Bispecific agonist antibodies against activin A receptor-like type 1 (ALK1)

Abstract

Provided herein are bispecific agonist antibodies that bind to ALK1, BMPR2, ActRIIA, and / or ActRIIB, as well as methods for using them.

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 458,044, filed Apr. 7, 2023; U.S. Provisional Patent Application No. 63 / 537,318, filed Sep. 8, 2023; and U.S. Provisional Patent Application No. 63 / 596,899, filed Nov. 7, 2023. The entire disclosure content of these documents is incorporated herein by reference.

Background Art

[0002] Hereditary hemorrhagic telangiectasia (HHT), also known as Osler-Weber-Landu disease, is an autosomal dominant genetic disorder characterized by vascular malformations (arteriovenous malformations, AVMs) in multiple organs caused by a lack of capillary reticular structure. The most common symptoms of HHT are epistaxis (nosebleeds), telangiectasia, and visceral lesions. Approximately 25–40% of patients have progressive disease, and AVMs can lead to acute, fatal bleeding and embolism. The majority of HHT patients (>85%) are heterozygous for loss-of-function (LOF) mutations in the endoglin (ENG, HHT1) or activin A receptor-like type 1 (ALK1, HHT2) gene. Patients with HHT1 and HHT2 develop very similar clinical symptoms due to sporadic vascular malformations, but the affected tissues differ. HHT1 patients, who account for approximately 61% of HHT cases, are prone to pulmonary arteriovenous malformations (PAVMs) and cerebral arteriovenous malformations (CAVMs), while HHT2 patients, who account for about one-third (37%) of patients, are prone to complications from hepatic AVMs and pulmonary hypertension. Liver damage can lead to secondary portal hypertension, which may necessitate liver transplantation and heart failure. Lung damage in these patients can lead to pulmonary hypertension (PAH). Activin receptor-like kinase 1 (ALK1) and endoglin are endothelial cell (EC)-specific receptors of the large TGF-β family. Members of the TGF-β family act on many, though not all, cell types in the body, resulting in diverse and complex cellular outcomes, such as growth arrest, immunosuppression, differentiation, apoptosis, and the determination of developmental cell fate during embryogenesis and disease development. Activation of the endothelial cell-specific TGF-β type I receptor ALK1 is due to the binding of several different ligands of the TGF-β family, including bone morphogenetic protein (BMP) 9, BMP10, and TGF-β.

[0003] TGF-beta signaling requires the recruitment of type I and type II receptors into a multimer to initiate signaling. Endoglin is a type III receptor that delivers BMP9 and 10 to type I and type II receptors on the endothelial cell membrane. The dimeric ligand molecule promotes the association of heteromeric complexes of type II and type I receptors, where the constitutively active kinase domain of the type II receptor is transphosphorylated and the kinase domain of the type I receptor is activated. The type I receptor can then mimic signaling through multiple signaling cascades, including SMAD, which translocates to the nucleus and activates the transcription of target genes.

[0004] Deficiencies in signaling pathways mediated by ALK1 are characteristic of both familial and sporadic PAH patients, leading to endothelial dysfunction, i.e., apoptosis, proliferation, interaction with smooth muscle cells (SMCs), and transdifferentiation. Over time, remodeling of vascular structures occludes pulmonary arterioles, increasing pulmonary vascular resistance and pulmonary artery pressure. This leads to decreased cardiac output, right heart failure, and ultimately death. [Overview of the project]

[0005] This disclosure improves upon the prior art by providing a heteromeric antibody that can activate SMAD signaling by effectively crosslinking the ALK1 receptor to a receptor selected from BMPRII, ActRIIA, and ActRIIB.

[0006] In one embodiment, the Specified Publicly Provided is a polyspecific binding protein comprising a first binding site that specifically binds to human ALK1, and a second binding site that specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB, wherein (a) the polyspecific binding protein is capable of inducing signal transduction by inducing proximity between ALK1 and BMPRII, ActRIIA, or ActRIIB, and (b) at least one modified hinge region.

[0007] In some embodiments, the first modified hinge region includes (a) an upper hinge region that is up to 7 amino acids long or absent, and (b) a lower hinge region, the lower hinge region being ligated to the N-terminus of the first constant region. In some embodiments, the polyspecific binding protein further includes a second modified hinge region ligated to the N-terminus of the second constant region. In some embodiments, the second modified hinge region includes (a) an upper hinge region that is up to 7 amino acids long or absent, and (b) a lower hinge region, the lower hinge region being ligated to the N-terminus of the second constant region. In some embodiments, the upper hinge regions of the first and second modified hinge regions have the same sequence. In some embodiments, the upper hinge regions of the first and second modified hinge regions have different sequences.

[0008] In some embodiments, the upper hinge region includes an amino acid sequence derived from the upper hinge region of a human IgG antibody. In some embodiments, the IgG antibody is selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the IgG antibody is IgG1. In some embodiments, the upper hinge region includes the amino acid sequence of SEQ ID NO: 1. In some embodiments, the upper hinge region includes the amino acid sequence of SEQ ID NO: 2. In some embodiments, the IgG antibody is IgG4. In some embodiments, the upper hinge region includes the amino acid sequence of SEQ ID NO: 3. In some embodiments, the upper hinge is absent.

[0009] In some embodiments, the first heavy chain constant region and / or the second heavy chain constant region comprises human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the first heavy chain constant region and / or the second heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 10.

[0010] In some embodiments, at least one heavy chain constant region includes a substitution at the 234th amino acid position according to EU numbering. In some embodiments, the substitution at the 234th amino acid position is alanine(A). In some embodiments, at least one heavy chain constant region includes a substitution at the 235th amino acid position according to EU numbering. In some embodiments, the substitution at the 235th amino acid position is alanine(A). In some embodiments, at least one heavy chain constant region includes a substitution at the 237th amino acid position according to EU numbering. In some embodiments, the substitution at the 237th amino acid position is alanine(A). In some embodiments, at least one heavy chain constant region includes one or more substitutions at the 234th, 235th, or 237th amino acid positions according to EU numbering. In some embodiments, the substitution at the 234th amino acid position is alanine(A), the substitution at the 235th amino acid position is alanine(A), and the substitution at the 237th amino acid position is alanine(A).

[0011] In some embodiments, the heavy chain constant region includes a heterodimerizing mutation that promotes heterodimerization between the first and second binding sites. In some embodiments, the heterodimerizing mutation is a knob-in-hole (KIH) mutation. In some embodiments, the first heavy chain constant region includes an amino acid substitution that generates a hole at position 366, 368, or 407, and the second heavy chain constant region includes an amino acid substitution that generates a knob at position 366. In some embodiments, the first heavy chain constant region includes the amino acid substitution T366S, L368A, or Y407V, and the second heavy chain constant region includes the amino acid substitution T366W.

[0012] In some embodiments, the heterodimerizing mutation is a charge-stabilizing mutation. In some embodiments, the first heavy chain constant region includes the amino acid substitution N297K, and the second heavy chain constant region includes the amino acid substitution N297D. In some embodiments, the first heavy chain constant region includes the amino acid substitution T299K, and the second heavy chain constant region includes the amino acid substitution T299D.

[0013] In some embodiments, the heterodimerizing mutation includes an engineered disulfide bond. In some embodiments, the engineered disulfide bond is formed by a first heavy chain constant region containing the amino acid substitution Y349C and a second heavy chain constant region containing the amino acid substitution S354C. In some embodiments, the engineered disulfide bond is formed by a C-terminal extension peptide fused to the C-terminus of each of the first and second heavy chain constant regions. In some embodiments, the C-terminal extension of the first heavy chain constant region contains the amino acid sequence GEC, and the C-terminal extension of the second heavy chain constant region contains the amino acid sequence SCDKT.

[0014] In some embodiments, at least one heavy chain constant region contains one or more mutations that promote half-life extension. In some embodiments, at least one heavy chain constant region contains one or more substitutions at amino acid positions 252, 254, or 256 according to EU numbering. In some embodiments, the substitution at amino acid position 252 is tyrosine (Y), the substitution at amino acid position 254 is threonine (T), and the substitution at amino acid position 256 is glutamic acid (E).

[0015] In some embodiments, the first binding site that specifically binds to human ALK1 is selected from single-chain Fv(scFv), VHH, Fab, F(ab')2, or a single-domain antibody. In some embodiments, the second binding site that specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB is selected from single-chain Fv(scFv), VHH, Fab, F(ab')2, or a single-domain antibody.

[0016] In some embodiments, the polyspecific binding protein comprises, from N-terminus to C-terminus, (ai) a first polypeptide chain comprising a first antigen-binding domain, a first modified hinge region, and a first constant region, and (bi) a second polypeptide chain comprising a second antigen-binding domain, a second modified hinge region, and a second constant region, (aii) a first polypeptide chain comprising a second antigen-binding domain, a first antigen-binding domain, a first modified hinge region, and a first constant region, and (bii) a second polypeptide chain comprising a second modified hinge region, and a second constant region, or (aiii) a first polypeptide chain comprising a first modified hinge region, and a first constant region, and (biii) a second polypeptide chain comprising a second antigen-binding domain, a first antigen-binding domain, a second modified hinge region, and a second constant region. In some embodiments, (a) the first binding portion includes a VHH domain and the second portion includes a VHH domain; (b) the first binding portion includes a Fab domain and the second binding portion includes a VHH domain; (c) the first binding portion includes a VHH domain and the second binding portion includes a Fab domain; (d) the first binding portion includes a Fab domain and the second binding portion includes a Fab domain; (e) the first binding portion includes a Fab domain and the second binding portion includes scFv; (f) the first binding portion includes scFv and the second binding portion includes a Fab domain; (g) the first binding portion includes scFv and the second binding portion includes scFv; (h) the first binding portion includes scFv and the second binding portion includes VHH; or (i) the first binding portion includes VHH and the second binding portion includes scFv.

[0017] In some embodiments, the polyspecific binding protein comprises a first and a second polypeptide chain, the first polypeptide chain comprising VH1-(HX1)n-VH2-C-(HX2)n, where VH1 is a first heavy chain variable domain, VH2 is a second heavy chain variable domain, C is a heavy chain constant domain, HX1 is a linker, HX2 is an Fc region, and n is independently 0 or 1; and the second polypeptide chain comprising VL1-(LX1)n-VL2-C-(LX2)n, where VL1 is a first light chain variable domain, VL2 is a second light chain variable domain, C is a light chain constant domain, LX1 is a linker, LX2 does not contain an Fc region, and n is independently 0 or 1.

[0018] In some embodiments, VH1 specifically binds to human ALK1, and VH2 specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB.

[0019] In some embodiments, VL1 specifically binds to human ALK1, and VL2 specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB.

[0020] In some embodiments, VH1 specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB, and VH2 specifically binds to human ALK1.

[0021] In some embodiments, VL1 specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB, and VL2 specifically binds to human ALK1.

[0022] In some embodiments, the linker HX1 comprises the amino acid sequence PLAP or PAPNLLGGP.

[0023] In some embodiments, the linker LX1 comprises the amino acid sequence PLAP or PAPNLLGGP.

[0024] In some embodiments, linker HX1 comprises the amino acid sequence PLAP, and linker LX1 comprises the amino acid sequence PLAP or PAPNLLGGP.

[0025] In some embodiments, the first and / or second antigen-binding domains are cleaved at the C-terminus adjacent to the upper hinge domain. In some embodiments, at least one residue is cleaved at the C-terminus adjacent to the upper hinge domain. In some embodiments, at least two residues are cleaved at the C-terminus adjacent to the upper hinge domain. In certain embodiments, the SS amino acid at the C-terminus of the VH domain is cleaved.

[0026] In some embodiments, the polyspecific binding protein comprises a first polypeptide chain from any one of SEQ ID NOs: 136-141 and a second polypeptide chain from any one of SEQ ID NOs: 142-145.

[0027] In one embodiment, the present disclosure provides a polyspecific binding protein comprising at least a first polypeptide chain, The first polypeptide chain comprises a first variable heavy chain domain (VH1) linked to a second variable heavy chain domain (VH2) via at least one modified hinge region, VH1 specifically binds to ALK1, while VH2 specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB.

[0028] In some embodiments, one or both of VH1 and VH2 are VH domains or VHH domains.

[0029] In some embodiments, the polyspecific binding protein further comprises a second polypeptide chain, the second polypeptide chain comprising a first variable light chain domain (VL1) linked to a second variable light chain domain (VL2), the VL1 specifically binding to ALK1, and the VL2 specifically binding to a target selected from BMPRII, ActRIIA, and ActRIIB.

[0030] In some embodiments, VL1 is connected to VL2 via at least one modified hinge region.

[0031] In some embodiments, one or both of VH1 and VH2 are cleaved at the C-terminus.

[0032] In some embodiments, the C-terminus is cleaved at least one residue.

[0033] In some embodiments, the C-terminus is cleaved at least two residues.

[0034] In some embodiments, the C-terminal SS amino acid residue is removed.

[0035] In some embodiments, the polyspecific binding protein is VH1-HX1-VH2-C-Fc of the first polypeptide chain, however, VH1 is the first heavy chain variable domain, VH2 is the second heavy chain variable domain, C is a heavy chain constant domain, HX1 is a linker in the modified hinge region, Fc is an Fc region, and The second polypeptide chain is VL1-LX1-VL2-C, however, VL1 is the first light chain variable domain, VL2 is the second light chain variable domain, C is a light chain constant domain, LX1 includes the linker in the modified hinge region.

[0036] In some embodiments, the modified hinge region includes or consists of the amino acid sequence PLAP or PAPNLLGGP.

[0037] In some embodiments, the binding site that specifically binds to ALK1 cross-reacts with human ALK1 and mouse ALK1.

[0038] In some embodiments, the binding site that specifically binds to ActRIIA cross-reacts with ActRIIB.

[0039] In another embodiment, the Specified herein provides a polyspecific binding protein comprising a first binding site that specifically binds to ALK1, and a second binding site that specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB, wherein (a) the polyspecific binding protein is capable of inducing signal transduction by inducing proximity between ALK1 and BMPRII, ActRIIA, or ActRIIB, and (b) at least one modified hinge region comprising (i) an upper hinge region that is up to 7 amino acids long or absent, and (ii) a lower hinge region, the lower hinge region being ligated to the N-terminus of a first heavy chain constant region.

[0040] In another embodiment, the herein provides a polyspecific binding protein comprising at least a first polypeptide chain, the first polypeptide chain comprising a first variable heavy chain domain (VH1) linked to a second variable heavy chain domain (VH2) via at least one modified hinge region, wherein VH1 specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB, and VH2 specifically binds to ALK1, or VH1 specifically binds to ALK1 and VH2 specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB.

[0041] In some embodiments, one or both of VH1 and VH2 are VH domains or VHH domains.

[0042] In some embodiments, the polyspecific binding protein further comprises a second polypeptide chain, the second polypeptide chain comprising a first variable light chain domain (VL1) linked to a second variable light chain domain (VL2), wherein VL1 specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB, and VL2 specifically binds to ALK1, or VL1 specifically binds to ALK1 and VL2 specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB.

[0043] In some embodiments, VL1 is connected to VL2 via at least one modified hinge region.

[0044] In some embodiments, one or both of VH1 and VH2 are cleaved at the C-terminus.

[0045] In some embodiments, the C-terminus is cleaved at least one residue.

[0046] In some embodiments, the C-terminus is cleaved at least two residues.

[0047] In some embodiments, the C-terminal SS amino acid residue is removed.

[0048] In some embodiments, the polyspecific binding protein is VH1-HX1-VH2-C-Fc of the first polypeptide chain, however, VH1 is the first heavy chain variable domain, VH2 is the second heavy chain variable domain, C is a heavy chain constant domain, HX1 is a linker in the modified hinge region, Fc is an Fc region, and The second polypeptide chain is VL1-LX1-VL2-C, however, VL1 is the first light chain variable domain, VL2 is the second light chain variable domain, C is a light chain constant domain, LX1 includes the linker in the modified hinge region.

[0049] In some embodiments, the modified hinge region includes i) an upper hinge region that is up to 7 amino acids long or does not exist, and ii) a lower hinge region.

[0050] In some embodiments, the modified hinge region includes or consists of the amino acid sequence PLAP or PAPNLLGGP.

[0051] In some embodiments, the VH that binds to ALK1 includes the amino acid sequence SIAMS of HCDR1, the amino acid sequence NINQDGSEKNYVDSMRG of HCDR2, and the amino acid sequence EFDY of HCDR3, and the VL that binds to ALK1 includes the amino acid sequence SGSSSNIGSNYVY of LCDR1, the amino acid sequence GNKRPS of LCDR2, and the amino acid sequence AAWDDSLNGRV of LCDR3.

[0052] In some embodiments, the VH that binds to ALK1 includes the amino acid sequence SYWMS of HCDR1, the amino acid sequence NINQDGSEKYYVDSMRG of HCDR2, and the amino acid sequence EYDY of HCDR3, and the VL that binds to ALK1 includes the amino acid sequence SGSSSNIGSNYVY of LCDR1, the amino acid sequence GNKRPS of LCDR2, and the amino acid sequence AAWDDSLNGRV of LCDR3.

[0053] In some embodiments, the VH that binds to ALK1 includes the amino acid sequence SYWMS for HCDR1, the amino acid sequence NIKQDGSEKNYVDSMRG for HCDR2, and the amino acid sequence EFDF for HCDR3, and the VL that binds to ALK1 includes the amino acid sequence SGSSSNIGSNYVY for LCDR1, the amino acid sequence GNKRPS for LCDR2, and the amino acid sequence AAWDDSLNGRV for LCDR3.

[0054] In some embodiments, the VH that binds to BMPRII includes the amino acid sequence DYYMT of HCDR1, the amino acid sequence SISGGSTYYADSRKG of HCDR2, and the amino acid sequence DFGVAGWFGQYGMDV of HCDR3, and the VL that binds to BMPRII includes the amino acid sequence TGSSSNIGAGYDVH of LCDR1, the amino acid sequence RSNQRPS of LCDR2, and the amino acid sequence SSYAGNYNLV of LCDR3.

[0055] In some embodiments, the VH that binds to BMPRII includes the amino acid sequence DYYMN of HCDR1, the amino acid sequence SISGGSTYYADSVKG of HCDR2, and the amino acid sequence DFGVAGWFGQFGMDV of HCDR3, and the VL that binds to BMPRII includes the amino acid sequence TGSSSNIGAGYDVH of LCDR1, the amino acid sequence RSNQRPS of LCDR2, and the amino acid sequence SSYAGNYNLV of LCDR3.

[0056] In some embodiments, the VH that binds to BMPRII includes the amino acid sequence DYYMN of HCDR1, the amino acid sequence SISGGSTYYADSVKG of HCDR2, and the amino acid sequence DFGVAGWFGYYGMDV of HCDR3, and the VL that binds to BMPRII includes the amino acid sequence TGSSSNIGAGYDVH of LCDR1, the amino acid sequence RSNQRPS of LCDR2, and the amino acid sequence SSYAGNYNLV of LCDR3.

[0057] In some embodiments, the VH that binds to ALK1 includes the amino acid sequence SIAMS of HCDR1, the amino acid sequence NINQDGSEKNYVDSMRG of HCDR2, and the amino acid sequence EFDY of HCDR3; the VL that binds to ALK1 includes the amino acid sequence SGSSSNIGSNYVY of LCDR1, the amino acid sequence GNKRPS of LCDR2, and the amino acid sequence AAWDDSLNGRV of LCDR3; the VH that binds to BMPRII includes the amino acid sequence DYYMT of HCDR1, the amino acid sequence SISGGSTYYADSRKG of HCDR2, and the amino acid sequence DFGVAGWFGQYGMDV of HCDR3; and the VL that binds to BMPRII includes the amino acid sequence TGSSSNIGAGYDVH of LCDR1, the amino acid sequence RSNQRPS of LCDR2, and the amino acid sequence SSYAGNYNLV of LCDR3.

[0058] In some embodiments, the VH that binds to ALK1 includes the amino acid sequence SYWMS of HCDR1, the amino acid sequence NINQDGSEKYYVDSMRG of HCDR2, and the amino acid sequence EYDY of HCDR3; the VL that binds to ALK1 includes the amino acid sequence SGSSSNIGSNYVY of LCDR1, the amino acid sequence GNKRPS of LCDR2, and the amino acid sequence AAWDDSLNGRV of LCDR3; the VH that binds to BMPRII includes the amino acid sequence DYYMN of HCDR1, the amino acid sequence SISGGSTYYADSVKG of HCDR2, and the amino acid sequence DFGVAGWFGQFGMDV of HCDR3; and the VL that binds to BMPRII includes the amino acid sequence TGSSSNIGAGYDVH of LCDR1, the amino acid sequence RSNQRPS of LCDR2, and the amino acid sequence SSYAGNYNLV of LCDR3.

[0059] In some embodiments, the VH that binds to ALK1 includes the amino acid sequence SYWMS of HCDR1, the amino acid sequence NIKQDGSEKNYVDSMRG of HCDR2, and the amino acid sequence EFDF of HCDR3; the VL that binds to ALK1 includes the amino acid sequence SGSSSNIGSNYVY of LCDR1, the amino acid sequence GNKRPS of LCDR2, and the amino acid sequence AAWDDSLNGRV of LCDR3; the VH that binds to BMPRII includes the amino acid sequence DYYMN of HCDR1, the amino acid sequence SISGGSTYYADSVKG of HCDR2, and the amino acid sequence DFGVAGWFGYYGMDV of HCDR3; and the VL that binds to BMPRII includes the amino acid sequence TGSSSNIGAGYDVH of LCDR1, the amino acid sequence RSNQRPS of LCDR2, and the amino acid sequence SSYAGNYNLV of LCDR3.

[0060] In some embodiments, VH bound to ALK1 is an amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVANINQDGSEKNYVDSMRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREFDYWGQGTLVTVSS, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. VL, which contains an amino acid sequence and binds to ALK1, includes the amino acid sequence QSVLAQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYGNNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGRVFGGGTKLTVL, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0061] In some embodiments, the VH bound to ALK1 is an amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINQDGSEKYYVDSMRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREYDYWGQGTLVTVSS, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. VL, which contains an amino acid sequence and binds to ALK1, includes the amino acid sequence QSVLAQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYGNNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGRVFGGGTKLTVL, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0062] In some embodiments, VH bound to ALK1 is an amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKNYVDSMRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREFDFWGQGTLVTVSS, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to it. VL, which contains an amino acid sequence and binds to ALK1, includes the amino acid sequence QSVLAQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYGNNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGRVFGGGTKLTVL, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0063] In some embodiments, VH bound to BMPRII has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMTWIRQAPGKGLEWVSSISGGSTYYADSRKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDFGVAGWFGQYGMDVWGQGTLVTVSS, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to it. The VL that binds to BMPRII contains an amino acid sequence having the amino acid sequence QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYRSNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCSSYAGNYNLVFGGGTKLTVL, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to it.

[0064] In some embodiments, VH bound to BMPRII has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMNWIRQAPGKGLEWVSSISGGSTYYADSVKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDFGVAGWFGQFGMDVWGQGTLVTVSS, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to it. The VL that binds to BMPRII contains an amino acid sequence having the amino acid sequence QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYRSNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCSSYAGNYNLVFGGGTKLTVL, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to it.

[0065] In some embodiments, VH bound to BMPRII has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMNWIRQAPGKGLEWVSSISGGSTYYADSVKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDFGVAGWFGYYGMDVWGQGTLVTVSS, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to it. The VL that binds to BMPRII contains an amino acid sequence having the amino acid sequence QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYRSNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCSSYAGNYNLVFGGGTKLTVL, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to it.

[0066] In some embodiments, the VH that binds to ALK1 is the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVANINQDGSEKNYVDSMRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREFDYWGQGTLVTVSS, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. VL, which contains an amino acid sequence having % identity and binds to ALK1, is the amino acid sequence QSVLAQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYGNNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGRVFGGGTKLTVL, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 9% VH that contains an amino acid sequence with 9% identity and binds to BMPRII is the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMTWIRQAPGKGLEWVSSISGGSTYYADSRKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDFGVAGWFGQYGMDVWGQGTLVTVSS, or, relative to it, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, and less Both contain amino acid sequences having 98% or at least 99% identity, and the VL that binds to BMPRII is the amino acid sequence QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYRSNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCSSYAGNYNLVFGGGTKLTVL, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%,It contains an amino acid sequence with at least 98% or at least 99% identity.

[0067] In some embodiments, the VH that binds to ALK1 is the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINQDGSEKYYVDSMRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREYDYWGQGTLVTVSS, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. VL, which contains an amino acid sequence having % identity and binds to ALK1, is the amino acid sequence QSVLAQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYGNNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGRVFGGGTKLTVL, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 9% VH that contains an amino acid sequence with 9% identity and binds to BMPRII is the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMNWIRQAPGKGLEWVSSISGGSTYYADSVKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDFGVAGWFGQFGMDVWGQGTLVTVSS, or, relative to it, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, and less Both contain amino acid sequences having 98% or at least 99% identity, and the VL that binds to BMPRII is the amino acid sequence QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYRSNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCSSYAGNYNLVFGGGTKLTVL, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%,It contains an amino acid sequence with at least 98% or at least 99% identity.

[0068] In some embodiments, the VH that binds to ALK1 is the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKNYVDSMRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREFDFWGQGTLVTVSS, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. VL, which contains an amino acid sequence having % identity and binds to ALK1, is the amino acid sequence QSVLAQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYGNNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGRVFGGGTKLTVL, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 9% VH that contains an amino acid sequence with 9% identity and binds to BMPRII is the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMNWIRQAPGKGLEWVSSISGGSTYYADSVKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDFGVAGWFGYYGMDVWGQGTLVTVSS, or, relative to it, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, and less Both contain amino acid sequences having 98% or at least 99% identity, and the VL that binds to BMPRII is the amino acid sequence QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYRSNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCSSYAGNYNLVFGGGTKLTVL, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%,It contains an amino acid sequence with at least 98% or at least 99% identity.

[0069] In some embodiments, the first polypeptide chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 136-142, and the second polypeptide chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 143-146.

[0070] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0071] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0072] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0073] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0074] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0075] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 142, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0076] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0077] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0078] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 73, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0079] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 74, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0080] In one embodiment, the Disclosure provides a polyspecific binding protein comprising a first polypeptide chain and a second polypeptide chain, each comprising a first single-chain variable fragment (scFv) linked from the N-terminus to the C-terminus of a second scFv, wherein the first scFv specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB, and the second scFv specifically binds to ALK1, or the first scFv specifically binds to ALK1 and the second scFv specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB.

[0081] In some embodiments, the first scFv is connected to the second scFv via at least one modified hinge region.

[0082] In some embodiments, the scFv that binds to ALK1 includes a VH domain comprising the amino acid sequence SIAMS of HCDR1, the amino acid sequence NINQDGSEKNYVDSMRG of HCDR2, and the amino acid sequence EFDY of HCDR3, and the VL that binds to ALK1 includes the amino acid sequence SGSSSNIGSNYVY of LCDR1, the amino acid sequence GNKRPS of LCDR2, and the amino acid sequence AAWDDSLNGRV of LCDR3.

[0083] In some embodiments, the scFv that binds to ALK1 includes a VH domain comprising the amino acid sequence SYWMS of HCDR1, the amino acid sequence NINQDGSEKYYVDSMRG of HCDR2, and the amino acid sequence EYDY of HCDR3, as well as a VL domain comprising the amino acid sequence SGSSSNIGSNYVY of LCDR1, the amino acid sequence GNKRPS of LCDR2, and the amino acid sequence AAWDDSLNGRV of LCDR3.

[0084] In some embodiments, the scFv that binds to ALK1 includes a VH domain comprising the amino acid sequence SYWMS of HCDR1, the amino acid sequence NIKQDGSEKNYVDSMRG of HCDR2, and the amino acid sequence EFDF of HCDR3, as well as a VL domain comprising the amino acid sequence SGSSSNIGSNYVY of LCDR1, the amino acid sequence GNKRPS of LCDR2, and the amino acid sequence AAWDDSLNGRV of LCDR3.

[0085] In some embodiments, the scFv that binds to BMPRII includes a VH domain comprising the amino acid sequence DYYMT of HCDR1, the amino acid sequence SISGGSTYYADSRKG of HCDR2, and the amino acid sequence DFGVAGWFGQYGMDV of HCDR3, as well as a VL domain comprising the amino acid sequence TGSSSNIGAGYDVH of LCDR1, the amino acid sequence RSNQRPS of LCDR2, and the amino acid sequence SSYAGNYNLV of LCDR3.

[0086] In some embodiments, the scFv that binds to BMPRII includes a VH domain containing the amino acid sequence DYYMN of HCDR1, the amino acid sequence SISGGSTYYADSVKG of HCDR2, and the amino acid sequence DFGVAGWFGQFGMDV of HCDR3, as well as a VL domain containing the amino acid sequence TGSSSNIGAGYDVH of LCDR1, the amino acid sequence RSNQRPS of LCDR2, and the amino acid sequence SSYAGNYNLV of LCDR3.

[0087] In some embodiments, the scFv that binds to BMPRII includes a VH domain comprising the amino acid sequence DYYMN of HCDR1, the amino acid sequence SISGGSTYYADSVKG of HCDR2, and the amino acid sequence DFGVAGWFGYYGMDV of HCDR3, as well as a VL domain comprising the amino acid sequence TGSSSNIGAGYDVH of LCDR1, the amino acid sequence RSNQRPS of LCDR2, and the amino acid sequence SSYAGNYNLV of LCDR3.

[0088] In some embodiments, the scFv that binds to ALK1 includes a VH domain containing the amino acid sequence SIAMS of HCDR1, NINQDGSEKNYVDSMRG of HCDR2, and EFDY of HCDR3; the VL that binds to ALK1 includes the amino acid sequence SGSSSNIGSNYVY of LCDR1, GNKRPS of LCDR2, and AAWDDSLNGRV of LCDR3; the scFv that binds to BMPRII includes a VH domain containing the amino acid sequence DYYMT of HCDR1, SISGGSTYYADSRKG of HCDR2, and DFGVAGWFGQYGMDV of HCDR3, as well as a VL domain containing the amino acid sequence TGSSSNIGAGYDVH of LCDR1, RSNQRPS of LCDR2, and SSYAGNYNLV of LCDR3.

[0089] In some embodiments, the scFv that binds to ALK1 includes a VH domain containing the amino acid sequence SYWMS of HCDR1, the amino acid sequence NINQDGSEKYYVDSMRG of HCDR2, and the amino acid sequence EYDY of HCDR3, as well as a VL domain containing the amino acid sequence SGSSSNIGSNYVY of LCDR1, the amino acid sequence GNKRPS of LCDR2, and the amino acid sequence AAWDDSLNGRV of LCDR3. The scFv that binds to BMPRII includes a VH domain containing the amino acid sequence DYYMN of HCDR1, the amino acid sequence SISGGSTYYADSVKG of HCDR2, and the amino acid sequence DFGVAGWFGQFGMDV of HCDR3, as well as a VL domain containing the amino acid sequence TGSSSNIGAGYDVH of LCDR1, the amino acid sequence RSNQRPS of LCDR2, and the amino acid sequence SSYAGNYNLV of LCDR3.

[0090] In some embodiments, the scFv that binds to ALK1 includes a VH domain containing the amino acid sequence SYWMS of HCDR1, the amino acid sequence NIKQDGSEKNYVDSMRG of HCDR2, and the amino acid sequence EFDF of HCDR3, as well as a VL domain containing the amino acid sequence SGSSSNIGSNYVY of LCDR1, the amino acid sequence GNKRPS of LCDR2, and the amino acid sequence AAWDDSLNGRV of LCDR3. The scFv that binds to BMPRII includes a VH domain containing the amino acid sequence DYYMN of HCDR1, the amino acid sequence SISGGSTYYADSVKG of HCDR2, and the amino acid sequence DFGVAGWFGYYGMDV of HCDR3, as well as a VL domain containing the amino acid sequence TGSSSNIGAGYDVH of LCDR1, the amino acid sequence RSNQRPS of LCDR2, and the amino acid sequence SSYAGNYNLV of LCDR3.

[0091] In some embodiments, the scFv that binds to ALK1 is an amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVANINQDGSEKNYVDSMRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREFDYWGQGTLVTVSS, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. The material comprises a VH domain containing an amino acid sequence, and a VL domain containing an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the same.

[0092] In some embodiments, the scFv bound to BMPRII has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMTWIRQAPGKGLEWVSSISGGSTYYADSRKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDFGVAGWFGQYGMDVWGQGTLVTVSS, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to it. The VH domain comprises an amino acid sequence having the amino acid sequence QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYRSNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCSSYAGNYNLVFGGGTKLTVL, or a VL domain comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0093] In some embodiments, the scFv that binds to ALK1 includes the amino acid sequence of SEQ ID NO: 120, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0094] In some embodiments, the scFv that binds to ALK1 includes the amino acid sequence of SEQ ID NO: 122, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0095] In some embodiments, the scFv that binds to BMPRII includes the amino acid sequence of SEQ ID NO: 121, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0096] In some embodiments, the scFv that binds to ALK1 includes the amino acid sequence of SEQ ID NO: 123, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0097] In some embodiments, the first and second polypeptide chains each include one amino acid sequence of any one of SEQ ID NOs. 60 to 63, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0098] In some embodiments, the multispecific binding protein can induce signal transduction by inducing proximity between ALK1 and BMPRII, ActRIIA, or ActRIIB.

[0099] In some embodiments, the polyspecific binding protein exhibits higher agonist activity compared to the polyspecific binding protein lacking at least one modified hinge region.

[0100] In some embodiments, the polyspecific binding protein induces at least about 35% of BMP9 activity.

[0101] In some embodiments, BMP9 activity is identified by measuring phosphorylated SMAD1 (pSMAD1) levels in cells incubated with a multispecific binding protein and / or cells incubated with BMP9.

[0102] In some embodiments, the melting onset temperature (Tonset) for the unfolding of the polyspecific binding protein is at least about 55°C.

[0103] In some embodiments, the thermal transition midpoint (Tm) of the melting temperature of the polyspecific binding protein is at least about 64°C.

[0104] In some embodiments, the polyspecific binding proteins Tonset and Tm are identified by differential scanning calorimetry (DSC).

[0105] In some embodiments, the polyspecific binding protein can stimulate the expression of ID1 in cells.

[0106] In some embodiments, the expression of ID1 in these cells is at least 50% of the expression of ID1 in cells incubated with BMP9.

[0107] In some embodiments, the first polypeptide chain further includes a heavy chain constant region.

[0108] In some embodiments, the heavy chain constant region includes a substitution at the 234th amino acid position according to EU numbering.

[0109] In some embodiments, the amino acid substitution at position 234 is alanine (A).

[0110] In some embodiments, the heavy chain constant region includes a substitution at the 235th amino acid position according to EU numbering.

[0111] In some embodiments, the amino acid substitution at position 235 is alanine (A).

[0112] In some embodiments, the heavy chain constant region includes a substitution at the 237th amino acid position according to EU numbering.

[0113] In some embodiments, the amino acid substitution at position 237 is alanine (A).

[0114] In some embodiments, the heavy chain constant region includes one or more substitutions at the EU numbered amino acid positions 234, 235, or 237.

[0115] In some embodiments, the substitution at amino acid position 234 is alanine(A), the substitution at amino acid position 235 is alanine(A), and the substitution at amino acid position 237 is alanine(A).

[0116] In some embodiments, the heavy chain constant region includes a heterodimerizing mutation that promotes heterodimerization between the first binding site and the second binding site.

[0117] In some embodiments, the heterodimerizing mutation is a knob-in-hole (KIH) mutation.

[0118] In some embodiments, the first heavy chain constant region includes an amino acid substitution that generates a hole at position 366, 368, or 407, and the second heavy chain constant region includes an amino acid substitution that generates a knob at position 366.

[0119] In some embodiments, the first heavy chain constant region includes the amino acid substitution T366S, L368A, or Y407V, and the second heavy chain constant region includes the amino acid substitution T366W.

[0120] In some embodiments, heterodimerizing mutations are charge-stabilizing mutations.

[0121] In some embodiments, the first heavy chain constant region includes the amino acid substitution N297K, and the second heavy chain constant region includes the amino acid substitution N297D.

[0122] In some embodiments, the first heavy chain constant region includes the amino acid substitution T299K, and the second heavy chain constant region includes the amino acid substitution T299D.

[0123] In some embodiments, the heterodimerizing mutation includes an engineered disulfide bond.

[0124] In some embodiments, the engineered disulfide bond is formed by a first heavy chain constant region containing the amino acid substitution Y349C and a second heavy chain constant region containing the amino acid substitution S354C.

[0125] In some embodiments, the engineered disulfide bond is formed by a C-terminal extension peptide fused to the C-terminus of the first and second heavy chain constant regions, respectively.

[0126] In some embodiments, the C-terminal extension of the first heavy chain constant region includes the amino acid sequence GEC, and the C-terminal extension of the second heavy chain constant region includes the amino acid sequence SCDKT.

[0127] In some embodiments, at least one heavy chain constant region contains one or more mutations that promote half-life extension.

[0128] In some embodiments, at least one heavy chain constant region includes one or more substitutions at the EU numbered amino acid positions 252, 254, or 256.

[0129] In some embodiments, the substitution at amino acid position 252 is tyrosine (Y), the substitution at amino acid position 254 is threonine (T), and the substitution at amino acid position 256 is glutamic acid (E).

[0130] In some embodiments, at least one heavy chain constant region includes one or more substitutions at the 428th or 434th amino acid position according to EU numbering.

[0131] In some embodiments, at least one heavy chain constant region includes substitution of M428L and N434S according to EU numbering.

[0132] In one embodiment, the present disclosure provides a pharmaceutical composition comprising a polyspecific binding protein and a pharmaceutically acceptable carrier as described herein.

[0133] In one embodiment, the present disclosure provides an isolated nucleic acid molecule encoding a polyspecific binding protein described herein.

[0134] In one embodiment, the present disclosure provides an expression vector comprising a nucleic acid molecule described herein.

[0135] In one embodiment, the present disclosure provides a host cell containing the expression vector described herein.

[0136] In one embodiment, the present disclosure provides a method for treating a disease or disorder in a subject, the method comprising administering a polyspecific binding protein described herein to a subject in need thereof.

[0137] In some embodiments, the disease or disorder is a disease or disorder of the vascular system.

[0138] In some embodiments, the vascular disease or disorder is hereditary hemorrhagic telangiectasia (HHT).

[0139] In some embodiments, the vascular disease or disorder is pulmonary hypertension (PAH).

[0140] In some embodiments, the polyspecific binding protein is used as a pharmaceutical product.

[0141] In one embodiment, the Disclosure provides a method for inducing signaling between ALK1 and BMPRII, ActRIIA, or ActRIIB in a subject, the method comprising administering a multispecific binding protein described herein to the subject.

[0142] In some embodiments, the multispecific binding protein can induce signal transduction by inducing proximity between ALK1 and BMPRII, ActRIIA, or ActRIIB.

[0143] In some embodiments, the polyspecific binding protein exhibits higher agonist activity compared to the polyspecific binding protein lacking at least one modified hinge region.

[0144] In some embodiments, the polyspecific binding protein induces at least about 35% of BMP9 activity.

[0145] In some embodiments, BMP9 activity is identified by measuring phosphorylated SMAD1 (pSMAD1) levels in cells incubated with a multispecific binding protein and / or cells incubated with BMP9. [Brief explanation of the drawing]

[0146] [Figure 1] This figure shows a specific exemplary embodiment of the bispecific antibody format described herein. [Figure 2] This is a schematic diagram illustrating the workflow for characterizing the bispecific antibodies of this disclosure. [Figure 3]Graphs A-C show arteriovenous malformations (AVMs) in the retina of HHT mouse models. Graph A compares mice treated with a control (no bispecific antibody) to those treated with DGL288 (15 mg / kg / day). Mice treated with DGL288 did not develop detectable AVMs compared to the control. Graph B shows that mice treated with 1 mg / kg / day of DGL292 did not develop AVMs compared to mice treated with the control. Graph C demonstrates that DGL288 administered at a dose of 1 mg / kg / day also did not develop AVMs compared to mice treated with the control. [Figure 4] This graph shows arteriovenous malformations (AVMs) in the retina of an HHT mouse model. Mice were treated with a control (no bispecific antibody) and compared with those treated with DGL292, DGL945, and DGL947 (1 mg / kg / day). Mice treated with DGL292, DGL945, and DGL947 did not develop detectable AVMs compared to the control. [Modes for carrying out the invention]

[0147] Before describing this disclosure, it should be understood that this disclosure is not limited to the specific methods and experimental conditions described, as methods and conditions can vary. Furthermore, since the scope of this disclosure is limited only by the appended claims, it should be understood that the terminology used herein is for illustrative purposes only and not intended to limit any particular embodiment.

[0148] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure pertains.

[0149] In carrying out this disclosure, any methods and materials similar to or equivalent to those described herein may be used, but exemplary methods and materials are described below. All published documents referenced herein are incorporated herein by reference to their entirety.

[0150] As used herein, the term “antibody” (singular and plural) includes full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules containing the CDR, VH, and / or VL regions of an antibody. Examples of antibodies include, but are not limited to, monoclonal antibodies, recombinant antibodies, monospecific antibodies, polyspecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies containing two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, antibody-drug conjugates, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fvs(scFv), camelized antibodies, aphibodies, common light chain antibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs(sdFv), anti-idiotype (anti-Id) antibodies (e.g., anti-anti-Id antibodies), bivariable domains (DVDs), and any of the antigen-binding fragments described above. In certain embodiments, the antibodies described herein refer to a population of polyclonal antibodies. Antibodies can be immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a, or IgG2b). In certain embodiments, the antibodies described herein are IgG antibodies, or their class (e.g., human IgG1, or IgG4), or subclasses. As used herein, the terms "VH" and "VL" refer to the variable domains of the heavy and light chains of an antibody, respectively, as described in Kabat et al., (1991) Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242, Bethesda) (which is incorporated herein by reference in its entirety).

[0151] As used herein, the term "VHH" refers to the heavy chain variable domain of camelid heavy chain-only antibodies (HCAbs) and their humanized variants, as described in Hamers-Casterman C. et al., Nature (1993) 363:446-8.10.1038 / 363446a0 (which is incorporated herein by reference in its entirety).

[0152] As used herein, the term "VH / VL pair" refers to a combination of VH and VL that, together, form a binding site for an antigen.

[0153] As used herein, the term “heavy chain” as used in relation to antibodies may refer to any of the specific types, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain that gives rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, including subclasses of IgG, such as IgG1, IgG2, IgG3, and IgG4.

[0154] As used herein, the term "full-length antibody heavy chain" refers to an antibody heavy chain comprising the VH, CH1 region, hinge region, CH2 domain, and CH3 domain from the N-terminus to the C-terminus.

[0155] As used herein, the term “light chain” as used in reference to an antibody may refer to one of a specific type, such as kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In certain embodiments, the light chain is a human light chain. As used herein, the term “complementarity-determining region” or “CDR” refers to the sequence of amino acids within the antibody variable region that confer antigen specificity and binding affinity. Generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, CDR-H3), and each light chain variable region has three CDRs (CDR-L1, CDR-L2, CDR-L3). The “framework region” or “FR” is known in the art to refer to the portion of the heavy chain and light chain variable regions other than the CDRs. Generally, each heavy chain variable region has four FRs (FR-H1, FR-H2, FR-H3, and FR-H4), and each light chain variable region has four FRs (FR-L1, FR-L2, FR-L3, and FR-L4).

[0156] The precise amino acid sequence boundaries of specific CDRs or FRs are determined using the following schemes: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J.Mol.Biol.262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J.Mol.Biol.262, 732-745. (“Contact” numbering scheme); and Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like The domains can be easily determined using one of several well-known schemes, including the scheme described in "domains," Dev.Comp.Immunol., 2003 January;27(1):55-77 ("IMGT" numbering scheme) and the scheme described in Honegger A. and Pluckthun A., "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J.Mol.Biol., 2001 Jun.8;309(3):657-70 (AHo numbering scheme).

[0157] The boundaries of specific CDRs or FRs can differ depending on the scheme used for identification. For example, the Kabat scheme is based on sequence alignment, while the Chothia scheme is based on structural information. The numbering in both the Kabat and Chothia schemes is based on the most common antibody region sequence length, and some antibodies have insertions and deletions given by insertion letters, e.g., "30a". The difference in numbering arises because these two schemes place specific insertions and deletions ("indels") in different positions. The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme.

[0158] As used herein, the term “single-chain variable fragment” (scFv) refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are adjacently linked by a short, flexible polypeptide linker, and the scFv is expressible as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, the scFv as used herein may have the VL and VH variable regions in any order, for example, with respect to the N-terminus and C-terminus of the polypeptide, and the scFv may contain VL·linker·VH or VH·linker·VL.

[0159] As used herein, the term “human antibody” is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs of this disclosure may include, for example, amino acid residues not encoded by human germline immunoglobulin sequences in the CDR, particularly CDR3 (mutations introduced, for example, by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo). However, as used herein, the term “human antibody” is not intended to include mAbs in which a CDR sequence derived from the germline of another mammalian species (e.g., mouse) is grafted onto a human FR sequence. This term includes antibodies recombinantly produced in or from non-human mammals. This term is not intended to include antibodies isolated from or generated in human subjects.

[0160] As used herein, the term “multispecific antigen-binding molecule” refers to dispecific, trispecific, or multispecific antigen-binding molecules and their antigen-binding fragments. A multispecific antigen-binding molecule may be specific to a different epitope in one target polypeptide, or it may contain antigen-binding domains specific to epitopes in multiple target polypeptides. A multispecific antigen-binding molecule may be a single polyfunctional polypeptide, or it may be a multimeric complex of two or more polypeptides covalently or non-covalently bonded to each other. The term “multispecific antigen-binding molecule” includes antibodies of this disclosure that can be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof may be functionally linked to one or more other molecular entities, such as a protein or fragment thereof, to produce a dispecific or multispecific antigen-binding molecule having a second binding specificity (e.g., by chemical bonding, gene fusion, non-covalent bonding, or otherwise). According to this disclosure, the term “multispecific antigen-binding molecule” also includes dispecific, trispecific, or multispecific antibodies or their antigen-binding fragments. In certain exemplary embodiments, the antibody of the present disclosure is functionally ligated to another antibody or its antigen-binding fragment in order to produce a bispecific antibody having a second binding specificity.

[0161] As used herein, the terms "valence" or "valency" refer to the number of binding sites in an antibody molecule. For example, the term "bivalent" refers to the presence of two binding sites. In some embodiments, an antibody molecule may be polyvalent. Thus, the term "trivalent" refers to three binding sites, and the term "tetravalent" refers to four binding sites. In some embodiments, more than four binding sites may be present. In some embodiments, binding sites may bind to the same antigen. In some embodiments, binding sites may bind to different antigens.

[0162] In some embodiments, the polyvalent antibody molecule of the present invention is a multichain molecule having one or more binding sites on each chain.

[0163] For example, in one embodiment, the polyvalent molecule is a divalent molecule having one binding site (e.g., VHH or scFV) on the first chain and a second binding site on the second chain. In another embodiment, the polyvalent molecule is a divalent molecule having two binding sites on the first chain and no binding sites on the second chain.

[0164] In another embodiment, the polyvalent molecule is a trivalent molecule having one binding site (e.g., VHH or scFV) on the first chain and second and third binding sites on the second chain. In yet another embodiment, the polyvalent molecule is a trivalent molecule having three binding sites on the first chain and no binding sites on the second chain.

[0165] In another embodiment, the polyvalent molecule is a tetravalent molecule having two binding sites on the first chain and two binding sites on the second chain. In another embodiment, the polyvalent molecule is a tetravalent molecule having three binding sites on the first chain and one binding site on the second chain. In yet another embodiment, the polyvalent molecule is a tetravalent molecule having four binding sites on the first chain and no binding sites on the second chain.

[0166] In exemplary embodiments, the heteromeric antibody of the present disclosure is a bispecific antibody. The bispecific antibody may be a monoclonal antibody, such as a human antibody or a humanized antibody, having binding specificity to at least two different antigens.

[0167] Methods for producing bispecific antibodies are well known. Traditionally, recombinant production of bispecific antibodies has been based on the co-expression of two immunoglobulin heavy / light chain pairs, each with two heavy chains having different specificities (Milstein et al., Nature 305:537 (1983)). Because the heavy and light chains of immunoglobulins are combined randomly, a hybridoma (quadroma) can produce a mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually achieved by affinity chromatography. More modern techniques for generating bispecific antibodies utilize heterodimerizing domains that favor the desired pairing of the heavy chain of an antibody with primary specificity with the heavy chain of an antibody with secondary specificity.

[0168] An antibody variable domain with desired binding specificity can be fused to an immunoglobulin constant domain sequence. This fusion is typically with an immunoglobulin heavy chain constant domain containing at least a portion of the hinge, CH2, and CH3 regions. This may have a first heavy chain constant region (CH1) containing a site necessary for light chain binding, present in at least one of the fusions. DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain is inserted into a separate expression vector and co-transfected into a suitable host organism. For further details on the generation of bispecific antibodies, see, for example, Suresh et al., Meth. Enzymol. 121:210 (1986).

[0169] As used herein, the term "Fc" refers to a polypeptide comprising a CH2 domain and a CH3 domain, wherein the C-terminus of the CH2 domain is (directly or indirectly) linked to the N-terminus of the CH3 domain. The term "Fc polypeptide" includes antibody heavy chains (e.g., those forming half-antibodies) linked to antibody light chains by disulfide bonds.

[0170] In certain embodiments, the Fc chain begins in a hinge region immediately upstream of the papain cleavage site of the antibody and ends at the C-terminus. Thus, a complete Fc chain includes at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, the Fc chain includes at least one of the hinge domains (e.g., upper, central, and / or lower hinge regions), a CH2 domain, a CH3 domain, a CH4 domain, or a variant, part, or fragment thereof. In certain embodiments, the Fc domain includes a complete Fc chain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In certain embodiments, the Fc chain includes a hinge domain (or part thereof) fused to a CH3 domain (or part thereof). In certain embodiments, the Fc chain includes a CH2 domain (or part thereof) fused to a CH3 domain (or part thereof). In certain embodiments, the Fc chain consists of a CH3 domain or part thereof. In certain embodiments, the Fc chain consists of a hinge domain (or part thereof) and a CH3 domain (or part thereof). In certain embodiments, the Fc chain consists of a CH2 domain (or a portion thereof) and a CH3 domain. In certain embodiments, the Fc chain consists of a hinge domain (or a portion thereof) and a CH2 domain (or a portion thereof). In certain embodiments, the Fc chain lacks at least a portion of the CH2 domain (e.g., all or a portion of the CH2 domain). As used herein, the Fc chain generally refers to a polypeptide comprising all or a portion of the Fc chain of an immunoglobulin heavy chain. This includes, but is not limited to, polypeptides comprising the entire CH1, hinge, CH2, and / or CH3 domains, as well as fragments of such peptides comprising only, for example, the hinge, CH2, and CH3 domains. The Fc chain may originate from any species and / or any subtype of immunoglobulin, including but not limited to human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. The Fc domain encompasses native Fc and Fc variant molecules. Similar to Fc variants and natural Fc, the term Fc chain includes molecules in monomeric or polymeric form, whether digested from the whole antibody or produced by other means.In some embodiments, the Fc chain includes the carboxyl terminal portions of both heavy chains linked by disulfide bonds. In certain embodiments, the Fc chain consists of a CH2 domain and a CH3 domain.

[0171] In some embodiments, the Fc polypeptide contains some or all of the wild-type hinge sequence (generally at its N-terminus). In some embodiments, the Fc polypeptide does not contain the functional or wild-type hinge sequence.

[0172] As used herein, the term “CH1 domain” refers to the first constant domain of the antibody heavy chain (e.g., amino acid positions 118–215 of human IgG1, according to the EU index). This term includes the naturally occurring CH1 domain and engineered variants of the naturally occurring CH1 domain (e.g., CH1 domains that include one or more amino acid insertions, deletions, substitutions, or modifications compared to the naturally occurring CH1 domain).

[0173] As used herein, the term “CH2 domain” refers to the second constant domain of the antibody heavy chain (e.g., amino acid positions 231–340 of human IgG1, according to the EU index). This term includes both the naturally occurring CH2 domain and engineered variants of the naturally occurring CH2 domain (e.g., a CH2 domain that includes one or more amino acid insertions, deletions, substitutions, or modifications compared to the naturally occurring CH2 domain).

[0174] As used herein, the term “CH3 domain” refers to the third constant domain of the antibody heavy chain (e.g., amino acid positions 341–447 of human IgG1, according to the EU index). This term includes both the naturally occurring CH3 domain and engineered variants of the naturally occurring CH3 domain (e.g., a CH3 domain that includes one or more amino acid insertions, deletions, substitutions, or modifications compared to the naturally occurring CH3 domain).

[0175] As used herein, the term “hinge region” refers to a portion of an antibody heavy chain that contains cysteine ​​residues mediating a disulfide bond between two heavy chains in an intact antibody (e.g., the cysteine ​​residues at positions 226 and 229 of human IgG1 according to the EU index). This term includes spontaneously occurring hinge regions and engineered variants of spontaneously occurring hinge regions (e.g., hinge regions involving one or more amino acid insertions, deletions, substitutions, or modifications compared to a spontaneously occurring hinge region). An exemplary hinge region of full-length IgG1 contains amino acids 216–230 of human IgG1 according to the EU index. A hinge region may consist of at least two (e.g., five, ten, fifteen, twenty, forty, sixty, or more) amino acids that result in a flexible or semiflexible bond between adjacent variable regions and / or constant domains in a single polypeptide molecule. In some embodiments, the hinge region is an immunoglobulin-like hinge region. In some embodiments, the immunoglobulin-like hinge region may be or be derived from any IgG1, IgG2, IgG3, or IgG4 subtype, or IgA, IgE, IgD, or IgM (including their chimeric forms, e.g., a chimeric IgG1 / 2 hinge region).

[0176] In some embodiments, the hinge region may be of a human IgG1 subtype, extending from amino acid 216 to amino acid 230 according to the EU Index numbering system, or from amino acid 226 to amino acid 243 according to the Kabat numbering system. Those skilled in the art may have different understandings of the exact amino acids corresponding to various domains of the IgG molecule. Therefore, the N-terminus or C-terminus of the domains outlined above may be extended or shortened by one, two, three, four, five, six, seven, eight, nine, or even ten amino acids.

[0177] As used herein, the term “upper hinge” typically refers to the last residue of the CH1 domain up to, but excluding, the first interchain cysteine. The upper hinge can sometimes be defined as the N-terminal sequence from position 216 to 225 according to the Kabat EU numbering system for IgG1 antibodies (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, Md., 1991). The term “central hinge” refers to the region extending from the first interchain cysteine ​​to the proline residue adjacent to the carboxyl terminus of the last central hinge cysteine. The central hinge can be the N-terminal sequence from position 226 to 230 according to the Kabat EU numbering system. The term “lower hinge” refers to a highly conserved sequence of 7-8 amino acids. The lower hinge can be defined as the sequence from position 231-238 according to the Kabat EU numbering system for IgG1 antibodies. In some embodiments, the antibody according to the present invention substantially comprises upper, central, and lower hinges.

[0178] As used herein, the term “modified hinge region” refers to a hinge region that has been altered in one or more of the hinge characteristics, including but not limited to, flexibility, length, conformation, charge, and hydrophobicity, compared to a wild-type hinge. Modified hinge regions disclosed herein can be generated by methods well known in the art, such as introducing modifications to a wild-type hinge. In some embodiments, the hinge region may be modified by one or more amino acids. Modifications that may be used to generate a modified hinge region include, but are not limited to, insertions, deletions, substitutions, and rearrangements of amino acids. The above modifications to the disclosed hinges and modified hinge regions are collectively referred herein to as “hinge modifications of the present invention,” “modified hinges of the present invention,” or simply “hinge modifications” or “modified hinges.” Modified hinge regions disclosed herein can be incorporated into optimal molecules, including, but not limited to, antibodies and fragments thereof. In some embodiments, the hinge region may be shortened and may include only a portion of the complete hinge region.

[0179] As described herein, molecules containing a modified hinge may exhibit altered (e.g., enhanced) agonist activity compared to molecules having the same amino acid sequence except for the modified hinge, such as a molecule having the same amino acid sequence except for the inclusion of a wild-type hinge. In some embodiments, the antibody contains a modified hinge region, the upper hinge region being up to 7 amino acids long. In some embodiments, the upper hinge region is absent. In some embodiments, the modified hinge is a modified IgG1 linker. In some embodiments, the modified IgG1 hinge is derived from the sequence PLAPDKTHT (SEQ ID NO: 1). In some embodiments, the modified IgG1 hinge contains the sequence PLAP (SEQ ID NO: 2). In some embodiments, the modified IgG1 hinge contains the sequence DKTHT (SEQ ID NO: 5). In some embodiments, the modified hinge is a modified IgG4 hinge. In some embodiments, the modified IgG1 hinge contains the sequence EKSYGPP (SEQ ID NO: 4). In some embodiments, the modified hinge is a Gly / Ser hinge. In some embodiments, the Gly / Ser hinge contains the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 3). In some embodiments, the C-terminal residue of the variable domain adjacent to the upper hinge is cleaved. In some embodiments, at least one residue of the variable domain adjacent to the upper hinge is cleaved. In some embodiments, at least two residues of the variable domain adjacent to the upper hinge are cleaved.

[0180] The modified hinge regions of this disclosure may be used as linkers for linking one or more antigen-binding domains of this disclosure. In certain embodiments, a first variable heavy chain domain (VH1) is linked to a second variable heavy chain domain (VH2) via at least one modified hinge region. In certain embodiments, a first variable light chain domain (VL1) is linked to a second variable light chain domain (VL2) via at least one modified hinge region. VH1 and VL1 associate to form a first antigen-binding domain, and VH2 and VL2 associate to form a second antigen-binding domain. In other embodiments, a first scFv is linked to a second scFv via at least one modified hinge region.

[0181] In certain embodiments, the polyspecific binding proteins of this disclosure (i.e., polyspecific binding proteins having at least a first antigen-binding protein and a second antigen-binding protein) have higher agonist activity compared to polyspecific binding proteins lacking at least one modified hinge region. For example, a polyspecific binding protein having VH1 linked to VH2 via at least one modified hinge region and / or VL1 linked to VL2 via at least one modified hinge region may have higher agonist activity of a target receptor pair (e.g., ALK1 and one of BMPRII, ActRIIA, and ActRIIB) than the same polyspecific binding protein lacking at least one modified hinge region.

[0182] As used herein, the term “EU index” refers to the EU numbering rules for the constant region of an antibody, as described in Edelman, GM. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, USDept. Health and Human Services, 5th edition, 1991 (each reference is incorporated herein by reference in whole). All numbering of amino acid positions of Fc polypeptides or fragments as used herein follows the EU index. As used herein, the term “linker” refers to a sequence of 0 to 100 consecutive amino acid residues. Linkers may or may not be present, identical or different. Linkers contained in a protein or polypeptide may all have the same amino acid sequence or may have different amino acid sequences.

[0183] In some embodiments, the term “linker” refers to a sequence of 1 to 100 consecutive amino acid residues. Typically, linkers provide flexibility and spatial separation between two amino acids or between two polypeptide domains. Depending on the molecular format, linkers may be inserted between VH, VL, CH, and / or CL domains to provide sufficient flexibility and mobility to the light and heavy chain domains. Linkers are typically inserted at the amino sequence level, between variable domains (between a variable domain and a knockout domain) or between a variable domain and a constant domain. Because the approximate size of immunoglobulin domains is well understood, inter-domain transitions can be identified. The precise location of domain transitions can be determined by the positioning of peptide segments that do not form secondary structural elements such as beta sheets or alpha helices, as demonstrated by experimental data or determined by modeling or secondary structure prediction techniques.

[0184] As used herein, the terms "specifically binds," "specific binding," "binding specificity," or "specifically recognized" refer to an antigen-binding protein or an antigen-binding fragment thereof that exhibits a detectable affinity for an antigen (e.g., a BMPRI receptor or a BMPRII receptor antigen) and does not exhibit significant cross-reactivity with targets that are not BMPRI receptor or BMPRII receptor proteins. As used herein, the term "affinity" refers to the strength of the interaction between the antigen-binding site of an antigen-binding protein or an antigen-binding fragment thereof and the epitope to which it binds. In certain exemplary embodiments, affinity is measured by surface plasmon resonance (SPR) on, for example, a Biacore instrument. As will be readily appreciated by those skilled in the art, the affinity of an antigen-binding protein can be reported in molar concentration units (M) as a dissociation constant (KD). The antigen-binding proteins or antigen-binding fragments thereof of the present disclosure have a KD value in the range of about 10

[0185] , M to about 10 -12 M (i.e., in the range of low micromolar to picomolar), about 10 -7 M to 10 -11 M, about 10 -8 M to about 10 -10 M, about 10 -9 M. In certain embodiments, the antigen-binding protein or an antigen-binding fragment thereof has a binding affinity of about 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M. In certain embodiments, the antigen-binding protein or an antigen-binding fragment thereof has a binding affinity of about 10 -7 M to about 10 -9 M (nanomolar range).

[0185] Specific binding may be determined by any means recognized in the art for determining such binding. In some embodiments, specific binding is determined by a competitive binding assay (e.g., ELISA) or a Biacore assay. In certain embodiments, the assay is performed at approximately 20°C, 25°C, 30°C, or 37°C.

[0186] As used herein, “administer” or “dosage” means the act of physically delivering an extracorporeal substance (e.g., an isolated bound polypeptide provided herein) to a patient by injection or other means, for example, by pulmonary delivery (e.g., inhalation), mucosal delivery (e.g., intranasal delivery), intradermal delivery, intravenous delivery, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When managing or treating a disease or its symptoms, administration of the substance is generally performed after the onset of the disease or its symptoms. When preventing a disease or its symptoms, administration of the substance is generally performed before the onset of the disease or its symptoms and may be continued for a long period to delay the appearance or reduce the severity of disease-related symptoms.

[0187] As used herein, the term “composition” is intended to include not only products that optionally contain a given component (e.g., an isolated bound polypeptide provided herein) in a predetermined amount, but also any products that result directly or indirectly from the combination of a given component in a predetermined amount, either optionally.

[0188] "Effective amount" means the amount of active pharmaceutical agent (e.g., the isolated bound polypeptide of this disclosure) that is sufficient to produce the desired physiological outcome in an individual requiring the drug. The effective amount may vary between individuals depending on the health and physical condition of the individual being treated, the taxonomic group of the individual being treated, the method of preparing the composition, the assessment of the individual's medical condition, and other relevant factors.

[0189] As used herein, the terms “subject” and “patient” are used synonymously. As used herein, the subject may be an animal other than a primate (e.g., cattle, pigs, horses, cats, dogs, rats, mice, etc.) or a mammal such as a primate (e.g., monkeys and humans). In certain embodiments, the term “subject” as used herein refers to a vertebrate such as a mammal. Mammals include, but are not limited to, humans, non-human primates, wild animals, feral animals, livestock, sports animals, and pets.

[0190] As used herein, the term “therapy” refers to any protocol, method and / or agent that may be used in the prevention, management, treatment and / or improvement of a disease or its associated symptoms. In some embodiments, the term “therapy” refers to any protocol, method and / or agent that may be used in the modulation of the immune response to an infection or its associated symptoms in a subject. In some embodiments, the term “therapy” (singular and plural) refers to biological therapies, supportive therapies and / or other therapies known to those skilled in the art, such as healthcare workers, that are useful in the prevention, management, treatment and / or improvement of a disease or its associated symptoms. In other embodiments, the term “therapy” (singular and plural) refers to biological therapies, supportive therapies and / or other therapies known to those skilled in the art, such as healthcare workers, that are useful in the modulation of the immune response to an infection or its associated symptoms in a subject.

[0191] As used herein, the terms “to treat,” “treatment,” and “to treat” mean a reduction or improvement in the progression, severity, and / or duration of a disease or its associated symptoms, resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents, such as isolated conjugated polypeptides provided herein). The term “to treat” may also mean, as used herein, an alteration of the disease process of the subject being treated. The therapeutic effects of treatment include, but are not limited to, prevention of the onset or recurrence of the disease, relief of symptoms, reduction of direct or indirect pathological consequences of the disease, slowing of the rate of disease progression, improvement or mitigation of the disease state, and remission or improved prognosis.

[0192] The terms "about" or "approximately" mean within about 20% of a given value or range, for example, within about 10%, within about 5%, or within about 1%.

[0193] BMPRI receptor and BMPRII receptor Bone morphogenetic protein (BMP) type I and II receptors are serine-threonine kinase transmembrane signaling proteins that regulate a wide range of ligand-dependent cell fate with temporal and spatial fidelity during development and postnatally. Receptor activation, induced by initial binding to their ligands (BMPs) and subsequent heterodimerization, triggers intracellular signaling initiated by the phosphorylation of receptor-regulated SMAD1, 5, and 8 (R-SMAD). These activated R-SMADs form heteromeric complexes with SMAD4, which are involved in specific transcriptional responses.

[0194] As used herein, the term “ALK1” refers to the activin A receptor-like type 1, BMPI-type receptor. Other terms for ALK1 include ACVRLK1, serine / threonine-protein kinase receptor R3, TGF-B superfamily receptor type I, and HHT2. The ALK1 protein is encoded by the gene ACVRL1. The ALK1 protein includes human, mouse, and further mammalian homologs. Sequences of human ALK1 are available using the UniProt identifier P37023 (ACVL1 HUMAN), e.g., human isoform P37023-1. Sequences of mouse ALK1 are available using the UniProt identifier Q61288 (ACVL1 MOUSE). The term “ALK1” may encompass different isoforms and variants that may exist across different species, all of which are included in the term ALK1. Furthermore, the term "ALK1" may include synthetic variants of the ALK1 protein produced, for example, by introducing at least one mutation. The protein ALK1 may also undergo various modifications, such as synthetic or spontaneous modifications. Spontaneous mutations in the ALK1 gene are associated with hereditary hemorrhagic telangiectasia (HHT) type 2, in which patients have pulmonary hypertension, routine epistaxis, stroke, and embolism.

[0195] The term "BMPRII" refers to the osteogenesis imperfecta receptor type 2 protein. Other names include BMP2 receptor, osteogenesis imperfecta receptor type II, BMPII receptor, BMR2, PPH1, BMPR3, BRK-3, POVD1, T-ALK, BMPRII, and BMPR-II. The BMPRII protein is encoded by the gene BMPR2. The BMPRII protein includes human, mouse, and further mammalian homologs. Sequences of human BMPRII are available using UniProt identifier Q13873 (BMPRII HUMAN), e.g., human isoform 1 (identifier: Q13873-1) and human isoform 2 (identifier: Q13873-2). Sequences of mouse BMPRII are available using UniProt identifier 035607 (BMPRII MOUSE). Different isoforms and variants may exist across different species, and these are all included in the term BMPRII. Furthermore, synthetic variants of the BMPRII protein may be generated, for example, by introducing at least one mutation, and these are included in the term BMPRII. The BMPRII protein may also undergo various modifications, such as synthetic or spontaneous modifications.

[0196] As used herein, the term “ActRIIA” refers to the family of activin receptor type IIA (ActRIIA) proteins derived from any species, and variants obtained from such ActRIIA proteins by mutagenesis or other modifications. References to ActRIIA herein are understood to refer to any one of the currently identified forms. Members of the ActRIIA family are generally transmembrane proteins comprising a ligand-binding extracellular domain containing a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predictive serine / threonine kinase activity. The term “ActRIIA” includes polypeptides, including any spontaneously occurring polypeptide of ActRIIA family members, and any variants thereof (including mutants, fragments, fusions, and peptide-mimicking forms) that retain useful activity.

[0197] As used herein, the term “ActRIIB” refers to the family of activin receptor type IIB (ActRIIB) proteins derived from any species, and variants obtained from such ActRIIB proteins by mutagenesis or other modifications. References to ActRIIB herein are understood to refer to any of the currently identified forms. Members of the ActRIIB family are generally transmembrane proteins comprising a ligand-binding extracellular domain containing a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain having predictive serine / threonine kinase activity. The term “ActRIIA” includes polypeptides, including any spontaneously occurring polypeptides of ActRIIB family members, and any variants thereof (including variants, fragments, fusions, and peptide-mimicking forms) that retain useful activity. Examples of such variant ActRIIB polypeptides are provided throughout this disclosure and in International Patent Application Publications WO2006 / 012627 and WO2008 / 097541, which are incorporated herein by reference in their entirety.

[0198] ALK1 / BMPRII, ActRIIA, or ActRIIB bispecific antibodies The bispecific antibodies provided herein promote heterodimerization between ALK1 and BMPII receptors, such as BMPRII, ActRIIA, and ActRIIB. The bispecific antibodies according to the present invention can be produced in high yield. The bispecific antibodies or their binding domains can be readily matured, or a screening approach can be used to detect binders with optimized binding ability. In the case of bispecific antibodies, each binding site can be individually optimized. Finally, even in cases where downstream signaling is absent due to, for example, a genetic defect, the antibody approach may still be able to rescue the ALK1 / BMPRII, ALK1 / ActRIIA, or ALK1 ActRIIB signaling cascade.

[0199] The antibodies disclosed herein specifically bind to ALK1 and BMPRII, ActRIIA, or ActRIIB. That is, they bind to their targets with a higher affinity (e.g., at least twice as high) than their binding affinity to unrelated antigens (e.g., bovine serum albumin (BSA), casein).

[0200] As used herein, the term “inducing proximity” between ALK1 and BMPRII, ActRIIA, or ActRIIB means matching ALK1 with one of BMPRII, ActRIIA, or ActRIIB so that the ALK1 / BMPRII, ALK1 / ActRIIA, or ALK1 / ActRIIB signaling cascade is stimulated. In certain embodiments, the proximity induced by the multispecific binding proteins of this disclosure is the same as or similar to the proximity induced when BMP9 matches ALK1 and BMPRII. Stimulation of the ALK1 / BMPRII, ALK1 / ActRIIA, or ALK1 / ActRIIB signaling cascade may be detected through the detection of phosphorylated SMAD proteins (e.g., pSMAD1, pSMAD5, and / or pSMAD8) and any downstream consequences of the signaling cascade, including but not limited to the detection of gene expression associated with the signaling cascade. Genes that have been shown to be upregulated by the ALK1 / BMPRII, ALK1 / ActRIIA, or ALK1 / ActRIIB signaling cascades include, but are not limited to, ID1, ID3, and TMEM100.

[0201] The bispecific antibodies of this disclosure are exemplified in the examples by numerous ALK1 / BMPRII bispecific antibodies, but it is expected that the technical effect of the exemplified bispecific antibodies (i.e., inducing agonism) will similarly extend to ALK1 / ActRIIA and ALK1 / ActRIIB bispecific antibodies. Those skilled in the art will understand that the technical effect of inducing proximity between ALK1 and BMPRII using the ALK1 / BMPRII bispecific antibody, and the subsequent activation of the receptor complex, extends to ALK1 / ActRIIA and ALK1 / ActRIIB bispecific antibodies, which in turn induces proximity between ALK1 and ActRIIA, and between ALK1 and ActRIIB.

[0202] The bispecific antibodies of this disclosure may utilize at least one modified hinge region. The modified hinge region functions as a linker for connecting different domains of the bispecific antibody. In certain embodiments, the modified hinge region links a first variable heavy chain domain (VH1) to a second variable heavy chain domain (VH2), and / or the modified hinge region links a first variable light chain domain (VL1) to a second variable light chain domain (VL2). In another embodiment, the modified hinge region links a first scFv to a second scFv. In certain embodiments, the modified hinge region includes i) an upper hinge region that is or is absent and is up to 7 amino acids long, and ii) a lower hinge region. In certain embodiments, the modified hinge region includes or consists of the amino acid sequence PLAP or PAPNLLGGP.

[0203] The bispecific antibodies (e.g., multispecific binding proteins) of this disclosure exhibit higher agonist activity compared to bispecific antibodies lacking at least one modified hinge region. Agonist activity can be measured using specific receptor efficacy assays (e.g., Pathhunter U2OS dimerization assay (DiscoverX) efficacy assay (e.g., Pathhunter)) including cell lines expressing the target receptor of interest (e.g., U2OS). Binding of the bispecific antibody to the receptor triggers a signaling cascade, resulting in the expression of a quantifiable reporter gene.

[0204] The bispecific antibodies (e.g., polyspecific binding proteins) of this disclosure induce at least about 35% of BMP9 activity. In certain embodiments, the bispecific antibodies (e.g., polyspecific binding proteins) of this disclosure induce at least about 40% of BMP9 activity. In certain embodiments, the bispecific antibodies (e.g., polyspecific binding proteins) of this disclosure induce at least about 40% of BMP9 activity. In certain embodiments, the bispecific antibodies (e.g., polyspecific binding proteins) of this disclosure induce at least about 45% of BMP9 activity. In certain embodiments, the bispecific antibodies (e.g., polyspecific binding proteins) of this disclosure induce at least about 50% of BMP9 activity. In certain embodiments, the bispecific antibodies (e.g., polyspecific binding proteins) of this disclosure induce at least about 55% of BMP9 activity. In certain embodiments, the bispecific antibodies (e.g., polyspecific binding proteins) of this disclosure induce at least about 60% of BMP9 activity. In certain embodiments, the bispecific antibody (e.g., a polyspecific binding protein) of the Disclosure induces at least about 65% of the activity of BMP9. In certain embodiments, the bispecific antibody (e.g., a polyspecific binding protein) of the Disclosure induces at least about 70% of the activity of BMP9. In certain embodiments, the bispecific antibody (e.g., a polyspecific binding protein) of the Disclosure induces at least about 75% of the activity of BMP9. In certain embodiments, the bispecific antibody (e.g., a polyspecific binding protein) of the Disclosure induces at least about 80% of the activity of BMP9.

[0205] In certain embodiments, BMP9 activity is identified by measuring phosphorylated SMAD1 (pSMAD1) levels, phosphorylated SMAD5 (pSMAD5) levels, and / or phosphorylated SMAD8 (pSMAD8) levels in cells incubated with a multispecific binding protein and / or BMP9. Phosphorylated SMAD levels (i.e., pSMAD1, pSMAD5, and pSMAD8) can be detected using an enzyme-linked immunosorbent assay (ELISA). Briefly, a first population of cells (e.g., HUVEC cells) is incubated with the bispecific antibody of this disclosure, and a second population of cells (e.g., HUVEC cells) is incubated with BMP9. After incubation time, the cells are lysed, and the cell lysates are analyzed using an antibody against the phosphorylated SMAD protein (i.e., pSMAD1, pSMAD5, or pSMAD8). Antibody binding is detected (e.g., via a fluorescence signal) and quantified. Next, the level of phosphorylated SMAD protein in the first cell population is compared to the level of phosphorylated SMAD protein in the second cell population to determine the activity percentage of the bispecific antibody against BMP9.

[0206] The bispecific antibodies (e.g., multispecific binding proteins) of this disclosure can stimulate the expression of genes selected from ID1, ID3, and TMEM100 in cells. The expression of ID1, ID3, and / or TMEM100 in such cells is at least 50% of the expression of ID1, ID3, and / or TMEM100 from cells incubated with BMP9. In certain embodiments, the expression of ID1, ID3, and / or TMEM100 in such cells is at least equal to the expression of ID1, ID3, and / or TMEM100 from cells incubated with BMP9. In certain embodiments, the expression of ID1, ID3, and / or TMEM100 in such cells is at least 1.5 times higher than the expression of ID1, ID3, and / or TMEM100 from cells incubated with BMP9. In certain embodiments, the expression of ID1, ID3, and / or TMEM100 in the cells is at least twice as high as the expression of ID1, ID3, and / or TMEM100 from cells incubated with BMP9. In certain embodiments, the expression of ID1, ID3, and / or TMEM100 in the cells is at least three times higher than the expression of ID1, ID3, and / or TMEM100 from cells incubated with BMP9. In certain embodiments, the expression of ID1, ID3, and / or TMEM100 in the cells is at least four times higher than the expression of ID1, ID3, and / or TMEM100 from cells incubated with BMP9. In certain embodiments, the expression of ID1, ID3, and / or TMEM100 in the cells is at least five times higher than the expression of ID1, ID3, and / or TMEM100 from cells incubated with BMP9. In certain embodiments, the expression of ID1, ID3, and / or TMEM100 in the cells is at least 6 times higher than the expression of ID1, ID3, and / or TMEM100 in cells incubated with BMP9.

[0207] The detection of ID1, ID3, and TMEM100 expression can be achieved using standard molecular biology techniques and PCR. Briefly, a first population of cells (e.g., HUVEC cells or HMEC-1 cells) is incubated with the bispecific antibodies of this disclosure, and a second population of cells (e.g., HUVEC cells or HMEC-1 cells) is incubated with BMP9. After incubation time, mRNA is isolated from the cells, cDNA is generated, and PCR is performed to detect the levels of ID1, ID3, and / or TMEM100 against a control gene, e.g., GAPDH. Subsequently, the levels of ID1, ID3, and / or TMEM100 in the first population of cells are compared to the levels of ID1, ID3, and / or TMEM100 in the second population of cells.

[0208] thermal stability Certain bispecific antibodies (e.g., polyspecific binding proteins) of this disclosure have improved thermal stability compared to other antibodies of this disclosure. For example, the bispecific antibodies called DGL947 (containing the first polypeptide chain of SEQ ID NO: 139 and the second polypeptide chain of SEQ ID NO: 146) and DGL949 (containing the first polypeptide chain of SEQ ID NO: 141 and the second polypeptide chain of SEQ ID NO: 146) have improved thermal stability compared to the bispecific antibodies called DGL945 and DGL1146. As used herein, “improved thermal stability” refers to a higher melting temperature. The melting temperature may be the melting onset temperature (Tonset) and / or the thermal transition midpoint (Tm) of the melting temperature.

[0209] In certain embodiments, the melting onset temperature (Tonset) for unfolding of the bispecific antibodies of the Disclosure is at least about 50°C, at least about 51°C, at least about 52°C, at least about 53°C, at least about 54°C, at least about 55°C, at least about 56°C, at least about 57°C, at least about 58°C, at least about 59°C, or at least about 60°C.

[0210] In certain embodiments, the thermal transition midpoint (Tm) of the melting temperature of the bispecific antibody of the present disclosure is at least about 63°C, at least about 64°C, at least about 65°C, at least about 66°C, at least about 67°C, at least about 68°C, at least about 69°C, at least about 70°C, at least about 71°C, or at least about 72°C.

[0211] The Tonset and Tm of the two-specific antibodies of this disclosure are determined by differential scanning calorimetry (DSC).

[0212] In some embodiments of the first aspect, the bispecific antibody specifically binds to the extracellular domain of ALK1 and / or the extracellular domain of BMPRII, ActRIIA, or ActRIIB. In some embodiments, ALK1 is human ALK1 or a fragment thereof, and / or BMPRII, ActRIIA, or ActRIIB is human BMPRII, ActRIIA, or ActRIIB or a fragment thereof. In some embodiments, the bispecific antibody binds to the extracellular domain of human ALK1 or a fragment thereof and / or the extracellular domain of human BMPRII or a fragment thereof.

[0213] In some embodiments, the bispecific antibody reacts with ALK1 up to approximately 10 times -4 M ~ about 10 -13 M (for example, 10 -4 M, 10 -4.5 M, 10 -5 M, 10 -5.5 M, 10 -6 M, 10 -6.5 M, 10 -7 M, 10 -7.5 M, 10 -8 M, 10 -8.5 M, 10 -9 M, 10 -9.5 M, 10 -10 M, 10 -10.5 M, 10 -11 M, 10 -11.5 M, 10 -12 M, 10 -12.5 M, 10 -13 Join at Kd of M).

[0214] In some embodiments, the bispecific antibody is added to BMPRII, ActRIIA, or ActRIIB, up to approximately 10 times. -4 M ~ about 10 -13 M (for example, 10 -4 M, 10 -4.5 M, 10 -5 M, 10 -5.5 M, 10 -6 M, 10 -6.5 M, 10 -7 M, 10 -7.5 M, 10 -8 M, 10 -8.5 M, 10 -9 M, 10 -9.5 M, 10 -10 M, 10 -10.5 M, 10 -11 M, 10 -11.5 M, 10 -12 M, 10 -12.5 M, 10 -13 Join at Kd of M).

[0215] In some embodiments, the bispecific antibodies are used with ALK1 and BMPRII, or ALK1 and ActRIIA, or ALK1 and ActRIIB, up to approximately 10 -4 M ~ about 10 -13 M (for example, 10 -4 M, 10 -4.5 M, 10 -5 M, 10 -5.5 M, 10 -6 M, 10 -6.5 M, 10 -7 M, 10 -7.5 M, 10 -8 M, 10 -8.5 M, 10 -9 M, 10 -9.5 M, 10 -10 M, 10 -10.5 M, 10 -11 M, 10 -11.5 M, 10 -12 M, 10 -12.5 M, 10 -13 Join at Kd of M).

[0216] The Kd of an antibody that binds to an antigen can be assayed using any method known in the art for quantifying antibody binding to antigen-expressing cells, including, for example, immunoassays, such as enzyme-linked immunosorbent assay (ELISA), bimolecular interaction analysis (BIA) (e.g., Sjolander & Urbaniczky; Anal. Chem. 63:2338-2345, 1991; Szabo, et al., Curr. Opin. Struct. Biol. 5:699-705, 1995), and fluorescence-activated cell classification (FACS). BIA is a technique for analyzing bispecific interactions in real time without labeling any of the reactants (e.g., BIACORE®). Changes in surface plasmon resonance (SPR) optical phenomena can be used as an indicator of real-time reactions between biomolecules.

[0217] In some embodiments, the antibody according to the present invention includes, in addition to binding domains to ALK1 and BMPRII, ActRIIA, or ActRIIB, a binding domain to a ligand of the ALK1 / BMPRII, ALK1 / ActRIIA, or ALK1 / ActRIIB receptor, or a binding domain to another molecule involved in the signaling of ALK1 / BMPRII, ALK1 / ActRIIA, or ALK1 / ActRIIB.

[0218] In some embodiments, the binding site that specifically binds to ALK1 cross-reacts with human ALK1 and mouse ALK1.

[0219] In some embodiments, the binding site that specifically binds to ActRIIA cross-reacts with ActRIIB.

[0220] Unless there is an obvious incompatibility to those skilled in the art, each embodiment illustrating binding ability can be combined with each embodiment illustrating antibody format.

[0221] Joint domain One component of the polyspecific binding protein of this disclosure is a binding domain or binding specificity that binds to a first cell surface target and a second cell surface target. In a particular embodiment, the first cell surface target is a first receptor subunit, and the second cell surface target is the same receptor subunit.

[0222] The polyspecific binding proteins disclosed herein may utilize any type of binding moiety that specifically binds to a particular receptor subunit. In certain embodiments, the binding moiety includes an antibody-variable domain. Exemplary binding moieties containing an antibody-variable domain include, but are not limited to, VH, VL, VHH, VH / VL pairs, scFv, diabody, or Fab. Other preferred binding moiety formats include lipocalin (see, e.g., Gebauer M. et al., 2012, Method Enzymol. 503:157-188, which is incorporated herein by reference in its entirety), adonectin (see, e.g., Lipovsek D., 2011, Protein Eng. Des. Sel. 24:3-9, which is incorporated herein by reference in its entirety), avimer (see, e.g., Silverman J, et al., 2005, Nat. Biotechnol. 23:1556-1561, which is incorporated herein by reference in its entirety), finomer (see, e.g., Schlatter D, et al., 2012, mAbs 4:497-508, which is incorporated herein by reference in its entirety), and kunitz domain (see, e.g., Hosse RJet). See al., 2006, Protein Sci. 15:14-27 (this document is incorporated herein by reference in its entirety), knottin (see, for example, Kintzing JR et al., 2016, Curr. Opin. Chem. Biol. 34:143-150 (this document is incorporated herein by reference in its entirety)), afibody (see, for example, Feldwisch J. et al., 2010 J. Mol. Biol. 398:232-247 (this document is incorporated herein by reference in its entirety)), and DARPin (see, for example, Pluckthun A., 2015, Annu. Rev. Pharmacol. Toxicol. 55:489-511 (this document is incorporated herein by reference in its entirety)).

[0223] In certain embodiments, the binding domain comprises variable regions of the heavy and / or light chains of a conventional antibody or its antigen-binding fragment (e.g., Fab or scFv). The term “conventional antibody” is used herein to refer to a heterotetrameric antibody containing heavy and light chains of immunoglobulin arranged according to a “Y” configuration. Such conventional antibodies may originate from any suitable species, including but not limited to antibodies of llama, alpaca, camel, mouse, rat, rabbit, goat, hamster, chicken, monkey, or human origin. In certain exemplary embodiments, the conventional antibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), where the VH domain and / or VL domain, or one or more complementarity-determining regions (CDRs) thereof, originate from the same antibody. In certain embodiments, the antigen-binding region of a conventional antibody may be referred to as “Fab” (Fragment antigen-binding). Fab comprises one constant domain and one variable domain from each of the heavy and light chains. The variable heavy and light chains contain CDRs involved in antigen binding.

[0224] In other embodiments, the binding subunit of a particular receptor subunit includes at least a CDR or VHH domain of the VHH antibody or Nanobody®. The VHH antibody, a heavy-chain antibody derived from camelids, consists of two heavy chains and lacks a light chain (Hamers-Casterman, et al. Nature. 1993;363;446-8). Each heavy chain of the VHH antibody has a variable domain at its N-terminus, which is referred to in the art as the "VHH" domain to distinguish it from the variable domain of the heavy chain of conventional antibodies, i.e., the VH domain. Similar to conventional antibodies, the VHH domain of this molecule includes HCDR1, HCDR2, and HCDR3 regions that confer antigen-binding specificity, and therefore fragments such as the VHH antibody or isolated VHH domain are suitable as components of the multispecific binding protein of this disclosure.

[0225] Multispecific binding proteins In certain embodiments, the first and second binding domains disclosed herein may be paired together or operably ligated to generate a polyspecific binding protein capable of crosslinking the first and second subunits of a given receptor (e.g., a BMPI-type receptor and a BMPII-type receptor). In some embodiments, the first specific binding domain (e.g., VHH or scFv) is operably ligated (directly or indirectly) to the N-terminus and / or C-terminus of the first Fc domain or polypeptide, and the second specific binding domain is operably ligated to the N-terminus and / or C-terminus of the second Fc domain or polypeptide, so as to facilitate heterodimerization of the first and second specific binding domains.

[0226] In certain exemplary embodiments, the polyspecific binding proteins of this disclosure exhibit agonism to any given signaling pathway, i.e., no antagonistism to the ALK1 pathway. In some embodiments, agonism can be measured using a specific receptor efficacy assay (e.g., Pathhunter U2OS dimerization assay (DiscoverX) efficacy assay (e.g., Pathhunter)) including a cell line expressing the target receptor of interest (e.g., U2OS). Binding of the bispecific antibody to the receptor triggers a signaling cascade, resulting in the expression of a quantifiable reporter gene.

[0227] In certain embodiments, the polyspecific binding protein comprises a dual variable domain format. The “dual variable domain” (“DVD”) binding proteins of this disclosure comprise two or more antigen-binding sites and are tetravalent or polyvalent binding proteins. The DVDs of this disclosure are polyspecific, i.e., capable of binding to ALK1 and one of BMPRII, ActRIIA, and ActRIIB. A DVD binding protein comprising two heavy-chain DVD polypeptides and two light-chain DVD polypeptides is referred to as “DVD immunoglobulin” or “DVD-Ig.” Each half of DVD-Ig comprises a heavy-chain DVD polypeptide and a light-chain DVD polypeptide, as well as two or more antigen-binding sites. Each binding site comprises a heavy-chain variable domain and a light-chain variable domain, and each antigen-binding site has a total of six CDRs involved in antigen binding.

[0228] Descriptions of the design, expression, and characterization of the DVD-Ig molecule are provided in PCT Publication No. WO2007 / 024715, U.S. Patent No. 7,612,181, and Wu et al., Nature Biotechnol., 25:1290-1297 (2007). Examples of such DVD-Ig molecules include a heavy chain containing the structural formula VD1-(X1)n-VD2-C-(X2)n (wherein VD1 is the first heavy chain variable domain, VD2 is the second heavy chain variable domain, C is the heavy chain constant domain, X1 is a linker, but not CH1, X2 is an Fc region, and n is 0 or 1), and a light chain containing the structural formula VD1-(X1)n-VD2-C-(X2)n (wherein VD1 is the first light chain variable domain, VD2 is the second light chain variable domain, C is the light chain constant domain, X1 is a linker, but not CH1, X2 does not contain an Fc region, and n is 0 or 1). Such DVD-Ig may comprise two such heavy chains and two such light chains, in which case each chain comprises variable domains linked in series without constant regions interposing between the variable domains, the heavy chains and light chains associate to form a series-type functional antigen-binding site, and the heavy-light chain pair may associate with another heavy-light chain pair to form a tetramer-binding protein having four functional antigen-binding sites. In another example, the DVD-Ig molecule may comprise heavy chains and light chains, each comprising three variable domains (VD1, VD2, VD3) linked in series without constant regions interposing between the variable domains, in which case the heavy-light chain pair may associate to form three antigen-binding sites, or the heavy-light chain pair may associate with another heavy-light chain pair to form a tetramer-binding protein having six antigen-binding sites.

[0229] In embodiments, the Disclosure provides a binding protein comprising first and second polypeptide chains, wherein the first polypeptide chain comprises a first VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker but not CH1, X2 is an Fc region, and n is independently 0 or 1; and the second polypeptide chain comprises a second VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first light chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker but not CH1, X2 does not contain an Fc region, and n is independently 0 or 1.

[0230] In relation to the construction of DVD-Ig or other binding protein molecules, the term "linker" is used to indicate a polypeptide ("linker polypeptide") containing a single amino acid or two or more amino acid residues linked by a peptide bond, and is used to link one or more antigen-binding sites. Such linker polypeptides are well known in the art (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993), Poljak, RJ, Structure, 2:1121-1123 (1994)). Generally, flexible linkers composed of small nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids can be used. Exemplary flexible linkers include, but are not limited to, GGGGSG(sequence ##), GGSGG(sequence ##), GGGGSGGGGS(sequence ##), GGSGGGGSG(sequence ##), GGSGGGGSGGGGS(sequence ##), GGGGSGGGGSGGGG(sequence ##), GGGGSGGGGSGGGGS(sequence ##), and RADAAAAGGGGSGGGGSGGGGSGGGGS(sequence ##).

[0231] Alternatively, a rigid linker may be used to link one or more antigen-binding proteins. This rigid linker allows for the maintenance of a certain distance between the linked antigen-binding proteins, thereby potentially enhancing the activity of the individual proteins. The rigid linker may use one or more proline amino acids to confer rigidity. Examples of rigid linkers include ASTKGP (SEQ ID NO: ##), ASTKGPSVFPLAP (SEQ ID NO: ##), TVAAP (SEQ ID NO: ##), RTVAAP (SEQ ID NO: ##), TVAAPSVFIFPP (SEQ ID NO: ##), RTVAAPSVFIFPP (SEQ ID NO: ##), AKTTPKLEEGEFSEAR (SEQ ID NO: ##), AKTTPKLEEGEFSEARV (SEQ ID NO: ##), AKTTPKLGG (SEQ ID NO: ##), SAKTTPKLGG (SEQ ID NO: ##), SAKTTP (SEQ ID NO: ##), RADAAP (SEQ ID NO: ##), RADAAPTVS (SEQ ID NO: ##), and RADAAAAGGPGS. Examples include, but are not limited to, (sequence number ##), SAKTTPKLEEGEFSEARV(sequence number ##), ADAAP(sequence number ##), ADAAPTVSIFPP(sequence number ##), QPKAAP(sequence number ##), QPKAAPSVTLFPP(sequence number ##), AKTTPP(sequence number ##), AKTTPPSVTPLAP(sequence number ##), AKTTAP(sequence number ##), AKTTAPSVYPLAP(sequence number ##), GENKVEYAPALMALS(sequence number ##), GPAKELTPLKEAKVS(sequence number ##), and GHEAAAVMQVQYPAS(sequence number ##).

[0232] In certain embodiments, the linker includes the modified hinge region described herein.

[0233] In certain embodiments, the linker includes or consists of PLAP, PAPNLLGGP, PLAPDKTHT, EKSYGPP, or DKTHT.

[0234] In certain embodiments, the polyspecific binding protein comprises first and second polypeptide chains, The first polypeptide chain comprises VH1-(HX1)n-VH2-C-(HX2)n, VH1 is the first heavy chain variable domain, VH2 is the second heavy chain variable domain, C is the heavy chain constant domain, HX1 is the linker, HX2 is the Fc region, and n is independently 0 or 1. The second polypeptide chain comprises VL1-(LX1)n-VL2-C-(LX2)n, VL1 is the first light chain variable domain, VL2 is the second light chain variable domain, C is the light chain constant domain, LX1 is the linker, LX2 does not contain the Fc region, and n is independently 0 or 1.

[0235] In certain embodiments, VH1 specifically binds to human ALK1, and VH2 specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB.

[0236] In certain embodiments, VL1 specifically binds to human ALK1, and VL2 specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB.

[0237] In certain embodiments, VH1 specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB, and VH2 specifically binds to human ALK1.

[0238] In certain embodiments, VL1 specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB, and VL2 specifically binds to human ALK1.

[0239] In certain embodiments, the linker HX1 comprises the amino acid sequence PLAP or PAPNLLGGP.

[0240] In certain embodiments, the linker LX1 comprises the amino acid sequence PLAP or PAPNLLGGP.

[0241] In certain embodiments, linker HX1 comprises the amino acid sequence PLAP, and linker LX1 comprises the amino acid sequence PLAP or PAPNLLGGP.

[0242] In certain embodiments, the polyspecific binding protein comprises two VH1-(HX1)n-VH2-C-(HX2)n polypeptide chains and two VL1-(LX1)n-VL2-C-(LX2)n polypeptide chains.

[0243] In a particular embodiment, with respect to (HX1)n, n is 1, and with respect to (HX2)n, n is 1.

[0244] In a particular embodiment, with respect to (LX1)n, n is 1, and with respect to (LX2)n, n is 0.

[0245] In certain embodiments, the polyspecific binding protein comprises first and second polypeptide chains, The first polypeptide chain comprises VH1-(HX1)n-VH2-C-Fc, VH1 is the first heavy chain variable domain, VH2 is the second heavy chain variable domain, C is the heavy chain constant domain, HX1 is the linker, Fc is the Fc region, and n is independently 0 or 1. The second polypeptide chain comprises VL1-(LX1)n-VL2-C, VL1 is the first light chain variable domain, VL2 is the second light chain variable domain, C is the light chain constant domain, LX1 is the linker, and n is independently 0 or 1.

[0246] Non-DVD-IG format In another embodiment of this disclosure, the polyspecific binding protein is oriented from the N-terminus to the C-terminus. ai) A first polypeptide chain comprising a first antigen-binding domain, a first linker (e.g., a modified hinge region), and a first constant region, and bi) A second polypeptide chain comprising a second antigen-binding domain, a second linker (e.g., a modified hinge region), and a second constant region, aii) A first polypeptide chain comprising a second antigen-binding domain, a first antigen-binding domain, a first linker (e.g., a modified hinge region), and a first constant region, and bii) A second polypeptide chain comprising a second constant region, with or without a second linker (e.g., a modified hinge region), aiii) A first polypeptide chain comprising a first constant region, with or without a first linker (e.g., a modified hinge region), and biii) A second polypeptide chain comprising a second antigen-binding domain, a first antigen-binding domain, a second linker (e.g., a modified hinge region), and a second constant region, or aiv) A first polypeptide chain comprising a first antigen-binding domain, optionally a first linker (e.g., a modified hinge region), a second antigen-binding domain, optionally a second linker (e.g., a modified hinge region), and a first constant region, and biv) A second polypeptide chain comprising a third antigen-binding domain, optionally a third linker (e.g., a modified hinge region), a fourth antigen-binding domain, optionally a fourth linker (e.g., a modified hinge region), and a second constant region.

[0247] In certain embodiments, the first antigen-binding domain comprises an scFv, VHH, Fab, F(ab’)2, or a single-domain antibody.

[0248] In certain embodiments, the second antigen-binding domain comprises an scFv, VHH, Fab, F(ab’)2, or a single-domain antibody. <​​​​​​In certain embodiments, the fourth antigen-binding domain comprises a scFv, VHH, Fab, F(ab’)2, or single-domain antibody.

[0251] In certain embodiments, any one or more of the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain, and the fourth antigen-binding domain comprises a scFv, VHH, Fab, F(ab’)2, or single-domain antibody.

[0252] In certain embodiments, the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain, and the fourth antigen-binding domain each comprise a scFv.

[0253] The Fc polypeptides used in the polyspecific binding proteins of this disclosure generally comprise a CH2 domain and a CH3 domain, with the C-terminus of the CH2 domain being (directly or indirectly) ligated to the N-terminus of the CH3 domain. Any naturally occurring or variant CH2 and / or CH3 domains may be used. For example, in certain embodiments, the CH2 and / or CH3 domains are spontaneously occurring CH2 or CH3 domains derived from an IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 antibody heavy chain, e.g., a human IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 antibody heavy chain. The CH2 and CH3 domains may originate from the same or different antibody heavy chains. In certain embodiments, the Fc polypeptide comprises CH2 and CH3 domain-containing portions from a single antibody heavy chain. In certain embodiments, the CH2 and / or CH3 domains are variants of the spontaneously occurring CH2 or CH3 domain, respectively. In certain embodiments, the CH2 and / or CH3 domains are variants comprising one or more amino acid insertions, deletions, substitutions, or modifications compared to the naturally occurring CH2 or CH3 domain. In certain embodiments, the CH2 and / or CH3 domains are chimeras of one or more CH2 or CH3 domains. In certain embodiments, the CH2 domain comprises amino acids 231-340 of the naturally occurring hinge region (e.g., human IgG1) according to the EU index. In certain embodiments, the CH3 domain comprises amino acids 341-447 of the naturally occurring hinge region (e.g., human IgG1) according to the EU index.

[0254] In certain embodiments, the Fc polypeptide further comprises a hinge region, the C-terminus of which is (directly or indirectly) linked to the N-terminus of the CH2 domain. For example, in certain embodiments, the hinge region is a spontaneously occurring hinge region derived from an IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 antibody heavy chain, e.g., a human IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 antibody heavy chain. The hinge region may be derived from the same or a different antibody heavy chain as the CH2 domain and / or the CH3 domain. In certain embodiments, the hinge region is a variant comprising one or more amino acid insertions, deletions, substitutions, or modifications compared to a spontaneously occurring hinge region. In certain embodiments, the hinge region is a chimera of one or more hinge regions. In certain embodiments, the hinge region comprises amino acid positions 226-229 of a spontaneously occurring hinge region (e.g., human IgG1) according to the EU index. In certain embodiments, the hinge region includes amino acids 216-230 of the naturally occurring hinge region (e.g., human IgG1) according to the EU index. In certain embodiments, the hinge region includes amino acids 216-230 of the naturally occurring hinge region (e.g., human IgG1) according to the EU index. In certain embodiments, the hinge region is a variant IgG4 hinge region containing serine (S) at amino acid position 228 according to the EU index.

[0255] In certain embodiments, the Fc polypeptide further comprises a CH1 domain, the C-terminus of which is (directly or indirectly) linked to the N-terminus of the hinge region. For example, in certain embodiments, the CH1 domain is a spontaneously occurring CH1 domain derived from an IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 antibody heavy chain, e.g., a human IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 antibody heavy chain. The CH1 domain may be derived from the same or a different antibody heavy chain as the hinge region, the CH2 domain, and / or the CH3 domain. In certain embodiments, the CH1 domain is a variant comprising one or more amino acid insertions, deletions, substitutions, or modifications compared to a spontaneously occurring CH1 domain. In certain embodiments, the CH1 domain is a chimera of one or more CH1 domains. In certain embodiments, the CH1 domain comprises amino acid positions 118-215 of a spontaneously occurring hinge region (e.g., human IgG1) according to the EU index.

[0256] In certain embodiments, the Fc polypeptide lacks a CH1 domain or contains a mutation in the CH1 domain or heavy chain variable domain that prevents association between the antibody light chain and heavy chain. In certain embodiments, the antibody heavy chain lacks a portion of the hinge region.

[0257] Heterodimeration motif In certain exemplary embodiments, further engineering of the first and second Fc domains enhances heterodimerization of the first and second specific binding domains, minimizing the effects of improper chain pairing (i.e., pairing of a BMPI-type receptor with a BMPII-type receptor).

[0258] The production of desired polyspecific antibodies can be improved by using any known technique in the art to address the problem of improper chain pairing. For example, US2010 / 0254989A1 describes the construction of a bispecific cMet-ErbB1 antibody in which the VH and VL of individual antibodies are genetically fused via a GlySer linker. In the case of bispecific antibodies containing an Fc domain, mutations can be introduced into the Fc to promote proper heterodimerization of the Fc portion. Several such techniques are outlined in Klein et al. (mAbs(2012)4:6,1-11), the contents of which are incorporated herein by reference in their entirety.

[0259] In certain embodiments, the first and second specific binding specificities of a polyspecific antibody are heterodimerized via knob-into-hole (KiH) pairing of the Fc domain. This dimerization technique utilizes engineered “protrusions” or “knobs” and “cavitations” or “holes” at the interface of the CH3 domain. If a knob or hole of suitable positioning and size exists at the interface of either the first or second CH3 domain, then only the corresponding hole or knob at the adjacent interface needs to be engineered, thereby promoting and enhancing the pairing of Fc domains at the CH3 / CH3 domain interface. An IgG Fc domain fused to a VHH has a knob, and an IgG Fc domain of a conventional antibody has a hole designed to accommodate a knob, or vice versa. The “knob” refers to at least one amino acid side chain, typically a large side chain, protruding from the interface of the CH3 portion of the first Fc domain. This protrusion is complementary to the “hole” at the CH3 portion of the second Fc domain, forming a “knob” that is received by the “hole”. A "hole" is at least one amino acid side chain, typically a small side chain, that is recessed from the interface of the CH3 portion of the second Fc domain. This technique is described, for example, in U.S. Patents 5,821,333, 5,731,168, and 8,216,805, Ridgway et al. Protein Engineering (1996) 9:617-621), and Carter PJImmunol. Methods (2001) 248:7-15, which are incorporated herein by reference.

[0260] Exemplary amino acid residues that can function as knobs include arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W). Amino acid residues present in the CH3 domain may be exchanged or substituted with amino acid residues of the knob. Preferred amino acids for substitution may include any amino acid with a small side chain, such as alanine (A), asparagine (N), aspartic acid (D), glycine (G), serine (S), threonine (T), or valine (V).

[0261] Exemplary amino acid residues that can function as holes include alanine (A), serine (S), threonine (T), or valine (V). Amino acid residues present in the CH3 domain can be exchanged or substituted for the hole amino acid residue. Preferred amino acids for substitution may include any amino acid with a large side chain, such as arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W).

[0262] The CH3 domain is preferably derived from a human IgG1 antibody. Exemplary amino acid substitutions for the CH3 domain include Y349C, S354C, T366S, T366Y, T366W, F405A, F405W, Y407T, Y407A, Y407V, T394S, or combinations thereof. Preferred exemplary combinations are S354C, T366Y, or T366W for the knob mutation of the first CH3 domain, and Y349C, T366S, L368A, Y407T, or Y407V for the hole mutation of the second CH3 domain.

[0263] In certain embodiments, two Fc domains of an antigen-binding construct are heterodimerized by Fab-arm exchange (FAE). Human IgG1 carrying the P228S hinge mutation may contain the F405L or K409R CH3 domain mutation. Mixing the two antibodies with a reducing agent yields the FAE. This technique is described in U.S. Patent No. 9,212,230 and Labrijn AFPNAS (2013) 110(13):5145-5150, which are incorporated herein by reference.

[0264] In other embodiments, the two Fc domains of the antigen-binding construct are heterodimerized by an electrostatic steering effect. This dimerization technique utilizes electrostatic steering to promote and enhance the pairing of Fc domains at the CH3 / CH3 domain interface. The charge complementarity between the two CH3 domains is modified so that heterodimerization (pairing of opposite charges) is preferred over homodimerization (pairing of same charges). In this method, homodimerization is prevented by electrostatic repulsion. Specific exemplary amino acid residue substitutions that result in the electrostatic steering effect include K409D, K392D, and / or K370D in the first CH3 domain, and D399K, E356K, and / or E357K in the second CH3 domain. This technology is described in U.S. Patent Publication No. 2014 / 0154254A1 and Gunasekaran K. JBC (2010) 285(25):19637–19646, which are incorporated herein by reference.

[0265] In other embodiments, charge complementarity is formed by a first Fc domain containing mutations N297K and / or T299K, and a second Fc domain containing mutations N297D and / or T299D.

[0266] In one embodiment of the present invention, the two Fc domains of an antigen-binding construct are heterodimerized by a hydrophobic interaction effect. This dimerization technique utilizes hydrophobic interactions instead of electrostatic interactions to promote and enhance the pairing of Fc domains at the CH3 / CH3 domain interface. Exemplary amino acid residue substitutions may include K409W, K360E, Q347E, Y349S, and / or S354C in the first CH3 domain, and D399V, F405T, Q347R, E357W, and / or Y349C in the second CH3 domain. Preferred pairs of amino acid residue substitutions between the first and second CH3 domains include K409W:D399V, K409W:F405T, K360E:Q347R, Y349S:E357W, and S354C:Y349C. This technology is described in U.S. Patent Publication No. 2015 / 0307628A1.

[0267] In one embodiment of the present invention, heterodimerization can be mediated by the use of a leucine zipper fusion. Heterodimerization is forced by a leucine zipper domain fused to the C-terminus of each CH3 domain in the antibody chain. This technique is described in Wranik B. JBC (2012) 287(52):43331-43339.

[0268] In one embodiment of the present invention, heterodimerization can be mediated by the use of a Strand Exchange Engineered Domain (SEED) body. Heterodimerization is forced by CH3 domains derived from IgG and IgA formats. This technique is described in Muda M. PEDS (2011) 24(5):447-454.

[0269] In other embodiments, the heterodimerization motif may include a non-native disulfide bond formed by engineered cysteine ​​residues. In certain embodiments, the first set of disulfides may include a Y349C mutation in the first Fc domain and an S354C mutation in the second Fc domain. In other embodiments, the engineered disulfide bond may be introduced by fusing a C-terminal extension peptide containing engineered cysteine ​​residues to the C-terminus of each of the two Fc domains. In certain embodiments, the first Fc domain may include a substitution of the carboxyl terminus with "GEC" as "PGK", and the second Fc domain may include a substitution of the carboxyl terminal amino acid "PGK" with "KSCDKT".

[0270] In yet another approach, the multispecific antibody can use the principle of CrossMab (outlined in Klein et al.), which involves domain exchange between the heavy and light chains, to promote the formation of proper pairing. Yet another approach involves engineering the interface between pairs of VH-VL domains or CH1-CL domains of the heavy and light chains to increase the affinity between the heavy chain and its cognate light chain (Lewis et al. Nature Biotechnology (2014) 32:191-198).

[0271] An alternative to producing a multispecific antibody preparation with proper antigen specificity is the development of methods for enriching antibodies with proper heavy-chain-light-chain pairing. For example, Spiess et al. (Nature Biotechnology (2013) 31:753-758) reported a method for producing a MET-EGFR bispecific antibody from co-cultures of bacteria expressing two different half-antibodies.

[0272] Methods have also been reported for mutating at least one constant region of the heavy chain of a bispecific antibody to change its affinity factor, such as its binding affinity for protein A. This enables the isolation of properly paired heavy-chain heterodimers based on purification techniques that utilize the difference in binding of the two heavy chains to the affinity factor (see US2010 / 0331527, WO2013 / 136186).

[0273] International Patent Application No. PCT / EP2012 / 071866 (WO2013 / 064701) addresses the problem of improper chain pairing by using a method for isolating a multispecific antibody based on the use of anti-idiotype binding agents, particularly anti-idiotype antibodies. The anti-idiotype binding agents are used in a two-step selection method in which a first agent is used to capture an antibody having a VH-VL domain pairing specific for a first antigen, and then a second agent is used to capture an antibody also having a second VH-VL domain pairing specific for a second antigen.

[0274] In yet another embodiment, the polyspecific antibody employs a first binding specificity having a conventional Fab binding region and a second binding specificity including a single-domain antibody (VHH) binding region. The heterodimerization method used forces binding of the Fab heavy chain region to only the complete heavy chain of the VHH. Since the VHH chain does not associate with the light chain, the light chain region of the Fab portion associates only with its corresponding heavy chain.

[0275] In certain other embodiments, the polyspecific binding proteins described herein further include a common light chain. As used herein, the term “common light chain” refers to a light chain that, by pairing with a first light chain of an antibody that binds to a first antigen, can form a binding site that specifically binds to the first antigen, and by pairing with a second heavy chain of an antibody that binds to a second antigen, can form a binding site that specifically binds to the second antigen. The common light chain is a polypeptide comprising an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL) in the direction from the N-terminus to the C-terminus, and is also abbreviated herein as “VL-CL”. Polyspecific binding proteins having a common light chain require heterodimerization of different heavy chains. In certain embodiments, the heterodimerization method described above may be used with a common light chain. In certain exemplary embodiments, the heterodimerization motif may include a non-natural disulfide bond formed by engineered cysteine ​​residues. It has been shown that adding a disulfide bond between both the heavy and light chains of the antibody improves stability. Furthermore, disulfide bonds have also been used as a solution to improve light chain pairing in bispecific antibodies (Geddie M. Let al, mABs(2022)14(1)).

[0276] Unless otherwise stated, all antibody constant region numbering used herein corresponds to the EU numbering scheme described in Edelman et al. (Proc.Natl.Acad.Sci.63(1):78-85.1969).

[0277] Further methods for the heterodimerization of heavy and / or light chains and the production and purification of asymmetric antibodies are known in the art. See, for example, Klein C. mAbs (2012) 4(6):653-663 and U.S. Patent No. 9,499,634, which are incorporated herein by reference.

[0278] Effector function variation As described above, the polyspecific binding proteins of this disclosure can be prepared in various isotypes and using different constant regions. The Fc region of the polyspecific binding primarily determines its effector function with respect to Fc binding, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and antibody-dependent phagocytic activity (ADCP). These "cellular effector functions," unlike effector T cell functions, involve recruiting Fc receptor-possessing cells to the site of target cells to result in the killing of antibody-bound cells.

[0279] The antibodies according to the present invention may exhibit reduced effector function. In certain embodiments, one or more mutations reduce one or more of the following: antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), or complement-dependent cell-mediated cytotoxicity (CDC). In certain embodiments, the antibodies according to the present invention may lack ADCC, ADCP, and / or CDC activity. In any case, the antibodies according to the present invention may contain or optionally lack an Fc region that binds to one or more types of Fc receptors. The use of different antibody formats, as well as the presence or absence of FcR binding and cell effector function, allows for the modification of the antibodies to suit use for specific therapeutic purposes as described elsewhere in this specification.

[0280] In certain embodiments, the first and second Fc domains contain one or more mutations that reduce Fc effector function. In certain embodiments, the first and second Fc domains each contain the L234A and L235A mutations. These IgG1 mutations are also known as "LALA" mutations and are described in further detail in Xu et al. (Cell Immunol. 2000;200:16-26). In certain embodiments, the first and second Fc domains each contain the L234A, L235A, G237A, and / or P329G mutations. The amino acid positions of the Fc domains referred to herein are based on EU antibody numbering. Alternatively, the antibody may have a constant region that is effector null. The antibody may have a heavy chain constant region that does not bind to the Fcγ receptor, for example, this constant region may contain the L235E mutation. Another optional mutation in the heavy chain constant region is S228P, which increases stability. The heavy chain constant region may be IgG4 containing both the L235E and S228P mutations. This “IgG4-PE” heavy chain constant region is effector null. The invalidated IgG1 heavy chain constant region is also effector null. The invalidated IgG1 heavy chain constant region may contain alanine at positions 234, 235, and / or 237 (EU index numbering) and may be an IgG1 sequence containing, for example, the L234A, L235A, and / or G237A mutations ("LALAGA").

[0281] Human IgG1 constant regions containing specific mutations or altered glycosylations at residue Asn297 (e.g., N297Q, N297D, and N297K, EU index numbering) have been shown to reduce binding to the Fc receptor.

[0282] In other embodiments, it may be desirable to enhance the binding of the Fc region of a multispecific antibody to human Fc gamma receptor IIIA (FcgRIIIA) compared to that of the Fc region of the corresponding naturally occurring antibody. In certain embodiments, the constant region may be engineered for the enhancement of ADCC and / or CDC and / or ADCP. The potency of the Fc-mediated effect may be enhanced by engineering the Fc domain by various established techniques. Such methods increase affinity to specific Fc receptors and thus create a potentially diverse profile of enhanced activation. This may be achieved by modification of one or more amino acid residues. Examples of mutations are one or more residues (or equivalent positions in other IgG isotypes) selected from 239, 332, and 330 for the human IgG1 constant region. Thus, an antibody may contain a human IgG1 constant region with one or more mutations independently selected from S239D, I332E, and A330L (EU index numbering).

[0283] Increased affinity for Fc receptors can also be achieved by altering the innate glycosylation profile of the Fc domain, for example, by generating low-fucosylated or defucosylated variants. Non-fucosylated antibodies encapsulate a tri-mannosyl core structure of a complex N-glycan of Fc that does not contain fucose residues. These glycoengineered antibodies, lacking core fucose residues derived from the Fc N-glycan, may exhibit stronger ADCC than their fucosylated counterparts due to enhanced FcγRIIIA binding ability. For example, to increase ADCC, residues in the hinge region can be altered to increase binding to FcγRIIIA. Thus, antibodies may contain a human IgG heavy chain constant region, which is a variant of the wild-type human IgG heavy chain constant region. In certain embodiments, the variant human IgG heavy chain constant region binds to a human Fcγ receptor selected from the group consisting of FcγRIIB and FcγRIIA with higher affinity than the wild-type human IgG heavy chain constant region binds to human FcγRIIIA. This antibody may contain a variant human IgG heavy chain constant region that is a variant of the wild-type human IgG heavy chain constant region, and which binds to human FcγRIIB with higher affinity than the wild-type human IgG heavy chain constant region binds to human FcγRIIB. The variant human IgG heavy chain constant region may be the heavy chain constant region of variant human IgG1, variant human IgG2, or variant human IgG4. In one embodiment, the variant human IgG heavy chain constant region contains one or more amino acid mutations selected from G236D, P238D, S239D, S267E, L328F, and L328E (EU index numbering system).In another embodiment, the variant human IgG heavy chain constant region comprises a set of amino acid mutations selected from the group consisting of S267E and L328F; P238D and L328E; P238D, and one or more substitutions selected from the group consisting of E233D, G237D, H268D, P271G, and A330R; P238D, E233D, G237D, H268D, P271G, and A330R; G236D and S267E; S239D and S267E; V262E, S267E, and L328F; and V264E, S267E, and L328F (EU index numbering system).

[0284] Enhancement of CDC can be achieved by amino acid changes that increase affinity to C1q, the first component of the classical complement activation cascade. Another approach is to create a chimeric Fc domain created from human IgG1 and human IgG3 segments that takes advantage of IgG3's higher affinity for C1q. The antibodies of the present invention may contain amino acids mutated at residues 329, 331, and / or 322 to alter C1q binding and / or reduced or lost CDC activity. In another embodiment, the antibodies or antibody fragments disclosed herein may contain an Fc region having modifications at residues 231 and 239 that replace amino acids to alter the antibody's ability to immobilize complement. In one embodiment, the antibody or fragment has a constant region containing one or more mutations selected from E345K, E430G, R344D, and D356R, in particular a double mutation including R344D and D356R (EU index numbering system).

[0285] The functional properties of polyspecific binding proteins can be further modified by combining amino acid substitutions that alter Fc binding affinity with amino acid substitutions that affect binding to FcRn. Binding proteins with amino acid substitutions that affect FcRn binding (also referred to herein as "FcRn variants") may, under certain circumstances, extend the in vivo serum half-life compared to unmodified binding proteins. Naturally, any combination of Fc variants and FcRn variants can be used to modulate antigen-antibody complex clearance. Preferred FcRn variants that can be combined with any of the Fc variants described herein include, but are not limited to, N434A, N434S, M428L, V308F, V259I, M428L / N434S, V259I / V308F, Y436I / M428L, Y436I / N434S, Y436V / N434S, Y436V / M428L, M252Y, M252Y / S254T / T256E, and V259I / V308F / M428L.

[0286] Expression of antigen-binding proteins In one embodiment, polynucleotides encoding binding proteins (e.g., antigen-binding proteins and antigen-binding fragments thereof) disclosed herein are provided. Methods for producing binding proteins, including expressing these polynucleotides, are also provided.

[0287] The polynucleotides encoding the binding proteins disclosed herein are typically inserted into expression vectors for introduction into host cells that can be used to produce a desired amount of the binding protein. Thus, in certain embodiments, this disclosure provides expression vectors comprising the polynucleotides disclosed herein, as well as host cells comprising these vectors and polynucleotides.

[0288] The terms “vector” or “expression vector” are used herein to mean a vector used in accordance with this disclosure as a medium for introducing and expressing a desired gene in a 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, vectors suitable for this disclosure include a selection marker, appropriate restriction sites to facilitate the cloning of the desired gene, and the ability to enter and / or replicate within eukaryotic or prokaryotic cells.

[0289] For the purposes of this disclosure, numerous expression vector systems may be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vacciniavirus, baculovirus, retrovirus (RSV, MMTV, or MOMLV), or SV40 virus. Others involve the use of polycistrone systems including an internal ribosome binding site. Furthermore, cells into which the DNA has been integrated into the chromosome may be selected by introducing one or more markers that enable the selection of transfected host cells. The markers may confer prototrophicity to a nutrient-demanding host, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selection marker gene may be directly ligated to the DNA sequence to be expressed, or it may be introduced into the same cell by co-transformation. Additional elements may be required for optimal mRNA synthesis. These elements may include signal sequences, splice signals, and further, transcription promoters, enhancers, and termination signals. In some embodiments, the cloned variable region gene is inserted into an expression vector together with the heavy chain and light chain constant region genes (e.g., human constant region genes) synthesized as described above.

[0290] In other embodiments, the binding protein may be expressed using a polycistronic construct. In such an expression system, multiple target gene products, such as the heavy and light chains of an antibody, may be produced from a single polycistronic construct. Such a system advantageously utilizes an intrasequence ribosome entry site (IRES) to provide relatively high levels of polypeptide in eukaryotic host cells. A suitable IRES sequence is disclosed in U.S. Patent No. 6,193,980, which is incorporated herein by reference in whole for all purposes. Those skilled in the art will understand that any polypeptide disclosed herein can be effectively produced using such an expression system.

[0291] More generally, once a vector or DNA sequence encoding a binding protein, such as an antibody or a fragment thereof, is prepared, the expression vector can be introduced into a suitable host cell. That is, the host cell can be transformed. The introduction of plasmids into host cells can be achieved by various techniques well known to those skilled in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with envelope DNA, microinjection, and infection with intact viruses. See Ridgway, AAG “Mammalian Expression Vectors” Chapter 24.2, pp.470-472; Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). Plasmid introduction into the host may be by electroporation. Transformed cells are grown under conditions suitable for light and heavy chain production, and assays are performed for the synthesis of heavy and / or light chain proteins. Examples of assay techniques include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), fluorescence-activated cell sequencing (FACS), and immunohistochemistry.

[0292] As used herein, the term “transformation” shall be used in a broad sense to refer to the introduction of DNA into recipient host cells that alters their genotype.

[0293] Similarly, “host cells” refer to cells constructed using recombinant DNA technology and transformed with a vector encoding at least one heterologous gene. In descriptions of the process for isolating polypeptides from recombinant hosts, the terms “cells” and “cell culture” are used synonymously to indicate the source of antibodies unless otherwise specified. In other words, the recovery of polypeptides from “cells” may mean recovery from centrifugated whole cells, recovery from the supernatant of a lysed cell culture, or recovery from a cell culture containing both culture medium and suspension cells.

[0294] In one embodiment, the host cell line used for antibody expression is of mammalian origin. Those skilled in the art can determine the specific host cell line best suited to expressing the desired gene product. Exemplary host cell lines include, but are not limited to, GS-CHO and CHO-K1 (Chinese hamster ovary cell line), DG44 and DUXB11 (Chinese hamster ovary cell line, DHFR-negative), HELA (human cervical cancer), CV-1 (monkey kidney cell line), COS (a derivative of CV-1 containing the SV40 T antigen), R1610 (Chinese hamster fibroblast), BALBC / 3T3 (mouse fibroblast), HEK (human kidney cell line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cell), RAJI (human lymphocyte), and 293 (human kidney). In one embodiment, the cell line undergoes altered glycosylation of the antibodies expressed from the cell line, e.g., defucosylation (e.g., PER.C6® (Crucell) or FUT8 knockout CHO cell line (POTELLIGENT® cells) (Biowa, Princeton, NJ)). In one embodiment, NS0 cells may be used. CHO cells are particularly useful. Host cell lines are typically available through commercial services, e.g., the American Tissue Culture Collection, or from authors of published literature.

[0295] In vitro production allows for scale-up to obtain large quantities of the desired polypeptide. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include, for example, homogeneous suspension culture in airlift reactors or continuous agitation reactors, or immobilized or encapsulated cell culture in, for example, hollow fibers, microcapsules, agarose microbeads, or ceramic cartridges. If necessary and / or desired, polypeptide solutions may be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, chromatography in DEAE cellulose, and / or (immuno) affinity chromatography.

[0296] The genes encoding the binding proteins discussed in this disclosure can also be expressed in non-mammalian cells such as bacteria or yeast, or in plant cells. In this regard, it will be understood that various single-celled non-mammalian microorganisms, such as bacteria, i.e., microorganisms that can grow under culture or fermentation, can also be transformed. Easily transformable bacteria include members of the Enterobacteriaceae, e.g., strains of Escherichia coli or Salmonella; Bacillaceae, e.g., Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. When expressed in bacteria, it will also be understood that the binding proteins may become part of an inclusion body. In some embodiments, the binding proteins are subsequently isolated, purified, and assembled into a functional molecule. In some embodiments, the binding proteins of this disclosure are expressed in bacterial host cells. In some embodiments, bacterial host cells are transformed with an expression vector containing a nucleic acid molecule encoding the binding proteins of this disclosure.

[0297] In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used eukaryotic microorganism, but several other strains are also commonly available. For expression in Saccharomyces, for example, plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)) 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, such as ATCC number 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). Therefore, the presence of trpl damage, a characteristic of the yeast host cell genome, provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0298] Pharmaceutical formulations / pharmaceutical compositions In certain embodiments, a pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an antigen-binding protein described herein. Some embodiments include a pharmaceutical composition comprising a therapeutically effective amount of any one of the binding proteins described herein, or a binding protein-drug conjugate, mixed with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration.

[0299] Acceptable formulation materials are typically non-toxic to the recipient at the dosage and concentration used.

[0300] In some embodiments, the pharmaceutical composition may contain formulation materials for modifying, maintaining, or preserving the composition's pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution rate or release rate, adsorption, or osmosis.Suitable formulation materials include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (e.g., boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids), fillers (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), bulking agents, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin), proteins (e.g., serum albumin, gelatin, or immunoglobulin), colorants, flavoring agents, and diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (e.g., sodium), and preservatives (e.g., benzalcohol chloride). (e.g., sodium benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvent (e.g., glycerin, propylene glycol, or polyethylene glycol), sugar alcohol (e.g., mannitol or sorbitol), suspending agent, surfactant or wetting agent (e.g., Pluronic acid; PEG; sorbitan ester; polysorbates such as polysorbate 20 or polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancer (e.g., sucrose or sorbitol), tonicity enhancer (e.g., alkali metal halides such as sodium chloride or potassium chloride, or mannitol sorbitol), delivery medium, diluent, excipient and / or pharmaceutically adjuvant (e.g., REMINGTON'S, incorporated herein by reference for any purpose) This includes, but is not limited to, PHARMACEUTICAL SCIENCES (18th Ed., Argennaro, ed., Mack Publishing Company 1990) and subsequent editions of the same document.

[0301] In some embodiments, the optimal pharmaceutical composition is determined by those skilled in the art, for example, depending on the intended route of administration, the mode of delivery, and the desired dose. Such a composition may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the binding protein.

[0302] In some embodiments, the primary medium or carrier in the pharmaceutical composition may be either aqueous or non-aqueous. For example, suitable mediums or carriers for injection may be water, saline solution, or artificial cerebrospinal fluid, and other materials common in compositions for parenteral administration may be added. Neutral buffered saline, or saline mixed with serum albumin, are further exemplary mediums. Other exemplary pharmaceutical compositions may comprise Tris buffer at pH approximately 7.0–8.5, or acetate buffer at pH approximately 4.0–5.5, and may further comprise sorbitol or a suitable substitute. In one embodiment of the present disclosure, a binding protein composition may be prepared for storage by mixing a selected composition having a desired purity with an optional formulation agent in the form of a lyophilized cake or aqueous solution. Furthermore, the binding protein may be formulated as a lyophilized product using a suitable excipient such as sucrose.

[0303] In some embodiments, the pharmaceutical compositions of the present disclosure may be selected for parenteral or subcutaneous delivery. Alternatively, the compositions may be selected for inhalation or for delivery via the gastrointestinal tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the scope of the art of those skilled in the art.

[0304] In some embodiments, the components of the formulation are present at the administration site at an acceptable concentration. For example, a buffer is used to maintain the composition at a physiological pH or slightly lower, typically within a pH range of about 5 to about 8.

[0305] When parenteral administration is intended, the therapeutic composition for use may be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired binding protein in a pharmaceutically acceptable medium. A particularly preferred medium for parenteral injection is sterile distilled water, which is properly stored and contains the binding protein as a sterile isotonic solution. Further preparations may involve formulating the desired molecule using agents such as injection microspheres, biodegradable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, resulting in controlled or sustained release of the product, which can then be delivered by depot injection. Hyaluronic acid may also be used, which may have the effect of promoting the duration of action in the circulatory system. Other preferred means for introducing the desired molecule include implantable drug delivery devices.

[0306] In one embodiment, the pharmaceutical composition may be formulated for inhalation. For example, the binding protein may be formulated as a dry powder for inhalation. The inhalation solution of the binding protein may also be formulated using a propellant for aerosol delivery. In yet another embodiment, the solution may be sprayed.

[0307] Furthermore, it is intended that certain formulations may be administered orally. In one embodiment of this disclosure, the polyspecific binding protein administered in this manner may be formulated with or without carriers conventionally used in the preparation of solid dosage forms such as tablets and capsules. For example, a capsule may be designed to release the active portion of the formulation at a point when bioavailability is maximized in the gastrointestinal tract and pre-circulatory degradation is minimized. Additional agents may be included to facilitate the absorption of the binding protein. Diluents, flavorings, low-melting-point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders may also be used.

[0308] Another pharmaceutical composition may contain an effective amount of polyspecific binding protein in a mixture with a non-toxic excipient suitable for the manufacture of tablets. The solution can be prepared in unit dose form by dissolving the tablets in sterile water or another suitable medium. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose, or calcium phosphate; binders such as starch, gelatin, or acacia; or lubricants such as magnesium stearate, stearic acid, or talc.

[0309] Further pharmaceutical compositions of the present disclosure, including formulations containing binding proteins to sustained-release or controlled-release formulations, will be apparent to those skilled in the art. Techniques for formulating various other sustained-release or controlled-release means, such as liposome carriers, biodegradable microparticles or porous beads and depot injections, are also known to those skilled in the art. Further examples of sustained-release preparations include semipermeable polymer matrices in the form of articles, e.g., films or microcapsules. Controlled-release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gammaethyl-L-glutamic acid, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained-release compositions may also include liposomes, which may be prepared by any of several methods known in the art.

[0310] In some embodiments, pharmaceutical compositions used for in vivo administration must typically be sterile. This can be achieved by filtration through a sterile filtration membrane. If the composition is lyophilized, sterilization using this method may be performed either before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or as a solution. Furthermore, parenteral compositions are generally placed in containers with sterile access ports, such as intravenous solution bags or vials with stoppers that can be punctured by a subcutaneous needle.

[0311] Once formulated, pharmaceutical compositions may be stored in sterile vials as solutions, suspensions, gels, emulsions, solids, or dehydrated or lyophilized powders. Such formulations may be stored in a form ready for immediate use or in a form that requires reconstitution before administration (e.g., lyophilized).

[0312] This disclosure also encompasses kits for producing single-dose dosing units. Each kit may include both a first container containing a dry, multispecific binding protein and a second container containing an aqueous formulation. Kits including single-chamber and multi-chamber prefilled syringes (e.g., liquid syringes and lyosyringes) are also included within the scope of this disclosure.

[0313] The effective amount of a binding protein pharmaceutical composition used in treatment depends, for example, on the situation and purpose of the treatment. Therefore, it will be understood by those skilled in the art that the appropriate dosage level for a treatment depends to some extent on the molecules being delivered, the indications for which the binding protein is intended, the route of administration, and the patient's size (body weight, body surface or organ size) and condition (age and overall health). Accordingly, clinicians may titrate the dosage and modify the route of administration to achieve the optimal therapeutic effect.

[0314] The frequency of administration is determined by the pharmacokinetic parameters of the binding protein in the formulation used. Typically, the clinician will administer the composition until the desired effect is achieved. Therefore, the composition may be administered as a single dose, as two or more doses over time (with or without equal amounts of the desired molecule), or as a continuous infusion via an implantable device or catheter. Further refinement of the appropriate dosage is routinely performed by those skilled in the art and falls within the scope of routine tasks performed by those skilled in the art. The appropriate dosage can be confirmed by using appropriate dose-response data.

[0315] The administration route of the pharmaceutical composition follows known methods, such as by oral, intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intrafocal injection, by continuous release system, or by implantable device. If desired, the composition may be administered by bolus injection, continuously by infusion, or by implantable device.

[0316] In some embodiments, the composition can also be administered topically by implanting a membrane, sponge, or other suitable material that absorbs or encapsulates the desired molecule. When an implantable device is used, the device can be implanted in any suitable tissue or organ, and the desired molecule can be delivered by diffusion, sustained-release bolus, or continuous administration.

[0317] The polyspecific binding proteins disclosed herein can be formulated as aerosols for topical application, such as by inhalation (see, for example, U.S. Patents 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for the delivery of steroids useful in the treatment of inflammatory diseases, particularly asthma, and are incorporated herein by reference in their entirety). These formulations for administration to the airways may be in the form of aerosols or solutions for nebulizers, or ultrafine powders for blowing, either alone or in combination with an inert carrier such as lactose. In such cases, the particles of the formulations may have a diameter of less than 50 microns in one embodiment and less than 10 microns in another embodiment.

[0318] The polyspecific binding proteins disclosed herein may be formulated in the form of gels, creams, and lotions for topical or local application, e.g., to the skin and mucous membranes, e.g., to the eyes, and for application to the eyes, or to the capsule or intrathecal cavity. Topical administration is intended for transdermal delivery and for administration to the eyes or mucous membranes, or for inhalation therapy. Heterodimeric proteins may also be administered as nasal drops, either alone or in combination with other pharmaceutically acceptable excipients.

[0319] Transdermal patches, including ionophoresis and electrophoresis devices, are well known to those skilled in the art and can be used to administer heterodimeric proteins. For example, such patches are disclosed in U.S. Patents 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010,715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, all of which are incorporated herein by reference.

[0320] In certain embodiments, the pharmaceutical composition comprising the polyspecific binding protein described herein is a lyophilized powder that can be reconstituted for administration as a solution, emulsion, and other mixture. It can also be reconstituted and formulated as a solid or gel. The lyophilized powder is prepared by dissolving the heterodimeric protein or a pharmaceutically acceptable derivative thereof described herein in a suitable solvent. In certain embodiments, the lyophilized powder is sterile. The solvent may contain excipients to improve stability, or other pharmacological components of the powder or the reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may also contain a buffer such as citric acid, sodium phosphate, or potassium phosphate, or other such buffers known to those skilled in the art, at a nearly neutral pH in one embodiment. The solution is then sterile filtered under standard conditions known to those skilled in the art, followed by lyophilization, to yield the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial contains a single-dose or multi-dose dose of the compound. The lyophilized powder can be stored under suitable conditions, such as approximately 4°C to room temperature. Reconstitution of this lyophilized powder with sterile water for injection yields a formulation for parenteral administration. When reconstituting, the lyophilized powder is added to sterile water or another suitable carrier. The exact amount depends on the selected compound. Such amounts can be determined empirically. The polyspecific binding proteins provided herein can also be formulated to target specific tissues, receptors, or other areas of the body being treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods are contemplated herein for use in the present composition.For non-limiting examples of targeting methods, see, for example, U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, and 6,0 See also Patent Nos. 60,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874. All of these are incorporated herein by reference as a whole. In certain embodiments, the heterodimer proteins described herein target tumors.

[0321] Treatment / Usage Instructions Another aspect of this disclosure is the multispecific antibodies and / or antigen-binding proteins described herein for use as pharmaceuticals.

[0322] In certain embodiments, a method is provided for treating a disorder by activation of BMPI-type receptors and BMPII-type receptors, the method comprising administering an effective amount of the antigen-binding protein described herein to a subject requiring such treatment.

[0323] The binding protein can be used in any known assay method, including competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays, for the detection and quantification of one or more target antigens. The binding protein binds to one or more target antigens with an affinity appropriate to the assay method used.

[0324] For diagnostic applications, in some embodiments, the binding protein can be labeled with a detectable region. The detectable region can be anything that can produce a detectable signal, directly or indirectly. For example, the detectable region is 3 H, 14 C, 32 P, 35 S, 125 I,99 Tc, 111 In, or 67 It may be a radioactive isotope such as Ga; a fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase.

[0325] Binding proteins are also useful for in vivo imaging. Binding proteins labeled at a detectable region can be administered to animals (e.g., into the bloodstream) to assay the presence and location of the labeled antibody in the host. Binding proteins can be labeled at any region detectable in animals, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.

[0326] This disclosure also relates to a kit comprising a binding protein and other reagents useful for detecting the level of a target antigen in a biological sample. Such reagents may include detectable labels, blocking serum, positive and negative control samples, and detection reagents. In some embodiments, the kit comprises a composition comprising any of the binding proteins, polynucleotides, vectors, vector systems, and / or host cells described herein. In some embodiments, the kit comprises a container and a label or accompanying documentation attached to or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, and IV solution bags. Containers may be formed from a variety of materials, such as glass or plastic. The container may hold the composition alone or in combination with another composition effective for treating, preventing, and / or diagnosing a disease, and may have a sterile access port (for example, the container may be an IV solution bag or vial with a stopper puncturable by a subcutaneous needle). In some embodiments, the label or accompanying documentation indicates that the composition is used for the prevention, diagnosis, and / or treatment of a selected disease. Alternatively, or furthermore, the product or kit may further comprise a second (or third) container containing pharmaceutically acceptable buffers such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further comprise other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0327] In some embodiments, this disclosure relates to methods for preventing and / or treating a disease or disorder (e.g., cancer). In some embodiments, the method comprises administering to a patient a therapeutically effective amount of at least one of the binding proteins described herein, or a pharmaceutical composition related thereto. In some embodiments, the patient is human.

[0328] The content of any papers, patents, and patent applications, as well as all other documents and electronically available information, that are referenced or cited herein are incorporated herein by reference in whole to the same extent as each individual publication specifically and individually indicates that it is incorporated by reference. The applicant reserves the right to physically incorporate into this application any material and information from any such papers, patents, patent applications, or other physical and electronic documents.

[0329] While this disclosure has been described in relation to its specific embodiments, those skilled in the art will understand that various modifications may be made and equivalents may be substituted without departing from the true spirit and scope of this disclosure. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein can be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt specific circumstances, materials, compositions, processes, or treatment steps to the purpose, spirit, and scope of this disclosure. All such modifications are intended to fall within the scope of the claims appended herein. While specific embodiments have been described in detail so far, embodiments will be better understood by referring to the following examples. These examples are included for illustrative purposes only and are not intended to be limiting. [Examples]

[0330] Example 1. Bispecific antibodies against BMPRI and type II receptors having optimized hinges, linkers, and valencies. Bispecific antibodies targeting ALK1, a BMRPI-type receptor, and BMPRII, a BMPRII-type receptor, were designed with the sequences shown below. Several constructs include upper hinge variants: Hinge 1 = no upper hinge, Hinge 3 = upper hinge sequence PLAP (SEQ ID NO: 2), Hinge 6 = upper hinge sequence DKTHT (SEQ ID NO: 5).

[0331] The three-dimensional structure of BMP10 (PDB ID 7PPC) complexed with ALK1 and BMPRII was used in combination with the structural models of AlphaFold2 AF-P37023-F1-model_v4 (ALK1) and AF-Q13873-F1-model_v4 (BMPRII), and the model of Agnew et al. (DOI:10.1038 / s41467-021-25248-5) to construct models of the intracellular and extracellular domains of the BMPRII / ALK1 / BMP9 active tetramer receptor complex, enabling phosphorylation of the GS domain and activation of SMAD. The inventors predicted that the tetravalent format of the agonist antibody would promote the predicted tetramer receptor assembly required for signal transduction of the ALK1 / BMPRII complex.

[0332] The DIAGONAL platform predicted epitopes on ALK1 and BMPRII that could be targeted for binder binding to the receptor in this tetravalent format. Using these predictions, we designed a linker that fits the geometric constraints of the CDR and tetravalent antibody formats of the DGL molecule's binding module.

[0333] Antibodies were transiently transfected using the Expi293 (Thermo) system according to the manufacturer's instructions. Cells were harvested 6 days after transfection and collected using batch purification with mabSelect resin. The purity of the final product was evaluated using SDS-PAGE and analytical gel filtration. [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] [Table 2-14] [Table 2-15] [Table 2-16] [Table 2-17]

[0334] Example 2. Screening of agonist activity Bispecific antibodies were screened for agonist activity. The PathHunter U2Os ALK-1 / BMPR-2 dimerization assay was obtained from DiscoverX Corporation (93-0962C3). These cells were prepared using enzyme fragment complementation (EFC) technology, employing β-galactosidase fragments to evaluate protein-protein interactions. Reporter cells were revived and cultured according to the supplier's recommendations. Bispecific antibodies were compared to their native ligands, BMP9 and BMP10.

[0335] To perform the assay, cells were separated from the flask using a cell separation reagent (DiscoverX, 92-0009) and removed. The cells were centrifuged at 300 g for 4 minutes and resuspended in assay plating medium (DiscoverX 93-0563R22A) at a density of 250 K / ml. 20 μl of this suspension was plated into the wells of a 384-well plate and incubated at 37°C for 24 hours. Bispecific antibodies were prepared at 5-fold the final concentration. A 12-stop titration was performed using a 1:10 dilution to generate a curve. 5 μl of bispecific antibody was added to the 384-well plate and incubated for 3 hours. 25 μl of flash detection reagent (DiscoverX, 93-0247) was added to the wells, and the plate was read using Verilux Skan after 60 minutes. Data were analyzed using PRISM. [Table 3]

[0336] It was observed that tetravalent bispecific antibodies (i.e., two binding domains for ALK1 and two binding domains for BMPRII) induced stronger agonism than bivalent bispecific antibodies (i.e., one binding domain for ALK1 and one binding domain for BMPRII). These bivalent bispecific antibodies, DGL266-DGL271, exhibited 0-46% of BMP9 activity, while tetravalent bispecific antibodies, such as DGL285-DGL292, consistently showed higher values. Surprisingly, tetravalent bispecific antibodies with a BMPRII-binding domain followed by an ALK1-binding domain from the N-terminus to the C-terminus exhibited substantially higher agonism than tetravalent bispecific antibodies with an ALK1-binding domain followed by a BMPRII-binding domain from the N-terminus to the C-terminus. The above data is summarized below to compare the two bispecific antibodies with different orientations. JPEG2026515704000022.jpg71164

[0337] This effect was observed in both the dual scFv tetravalent format and the DVD-Ig format. The bispecific antibodies DGL285-288 were in the dual scFv tetravalent format, while DGL289-292 were in the DVD-Ig format.

[0338] The dual scFv tetravalent format comprises two polypeptide chains, each containing, from N-terminus to C-terminus, a first scFv for a first target, either ALK1 or BMPRII; a second scFv for a second target, either ALK1 or BMPRII; and an Fc domain. The first and second targets are different; if the first target is BMPRII, then the second target is ALK1. The first scFv may be linked to the second scFv using a linker such as a modified hinge as described herein.

[0339] This DVD-Ig format comprises four polypeptide chains. The first and second polypeptide chains each contain a first VH (VH1), a second VH (VH2), and an Fc domain, from the N-terminus to the C-terminus. The third and fourth polypeptide chains each contain a first VL (VL1) and a second VL (VH2), from the N-terminus to the C-terminus. VH1 and VL1 form a first binding domain to a first target, either ALK1 or BMPRII, and VH2 and VL2 form a second binding domain to a second target, either ALK1 or BMPRII. The first and second targets are different; if the first target is BMPRII, then the second target is ALK1. Linkers such as the modified hinges described herein may be used to link VH1 to VH2 and / or VL1 to VL2.

[0340] Example 3: Measurement of pSMAD in HUVEC cells HUVEC cells (CRL-1730) from ATCC were cultured in 15K cells per well in a 96-well plate, in 100 μl of complete HUVEC medium (F12K (Corning, 10-025-CV), 10% FBS (Gibco, A31605-02), ECGS (30 μg / ml, Corning, 356006), 0.1 mg / ml heparin (Sigma, H3393), 1 x Pen / Strep (Gibco, Cells were plated overnight in 15140-122). The following morning, cells were starved for 4 hours by replacing the medium with 50 μl of serum-free / ECGS-free F12K medium. Cells were then treated with 50 μl of serum-free / ECGS-free medium containing a dose curve of 2x concentrations of either a bispecific antibody or a BMP ligand. The medium was removed from the cells at various time points (5 min, 15 min, 30 min, 60 min), and 50 μl of lysis buffer (Abcam ELISA kit, AB186037) was added per well. After lysis, the buffer from the four wells was pooled for a single 200 μl lysed sample for each condition, frozen, and later subjected to ELISA to measure either total SMAD1 (Abcam, AB186037) or pSMAD1 (Abcam, AB186036). An anti-HEL antibody with the LALA-PG mutation (BioXCell, CP149) was used as a negative control. [Table 4]

[0341] Example 4: Measurement of in vivo activity The antibody was measured for agonist activity in a mouse model of HHT in which circulating BMP9 / BMP10 was neutralized by an anti-BMP9 / 10 antibody (Ruiz S, et al, Scientific Reports, 2016 Nov 22:5:37366). These mice developed retinal vascular damage after birth. Three animals were administered either DGL288 or a negative control antibody (anti-HEL, LALA-PG, BioXCell, CP149) at a dose of 15 mg / kg / day for two days in phases 3 and 4. The BMP9 / 10 antibody was administered on the same day. Analysis was completed in phase 6. The retina was dissected, whole tissue specimens were prepared, and then the retinal vascular system was labeled and arteriovenous malformations (AVMs) were detected by staining with both isolectin B4 and SMA. The results are shown in Figure 3A. Mice treated with DGL288 did not show AVM formation, while negative controls showed a mean of 4.8 AVMs / retina.

[0342] In the second series of experiments, all animals were administered BMP9 / 10 antibodies at P3 and P4. DGL288, DGL292, or PBS control were administered at 1 mg / kg / day at P4 and P5. Analysis was completed at P6 for DGL288 and littermates receiving negative control, and at P7 for littermates receiving DGL292 and PBS control. The retinas were dissected, whole tissue specimens were prepared, and then stained with both isolectin B4 and SMA to detect AVMs. Mice administered DGL292 did not form AVMs, compared to a mean of 5.7 / retina in controls (Figure 3B). Mice administered DGL288 did not form AVMs, compared to a mean of 4.5 / retina in controls (Figure 3C). No difference in body weight was observed, suggesting good tolerability of the agonists.

[0343] Example 5: Further Engineering of the Binder Based on the structural modeling of the receptor / antibody complex, the binder was engineered to further optimize the complementary region for binding to the antigen. Both ALK1 and BMPRII variants were designed to improve potency and / or stability. Table 5-1 Table 5-2 Table 5-3 Table 5-4 Table 5-5 Table 5-6 Table 5-7 Table 5-8 Table 5-9 Table 5-10 Table 5-11 Table 5-12 Table 6-1 Table 6-2 Table 6-3 Table 7-1 Table 7-2 Table 7-3 [Table 7-4]

[0344] These binders were then tested using an ELISA assay. High-binding plates (Corning, 9018) were coated overnight at 4C with either 2 ug / ml human BMPRII protein (Sino Biological, #10551-H08H) or 2 ug / ml human ALK1 protein (Sino Biological, #10066-H08H). The plates were then washed three times with wash buffer (R&D Systems, WA126). The plates were blocked at room temperature for 1 hour with PBS containing 1% BSA, and then further blocked at room temperature for 1 hour with PBS containing 1% BSA and 2 ug / ml goat anti-human IgG (Jackson ImmunoResearch, 109-005-190). The plates were then washed three times with wash buffer and DGL antibody or control diluted in PBS and 0.1% BSA. After incubating these antibodies at room temperature for 1 hour, the plates were washed three times with wash buffer. Next, the plates were incubated with mouse anti-human IgG Fc secondary-HRP (diluted with PBS / 0.1% BSA). Specifically, they were incubated at 2 ug / ml, 100 ul per well, at room temperature for 1 hour. After washing the plates three times with washing buffer, they were washed again with 100 ul of TMB (R&D Systems, DY9998B, substrate reagent pack). After the wells turned blue, 50 ul of stop solution (R&D Systems, DY994) was added to each well, and the absorbance of the plates was read at 450 nm. The results are shown in Table 8. [Table 8]

[0345] Example 6. DiscoverX data of the variant The bispecific antibodies were screened for agonist activity as described in Example 2. The reported data (RLU) are the average of two replicate tests at the highest concentration tested. The antibodies were compared to the natural ligand BMP9 in all plates. [Table 9-1] [Table 9-2] [Table 10] [Table 11-1] [Table 11-2]

[0346] Example 7: Engineering of scFv containing a bispecific agonist antibody with an optimized hinge The agonist activity of heteromer antibodies with modified hinges identified on the DIAGONAL platform was also tested. A variant of DGL288, DGL809, was designed with hinge 1. DGL809 was designed, expressed, and purified as described above. The heteromer antibody was tested using the DiscoverX assay. As shown in Table 12 (mean values ​​across two different experiments) which shows the activity level against BMP9 at an antibody concentration of 100 nM, DGL809 was superior to the parent DGL288. [Table 12]

[0347] Example 8. Engineering of a bispecific agonist antibody using an optimized linker in DVD-Ig format. Another way to stiffen agonist antibodies is to optimize the linker between IgG and further variable domains in DVD-Ig format. To pursue this pathway, we tested the agonist activity of heteromeric antibodies with modified hinged linkers of VH and IgG identified on the DIAGONAL platform. Variants of DGL292, DGL810, DGL811, and DGL812, were designed, expressed, and purified as described above. When the heteromeric antibodies were tested using the DiscoverX assay, the variants outperformed the parent DGL292, as seen in Table 14 (showing mean values ​​across two different experiments). [Table 13] [Table 14] [Table 15-1] [Table 15-2] [Table 15-3] [Table 16-1] [Table 16-2] [Table 16-3] [Table 17-1] [Table 17-2]

[0348] Example 9. Screening of agonist activity Bispecific antibodies were screened for agonist activity. The PathHunter U2Os ALK-1 / BMPR-2 dimerization assay was obtained from DiscoverX Corporation (93-0962C3). These cells were prepared using enzyme fragment complementation (EFC) technology, employing β-galactosidase fragments to evaluate protein-protein interactions. Reporter cells were revived and cultured according to the supplier's recommendations. Bispecific antibodies were compared to their native ligands, BMP9 and BMP10.

[0349] To perform the assay, cells were separated from the flask using a cell separation reagent (DiscoverX, 92-0009) and removed. The cells were centrifuged at 300 g for 4 minutes and resuspended in assay plating medium (DiscoverX 93-0563R22A) at a density of 250 K / ml. 20 μl of this suspension was plated into the wells of a 384-well plate and incubated at 37°C for 24 hours. Bispecific antibodies were prepared at 5-fold the final concentration. A 12-stop titration was performed using a 1:10 dilution to generate a curve. 5 μl of bispecific antibody was added to the 384-well plate and incubated for 3 hours. 25 μl of flash detection reagent (DiscoverX, 93-0247) was added to the wells, and the plate was read using Verilux Skan after 60 minutes. Data were analyzed using PRISM. The results are shown in Table 17 below. This data indicates that each of the bispecific antibodies tested possessed potent agonist activity. [Table 17-3]

[0350] Example 10. Measurement of agonist activity in endothelial cells HMEC-1 cells were plated in 200 μl of complete 10% MCDB growth medium at 30K cells / well in a 96-well plate and incubated overnight. After approximately 16 hours, the complete medium was replaced with 50 μl of serum-free MCDB medium. After incubating the cells in serum-free medium for 4 hours, 2x DGL tool was added to 50 μl of serum-free MCDB medium. After 45 minutes, the medium was removed, the cells were washed once with PBS, and then the lysis buffer from the ELISA kit was added. The lysates were then analyzed by ELISA according to the manufacturer's instructions (Abcam pSMAD1 ELISA AB186036). A 12-stop titration using a 1:10 dilution was performed to generate curves. An anti-HEL antibody with the LALA-PG mutation (BioXCell, CP149) was used as a negative control. Data were analyzed using PRISM. The reported data are the average of two experiments. The results are shown in Table 18 below. This data indicates that each of the two specific antibodies tested possessed potent agonist activity, as measured through pSMAD1 levels. [Table 18]

[0351] Example 11. Measurement of in vivo activity The antibody was measured for agonist activity in a mouse model of HHT in which circulating BMP9 / BMP10 was neutralized by an anti-BMP9 / 10 antibody (Ruiz S, et al, Scientific Reports, 2016 Nov 22:5:37366). These mice developed retinal vascular damage after birth. Three animals were administered DGL292, DGL945, DGL947, or a negative control antibody (anti-HEL, LALA-PG, BioXCell, CP149) at a dose of 1 mg / kg / day for two days in phases 3 and 4. The BMP9 / 10 antibody was administered on the same day. Analysis was completed in phase 6. The retina was dissected, whole tissue specimens were prepared, and then the retinal vascular system was labeled and arteriovenous malformations (AVMs) were detected by staining with both isolectin B4 and SMA. The results are shown in Figure 4. Mice treated with any ALK1-BMPRII agonist showed a significant reduction in AVM formation, while negative controls averaged 4.5 AVMs / retina.

[0352] Example 12. Analysis of thermal stability Differential scanning calorimetry (DSC) is a thermal analysis technique used to characterize the thermal stability of protein samples and evaluate the differences in their three-dimensional structures. Measurements were performed using MicroCal PEAQ DSC (Malvern) for thermal transition midpoint (Tm) and unfolding onset (TOnset) tests. Samples were diluted to 1 mg / mL in reference buffer (20 mM histidine, 8% (w / v) sucrose, 0.02% (w / v) PS80, pH 6.0). 400 μL of each reference buffer was added to the odd-numbered wells of a 96-well plate, and 400 μL of the sample was added to the even-numbered wells of the same plate. Experimental parameters were set so that the scan temperature ramped from 10°C to 95°C at a scanning rate of 200°C / hour. Data analysis was performed using MicroCal The analysis was performed using PEAQ-DSC automated data analysis software. The melting temperature data is shown in Table 19 below. Surprisingly, DGL947 and DGL949 were found to be more stable than DGL945 and DGL1146, as indicated by an increase in both the thermal unfolding onset temperature (Tonset) and the first unfolding event (Tm1). The variable domains of DGL947 and DGL949 differ from those of DGL945 and DGL1146 only within the CDR. [Table 19]

[0353] Example 13. Gene expression analysis of HMEC-1 cells HMEC-1 cells from ATCC were plated overnight in 100 μl of complete growth medium (MCDB-based, +10% FBS, Pen / Strep, L-glutamine, hydrocortisone, EGF) at a concentration of 30K cells / well in 96-well plates. After overnight incubation, the medium was removed and replaced with 50 μl of low-serum medium (same as growth medium but with 1% FBS). Cells were incubated for approximately 4 hours, during which time standard curves of agonists were prepared in low-serum medium at 2X final concentrations. After 4 hours, 50 μl of antibody or BMP9 was added to the cells and incubated overnight. After overnight incubation, the medium was removed and ZYMO RNA lysis buffer was added. RNA was isolated from cell lysates using the ZYMO 96 RNA Isolation Kit and RT reaction was performed using the Quanta Biosciences Kit. qPCR was performed on cDNA using the Thermo-designed Taqman assay for ID1, Serpine1, and GAPDH as housekeeping controls. The magnification change was calculated as DD ct. As shown in Tables 20-22, the results of the gene expression analysis show that the bispecific antibody stimulates the gene expression of the ALK1 target (ID1) using GAPDH as a housekeeping gene. Tables 23-25 ​​show the results of a second experiment using RPL36AL as the housekeeping gene. [Table 20] [Table 21] [Table 22] [Table 23] [Table 24] [Table 25]

[0354] Another cell line, the TIME cell line, was also used for gene expression analysis. TIME cells (ATCC), hTERT immortalized cells exhibiting endothelial morphology, were plated overnight in 100 μl of complete growth medium (vascular cell basal medium plus microvascular endothelial cell growth kit - VEGF) at a concentration of 30K cells / well in 96-well plates. After overnight incubation, the medium was removed and replaced with 50 μl of low-serum medium (growth medium diluted 1:10 with vascular cell basal medium). The cells were incubated for approximately 4 hours, during which time standard curves for agonists were prepared in low-serum medium at 2X final concentration. After 4 hours, 50 μl of agonist was added to the cells and incubated overnight. After overnight incubation, the medium was removed and ZYMO RNA lysis buffer was added. RNA was isolated from the cell lysates using the ZYMO 96 RNA isolation kit, and the RT reaction was performed using the Quanta Biosciences kit. qPCR was performed on CDNA using ID1, Serpine1, and a Taqman assay designed by Thermo for GAPDH or RPL36AL as housekeeping controls. The digit change was calculated as DD ct. Tables 26-27 show the results using RPL36AL as a housekeeping control. [Table 26] [Table 27] [Table 28]

[0355] Example 14. Stabilization of ALK1 receptor on the cell surface. The bispecific antibodies of this disclosure can stabilize the ALK1 receptor, which is complexed with one of BMPRII, ActRIIA, and ActRIIB on the cell surface. Through receptor stabilization, signal transduction can be sustained for a longer period of time.

[0356] To evaluate the stabilization of the ALK1 receptor complex on the cell surface, staining may be performed for ALK1 and one of BMPRII, ActRIIA, and ActRIIB. An exemplary protocol is described below, but those skilled in the art will readily recognize alternative approaches for detecting proteins on the cell surface. Furthermore, certain parameters outlined in the exemplary protocol (e.g., buffer selection, buffer component concentrations, cell line selection, total cells, antibody concentration, time, temperature, etc.) may be adjusted as needed to optimize the assay.

[0357] Staining of ALK1 and BMPRII in MS1 ​​cells: Autoclaved coverslips are placed in 24-well cell culture plates, and MS1 cells are seeded onto the coverslips in complete medium and allowed to adhere overnight. The cells are then starved for approximately 3 hours, followed by treatment with a bispecific antibody (e.g., DGL288) or IgG control disclosed herein at a concentration of approximately 1 μg / mL for 2 hours. After treatment, the coverslips are rinsed twice with PBS for approximately 5 minutes each time, fixed in 4% paraformaldehyde for approximately 10 minutes, and then washed again with PBS.

[0358] Next, the cells were permeabilized for approximately 15 minutes using PBS containing 0.25% Triton X-100, and then blocked for approximately 1 hour in a PBS solution containing 5% normal goat serum (Sigma-Aldrich, #G9023-10ML) and 0.25% Triton X-100. Subsequently, primary antibodies containing ALK1 (1:100 dilution, Santacruz #sc-101556), BMPRII (1:100 dilution, Invitrogen #MA5-15827), and CD31-AF667 (1:50 dilution, Miltenyi #130-128-736), diluted in a PBS solution of 1% NGS and 0.25% Triton X-100, were added, and the cells were incubated overnight at 4°C. The following day, the coverslips were washed twice with PBS for 5 minutes each time, and then incubated with secondary antibodies (goat anti-rat IgG H+L AF568, Thermo Fisher Scientific, #A-11077; goat anti-mouse IgG1 AF488, Thermo Fisher Scientific, #A-21121) diluted 1:1000 in a PBS solution of 1% NGS and 0.25% Triton X-100. After three further washes with PBS, the cells were stained with DAPI (BD Biosciences, #564907) and then washed three more times with PBS. Finally, the coverslips were mounted on glass slides using ProLong® Diamond Antifade Mountant (Thermo Fisher #P36965). Cell imaging was performed at 63x magnification using a confocal Zeiss LSM900 microscope, and image analysis was performed using Zenblue Zeiss software.

Claims

1. A polyspecific binding protein comprising at least a first polypeptide chain, wherein the first polypeptide chain comprises a first variable heavy chain domain (VH1) linked to a second variable heavy chain domain (VH2) via at least one modified hinge region, The VH1 specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB, and the VH2 specifically binds to ALK1, or The multispecific binding protein wherein VH1 specifically binds to ALK1, and VH2 specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB.

2. The multispecific binding protein according to claim 1, wherein one or both of the VH1 and VH2 are a VH domain or a VHH domain.

3. Furthermore, the present invention comprises a second polypeptide chain, wherein the second polypeptide chain comprises a first variable light chain domain (VL1) linked to a second variable light chain domain (VL2), The VL1 specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB, and the VL2 specifically binds to ALK1, or The multispecific binding protein wherein VL1 specifically binds to ALK1, and VL2 specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB.

4. The multispecific binding protein according to claim 3, wherein the VL1 is linked to the VL2 via at least one modified hinge region.

5. The polyspecific binding protein according to any one of claims 1 to 4, wherein one or both of the VH1 and VH2 are cleaved at the C-terminus.

6. The multispecific binding protein according to claim 5, wherein at least one residue is cleaved at the C-terminus.

7. The multispecific binding protein according to claim 5 or 6, wherein at least two residues are cleaved at the C-terminus.

8. The polyspecific binding protein according to any one of claims 5 to 7, wherein the C-terminal SS amino acid residue is deleted.

9. A polyspecific binding protein according to any one of claims 1 to 8, wherein the first polypeptide chain is VH1-HX1-VH2-C-Fc, provided that, VH1 is the first heavy chain variable domain, VH2 is the second heavy chain variable domain, C is a heavy chain constant domain, HX1 is a linker in the modified hinge region, Fc is an Fc region, and The second polypeptide chain is VL1-LX1-VL2-C, however, VL1 is the first light chain variable domain, VL2 is the second light chain variable domain, C is a light chain constant domain, LX1 includes a linker in the modified hinge region. The multispecific binding protein.

10. A polyspecific binding protein according to any one of claims 1 to 9, wherein the modified hinge region is i) Upper hinge region that is or does not exist and is at most 7 amino acids long, ii) The multispecific binding protein including the lower hinge region.

11. The polyspecific binding protein according to any one of claims 1 to 10, wherein the modified hinge region comprises or consists of the amino acid sequence PLAP or PAPNLLGGGP.

12. A polyspecific binding protein according to any one of claims 1 to 11, A) The VH that binds to ALK1 includes the amino acid sequence SYAMS of HCDR1, the amino acid sequence NINQDGSEKNYVDSMRG of HCDR2, and the amino acid sequence EFDY of HCDR3, and the VL that binds to ALK1 includes the amino acid sequence SGSSSNIGSNYVY of LCDR1, the amino acid sequence GNNKRPS of LCDR2, and the amino acid sequence AAWDDSLNGRV of LCDR3, or B) The VH that binds to ALK1 includes the amino acid sequence SYWMS of HCDR1, the amino acid sequence NINQDGSEKYYVDSMRG of HCDR2, and the amino acid sequence EYDY of HCDR3, and the VL that binds to ALK1 includes the amino acid sequence SGSSSNIGSNYVY of LCDR1, the amino acid sequence GNNKRPS of LCDR2, and the amino acid sequence AAWDDSLNGRV of LCDR3, or C) The polyspecific binding protein wherein VH that binds to ALK1 comprises the amino acid sequence SYWMS of HCDR1, the amino acid sequence NIKQDGSEKNYVDSMRG of HCDR2, and the amino acid sequence EFDF of HCDR3, and VL that binds to ALK1 comprises the amino acid sequence SGSSSNIGSNYVY of LCDR1, the amino acid sequence GNNKRPS of LCDR2, and the amino acid sequence AAWDDSLNGRV of LCDR3.

13. A polyspecific binding protein according to any one of claims 1 to 12, A) The VH that binds to BMPRII includes the amino acid sequence DYYMT of HCDR1, the amino acid sequence SISGGGSTYYADSRKG of HCDR2, and the amino acid sequence DFGVAAGWFGQYGMDV of HCDR3, and the VL that binds to BMPRII includes the amino acid sequence TGSSSNIGAGYDVH of LCDR1, the amino acid sequence RSNQRPS of LCDR2, and the amino acid sequence SSYAGNYNLV of LCDR3, or B) The VH that binds to BMPRII includes the amino acid sequence DYYMN of HCDR1, the amino acid sequence SISGGGSTYYAADSVKG of HCDR2, and the amino acid sequence DFGVAAGWFGQFGMDV of HCDR3, and the VL that binds to BMPRII includes the amino acid sequence TGSSSNIGAGYDVH of LCDR1, the amino acid sequence RSNQRPS of LCDR2, and the amino acid sequence SSYAGNYNLV of LCDR3, or C) The polyspecific binding protein wherein VH that binds to BMPRII includes the amino acid sequence DYYMN of HCDR1, SISGGGSTYYADSVKG of HCDR2, and the amino acid sequence DFGVAAGWFGYYGMDV of HCDR3, and VL that binds to BMPRII includes the amino acid sequence TGSSSNIGAGYDVH of LCDR1, RSNQRPS of LCDR2, and the amino acid sequence SSYAGNYNLV of LCDR3.

14. A polyspecific binding protein according to any one of claims 1 to 13, A) The VH that binds to ALK1 contains the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSSYAMSWVRQAPGKGLEWVANINQDGSEKNYVDSMRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREFDYWGQGTLVTVSS, or an amino acid sequence having at least 90% identity thereto. The VL that binds to ALK1 contains the amino acid sequence QSVLAQPPSASGTPGQRVTISCSGSSSSNIGSNYVYWYQQLPGTAPKLLLIYGNNKRPSGVPDRFSGSKSGTSASLAIISGLRSSEDEADYYCAAWDDSLNGRVFGGGTKLTVL, or an amino acid sequence having at least 90% identity thereto, or B) The VH that binds to ALK1 contains the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSSYWMSWVRQAPGKGLEWVANINQDGSEKYYVDSMRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREYDYWGQGTLVTVSS, or an amino acid sequence having at least 90% identity thereto. The VL that binds to ALK1 contains the amino acid sequence QSVLAQPPSASGTPGQRVTISCSGSSSSNIGSNYVYWYQQLPGTAPKLLLIYGNNKRPSGVPDRFSGSKSGTSASLAIISGLRSSEDEADYYCAAWDDSLNGRVFGGGTKLTVL, or an amino acid sequence having at least 90% identity thereto, or C) The VH that binds to ALK1 contains the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSSYWMSWVRQAPGKGLEWVANIKQDGSEKNYVDSMRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREFDFWGQGTLVTVSS, or an amino acid sequence having at least 90% identity thereto, and the AL The multispecific binding protein wherein the VL that binds to K1 contains the amino acid sequence QSVLAQPPSASGTPGQRVTISCSGSSSSNIGSNYVYWYQQLPGTAPKLLLIYGNNKRPSGVPDRFSGSKSGTTSASLAISGLRSSEDEADYYCAAWDDSLNGRVFGGGTKLTVL, or an amino acid sequence having at least 90% identity thereto.

15. A polyspecific binding protein according to any one of claims 1 to 14, A) The VH that binds to BMPRII is an amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMTWIRQAPGKGLEWVSSISGGGSTYYADSRKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDFGVAAGWFGQYGMDVWGQGTLVTVSS, or an amino acid having at least 90% identity thereto. The VL containing the sequence and bound to BMPRII contains the amino acid sequence QSVLTQPPSASGTPGQRVTISCTGSSSSNIGAGYDVHWYQQLPGTAPKLLLIYRSNQRPSGVPDRFGSGSKSGTTSASLAISGLRSSEDEADYYYCSSYAGNYNLVFGGGTKLTVL, or an amino acid sequence having at least 90% identity thereto, or B) The VH that binds to BMPRII is an amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMNWIRQAPGKGLEWVSSISGGGSTYYADSVKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDFGVAAGWFGQFGMDVWGQGTLVTVSS, or an amino acid having at least 90% identity thereto. The VL containing the sequence and bound to BMPRII contains the amino acid sequence QSVLTQPPSASGTPGQRVTISCTGSSSSNIGAGYDVHWYQQLPGTAPKLLLIYRSNQRPSGVPDRFGSGSKSGTTSASLAISGLRSSEDEADYYYCSSYAGNYNLVFGGGTKLTVL, or an amino acid sequence having at least 90% identity thereto, or C) The VH that binds to BMPRII contains the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMNWIRQAPGKGLEWVSSISGGGSTYYADSVKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDFGVAGWFGYYGMDVWGQGTLVTVSS, or an amino acid sequence having at least 90% identity thereto. The multispecific binding protein wherein the VL that binds to BMPRII contains the amino acid sequence QSVLTQPPSASGTPGQRVTISCTGSSSSNIGAGYDVHWYQQLPGTAPKLLLIYRSNQRPSGVPDRFGSGSKSGTTSASLAISGLRSSEDEADYYYCSSYAGNYNLVFGGGTKLTVL, or an amino acid sequence having at least 90% identity thereto.

16. The polyspecific binding protein according to any one of claims 3 to 15, wherein the first polypeptide chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 136 to 142, and the second polypeptide chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 143 to 146.

17. The polyspecific binding protein according to any one of claims 3 to 15, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:

146.

18. The polyspecific binding protein according to any one of claims 3 to 15, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:

146.

19. The polyspecific binding protein according to any one of claims 3 to 15, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:

146.

20. The polyspecific binding protein according to any one of claims 3 to 15, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:

146.

21. The polyspecific binding protein according to any one of claims 3 to 15, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:

146.

22. The polyspecific binding protein according to any one of claims 3 to 15, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 142, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:

146.

23. The polyspecific binding protein according to any one of claims 3 to 15, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 68, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:

69.

24. The polyspecific binding protein according to any one of claims 3 to 15, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 70, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:

71.

25. The polyspecific binding protein according to any one of claims 3 to 15, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 72, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:

73.

26. The polyspecific binding protein according to any one of claims 3 to 15, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 74, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:

75.

27. A polyspecific binding protein comprising a first polypeptide chain and a second polypeptide chain, wherein each of the first and second polypeptide chains includes a first single-chain variable fragment (scFv) linked to a second scFv from the N-terminus to the C-terminus, The first scFv specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB, and the second scFv specifically binds to ALK1, or The polyspecific binding protein wherein the first scFv specifically binds to ALK1, and the second scFv specifically binds to a target selected from BMPRII, ActRIIA, and ActRIIB.

28. The polyspecific binding protein according to claim 27, wherein the first scFv is linked to the second scFv via at least one modified hinge region.

29. The scFv coupled to ALK1 is A): The VH domain includes the amino acid sequence SYAMS of HCDR1, the amino acid sequence NINQDGSEKNYVDSMRG of HCDR2, and the amino acid sequence EFDY of HCDR3. The VL that binds to ALK1 includes the amino acid sequence SGSSSNIGSNYVY of LCDR1, the amino acid sequence GNNKRPS of LCDR2, and the amino acid sequence AAWDDSLNGRV of LCDR3, or B): The VH domain containing the amino acid sequence SYWMS of HCDR1, the amino acid sequence NINQDGSEKYYVDSMRG of HCDR2, and the amino acid sequence EYDY of HCDR3, and It contains a VL domain comprising the amino acid sequence SGSSSNIGSNYVY of LCDR1, the amino acid sequence GNNKRPS of LCDR2, and the amino acid sequence AAWDDSLNGRV of LCDR3, or C): The VH domain containing the amino acid sequence SYWMS of HCDR1, the amino acid sequence NIKQDGSEKNYVDSMRG of HCDR2, and the amino acid sequence EFDF of HCDR3, and The polyspecific binding protein according to claim 27 or 28, comprising a VL domain containing the amino acid sequence SGSSSNIGSNYVY of LCDR1, the amino acid sequence GNNKRPS of LCDR2, and the amino acid sequence AAWDDSLNGRV of LCDR3.

30. The scFv coupled to the BMPRII is A): The VH domain containing the amino acid sequence DYYMT of HCDR1, the amino acid sequence SISGGSTYYADSRKG of HCDR2, and the amino acid sequence DFGVAGWFGQYGMDV of HCDR3, and It contains a VL domain comprising the amino acid sequence TGSSSNIGAGYDVH of LCDR1, the amino acid sequence RSNQRPS of LCDR2, and the amino acid sequence SSYAGNYNLV of LCDR3, or B): The VH domain containing the amino acid sequence DYYMN of HCDR1, the amino acid sequence SISGGSTYYADSVKG of HCDR2, and the amino acid sequence DFGVAGWFGQFGMDV of HCDR3, and It contains a VL domain comprising the amino acid sequence TGSSSNIGAGYDVH of LCDR1, the amino acid sequence RSNQRPS of LCDR2, and the amino acid sequence SSYAGNYNLV of LCDR3, or C): The VH domain containing the amino acid sequence DYYMN of HCDR1, the amino acid sequence SISGGGSTYYAADSVKG of HCDR2, and the amino acid sequence DFGVAGWFGYYGMDV of HCDR3, and A polyspecific binding protein according to any one of claims 27 to 29, comprising a VL domain containing the amino acid sequence TGSSSNIGAGYDVH of LCDR1, the amino acid sequence RSNQRPS of LCDR2, and the amino acid sequence SSYAGNYNLV of LCDR3.

31. The scFv coupled to ALK1 is A VH domain comprising the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSSYAMSWVRQAPGKGLEWVANINQDGSEKNYVDSMRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREFDYWGQGTLVTVSS, or an amino acid sequence having at least 90% identity thereto, and A polyspecific binding protein according to any one of claims 27 to 30, comprising a VL domain containing the amino acid sequence QSVLAQPPSASGTPGQRVTISCSGSSSNIGGSNYVYWYQQLPGTAPKLLLIYGNNKRPSGVPDRFSGSKSGTTSASLAISGLRSSEDEADYYCAAWDDSLNGRVFGGGTKLTVL, or an amino acid sequence having at least 90% identity thereto.

32. The scFv coupled to the BMPRII is A VH domain comprising the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMTWIRQAPGKGLEWVSSISGGGSTYYADSRKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDFGVAAGWFGQYGMDDVWGQGTLVTVSS, or an amino acid sequence having at least 90% identity thereto, and A polyspecific binding protein according to any one of claims 27 to 31, comprising a VL domain containing the amino acid sequence QSVLTQPPSASGTPGQRVTISCTGGSSSSNIGAGAGYDVHWYQQLPGTAPKLLLIYRSNQRPSGVPDRFGSGSKSGTTSASLAISGLRSSEDEADYYYCSSYAGNYNLVFGGGTKLTVL, or an amino acid sequence having at least 90% identity thereto.

33. The polyspecific binding protein according to any one of claims 27 to 30, wherein the scFv that binds to ALK1 includes the amino acid sequence of SEQ ID NO: 120, or an amino acid sequence having at least 90% identity thereto.

34. The polyspecific binding protein according to any one of claims 27 to 30, wherein the scFv that binds to ALK1 includes the amino acid sequence of SEQ ID NO: 122, or an amino acid sequence having at least 90% identity thereto.

35. The polyspecific binding protein according to any one of claims 27 to 30, wherein the scFv that binds to BMPRII comprises the amino acid sequence of SEQ ID NO: 121, or an amino acid sequence having at least 90% identity thereto.

36. The polyspecific binding protein according to any one of claims 27 to 30, wherein the scFv that binds to ALK1 includes the amino acid sequence of SEQ ID NO: 123, or an amino acid sequence having at least 90% identity thereto.

37. The polyspecific binding protein according to any one of claims 27 to 30, wherein each of the first and second polypeptide chains comprises one amino acid sequence from any one of SEQ ID NOs. 60 to 63.

38. A multispecific binding protein according to any one of claims 1 to 37, which can induce signal transduction by inducing proximity between ALK1 and BMPRII, ActRIIA, or ActRIIB.

39. A multispecific binding protein according to any one of claims 1 to 38, having higher agonist activity compared to a multispecific binding protein lacking at least one modified hinge region.

40. A multispecific binding protein according to any one of claims 1 to 39, which induces at least about 35% of the activity of BMP9.

41. The multispecific binding protein according to claim 40, wherein the activity of BMP9 is identified by measuring the level of phosphorylated SMAD1 (pSMAD1) in cells incubated with the multispecific binding protein and / or in cells incubated with BMP9.

42. The polyspecific binding protein according to any one of claims 1 to 41, wherein the unfolding melt onset temperature (Tonset) is at least about 55°C.

43. A polyspecific binding protein according to any one of claims 1 to 42, wherein the thermal transition midpoint (Tm) of the melting temperature is at least about 64°C.

44. The polyspecific binding protein according to claim 42 or 43, wherein Tonset and Tm are identified by differential scanning calorimetry (DSC).

45. A multispecific binding protein according to any one of claims 1 to 44, which is capable of stimulating the expression of ID1 in cells.

46. The multispecific binding protein according to claim 45, wherein the expression of ID1 in the cells is at least 50% of the expression of ID1 from cells incubated with BMP9.

47. The polyspecific binding protein according to any one of claims 1 to 46, wherein the first polypeptide chain further comprises a heavy chain constant region.

48. The polyspecific binding protein according to claim 47, wherein the heavy chain constant region includes a substitution at the 234th amino acid position according to EU numbering.

49. The polyspecific binding protein according to claim 48, wherein the substitution at the 234th amino acid position is alanine (A).

50. The polyspecific binding protein according to claim 47, wherein the heavy chain constant region includes a substitution at the 235th amino acid position according to EU numbering.

51. The polyspecific binding protein according to claim 50, wherein the substitution at the 235th position of the amino acid is alanine (A).

52. The polyspecific binding protein according to claim 47, wherein the heavy chain constant region includes a substitution at the 237th amino acid position according to EU numbering.

53. The polyspecific binding protein according to claim 52, wherein the substitution at the 237th amino acid position is alanine (A).

54. The polyspecific binding protein according to claim 47, wherein the heavy chain constant region includes one or more substitutions at the 234th, 235th, or 237th amino acid positions according to EU numbering.

55. A polyspecific binding protein according to claim 54, The aforementioned substitution at the 234th position of the amino acid is alanine (A), The aforementioned substitution at the 235th position of the amino acid is alanine (A), and The aforementioned substitution at the 237th position of the amino acid is alanine (A). The aforementioned multispecific binding protein.

56. The polyspecific binding protein according to any one of the prior claims, wherein the heavy chain constant region includes a heterodimerizing mutation that promotes heterodimerization between the first binding portion and the second binding portion.

57. The polyspecific binding protein according to claim 56, wherein the heterodimerizing mutation is a knob-in-hole (KIH) mutation.

58. The polyspecific binding protein according to claim 57, wherein the first heavy chain constant region includes an amino acid substitution that generates a hole at position 366, 368, or 407, and the second heavy chain constant region includes an amino acid substitution that generates a knob at position 366.

59. The polyspecific binding protein according to claim 58, wherein the first heavy chain constant region comprises an amino acid substitution T366S, L368A, or Y407V, and the second heavy chain constant region comprises an amino acid substitution T366W.

60. The polyspecific binding protein according to claim 56, wherein the heterodimerizing mutation is a charge-stabilizing mutation.

61. The polyspecific binding protein according to claim 60, wherein the first heavy chain constant region comprises the amino acid substitution N297K, and the second heavy chain constant region comprises the amino acid substitution N297D.

62. The polyspecific binding protein according to claim 60, wherein the first heavy chain constant region comprises the amino acid substitution T299K, and the second heavy chain constant region comprises the amino acid substitution T299D.

63. The polyspecific binding protein according to claim 56, wherein the heterodimerizing mutation comprises an engineered disulfide bond.

64. The polyspecific binding protein according to claim 63, wherein the engineered disulfide bond is formed by a first heavy chain constant region containing the amino acid substitution Y349C and a second heavy chain constant region containing the amino acid substitution S354C.

65. The polyspecific binding protein according to claim 63 or 64, wherein the engineered disulfide bond is formed by a C-terminal extension peptide fused to the C-terminus of the first heavy chain constant region and the second heavy chain constant region, respectively.

66. The polyspecific binding protein according to claim 65, wherein the C-terminal extension of the first heavy chain constant region comprises the amino acid sequence GEC, and the C-terminal extension of the second heavy chain constant region comprises the amino acid sequence SCDKT.

67. A polyspecific binding protein according to any one of the prior claims, wherein at least one heavy chain constant region contains one or more mutations that promote half-life extension.

68. The polyspecific binding protein according to claim 67, wherein at least one heavy chain constant region comprises one or more substitutions at the EU numbered amino acid positions 252, 254, or 256.

69. A polyspecific binding protein according to claim 68, The aforementioned substitution at the 252nd position of the amino acid is tyrosine (Y), The aforementioned substitution at the 254th position of the amino acid is threonine (T), and The aforementioned substitution at the 256th position of the amino acid is glutamic acid (E). The aforementioned multispecific binding protein.

70. The polyspecific binding protein according to claim 67, wherein at least one heavy chain constant region comprises one or more substitutions at the 428th or 434th amino acid position according to EU numbering.

71. The polyspecific binding protein according to claim 70, wherein at least one heavy chain constant region comprises substitutions of M428L and N434S according to EU numbering.

72. A pharmaceutical composition comprising a polyspecific binding protein according to any one of the prior claims and a pharmaceutically acceptable carrier.

73. An isolated nucleic acid molecule encoding a polyspecific binding protein according to any one of claims 1 to 71.

74. An expression vector comprising the nucleic acid molecule described in claim 73.

75. A host cell comprising the expression vector described in claim 74.

76. A method for treating a disease or disorder in a subject, comprising administering a multispecific binding protein according to any one of claims 1 to 71 to a subject in need thereof.

77. The method according to claim 76, wherein the disease or disorder is a disease or disorder of the vascular system.

78. The method according to claim 77, wherein the disease or disorder of the vascular system is hereditary hemorrhagic telangiectasia (HHT).

79. The method according to claim 77, wherein the disease or disorder of the vascular system is pulmonary hypertension (PAH).

80. A multispecific binding protein according to any one of claims 1 to 71, for use as a pharmaceutical product.

81. A method for inducing signal transduction between ALK1 and BMPRII, ActRIIA, or ActRIIB in a subject, comprising administering a multispecific binding protein according to any one of claims 1 to 71 to the subject.

82. The method according to any one of claims 76 to 81, wherein the polyspecific binding protein can induce signal transduction by inducing proximity between ALK1 and BMPRII, ActRIIA, or ActRIIB.

83. The method according to any one of claims 76 to 82, wherein the polyspecific binding protein has higher agonist activity compared to a polyspecific binding protein lacking at least one modified hinge region.

84. The method according to any one of claims 76 to 83, wherein the polyspecific binding protein induces at least about 35% of the activity of BMP9.

85. The method according to claim 84, wherein the activity of BMP9 is identified by measuring the level of phosphorylated SMAD1 (pSMAD1) in cells incubated with the multispecific binding protein and / or cells incubated with BMP9.

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