[0049] The term “polynucleotide” is intended to encompass both singular and plural nucleic acids and refers to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA) or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphate diester bonds or non-conventional bonds (e.g., amide bonds, as found in peptide nucleic acids (PNA)). The term “nucleic acid” refers to any one or more nucleic acid segments present in a polynucleotide, such as DNA or RNA fragments. “Isolated” nucleic acid or polynucleotide refers to nucleic acid molecules, DNA or RNA that have been removed from their natural environment. For example, recombinant polynucleotides encoding antibodies contained in a vector are considered to be isolated for the purposes described herein. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or (partially or substantially) purified polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides described herein. Isolated polynucleotides or nucleic acids described herein further include such molecules produced synthetically. In addition, polynucleotides or nucleic acids may be regulatory elements such as promoters, ribosome binding sites, or transcription terminators, or may include them.

[0050] As used herein, “coding region” is a portion of a nucleic acid consisting of codons that are translated into amino acids. A “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid but may be considered part of a coding region, while any adjacent sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of a coding region. Two or more coding regions described herein may reside on a single polynucleotide construct, for example, on a single vector, or on separate polynucleotide constructs, for example, on separate (different) vectors. Furthermore, any vector may contain a single coding region or two or more coding regions; for example, a single vector may separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, vectors, polynucleotides, or nucleic acids described herein may encode heterogeneous coding regions that are fused to or unfused to nucleic acids encoding a binding molecule, antibody, or a fragment, variant, or derivative thereof. Heterogeneous coding regions include, without limitation, special elements or motifs such as secretory signaling peptides or heterogeneous functional domains.

[0051] In some embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide containing a nucleic acid encoding a polypeptide may typically include a promoter and / or other transcriptional or translational regulatory elements operably bound to one or more coding regions. An operable binding, in the case of a coding region for a gene product, e.g., a polypeptide, binds to one or more regulatory sequences in such a manner that the expression of the gene product occurs under the influence or regulation of the regulatory sequences. Two DNA fragments (such as a polypeptide coding region and a promoter bound to it) are “operably bound” or “operably ligated” if the induction of promoter function results in the transcription of mRNA encoding the desired gene product, and the nature of the binding between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product, or with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably bound to a nucleic acid encoding a polypeptide if the promoter had the ability to act on the transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of DNA only in predetermined cells. Other transcriptional regulatory elements, in addition to promoters such as enhancers, operators, repressors, and transcription termination signals, can operably bind to polynucleotides to direct cell-specific transcription. Suitable promoters and other transcriptional regulatory regions are disclosed herein.

[0052] Various transcriptional regulatory regions are known to those skilled in the art. These include, but are not limited to, transcriptional regulatory regions that function in vertebrate cells, such as cytomegalovirus (early promoter, combined with intron-A), simian virus 40 (early promoter), and promoter and enhancer segments derived from retroviruses (e.g., Roussarcoma virus). Other transcriptional regulatory regions include those derived from vertebrate genes such as actin, heat shock proteins, bovine growth hormone, and rabbit β-globin, as well as other sequences capable of regulating gene expression in eukaryotic cells. Further suitable transcriptional regulatory regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., promoters that can be induced by interferon or interleukin).

[0053] Similarly, various translational regulatory elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation start and stop codons, and picornavirus-derived elements (specifically, intra-sequence ribosome entry sites, or IRESs, also referred to as CITE sequences).

[0054] In some embodiments, the polynucleotides described herein are RNA, for example, RNA in the form of messenger RNA (mRNA).

[0055] The polynucleotides and nucleic acid coding regions described herein may be coupled with further coding regions encoding secretory or signal peptides that direct the secretion of polypeptides encoded by the polynucleotides described herein. According to the signaling hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein when efflux of the growing protein chain beyond the rough endoplasmic reticulum is initiated. Those skilled in the art will understand that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide that is cleaved from the complete or "full-length" polypeptide to produce a secretory or "mature" form of polypeptide. In some embodiments, native signal peptides, such as immunoglobulin heavy chain or light chain signal peptides, or functional derivatives sequentially therewith, retain the ability to direct the secretion of polypeptides operably bound to them. Alternatively, heterologous mammalian signal peptides, or functional derivatives thereof, may be used. For example, the wild-type leader sequence can be replaced with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0056] Unless otherwise stated, the terms “disorder” and “disease” shall be used interchangeably herein.

[0057] As used herein, “binding molecule” primarily relates to antibodies and their fragments, but may also refer to other non-antibody molecules that bind to BMPRI / BMPRII, including, but not limited to, hormones, receptors, ligands, major histocompatibility complex (MHC) molecules, chaperones such as heat shock proteins (HSPs), and cell-to-cell adhesion molecules such as cadherins, integrins, type C lectins, and members of the immunoglobulin (Ig) superfamily. Therefore, without limiting the scope of this disclosure, for clarification purposes only, most of the embodiments described below are considered in relation to antibodies and antibody-like molecules and represent specific embodiments of binding molecules for the development of therapeutic and diagnostic agents.

[0058] The terms “antibody” and “immunoglobulin” are used interchangeably herein. An antibody or immunoglobulin is a BMPRI / BMPRII-binding molecule that contains, and usually contains, at least a variable domain of the heavy chain and at least a variable domain of the light chain. The basic immunoglobulin structures in vertebrate systems are relatively well understood; see, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). As used herein, the term “antibody” refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementarity-determining regions (CDRs) (e.g., any of the three CDRs derived from the immunoglobulin light chain or any of the three CDRs derived from the immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, antibodies may contain the Fc region of human antibodies. The term also includes derivatives, such as bispecific antibodies, single-chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0059] As will be discussed in more detail below, the term “immunoglobulin” encompasses a broad range of polypeptides that can be biochemically distinguished. Those skilled in the art will understand that the heavy chain is classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), among several subclasses, including (e.g., γ1-γ4). It is the nature of this chain that determines the “class” of an antibody as IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc., are well-characterized and known to confer functional specialization. The modified versions of each of these classes and isotypes are readily identifiable to those skilled in the art in consideration of this disclosure and are therefore within the scope of this disclosure. All immunoglobulin classes are clearly within the scope of this disclosure, and the following discussion generally concerns the IgG class of immunoglobulin molecules. Regarding IgG, a standard immunoglobulin molecule contains two identical light-chain polypeptides with a molecular weight of approximately 23,000 daltons, and two identical heavy-chain polypeptides with a molecular weight of 53,000–70,000. The four chains are typically linked by disulfide bonds in a "Y" configuration, with the light chains beginning at the "Y" entry point and flanking the heavy chains that continue through the variable region.

[0060] Light chains are classified as either copper or lambda (κ, λ). Each heavy chain class can bind to either a copper or lambda light chain. Generally, light and heavy chains are covalently bonded to each other, and the "tails" of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when the immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell. In the heavy chains, the amino acid sequence runs from the N-terminus at the fork end of the Y configuration to the C-terminus at the bottom of each chain.

[0061] Both the light and heavy chains are divided into regions of structural and functional homology. The terms “constant” and “variable” are used functionally. In this regard, it will be understood that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains (CL) of the light chain and the constant domains (CH1, CH2, or CH3) of the heavy chain confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, and complement binding. Traditionally, the numbering of constant region domains increases as they become more distal to the antigen-binding site or the amino terminus of the antibody. The N-terminal portion is the variable region, the C-terminal portion is the constant region, and the CH3 and CL domains actually contain the carboxyl terms of the heavy and light chains, respectively.

[0062] As shown above, the variable region enables the antibody to selectively recognize and specifically bind to epitopes on the antigen. Specifically, a combination of the antibody's VL and VH domains, or subsets of complementarity-determining regions (CDRs), forms a variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site located at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs located in each of the VH and VL chains. Any antibody or immunoglobulin fragment containing a structure sufficient to specifically bind to BMPRI / BMPRII is referred to herein interchangeably as a “binding fragment” or “immunospecific fragment.”

[0063] In naturally occurring antibodies, each antigen-binding domain contains six hypervariable regions, sometimes called "complementarity-determining regions" or "CDRs," which are short, discontinuous sequences of amino acids that are specifically positioned to form the antigen-binding domain when the antibody takes its three-dimensional configuration in an aqueous solution environment. The "CDRs" are flanked by four relatively conserved "framework" regions or "FRs" that exhibit little intermolecular variability. The framework regions largely conform to the β-sheet structure, and the CDRs form binding loops and, in some cases, form part of the β-sheet structure. Thus, the framework regions act to form a scaffold that positions the CDRs in the correct orientation through non-covalent interactions between the chains. The antigen-binding domain formed by the positioned CDRs defines a complementary surface to the epitope on the immunoreactive antigen. This complementary surface facilitates the non-covalent binding of the antibody to its cognitive epitope. Since the amino acids containing the CDR and framework regions are precisely defined, they can be readily defined for any given heavy or light chain variable region by those skilled in the art, and the whole is incorporated herein by reference in “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196(1987), 901–917.

[0064] Where there are two or more definitions of a term used and / or accepted in the art, the definitions of the terms used herein are intended to include all such meanings unless explicitly stated otherwise. A specific example is the use of the term “complementarity-determining region” (“CDR”) to describe a non-contiguous antigen that combines sites found within the variable regions of both heavy-chain and light-chain polypeptides. This particular region is described by Kabat et al., US Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and Chothia and Lesk, J. Mol. Biol., 196(1987), 901–917, which are incorporated herein by reference, and when compared to each other, the definitions include overlaps or subsets of amino acid residues. Nevertheless, the application of any definition to refer to a CDR of an antibody or its variant is intended to be within the scope of the terms defined and used herein. The appropriate amino acid residues encompassing the CDR as defined by each of the references cited above are listed below in Table A for comparison. The exact number of residues that constitute a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues constitute a particular hypervariable region or CDR of a human IgG subtype of an antibody that has been conferred with the variable region amino acid sequence of the antibody.

[0065] [Table 1]

[0066] Kabat et al. also defined a numbering system for variable domain sequences applicable to any antibody. Those skilled in the art can clearly assign this system of “Kabat numbering” to any variable domain sequence without relying on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system described by Kabat et al., US Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in the antibodies or their antigen-binding fragments, variants, or derivatives described herein are based on the Kabat numbering system, but are theoretical and not equally applicable to all antibodies described herein. For example, depending on the position of the first CDR, the following CDRs may be shifted in either direction.

[0067] To avoid confusion, when the term CDR is used without specifying the method used to identify the CDR, the term refers to a CDR identified using the Paratome prediction method, for example, as shown in Table 1.

[0068] The antibodies or their antigen-binding fragments, immunospecific fragments, variants, or derivatives described herein include, but are not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, primate-like antibodies, murine or chimeric antibodies, single-chain antibodies, epitope-binding fragments, such as Fab, Fab' and F(ab')2, Fd, Fv, single-chain Fv(scFv), single-chain antibodies, disulfide-linked Fv(sdFv), fragments containing any of the VL or VH domains, fragments generated by Fab expression libraries, and anti-idiotype (anti-Id) antibodies (e.g., anti-Id antibodies against antibodies disclosed herein). The ScFv molecule is known in the art and is described, for example, in U.S. Patent No. 5,892,019. The immunoglobulin or antibody molecules described herein may be immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0069] In some embodiments, the antibody is not IgM or a derivative thereof having a pentavalent structure. In particular, in specific applications, especially in therapeutic use, IgM is not as useful as IgG and other bivalent antibodies or their corresponding conjugate molecules, as they often exhibit nonspecific cross-reactivity and very low affinity due to their pentavalent structure and lack of affinity maturation.

[0070] In some embodiments, the antibody is IgM or a derivative thereof having a pentavalent structure.

[0071] In some embodiments, the antibody is not a polyclonal antibody; that is, it consists of substantially one specific antibody species rather than a mixture obtained from a plasma immunoglobulin sample.

[0072] Antibody fragments containing single-chain antibodies may include the variable region alone or in combination with all or part of the following hinge regions, CH1, CH2, and CH3 domains. BMPRI / BMPRII-binding fragments also containing any combination of the variable region with the hinge regions, CH1, CH2, and CH3 domains are also provided herein. The antibodies or their immunospecific fragments described herein may be of any animal origin, including birds and mammals. In some embodiments, the antibody is a human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or bird antibody. In other embodiments, the variable region may be of cartilaginous fish origin (e.g., shark).

[0073] In some embodiments, the antibody is a human monoclonal antibody isolated from a human. Optionally, the framework region of the human antibody is aligned and applied according to the relevant human germline variable region sequence in a database, e.g., see Vbase (vbase.mrc-cpe.cam.ac.uk) hosted by the MRC Centre for Protein Engineering (Cambridge, UK). For example, amino acids considered to deviate from the true germline sequence may be due to PCR primer sequences incorporated during the cloning process. Compared to artificially generated human-like antibodies or heterologous mice, such as single-chain antibody fragments (scFv) derived from phage display antibody libraries, the human monoclonal antibodies described herein are characterized by (i) being obtained using a human immune response rather than an animal substitute, i.e., the antibody is produced in response to the native BMPRI / BMPRII of its relevant conformation in the human body; (ii) protecting the individual, or at least the presence of BMPRI / BMPRII being important; and (iii) minimizing the risk of cross-reactivity to autoantigens because the antibody is of human origin. Therefore, as used herein, terms such as “human monoclonal antibody,” “human monoclonal autoantibody,” and “human antibody” are used to refer to BMPRI / BMPRII binding molecules of human origin, i.e., isolated from human cells such as B cells or their hybridomas, or isolated from cDNA directly cloned from the mRNA of human cells, for example, human memory B cells. A human antibody remains “human” even if amino acid substitutions are made in the antibody to improve its binding characteristics, for example.

[0074] Below, for example, antibodies derived from a human immunoglobulin library or transgenic of one or more human immunoglobulins and derived from animals that do not express endogenous immunoglobulins, as described in U.S. Patent No. 5,939,598 by Kucherlapati et al., are referred to as human-like antibodies to distinguish them from true human antibodies as described herein.

[0075] For example, heavy-light chain pairing of human-like antibodies, such as synthetic and semi-synthetic antibodies typically isolated from phage displays, does not necessarily reflect the intrinsic pairing that occurs in true human B cells. Therefore, Fab and scFv fragments obtained from recombinant expression libraries commonly used in the prior art can be considered artificial, with all possible related effects on immunogenicity and stability.

[0076] As used herein, the term “heavy chain portion” includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain portion comprises at least one of the following: a CH1 domain, a hinge domain (e.g., upper, middle, and / or lower hinge region), a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, a conjugated polypeptide for use in the methods described herein may include: a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide described herein comprises a polypeptide chain comprising a CH3 domain. Furthermore, a conjugated polypeptide for use in the methods described herein may lack at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As described above, it will be understood by those skilled in the art that these domains (e.g., the heavy chain portion) may be modified so that their amino acid sequences differ from those of naturally occurring immunoglobulin molecules.

[0077] In certain antibodies, or their antigen-binding fragments, variants, or derivatives disclosed herein, the heavy chain portion of one polypeptide chain of the polymer is identical to that of the second polypeptide chain of the polymer. Alternatively, monomers containing the heavy chain portions described herein are not identical. For example, each monomer may contain different target-binding sites that form, for example, a bispecific antibody or diabody.

[0078] In some embodiments, the antibodies, or their antigen-binding fragments, variants, or derivatives disclosed herein, consist of a single polypeptide chain such as an scFv and are to be expressed intracellularly (intrabody) for possible in vivo therapeutic and diagnostic applications.

[0079] The heavy chain portion of the conjugated polypeptide for use in the diagnostic and treatment methods disclosed herein may be derived from different immunoglobulin molecules. For example, the heavy chain portion of the polypeptide may include a CH1 domain derived from the IgG1 molecule and a hinge region derived from the IgG3 molecule. In another example, the heavy chain portion may include a hinge region partially derived from the IgG1 molecule and a hinge region partially derived from the IgG3 molecule. In yet another example, the heavy chain portion may include a chimeric hinge partially derived from the IgG1 molecule and a chimeric hinge partially derived from the IgG4 molecule.

[0080] As used herein, the term “light chain moiety” includes an amino acid sequence derived from an immunoglobulin light chain. In some embodiments, the light chain moiety includes at least one VL or CL domain.

[0081] The minimum size of a peptide or polypeptide epitope for an antibody is considered to be about 4–5 amino acids. A peptide or polypeptide epitope may contain at least 7, at least 9, or at least about 15–about 30 amino acids. Since a CDR can recognize its tertiary form of the antigen peptide or polypeptide, the amino acids containing the epitope do not need to be consecutive and, in some cases, do not need to be on the same peptide chain. In some embodiments, a peptide or polypeptide epitope recognized by the antibody described herein contains a sequence of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or about 5–about 30, about 10–about 30, or about 15–about 30 consecutive or discontinuous amino acid sequences of BMPRI / BMPRII.

[0082] In this specification, the terms “specifically binding” or “specifically recognizing” are used interchangeably and generally mean that a binding molecule, such as an antibody, binds to an epitope via its antigen-binding domain, and that the binding requires some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” to an epitope if it binds to that epitope via its antigen-binding domain more readily than it would to bind to a random, unrelated epitope. Those skilled in the art will understand that an antibody may specifically bind to or specifically recognize an isolated polypeptide containing or consisting of amino acid residues corresponding to a linear portion of a non-contiguous epitope. The term “specificity” is used herein to describe the relative affinity of a particular antibody to a particular epitope. For example, antibody “A” may be considered to have higher specificity than antibody “B” for a given epitope, or antibody “A” may be said to bind to epitope “C” with higher specificity than it has for the relevant epitope “D”.

[0083] If present, all grammatical forms of the term “immunological binding features” or other binding features refer to the specificity, affinity, cross-reactivity, and other binding features of an antibody with an antigen.

[0084] "Preferential binding" means that the binding molecule, such as an antibody, binds specifically to the epitope more easily than to related, similar, homologous, or similar epitopes. Therefore, an antibody that "preferentially binds" to a given epitope may cross-react with related epitopes, but is more likely to bind to that epitope than to related epitopes.

[0085] As a non-limiting example, a binding molecule, such as an antibody, can have a dissociation station (K) of the antibody with respect to a second epitope. D K is less than ) DIf the antibody binds to the first epitope, it can be considered to preferentially bind to the first epitope. In another non-limiting example, the antibody's K for the second epitope D If an antibody binds to the first epitope with an affinity at least an order of magnitude lower, it can be considered to preferentially bind to the first antigen. In another non-limiting example, an antibody's K for the second epitope D If a molecule binds to the first epitope with an affinity at least two orders of magnitude lower, it can be considered to preferentially bind to the first epitope.

[0086] In another non-limiting example, a binding molecule, such as an antibody, may be considered to preferentially bind to the first epitope if it binds to the first epitope with an affinity (k(off)) that is less than the antibody's off-rate (k(off)) for the second epitope. In yet another non-limiting example, an antibody may be considered to preferentially bind to the first epitope if it binds to the first epitope with an affinity (k(off)) that is at least one order of magnitude lower than the antibody's k(off) for the second epitope. In yet another non-limiting example, an antibody may be considered to preferentially bind to the first epitope if it binds to the first epitope with an affinity (k(off)) that is at least two orders of magnitude lower than the antibody's k(off) for the second epitope.

[0087] A binding molecule, such as an antibody, is said to competitively inhibit the binding of a reference antibody to a given epitope if it preferentially binds to that epitope to such an extent that it blocks the binding of the reference antibody to that epitope to some degree. Competitive inhibition can be determined by any method known in the art, such as a competitive ELISA assay. An antibody may be said to competitively inhibit the binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%. Those skilled in the art will understand that the binding of an antibody to its epitope can also be competitively inhibited by a binding molecule other than an antibody.

[0088] As used herein, the term “affinity” refers to a measure of the strength of binding of an individual epitope to a binding molecule, such as a CDR of an immunoglobulin molecule; see, for example, Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988), pp. 27-28. As used herein, the term “avidity” refers to the overall stability of the complex between a population of immunoglobulins and antigens, i.e., the functional binding strength of an immunoglobulin mixture to an antigen; see, for example, Harlow, pp. 29-34. Avidity relates to both the affinity of individual immunoglobulin molecules in a population to a particular epitope and the binding titer of the immunoglobulin to the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repeatable epitope structure, such as a polymer, would be one example of high avidity. The affinity or avidity of an antibody to an antigen can be determined experimentally using any suitable method, see, for example, Berzofsky et al., “Antibody-Antigen Interactions” In Fundamental Immunology, Paul, WE, Ed., Raven Press New York, NY (1984), Kuby, Janis Immunology, WH Freeman and Company New York, NY (1992), and the methods described herein. Common techniques for measuring the affinity of an antibody to an antigen include ELISA, RIA, and surface plasmon resonance. The measured affinity of a particular antibody-antigen interaction may vary when measured under different conditions, e.g., salt concentration, pH. Therefore, affinity and other antigen-binding parameters, e.g., K D The IC50 measurement is preferably obtained using a standardized solution of the antibody and antigen, and a standardized buffer.

[0089] Binding molecules, such as antibodies or their antigen-binding fragments, variants, or derivatives described herein, may also be described or specified in terms of their cross-reactivity. As used herein, the term “cross-reactivity” refers to the ability of an antibody specific to one antigen to react with a second antigen; a measure of relevance between two different antigenic substances. Thus, an antibody is cross-reactive if it binds to an epitope other than the one that induced its formation. Cross-reactive epitopes generally have many of the same complementary structural properties as the inducible epitope, and in some cases may actually fit better than the original.

[0090] For example, a particular antibody may have some degree of cross-reactivity and bind to an epitope that is associated with, but not identical to, a reference epitope, such as an epitope that has at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity with the reference epitope (calculated using methods known in the art described herein). An antibody may have little to no cross-reactivity if it does not bind to an epitope with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity with the reference epitope (calculated using methods known in the art described herein). An antibody may be considered "highly specific" to a particular epitope if it does not bind to any other analog, ortholog, or homolog of that epitope.

[0091] The binding molecules, such as the antibodies or their antigen-binding fragments, variants, or derivatives described herein, may also be described or specified in relation to their binding affinity to BMPRI / BMPRII.

[0092] As previously shown, the subunit structures and three-dimensional configurations of the constant regions of various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the variable domain at the amino terminus of an immunoglobulin heavy chain, and the term "CH1 domain" includes the primary (mostly amino-terminus) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino-terminus relative to the hinge region of the immunoglobulin heavy chain molecule.

[0093] As used herein, the term “CH2 domain” includes, for example, the portion of a heavy chain molecule extending from approximately residues 244–360 of an antibody using a conventional numbering scheme in the Fc domain (residues 244–360, Kabat numbering system; and residues 231–340, EU numbering system; see Kabat EA et al. previously mentioned). The CH2 domain is unique in that it does not form a close pair with another domain. Rather, two N-linked branched carbohydrate chains are sandwiched between the two CH2 domains of an intact, native IgG molecule. It has also been well demonstrated that the CH3 domain extends from the CH2 domain of the IgG molecule to the C-terminus and contains approximately 108 residues.

[0094] As used herein, the term “hinge region” includes the portion of the heavy chain molecule that links the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 residues and is mobile, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be divided into three distinct domains: upper, middle, and lower hinge domains; see Roux et al., J. Immunol. 161(1998), 4083.

[0095] As used herein, the term “disulfide bond” includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by a disulfide bond, and the two heavy chains are linked by two disulfide bonds at positions 239 and 242 (positions 226 or 229, EU numbering system) using the Kabat numbering system.

[0096] As used herein, the terms “combined,” “fused,” and “fused” are used interchangeably. These terms refer to the joining of two or more elements or components to one another by any means, including chemical bonding or recombination. “In-frame fusion” refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a longer, consecutive ORF in a manner that maintains the correct translational reading frame of the original ORF. Thus, a recombinant fusion protein is a single protein containing two or more segments corresponding to polypeptides encoded by the original ORF (whose segments are not typically joined in nature). Thus, the reading frame is made consecutively within the fused segments, although the segments may be physically or spatially separated, for example, by an in-frame linker sequence. For example, polynucleotides encoding an immunoglobulin variable region CDR may be fused in-frame, but may be separated by at least one polynucleotide encoding an immunoglobulin framework region or further CDR regions, insofar as the “fused” CDR is co-translated as part of a consecutive polypeptide.

[0097] As used herein, the term “expression” refers to the process by which a gene produces a biochemical substance, such as RNA or polypeptide. The process includes, but is not limited to, gene knockdown and any emergence of a functional presence of a gene within a cell, including both transient and stable expression. It includes, but is not limited to, the transcription of a gene into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and the translation of such mRNA into polypeptides. If the final desired product is a biochemical substance, expression includes the production of that biochemical substance and any precursors. Gene expression produces a “gene product.” As used herein, a gene product may be a nucleic acid, such as messenger RNA produced by the transcription of a gene, or a polypeptide translated from the transcript. The gene products described herein further include nucleic acids having post-transcriptional modifications, such as polyadenylation, or polypeptides having post-translational modifications, such as methylation, glycosylation, lipid addition, binding to other protein subunits, or protein cleavage.

[0098] As used herein, the term “sample” refers to any biological material obtained from a subject or patient. In one embodiment, the sample may include blood, plasma, or urine. In other embodiments, the sample may include whole blood, plasma, B cells concentrated from a blood sample, and cultured cells (e.g., B cells derived from the subject). The sample may also include biopsy or tissue samples, including nerve tissue. In other embodiments, the sample may include whole cells and / or cell lysates. Blood samples can be collected by methods known in the art. In one embodiment, the pellet can be resuspended in 200 μl of buffer (20 mM Tris, pH 7.5, 0.5% Nonidet, 1 mM EDTA, 1 mM PMSF, 0.1 M NaCl, IX Sigma protease inhibitor, and IX Sigma phosphatase inhibitors 1 and 2) by vortexing at 4°C. The suspension can be maintained on ice for 20 minutes using intermittent vortexing. After spinning down at 15,000 × g for 5 minutes at approximately 4°C, the supernatant aliquots can be stored at approximately -70°C.

[0099] As used herein, the terms “to treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventive measures, the purpose of which is to prevent or delay (mitigate) any undesirable physiological changes or impairments described herein. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction of disease severity, stabilization (i.e., non-exacerbating) of disease, delay or slowing of disease progression, remission or mitigation of disease status, and reduction (either partial or total), whether detectable or undetectable. “Treatment” may also mean extended survival compared to survival predicted without treatment. Those requiring treatment include those who already have a condition or impairment, as well as those who are prone to developing a condition or impairment, or whose onset of a condition or impairment should be prevented.

[0100] "Subject," "individual," "animal," "patient," or "mammal" refers to any subject for which diagnosis, prognosis, prevention, or treatment is desired, specifically a mammalian subject, such as a human patient.

[0101] Compositions or methods described herein that include components or steps may also essentially consist of or comprise those components or steps.

[0102] II. Antibodies Anti-BMPRI / II antibodies and their antigen-binding fragments, such as their antigen-binding domains, and fusions / multimers are provided. Derivatives or variants of anti-BMPRI / II antibodies or their antigen-binding fragments are also provided herein. Conjugates comprising such antibodies or their BMPRI / BMPRII-binding fragments or their derivatives or variants are also provided herein. In some embodiments, the antibodies or their BMPRI / BMPRII-binding fragments described herein exhibit binding characteristics and / or biological properties outlined for the antibodies described in the following Examples section.

[0103] An antibody and its antigen-binding fragment, e.g., the antigen-binding domain, may be characterized by including at least one complementarity-determining region (CDR) of the VH and / or VL variable region containing any one of the amino acid sequences shown herein. Exemplary corresponding nucleotide sequences encoding the variable regions identified above are described herein (see below). Exemplary sets of CDRs for the above amino acid sequences of the VH and / or VL regions are provided below. Exemplary corresponding framework regions for CDRs are also provided herein. However, as will be discussed below, those skilled in the art will be well aware that, in addition to or instead of those amino acid sequences described herein, different CDRs may be used with one, two, three amino acids or, in the case of CDR2 and CDR3, still further amino acids.

[0104] In some embodiments, the antibody or its antigen-binding fragment, for example, the antigen-binding domain described herein, comprises at least one CDR containing or consisting of the amino acid sequences shown in Examples, Table 1, or Figure 7. In some embodiments, the antibody or its antigen-binding fragment described herein comprises one, two, three, four, five, or six CDRs containing or consisting of the amino acid sequences shown in Examples, Table 1, or Figure 7. The following table provides exemplary scFv sequences.

[0105] [Table 2-1]

[0106] [Table 2-2]

[0107] In some embodiments, the antibody or its antigen-binding fragment, for example, the antigen-binding domain, comprises a heavy chain variable region (VH) complementarity-determining region 1 (CDR1), VH CDR2, and VH CDR3, including the amino acid sequences shown herein, for example, in Examples, Figure 7, and / or Table 1, and optionally further comprises light chain variable region (VL) CDR1, VL CDR2, and VL CDR3 amino acid sequences, as shown herein, for example, in Examples, Figure 7, and / or Table 1.

[0108] In some embodiments, the antibody or its antigen-binding fragment, for example, the antigen-binding domain, as described herein, includes a VH containing the heavy chain framework region 1 (FR1) as described herein (i.e., some or all of the framework portion of SEQ ID NOs: 146, 148, 150, 152, 154, 160, 162, 164, 166, 168, 156, 158, 178, 180, 182, 184, 186, 188, 170, 172, 174, 176, or 212).

[0109] In some embodiments, the antibody or its antigen-binding fragment, e.g., antigen-binding domain, described herein, comprises a CDR sequence that is at least 95% identical to the CDR sequence in Examples, Figure 7, and / or Table 1; VH CDR1, 2, 3 and VL CDR1, 2, 3 that are identical to the complementarity-determining regions in Examples, Figure 7, and / or Table 1; VH and / or VL sequences that are at least 95% identical to the sequence in Figure 7; or sequences that are at least 95% identical to the sequence in Examples, Figure 7, and / or Table 1.

[0110] In some embodiments, the antigen-binding domain, antibody or antigen-binding fragment described herein includes VH CDR1, CDR2, or CDR2, or VL CDR1, CDR2, or CDR3, which comprises or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence described herein.

[0111] In some embodiments, the antibody or its antigen-binding fragment, e.g., the antigen-binding domain, contains a sequence with 0 or 1 to 10 (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) modifications, i.e., amino acid substitutions, additions, or deletions, compared to the reference amino acid sequence, e.g., the reference amino acid sequence in the VH or LH. In some embodiments, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the modifications are not in the CDR, but rather one or two modifications in one of the CDRs, e.g., up to two modifications in one CDR, e.g., one light chain CDR. Such variants retain the ability to bind to the same antigen as the parent antibody or its antigen-binding fragment.

[0112] A "conservative amino acid substitution" is one in which an amino acid residue is substituted with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0113] In some embodiments, variants of the antibodies described herein (e.g., antibodies or antigen-binding fragments having amino acid substitutions, additions, or deletions compared to the VH and / or VL of the amino acid sequence described herein) retain the ability to bind to BMPRI / BMPRII and / or retain one or more characteristics of the antibodies described in the following examples.

[0114] In some embodiments, the antibody or antigen-binding fragment described herein includes a heavy chain variable region (VH) comprising or consisting of the amino acid sequence described herein. In some embodiments, the antibody or antigen-binding fragment described herein includes a light chain variable region (VL) comprising or consisting of the amino acid sequence described herein. In some embodiments, the antibody or antigen-binding fragment described herein includes a heavy chain variable region (VH) comprising or consisting of the amino acid sequence described herein, and further includes a light chain variable region (VL) comprising or consisting of the amino acid sequence described herein. In some embodiments, the antibody or antigen-binding fragment includes VH and VL of the sequence described herein.

[0115] In some embodiments, the antibodies described herein include a heavy chain containing VH as described herein. In some examples, the heavy chain includes an IgG constant region. In some examples, the heavy chain includes a human IgG constant region.

[0116] In some embodiments, the antibodies described herein include a light chain comprising a VL as described herein. In some examples, the light chain includes a copper constant region. In some examples, the light chain includes a human copper constant region. In some examples, the light chain includes a lambda constant region. In some examples, the light chain includes a human lambda constant region.

[0117] In some embodiments, the antibody described herein comprises a heavy chain containing VH as described herein and a light chain containing VL as described herein, wherein the heavy chain and / or light chain contain an IgG (e.g., human IgG) constant region.

[0118] Alternatively, the antibodies described herein are antibodies or their antigen-binding fragments, derivatives, or variants that compete with the antibodies having VH CDR1-3 described herein for binding to BMPRI / BMPRII. Alternatively, the antibodies described herein are antibodies or their antigen-binding fragments, derivatives, or variants that compete with the antibodies having VL CDR1-3 described herein for binding to BMPRI / BMPRII. Alternatively, the antibodies described herein are antibodies or their antigen-binding fragments, derivatives, or variants that compete with the antibodies having VH CDR1-3 and VL CDR1-3 described herein for binding to BMPRI / BMPRII. Alternatively, the antibodies described herein are antibodies or their antigen-binding fragments, derivatives, or variants that compete with the antibodies having VH and / or VL described herein for binding to BMPRI / BMPRII. These antibodies may be human, rodent (e.g., mouse), chimeric, or humanized, particularly for therapeutic applications.

[0119] Alternatively, the antibodies described herein are antibodies or their antigen-binding fragments, derivatives, or variants that bind to the same epitopes as the anti-BMPRI / BMPRII antibodies, including VH and VL, described herein.

[0120] Competition between antibodies is determined by an assay in which the immunoglobulin being tested inhibits the specific binding of the reference antibody to a common antigen, such as BMPRI / BMPRII. Numerous types of competitive binding assays are known, e.g., solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), sandwich competitive assays; see Stahli et al., Methods in Enzymology 9 (1983), 242-253; solid-phase direct biotin-avidin EIA; see Kirkland et al., J. Immunol. 137 (1986), 3614-3619 and Cheung et al., Virology 176 (1990), 546-552; solid-phase direct labeling assays, solid-phase direct labeling sandwich assays; see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press (1988); solid-phase direct labeling RIA using I125 labeling; see Morel et al., Molec. Immunol. 25 (1988), 7-15 and Moldenhauer et al. See al., Scand. J. Immunol. 32(1990), 77-82. Typically, such assays involve the use of purified BMPRI / BMPRII or aggregates thereof, bound to a solid surface or cell, producing either an unlabeled test immunoglobulin or a labeled reference immunoglobulin, i.e., a human monoclonal antibody as described herein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cell in the presence of the test immunoglobulin. Typically, the test immunoglobulin is present in excess. In some embodiments, the competitive binding assay is performed under conditions known in the art. Antibodies identified by the competitive assay (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that, due to the resulting steric hindrance, are sufficiently close to the epitope bound by the reference antibody.Typically, when competing antibodies are present in excess, they will inhibit the specific binding of the reference antibody to a common antigen by at least 50% or 75%. Therefore, the present invention further relates to antibodies, or their antigen-binding fragments, variants, or derivatives, that competitively inhibit reference antibodies, including VH and VL of the Sequence ID numbers described herein, from binding to BMPRI / BMPRII.

[0121] In some embodiments, isolated polypeptides comprising immunoglobulin heavy chain variable regions (VHs) are provided herein, where at least one or at least two VH-CDRs of the heavy chain variable region are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the reference heavy chain VH-CDR1, VH-CDR2, or VH-CDR3 amino acid sequences from antibodies disclosed herein (see, for example, Table 1 and Examples for VH CDR sequences). Alternatively, the VH-CDR1, VH-CDR2, and VH-CDR3 regions of VH are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the reference heavy chain VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences of antibodies disclosed herein (see, for example, Table 1, Figure 7, and Examples for VH CDR sequences). Therefore, the heavy chain variable regions described herein may have VH-CDR1, VH-CDR2, and VH-CDR3 polypeptide sequences related to the sequences in Table 1, Figure 7, and Examples.

[0122] CDRs are defined by various methods / systems according to those skilled in the art. These systems and / or definitions have been developed and refined over many years and include Kabat, Chothia, IMGT, AbM, and Contact. The Kabat definition is based on sequence variability and is generally the most commonly used. The Chothia definition is based on the location of the structural loop region. The IMGT system is based on the sequence variability and location within the structure of the variable domain. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on the analysis of available antibody crystal structures. An exemplary system is a combination of Kabat and Chothia. Software programs for antibody sequence analysis and CDR determination (e.g., abYsis (bioinf.org.uk / abysis / sequence_input / key_annotation / key_annotation.cgi) and Paratome, Kunik et al. PLoS Comput Biol 8(2): e1002388(2012); Kunik et al., Nucleic Acids Res. 2012 Jul;40(Web Server issue):W521-4(2012)) are available and known to those skilled in the art.

[0123] The specific CDR sequences defined in Table 1 are generally based on Paratome predictions. However, it will be understood that references to the heavy chain CDR or multiple CDRs and / or light chain CDR or multiple CDRs of a particular antibody encompass all CDR definitions known to those skilled in the art.

[0124] Table 1 shows the CDRs defined by the Paratome system, while other CDR definitions, such as those defined by the Kabat, AbM, Contact, IMGT, or Chothia systems shown in Figure 7, are also included in the present invention and can be readily identified by those skilled in the art using the sequences presented in Figure 7 and the examples.

[0125] The isolated polypeptides described herein may include immunoglobulin heavy chain variable regions (VH) having polypeptide sequences in which the VH-CDR1, VH-CDR2, and VH-CDR3 regions are identical to the VH-CDR1, VH-CDR2, and VH-CDR3 sequences shown herein.

[0126] Furthermore, the isolated polypeptides may contain immunoglobulin heavy chain variable regions (VHs) having polypeptide sequences in which the VH-CDR1, VH-CDR2, and VH-CDR3 regions are identical to the VH-CDR1, VH-CDR2, and VH-CDR3 sequences shown in Table 1 or the Examples, except for one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in any one of the VH-CDRs. In some embodiments, the amino acid substitutions are conserved.

[0127] The isolated polypeptide may contain immunoglobulin light chain variable regions (VLs) in which at least one or at least two VL-CDRs of the light chain variable region are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of antibody-derived reference light chain VL-CDR1, VL-CDR2, or VL-CDR3 disclosed herein (for example, see Examples, Table 1, and Figure 7 for VL CDR sequences). Alternatively, the VL-CDR1, VL-CDR2, and VL-CDR3 regions of VL may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the reference light chain VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences of antibodies disclosed herein (see, for example, Examples, Table 1, and Figure 7 for VL CDR sequences). Thus, the light chain variable regions may have VL-CDR1, VL-CDR2, and VL-CDR3 polypeptide sequences related to the sequences in Table 1 or Examples. While Table 1 shows VL-CDR, other CDR definitions, such as VL-CDR as defined by the Kabat or Chothia systems, are also included in the present invention.

[0128] Isolated polypeptides containing immunoglobulin light chain variable regions (VLs) having polypeptide sequences in which the VL-CDR1, VL-CDR2, and VL-CDR3 regions are identical to the VL-CDR1, VL-CDR2, and VL-CDR3 sequences shown in Table 1 and Figure 7 are also provided herein.

[0129] The isolated polypeptides may also contain immunoglobulin light chain variable regions (VLs) in which the VL-CDR1, VL-CDR2, and VL-CDR3 regions have polypeptide sequences identical to the VL-CDR1, VL-CDR2, and VL-CDR3 sequences in Table 1 or Figure 7, except for one, two, three, four, five, six, seven, eight, nine, and ten amino acid substitutions in any one of the VL-CDRs. In some embodiments, the amino acid substitutions are conserved.

[0130] Generally, each antigen-binding domain will contain all of the single-clonal CDRs listed in Table 1 or Figure 7. In some embodiments, the antigen-binding domain may contain multiple clone-derived CDRs, as long as they retain their antigen-binding ability.

[0131] Immunoglobulins or the cDNA encoding them can be modified. Therefore, the methods described herein may include any one of the steps of generating a chimeric antibody, a humanized antibody, a single-chain antibody, a Fab-fragment, a bispecific antibody, a fusion antibody, or a labeled antibody or analog thereof. Corresponding methods are known to those skilled in the art and are described, for example, in Harlow and Lane, “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor (1988). When derivatives of such antibodies are obtained by phage display technology, the efficiency of phage antibodies binding to the same epitope as any of the antibodies described herein can be increased using surface plasmon resonance utilized in the BIAcore system (Schier, Human Antibodies Hybridomas 7(1996), 97-105; Malmborg, J. Immunol. Methods 183(1995), 7-13). The generation of chimeric antibodies is described, for example, in International Publication No. 89 / 09622. Methods for producing humanized antibodies are described, for example, in European Patent Application Publication No. 0239400 and International Publication No. 90 / 07861. Further sources of antibodies to be used in accordance with the present invention are so-called heterologous antibodies. General principles for the production of heterologous antibodies, such as human-like antibodies in mice, are described, for example, in International Publication No. 91 / 10741, International Publication No. 94 / 02602, International Publication No. 96 / 34096 and International Publication No. 96 / 33735. As discussed above, the antibodies described herein may exist in various forms, including, for example, Fv, Fab and F(ab)2, as well as single chains, in addition to complete antibodies; see, for example, International Publication No. 88 / 09344. Antibodies produced by such methods are also provided herein.

[0132] The antibodies described herein or their corresponding immunoglobulin chains may be further modified using prior art known in the art, for example, by using amino acid deletions, insertions, substitutions, additions, and / or recombinations and / or any other modifications known in the art, either alone or in combination. Methods for introducing such modifications into the DNA sequence underlying the amino acid sequence of the immunoglobulin chain are well known to those skilled in the art; see, for example, Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) NY and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1994). Modifications of antibodies described herein include side-chain modifications, skeletal modifications, and N- and C-terminal modifications including acetylation, hydroxylation, methylation, and amidation, as well as chemical and / or enzymatic derivatization of one or more constituent amino acids, including the attachment of carbohydrate or lipid moieties and cofactors. Similarly, the present invention encompasses the creation of chimeric proteins comprising an antibody or a fragment of a part thereof, described at the amino terminus, fused at the carboxyl terminus to a heterogeneous molecule such as an immunostimulant ligand; for corresponding technical details, see, for example, International Publication No. 00 / 30680.

[0133] In addition, since heavy chain CDR3 (HCDR3) is often observed to be the region with a higher degree of variability and the primary involvement in antigen-antibody interaction, peptides containing the above-mentioned binding molecules at a specific CDR3 of the heavy chain, for example, those containing the CDR3 region of any one of the variability regions of the above-mentioned antibodies, are provided herein. Such peptides can be readily synthesized or produced by recombinant means for producing the binding agents described herein. Such methods are well known to those skilled in the art. The peptides can be synthesized, for example, using commercially available automated peptide synthesizers. The peptides can also be produced by recombinant techniques, which involve incorporating peptide-expressing DNA into an expression vector and transforming cells using the expression vector to produce the peptide.

[0134] Therefore, the following are described herein: a binding molecule, for example, an antibody or its binding fragment that binds to the anti-BMPRI / BMPRII antibody described herein and exhibits the above-mentioned properties, i.e., specifically recognizes (binds to) BMPRI / BMPRII. Such antibodies and binding molecules can be tested for their binding specificity and affinity by ELISA, Western blotting, and immunohistochemistry as described herein (see, for example, the examples).

[0135] Instead of directly obtaining immunoglobulins from cultures of immortalized B cells or B memory cells, the immortalized cells described herein can be used as a source of rearranged heavy and light chain loci for subsequent expression and / or genetic manipulation. The rearranged antibody genes can be reverse transcribed from suitable mRNA to produce cDNA. If necessary, the heavy chain constant region can be replaced with that of a different isotype or removed. Variable regions can be ligated to encode single-chain Fv regions. Multiple Fv regions can be ligated to confer the ability to bind to more than one target, or chimeric heavy and light chain combinations can be utilized. Given that the genetic material is available, designing the analogs described above, while retaining both their ability to bind to desired targets, is straightforward. Methods for cloning antibody variable regions and generating recombinant antibodies are known to those skilled in the art and are described, for example, in Gilliland et al., Tissue Antigens 47(1996), 1-20; and Doenecke et al., Leukemia 11(1997), 1787-1792.

[0136] Once suitable genetic material is obtained and, if necessary, modified to encode analogs, the coding sequence, including those encoding minimal variable regions of the heavy and light chains, can be inserted into an expression system contained in a vector that can be introduced into standard recombinant host cells. A variety of such host cells can be used for efficient processing, but mammalian cells may also be considered. Typical mammalian cell lines useful for this purpose include, but are not limited to, CHO cells, HEK293 cells, or NSO cells.

[0137] Next, antibody or analog production is carried out by culturing a modified recombinant host under culture conditions appropriate for host cell growth and coding sequence expression. The antibody is then recovered by isolation from the culture. The expression system is designed to include a signal peptide so that the resulting antibody is secreted into the culture medium, but intracellular production is also possible.

[0138] Accordingly, the present invention also relates to polynucleotides encoding antibodies or equivalent binding molecules as described herein. In some embodiments, the polynucleotide encodes at least a variable region of the immunoglobulin chain of the antibody described above. Typically, the variable region encoded by the polynucleotide includes at least one complementarity-determining region (CDR) of the VH and / or VL of the variable region of the antibody.

[0139] Those skilled in the art will readily understand that the variable domains of antibodies having the variable domains described above can be used to construct other polypeptides or antibodies with desired specificity and biological function. Accordingly, polypeptides and antibodies comprising at least one or more CDRs of the variable domains described above, e.g., all of the CDRs, and advantageously having substantially the same or similar binding properties as the antibodies described in the accompanying examples are provided herein. Those skilled in the art know that binding affinity can be enhanced by causing amino acid substitutions within the CDRs or within the hypervariable loops, partially overlapping with the CDRs as defined by Kabat (Chothia and Lesk, J. Mol. Biol. 196(1987), 901-917), see, for example, Riechmann, et al, Nature 332(1988), 323-327. Accordingly, antibodies having one or more of the above-mentioned CDRs with one or more amino acid substitutions, or two or fewer amino acid substitutions, are also provided herein. In some embodiments, the antibodies described herein include two or all three CDRs of the variable regions listed in Table 1 in one or both of their immunoglobulin chains.

[0140] Binding molecules known to those skilled in the art, such as antibodies, or their antigen-binding fragments, variants, or derivatives, may include a constant region mediating one or more effector functions. For example, binding of the complement C1 component to the antibody constant region can activate the complement system. Complement activation is important in the opsonization and lysis of cellular pathogens. Complement activation can also stimulate inflammatory responses and may be involved in autoimmune allergies. Furthermore, antibodies bind to various cell receptors via Fc regions having Fc receptor binding sites on the antibody Fc region that bind to Fc receptors (FcRs) on cells. There are numerous Fc receptors specific to different classes of antibodies, including IgG (gamma receptor), IgE (epsilon receptor), IgA (alpha receptor), and IgM (mu receptor). The binding of antibodies to Fc receptors on the cell surface triggers numerous important and diverse biological responses, including engraftment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (known as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and regulation of immunoglobulin production.

[0141] Accordingly, some embodiments described herein include antibodies, antigen-binding fragments, variants, or derivatives thereof in which at least a portion of one or more constant region domains is deleted or otherwise modified to result in desired biochemical characteristics, such as reduced effector function, non-covalent dimerization ability, increased ability to localize at the BMPRI / BMPRII site, shortened serum half-life, or lengthened serum half-life, compared to an unmodified antibody of approximately the same immunogenicity. For example, some antibodies for use in the diagnostic and treatment methods described herein are domain deletion antibodies that contain a polypeptide chain similar to an immunoglobulin heavy chain but lack at least a portion of one or more heavy chain domains. For example, in a particular antibody, one entire domain of the constant region of the modified antibody may be deleted, for example, all or part of the CH2 domain may be deleted. In other embodiments, certain antibodies for use in the diagnostic and treatment methods described herein have a constant region modified to remove glycosylation, e.g., an IgM heavy chain constant region, which is referred elsewhere herein as a non-glycosylated or "agly" antibody. Such "agly" antibodies may be prepared enzymatically and by manipulating consensus glycosylation sites in the constant region. While not constrained by theory, it is believed that "agly" antibodies may have improved safety and stability profiles in vivo. Methods for generating non-glycosylated antibodies with desired effector function can be found, for example, in International Publication No. 2005 / 018572, which is incorporated entirely by reference.

[0142] In certain antibodies, or their antigen-binding fragments, variants, or derivatives described herein, the Fc moiety may be mutated to reduce effector function using techniques known in the art. For example, deletion or inactivation of the constant region domain (through point mutation or other means) may reduce Fc receptor binding of the circulating modified antibody, thereby increasing BMPRI / BMPRII localization. In other cases, the constant region modification consistent with the present invention may mediate complement binding, thereby reducing the serum half-life and nonspecific binding of the conjugated cytotoxin. Further modifications of the constant region may be used to modify disulfide bonds or oligosaccharide moieties that enable enhanced localization due to increased antigen specificity or antibody mobility. The resulting physiological profiles, bioavailability, and other biochemical effects of the modifications, such as BMPRI / BMPRII localization, in vivo distribution, and serum half-life, can be readily measured and quantified using well-known immunological techniques without excessive experimentation.

[0143] Some antibodies, or their antigen-binding fragments, variants, or derivatives described herein, may have their Fc portion mutated or replaced with an alternative protein sequence to enhance receptor-mediated endocytosis of the antibody, for example, via the Fcγ receptor, LRP, or Thy1 receptor, or to increase antibody uptake by cells through "SuperAntibody technology," which is said to allow the antibody to shut out living cells without harming them (Expert Opin. Biol. Ther. (2005), 237-241). For example, the production of bispecific or multispecific antibodies having a fusion protein of an antibody-binding region of a cell surface receptor and a cognitive protein ligand, or BMPRI / BMPRII, as well as a specific sequence that binds to the cell surface receptor, may be carried out by manipulation using techniques known in the art.

[0144] In some of the antibodies, or their antigen-binding fragments, variants, or derivatives described herein, the Fc portion may be mutated, replaced with an alternative protein sequence, or the antibody may be chemically modified to increase its passage across the blood-brain barrier.

[0145] Modified forms of the antibodies described herein, or their antigen-binding fragments, variants, or derivatives, can be prepared from the precursor or the whole parent antibody using techniques known in the art. Typical techniques are discussed in more detail herein. The antibodies described herein, or their antigen-binding fragments, variants, or derivatives, can be prepared or manufactured using techniques known in the art. In some embodiments, the antibody molecule or its fragment is “recombinantly produced,” i.e., produced using recombinant DNA technology. Exemplary techniques for preparing the antibody molecule or its fragment are discussed in more detail elsewhere herein.

[0146] The antibodies, or their antigen-binding fragments, variants, or derivatives described herein, also include derivatives modified by covalent bonding to the antibody, for example, of any type of molecule, such that the covalent bond does not prevent the antibody from specifically binding to its cognitive epitope. Examples, but not limited to, antibody derivatives include antibodies modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, protein cleavage, or binding to cellular ligands or other proteins. Any of the numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, or metabolic synthesis of tunicamycin. In addition, derivatives may contain one or more non-classical amino acids.

[0147] In some embodiments, the antibodies, or their antigen-binding fragments, variants, or derivatives described herein will not induce an adverse immune response in the animal to be treated, e.g., a human. In some embodiments, the binding molecules, e.g., the antibodies, or their antigen-binding fragments described herein, are derived from a patient, e.g., a human patient, and subsequently used in the same species from which they originate, e.g., humans, thereby mitigating or minimizing the occurrence of an adverse immune response.

[0148] Deimmunization can also be used to reduce the immunogenicity of an antibody. As used herein, the term “deimmunization” includes modification of an antibody to modify T cell epitopes; see, for example, International Publication No. 98 / 52976 and International Publication No. 00 / 34317. For example, VH and VL sequences derived from a starting antibody are analyzed, and human T cell epitopes are “mapped” from their respective V regions, thereby indicating the location of the epitope in relation to complementarity-determining regions (CDRs) and other key residues in the sequence. Individual T cell epitopes derived from the T cell epitope map are analyzed to identify alternative amino acid substitutions that carry a low risk of altering the activity of the final antibody. A wide range of alternative VH and VL sequences, including combinations of amino acid substitutions, are designed, and these sequences are subsequently incorporated into a wide range of conjugating polypeptides, e.g., BMPRI / BMPRII-specific antibodies or their immunospecific fragments, for use in the diagnostic and therapeutic methods disclosed herein, and then tested for function. Typically, 12–24 variant antibodies are generated and tested. The complete heavy and light chain genes, including the modified V and human C regions, are then cloned into expression vectors, and the plasmids are subsequently introduced into cell lines for the production of the entire antibody. The antibodies are then compared in appropriate biochemical and biological assays to identify the optimal variant.

[0149] Monoclonal antibodies can be prepared using a wide range of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma technologies, including those known in the art and those taught in, for example, Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas Elsevier. NY, 563-681 (1981), the aforementioned references being incorporated by reference in their entirety. As used herein, the term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryote, prokaryote, or phage clone, rather than the method by which it is produced. Thus, the term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology.

[0150] In the well-known hybridoma process (Kohler et al., Nature 256(1975), 495), relatively short-lived or dying lymphocytes of mammalian origin, such as B cells from the mouse subjects described herein, are fused with immortal tumor cell lines (e.g., myeloma cell lines) to produce hybrid cells or "hybridomas" that are both immortal and capable of producing antibodies encoded by the B cells' genes. The resulting hybrids are isolated into single gene lines by sorting, dilution, and regrowth using each individual line containing genes specific to the formation of a single antibody. These produce allogeneic antibodies against the desired antigen and are referred to as "monoclonals" by reference to their pure genetic origin.

[0151] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium containing one or more substances that inhibit the growth or survival of unfused parent myeloma cells. Those skilled in the art will understand that reagents, cell lines, and media for hybridoma formation, selection, and growth are commercially available from numerous sources, and that standardized protocols are well established. Generally, the culture medium in which the hybridoma cells are grown is assayed for the production of monoclonal antibodies against the desired antigen. The binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by in vitro assays such as immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA) as described herein. After hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, clones may be subcloned by limiting dilution techniques and grown by standard methods, see, for example, Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp 59-103 (1986). It will be further understood that monoclonal antibodies secreted from subclones may be isolated from culture media, ascites fluid, or serum by conventional purification methods such as protein A, hydroxyl apatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0152] In some embodiments, lymphocytes can be selected by microscopy and variable genes can be isolated. For example, peripheral blood mononuclear cells can be isolated from immunized or innately immune animals, such as humans, and cultured in vitro for about 7 days. The culture can be screened for specific immunoglobulins that meet screening criteria. Cells derived from positive wells can be isolated. Individual Ig-producing B cells can be isolated by FACS or by identifying them in a complement-mediated hemolytic plaque assay. Ig-producing B cells can be taken into a test tube by microscopy, and the VH and VL genes can be amplified, for example, using RT-PCR. The VH and VL genes can be cloned into antibody expression vectors and introduced into cells (e.g., eukaryotic or prokaryotic cells) for expression.

[0153] Alternatively, antibody-producing cell lines may be selected and cultured using techniques well known to those skilled in the art. Such techniques are described in various laboratory manuals and elementary publications. In this regard, techniques suitable for use in the methods and compositions described below are described in Current Protocols in Immunology, Coligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991), with supplements, and are incorporated herein by reference in their entirety.

[0154] Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, Fab and F(ab')2 fragments may be generated by recombinant DNA or by protein cleavage of immunoglobulin molecules using enzymes such as papain (to generate the Fab fragment) or pepsin (to generate the F(ab')2 fragment). The F(ab')2 fragment contains a variable region, a light chain constant region, and a heavy chain CH1 domain. Such fragments are sufficient for use in immunodiagnostic techniques, for example, which involve conjugating the immunospecific portion of an immunoglobulin to a detection reagent such as a radioisotope.

[0155] In some embodiments, the antibodies described herein include at least one heavy chain or light chain CDR of the antibody molecule. In other embodiments, the antibodies described herein include at least two CDRs derived from one or more antibody molecules. In other embodiments, the antibodies described herein include at least three CDRs derived from one or more antibody molecules. In other embodiments, the antibodies described herein include at least four CDRs derived from one or more antibody molecules. In other embodiments, the antibodies described herein include at least five CDRs derived from one or more antibody molecules. In other embodiments, the antibodies described herein include at least six CDRs derived from one or more antibody molecules. Exemplary antibody molecules containing at least one CDR that may be included in the target antibody are described herein.

[0156] The antibodies described herein can be produced by any method known in the art for the synthesis of antibodies, in particular by chemical synthesis or by the recombinant expression techniques described herein.

[0157] In some embodiments, the antibodies described herein, or their antigen-binding fragments, variants, or derivatives, include a synthetic constant region in which one or more domains are partially or entirely deleted ("domain-deleted antibodies"). In some embodiments, a compatible modified antibody would include a domain-deleted construct or variant in which the entire CH2 domain is removed. In other embodiments, a short bound peptide may be replaced with a deletion domain to provide mobility and freedom of movement for the variable region. Those skilled in the art will understand that such constructs are particularly preferred due to the regulatory properties of the CH2 domain over the metabolic rate of the antibody. Domain-deleted constructs can be produced using a vector encoding the IgG1 human constant domain; see, for example, International Publication No. 02 / 060955 and International Publication No. 02 / 096948. This vector is manipulated to delete the CH2 domain and produce a synthetic vector expressing the domain-deleted IgG1 constant region.

[0158] In some embodiments, the antibodies, or their antigen-binding fragments, variants, or derivatives described herein are minibodies. Minibodies can be prepared using methods described in the art, see, for example, U.S. Patent No. 5,837,821 or International Publication No. 94 / 09817.

[0159] In some embodiments, the antibodies described herein, or their antigen-binding fragments, variants, or derivatives, include immunoglobulin heavy chains having several amino acid deletions or substitutions, or even a single amino acid deletion, insofar as they enable binding between monomeric subunits and maintain binding to BMPRI / BMPRII. For example, a single amino acid mutation in a selected range of the CH2 domain may substantially reduce Fc binding, thereby increasing BMPRI / BMPRII localization. Similarly, it may be desirable to simply delete a portion of one or more constant region domains that modulate effector functions to be regulated (e.g., complement binding). Such partial deletions of a constant region may improve selected characteristics of the antibody (serum half-life) while retaining other desired functions associated with the constant region domain of the intact subject. Furthermore, as mentioned above, the constant region of the disclosed antibody may be synthetic through one or more amino acid mutations or substitutions that enhance the profile of the resulting construct. In this regard, it may be possible to disrupt the activity mediated by conserved binding sites (e.g., Fc binding) while substantially maintaining the conformation and immunogenicity profile of the modified antibody. Other embodiments may involve the addition of one or more amino acids to the constant region to enhance desired features such as effector function, or to introduce further cytotoxicity or carbohydrate attachment. In such embodiments, it may be desirable to insert or replicate specific sequences derived from selected constant region domains.

[0160] The present invention also provides an antibody comprising, essentially, a variant (including derivatives) of an antibody molecule described herein (e.g., the VH region and / or VL region), wherein the antibody or fragment binds immunospecifically to BMPRI / BMPRII. Mutations can be introduced in the nucleotide sequence encoding the antibody, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions, using standard techniques known to those skilled in the art. In some embodiments, the variant (including derivatives) encodes fewer than 50 amino acid substitutions, fewer than 40 amino acid substitutions, fewer than 30 amino acid substitutions, fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions compared to the reference VH region, VH-CDR1, VH-CDR2, VH-CDR3, VL region, VL-CDR1, VL-CDR2, or VL-CDR3. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity (e.g., the ability to bind to BMPRI / BMPRII).

[0161] For example, mutations can be induced only in the framework region or only in the CDR region of the antibody molecule. The introduced mutations may be silent or neutral missense mutations and may have little to no effect on the antibody's ability to bind to an antigen, for example; in fact, some such mutations do not change the amino acid sequence at all. These types of mutations may be useful for optimizing codon use or improving antibody production in hybridomas. The codon-optimized coding regions encoding the antibodies described herein are disclosed elsewhere herein. Alternatively, non-neutral missense mutations may alter the antibody's ability to bind to an antigen. The location of most silent and neutral missense mutations is probably in the framework region, while the location of most non-neutral missense mutations is probably in the CDR, but this is not an absolute requirement. Those skilled in the art will be able to design and test mutant molecules having desired properties, such as no change in antigen-binding activity or a change in binding activity (e.g., improvement of antigen-binding activity or change in antibody specificity). After mutagenesis, the encoded protein may be expressed routinely, and the functional and / or biological activity of the encoded protein (e.g., its ability to immunospecifically bind to at least one epitope of BMPRI / BMPRII) can be determined using the techniques described herein or by routinely modifying techniques known in the art.

[0162] BMPRI / BMPRII conjugates, for example, the BMPRI / BMPRII-conjugated antibodies described herein (not limited to), may be characterized using any in vivo or in vitro model. Those skilled in the art will readily understand that the BMPRI / BMPRII conjugates (e.g., antibodies) described herein may be characterized in the mouse models described herein.

[0163] III. Polynucleotides encoding antibodies Polynucleotides encoding antibodies, or their antigen-binding fragments, variants, or derivatives, as described herein are also provided herein. Polynucleotides encoding antibodies, or their antigen-binding fragments, variants, or derivatives, may consist of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides encoding antibodies, or their antigen-binding fragments, variants, or derivatives, may consist of hybrid molecules containing single-stranded and double-stranded DNA, DNA which is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA which is a mixture of single-stranded and double-stranded regions, single-stranded or more typically double-stranded DNA and RNA which may be a mixture of single-stranded and double-stranded regions, or DNA and RNA which may be a mixture of single-stranded or more typically double-stranded regions. In addition, polynucleotides encoding antibodies, or their antigen-binding fragments, variants, or derivatives, may consist of triple-stranded regions containing RNA or DNA or both RNA and DNA. Polynucleotides encoding antibodies, or their antigen-binding fragments, variants, or derivatives, may also contain one or more modified bases or a DNA or RNA backbone modified for stability or other reasons. "Modified" bases include, for example, unusual bases such as tritylated bases and inosine. Various modifications can be made to DNA and RNA, so that "polynucleotides" encompass chemically, enzymatically, or metabolically modified forms.

[0164] Isolated polynucleotides encoding non-natural variants of polypeptides derived from immunoglobulins (e.g., immunoglobulin heavy chain or light chain portions) can be generated by introducing one or more nucleotide substitutions, additions, or deletions into the nucleotide sequence of an immunoglobulin, such that one or more amino acid substitutions, additions, or deletions are introduced into the encoding protein. The mutations may be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. In some embodiments, the conservative amino acid substitutions are made at one or more non-essential amino acid residues.

[0165] As is well known, RNA can be isolated from intrinsic B cells, hybridoma cells, or other transformed cells by standard techniques such as guanidium isothiocyanate extraction and precipitation, followed by centrifugation or chromatography. If desired, mRNA may be isolated from total RNA by standard techniques such as chromatography on oligo-dT cellulose. Appropriate techniques are known in the art. In some embodiments, cDNA encoding the light and heavy chains of antibodies can be prepared simultaneously or separately using reverse transcriptase and DNA polymerase according to well known methods. PCR may be initiated with consensus constant-region primers or with more specific primers based on published heavy and light chain DNA and amino acid sequences. As discussed above, PCR may also be used to isolate DNA clones encoding the antibody light and heavy chains. In this case, the library may be screened with consensus primers or larger homologous probes, such as human constant-region probes.

[0166] DNA, typically plasmid DNA, may be isolated from cells using techniques known in the art and then restriction enzyme-mapped and sequenced according to standard, well-known techniques, for example, as described in detail in the aforementioned references relating to recombinant DNA techniques. Of course, the DNA may also be synthesized according to the present invention at any point during the isolation process or subsequent analysis.

[0167] In some embodiments, isolated polynucleotides comprising essentially or comprising a nucleic acid encoding an immunoglobulin heavy chain variable region (VH), wherein at least one CDR of the heavy chain variable region or at least two VH-CDRs of the heavy chain variable region are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the reference heavy chain VH-CDR1, VH-CDR2, or VH-CDR3 amino acid sequence derived from an antibody disclosed herein. Alternatively, the VH-CDR1, VH-CDR2, or VH-CDR3 region of VH is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the reference heavy chain VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences derived from an antibody disclosed herein. Therefore, according to this embodiment, the heavy chain variable region of the antibody or antigen-binding fragment described herein has a VH-CDR1, VH-CDR2, or VH-CDR3 polypeptide sequence related to the polypeptide sequence shown in the example.

[0168] In some embodiments, isolated polynucleotides comprising nucleic acids encoding immunoglobulin heavy chain variable regions (VHs) are provided herein, each having polypeptide sequences such that the VH-CDR1, VH-CDR2, and VH-CDR3 regions are identical to the VH-CDR1, VH-CDR2, and VH-CDR3 groups shown in the examples, except for one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in any one of the VH-CDRs. In some embodiments, the amino acid substitutions are conserved.

[0169] In another embodiment, isolated polynucleotides comprising essentially or comprising a nucleic acid encoding an immunoglobulin light chain variable region (VL) in which at least one VL-CDR of the light chain variable region or at least two VL-CDRs of the light chain variable region are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the reference light chain VL-CDR1, VL-CDR2, or VL-CDR3 amino acid sequence derived from an antibody disclosed herein are provided herein. Alternatively, the VL-CDR1, VL-CDR2, or VL-CDR3 region of the VL is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the reference light chain VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences derived from an antibody disclosed herein. Therefore, according to this embodiment, the light chain variable region of the antibody or antigen-binding fragment described herein has a VL-CDR1, VL-CDR2, or VL-CDR3 polypeptide sequence associated with the polypeptide sequence shown in the example.

[0170] In another embodiment, isolated polynucleotides comprising nucleic acids encoding immunoglobulin light chain variable regions (VLs) are provided herein, which have polypeptide sequences in which the VL-CDR1, VL-CDR2, and VL-CDR3 regions are identical to the VL-CDR1, VL-CDR2, and VL-CDR3 groups shown in the examples, except for one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in any one of the VL-CDRs. In some embodiments, the amino acid substitutions are conserved.

[0171] In another embodiment, isolated polynucleotides comprising essentially or comprising nucleic acids encoding immunoglobulin heavy chain variable regions (VH), wherein the VH-CDR1, VH-CDR2, and VH-CDR3 regions have polypeptide sequences identical to those of the VH-CDR1, VH-CDR2, and VH-CDR3 groups shown in the examples, are provided herein.

[0172] In another embodiment, isolated polynucleotides comprising essentially or comprising nucleic acids encoding immunoglobulin light chain variable regions (VLs), wherein the VL-CDR1, VL-CDR2, and VL-CDR3 regions have polypeptide sequences identical to those of the VL-CDR1, VL-CDR2, and VL-CDR3 groups shown in the examples, are provided herein.

[0173] As is known in the art, “sequence identity” between two polypeptides or two polynucleotides is determined by comparing the amino acid or nucleic acid sequence of one polypeptide or polynucleotide with the sequence of the second polypeptide or polynucleotide. When considered herein, whether any particular polypeptide is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to another polypeptide can be determined, without limitation, using methods and computer programs / software known in the art, such as the BESTFIT program (Wisconsin Sequence Analysis Package, version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). BESTFIT uses the local homology algorithm described in Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489, to find the best homology segment between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is 95% identical to, for example, a reference sequence described herein (e.g., an antibody or its antigen-binding fragment described herein), the parameters are, of course, set such that the percentage of identity is calculated over the entire length of the reference polypeptide sequence, and a homology gap of up to 5% of the total number of amino acids in the reference sequence is permitted.

[0174] In some embodiments, the polynucleotide essentially comprises or comprises a nucleic acid having a polynucleotide sequence of the VH or VL region of an anti-BMPRI / BMPRII antibody as shown herein. In this regard, it will be readily apparent to those skilled in the art that a polynucleotide encoding at least the variable domains of the light chain and / or heavy chain may encode the variable domains of both immunoglobulin chains and only one of them. Further provided herein are polynucleotides comprising or comprising a nucleotide sequence encoding the amino acid sequence of an antibody or its antigen-binding fragment as shown in, for example, the Examples, Figure 7, or Table 1.

[0175] In some embodiments, isolated polynucleotides comprising essentially or comprising a nucleic acid encoding an immunoglobulin heavy chain variable region that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or 95% identical to a reference heavy chain VH are provided herein. In some embodiments, the amino acid sequence of the reference heavy chain variable region is as shown herein.

[0176] In some embodiments, isolated polynucleotides comprising essentially or comprising a nucleic acid encoding an immunoglobulin light chain variable region that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or 95% identical to a reference light chain VL are provided herein. In some embodiments, the amino acid sequence of the reference light chain variable region is as shown herein.

[0177] Polynucleotide fragments described herein and elsewhere are also provided herein. In addition, polynucleotides encoding fusion polynucleotides, Fab fragments, and other derivatives described herein are also considered.

[0178] Polynucleotides may be generated or manufactured by any method known in the art. For example, if the nucleotide sequence of an antibody is known, the polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides, as described in Kutmeier et al., BioTechniques 17(1994), 242, which, for example, involves synthesizing the oligonucleotide-containing portions of the antibody-encoding sequence, annealing and ligating those oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0179] Alternatively, the polynucleotide encoding the antibody, or its antigen-binding fragment, variant, or derivative, may be generated from nucleic acids derived from a suitable source. If a clone containing the nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, the nucleic acid encoding the antibody may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library or a cDNA library derived therefrom, or nucleic acids isolated from any tissue or cell expressing a BMPRI / BMPRII-specific antibody, preferably polyA+RNA, such as selected hybridoma cells expressing the antibody) by PCR amplification using synthetic primers that can hybridize to the 3' and 5' ends of the sequence, or, for example, by cloning a cDNA clone from an antibody-encoding cDNA library using oligonucleotide probes specific to a particular gene sequence. The amplified nucleic acid, then produced by PCR, may be cloned into a replicable cloning vector using any method known in the art.

[0180] Once the nucleotide sequence and corresponding amino acid sequence of an antibody, or its antigen-binding fragment, variant, or derivative, are determined, the nucleotide sequence may be manipulated using methods well known in the art for manipulating the nucleotide sequence, such as recombinant DNA techniques, site-directed mutagenesis, PCR, etc., to generate antibodies having different amino acid sequences, for example, to generate amino acid substitutions, deletions, and / or insertions (see, for example, the techniques described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1990) and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, NY (1998), both of which are incorporated herein by reference in their entirety).

[0181] IV. Fusion Proteins and Conjugates In some embodiments, the antibody polypeptide includes an amino acid sequence or one or more moieties that do not normally bind to the antibody. Exemplary modifications are described in more detail below. For example, the single-chain fv antibody fragments described herein may include a mobile linker sequence or may be modified to include a functional moiety (e.g., PEG, drug, toxin, or labeling such as fluorescent, radioactive, enzyme, nuclear magnetic, or heavy metal).

[0182] The antibody polypeptides described herein may include, essentially consist of, or consist of a fusion protein. The fusion protein is, for example, a chimeric molecule containing an immunoglobulin BMPRI / BMPRII-binding domain together with at least one target binding site and at least one heterologous moiety, i.e., a moiety that does not spontaneously bind in nature. The amino acid sequences may typically be present in separate proteins that are brought together in the fusion polypeptide, or they may typically be present in the same protein but in a new configuration in the fusion polypeptide. The fusion protein may be produced, for example, by chemical synthesis, or by generating and translating polynucleotides in which the peptide region encodes in the desired relationship.

[0183] In application to polynucleotides or polypeptides, the term “heterogeneous” means that the polynucleotide or polypeptide originates from an entity distinct from the rest of the entity they are being compared to. For example, as used herein, a “heterogeneous polypeptide” to be fused to an antibody, or its antigen-binding fragment, variant, or analog, is derived from a non-immunoglobulin polypeptide of the same species or from an immunoglobulin or non-immunoglobulin polypeptide of a different species.

[0184] As will be discussed in more detail elsewhere herein, the antibodies, or their antigen-binding fragments, variants, or derivatives described herein, may further be recombinantly fused to heterologous polypeptides at their N or C terminus, or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, antibodies may be recombinantly fused to or conjugated to molecules useful as labeling and effector molecules in detection assays, such as heterologous polypeptides, drugs, radionuclides, or toxins; see, for example, International Publication No. 92 / 08495; International Publication No. 91 / 14438; International Publication No. 89 / 12624; U.S. Patent No. 5,314,995; and European Patent Application No. 0396387.

[0185] In some cases, an antibody or its antigen-binding fragment is conjugated to a drug. Methods for generating such drug conjugates are known in the art and are described herein (see, for example, the following examples). In some cases, the drug is an immunosuppressive drug (see, for example, the following section “Immunosuppressive or Immunomodulatory Drugs” for examples of immunosuppressive drugs). In some cases, the drug is Taxol. In some cases, the drug is directly conjugated to an antibody or its antigen-binding fragment. In some cases, the drug is conjugated to an antibody or its antigen-binding fragment via a linker. Examples of linkers that may be used to conjugate a drug to an antibody or its antigen-binding fragment as described herein include succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (mc-vc-PAB). In some cases, the drug is covalently conjugated to an antibody or its antigen-binding fragment. Covalent conjugation of a drug to an antibody or its antigen-binding fragment may be carried out as described in the Examples section below. For example, a drug may be chemically modified using, for example, glutaraldehyde to generate a carboxylic acid group, which is then activated using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) / N-hydroxysulfosuccinimide (NHS) chemical, and the resulting drug preferentially reacts with (i.e., conjugates) a primary amine on the antibody or its antigen-binding fragment (e.g., on lysine).

[0186] The antibodies, or their antigen-binding fragments, variants, or derivatives described herein, may consist of amino acids linked to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than those encoded by 20 genes. Antibodies may be modified by natural processes such as post-translational processing, or by chemical modification techniques well known in the art. Such modifications are well described in basic texts, more detailed monographs, and numerous research publications. Modifications may occur anywhere on the antibody, including the peptide backbone, amino acid side chains and amino or carboxyl termini, or carbohydrate portions. It will be understood that the same type of modification may be present in several sites of a given antibody to the same or varying degrees. Furthermore, a given antibody may contain many types of modifications. Antibodies may be branched, for example, as a result of ubiquitination, and may be cyclic, with or without branching. Cyclic, branched, and branched cyclic antibodies may arise from natural post-translational processes or be made by synthetic methods.Modifications include transfer-RNA-mediated addition of amino acids to proteins, such as acetylation, acylation, ADP-ribosylation, amidation, covalent bonding of flavins, covalent bonding of heme moieties, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, cysteine ​​formation, pyroglutamic acid formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodization, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfated, arginylation, and ubiquitination, for example, Proteins - Structure And Molecular Properties, TE Creighton, WH Freeman and Company, New York 2nd Ed., (1993); Posttranslational Covalent Modification Of See Proteins, BC Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182 (1990), 626-646; Rattan et al., Ann. NY Acad. Sci. 663 (1992), 48-62).

[0187] Fusion proteins comprising an antibody, or its antigen-binding fragment, variant, or derivative, and a heterologous polypeptide are also provided herein. In some embodiments, the fusion protein essentially comprises or comprises one or more amino acid sequences from any of the VH regions of the antibodies described herein, or one or more amino acid sequences from any of the VL regions of the antibodies described herein, or a fragment or variant thereof, and a polypeptide having a heterologous polypeptide sequence. In other embodiments, the fusion protein for use in the diagnostic and treatment methods disclosed herein essentially comprises or comprises one, two, or three amino acid sequences from any of the VH-CDRs of the antibody, or its fragment, variant, or derivative, or one, two, or three amino acid sequences from any of the VL-CDRs of the antibody, or its fragment, variant, or derivative, and a polypeptide having a heterologous polypeptide sequence. In some embodiments, the fusion protein comprises the amino acid sequence of VH-CDR3 of the antibody, or its fragment, derivative, or variant described herein, and the fusion protein comprises a polypeptide having a heterologous polypeptide sequence that specifically binds to BMPRI / BMPRII. In another embodiment, the fusion protein comprises the amino acid sequence of at least one VH region of an antibody described herein, the amino acid sequence of at least one VL region of an antibody or its fragment, derivative, or variant described herein, and a polypeptide having a heterologous polypeptide sequence. In some embodiments, the VH and VL regions of the fusion protein correspond to a single-source antibody (or scFv or Fab fragment) that specifically binds to BMPRI / BMPRII. In yet another embodiment, the fusion protein for use in the diagnostic and treatment methods disclosed herein comprises one, two, three or more amino acid sequences of any VH CDR of an antibody, and one, two, three or more amino acid sequences of any VL CDR of an antibody or its fragment or variant, and a polypeptide having a heterologous polypeptide sequence.In some embodiments, two, three, four, five, or six or more VH-CDRs or VL-CDRs correspond to single-source antibodies (or scFv or Fab fragments) described herein. Nucleic acid molecules encoding these fusion proteins are also provided herein.

[0188] Exemplary fusion proteins reported in the literature include T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84(1987), 2936-2940; CD4 (Capon et al., Nature 337(1989), 525-531; Traunecker et al., Nature 339(1989), 68-70; Zettmeissl et al., DNA Cell Biol. USA 9(1990), 347-353; and Byrn et al., Nature 344(1990), 667-670); L-selectin (homing receptor) (Watson et al., J. Cell. Biol. 110(1990), 2221-2229; and Watson et al., Nature 349(1991)), 164-167);CD44(Aruffo et al., Cell 61(1990), 1303-1313);CD28 and B7(Linsley et al., J. Exp. Med. 173(1991),721-730);CTLA-4(Lisley et al., J. Exp. Med. 174(1991), 561-569); CD22 (Stamenkovic et al., Cell 66(1991), 1133-1144); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88(1991), 10535-10539; Lesslauer et al., Eur. J. Immunol. 27(1991), 2883-2886; and Peppel et al. al., J. Exp. Med. 174(1991), 1483-1489(1991); and fusions of IgE receptor α (Ridgway and Gorman, J. Cell. Biol. 115(1991), Abstract No. 1448).

[0189] As discussed elsewhere in this specification, the antibodies described herein, or their antigen-binding fragments, variants, or derivatives, may be fused to heterologous polypeptides to extend the in vivo half-life of the polypeptides or used in immunoassays using methods known in the art. For example, in some embodiments, PEG can be conjugated to the antibodies described herein to extend their in vivo half-lives; see, e.g., Leong et al., Cytokine 16(2001), 106-119; Adv. in Drug Deliv. Rev. 54(2002), 531; or Weir et al., Biochem. Soc. Transactions 30(2002), 512.

[0190] Furthermore, antibodies, or their antigen-binding fragments, variants, or derivatives described herein, can be fused to marker sequences, such as peptides, to facilitate their proliferation or detection. In certain embodiments, the marker amino acid sequence is a hexahistidine peptide (HIS), such as the tag provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among many commercially available options. As described in Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86(1989), 821-824, for example, hexahistidine leads to the convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the "HA" tag (Wilson et al., Cell 37(1984), 767) and "flag" tags, which correspond to epitopes derived from influenza hemagglutinin protein.

[0191] The fusion protein can be prepared using methods well known in the art, see, for example, U.S. Patent Nos. 5,116,964 and 5,225,538. The exact site where the fusion is made may be empirically selected to optimize the secretion or binding characteristics of the fusion protein. The DNA encoding the fusion protein is then introduced into a host cell for expression.

[0192] The antibodies described herein may be used in an unconjugated form or conjugated to at least one of various molecules, for example, to improve the therapeutic properties of a molecule, to facilitate target detection, or for imaging or treatment of a patient. The antibodies described herein, or their antigen-binding fragments, variants, or derivatives, may be labeled or conjugated either before or after purification when purification is performed. In particular, the antibodies described herein, or their antigen-binding fragments, variants, or derivatives, may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical formulations, or PEGs.

[0193] Conjugates, which are immunotoxins containing conventional antibodies, are widely described in the art. The toxins may be conjugated to antibodies by conventional coupling techniques, or immunotoxins containing a protein-toxin portion may be produced as fusion proteins. Such immunotoxins can be obtained using the antibodies described herein in the corresponding methods. Descriptions of such immunotoxins are provided by Byers, Seminars Cell. Biol. 2(1991), 59-70 and Fanger, Immunol. Today 12(1991), 51-54.

[0194] Those skilled in the art will understand that conjugates may also be assembled using various techniques, depending on the selected agent to be conjugated. For example, conjugates with biotin are prepared, for instance, by reacting a BMPRI / BMPRII-conjugated polypeptide with an activated biotin ester, such as biotin N-hydroxysuccinimid ester. Similarly, conjugates with fluorescent markers may be prepared in the presence of a coupling agent, such as those listed herein, or by reaction with an isothiocyanate or fluorescein-isothiocyanate. Conjugates of antibodies, or their antigen-binding fragments, variants, or derivatives described herein, are prepared in a similar manner.

[0195] Antibodies described herein, or their antigen-binding fragments, variants, or derivatives, conjugated to diagnostic or therapeutic agents are also provided herein. Antibodies can be used diagnostically to, for example, indicate the presence of BMPRI / BMPRII-related disease, indicate the risk of developing BMPRI / BMPRII-related disease, or monitor the onset or progression of BMPRI / BMPRII-related disease; that is, they can be used as part of a clinical laboratory technique to determine, for example, the effectiveness of a given treatment and / or prophylactic regimen. Detection can be facilitated by coupling the antibody, or its antigen-binding fragment, variant, or derivative, to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals used in various positron emission tomography, and non-radioactive paramagnetic metal ions; see, for example, U.S. Patent No. 4,741,900, for metal ions that can be conjugated to antibodies for diagnostic use. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansilchloride, or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include 125I, 131I, 111In, or 99Tc.

[0196] Antibodies, or their antigen-binding fragments, variants, or derivatives, can also be detectably labeled by coupling them with chemiluminescent compounds. The presence of chemiluminescently tagged antibodies is then determined by detecting the presence of light emitted during the course of the chemical reaction. Particularly useful examples of chemiluminescent labeling compounds include luminol, isoluminol, ceromomatic acridinium esters, imidazole, acridinium salts, and oxalate esters.

[0197] One method for detecting and labeling antibodies, or their antigen-binding fragments, variants, or derivatives, is to conjugate them to an enzyme and use the conjugated product in an enzyme-mediated immunoassay (EIA) (Voller, A., “The Enzyme Linked Immunosorbent Assay (ELISA)” Microbiological Associates Quarterly Publication, Walkersville, Md., Diagnostic Horizons 2(1978), 1-7); Voller et al., J. Clin. Pathol. 31(1978), 507-520; Butler, Meth. Enzymol. 73(1981), 482-523; Maggio, E.(ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla., (1980); Ishikawa, E. et al.,(eds.), Enzyme Immunoassay, Kgaku Shoin, Tokyo(1981). The enzyme bound to the antibody will react with a suitable substrate, preferably a chromogenic substrate, in such a manner that it produces a chemical moiety that can be detected, for example, by spectrophotometric, fluorescence quantitative, or visual means. Enzymes that can be used to detectably label antibodies include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. In addition, detection can be achieved by colorimetric analysis methods that utilize the chromogenic substrate of the enzyme. Detection may also be achieved by visual comparison of the degree of enzymatic reaction of the substrate in comparison with a similarly prepared standard.

[0198] Detection may also be achieved using any of the various other immunoassays. For example, antibodies can be detected through the use of radioimmunoassays (RIAs) by radiolabeling the antibody, or its antigen-binding fragment, variant, or derivative (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, (March, 1986)), which is incorporated herein by reference). Radioisotopes can be detected by means including, but not limited to, gamma counters, scintillation counters, or autoradiography.

[0199] Antibodies, or their antigen-binding fragments, variants, or derivatives, can also be detectably labeled using fluorescent metals such as 152Eu or others in the lanthanide series. These metals can be conjugated to antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0200] Techniques for conjugating various parts of an antibody, or its antigen-binding fragment, variant, or derivative, are well known. For example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. See 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. 62 (1982), 119-158.

[0201] As described above, in some embodiments, a conjugating molecule, such as a conjugating polypeptide, such as a portion of an antibody or its immunospecific fragment that enhances safety or efficacy, can be conjugated. For example, in some embodiments, PEG can be conjugated to the conjugating molecules described herein to extend their in vivo half-lives. Leong et al., Cytokine 16(2001), 106; Adv. in Drug Deliv. Rev. 54(2002), 531; or Weir et al., Biochem. Soc. Transactions 30(2002), 512.

[0202] Bispecific and multispecific antibodies The bispecific and multispecific antibody reagents described herein can act as alternative ligands for assembling the type I and type II receptor complex in this pathway. These reagents initiate the assembly of type I and type II receptors, phosphorylate downstream SMAD proteins, and modulate signaling in a manner similar to that of endogenous ligands. In contrast to endogenous ligands, which can pair with a large number of distinct type I and type II ligands for the most part, the bispecific and multispecific reagents of this application target specific pairs of type I and type II receptors due to the overlapping specificity of the receptors. By targeting specific pairs of receptors, these reagents can induce specific downstream functions of those receptor pairs without resulting in the formation of other receptor pairs that would normally be brought about by endogenous ligands. This selective targeting of receptors allows for the recruitment of activity resulting from the activation of a specific pair of receptors while avoiding the activation of other receptor pairs that could otherwise be activated by a given endogenous ligand. This selective targeting of receptors can enhance the desired activity of a given TGF-beta superfamily ligand while eliminating undesirable effects for therapeutic purposes.

[0203] The bispecific antibodies possessing specificity for various combinations of surface BMP / TGF-beta receptors described herein can be used in a wide range of contexts, for example, therapeutically. Multispecific or bispecific antibodies that recognize type I and type II receptors of the TGF-beta superfamily can induce specific BMP or TGF-beta pathway-specific activation, gene transcription, and cell biology and fate of SMAD effectors, similar to endogenous BMPs, activins, GDFs, and TGF-beta ligands.

[0204] The signaling of bispecific antibodies does not involve the chaotic state of their native ligands, which provides a way to induce specific cellular effects in specific target tissues, regardless of systemic administration. Instead of binding to multiple type II and multiple type I receptors, as is often the case with native ligands, bispecificity targets a single pair of type II and type I receptors. This will limit the biological effects of the signaling and the tissues targeted. Unlike native ligands, which have a half-life in circulation ranging from minutes to hours, bispecific signaling molecules have pharmacokinetics that can be tuned depending on the desired biological effect. Bispecific IgG Fc molecules are generally expected to have a half-life of several weeks, as is the case with therapeutic IgG molecules or other IgG Fc fusion molecules. However, when expressed as a bispecific molecule without Fc, i.e., an F(ab')2 fragment, or a bispecific single-chain Fv(scFv) molecule, it can also be designed to have a shorter half-life on the order of minutes or hours. Bispecific IgG Fc can be directed to specific actions based on ADCC or complement fixation function, depending on the use of the native IgG Fc domain or a mutant IgG Fc domain.

[0205] Unlike natural ligands that possess a heparan sulfate-binding domain that binds to extracellular matrix glycosaminoglycans and thereby causes sequestering from circulating or target cells, engineered bispecific signaling molecules can be systemically administered to target diverse tissues due to their more favorable distribution and pharmacodynamics.

[0206] Mutant protein ligands of the BMP / TGF-beta signaling pathway, also known as mutant proteins, can be designed to exhibit more selective receptor targeting, tissue targeting, or pharmacokinetic and pharmacodynamic properties. While mutant proteins of the BMP / TGF-beta signaling pathway can be generated to enable some of their functions to work selectively, these mutant proteins will likely be immunogenic, and anti-drug antibody responses may not only neutralize the action of their therapeutic agents, reduce or render them ineffective with repeated use or exposure, but may also trigger an autoimmune response of pathogens against endogenous ligands, inhibiting their essential function and causing permanent dysfunction of endogenous physiological functions.

[0207] Manipulated multispecific and bispecific antibody reagents may elicit anti-drug antibody responses; however, as with other therapeutic antibodies, these responses and their corresponding effects can be minimized by sequence optimization to match endogenous antibody idiotypes and peptide sequences, as well as by optimizing protein expression and protein folding, organization, and delivery systems. The inventors anticipate that, as with other therapeutic antibodies, the occurrence of anti-drug antibody responses, if present, will not disrupt therapeutic efficacy for a number of reasons.

[0208] Many endogenous ligands in this pathway spontaneously bind to extracellular matrix proteins via heparan sulfate-binding domains, or to other extracellular matrix proteins that work to sequester these proteins at their potential sites of activity until activation by biochemical changes in those tissues that regulate their activity. These interactions can also limit the ability of administered endogenous ligands to act on target cells. Therefore, these proteins may not be suitable for systemic administration. Bispecific or multispecific antibody reagents targeting this pathway can be administered systemically and dispersed into diverse tissue compartments for therapeutic action. Manipulated bispecific signaling molecules may be administered systemically to target diverse tissues for more favorable distribution and pharmacodynamics. Bispecific or multispecific antibody reagents targeting this pathway may instead undergo targeting using homing domains to concentrate their action on specific tissue compartments.

[0209] As used herein, the term “antibody” refers to an immunoglobulin molecule or its immunologically active portion, i.e., the antigen-binding portion. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments that retain the ability to bind to an antigen. Such fragments can be obtained commercially or by methods known in the art. For example, an F(ab)2 fragment can be produced by treating an antibody with an enzyme such as pepsin, a nonspecific endopeptidase that typically produces one F(ab)2 fragment and numerous small peptides of the Fc portion. The resulting F(ab)2 fragment consists of two disulfide-bonded Fab units. The Fc fragment can be extensively degraded and separated from F(ab)2 by dialysis, gel filtration, or ion-exchange chromatography. An F(ab) fragment can be produced using papain, a nonspecific thiol-endopeptidase that degrades an IgG molecule into three fragments of similar size: two Fab fragments and one Fc fragment, in the presence of a reducing agent. When the Fc fragment is the target, papain is the enzyme of choice to produce 50,000 daltons of Fc fragment and isolate the F(ab) fragment, which can then be removed by affinity purification, for example, using protein A / G. Numerous kits for generating F(ab) fragments are commercially available. In addition, commercially available services for generating antigen-binding fragments, such as Bio Express, West Lebanon, and NH, can be used. Antibodies are at least bispecific (i.e., recognize two different epitopes on either the same or different antigen; see, e.g., Brinkmann and Kontermann, MAbs. 2017 Feb-Mar;9(2): 182-212) or multispecific (i.e., show recognition of more than two epitopes; see, e.g., Egan et al., MAbs. 2017 Jan;9(1): 68-84).

[0210] The antibody can be deimmunized or humanized, fully human, non-human, e.g., mouse, or single-chain antibody. In some embodiments, the antibody may have effector function and be able to immobilize complement. In some embodiments, the antibody may have or may not have reduced ability to bind to the Fc receptor. For example, the antibody may be an isotype or subtype, fragment or other variant that does not support binding to the Fc receptor, and may have, for example, a mutagenic or deficient Fc receptor binding region.

[0211] In preferred embodiments, the antibody is a single-chain antibody. Single-chain antibodies (scFVs) can be manipulated (see, for example, Colcher et al., Ann. NY Acad. Sci. 880:263-80(1999); and Reiter, Clin. Cancer Res. 2:245-52(1996); Ahmad et al., Clin Dev Immunol. 2012;2012:980250; Brinkmann and Kontermann, MAbs. 2017 Feb-Mar;9(2): 182-212). Single-chain antibodies can be dimerized or multimerized to produce polyvalent antibodies with specificity for different epitopes of the same target protein.

[0212] In some embodiments, the bispecific and multispecific reagents described herein each comprise two or more distinct scFVs that bind to different protein targets, each scFv comprising variable regions of heavy (VH) and light (VL) chains linked together by an intrabody mobile peptide linker. This intrabody linker should be long enough for the VH and VL chains to fold and interact correctly and should contain a hydrophilic domain. In some embodiments, the intrabody linker optionally comprises extensions of Gly and Ser residues, accompanied by charged Glu and Lys dispersed to enhance solubility. See, for example, Ahmad et al., Clin Dev Immunol. 2012;2012:980250.

[0213] The scFV is then coupled to a mobile interbody linker of, for example, 5–50, 15–40, or 20–30 amino acids (e.g., at least 5, 10, 15, or 20 amino acids, up to 25, 35, 40, or 50 amino acids, and all ranges including the aforementioned numbers as endpoints). The interbody linkers may include manipulated linkers, e.g., short alanine linkers (Ala3), hydrophilic linkers, glycine-serine-rich linkers, helical linkers, and native linkers derived from various immunoglobulin and non-immunoglobulin molecules. See Brinkmann and Kontermann, MAbs. 2017 Feb-Mar;9(2): 182-212. In some embodiments, a 20-residue (G4S) 4-linker or one or more examples such as GGGGSG (SEQ ID NO: 103); GGGGSGGGS (SEQ ID NO: 104); GSGGGGDGGGGSG (SEQ ID NO: 194); GGGGSGGGGSGDGSS (SEQ ID NO: 195), EGKSSGSGSDSKAS (SEQ ID NO: 105) or EGKSSGSGSESKAS (SEQ ID NO: 106); SGGGGSGGGGSSGSGGGGDGGGGSG (SEQ ID NO: 192); SGGGGSGGGGSSGSGGGGDGGGGSGGT (SEQ ID NO: 107); SGGSGGGGSSGGGGSGGGGSSGGGGDGGGGSG (SEQ ID NO: 193); GSGGGGDSGGGGSGGGGSSGGGGSG (SEQ ID NO: 195); GSGGGGDSGGGGSGGSGGSGGSGGGSGGGGSG (SEQ ID NO: 196); or SGGSGGGGSSGGGGSGGGGSSGGGGDGGGGSGGT (SEQ ID NO: 108). In some embodiments, one of the linkers also includes one or more histidine residues, for example, two, four, six or more histidine residues, e.g., GGHHHHHHHHGG (SEQ ID NO: 198); SGGGGSHHHHHHHHSGGGGS (SEQ ID NO: 199); or SGGGGSGGHHHHHHHHGGSGGGGS (SEQ ID NO: 200). The bispecific scFV can be arranged as VH-VL or VL-VH, and multiple higher-order ones can also be arranged in any order.

[0214] Antigen-binding domains for type I and type II receptors can be generated using methods known in the art. For example, the process may involve obtaining antibody-secreting immune cells (lymphocytes) from the spleen of an animal (e.g., a mouse) already immunized with the antigen of interest (e.g., all or part of a type I or II receptor), either in vivo or in vitro. The antibody-secreting lymphocytes are then indeterminately replicated in myeloma cells or cell cultures, thereby fusing with transformed cells capable of producing immortal, immunoglobulin-secreting cell lines. The resulting fused cells, or hybridomas, are cultured, and the resulting colonies are screened for the production of desired monoclonal antibodies. Colonies producing such antibodies are cloned and grown either in vivo or in vitro to produce large quantities of antibodies. A description of the theoretical basis and practical methodology for fusing such cells is found in Kohler and Milstein, Nature 256:495 (1975), which is incorporated by reference. The sequence of the antigen-binding region of the clone can be determined using known methods.

[0215] Exemplary sequences of human type I or type II receptor proteins are shown in Table A.

[0216] [Table 3]

[0217] Exemplary sequences of CDRs and scFVs are provided herein, for example, in Table 1. Sequences used in the antibody may be at least 80%, 85%, 90%, 95%, 97%, or 99% identical to sequences provided herein, insofar as they retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the level of activity of the reference antibody, for example. The calculation of "identity" between two sequences can be performed as follows: The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second nucleic acid sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). The length of the aligned sequences for comparison purposes is at least 70% (e.g., at least 80%, 90%, or 100%) of the length of the reference sequence. Nucleotides at the corresponding nucleotide positions are then compared. The molecules are identical at a given position when a position in a first sequence is occupied by the same nucleotide at the corresponding position in a second sequence. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that must be introduced for optimal alignment of the two sequences.

[0218] Sequence comparison and determination of percentage identity between two sequences can be achieved using numerical computation algorithms. In some embodiments, percentage identity between two nucleotide sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which is incorporated into the GAP program in the GCG software package (available at gcg.com), using either the Blossum62 matrix, the PAM250 matrix, or the NWSgapdna.CMP matrix. In some embodiments, percentage identity between two amino acid or nucleotide sequences can be determined using the E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) algorithm, which is incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, gap length penalty 12, and gap penalty 4.

[0219] In some embodiments, the bispecific and multispecific reagents described herein may also include, or may not include, one or more antigen-binding domains derived from known anti-type I or anti-type II receptor antibodies, for example, the anti-type I or anti-type II receptor antibodies provided in Table B or C.

[0220] [Table 4]

[0221] [Table 5-1]

[0222] [Table 5-2]

[0223] Bispecific and multispecific antibodies can be expressed, for example, in genetically engineered cells or animals from nucleic acids containing antibody-encoding sequences, and then purified. Therefore, antibody-encoding nucleic acids and vectors, preferably expression vectors, containing the antibody-encoding nucleic acids described herein, are also provided herein. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another bound nucleic acid, and may include plasmids, cosmids, or viral vectors. Vectors may be capable of self-replication and can integrate into host DNA. Viral vectors include, for example, replication-deficient retroviruses, adenoviruses, and adeno-associated viruses.

[0224] The vector may contain a nucleic acid encoding the antibody described herein, in a form suitable for nucleic acid expression in a host cell. Preferably, the recombinant expression vector includes one or more regulatory sequences operably ligated to the nucleic acid sequence to be expressed. The term “regulatory sequence” includes promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals). Regulatory sequences include those that direct the constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and / or inducible sequences. The design of the expression vector may depend on factors such as the selection of host cells to be transformed and the desired level of protein expression. The expression vector of the present invention can be introduced into a host cell to produce a protein or polypeptide comprising a fusion protein or polypeptide encoded by the nucleic acid described herein encoding the antibody described herein.

[0225] Recombinant expression vectors can be designed for the expression of the antibodies described herein in prokaryotic or eukaryotic cells. For example, the polypeptides of the present invention can be expressed in Escherichia coli (E. coli), insect cells (e.g., using a baculovirus expression vector), yeast cells, or mammalian cells. Suitable host cells are further discussed in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA. Alternatively, the recombinant expression vectors can be transcribed and translated in vitro, for example, using a T7 promoter control sequence and T7 polymerase. The antibodies described herein can be purified after expression or translation for use in the methods or compositions described herein using methods known in the art.

[0226] Alternatively, nucleic acids encoding antibodies described herein, or vectors (e.g., viral vectors) for nucleic acid expression, can be delivered to targets requiring them.

[0227] V. Expression of antibody polypeptide Following the manipulation of isolated genetic material to provide the antibodies, or their antigen-binding fragments, variants, or derivatives described herein, the polynucleotide encoding the antibody is typically inserted into an expression vector for introduction into host cells that can be used to produce a desired amount of antibody. Recombinant expression of antibodies, or their fragments, derivatives, or analogs, such as the heavy or light chain of an antibody that binds to a target molecule, is described herein. Once a polynucleotide encoding the antibody molecule or the heavy or light chain of an antibody, or a portion thereof (preferably containing the heavy or light chain variable domain), is obtained, a vector for producing the antibody molecule may be generated by recombinant DNA technology using techniques well known in the art. Thus, a method for preparing a protein by expressing a polynucleotide containing an antibody encoding a nucleotide sequence is described herein. Expression vectors containing the antibody-coding sequence and appropriate transcription and translation regulatory signals can be constructed using methods well known to those skilled in the art. These methods include, for example, in vitro recombinant DNA technology, synthesis technology, and in vivo genetic recombination. Accordingly, replicable vectors are provided herein that include a nucleotide sequence encoding an antibody molecule described herein, or its antigen-binding fragment, or its heavy or light chain, or its heavy or light chain variable domain, operably linked to a promoter. Such vectors may include a nucleotide sequence encoding the constant region of the antibody molecule (see, for example, International Publication No. 86 / 05807 and International Publication No. 89 / 01036; and U.S. Patent No. 5,122,464), and the variable domain of the antibody may be cloned into such vectors for the expression of the entire heavy or light chain.

[0228] The terms “vector” or “expression vector” are used herein to mean a vector used in accordance with the methods described herein as a vehicle for introducing and expressing a desired gene in a host cell. As is known to those skilled in the art, such vectors can be readily selected from the group consisting of plasmids, phages, viruses, and retroviruses. Generally, the vectors described herein will include a selection marker, appropriate restriction enzyme sites to facilitate the cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells. Numerous expression vector systems may be available. For example, one type of vector utilizes a DNA element derived from an animal virus such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV, or MOMLV), or SV40 virus. Others involve the use of polycistronic systems having an internal ribosome binding site. In addition, cells into which the DNA has been integrated into their chromosomes may be selected by introducing one or more markers that allow for the selection of the gene-transfected host cell. The markers may result in prototrophicity to a trophic-dependent host, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. Selectable marker genes can be directly bound to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Further elements may also be required for optimal mRNA synthesis. These elements may include signal sequences, splice signals, and transcription promoters, enhancers, and termination signals.

[0229] In certain embodiments, the cloned variable region gene is inserted into an expression vector along with the heavy and light chain constant region genes discussed above (e.g., human heavy and light chain constant region genes). Any expression vector capable of inducing expression in eukaryotic cells may be used. Examples of suitable vectors include, but are not limited to, plasmids pcDNA3, pHCMV / Zeo, pCR3.1, pEF1 / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAX1, and pZeoSV2 (available from Invitrogen, San Diego, CA), as well as plasmid pCI (available from Promega, Madison, WI). In general, screening a large number of transformed cells for those that adequately express high levels of immunoglobulin heavy and light chains is a routine experiment that can be performed, for example, by robot-assisted systems. The vector system is also taught in U.S. Patent No. 5,736,137 and U.S. Patent No. 5,658,570, which are incorporated herein by reference in their entirety. This system yields high expression levels, e.g., >30 pg / cell / day. Other exemplary vector systems are disclosed, for example, in U.S. Patent No. 6,413,777.

[0230] In other embodiments, the antibodies, or their antigen-binding fragments, variants, or derivatives described herein, may be expressed using a polycistronic construct, such as that disclosed in U.S. Patent Application Publication No. 2003-0157641, which is incorporated herein by reference. In these expression systems, the desired multiple gene products, such as the heavy and light chains of the antibody, may be generated from a single polycistronic construct. These systems advantageously utilize intra-sequence ribosome entry sites (IRESs) to yield relatively high levels of antibody. Equivalent IRES sequences are disclosed in U.S. Patent No. 6,193,980, which is also incorporated herein by reference. Those skilled in the art will understand that such expression systems can be used to efficiently generate the entire range of antibodies disclosed in this application.

[0231] More generally, once a vector or DNA sequence encoding a monomeric subunit of an antibody is prepared, the expression vector can be introduced into a suitable host cell. The introduction of a plasmid into a host cell can be achieved by various techniques well known to those skilled in the art. These include, but are not limited to, gene transfer, including, for example, lipotransfection using Fugene® or lipofectamine, protoplast fusion, calcium phosphate precipitation, cell fusion using enveloped DNA, microinjection, and infection with an intact virus. Typically, plasmid introduction into a host is via a standard calcium phosphate coprecipitation method. Host cells with the expression construct are grown under conditions suitable for light and heavy chain production and assayed for heavy chain and / or light chain protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), or fluorescence-labeled cell preparative analysis (FACS), immunohistochemistry, etc.

[0232] The expression vector is transferred to a host cell by the prior art, and the gene-transfected cell is then cultured by the prior art to produce an antibody for use in the method described herein. Thus, host cells containing polynucleotides encoding the antibody or its antigen-binding fragment, or its heavy or light chain, operably linked to a heterologous promoter, are also provided herein. In certain embodiments, vectors encoding both the heavy and light chains may be co-expressed in the host cell for the expression of a double-stranded antibody, as described in detail below, for the expression of an entire immunoglobulin molecule.

[0233] Host cells may be simultaneously transfected using two expression vectors described herein: a first vector encoding a heavy chain polypeptide and a second vector encoding a light chain polypeptide. The two vectors may contain identical selectable markers to enable equal expression of the heavy chain and light chain polypeptides. Alternatively, a single vector encoding both the heavy chain and light chain polypeptides may be used. In such circumstances, the light chain may be advantageously placed before the heavy chain to avoid excessive, non-toxic heavy chain expression (see Proudfoot, Nature 322(1986), 52; Kohler, Proc. Natl. Acad. Sci. USA 77(1980), 2197). The heavy chain and light chain coding sequences may include cDNA or genomic DNA.

[0234] As used herein, “host cell” refers to a cell having a vector, constructed using recombinant DNA technology, and encoding at least one heterologous gene. In describing the process of isolating antibodies from recombinant hosts, the terms “cell” and “cell culture” are used interchangeably to indicate the source of the antibody unless otherwise clearly specified. In other words, the recovery of polypeptides from “cells” may mean from either a spin-down of whole cells or from a cell culture containing both culture medium and suspended cells.

[0235] Various host expression vector systems may be used to express antibody molecules for use in the methods described herein. Such host expression systems represent a vehicle from which the desired coding sequence can be generated and subsequently purified, but also represent cells that, when transformed or transgenerated with an appropriate nucleotide coding sequence, can express the antibody molecules described herein in insights. These include bacteria (e.g., Escherichia coli, Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody-coding sequences. This includes, but is not limited to, microorganisms such as: yeast transformed with recombinant yeast expression vectors containing antibody-coding sequences (e.g., Saccharomyces, Pichia); insect cell systems infected with recombinant virus expression vectors containing antibody-coding sequences (e.g., baculovirus); plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors containing antibody-coding sequences (e.g., Ti plasmid); or mammalian cell systems having recombinant expression constructs containing promoters derived from mammalian cell genomes (e.g., metallothionein promoter) or promoters derived from mammalian viruses (e.g., late adenovirus promoter; vaccinia virus 7.5K promoter) (e.g., COS, CHO, NSO, BLK, 293, 3T3 cells). In some embodiments, bacterial cells, such as Escherichia coli, and more preferably eukaryotic cells, are used for the expression of the recombinant antibody molecule as a whole.For example, mammalian cells such as Chinese hamster ovary (CHO) cells, combined with vectors such as major intermediate early gene promoter elements derived from human cytomegalovirus, are an efficient antibody expression system. See, for example, Foecking et al., Gene 45 (1986), 101; and Cockett et al., Bio / Technology 8 (1990), 2.

[0236] Host cell lines used for protein expression are often of mammalian origin, and those skilled in the art demonstrate the ability to determine the specific host cell line best suited to the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, CHO (Chinese hamster ovary), DG44 and DUXB11 (Chinese hamster ovary cell line, DHFR-negative), HELA (human cervical cancer), CVI (monkey kidney cell line), COS (CVI derivative using the SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, WI38, R1610 (Chinese hamster fibroblasts), BALBC / 3T3 (mouse fibroblasts), HAK (hamster kidney cell line), SP2 / O (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), and 293 (human kidney). In certain embodiments, the host cell line is CHO or 293 cells. Host cell lines are typically available from the American Tissue Culture Collection, a commercially available service, or from published literature.

[0237] In addition, a host cell line may be selected that regulates the expression of the inserted sequence or modifies and processes the gene product in a desired specific manner. Such modification (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for protein function. Different host cells have characteristic and specific mechanisms for post-translational processing as well as modification of proteins and gene products. By selecting an appropriate cell line or host system, the correct modification and processing of the expressed foreign protein can be ensured. For this purpose, eukaryotic host cells with cellular mechanisms for correct processing of major transcripts, glycosylation of gene products, and phosphorylation may be used.

[0238] For the long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express antibody molecules may be constructed by manipulation. Rather than using expression vectors containing viral replication origins, host cells can be transformed using DNA regulated by appropriate expression regulatory elements (e.g., promoters, enhancers, sequences, transcriptional terminators, polyadenylation sites, etc.) and selectable markers. After the introduction of the foreign DNA, the manipulated cells may be grown in enriched medium for 1-2 days, and then switched to selective medium. Selectable markers in recombinant plasmids confer resistance to selection, allowing cells to stably integrate the plasmid into their chromosomes, grow to form foci, and subsequently be cloned and expanded into cell lines. This method may be advantageously used to construct cell lines that stably express antibody molecules by manipulation.

[0239] A number of selection systems may be used, including, but not limited to, the genes of herpes simplex virus thymidine kinase (Wigler et al., Cell 11(1977), 223), hypoxanthine guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48(1992), 202), and adenine phosphoribosyltransferase (Lowy et al., Cell 22(1980), 817), which can be utilized in tk-, hgprt-, or aprt- cells, respectively. Furthermore, the following genes confer resistance to anti-metabolites: dhfr (Wigler et al., Natl. Acad. Sci. USA 77(1980), 357; O'Hare et al., Proc. Natl. Acad. Sci. USA 78(1981), 1527), which confers resistance to methotrexate; gpt (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78(1981), 2072), which confers resistance to mycophenolate; and neo (Goldspiel et al., Clinical Pharmacy 12(1993), 488-505; Wu and Wu, Biotherapy 3(1991), 87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxicol.) 32(1993), 573-596; Mulligan, Science 260(1993), 926-932; and Morgan and Anderson, Ann. Rev. Biochem. 62(1993), 191-217; TIB TECH 11(1993), 155-215; and it can be used as a selective substrate for hygro (Santerre et al., Gene 30(1984), 147) to confer resistance to hygromycin.Methods of recombinant DNA technology that can be used that are generally known in the art are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); and Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), the entirety of which is incorporated herein by reference.

[0240] The expression level of antibody molecules can be increased by vector amplification; for an overview, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Academic Press, New York, Vol. 3 (1987). When a marker in an antibody-expressing vector system is amplified, increasing the level of the inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the antibody gene, antibody production will also be increased; see Crouse et al., Mol. Cell. Biol. 3 (1983), 257.

[0241] In vitro production allows for scale-up to obtain large quantities of the desired polypeptide. Mammalian cell culture techniques under tissue culture conditions are known in the art and include, for example, homogeneous suspension culture in an airlift reactor or a continuous stirring reactor, or immobilized or captured cell culture on, for example, hollow fibers, microcapsules, agarose microbeads, or ceramic cartridges. If necessary and / or desired, the polypeptide solution can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose, or (immuno-)affinity chromatography, for example, after preferential biosynthesis of the synthetic hinge region polypeptide or before or after the HIC chromatography step described herein.

[0242] The genes encoding antibodies, or their antigen-binding fragments, variants, or derivatives, described herein can also be expressed in bacteria or non-mammalian cells such as insects, yeasts, or plant cells. Bacteria that readily take up nucleic acids include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. When expressed in bacteria, heterologous polypeptides are typically found as part of inclusion bodies, which will be further understood. Heterologous polypeptides must be isolated, purified, and then assembled into functional molecules. If a tetravalent antibody is desired, the subunits will then self-assemble into a tetravalent antibody; see, for example, International Publication No. 02 / 096948.

[0243] In bacterial systems, numerous expression vectors may be advantageously selected depending on the intended use for the antibody molecule being expressed. For example, when large quantities of such proteins are to be produced, a vector directing high levels of expression of a readily purifiable fusion protein product may be desirable for the production of a pharmaceutical composition of the antibody molecule. Such vectors include, but are not limited to, the Escherichia coli (E. coli) expression vector pUR278 (Ruther et al., EMBO J. 2 (1983), 1791); the pIN vector (Inouye & Inouye, Nucleic Acids Res. 13 (1985), 3101-3109; Van Heeke & Schuster, J. Biol. Chem. 24 (1989), 5503-5509), in which the antibody-coding sequence can be individually ligated into the vector in-frame with the lacZ coding region so that a fusion protein is produced. pGEX vectors may also be used to express exogenous polypeptides as fusion proteins with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption and binding of glutathione-agarose beads to a matrix, followed by elution in the presence of free glutathione. pGEX vectors are designed to include a thrombin or factor Xa protease cleavage site so that the cloned target gene product can be released from the GST portion.

[0244] In addition to prokaryotes, eukaryotic microorganisms may also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms, but many other strains, such as Pichia pastoris, are also commonly available. For expression in the genus Saccharomyces, plasmid YRp7, e.g., (Stinchcomb et al., Nature 282(1979), 39; Kingsman et al., Gene 7(1979), 141; Tschemper et al., Gene 10(1980), 157) is commonly used. This plasmid already contains the TRP1 gene, which provides a selection marker for yeast mutants lacking the ability to grow in tryptophan, e.g., ATCC number 44076 or PEP4-1 (Jones, Genetics 85(1977), 12). Subsequently, the presence of trpl damage as a characteristic of the yeast host cell genome provides an environment effective for detecting transformation by growth in the absence of tryptophan.

[0245] In insect systems, the polyhedron virus (AcNPV) is typically used as a vector for expressing exogenous genes. The virus grows in Spodoptera frugiperda cells. Antibody-encoding sequences may be individually cloned into non-essential regions of the virus (e.g., polyhedrin genes) and placed under the regulation of an AcNPV promoter (e.g., a polyhedrin promoter).

[0246] When the antibodies described herein are recombinantly expressed, the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms described herein can be purified by following standard methods of the art, including, for example, chromatography (e.g., ion exchange, affinity, particularly affinity for specific antigens after protein A, and size classification column chromatography), centrifugation, differential solubility, for example, by ammonium sulfate precipitation, or by any other standard technique for protein purification, see, for example, Scopes, “Protein Purification”, Springer Verlag, NY (1982). Alternatively, another method for increasing the affinity of the antibodies described herein is disclosed in U.S. Patent Application Publication No. 2002-0123057.

[0247] VI.How to use This composition and method can be used in a variety of ways, including for research and therapeutic purposes.

[0248] For example, heterotopic ossification (HO), the formation of ectopic endochondral bone in skeletal muscle and soft tissue, is a significant cause of pathological conditions resulting from joint immobility and pain. The exact mechanisms involved in HO are unknown, but its association with trauma, inflammation, and biomechanical stress suggests impaired injury repair and homeostasis processes. We investigated the underlying mechanisms of a monogenic cause of HO in fibrodysplasia ossificans progressive (FOP), which is caused by activating mutations in the bone morphogenetic protein (BMP) type I receptor ALK2 (whereas trauma-induced HO appears to be regulated by ALK2, ALK3, and potentially ALK6). While FOP and acquired forms of HO share common mechanisms of improper BMP signaling, the mode in which BMP signaling is interpreted as regulating bone formation versus tissue regeneration remains incomplete.

[0249] BMP9 is a multifunctional ligand that can induce vascular endothelial quiescence and is protective against pulmonary vascular diseases in animal models of pulmonary hypertension. However, in some situations, treatment with animal BMP9 can lead to heterotopic ossification or the undesired formation of heterotopic bone in soft tissue at the injection site. BMP9 associates with type II receptors BMPRII, ACTRIIA, and ACTRIIB and with type I receptors ALK1 and ALK2. By associating with BMPR2 and ALK1, bispecific antibody reagents can induce the vascular endothelial quiescence effect of BMP9 resulting from the activation of either of these type II receptors using ALK2 in cells with osteogenic capacity, such as tissue resident fibroadipogenic progenitor cells, without causing heterotopic ossification resulting from the activation of ALK2 in these cells, and potentially without causing liver necrosis resulting from the activation of either of the type II receptors using ALK2 in the liver. Functional specificity is expressed in all tissues, particularly on mesenchymal stem cells involved in heterotopic ossification, and is controlled by targeting ALK1, which is limited to endothelium and associated with the endothelial BMP9 signaling function, in contrast to ALK2, which is associated with the signaling of highly osteogenic molecules such as BMP6 and BMP7.

[0250] Thus, exemplary bispecific antibodies are provided that recognize BMPR2 and ALK1 and recapitulate the signaling of BMP9 for the treatment of vascular conditions including pulmonary arterial hypertension and hereditary hemorrhagic telangiectasia (HHT) syndrome. Other examples are provided below in Table D.

[0251]

Table 6

[0252] Thus, for example, by avoiding the mobilization of ALK2 signaling, which is also associated with endogenous BMP9, without the ectopic ossification effect of BMP9 resulting from ALK2 signaling, and avoiding undesirable effects on liver necrosis and regeneration, to enhance the defective BMP9 signaling in this disease, a therapeutic molecule comprising an anti-BMPRII / anti-ALK1 bispecific antibody can be used herein for the treatment of pulmonary arterial hypertension via the anti-BMPRII / anti-ALK1 bispecific antibody.

[0253] The methods described herein also include the treatment of hereditary hemorrhagic telangiectasia via an anti-BMPRII / anti-ALK1 bispecific antibody to enhance defective BMP9 signaling in this disease; the treatment of acute respiratory distress syndrome (ARDS), acute lung injury (ALI) or other pulmonary vascular leakage syndromes via an anti-BMPRII / anti-ALK1 bispecific antibody to reproduce defective BMP9 signaling; the treatment of various other vascular disease states where endothelial dysfunction may be receptive to modulation by an anti-BMPRII / anti-ALK1 bispecific antibody to reproduce defective BMP9 signaling; and the treatment of liver fibrosis via an anti-BMPRII / anti-ALK1 bispecific antibody to reproduce defective BMP9 signaling in the liver.

[0254] The methods may also include the treatment of renal fibrosis via an anti-BMPRII / anti-ALK3, anti-BMPRII / anti-ALK2, anti-ACTRIIA / anti-ALK3, or anti-ACTRIIA / anti-ALK2 bispecific antibody. Identification of the ideal targeting molecule will likely depend on the identification of signaling molecules with mostly antifibrotic effects including minimal ectopic ossification effects.

[0255] The methods may also include the treatment of fractures, repair of fracture non-unions, or induction of bone fusion using an anti-BMPR2 / anti-ALK3 bispecific antibody to reproduce BMP2, BMP4, or BMP6 signaling, which promotes osteoblast differentiation and bone mineralization, thereby promoting endochondral bone formation.

[0256] The methods may also include treating fractures, repairing fracture pseudarthrosis, or inducing bone fusion using anti-BMPR2 or anti-ACTRIIA, along with anti-ALK2 bispecific antibodies to promote chondrocyte differentiation and thereby replicate BMP6, BMP7, or BMP8 signaling to promote endochondral ossogenesis.

[0257] The methods may also include ex vivo or in vitro induction of osteogenic or chondrogenic differentiation using anti-BMPR2 / anti-ALK3 or anti-BMPR2 / anti-ALK2 bispecific antibodies for the engineering of cartilage or bone tissue.

[0258] Therapeutic molecules include anti-BMPR2 / anti-ALK3, anti-BMPR2 / anti-ALK2, anti-ACTRIIA / anti-ALK3, or anti-ACTRIIA / anti-ALK2 bispecific antibodies that can be used to induce bone formation ex vivo via activation of BMPR2 and ALK3 (or ALK2), or via activation of ACTRIIA and ALK2 (or ALK3), for example, post-traumatic bone treatment engineering, reconstructive surgery, or orthopedic, spinal, or neurosurgical procedures. Therapeutic molecules including anti-BMPR2 / anti-ALK3 or anti-ACTRIIA / anti-ALK2 bispecific antibodies for inducing hepcidin expression in the liver by stimulating the activation of BMPR2 and ALK3, or ACTRIIA and ALK2, in the liver, and for reducing iron overload in transfusion-dependent anemia, beta-thalassemia, and hemochromatosis-like conditions are also provided herein.

[0259] Exemplary therapeutic molecules containing anti-ACTRIIB / anti-ALK7 bispecific antibodies can be used to induce adipogenesis via activation of ACTRIIB and ALK7 for reconstructive and aesthetic surgical applications. These agents can be used for the production of biological implants from patient-derived cells or human progenitor cells for plastic surgery, orthopedic and reconstructive surgery applications in Insights or ExVivo.

[0260] Therapeutic molecules containing anti-BMPR2 / anti-ALK2 or anti-ACTRIIA / anti-ALK2, targeting BMPR2 or ACTRIIA together with ALK2, can improve or correct metabolic syndrome, obesity, or diabetes and related disorders by promoting brown adipose tissue formation and improving energy utilization.

[0261] Therapeutic molecules containing anti-BMPR2 / anti-ALK6 or anti-ACTRIIA / anti-ALK6 bispecific antibodies can be used, for example, to induce tendinogenesis via activation of BMPR2 or ACTRIIA and ALK6 by stimulating the tissue-specific actions of GDF5, GDF6, and GDF7 in tendon progenitor tissue or mesenchymal stem cells with tendon potential. These agents can be used for orthopedic and reconstructive surgery applications in insights or ex vivo for the production of biological implants from patient-derived cells or human progenitor cells.

[0262] Therapeutic molecules containing anti-TGFBRII / anti-ALK5, anti-TGFBRII / anti-ALK4, anti-ACTRIIA / anti-ALK5 or anti-ACTRIIA / anti-ALK4, anti-ACTRIIB / anti-ALK5 or anti-ACTRIIB / anti-ALK4 bispecific antibodies can be used, for example, to induce growth arrest of tumor cells by activation of TGFBRII and ALK5, or by activation of TGFBRII and ALK4, or alternatively, by activation of ACTRIIA or ACTRIIB together with ALK4 or ALK5.

[0263] Therapeutic molecules containing anti-TGFBRII / anti-ALK5 and anti-TGFBRII / anti-ALK4 bispecific antibodies can be used, for example, to induce fibrotic tissue formation for reconstructive or cosmetic surgery applications via activation of TGFBRII and ALK5, or via activation of TGFBRII and ALK4, or for the treatment of Marfan disease, Loeys-Dietz syndrome, and other aortic disorders resulting from dysregulated or deficient TGFBRII signaling via activation of TGFBRII and ALK5, or via activation of TGFBRII and ALK4.

[0264] Therapeutic molecules containing anti-ACTRIIA / anti-ALK5 or anti-ACTRIIA / anti-ALK4 bispecific antibodies can be used to target ACTRIIA and ALK4, or ALK5, to inhibit pathological hypertrophy, such as that seen in hypertrophic cardiomyopathy or hypertensive heart disease.

[0265] Other receptor combinations can be targeted via bispecific antibodies to induce novel physiological effects in tissues and cells. For example, anti-TGFBR2 / anti-ALK3; anti-BMPR2 / anti-ALK4; anti-BMPR2 / anti-ALK5 can induce physiology (osteogenesis, anti-muscleogenesis, or fibroogenesis, respectively, stimulating BMP2 / 4 via ALK3, GDF8 / 11 via ALK4, and TGFb1 / 3 via ALK5) in highly restricted subsets of tissues that are directed by type I receptors but express this specific complement of type II and type I receptors. Alternatively, these novel or non-native signaling combinations may induce unique biologies not seen with endogenous ligands, which may be therapeutically useful. Such biologies may include chondrogenesis isolated from calcification, or calcification isolated from chondrogenesis.

[0266] In any of the bispecific antibodies or applications described above, anti-AMHRII, anti-ACTRIIA, or anti-ACTRIIB may be interchangeable with anti-BMPRII in combination with a predetermined anti-type I receptor antibody to achieve similar, more potent, or more tissue-selective action.

[0267] When used in this context, "to treat" means to restore at least one symptom of the disorder. Administration of a therapeutically effective dose of any compound described herein for the treatment of a condition will result in improvement of at least one symptom or clinical parameter.

[0268] VII. Pharmaceutical Compositions and Methods of Administration The methods described herein include the use of a pharmaceutical composition comprising a bispecific antibody described herein as an active ingredient.

[0269] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption retarders that are compatible with pharmaceutical administration. Supplementary active compounds may also be incorporated into the composition.

[0270] Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral administration (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.

[0271] Methods for formulating suitable pharmaceutical compositions are known in the art; see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, a solution or suspension used for parenteral, intradermal, or subcutaneous application may contain the following components: a sterile diluent such as water for injection, physiological saline solution, non-volatile oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvent; an antimicrobial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate; and an agent for adjusting osmotic pressure such as sodium chloride or glucose. The pH can be adjusted using an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0272] Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (if water-soluble) or dispersions or sterile powders for the immediate preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringe applicability exists. It must be stable under manufacturing and storage conditions and must be protected against microbial contamination such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or suitable mixtures thereof. Correct fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, mannitol, sorbitol, and polyalcohols such as sodium chloride. The absorption of the injectable composition can be extended by including absorption-delaying agents, such as aluminum monostearate and gelatin in the composition.

[0273] A sterile injectable solution can be prepared by incorporating the required amount of the active compound in a suitable solvent with one or a combination of the components listed above, and then, if necessary, by sterilized filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other components of the listed above, as required. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and lyophilization, from which powders of the active ingredient and any further desired components are obtained from the solution that has already been sterile filtered.

[0274] Oral compositions generally include inert diluents and edible carriers. For purposes of oral therapeutic administration, the active compounds can be incorporated with excipients and used in the form of tablets, troches, or capsules, such as gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, etc. can contain the following ingredients or compounds of similar nature: binders such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose, disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0275] For administration by inhalation, the compounds can be delivered in the form of a compressed container or dispenser containing a suitable high-pressure gas, such as a gas like carbon dioxide gas, or an aerosol spray from a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.

[0276] Systemic administration of the therapeutic compounds described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, suitable penetration enhancers for the barrier to be permeated are used in the formulation. Such penetration enhancers are generally known in the art and include, for example, for transmucosal administration, surfactants, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, plasters, gels, or creams generally known in the art.

[0277] The pharmaceutical composition may also be prepared in the form of suppositories (for example, with conventional suppository bases such as cocoa butter and other glycerides) or retained enemas for rectal delivery.

[0278] Therapeutic compounds that are nucleic acids or contain nucleic acids can be administered by any method suitable for the administration of nucleic acid agents, such as DNA vaccines. These methods include needle-free methods such as gene guns, bioinjectors, and skin patches, as well as microparticle DNA vaccine technology disclosed in U.S. Patent No. 6,194,389 and needle-free mammalian percutaneous vaccines, including vaccines in powder form disclosed in U.S. Patent No. 6,168,587. In addition, intranasal delivery is possible, as specifically described above in Hamajima et al., Clin. Immunol. Immunopathol., 88(2), 205-10 (1998). Liposomes (e.g., described in U.S. Patent No. 6,472,375) and microencapsulation can also be used. Biodegradable, targetable microparticle delivery systems can also be used (e.g., described in U.S. Patent No. 6,471,996).

[0279] In one embodiment, the therapeutic compound is prepared using a carrier that protects the therapeutic compound from rapid removal from the body, such as a sustained-release formulation, including implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations can be prepared using standard techniques or are commercially available, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes targeted to select cells using monoclonal antibodies against cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0280] The pharmaceutical composition may be contained in a container, pack, or dispenser, along with instructions for administration. [Examples]

[0281] The present invention is further described in the following embodiments, which do not limit the scope of the invention as set forth in the claims.

[0282] material and method The following materials and methods were used in the following examples.

[0283] Cell culture and Western blot analysis Pulmonary artery endothelial cells (PAECs, patient number 593089, Lonza) were seeded in complete medium (Lonza CC-3156 and CC-4178) at a rate of 400,000 cells per well in 12-well plates (Falcon® polystyrene microplates, Corning 353043). After overnight incubation at 37°C and 5.0% CO2, the cells were washed with PBS (GIBCO 14190-144), given fresh medium, and incubated for 24 hours. The PAECs were rinsed twice with PBS and starved for 24 hours in basal medium containing 0.1% FBS (GIBCO A3160402). Selected wells were pre-incubated for 60 minutes with 10 ng / mL each of two most promising biotinylated scFv combinations (ALK1.8 and BMPR2.12), followed by 1 hour pre-incubation with 1 ng / mL of rhBMP9 (R&D 3209BP-CF), 1 mg / mL of streptavidin (ThermoFisher 21122), and 10 ng / mL of monoclonal BMP9 neutralizing antibody (R&D MAB3209) in various concurrent treatments. The wells were washed with PBS and collected in 80 mL of 1× NuPAGE LDS sample buffer (ThermoFisher NP0007). Samples were incubated at 100°C for 5 minutes, then separated by electrophoresis. Total Smad1 (CST 6944) and GAPDH (Thermo Fisher MA5-15738-HRP) were used as housekeeping markers, and the samples were analyzed by Western blotting using SMAD1 / 3 (Abcam 52903), SMAD1 / 5 / 8 (Cell Signaling, Technology, CST 9516), and SMAD2 (CST 3108), which are specific antibodies that recognize phosphorylation forms. Western blots were detected using SuperSignal® West Femto highest sensitivity substrate (Thermo Fisher 34096) and the Kodak Carestream in vivo MS-FX Pro multispectral imaging system.

[0284] Pulmonary capillary endothelial cells (PMVECs) from wild-type and Acvrl1 KO mice were seeded in complete medium (Lonza CC-3156 and CC-4178) at a rate of 400,000 cells per well in 12-well plates (Falcon® polystyrene microplates, Corning 353043). After incubation overnight at 37°C and 5.0% CO2, the cells were washed with PBS (GIBCO 14190-144), given fresh medium, and incubated for 24 hours. The PMVECs were rinsed twice with PBS and starved for 24 hours in basal medium containing 0.1% FBS (GIBCO A3160402). Selected wells were pre-incubated for 60 minutes with 10 ng / mL each of the two most promising biotinylated scFv combinations (ALK1.8 and BMPR2.12), followed by 1 hour of pre-incubation with various concurrent treatments: 1 ng / mL of rhBMP9 (R&D 3209BP-CF), 1 ug / mL of streptavidin (ThermoFisher 21122), and 10 ng / mL of monoclonal BMP9 neutralizing antibody (R&D MAB3209). The wells were washed with PBS and collected in 80 mL of 1× NuPAGE LDS sample buffer (ThermoFisher NP0007). Samples were incubated at 100°C for 5 minutes, then separated by electrophoresis. Total SMAD1 (CST 6944) and GAPDH (Thermo Fisher MA5-15738-HRP) were used as housekeeping markers, and the samples were analyzed by Western blotting using SMAD1 / 3 (Abcam 52903), SMAD1 / 5 / 8 (CST 9516), and SMAD2 (CST 3108), which are specific antibodies that recognize phosphorylation forms. Western blots were detected using SuperSignal® West Femto highest sensitivity substrate (Thermo Fisher 34096) and the Kodak Carestream in vivo MS-FX Pro multispectral imaging system.

[0285] Biotinylation methods The most promising scFv clones were stimulated with streptavidin and biotinylated to crosslink the receptor. The samples were dialyzed using a 7 kDa cutoff Zeba® spin desalting column (ThermoFisher 89889) and then conjugated using a DSB-X® biotin protein labeling kit (ThermoFisher D20655).

[0286] The effectiveness of the biotinylation method was demonstrated by Western blotting using streptavidin-HRP (ThermoFisher 21130). Conservation of binding affinity and specificity was shown by biolayer interferometry (BLI, Octet Red) analysis to confirm the ability of the biotinylated scFv protein to bind to immobilized ALK1 and BMPR2 receptors in a specific manner.

[0287] Incell Western Telomerase-immobilized capillary endothelial (TIME) cell cultures were seeded in complete medium (ATCC 100-030 and 100-041) in high-binding 96-well plates (Corning 3340), grown to confluence, and serum depleted for 24 hours. TIME cells were treated with various concurrent treatments using rhBMP9 (20 pM), scFv protein (500 pg / mL), and streptavidin (1 mg / mL). Plates were washed twice with PBS, fixed with cold methanol (Fisher 412), washed, permeabilized, washed, and blocked with 2% BSA (Fisher BP1600) in TBS (ThermoFisher J75892). A primary antibody specific to p-SMAD1 / 5 (CST 9516) or p-SMAD2 (CST 8828) was added (1:1000 dilution), followed by a secondary antibody (HRP anti-rabbit IgG, CST 1:10000). After washing, the assay was developed using BioFx UltraSensitive chemiluminescent substrate (Surmodics) and eluted on a SpectraMax plate luminometer with an integration time of 0.25 seconds.

[0288] Luciferase assay Bovine aortic endothelial cells (BAEC, Sigma-Aldrich B304-05) were seeded in complete medium (Lonza, CC-3156 and CC-4176) on 96-well plates (Corning 3340) at a cell concentration of 31,000 cells per well. The following day, the cells were transfected using 60 ng / mL PolyJet (SignaGen, SL100688) and 20 ng / mL BRE-LUC plasmid per well, as described in the gene transduction reagent protocol. After 16 hours, the cells were washed twice with PBS and given serum-reduced medium for 24 hours of recovery. The following day, the BAECs were washed with PBS, starved for 6 hours (FBS 0.1%), treated with 20 pM rhBMP9, 20 nM ALK1-Fc (R&D 370AL), and 20 nM different scFv, and then incubated overnight at 37°C in 5.0% CO2.

[0289] The following day, the inventors tested cell viability using the MTS assay (Promega G3582) and performed a luciferase activity assay as described in the Promega kit (No. 1500). The plates were read on a SpectraMax plate luminometer using an integration of 0.25 seconds.

[0290] Octet Red To characterize the biochemical interactions of the scFv clones with recombinant human ALK1 and BMPR2 receptors, the inventors used the Biolayer Interferometry method (Octet Red 384, ForteBio). Receptors expressed as IgG Fc fusion proteins (i.e., ALK1-Fc, and BMPR2-Fc) were immobilized at a concentration of 10 μg / mL on an anti-human IgG Fc capture sensor (ForteBio, cat. no. 18-0015) in an equilibration buffer of 20 mM HEPES (GIBCO cat. no. 15630), 0.05% Tween-20 (Promega cat. no. H5151), and 25 mM imidazole (SIGMA cat. no. I0250) in PBS. All clones were tested at different concentrations in the equilibration buffer: 50 nM, 100 nM, 200 nM, and 400 nM. The ForteBio program methodology was set as follows: baseline equilibration 60 seconds, load time 60 seconds, wash time 120 seconds, bind time 300 seconds, and dissociation time 600 seconds. Binding kinetics were analyzed using ForteBio data analysis software.

[0291] [Example 1] Single-chain Fv (scFv) Abs of the extracellular domains of BMP / TGFβ receptors can regulate signal transduction and form functional ligands. The inventors performed screening to identify a panel of human scFv Abs that bind to the extracellular domains of ALK1 and BMPR2, which are type I and type II receptors that form the signal transduction complex for the endothelial-specific ligand BMP9. 6、7 . Human germline V H and V L Approximately 10 single-chain variable region (scFv) Abs derived from the genes were expressed 12A combinatorial diversity library of individual phage particles was screened for phage clones that bind to recombinant human ALK1-Fc or BMPR2-Fc extracellular domain-IgG Fc fusion proteins but not to IgG Fc itself, using sequential affinity purification of immobilized recombinant ALK1 or BMPR2 proteins and depletion of nonspecific clones, thereby revealing several clones of interest. Three rounds of enrichment yielded a panel of scFvs that bind to ALK1 or BMPR2 but not to both, and possess unique sequences. These phage sequences were subcloned into scFV expression vectors for scale-up and purification in Escherichia coli (E. coli). For each target, several scFv fragments were evaluated for moderate to high affinity (K) to their intended target based on Octet Red biolayer interferometry (BLI, Fortebio). D The CDR sequences were approximately 20-200 nM and maintained this affinity and specificity even after biotinylation with a low substitution ratio (average 1.5-2 sites per molecule). Binding to one receptor was maintained, but binding to the other was not, and no binding was observed to the control ACTRIIA-Fc protein. The CDR sequences were predicted using the PARATOME online tool (Kunik et al., PLoS Comput Biol 8(2): e1002388(2012); Kunik et al., Nucleic Acids Res. 2012 Jul;40(Web Server issue): W521-4(2012)). Therefore, several scFv candidates that bound to each antigen were selected, their activity was confirmed by ELISA, and their sequences were cloned into expression vectors and expressed as single-chain Fv proteins.

[0292] Screening of binding efficiency of single-chain variable fragment-positive clones to ALK1 and BMPR2 using biolayer interferometry (BLI). The affinity of scFv clones to human BMPR2 and ALK1 receptors was determined using biolayer interferometry. The binding dynamics of scFv clones ALK1.3 (Figure 1A) and ALK1.8 (Figure 1B) to immobilized ALK1, as well as the binding dynamics of BMPR2.12 (Figure 1C) and BMPR2.23 (Figure 1D) to BMPR2 are shown. The specificity of these clones to immobilized ALK1 (Figure 2A) versus BMPR2 (Figure 2B) was also tested, showing that each of these clones exhibited affinity for their intended target, but not for the highly homologous control protein, the human ACVR2A receptor (Figures 1 and 2C).

[0293] scFv against BMPR2 or ALK1 inhibits BMP9 signaling in vitro. To test the ability of selected single-chain antibodies to recognize the extracellular ligand-binding domains of human ALK1 and BMPR2 receptors, we used a cell-based assay of BMP-transcriptional activity with a BMP-responsive element luciferase (BRE-luciferase) reporter via a plasmid expressing firefly luciferase controlled by a BMP-responsive element promoter sequence cloned from the human ID1 gene. Transgenic cells were stimulated overnight with rhBMP9 (1 ng / mL) in the presence / absence of various concentrations of scFv protein. BMP9 ligand trap ALK1-Fc was also used as a positive control. scFV clones BMPR2.12, BMPR2.23, ALK1.3, and ALK1.8 showed the most efficient inhibition of BRE-luciferase activity, suggesting that these scFv proteins bind to the human ALK1 region and the BMPR2 receptor associated with ligand binding (Figure 3).

[0294] Biotinylation of scFv protein. ALK1.3, ALK1.8, BMPR2.12, and BMPR2.23 scFv were biotinylated as described in the Materials and Methods section. The resulting biotinylated scFv were tested by Western blot analysis to confirm their biotinylation success (Figure 4A).

[0295] Binding efficiency and specificity of biotinylated scFv to ALK1 and BMPR2. To verify that biotinylation of scFv does not interfere with their binding ability, positive biotinylated and wild-type clones were re-screened using biolayer interferometry. Biotinylated ALK1.3 and ALK1.8 scFv proteins (ALK1.3-Bio, ALK1.8-Bio, Figures 4B and 4D) and BMPR2.12 scFv protein (BMPR2.12-Bio, Figure 4C) retained affinity for their intended targets while maintaining specificity based on a lack of affinity for highly homologous control proteins.

[0296] Biotinylated scFV complexes targeting ALK1 and BMPR2 induce SMAD1 / 5 / 9 activation equivalent to BMP9 activation in endothelial cells. To demonstrate specific activation of BMPR2:ALK1 signaling in vitro, human and mouse capillary endothelial cell lines were pre-incubated with different combinations of biotinylated scFv and treated with streptavidin. Treatment of cultured human pulmonary capillary endothelial cells (PMVECs) with the biotinylated ALK1.8 / BMPR2.12 scFv complex in the presence of streptavidin at 37°C for 30 minutes induced phosphorylation of SMAD1 / 5 / 9, while incubation with individual biotinylated ALK1.8 or BMPR2.12 did not activate signaling (Figure 5). Signaling via the biotinylated ALK1.8 / BMPR2.12 streptavidin complex was similar to that observed after treatment with recombinant human BMP9, but, in contrast to rhBMP9, was not affected by the BMP9 neutralizing antibody (mAb3209) or the BMP9 ligand-trapped ALK1-Fc, as seen by Western blotting (Figure 5) and in-cell Western analysis (Figure 6B). Similar experiments were repeated using wild-type (WT) and Acvrl1 KO mouse PMVECs, which showed activation of SMAD1 / 5 / 9 signaling in response to the ALK1.8 / BMPR2.12 complex in WT mouse cells, comparable to the results in human PMVECs, but not in Acvrl1 KO PMVECs (Figure 6A), which is consistent with the requirements of ALK1 for signaling via the bispecific scFv complex.

[0297] Table 1 shows exemplary sequences for some of the identified scFV CDRs.

[0298] [Table 7-1]

[0299] [Table 7-2]

[0300] [Table 7-3]

[0301] [Table 7-4]

[0302] An example of an anti-BMPRI / BMPRII antibody sequence is shown below. The sequence is shown as VL-linker-VH, and the linker is in bold.

[0303] Clone ALK1.2 (anti-ALK1 (ACVRL1)) Nucleotide sequence (SEQ ID NO: 145)

[0304] [ka]

[0305] Amino acid sequence (SEQ ID NO: 146)

[0306] [ka]

[0307] Clone ALK1.3 (anti-ALK1 (ACVRL1)) Nucleotide sequence (SEQ ID NO: 147)

[0308] [ka]

[0309] Amino acid sequence (SEQ ID NO: 148)

[0310] [ka]

[0311] Clone ALK1.8 (anti-ALK1 (ACVRL1)) Nucleotide sequence (SEQ ID NO: 149)

[0312] [ka]

[0313] Amino acid sequence (SEQ ID NO: 150)

[0314] [ka]

[0315] Clone ALK1.8b (anti-ALK1 (ACVRL1)) Nucleotide sequence (SEQ ID NO: 151)

[0316] [ka]

[0317] Amino acid sequence (SEQ ID NO: 152)

[0318] [ka]

[0319] Clone BMPR2.12 (anti-BMPR2 (BMPRII)) Nucleotide sequence (SEQ ID NO: 153)

[0320] [ka]

[0321] Amino acid sequence (SEQ ID NO: 154)

[0322] [ka]

[0323] Clone BMPR2.23 (anti-BMPR2 (BMPRII)) Nucleotide sequence (SEQ ID NO: 211)

[0324] [ka]

[0325] Amino acid sequence (SEQ ID NO: 212)

[0326] [ka]

[0327] Clone B15: (Anti-ALK3 (BMPRIA)) Nucleotide sequence (SEQ ID NO: 155)

[0328] [ka]

[0329] Amino acid sequence (SEQ ID NO: 156)

[0330] [ka]

[0331] Clone B16: (Anti-ALK3 (BMPRIA)) Nucleotide sequence (SEQ ID NO: 157)

[0332] [ka]

[0333] Amino acid sequence (SEQ ID NO: 158)

[0334] [ka]

[0335] Clone RI-2: (Anti-ALK2 (ACVR1)) Nucleotide sequence (SEQ ID NO: 159)

[0336] [ka]

[0337] Amino acid sequence (SEQ ID NO: 160)

[0338] [ka]

[0339] Clone RI-3: (Anti-ALK2 (ACVR1)) * Please note the different linker sequences. Nucleotide sequence (SEQ ID NO: 161)

[0340] [ka]

[0341] Amino acid sequence (SEQ ID NO: 162)

[0342] [ka]

[0343] Clone RI-4: (Anti-ALK2 (ACVR1)) Nucleotide sequence (SEQ ID NO: 163)

[0344] [ka]

[0345] Amino acid sequence (SEQ ID NO: 164)

[0346] [ka]

[0347] Clone RI-2-7 (anti-ALK2 (ACVR1)) Nucleotide sequence (SEQ ID NO: 165)

[0348] [ka]

[0349] Amino acid sequence (SEQ ID NO: 166)

[0350] [ka]

[0351] Clone RI-9: (Anti-ALK2 (ACVR1)) Nucleotide sequence (SEQ ID NO: 167)

[0352] [ka]

[0353] Amino acid sequence (SEQ ID NO: 168)

[0354] [ka]

[0355] Clone RII-19 (anti-ACTRIIA (ACVR2A)) Nucleotide sequence (SEQ ID NO: 169)

[0356] [ka]

[0357] Amino acid sequence (SEQ ID NO: 170)

[0358] [ka]

[0359] Clone H1-2 (anti-activin RIIA (ACVR2A)) Nucleotide sequence (SEQ ID NO: 171)

[0360] [ka]

[0361] Amino acid sequence (SEQ ID NO: 172)

[0362] [ka]

[0363] Clone H2-11 (anti-activin RIIB (ACVR2B)) Nucleotide sequence (SEQ ID NO: 173)

[0364] [ka]

[0365] Amino acid sequence (SEQ ID NO: 174)

[0366] [ka]

[0367] Clone H2-15 (anti-activin RIIB (ACVR2B)) Nucleotide sequence (SEQ ID NO: 175)

[0368] [ka]

[0369] Amino acid sequence (SEQ ID NO: 176)

[0370] [ka]

[0371] Clone H5-2 (anti-ALK4 (activin RIB)) Nucleotide sequence (SEQ ID NO: 177)

[0372] [ka]

[0373] Amino acid sequence (SEQ ID NO: 178)

[0374] [ka]

[0375] Clone H3-2 (anti-ALK6 (BMPR1B)) Nucleotide sequence (SEQ ID NO: 179)

[0376] [ka]

[0377] Amino acid sequence (SEQ ID NO: 180)

[0378] [ka]

[0379] Clone H4-1 (anti-ALK7 (ACVR1C)) Nucleotide sequence (SEQ ID NO: 181)

[0380] [ka]

[0381] Amino acid sequence (SEQ ID NO: 182)

[0382] [ka]

[0383] Clone H4-4 (anti-ALK7 (ACVR1C)) Nucleotide sequence (SEQ ID NO: 183)

[0384] [ka]

[0385] Amino acid sequence (SEQ ID NO: 184)

[0386] [ka]

[0387] Clone H4-7 (anti-ALK7 (ACVR1C)) Nucleotide sequence (SEQ ID NO: 185)

[0388] [ka]

[0389] Amino acid sequence (SEQ ID NO: 186)

[0390] [ka]

[0391] Clone H4-8 (anti-ALK7 (ACVR1C)) Nucleotide sequence (SEQ ID NO: 187)

[0392] [ka]

[0393] Amino acid sequence (SEQ ID NO: 188)

[0394] [ka]

[0395] [Example 2] Bispecific construct Several examples of bispecific constructs were generated from scFv clones that bind to ACVRL1 (ACVRL1.8) and BMPR2 (BMPR2.12). These intermediate complexes were constructed to obtain bispecific molecules for ALK1 / ACVRL1 and BMPR2. These complexes were constructed by expressing two scFv as single molecules separated by a linker with a C-terminal His tag, or by expressing each scFv separately as an Fc-fusion molecule and then combining them into a single scFv-Fc heterodimer.

[0396] Neither single-molecule bispecificity nor heterodimer bispecificity exhibited significant BMP transcriptional activity in in vitro assays. See Figure 8. Rather than possessing biological activity as single-molecule bispecificity molecules, these molecules exhibited activity when linked as tetramers by ligating His tags using Ni++, Ca++, or anti-His, thereby forming heterotetramers. These results indicate that heterotetramer artificial ligands drive signaling in this pathway, and this finding is key to enabling these BMP-mimicking signaling molecules to function.

[0397] Clone ACVRL1.8 (anti-ACVRL1) Amino acid sequence:

[0398] [ka]

[0399] Clone BMPR2.12 (anti-BMPR2) amino acid sequence

[0400] [ka]

[0401] Dual-specific construct: BMPR2.12-Medium-ACVRL1.8-His Amino acid sequence:

[0402] [ka]

[0403] ACVRL1.8-Long-BMPR2.12-His Amino acid sequence:

[0404] [ka]

[0405] BMPR2.12-Long-ACVRL1.8-His Amino acid sequence:

[0406] [ka]

[0407] [Example 3] tetravalent construct The tetravalent construct was generated from scFv clones that bind to ACVRL1 (ACVRL1.8) and BMPR2 (BMPR2.12). These were designed to form a tetravalent complex expressed as a single molecule with a spacer of specific, variable length. Alternatively, they were generated by two bispecific scFv expressing as an IgG-Fc fusion protein, and then designed as a tetravalent IgG-Fc protein bound to two disulfide-bonded homodimeric Fc complexes.

[0408] These constructs were able to induce potent BMP signaling in cells (see Figure 8). A tetravalent complex of anti-ACVRL1 and anti-BMPR2 induced BMP transcriptional activity in endothelial cells. TIME cells (human telomerase-immortalized capillary endothelial cells) were cultured and transiently transfected using a plasmid expressing a BMP response element (BRE-luciferase) reporter. BMP9 (1 ng / mL) induced BMP transcriptional activity in these cells, based on the detection of luciferase activity divided by the relative number of cells based on an MTS colorimetric viability assay (RLU / viability). A bispecific construct consisting of a single anti-ACVRL1 scFv and a single anti-BMPR2 scFv, bound by a spacer and possessing a C-terminal His-tagged domain ("ACVRL1.8-L-BMPR2.12-His"), did not induce signaling activity on its own. However, when combined with a monoclonal anti-His antibody, these bispecific molecules were complexed into a tetravalent complex, demonstrating activity. To illustrate the signaling of the tetravalent complex when expressed as a disulfide-linked heterotetramer, a tetravalent complex of the same bispecific molecules, expressed as an IgG Fc domain fusion protein and assembled into a disulfide-linked homodimer ("ACVRL1.8-L-BMPR2.12-Short-Fc"), induced potent signaling.

[0409] In vivo BMP signaling activity was demonstrated based on Id1 gene transcription activity assayed in whole lung tissue from 8-week-old mice 24 hours after tail vein injection of physiological saline (50 uL), recombinant human BMP9 (rhBMP9, 150 ug / kg in 50 uL), or recombinant tetramer Fc fusion protein ACVRL1.8-L-BMPR2.12-Short-Fc (150 ug / kg in 50 uL), consistent with in vitro signaling activity (Figure 9). Injection of BMP9 or ACVRL1.8-L-BMPR2.12-Short-Fc induced increased BMP transcription activity in whole lung tissue compared to physiological saline.

[0410] Demonstration of such signaling in vivo is achieved by intravenous, intramuscular, or subcutaneous injection of recombinant signaling proteins, such as ACVRL1.8-L-BMPR2.12-Short-Fc or similar molecules, into a suitable animal model such as a mouse or rat. 30 minutes to 24 hours later, the effects of these treatments versus saline on BMP signaling activity are assayed by immunohistochemistry on animal tissue for phosphorylation of SMAD1 / 5 / 9 effector proteins, by Western blotting of tissue extracts for phosphorylated SMAD1 / 5 / 9, by assaying the expression of ID1, ID2, and / or ID3 in target tissue by quantitative RT-PCR, or by assaying GFP expression in transgenic reporter mouse expression under the control of BMP transcription response elements (Monteiro et al., Genesis. 2008 Jul;46(7):335-46).

[0411] Example sequence of a tetravalent construct scFv clone BMPR2.12 (anti-BMPR2):

[0412] [ka]

[0413] A=scFv clone ACVRL1.8 (anti-ACVRL1):

[0414] [ka]

[0415] A moderate spacer between scFv clones within a M=2 valent complex: SGGGGSGGGGSSGSGGGGDGGGGSG (SEQ ID NO: 192)

[0416] Long spacers between ScFv clones in a L=2 valent complex: SGGSGGGGSSGGGGSGGGGSSGGGGDGGGGSG (SEQ ID NO: 193)

[0417] short = a short spacer before the IgG Fc domain in homodimer Fc molecules: GSGGGGDGGGGSG (SEQ ID NO: 194)

[0418] medium = A moderate spacer before the IgG Fc domain in a homodimer Fc molecule: GSGGGGDSGGGGSGGGGSSGGGGSG (SEQ ID NO: 195)

[0419] long = Long spacer before the IgG Fc domain in homodimer Fc molecules: GSGGGGDSGGGGSGGSGGSGGSGGGSGGGGSG (SEQ ID NO: 196)

[0420] Fc=IgG Fc constant domain:

[0421] [ka]

[0422] short(His) = A short spacer between bivalent arms with the His tag: GGHHHHHHHHGG (Sequence ID 198)

[0423] medium(His) = A medium-sized spacer between bivalent arms with the His tag: SGGGGSHHHHHHHHSGGGGS (Sequence ID 199)

[0424] long(His)=His tag, a long spacer between bivalent arms: SGGGGSGGHHHHHHHHGGSGGGGS (Sequence ID 200)

[0425] Bold = CDR area of ​​scFv

[0426] (1) BMA-short-Fc

[0427]

change

[0428] (2)BMA-medium-Fc

[0429]

change

[0430] (3)BMA-long-Fc

[0431]

change

[0432] (4)ALB-short-Fc

[0433]

change

[0434] (5)ALB-medium-Fc

[0435]

change

[0436] (6)ALB-long-Fc

[0437]

change

[0438] (7)BMA-'short(His)'-ALB

[0439]

change

[0440] (8) BMA-'medium(His)'-ALB

[0441] [ka]

[0442] (9) BMA-'long(His)'-ALB

[0443] [ka]

[0444] (10) BMA-'short(His)'-AMB

[0445] [ka]

[0446] References

[0447] [Table 8-1]

[0448] [Table 8-2]

[0449] Other Embodiments While the present invention is described in conjunction with its detailed description, it should be understood that the foregoing description is intended to be illustrative and does not limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. Various embodiments of the present invention are shown below. 1. At least The first antigen-binding domain that binds to bone morphogenetic protein receptor (BMPR) type I (BMPRI); and Second antigen-binding domain that binds to bone morphogenetic protein receptor (BMPR) type II (BMPRII) Multiple or bispecific antibody molecules containing, The first and second antigen-binding domains are linked to each other by a mobile linker, and may be in any order. Each antigen-binding domain can act as an agonist against the BMPR to which it binds. Preferably, each antigen-binding domain is an scFv. antibody molecule. 2. The antibody molecule described in 1 above, wherein the binding of the antibody molecule to a cell initiates BMP / TGF-beta / activin / GDF signaling. 3. The antibody molecule described in 1 above, wherein the first antigen-binding domain binds to BMPRI selected from the group consisting of ALK1(ACVRL1);ALK2(ACVR1A);ALK3(BMPR1A);ALK4(ACVR1B);ALK5(TGFBR1);ALK6(BMPR1B); orALK7(ACVR1C). 4. The antibody molecule according to 3 above, wherein the first antigen-binding domain binds to ALK1 and optionally includes a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; VH CDR1, 2, 3 and VL CDR1, 2, 3 that are identical to the complementarity-determining region in SEQ ID NOs. 146, 148, 150, or 152; VH and / or VL sequences that are at least 95% identical to the sequence in Figure 7; or sequences that are at least 95% identical to the sequence in SEQ ID NOs. 146, 148, 150, or 152. 5. The antibody molecule according to 3 above, wherein the first antigen-binding domain binds to ALK2 and optionally includes a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; includes VH CDR1, 2, 3 and VL CDR1, 2, 3 which are identical to the complementarity-determining regions in SEQ ID NOs. 160, 162, 164, 166, or 168; includes VH and / or VL sequences that are at least 95% identical to the sequence in Figure 7; or includes a sequence that is at least 95% identical to the sequence in SEQ ID NOs. 160, 162, 164, 166, or 168. 6. The antibody molecule described in 3 above, wherein the first antigen-binding domain binds to ALK3 and optionally includes a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; VH CDR1, 2, 3 and VL CDR1, 2, 3 that are identical to the complementarity-determining region in SEQ ID NO: 156 or 158; VH and / or VL sequences that are at least 95% identical to the sequence in Figure 7; or sequences that are at least 95% identical to the sequence in SEQ ID NO: 156 or 158. 7. The antibody molecule according to 3 above, wherein the first antigen-binding domain binds to ALK4 and optionally includes a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; VH CDR1, 2, 3 and VL CDR1, 2, 3 that are identical to the complementarity-determining region in SEQ ID NO: 178; VH and / or VL sequences that are at least 95% identical to the sequence in Figure 7; or sequences that are at least 95% identical to SEQ ID NO: 178. 8. The antibody molecule according to 3 above, wherein the first antigen-binding domain binds to ALK6 and optionally includes a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; VH CDR1, 2, 3 and VL CDR1, 2, 3 that are identical to the complementarity-determining region in SEQ ID NO: 180; VH and / or VL sequences that are at least 95% identical to the sequence in Figure 7; or a sequence that is at least 95% identical to SEQ ID NO: 180. 9. The antibody molecule according to 3 above, wherein the first antigen-binding domain binds to ALK7 and optionally includes a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; VH CDR1, 2, 3 and VL CDR1, 2, 3 that are identical to the complementarity-determining region in SEQ ID NOs. 182, 184, 186, or 188; VH and / or VL sequences that are at least 95% identical to the sequence in Figure 7; or sequences that are at least 95% identical to the sequence in SEQ ID NOs. 182, 184, 186, or 188. 10. The antibody molecule described in 1 above, wherein the second antigen-binding domain binds to BMPRII selected from the group consisting of ACTRIIA(ACVR2A); ACTRIIB(ACVR2B); BMPRII(BMPR2); TGFBRII(TGFBR2); or AMHRII(AMHR2). 11. The antibody molecule according to 10 above, wherein the second antigen-binding domain is bound to BMPR2 and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; contains VH CDR1, 2, 3 and VL CDR1, 2, 3 which are identical to the complementarity-determining region in SEQ ID NO: 154 or 212; contains a VH and / or VL sequence that is at least 95% identical to the sequence in Figure 7; or contains a sequence that is at least 95% identical to the sequence in SEQ ID NO: 154 or 212. 12. The antibody molecule according to 10 above, wherein the second antigen-binding domain is bound to ACTRIIA(ACVR2A) and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; contains VH CDR1, 2, 3 and VL CDR1, 2, 3 which are identical to the complementarity-determining regions in SEQ ID NOs. 170 and 172; contains a VH and / or VL sequence that is at least 95% identical to the sequence in Figure 7; or contains a sequence that is at least 95% identical to the sequence in SEQ ID NOs. 170 or 172. 13. The antibody molecule according to 10 above, wherein the second antigen-binding domain is bound to ACTRIIB(ACVR2B) and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; contains VH CDR1, 2, 3 and VL CDR1, 2, 3 which are identical to the complementarity-determining region in SEQ ID NO: 174 or 176; contains a VH and / or VL sequence that is at least 95% identical to the sequence in Figure 7; or contains a sequence that is at least 95% identical to the sequence in SEQ ID NO: 174 or 176. 14. (i) The first antigen-binding domain is bound to BMPR2 and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7, and / or the second antigen-binding domain is bound to ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7 and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; (ii) The first antigen-binding domain is bound to ACVR2A and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7, and / or the second antigen-binding domain is bound to ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7 and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; (iii) The first antigen-binding domain is bound to ACVR2B and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7, and / or the second antigen-binding domain is bound to ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7 and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; (iv) The first antigen-binding domain binds to TGFBR2 and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7, and / or the second antigen-binding domain binds to ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7 and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; (v) The antibody molecule according to item 1, wherein the first antigen-binding domain is bound to AMHR2 and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7, and / or the second antigen-binding domain is bound to ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7 and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7. 15. An antibody or its antigen-binding moiety that binds to ALK1 and optionally contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; contains VH CDR1, 2, 3 and VL CDR1, 2, 3 that are identical to the complementarity-determining regions in SEQ ID NOs. 146, 148, 150, or 152; contains VH and / or VL sequences that are at least 95% identical to the sequence in Figure 7; or contains a sequence that is at least 95% identical to the sequence in SEQ ID NOs. 146, 148, 150, or 152. 16. An antibody or its antigen-binding moiety that binds to ALK2 and optionally contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; contains VH CDR1, 2, 3 and VL CDR1, 2, 3 that are identical to the complementarity-determining regions in SEQ ID NOs. 160, 162, 164, 166, or 168; contains VH and / or VL sequences that are at least 95% identical to the sequence in Figure 7; or contains a sequence that is at least 95% identical to the sequence in SEQ ID NOs. 160, 162, 164, 166, or 168. 17. An antibody or its antigen-binding moiety that binds to ALK3 and optionally contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; contains VH CDR1, 2, 3 and VL CDR1, 2, 3 that are identical to the complementarity-determining region in SEQ ID NO: 156 or 158; contains a VH and / or VL sequence that is at least 95% identical to the sequence in Figure 7; or contains a sequence that is at least 95% identical to the sequence in SEQ ID NO: 156 or 158. 18. An antibody or its antigen-binding moiety that binds to ALK4 and optionally contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; contains VH CDR1, 2, 3 and VL CDR1, 2, 3 which are identical to the complementarity-determining region in SEQ ID NO: 178; contains a VH and / or VL sequence that is at least 95% identical to the sequence in Figure 7; or contains a sequence that is at least 95% identical to the sequence in SEQ ID NO: 178. 19. An antibody or its antigen-binding moiety that binds to ALK6 and optionally contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; contains VH CDR1, 2, 3 and VL CDR1, 2, 3 that are identical to the complementarity-determining region in SEQ ID NO: 180; contains a VH and / or VL sequence that is at least 95% identical to the sequence in Figure 7; or contains a sequence that is at least 95% identical to the sequence in SEQ ID NO: 180. 20. An antibody or its antigen-binding moiety that binds to ALK7 and optionally contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; contains VH CDR1, 2, 3 and VL CDR1, 2, 3 that are identical to the complementarity-determining regions in SEQ ID NOs. 182, 184, 186, or 188; contains VH and / or VL sequences that are at least 95% identical to the sequence in Figure 7; or contains a sequence that is at least 95% identical to the sequence in SEQ ID NOs. 182, 184, 186, or 188. 21. An antibody or its antigen-binding moiety that binds to BMPR2 and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; contains VH CDR1, 2, 3 and VL CDR1, 2, 3 which are identical to the complementarity-determining region in SEQ ID NO: 154 or 212; contains a VH and / or VL sequence that is at least 95% identical to the sequence in Figure 7; or contains a sequence that is at least 95% identical to the sequence in SEQ ID NO: 154 or 212. 22. An antibody or its antigen-binding moiety that binds to ACTRIIA(ACVR2A) and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; contains VH CDR1, 2, 3 and VL CDR1, 2, 3 which are identical to the complementarity-determining region in SEQ ID NO: 170 or 172; contains a VH and / or VL sequence that is at least 95% identical to the sequence in Figure 7; or contains a sequence that is at least 95% identical to the sequences in SEQ ID NOs: 170 and 172. 23. An antibody or its antigen-binding moiety that binds to ACTRIIB(ACVR2B) and contains a CDR sequence that is at least 95% identical to the CDR sequence in Table 1 or Figure 7; contains VH CDR1, 2, 3 and VL CDR1, 2, 3 which are identical to the complementarity-determining region in SEQ ID NO: 174 or 176; contains a VH and / or VL sequence that is at least 95% identical to the sequence in Figure 7; or contains a sequence that is at least 95% identical to the sequence in SEQ ID NO: 174 or 176. 24. An antibody molecule as described in 1 to 14 above, or an antibody or antigen-binding portion thereof as described in 15 to 23 above, for use in a method for treating the condition of blood vessels in a subject, wherein the vascular condition is optionally pulmonary arterial hypertension or hereditary hemorrhagic telangiectasia (HHT) syndrome. 25. An antibody molecule as described in 1 to 14 above, or an antibody or antigen-binding portion thereof as described in 15 to 23 above, for use in a method for treating pulmonary vascular leakage syndrome, wherein the antibody molecule or antibody or antigen-binding portion thereof optionally wherein the pulmonary vascular leakage syndrome is acute respiratory distress syndrome (ARDS) or acute lung injury (ALI). 26. An antibody molecule as described in 1 to 14 above, or an antibody as described in 15 to 23 above, or its antigen-binding portion, for use in a method for treating hepatic fibrosis in subjects with hepatic BMP9 signaling deficiency. 27. An antibody molecule as described in items 1 to 14 above, or a nucleic acid encoding an antibody or its antigen-binding portion as described in items 15 to 23 above. 28. A host cell containing the nucleic acid described in item 11 above, and optionally expressing an antibody molecule described in items 1 to 14 above, or an antibody or its antigen-binding portion described in items 15 to 23 above. 29. A pharmaceutical composition comprising the antibody molecule described in items 1 to 14 above, or the antibody described in items 15 to 23 above, or its antigen-binding portion. 30. A method for treating a vascular condition in a subject, wherein the vascular condition is optionally pulmonary arterial hypertension or hereditary hemorrhagic telangiectasia (HHT) syndrome, and the method comprises administering a therapeutically effective amount of an antibody molecule described in 1 to 14 above, or an antibody or antigen-binding portion thereof described in 15 to 23 above, to a subject in need of such treatment. 31. A method for treating pulmonary vascular leakage syndrome, wherein the pulmonary vascular leakage syndrome is optionally acute respiratory distress syndrome (ARDS) or acute lung injury (ALI), and the method comprises administering a therapeutically effective dose of an antibody molecule described in items 1 to 14 above, or an antibody or antigen-binding portion thereof described in items 15 to 23 above, to a subject in need thereof. 32. A method for treating hepatic fibrosis in a subject having a hepatic BMP9 signaling deficiency, comprising administering a therapeutically effective amount of the antibody molecule described in 1 to 14 above, or the antibody or antigen-binding portion thereof described in 15 to 23 above, to a subject in need.

Claims

1. A quadruple-specific construct containing pairs of molecules, where each molecule is The primary antigen-binding domain that binds to the bone morphogenetic protein receptor (BMPR) type I (BMPRI); A second antigen-binding domain that binds to the bone morphogenetic protein receptor (BMPR) type II (BMPRII); and FC Domain The first and second antigen-binding domains are linked to each other by a mobile linker, and may be in any order. Each antigen-binding domain can act as an agonist against the BMPR to which it binds. Each molecule is fused by a disulfide bond in the Fc domain, forming the quadruple specific construct. The first antigen-binding domain is bound to ALK1 and includes VH CDR1, 2, 3 and VL CDR1, 2, 3 which are identical to sequence numbers 544, 549, 554, 585, 588, and 590, respectively. The second antigen-binding domain is bound to BMPR2 and includes VH CDR1, 2, 3 and VL CDR1, 2, 3 which are identical to SEQ ID NOs. 503, 508, 513, 516, 519, and 521, respectively. Quadripartite specificity construct.

2. The quadruple-specific construct according to claim 1, wherein each antigen-binding domain is an scFv.

3. The quadruple-specific construct according to claim 1, wherein the binding of the quadruple-specific construct to a cell initiates BMP / TGF-beta / activin / GDF signaling.

4. The quadruple-specific construct according to any one of claims 1 to 3, wherein the first antigen-binding domain comprises a VH and / or VL sequence that is at least 95% identical to SEQ ID NO:

150.

5. The quadruple-specific construct according to any one of claims 1 to 4, wherein the second antigen-binding domain comprises a VH and / or VL sequence that is at least 95% identical to SEQ ID NO:

154.

6. A composition comprising the quadruple-specific construct according to claims 1 to 5 for use in a method for treating a vascular condition in a subject, wherein the vascular condition is pulmonary arterial hypertension or hereditary hemorrhagic telangiectasia (HHT) syndrome.

7. A composition comprising the quadruple-specific construct according to claims 1 to 5 for use in a method for treating pulmonary vascular leakage syndrome.

8. The composition according to claim 7, wherein the pulmonary vascular leakage syndrome is acute respiratory distress syndrome (ARDS) or acute lung injury (ALI).

9. A composition comprising the quadruspecific construct according to claims 1 to 5 for use in a method for treating hepatic fibrosis in subjects having intrahepatic BMP9 signaling deficiency.

10. A nucleic acid encoding the quadruple-specific construct according to claims 1 to 5.

11. A host cell containing the nucleic acid described in claim 10.

12. A host cell according to claim 11, which expresses the quadruple-specific construct according to claims 1 to 5.

13. A pharmaceutical composition comprising the quadruple-specific construct described in claims 1 to 5.

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