ALK1 / bmprii bispecific antibodies for treatment of ocular diseases

Abstract

Provided herein are methods of treating or preventing ocular diseases with bispecific antibodies that bind to ALK1, BMPRII, ActRIIA, and / or ActRIIB.

Description

[0001] Attorney Docket No. 769472: DGT9-010PC

[0002] ALK1 / BMPRII BISPECIFIC ANTIBODIES FOR TREATMENT OF OCULAR DISEASES

[0003] RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 717,824, filed November 7, 2024, the entire disclosure of which is hereby incorporated herein by reference.

[0005] BACKGROUND

[0006] Several debilitating ocular diseases are associated with vascular dysfunction, such as neovascularization and the formation of vascular lesions in the eye. These diseases, such as wet age-related macular degeneration (AMD), progress over time, as new blood vessels damage various regions of the eye. This progression ultimately leads to vision loss.

[0007] Vascular endothelial growth factor (VEGF) is a contributor to ocular disease progression. As such, several therapies exist for the treatment ocular diseases with VEGF inhibitors. However, VEGF inhibitor therapies carry several drawbacks, including resistance and numerous side effects.

[0008] Bone morphogenetic protein 9 (BMP9) is a ligand that stimulates activin receptor-like kinase 1 (ALK1) signaling, which, among other things, can prevent VEGF-induced angiogenesis (Ntumba et al. Oncotarget. 7(53): 55957-55969. 2016). However, no ALK1- targeting therapeutics currently exist for the treatment of ocular diseases.

[0009] As such, there remains a need in the art for ALK1 -targeting therapeutics for inhibiting or treating ocular diseases.

[0010] SUMMARY

[0011] The present disclosure provides methods of treating or preventing ocular diseases and disorders with heteromeric antibodies which can effectively cross-link the ALK1 receptor to a receptor selected from BMPRII, ActRIIA, and ActRIIB and thereby activate SMAD signaling.

[0012] In one aspect, the disclosure provides a method of treating or preventing an ocular disease in a subject, the method comprising administering to the subject a multispecific binding protein comprising at least a first binding moiety which binds specifically to human ALK1 and at least a second binding moiety which binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB, thereby treating or preventing the ocular disease in the subject.

[0013] In certain embodiments, the ocular disease is selected from the group consisting of age-related macular degeneration (AMD), retinitis pigmentosa (RP), diabetic retinopathy, diabetic macular edema (DME), and hypertensive retinopathy. In certain embodiments, the AMD is wet AMD.

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[0016] In certain embodiments, the multispecific binding protein reduces neovascularization in the subject.

[0017] In certain embodiments, the multispecific binding protein reduces vascular lesion volume.

[0018] In certain embodiments, the multispecific binding protein is capable of inducing signaling by inducing proximity between ALK1 and BMPRII, ActRIIA, or ActRIIB.

[0019] In certain embodiments, the multispecific binding protein is capable of inducing SMAD1 / 5 phosphorylation.

[0020] In certain embodiments, the multispecific binding protein is capable of inducing soluble VEGFR1 (sVEGFRI) expression.

[0021] In certain embodiments, the multispecific binding protein is capable of modulating the expression of one or more genes selected from the group consisting of inhibitor of DNA binding 1 (ID1), C-X-C chemokine receptor 4 (CXCR4), C-X-C motif chemokine 12 (CXCL12), angiopoietin 2 (ANGPT2), Selectin E (SELE), Selectin P (SELP), endothelial cell specific molecule 1 (ESM1), SMAD7, SMAD6, endoglin (ENG), BMPR2, serpinel , endothelin 1 (EDN1), apelin (APLN), apelin receptor (ALPNR), adrenomedullin (ADM), EPH receptor B4 (EPHB4), neuregulin 1 (NRG1), ectonucleotide pyrophosphatase / phosphodiesterase 2 (ENPP2), C-C motif chemokine ligand 2 (CCL2), vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor B (VEGFB), intercellular adhesion molecule 1 (ICAM1), vascular adhesion molecule 1 (VCAM1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), transforming growth factor beta-1 (TGFB1), Fms Related Receptor Tyrosine Kinase 1 (FLT1), C-X-C motif chemokine ligand 8 (CXCL8), matrix metallopeptidase 1 (MMP1), and activin A receptor like type 1 (ACVRL1).

[0022] In certain embodiments, the multispecific binding protein is capable of reducing the expression of one or more genes selected from the group consisting of inhibitor of DNA binding 1 (ID1), C-X-C chemokine receptor 4 (CXCR4), C-X-C motif chemokine 12 (CXCL12), angiopoietin 2 (ANGPT2), Selectin E (SELE), Selectin P (SELP), endothelial cell specific molecule 1 (ESM1), SMAD7, SMAD6, endoglin (ENG), BMPR2, serpinel , endothelin 1 (EDN1), apelin (APLN), apelin receptor (ALPNR), adrenomedullin (ADM), EPH receptor B4 (EPHB4), neuregulin 1 (NRG1), ectonucleotide pyrophosphatase / phosphodiesterase 2 (ENPP2), C-C motif chemokine ligand 2 (CCL2), vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor B (VEGFB), intercellular adhesion molecule 1 (ICAM1), vascular adhesion molecule 1 (VCAM1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), transforming growth factor beta-1 (TGFB1), Fms Related

[0023] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC

[0024] Receptor Tyrosine Kinase 1 (FLT1), C-X-C motif chemokine ligand 8 (CXCL8), matrix metallopeptidase 1 (MMP1), and activin A receptor like type 1 (ACVRL1).

[0025] In certain embodiments, the reduction in expression is relative to a subject that is not administered the multispecific binding protein.

[0026] In certain embodiments, the multispecific binding protein comprises at least one modified hinge region.

[0027] In certain embodiments, the first modified hinge region comprises: a. an upper hinge region of up to 7 amino acids in length or is absent; and b. a lower hinge region, wherein the lower hinge region is linked to the N-terminus of a first constant region.

[0028] In certain embodiments, the multispecific binding protein further comprises a second modified hinge region linked to the N-terminus of a second constant region.

[0029] In certain embodiments, the second modified hinge region comprises a. an upper hinge region of up to 7 amino acids in length or is absent; and b. a lower hinge region, wherein the lower hinge region is linked to the N-terminus of the second constant region.

[0030] In certain embodiments, the upper hinge region of the first and the second modified hinge region are the same sequence.

[0031] In certain embodiments, the upper hinge region of the first and the second modified hinge regions are different sequences.

[0032] In certain embodiments, the upper hinge region comprises an amino acid sequence derived from an upper hinge region of a human IgG antibody.

[0033] In certain embodiments, the IgG antibody is selected from lgG1 , lgG2, lgG3, and lgG4. In certain embodiments, the IgG antibody is lgG1.

[0034] In certain embodiments, the upper hinge region comprises an amino acid sequence of SEQ ID NO: 2.

[0035] In certain embodiments, the upper hinge region comprises an amino acid sequence of SEQ ID NO: 5.

[0036] In certain embodiments, the IgG antibody is lgG4.

[0037] In certain embodiments, the upper hinge region comprises an amino acid sequence of SEQ ID NO: 4.

[0038] In certain embodiments, the upper hinge is absent.

[0039] In certain embodiments, the binding protein is multivalent.

[0040] In certain embodiments, the binding protein is tetravalent.

[0041] In certain embodiments, the first heavy chain constant region and / or the second heavy chain constant region comprise a human lgG1 , lgG2, lgG3, or lgG4.

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[0044] In certain embodiments, the first heavy chain constant region and / or the second heavy chain constant region comprise an amino acid sequence of SEQ ID NO: 10.

[0045] In certain embodiments, at least one heavy chain constant region comprises a substitution at amino acid position 234, according to Ell numbering. In certain embodiments, the substitution at amino acid position 234 is an alanine (A).

[0046] In certain embodiments, at least one heavy chain constant region comprises a substitution at amino acid position 235, according to Ell numbering. In certain embodiments, the substitution at amino acid position 235 is an alanine (A).

[0047] In certain embodiments, at least one heavy chain constant region comprises a substitution at amino acid position 237 according to Ell numbering. In certain embodiments, the substitution at amino acid position 237 is an alanine (A).

[0048] In certain embodiments, at least one heavy chain constant region comprises one or more substitutions at amino acid positions 234, 235, or 237, according to Ell numbering. In certain embodiments, the substitution at amino acid position 234 is an alanine (A), the substitution at amino acid position 235 is an alanine (A), and the substitution at amino acid position 237 is an alanine (A).

[0049] In certain embodiments, the heavy chain constant region comprises heterodimerization mutations to promote heterodimerization of the first binding moiety with the second binding moiety.

[0050] In certain embodiments, the heterodimerization mutations are Knob-in-Hole (KIH) mutations. In certain embodiments, the first heavy chain constant region comprises an amino acid substitution at position 366, 368, or 407 which produced a hole, and the second heavy chain constant region comprises an amino acid substitution at position 366 which produce a knob. In certain embodiments, the first heavy chain constant region comprises the amino acid substitution T366S, L368A, or Y407V, and the second heavy chain constant region comprises the amino acid substitution T366W.

[0051] In certain embodiments, the heterodimerization mutations are charge stabilization mutations. In certain embodiments, 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. In certain embodiments, 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.

[0052] In certain embodiments, the heterodimerization mutations comprise an engineered disulfide bond. In certain embodiments, the engineered disulfide bond is formed by a first heavy chain constant region comprising the amino acid substitution Y349C, and a second

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[0054] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC heavy chain constant region comprising the amino acid substitution S354C. In certain embodiments, the engineered disulfide bond is formed by a C-terminal extension peptide fused to the C-terminus of each of the first heavy chain constant region and the second heavy chain constant region. In certain embodiments, the first heavy chain constant region C- terminal extension comprises the amino acid sequence GEC, and the second heavy chain constant region C-terminal extension comprises the amino acid sequence SCDKT.

[0055] In certain embodiments, at least one heavy chain constant region comprises one or more mutations to promote increased half-life. In certain embodiments, at least one heavy chain constant region comprises one or more substitutions at amino acid positions 252, 254, or 256, according to Ell numbering. In certain embodiments, the substitution at amino acid position 252 is a tyrosine (Y), the substitution at amino acid position 254 is a threonine (T), and the substitution at amino acid position 256 is a glutamic acid (E).

[0056] In certain embodiments, at least one heavy chain constant region comprises one or more substitutions at amino acid positions 428 or 434, according to Ell numbering.

[0057] In certain embodiments, the substitution at amino acid position 428 is a leucine (L), and wherein the substitution at amino acid position 434 is a serine (S).

[0058] In certain embodiments, the first binding moiety that binds specifically to human ALK1 is selected from a single chain Fv (scFv), VHH, Fab, F(ab’)2, or a single domain antibody.

[0059] In certain embodiments, the second binding moiety that binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB is selected from a single chain Fv (scFv), VHH, Fab, F(ab’)2, or a single domain antibody.

[0060] In certain embodiments, the multispecific 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; or aiv) a first polypeptide chain comprising a first antigen binding domain, a second antigen binding domain, a first modified hinge region, and a first constant region; and

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[0062] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC biv) a second polypeptide chain comprising a third antigen binding domain, a fourth antigen binding domain, a second modified hinge region, and a second constant region.

[0063] In certain embodiments, the first antigen binding domain, second antigen binding domain, third antigen binding domain, and fourth antigen binding domain comprise an scFv.

[0064] In certain embodiments, (a) the first binding moiety comprises an VHH domain and the second moiety comprises a VHH domain; (b) the first binding moiety comprises a Fab domain and the second binding moiety comprises a VHH domain; (c) the first binding moiety comprises a VHH domain and the second binding moiety comprises a Fab domain; (d) the first binding moiety comprises a Fab domain and the second binding moiety comprises a Fab domain; (e) the first binding moiety comprises a Fab domain and the second binding moiety comprises an scFv; (f) the first binding moiety comprises a scFv and the second binding moiety comprises a Fab domain; (g) the first binding moiety comprises a scFv and the second binding moiety comprises a scFv; (h) the first binding moiety comprises a scFv and the second binding moiety comprises a VHH; or (i) the first binding moiety comprises a VHH and the second binding moiety comprises a scFv.

[0065] In certain embodiments, the first and / or the second antibody binding domain is truncated at the C-terminal end adjacent to the upper hinge domain. In certain embodiments, the C-terminal end adjacent to the upper hinge domain is truncated by at least one residue. In certain embodiments, the C-terminal end adjacent to the upper hinge domain is truncated by at least two residues.

[0066] In certain embodiments, the multispecific binding protein comprises a first and a second polypeptide chain, wherein: said first polypeptide chain comprises VH1-(HX1)n-VH2- C-(HX2)n, wherein: 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 said second polypeptide chain comprises VL1-(LX1)n-VL2- C-(LX2)n, wherein: 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 comprise an Fc region; and n is independently 0 or 1.

[0067] In certain embodiments, the VH1 binds specifically to human ALK1 and VH2 binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB.

[0068] In certain embodiments, the VL1 binds specifically to human ALK1 and VL2 binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB.

[0069] In certain embodiments, the VH1 binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB and VH2 binds specifically to human ALK1.

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[0072] In certain embodiments, the VL1 binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB and VL2 binds specifically to human ALK1.

[0073] In certain embodiments, the linker HX1 comprises an amino acid sequence of PI_AP or PAPNLLGGP.

[0074] In certain embodiments, the linker LX1 comprises an amino acid sequence of PI_AP or PAPNLLGGP.

[0075] In certain embodiments, the linker HX1 comprises an amino acid sequence of PLAP and linker LX1 comprises an amino acid sequence of PLAP or PAPNLLGGP.

[0076] In certain embodiments, the linker HX1 comprises an amino acid sequence of PAPNLLGGP and linker LX1 comprises an amino acid sequence of PLAP.

[0077] In certain embodiments, the multispecific binding protein comprises at least a first polypeptide chain, wherein: said 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; and the VH1 binds specifically to ALK1 and the VH2 binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB.

[0078] In certain embodiments, one or both of VH1 and VH2 are VH domains or VHH domains.

[0079] In certain embodiments, the multispecific binding protein further comprises a second polypeptide chain, wherein said second polypeptide chain comprises a first variable light chain domain (VL1) linked to a second variable light chain domain (VL2), and wherein VL1 binds specifically to ALK1 and the VL2 binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB.

[0080] In certain embodiments, the VL1 is linked to the VL2 via at least one modified hinge region.

[0081] In certain embodiments, one or both of VH1 and VH2 is truncated at the C-terminal end.

[0082] In certain embodiments, the C-terminal end is truncated by at least one residue.

[0083] In certain embodiments, the C-terminal end is truncated by at least two residues.

[0084] In certain embodiments, the SS amino acid residues of the C-terminal end are deleted.

[0085] In certain embodiments, the multispecific binding protein comprises a first polypeptide chain of VH1-HX1-VH2-C-Fc, wherein: 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 modified hinge region linker; and Fc is an Fc region; and a second polypeptide chain of VL1-LX1-VL2- C, wherein: VL1 is a first light chain variable domain; VL2 is a second light chain variable domain; C is a light chain constant domain; and LX1 is a modified hinge region linker.

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[0087] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC

[0088] In certain embodiments, the modified hinge region comprises or consists of an amino acid sequence of PLAP or PAPNLLGGP.

[0089] In certain embodiments, A) the VH binding to ALK1 comprises an HCDR1 amino acid sequence of SYAMS, an HCDR2 amino acid sequence of NINQDGSEKNYVDSMRG, and an HCDR3 amino acid sequence of EFDY; and the VL binding to ALK1 comprises an LCDR1 amino acid sequence of SGSSSNIGSNYVY, an LCDR2 amino acid sequence of GNNKRPS, and an LCDR3 amino acid sequence of AAWDDSLNGRV; or B) the VH binding to ALK1 comprises an HCDR1 amino acid sequence of SYWMS, an HCDR2 amino acid sequence of NINQDGSEKYYVDSMRG, and an HCDR3 amino acid sequence of EYDY; and the VL binding to ALK1 comprises an LCDR1 amino acid sequence of SGSSSNIGSNYVY, an LCDR2 amino acid sequence of GNNKRPS, and an LCDR3 amino acid sequence of AAWDDSLNGRV; or C) the VH binding to ALK1 comprises an HCDR1 amino acid sequence of SYWMS, an HCDR2 amino acid sequence of NIKQDGSEKNYVDSMRG, and an HCDR3 amino acid sequence of EFDF; and the VL binding to ALK1 comprises an LCDR1 amino acid sequence of SGSSSNIGSNYVY, an LCDR2 amino acid sequence of GNNKRPS, and an LCDR3 amino acid sequence of AAWDDSLNGRV.

[0090] In certain embodiments, A) the VH binding to BMPRII comprises an HCDR1 amino acid sequence of DYYMT, an HCDR2 amino acid sequence of SISGGSTYYADSRKG, and an HCDR3 amino acid sequence of DFGVAGWFGQYGMDV; and the VL binding to BMPRII comprises an LCDR1 amino acid sequence of TGSSSNIGAGYDVH, an LCDR2 amino acid sequence of RSNQRPS, and an LCDR3 amino acid sequence of SSYAGNYNLV; or B) the VH binding to BMPRII comprises an HCDR1 amino acid sequence of DYYMN, an HCDR2 amino acid sequence of SISGGSTYYADSVKG, and an HCDR3 amino acid sequence of DFGVAGWFGQFGMDV; and the VL binding to BMPRII comprises an LCDR1 amino acid sequence of TGSSSNIGAGYDVH, an LCDR2 amino acid sequence of RSNQRPS, and an LCDR3 amino acid sequence of SSYAGNYNLV; or C) the VH binding to BMPRII comprises an HCDR1 amino acid sequence of DYYMN, an HCDR2 amino acid sequence of SISGGSTYYADSVKG, and an HCDR3 amino acid sequence of DFGVAGWFGYYGMDV; and the VL binding to BMPRII comprises an LCDR1 amino acid sequence of TGSSSNIGAGYDVH, an LCDR2 amino acid sequence of RSNQRPS, and an LCDR3 amino acid sequence of SSYAGNYNLV.

[0091] In certain embodiments, A) the VH binding to ALK1 comprises an amino acid sequence of EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVANINQDGSEKN YVDSMRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREFDYWGQGTLVTVSS, or an

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[0093] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC amino acid sequence with at least 90% identity thereto; and the VL binding to ALK1 comprises an amino acid sequence of

[0094] QSVLAQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYGNNKRPSGVPD RFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGRVFGGGTKLTVL, or an amino acid sequence with at least 90% identity thereto; or B) the VH binding to ALK1 comprises an amino acid sequence of

[0095] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINQDGSEKY YVDSMRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREYDYWGQGTLVTVSS, or an amino acid sequence with at least 90% identity thereto; and the VL binding to ALK1 comprises an amino acid sequence of

[0096] QSVLAQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYGNNKRPSGVPD RFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGRVFGGGTKLTVL, or an amino acid sequence with at least 90% identity thereto; or C) the VH binding to ALK1 comprises an amino acid sequence of

[0097] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKN YVDSMRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREFDFWGQGTLVTVSS, or an amino acid sequence with at least 90% identity thereto; and the VL binding to ALK1 comprises an amino acid sequence of

[0098] QSVLAQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYGNNKRPSGVPD RFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGRVFGGGTKLTVL, or an amino acid sequence with at least 90% identity thereto.

[0099] In certain embodiments, A) the VH binding to BMPRII comprises an amino acid sequence of

[0100] EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMTWIRQAPGKGLEWVSSISGGSTYYAD SRKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDFGVAGWFGQYGMDVWGQGTLVT VSS, or an amino acid sequence with at least 90% identity thereto; and the VL binding to BMPRII comprises an amino acid sequence of QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYRSNQRPSGVP DRFSGSKSGTSASLAISGLRSEDEADYYCSSYAGNYNLVFGGGTKLTVL, or an amino acid sequence with at least 90% identity thereto; or B) the VH binding to BMPRII comprises an amino acid sequence of

[0101] EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMNWIRQAPGKGLEWVSSISGGSTYYAD SVKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDFGVAGWFGQFGMDVWGQGTLVT VSS, or an amino acid sequence with at least 90% identity thereto; and the VL binding to BMPRII comprises an amino acid sequence of

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[0104] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYRSNQRPSGVP DRFSGSKSGTSASLAISGLRSEDEADYYCSSYAGNYNLVFGGGTKLTVL, or an amino acid sequence with at least 90% identity thereto; or C) the VH binding to BMPRII comprises an amino acid sequence of

[0105] EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMNWIRQAPGKGLEWVSSISGGSTYYAD SVKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDFGVAGWFGYYGMDVWGQGTLVT VSS, or an amino acid sequence with at least 90% identity thereto; and the VL binding to BMPRII comprises an amino acid sequence of QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYRSNQRPSGVP DRFSGSKSGTSASLAISGLRSEDEADYYCSSYAGNYNLVFGGGTKLTVL, or an amino acid sequence with at least 90% identity thereto.

[0106] In certain embodiments, the first polypeptide chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 136-141 , and the second polypeptide chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 143- 146.

[0107] In certain embodiments, the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 137, and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 146.

[0108] In certain embodiments, the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 138, and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 146.

[0109] In certain embodiments, the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 139, and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 146.

[0110] In certain embodiments, the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 140, and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 146.

[0111] In certain embodiments, the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 141 , and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 146. In certain embodiments, the multispecific binding protein is administered intraocularly to the subject.

[0112] In certain embodiments, the multispecific binding protein is administered intravenously to the subject.

[0113] In certain embodiments, the multispecific binding protein is administered subcutaneously to the subject.

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[0116] BRIEF DESCRIPTION OF THE DRAWINGS

[0117] FIG. 1A-1C are graphs depicting arteriovenous malformations (AVMs) in the retina in a hereditary hemorrhagic telangiectasia (HHT) mouse model. FIG. 1A illustrates mice treated with control (no bispecific antibody) compared to DGL288 (15 mg / kg / day). Mice treated with DGL288 did not form detectable AVMs compared to control. FIG. 1 B illustrates that mice treated with 1 mg / kg / day of DGL292 did not form AVMs compared to the mice treated with control. FIG. 1C demonstrates that DGL288 given at a dose of 1 mg / kg / day also did not form AVMs compared to mice treated with control.

[0118] DETAILED DESCRIPTION

[0119] Before the present disclosure is described, it is to be understood that this disclosure is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0120] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0121] Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure, exemplary methods and materials are now described. All publications mentioned herein are incorporated herein by reference to describe in their entirety.

[0122] As used herein, the terms “antibody” and “antibodies” include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules comprising antibody CDRs, VH regions, and / or VL regions. Examples of antibodies include, without limitation, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain- antibody heavy chain pair, intrabodies, heteroconjugate antibodies, antibody-drug conjugates, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain Fvs (scFv), camelized antibodies, affibodies, common light chain antibodies, Fab fragments, F(ab’)2 fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti- Id) antibodies (including, e.g., anti-anti-ld antibodies), dual variable domains (DVD), and

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[0124] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC antigen-binding fragments of any of the above. In certain embodiments, antibodies described herein refer to polyclonal antibody populations. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class (e.g., lgG1 , lgG2, lgG3, lgG-4, lgA1 or lgA2), or any subclass (e.g., lgG2a or lgG2b) of immunoglobulin molecule. In certain embodiments, antibodies described herein are IgG antibodies, or a class (e.g., human lgG1 or lgG4) or subclass thereof. As used herein, the terms “VH” and “VL” refer to antibody heavy and light chain variable domain, respectively, as described in Kabat et al., (1991) Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242, Bethesda), which is herein incorporated by reference in its entirety.

[0125] As used herein, the term “VHH” refers to the heavy chain variable domain of a camelid heavy chain-only antibody (HCAb) and humanized variants thereof, as described in Hamers- Casterman C. et al., Nature (1993) 363:446-8.10.1038 / 363446a0, which is incorporated by reference herein in its entirety.

[0126] As used herein, the term “VH / VL Pair” refers to a combination of a VH and a VL that together form the binding site for an antigen.

[0127] As used herein, the term “heavy chain” when used in reference to an antibody can refer to any distinct type, e.g., alpha (a), delta (5), epsilon (E), gamma (y), and mu (p), based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., lgG1 , lgG2, lgG3, and lgG4.

[0128] As used herein, the term “full-length antibody heavy chain” refers to an antibody heavy chain comprising, from N to C terminal, a VH, a CH1 region, a hinge region, a CH2 domain and a CH3 domain.

[0129] As used herein, the term “light chain” when used in reference to an antibody can refer to any distinct type, e.g., kappa (K) or lambda (A) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In specific embodiments, the light chain is a human light chain. As used herein, the term “complementarity determining region” or “CDR” refers to sequences of amino acids within antibody variable regions, which confer antigen specificity and binding affinity. In general, there are three CDRs in each heavy chain variable region (CDR-H1 , CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1 , CDR-L2, CDR-L3). “Framework regions” or “FR” are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each heavy chain variable region (FR-H1 , FR-H2, FR-H3, and FR-H4), and four FRs in each light chain variable region (FR-L1 , FR-L2, FR-L3, and FR-L4).

[0130] 12

[0131] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC

[0132] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by 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), Lefranc M. P. et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev. Comp. Immunol., 2003 January; 27(1):55-77 (“IMGT” numbering scheme), and 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).

[0133] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on sequence alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.

[0134] As used herein, the term “single chain variable fragment” (scFv) refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0135] The term “human antibody,” as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human mAbs of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “human antibody,” as used herein, is not intended to include mAbs

[0136] 13

[0137] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC in which CDR sequences derived from the germline of another mammalian species (e.g., mouse), have been grafted onto human FR sequences. The term includes antibodies recombinantly produced in a non-human mammal, or in cells of a non-human mammal. The term is not intended to include antibodies isolated from or generated in a human subject.

[0138] The term “multispecific antigen-binding molecules,” as used herein refers to bispecific, tri-specific or multispecific antigen-binding molecules, and antigen-binding fragments thereof. Multispecific antigen-binding molecules may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for epitopes of more than one target polypeptide. A multispecific antigen-binding molecule can be a single multifunctional polypeptide, or it can be a multimeric complex of two or more polypeptides that are covalently or non-covalently associated with one another. The term “multispecific antigen-binding molecules” includes antibodies of the present disclosure that may be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non- covalent association or otherwise) to one or more other molecular entities, such as a protein or fragment thereof to produce a bi-specific or a multispecific antigen-binding molecule with a second binding specificity. According to the present disclosure, the term “multispecific antigen-binding molecules” also includes bispecific, trispecific or multispecific antibodies or antigen-binding fragments thereof. In certain exemplary embodiments, an antibody of the present disclosure is functionally linked to another antibody or antigen-binding fragment thereof to produce a bispecific antibody with a second binding specificity.

[0139] The term “valency” or “valent”, as used herein, denotes the presence of a number of binding sites in an antibody molecule. For example, the term bivalent indicates the presence of two binding sites. In some embodiments, the antibody molecule could be multivalent. As such, the term trivalent indicates three binding sites; the term tetravalent indicates four binding sites. In some embodiments, there may be more than four binding sites. In some embodiments, the binding sites may bind to the same antigen. In some embodiments, the binding sites bind to different antigens.

[0140] In some embodiments, the multivalent antibody molecules of the invention are multichain molecules with one or more binding sites in each chain.

[0141] For example, in one embodiment, the multivalent binding molecule is a bivalent molecule with one binding site (e.g., a VHH or scFV) in a first chain and a second binding site in a second chain. In another embodiment, the multivalent binding molecule is a bivalent molecule with two binding sites in a first chain and no binding sites in the second chain.

[0142] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC

[0143] In another embodiment, the multivalent binding molecule is a trivalent molecule with one binding site (e.g., a VHH or scFV) in a first chain and a second and third binding site in a second chain. In another embodiment, the multivalent binding molecule is a trivalent molecule with three binding sites in a first chain and no binding sites in a second chain.

[0144] In another embodiment, the multivalent binding molecule is a tetravalent molecule with two binding sites in a first chain and two binding sites in a second chain. In another embodiment, the multivalent binding molecule is a tetravalent molecule with three binding sites in a first chain and one binding site in a second chain. In another embodiment, the multivalent binding molecule is a tetravalent molecule with four binding sites in a first chain and no binding sites in a second chain.

[0145] In exemplary embodiments, the heteromeric antibodies of the present disclosure are bispecific antibodies. Bispecific antibodies can be monoclonal, e.g., human or humanized, antibodies that have binding specificities for at least two different antigens.

[0146] Methods for making bispecific antibodies are well-known. Traditionally, the recombinant production of bispecific antibodies was based on the co-expression of two immunoglobulin heavy chain / light chain pairs, where the two heavy chains have different specificities (Milstein et al., Nature 305:537 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, the hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. More modern techniques for generating bispecific antibodies employ heterodimerization domains that favor desired pairing of heavy chain from the antibody with a first specificity to the heavy chain of an antibody with a second specificity.

[0147] Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences. The fusion typically is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It may have the first heavy chain constant region (CH1) containing the site necessary for light chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transformed into a suitable host organism. For further details of generating bispecific antibodies see, for example Suresh et al., Meth. Enzymol. 121 :210 (1986).

[0148] 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 linked (directly or indirectly) to the

[0149] 15

[0150] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC

[0151] N-terminus of the CH3 domain. The term “Fc polypeptide” includes an antibody heavy chain linked to an antibody light chain by disulfide bonds (e.g., to form a half-antibody).

[0152] In certain embodiments, an Fc chain begins in the hinge region just upstream of the papain cleavage site and ends at the C-terminus of the antibody. Accordingly, a complete Fc chain comprises at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, an Fc chain comprises at least one of: a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof. In certain embodiments, an Fc domain comprises a complete Fc chain (i.e. , a hinge domain, a CH2 domain, and a CH3 domain). In certain embodiments, an Fc chain comprises a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, an Fc chain comprises a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, an Fc chain consists of a CH3 domain or portion thereof. In certain embodiments, an Fc chain consists of a hinge domain (or portion thereof) and a CH3 domain (or portion thereof). In certain embodiments, an Fc chain consists of a CH2 domain (or portion thereof) and a CH3 domain. In certain embodiments, an Fc chain consists of a hinge domain (or portion thereof) and a CH2 domain (or portion thereof). In certain embodiments, an Fc chain lacks at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). An Fc chain herein generally refers to a polypeptide comprising all or part of the Fc chain of an immunoglobulin heavy-chain. This includes, but is not limited to, polypeptides comprising the entire CHI, hinge, CH2, and / or CH3 domains as well as fragments of such peptides comprising only, e.g., the hinge, CH2, and CH3 domain. The Fc chain may be derived from an immunoglobulin of any species and / or any subtype, including, but not limited to, a human IgGI, lgG2, lgG3, lgG4, IgD, IgA, IgE, or IgM antibody. The Fc domain encompasses native Fc and Fc variant molecules. As with Fc variants and native Fc's, the term Fc chain includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means. In some embodiment, the Fc chain comprises the carboxy-terminal portions of both heavy chains held together by disulfides. In certain embodiments, an Fc chain consists of a CH2 domain and a CH3 domain.

[0153] In some embodiments, an Fc polypeptide comprises part or all of a wild-type hinge sequence (generally at its N-terminal). In some embodiments, an Fc polypeptide does not comprise a functional or wild-type hinge sequence.

[0154] As used herein, the term “CH1 domain” refers to the first constant domain of an antibody heavy chain (e.g., amino acid positions 118-215 of human lgG1 , according to the Ell index). The term includes naturally occurring CH1 domains and engineered variants of naturally occurring CH1 domains (e.g., CH1 domains comprising one or more amino acid

[0155] 16

[0156] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC insertions, deletions, substitutions, or modifications relative to a naturally occurring CH1 domain).

[0157] As used herein, the term “CH2 domain” refers to the second constant domain of an antibody heavy chain (e.g., amino acid positions 231-340 of human lgG1 , according to the Ell index). The term includes naturally occurring CH2 domains and engineered variants of naturally occurring CH2 domains (e.g., CH2 domains comprising one or more amino acid insertions, deletions, substitutions, or modifications relative to a naturally occurring CH2 domain).

[0158] As used herein, the term “CH3 domain” refers to the third constant domain of an antibody heavy chain (e.g., amino acid positions 341-447 of human lgG1 , according to the Ell index). The term includes naturally occurring CH3 domains and engineered variants of naturally occurring CH3 domains (e.g., CH3 domains comprising one or more amino acid insertions, deletions, substitutions, or modifications relative to a naturally occurring CH3 domain).

[0159] As used herein, the term “hinge region” refers to the portion of an antibody heavy chain comprising the cysteine residues (e.g., the cysteine residues at amino acid positions 226 and 229 of human lgG1 , according to the Ell index) that mediate disulfide bonding between two heavy chains in an intact antibody. The term includes naturally occurring hinge regions and engineered variants of naturally occurring hinge regions (e.g., hinge regions comprising one or more amino acid insertions, deletions, substitutions, or modifications relative to a naturally occurring hinge regions). An exemplary full-length lgG1 hinge region comprises amino acid positions 216-230 of human lgG1 , according to the Ell index. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semiflexible linkage 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 can be from or derived from any lgG1 , lgG2, lgG3, or lgG4 subtype, or from IgA, IgE, IgD or IgM, including chimeric forms thereof, e.g., a chimeric lgG1 / 2 hinge region.

[0160] In some embodiments, the hinge region can be from the human lgG1 subtype extending from amino acid 216 to amino acid 230 according to the numbering system of the Ell index, or from amino acid 226 to amino acid 243 according to the numbering system of Kabat. Those skilled in the art may differ in their understanding of the exact amino acids corresponding to the various domains of the IgG molecule. Thus, the N-terminal or C-terminal of the domains outlined above may extend or be shortened by 1 , 2, 3, 4, 5, 6, 7, 8, 9, or even 10 amino acids.

[0161] 17

[0162] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC

[0163] The term “upper hinge” as used herein typically refers to the last residue of the CH1 domain up to but not including the first inter-heavy chain cysteine. The upper hinge can sometimes be defined as the N-terminal sequence from position 216 to position 225 according to the Kabat Ell numbering system of an IgG 1 antibody (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, Md., 1991). The term “middle hinge” refers to the region extending from the first inter-heavy chain cysteine to a proline residue adjacent to the carboxyl-end of the last middle hinge cysteine. The middle hinge can be the N-terminal sequence from position 226 to position 230 according to the Kabat Ell numbering system. The term “lower hinge” refers to a highly conserved 7-8 amino acids. The lower hinge can be defined as the sequence from position 231 to 238 according the Kabat Ell numbering system of an IgG 1 antibody. In some embodiments, the antibody according to the present invention effectively comprises an upper, a middle, and a lower hinge.

[0164] As used herein, the term "a modified hinge region" refers to a hinge region in which alterations are made in one or more of the characteristics of the hinge, including, but not limited to, flexibility, length, conformation, charge and hydrophobicity relative to a wildtype hinge. The modified hinge regions disclosed herein may be generated by methods well known in the art, such as, for example introducing a modification into a wild-type hinge. In some embodiments, the hinge region may be modified by one or more amino acids. Modifications which may be utilized to generate a modified hinge region include, but are not limited to, amino acid insertions, deletions, substitutions, and rearrangements. Said modifications of the hinge and the modified hinge regions disclosed are referred to herein jointly as “hinge modifications of the invention”, “modified hinge(s) of the invention” or simply “hinge modifications” or “modified hinge(s).” The modified hinge regions disclosed herein may be incorporated into a molecule of choice including, but not limited to, antibodies and fragments thereof. In some embodiments, the hinge region may be truncated and contain only a portion of the full hinge region.

[0165] As demonstrated herein, molecules comprising a modified hinge may exhibit altered (e.g., enhanced) agonistic activity when compared to a molecule having the same amino acid sequence except for the modified hinge, such as, for example, a molecule having the same amino acid sequence except comprising a wild type hinge. In some embodiments, the antibody comprises a modified hinge region wherein the upper hinge region is up to 7 amino acids in length. In some embodiments, the upper hinge region is absent. In some embodiments, the modified hinge is a modified lgG1 linker. In some embodiments, the modified lgG1 hinge is derived from the sequence PLAPDKTHT (SEQ ID NO: 1). In some

[0166] 18

[0167] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC embodiments, the modified lgG1 hinge comprises the sequence PLAP (SEQ ID NO: 2). In some embodiments, the modified lgG1 hinge comprises the sequence DKTHT (SEQ ID NO: 5). In some embodiments, the modified hinge is a modified lgG4 hinge. In some embodiments, the modified lgG1 hinge comprises 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 comprises the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 3). In some embodiments, the C-terminal residues of the variable domain adjacent to the upper hinge are truncated. In some embodiments, at least one residue of the variable domain adjacent to the upper hinge is truncated. In some embodiments, at least two residues of the variable domain adjacent to the upper hinge is truncated.

[0168] As used herein, the term “Ell index” refers to the Ell numbering convention for the constant regions 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, U.S. Dept. Health and Human Services, 5th edition, 1991 , each of which is herein incorporated by reference in its entirety. All numbering of amino acid positions of the Fc polypeptides, or fragments thereof, used herein is according to the EU index. As used herein, the term “linker” refers to 0-100 contiguous amino acid residues. The linkers are, present or absent, and same or different. Linkers comprised in a protein or a polypeptide may all have the same amino acid sequence or may have different amino acid sequences.

[0169] In some embodiments, the term “linker” refers to 1-100 contiguous amino acid residues. Typically, a linker provides flexibility and spatial separation between two amino acids or between two polypeptide domains. A linker may be inserted between VH, VL, CH and / or CL domains to provide sufficient flexibility and mobility for the domains of the light and heavy chains depending on the format of the molecule. A linker is typically inserted at the transition between variable domains between variable and knockout domain, or between variable and constant domains, respectively, at the amino sequence level. The transition between domains can be identified because the approximate sizes of the immunoglobulin domains are well understood. The precise location of a domain transition can be determined by locating peptide stretches that do not form secondary structural elements such as beta-sheets or alpha-helices as demonstrated by experimental data or as can be determined by techniques of modeling or secondary structure prediction.

[0170] As used herein, the term “specifically binds,” “specifically binding,” “binding specificity” or “specifically recognized” refers that an antigen binding protein or antigen-binding fragment thereof that exhibits appreciable affinity for an antigen (e.g., a BMPR Type I receptor or BMPR Type II receptor antigen) and does not exhibit significant cross reactivity to a target that is not

[0171] 19

[0172] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC a BM PR Type I receptor or a BM PR Type II receptor protein. As used herein, the term “affinity” refers to the strength of the interaction between an antigen binding protein or antigen-binding fragment thereof antigen binding site and the epitope to which it binds. In certain exemplary embodiments, affinity is measured by surface plasmon resonance (SPR), e.g., in a Biacore instrument. As readily understood by those skilled in the art, an antigen binding protein affinity may be reported as a dissociation constant (KD) in molarity (M). The antigen binding protein or antigen-binding fragment thereof of the disclosure have KD values in the range of about 1 O'5M to about 10'12M (i.e., low micromolar to picomolar range), about 10'7M to 10'11M, about 10'8M to about 10'10M, about 10'9M. In certain embodiments, the antigen binding protein or antigen-binding fragment thereof has a binding affinity of about 10'5M ,10'6M, 10'7M, 10'8M, 10'9M, 10'10M, 10'11M, or 10'12M. In certain embodiments, the antigen binding protein or antigen-binding fragment thereof has a binding affinity of about 10'7M to about 10'9M (nanomolar range).

[0173] Specific binding can be determined according to any art-recognized means for determining such binding. In some embodiments, specific binding is determined by competitive binding assays (e.g., ELISA) or Biacore assays. In certain embodiments, the assay is conducted at about 20°C, 25°C, 30°C, or 37°C.

[0174] As used herein, “administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an isolated binding polypeptide provided herein) into a patient, such as by, but not limited to, pulmonary (e.g., inhalation), mucosal (e.g., intranasal), intradermal, intravenous, intramuscular delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being managed or treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease, or symptom thereof, is being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof and may be continued chronically to defer or reduce the appearance or magnitude of disease-associated symptoms.

[0175] As used herein, the term “composition” is intended to encompass a product containing the specified ingredients (e.g., an isolated binding polypeptide provided herein) in, optionally, the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in, optionally, the specified amounts.

[0176] “Effective amount” means the amount of active pharmaceutical agent (e.g., an isolated binding polypeptide of the present disclosure) sufficient to effectuate a desired physiological outcome in an individual in need of the agent. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the

[0177] 20

[0178] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.

[0179] As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, a subject can be a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, mice, etc.) or a primate (e.g., monkey and human). In certain embodiments, the term “subject,” as used herein, refers to a vertebrate, such as a mammal. Mammals include, without limitation, humans, non-human primates, wild animals, feral animals, farm animals, sport animals, and pets.

[0180] As used herein, the term “therapy” refers to any protocol, method and / or agent that can be used in the prevention, management, treatment and / or amelioration of a disease or a symptom related thereto. In some embodiments, the term “therapy” refers to any protocol, method and / or agent that can be used in the modulation of an immune response to an infection in a subject or a symptom related thereto. In some embodiments, the terms “therapies” and “therapy” refer to a biological therapy, supportive therapy, and / or other therapies useful in the prevention, management, treatment and / or amelioration of a disease or a symptom related thereto, known to one of skill in the art such as medical personnel. In other embodiments, the terms “therapies” and “therapy” refer to a biological therapy, supportive therapy, and / or other therapies useful in the modulation of an immune response to an infection in a subject or a symptom related thereto known to one of skill in the art such as medical personnel.

[0181] As used herein, the terms “treat,” “treatment” and “treating” refer to the reduction or amelioration of the progression, severity, and / or duration of a disease or a symptom related thereto, 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 an isolated binding polypeptide provided herein). The term “treating,” as used herein, can also refer to altering the disease course of the subject being treated. Therapeutic effects of treatment include, without limitation, preventing occurrence or recurrence of disease, alleviation of symptom(s), diminishment of direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0182] The term “about” or “approximately” means within about 20%, such as within about 10%, within about 5%, or within about 1 % or less of a given value or range.

[0183] BMPR Type I Receptors and BMPR Type II Receptors

[0184] Bone morphogenetic protein (BMP) Type I and Type II receptors are serine-threonine kinase transmembrane signal transduction proteins that regulate a vast array of ligand-

[0185] 21

[0186] 81386151 ,v1 Attorney Docket No. 769472: DGT9-010PC dependent cell-fate decisions with temporal and spatial fidelity during development and postnatal life. The activation of the receptors, induced by first binding to their ligand (BMPs) and then heterodimerizing, triggers intracellular signaling that is initiated by phosphorylation of receptor-regulated SMAD1 , 5, and 8 (R-SMADs). These activated R-SMADs form heteromeric complexes with SMAD4, which engage in specific transcriptional responses.

[0187] As used herein, the term “ALK1” refers to the activin A receptor like type 1 , a BMP Type I receptor. Alternative 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 ACVRLI. The ALK1 protein comprises human, murine, and further mammalian homologues. Sequence(s) for human ALK1 are accessible via UniProt Identifier P37023 (ACVL1 HUMAN), for instance human isoform P37023-1. Sequence(s) for murine ALK1 are accessible via UniProt Identifier Q61288 (ACVL1 MOUSE). The term “ALK1” may encompass different isoforms and variants that may exist for different species and are all comprised by the term ALK1. In addition, the term “ALK1” may include synthetic variants of the ALK1 protein produced, e.g. by introducing at least one mutation. The protein ALK1 may furthermore be subject to various modifications, e.g, synthetic or naturally occurring modifications. Naturally occurring mutations in the ALK1 gene are associated with hereditary hemorrhagic telangiectasia (HHT) type 2, wherein patients suffer pulmonary hypertension, daily epistaxis, strokes, and emboli.

[0188] The term "BMPRH" refers to the protein Bone morphogenetic protein receptor type 2. Alternative names comprise BMP type-2 receptor, Bone morphogenetic protein receptor type II, BMP type II receptor, BMR2, PPH1 , BMPR3, BRK-3, POVD1 , T-ALK, BMPR2 and BMPR- II. The BMPRII protein is encoded by the gene BMPR2. The BMPRH protein comprises human, murine, and further mammalian homologues. Sequence(s) for human BMPRII are accessible via UniProt Identifier Q13873 (BMPRII HUMAN), for instance human isoform 1 (identifier: QI 3873-1), and human isoform 2 (identifier: Q13873-2). Sequence(s) for murine BMPRII are accessible via UniProt Identifier 035607 (BMPRII MOUSE). Different isoforms and variants may exist for the different species and are all comprised by the term BMPRII. In addition, synthetic variants of the BMPRII protein may be generated, e.g. by introducing at least one mutation, and are comprised by the term BMPRII. The protein BMPRII may furthermore be subject to various modifications, e.g., synthetic or naturally occurring modifications.

[0189] As used herein, the term “ActRIIA” refers to a family of activin receptor type HA (ActRHA) proteins from any species and variants derived from such ActRIIA proteins by mutagenesis or other modification. Reference to ActRHA herein is understood to be a

[0190] 22

[0191] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC reference to any one of the currently identified forms. Members of the ActRIIA family are generally transmembrane proteins, composed of a ligand-binding extracellular domain comprising a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine kinase activity. The term “ActRIIA” includes polypeptides comprising any naturally occurring polypeptide of an ActRIIA family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity.

[0192] As used herein, the term “ActRIIB” refers to a family of activin receptor type IIB (ActRIIB) proteins from any species and variants derived from such ActRIIB proteins by mutagenesis or other modification. Reference to ActRIIB herein is understood to be a reference to any one of the currently identified forms. Members of the ActRIIB family are generally transmembrane proteins, composed of a ligand-binding extracellular domain comprising a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine kinase activity. The term “ActRIIA” includes polypeptides comprising any naturally occurring polypeptide of an ActRIIB family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity. Examples of such variant ActRIIB polypeptides are provided throughout the present disclosure as well as in International Patent Application Publication Nos. WO 2006 / 012627 and WO 2008 / 097541 , which are incorporated herein by reference in its entirety.

[0193] ALK1 / BMPRII, ActRIIA or ActRIIB Bispecific Antibodies

[0194] Bispecific antibodies as provided herein promote the heterodimerization of ALK1 and a BMP Type II receptor, such as BMPRII, ActRIIA, and ActRIIB. Bispecific antibodies according to the current invention can be produced with high yields. The bispecific antibodies or their binding domains can be easily maturated, or screening approaches can be used to detect binders with optimized binding capabilities. For bispecific antibodies, each binding site can be optimized individually. Finally, even in the absence of downstream signaling, e.g. due to a genetic defect, an antibody approach could still be able to rescue the ALK1 / BMPRII, ALK1 / ActRIIA, or the ALK1 ActRIIB signaling cascade.

[0195] The antibodies disclosed herein specifically bind to ALK1 and BMPRII, ActRIIA, or ActRIIB; i.e., they bind to their targets with an affinity that is higher (e.g., at least two-fold higher) than their binding affinity for an irrelevant antigen (e.g., bovine serum albumin (BSA), casein).

[0196] As used herein, the term “inducing proximity” between ALK1 and BMPRII, ActRIIA, or ActRIIB refers to bringing ALK1 and any one of BMPRII, ActRIIA, or ActRIIB together such

[0197] 23

[0198] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC that the ALK1 / BMPRII, ALK1 / ActRIIA, or the ALK1 ActRIIB signaling cascade is stimulated. In certain embodiments, the proximity induced by the multispecific binding proteins of the disclosure is the same or similar to the proximity induced when BMP9 brings ALK1 and BMPRII together.

[0199] In some embodiments according to the first aspect, the bispecific antibodies specifically binds an extracellular domain of ALK1 and / or an extracellular domain of BMPRII, ActRIIA, or ActRIIB. In some embodiments, the ALK1 is human ALK1 or a fragment thereof, and / or the BMPRII, ActRIIA, or ActRIIB is human BMPRII, ActRIIA, or ActRIIB or a fragment thereof. In some embodiments, the bispecific antibody binds an extracellular domain of human ALK1 or a fragment thereof and / or an extracellular domain of human BMPRII or a fragment thereof.

[0200] In some embodiments, the bispecific antibody binds to ALK1 with a Kd of at most about 10’4M to about 10’13M (e.g., 10’4M, 10’4 5M, 10’5M, 10’55M, 10’6M, 10’65M, 10’7M, 10’75M, 10’8M, 10’8 5M, 10’9M, 10’9 5M, 1O’10M, 1O’10 5M, 10’11M, 10’11 5M, 10’12M, 10’12 5M, 10’13M).

[0201] In some embodiments, the bispecific antibody binds to BMPRII, ActRIIA, or ActRIIB with a Kd of at most about 10'4M to about 10'13M (e.g., 10'4M, 10'45M, 10'5M, 10'55M, 10'6

[0202] In some embodiments, the bispecific antibody binds to ALK1 and BMPRII or ALK1 and ActRIIA or ALK1 and ActRIIB with a Kd of at most about about 10'4M to about 10'13M (e.g.,

[0203] The Kd of antibody binding to an antigen can be assayed using any method known in the art including, for example, immunoassays such as enzyme-linked immununospecific 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 sorting (FACS) for quantification of antibody binding to cells that express an antigen. BIA is a technology for analyzing bispecific interactions in real time, without labeling any of the interactants (e.g., BIACORETM). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

[0204] In some embodiments, the antibody according to the current invention, in addition to binding domains for ALK1 and BMPRII, ActRIIA, or ActRIIB further comprises a binding

[0205] 24

[0206] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC domain for a ligand of the ALK1 / BMPRII, ALK1 / ActRIIA, or ALK1 / ActRIIB receptor, or for another molecule involved in ALK1 / BMPRII, ALK1 / ActRIIA, or ALK1 / ActRIIB signaling.

[0207] In some embodiments, the binding moiety which binds specifically to ALK1 is cross reactive with human ALK1 and mouse ALK1.

[0208] In some embodiments, the binding moiety which binds specifically to ActRIIA is cross reactive with ActRIIB.

[0209] Except if there is an obvious incompatibility for a person skilled in the art, each of the embodiments describing the binding capabilities can be combined with each of the embodiments describing the format of the antibody.

[0210] METHODS OF TREATING OR PREVENTING OCULAR DISEASES

[0211] The instant disclosure describes methods of treating or preventing ocular diseases with the ALK1 and a BMP Type II receptor multispecific binding proteins described herein. The multispecific binding proteins described herein are capable of reducing or preventing neovascularization, which may be useful in the treatment and prevention of ocular diseases, such as ocular diseases associated with vascular dysfunction. As used herein, the term “neovascularization” refers to the growth of new blood vessels. In the context of an ocular disease, neovascularization can lead to vision loss.

[0212] In one aspect, the disclosure provides a method of treating or preventing an ocular disease in a subject, the method comprising administering to the subject a multispecific binding protein comprising at least a first binding moiety which binds specifically to human ALK1 and at least a second binding moiety which binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB, thereby treating or preventing the ocular disease in the subject.

[0213] In certain embodiments, the ocular disease is selected from the group consisting of age-related macular degeneration (AMD), retinitis pigmentosa (RP), diabetic retinopathy, diabetic macular edema (DME), and hypertensive retinopathy. In certain embodiments, the AMD is wet AMD.

[0214] In certain embodiments, the multispecific binding protein reduces neovascularization in the subject.

[0215] In certain embodiments, the multispecific binding protein reduces vascular lesion volume.

[0216] In certain embodiments, the multispecific binding protein is capable of inducing signaling by inducing proximity between ALK1 and BMPRII, ActRIIA, or ActRIIB.

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[0218] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC

[0219] In certain embodiments, the multispecific binding protein is capable of inducing SMAD1 / 5 phosphorylation.

[0220] In certain embodiments, the multispecific binding protein is capable of inducing soluble VEGFR1 (sVEGFRI) expression.

[0221] In certain embodiments, the multispecific binding protein is capable of modulating the expression of one or more genes selected from the group consisting of inhibitor of DNA binding 1 (ID1), C-X-C chemokine receptor 4 (CXCR4), C-X-C motif chemokine 12 (CXCL12), angiopoietin 2 (ANGPT2), Selectin E (SELE), Selectin P (SELP), endothelial cell specific molecule 1 (ESM1), SMAD7, SMAD6, endoglin (ENG), BMPR2, serpinel , endothelin 1 (EDN1), apelin (APLN), apelin receptor (ALPNR), adrenomedullin (ADM), EPH receptor B4 (EPHB4), neuregulin 1 (NRG1), ectonucleotide pyrophosphatase / phosphodiesterase 2 (ENPP2), C-C motif chemokine ligand 2 (CCL2), vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor B (VEGFB), intercellular adhesion molecule 1 (ICAM1), vascular adhesion molecule 1 (VCAM1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), transforming growth factor beta-1 (TGFB1), Fms Related Receptor Tyrosine Kinase 1 (FLT1), C-X-C motif chemokine ligand 8 (CXCL8), matrix metallopeptidase 1 (MMP1), and activin A receptor like type 1 (ACVRL1).

[0222] In certain embodiments, the multispecific binding protein is capable of reducing the expression of one or more genes selected from the group consisting of inhibitor of DNA binding 1 (ID1), C-X-C chemokine receptor 4 (CXCR4), C-X-C motif chemokine 12 (CXCL12), angiopoietin 2 (ANGPT2), Selectin E (SELE), Selectin P (SELP), endothelial cell specific molecule 1 (ESM1), SMAD7, SMAD6, endoglin (ENG), BMPR2, serpinel , endothelin 1 (EDN1), apelin (APLN), apelin receptor (ALPNR), adrenomedullin (ADM), EPH receptor B4 (EPHB4), neuregulin 1 (NRG1), ectonucleotide pyrophosphatase / phosphodiesterase 2 (ENPP2), C-C motif chemokine ligand 2 (CCL2), vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor B (VEGFB), intercellular adhesion molecule 1 (ICAM1), vascular adhesion molecule 1 (VCAM1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), transforming growth factor beta-1 (TGFB1), Fms Related Receptor Tyrosine Kinase 1 (FLT1), C-X-C motif chemokine ligand 8 (CXCL8), matrix metallopeptidase 1 (MMP1), and activin A receptor like type 1 (ACVRL1).

[0223] In certain embodiments, the reduction in expression is relative to a subject that is not administered the multispecific binding protein.

[0224] In certain embodiments, the multispecific binding protein is administered intraocularly to the subject.

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[0226] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC

[0227] In certain embodiments, the multispecific binding protein is administered intravenously to the subject.

[0228] In certain embodiments, the multispecific binding protein is administered subcutaneously to the subject.

[0229] BINDING DOMAINS

[0230] One component of the multispecific binding protein of the present disclosure is a binding domain or binding specificity which binds a first cell surface target and a second cell surface target. In certain embodiments, the first cell surface target is a first receptor subunit, and the second cell surface target is the receptor subunit.

[0231] Any type of binding moiety that specifically binds to a specific receptor subunit can be employed in the multispecific binding proteins disclosed herein. In certain embodiments, the binding moiety comprises an antibody variable domain. Exemplary binding moieties comprising an antibody variable domain include, without limitation, a VH, a VL, a VHH, a VH / VL pair, an scFv, a diabody, or a Fab. Other suitable binding moiety formats include, without limitation, lipocalins (see e.g., Gebauer M. et al., 2012, Method Enzymol. 503:157- 188, which is incorporated by reference herein in its entirety), adnectins (see e.g., Lipovsek D., 2011 , Protein Eng. Des. Sei. 24:3-9, which is incorporated by reference herein in its entirety), avimers (see e.g., Silverman J, et al., 2005, Nat. Biotechnol. 23:1556-1561 , which is incorporated by reference herein in its entirety), fynomers (see e.g., Schlatter D, et al., 2012, mAbs 4:497-508, which is incorporated by reference herein in its entirety), kunitz domains (see e.g., Hosse R.J. et al., 2006, Protein Sci. 15:14-27, which is incorporated by reference herein in its entirety), knottins (see e.g., Kintzing J.R. et al., 2016, Curr. Opin. Chem. Biol. 34:143-150, which is incorporated by reference herein in its entirety), affibodies (see e.g., Feldwisch J. et al., 2010 J. Mol. Biol. 398:232-247, which is incorporated by reference herein in its entirety), and DARPins (see e.g., Pluckthun A., 2015, Annu. Rev. Pharmacol. Toxicol. 55:489-511 , which is incorporated by reference herein in its entirety).

[0232] In certain embodiments, the binding domain comprises the heavy and / or light chain variable regions of a conventional antibody or antigen binding fragment thereof (e.g., a Fab or scFv), wherein the term “conventional antibody” is used herein to describe heterotetrameric antibodies containing heavy and light immunoglobulin chains arranged according to the “Y” configuration. Such conventional antibodies may derive 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) wherein

[0233] 27

[0234] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC the VH and / or VL domains or one or more complementarity determining regions (CDRs) thereof are derived from the same antibodies. In certain embodiments, the conventional antibody antigen binding region may be referred to as a “Fab” (Fragment antigen-binding). The Fab comprises one constant and one variable domain from each of heavy chain and light chain. The variable heavy and light chains contain the CDRs responsible for antigen binding.

[0235] In other embodiments, the specific receptor subunit binding subunit comprises at least a CDR or VHH domain of a VHH antibody or Nanobody. VHH antibodies, which are camelid- derived heavy chain antibodies, are composed of two heavy chains and are devoid of light chains (Hamers-Casterman, et al. Nature. 1993; 363; 446-8). Each heavy chain of the VHH antibody has a variable domain at the N-terminus, and these variable domains are referred to in the art as “VHH” domains in order to distinguish them from the variable domains of the heavy chains of the conventional antibodies i.e., the VH domains. Similar to conventional antibodies, the VHH domains of the molecule comprise HCDR1 , HCDR2 and HCDR3 regions which confer antigen binding specificity and therefore VHH antibodies or fragments such as isolated VHH domains, are suitable as components of the multispecific binding proteins of the present disclosure.

[0236] MULTISPECIFIC BINDING PROTEINS

[0237] In certain embodiments, the first and second binding domains disclosed herein can be paired together or operatively linked to generate a multispecific binding protein which is capable of cross-linking a first and a second subunits of the given receptor (e.g., a BMP Type I receptor and a BMP type II receptor). In some embodiments, the first specific binding domain (e.g., VHH or scFv) is operatively linked (directly or indirectly) to the N and / or C terminus of a first Fc domain or polypeptide, and the second specific binding domain is operatively linked to the N and / or C terminus of second Fc domain or polypeptide, such that the first Fc domain and the second Fc domain facilitate heterodimerization of the first and second specific binding domains.

[0238] In certain exemplary embodiments, the multispecific binding proteins of the disclosure are agonistic to any given signaling pathway, i.e., they are not antagonistic to the ALK1 pathway. In some embodiments, agonism may be measured using a specific receptor potency assay (e.g., Pathhunter U2OS dimerization assay (DiscoverX) Potency assays (e.g., Pathhunter) involve a cell line (e.g., U2OS) that expresses the target receptors of interest. The binding of the bispecific antibodies to the receptors triggers a signaling cascade leading to the expression of a reporter gene which can be quantified.

[0239] In certain embodiments, the multispecific binding protein comprises a dual variable domain format. “Dual variable domain” (“DVD”) binding proteins of the disclosure comprise

[0240] 28

[0241] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC two or more antigen binding sites and are tetravalent or multivalent binding proteins. The DVDs of the disclosure are multispecific, i.e., capable of binding 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 a “DVD immunoglobulin” or “DVD-lg”. Each half of a DVD-lg comprises a heavy chain DVD polypeptide and a light chain DVD polypeptide, and two or more antigen binding sites. Each binding site comprises a heavy chain variable domain and a light chain variable domain with a total of six CDRs involved in antigen binding per antigen binding site.

[0242] A description of the design, expression, and characterization of DVD-lg molecules is provided in PCT Publication No. WO 2007 / 024715; U.S. Pat. No. 7,612,181 ; and Wu et al., Nature Biotechnol., 25: 1290-1297 (2007). An example of such DVD-lg molecules comprises a heavy chain that comprises the structural formula VD1-(X1)n-VD2-C-(X2)n, wherein 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 with the proviso that it is not CH1 , X2 is an Fc region, and n is 0 or 1 ; and a light chain that comprises the structural formula VD1-(X1)n-VD2- C-(X2)n, wherein 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 with the proviso that it is not CH1 , and X2 does not comprise an Fc region; and n is 0 or 1. Such a DVD-lg may comprise two such heavy chains and two such light chains, wherein each chain comprises variable domains linked in tandem without an intervening constant region between variable regions, wherein a heavy chain and a light chain associate to form tandem functional antigen binding sites, and a pair of heavy and light chains may associate with another pair of heavy and light chains to form a tetrameric binding protein with four functional antigen binding sites. In another example, a DVD-lg molecule may comprise heavy and light chains that each comprise three variable domains (VD1 , VD2, VD3) linked in tandem without an intervening constant region between variable domains, wherein a pair of heavy and light chains may associate to form three antigen binding sites, and wherein a pair of heavy and light chains may associate with another pair of heavy and light chains to form a tetrameric binding protein with six antigen binding sites.

[0243] In an embodiment, the disclosure provides a binding protein comprising first and second polypeptide chains, wherein said first polypeptide chain comprises a first VD1-(X1)n- VD2-C-(X2)n, wherein: 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 with the proviso that it is not CH1 ; X2 is an Fc region; and n is independently 0 or 1 ; and wherein said second polypeptide chain comprises a second VD1-(X1)n-VD2-C-(X2)n, wherein: 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 with the proviso that it is not CH1 ; X2 does not comprise an Fc region; and n is independently 0 or 1.

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[0245] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC

[0246] With respect to constructing DVD-lg or other binding protein molecules, a “linker” is used to denote a single amino acid or a polypeptide (“linker polypeptide”) comprising two or more amino acid residues joined by peptide bonds and used to link one or more antigen binding portions. Such linker polypeptides are well known in the art (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993); Poljak, R. J., Structure, 2: 1121-1123 (1994)). Flexible linkers may be employed, which are generally composed of small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids. Exemplary flexible linkers include, but are not limited to, GGGGSG (SEQ ID NO: ##), GGSGG (SEQ ID NO: ##), GGGGSGGGGS (SEQ ID NO: ##), GGSGGGGSG (SEQ ID NO: ##), GGSGGGGSGS (SEQ ID NO: ##), GGSGGGGSGGGGS (SEQ ID NO: ##), GGGGSGGGGSGGGG (SEQ ID NO: ##), GGGGSGGGGSGGGGS (SEQ ID NO: ##), and

[0247] RADAAAAGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: ##).

[0248] Alternatively, rigid linkers may be employed to join one or more antigen binding proteins. Said rigid linkers may allow for the maintenance of fixed distances between linked antigen binding proteins, thereby promoting the activity of each individual protein. Rigid linkers may employ one of more proline amino acids to confer the rigidity. Exemplary rigid linkers include, but are not limited to, 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: ##), RADAAAAGGPGS (SEQ ID NO: ##), SAKTTPKLEEGEFSEARV (SEQ ID NO: ##), ADAAP (SEQ ID NO: ##), ADAAPTVSIFPP (SEQ ID NO: ##), QPKAAP (SEQ ID NO: ##), QPKAAPSVTLFPP (SEQ ID NO: ##), AKTTPP (SEQ ID NO: ##), AKTTPPSVTPLAP (SEQ ID NO: ##), AKTTAP (SEQ ID NO: ##), AKTTAPSVYPLAP (SEQ ID NO: ##), GENKVEYAPALMALS (SEQ ID NO: ##), GPAKELTPLKEAKVS (SEQ ID NO: ##), and GHEAAAVMQVQYPAS (SEQ ID NO: ##).

[0249] In certain embodiments, the linker comprises or consists of PI_AP, PAPNLLGGP, PLAPDKTHT, EKSYGPP, or DKTHT.

[0250] In certain embodiments, the multispecific binding protein comprises a first and a second polypeptide chain, wherein: said first polypeptide chain comprises VH1-(HX1)n-VH2-C-(HX2)n, wherein:

[0251] 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 said second polypeptide chain comprises VL1-(LX1)n-VL2-C-(LX2)n, wherein:

[0252] 30

[0253] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC

[0254] 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 comprise an Fc region; and n is independently 0 or 1.

[0255] In certain embodiments, VH1 binds specifically to human ALK1 and VH2 binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB.

[0256] In certain embodiments, VL1 binds specifically to human ALK1 and VL2 binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB.

[0257] In certain embodiments, VH1 binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB and VH2 binds specifically to human ALK1.

[0258] In certain embodiments, VL1 binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB and VL2 binds specifically to human ALK1.

[0259] In certain embodiments, linker HX1 comprises an amino acid sequence of PI_AP or PAPNLLGGP.

[0260] In certain embodiments, linker LX1 comprises an amino acid sequence of PI_AP or PAPNLLGGP.

[0261] In certain embodiments, linker HX1 comprises an amino acid sequence of PLAP and linker LX1 comprises an amino acid sequence of PLAP or PAPNLLGGP.

[0262] In certain embodiments, the multispecific binding protein comprises two polypeptide chains of VH1-(HX1)n-VH2-C-(HX2)n and two polypeptide chains of VL1-(LX1)n-VL2-C- (LX2)n.

[0263] In certain embodiments, for (HX1)n, n is 1 and for (HX2)n, n is 1.

[0264] In certain embodiments, for (LX1)n, n is 1 and for (LX2)n, n is 0.

[0265] In certain embodiments, the multispecific binding protein comprises a first and a second polypeptide chain, wherein: said first polypeptide chain comprises VH1-(HX1)n-VH2-C-Fc, wherein:

[0266] 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; Fc is an Fc region; and n is independently 0 or 1; and said second polypeptide chain comprises VL1-(LX1)n-VL2-C, wherein:

[0267] 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; and n is independently 0 or 1.

[0268] Non-DVD-lq Formats

[0269] In another aspect of the disclosure, the multispecific binding protein comprises from N-terminus to C-terminus:

[0270] 31

[0271] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC 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 linker (e.g., a modified hinge region) or the absence of a linker, and a second constant region; aiii) a first polypeptide chain comprising a first linker (e.g., a modified hinge region) or the absence of a linker, and a first constant 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, an optional first linker (e.g., a modified hinge region), a second antigen binding domain, an optional 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, an optional third linker (e.g., a modified hinge region), a fourth antigen binding domain, an optional fourth linker (e.g., a modified hinge region), and a second constant region.

[0272] In certain embodiments, the first antigen binding domain comprises an scFv, VHH, Fab, F(ab’)2, or a single domain antibody.

[0273] In certain embodiments, the second antigen binding domain comprises an scFv, VHH, Fab, F(ab’)2, or a single domain antibody.

[0274] In certain embodiments, the third antigen binding domain comprises an scFv, VHH, Fab, F(ab’)2, or a single domain antibody.

[0275] In certain embodiments, the fourth antigen binding domain comprises an scFv, VHH, Fab, F(ab’)2, or a single domain antibody.

[0276] In certain embodiments, any one or more of the first antigen binding domain, second antigen binding domain, third antigen binding domain, and fourth antigen binding domain comprise an scFv, VHH, Fab, F(ab’)2, or a single domain antibody.

[0277] In certain embodiments, the first antigen binding domain, second antigen binding domain, third antigen binding domain, and fourth antigen binding domain each comprise an scFv.

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[0280] The Fc polypeptides employed in the multispecific binding proteins of the disclosure generally comprise a CH2 domain and a CH3 domain, wherein the C-terminus of the CH2 domain is linked (directly or indirectly) to the N-terminus of the CH3 domain. Any naturally occurring or variant CH2 and / or CH3 domain can be used. For example, in certain embodiments, the CH2 and / or CH3 domain is a naturally occurring CH2 or CH3 domain from an lgG1 , lgG2, lgG3, lgG4, lgA1 , or lgA2 antibody heavy chain, e.g., a human lgG1 , lgG2, lgG3, lgG4, lgA1 , or lgA2 antibody heavy chain. The CH2 and CH3 domains can be from the same or different antibody heavy chains. In certain embodiments, the Fc polypeptide comprises a CH2 and CH3 domain-containing portion from a single antibody heavy chain. In certain embodiments, the CH2 and / or CH3 domain is a variant of a naturally occurring CH2 or CH3 domain, respectively. In certain embodiments, the CH2 and / or CH3 domain is a variant comprising one or more amino acid insertions, deletion, substitutions, or modifications relative to a naturally occurring CH2 or CH3 domain, respectively. In certain embodiments, the CH2 and / or CH3 domain is a chimera of one or more CH2 or CH3 domains, respectively. In certain embodiments, the CH2 domain comprises amino acid positions 231-340 of a naturally occurring hinge region (e.g., human lgG1), according to the Ell index. In certain embodiments, the CH3 domain comprises amino acid positions 341-447 of a naturally occurring hinge region (e.g., human lgG1), according to the Ell index.

[0281] In certain embodiments, the Fc polypeptides further comprise a hinge region, wherein the C-terminus of hinge region is linked (directly or indirectly) to the N-terminus of the CH2 domain. For example, in certain embodiments, the hinge region is a naturally occurring hinge region from an lgG1 , lgG2, lgG3, lgG4, lgA1 , or lgA2 antibody heavy chain, e.g., a human lgG1 , lgG2, lgG3, lgG4, lgA1 , or lgA2 antibody heavy chain. The hinge region can be from the same or different antibody heavy chain than the CH2 and / or CH3 domains. In certain embodiments, the hinge region is a variant comprising one or more amino acid insertions, deletion, substitutions, or modifications relative to a naturally 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 naturally occurring hinge region (e.g., human lgG1), according to the Ell index. In certain embodiments, the hinge region comprises amino acid positions 216-230 of a naturally occurring hinge region (e.g., human lgG1), according to the Ell index. In certain embodiments, the hinge region comprises amino acid positions 216-230 of a naturally occurring hinge region (e.g., human lgG1), according to the Ell index. In certain embodiments, the hinge region is a variant lgG4 hinge region comprising a serine (S) at amino acid position 228, according to the Ell index.

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[0284] In certain embodiments, the Fc polypeptides further comprise a CH1 domain, wherein the C-terminus of CH1 domain is linked (directly or indirectly) to the N-terminus of the hinge region. For example, in certain embodiments, the CH1 domain is a naturally occurring CH1 domain from an lgG1 , lgG2, lgG3, lgG4, lgA1 , or lgA2 antibody heavy chain, e.g., a human lgG1 , lgG2, lgG3, lgG4, lgA1 , or lgA2 antibody heavy chain. The CH1 domain can be from the same or different antibody heavy chain than the hinge region, CH2 domain and / or CH3 domain. In certain embodiments, the CH1 domain is a variant comprising one or more amino acid insertions, deletions, substitutions, or modifications relative to a naturally occurring CH1 domain. In certain embodiments, the CH1 domain is a chimera of one or more CH1 domain. In certain embodiments, the CH1 domain comprises amino acid positions 118-215 of a naturally occurring hinge region (e.g., human lgG1), according to the Ell index.

[0285] In certain embodiment, the Fc polypeptide lacks a CH1 domain or comprises mutations in a CH 1 domain or heavy chain variable domain that prevent association of the heavy chain with an antibody light chain. In certain embodiments, the antibody heavy chain lacks a portion of a hinge region.

[0286] Heterodimerization Motifs

[0287] In certain exemplary embodiments, the first and second Fc domains are further engineered to enhance heterodimerization of the first specific and second specific binding domains and minimize the effects of incorrect chain pairing (i.e., pairing of a BMP Type I receptor and a BMP Type II receptor).

[0288] Any art-recognized approach that addresses the problem of incorrect chain pairing can be employed to improve desired multispecific antibody production. For instance, LIS2010 / 0254989 A1 describes the construction of bispecific cMet - ErbB1 antibodies, where the VH and VL of the individual antibodies are fused genetically via a GlySer linker. For bispecific antibodies including an Fc domain, mutations may be introduced into the Fc to promote the correct heterodimerization of the Fc portion. Several such approaches are reviewed in Klein et al. (mAbs (2012) 4:6, 1 -11), the contents of which are incorporated herein by reference in their entirety.

[0289] In certain embodiments, the first specific and second specific binding specificities of the multispecific antibody are heterodimerized through knobs-into-holes (KiH) pairing of Fc domains. This dimerization technique utilizes “protuberances” or “knobs” with “cavities” or “holes” engineered into the interface of CH3 domains. Where a suitably positioned and dimensioned knob or hole exists at the interface of either the first or second CH3 domain, it is only necessary to engineer a corresponding hole or knob, respectively, at the adjacent

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[0291] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC interface, thus promoting and strengthening Fc domain pairing in the CH3 / CH3 domain interface. The IgG Fc domain that is fused to the VHH is provided with a knob, and the IgG Fc domain of the conventional antibody is provided with a hole designed to accommodate the knob, or vice-versa. A “knob” refers to an at least one amino acid side chain, typically a larger side chain, that protrudes from the interface of the CH3 portion of a first Fc domain. The protrusion creates a “knob” which is complementary to and received by a “hole” in the CH3 portion of a second Fc domain. The “hole” is an at least one amino acid side chain, typically a smaller side chain, which recedes from the interface of the CH3 portion of the second Fc domain. This technology is described, for example, in U.S. Pat. Nos. 5,821 ,333; 5,731 ,168 and 8,216,805; Ridgway et al. Protein Engineering (1996) 9:617-621); and Carter P. J. Immunol. Methods (2001) 248: 7-15, which are herein incorporated by reference.

[0292] Exemplary amino acid residues that may act as the knob include arginine (R), phenylalanine (F), tyrosine (Y) or tryptophan (W). An existing amino acid residue in the CH3 domain may be replaced or substituted with a knob amino acid residue. Preferred amino acids to substitute may include any amino acids with a small side chain, such as alanine (A), asparagine (N), aspartic acid (D), glycine (G), serine (S), threonine (T), or valine (V).

[0293] Exemplary amino acid residues that may act as the hole include alanine (A), serine (S), threonine (T), or valine (V). An existing amino acid residue in the CH3 domain may be replaced or substituted with a hole amino acid residue. Preferred amino acids to substitute may include any amino acids with a large side chain, such as arginine (R), phenylalanine (F), tyrosine (Y) or tryptophan (W).

[0294] The CH3 domain is preferably derived from a human IgG 1 antibody. Exemplary amino acid substitutions to the CH3 domain include Y349C, S354C, T366S, T366Y, T366W, F405A, F405W, Y407T, Y407A, Y407V, T394S, or combinations thereof. A preferred exemplary combination is S354C, T366Y or T366W for the knob mutation on a first CH3 domain and Y349C, T366S, L368A, Y407T or Y407V for the hole mutation on a second CH3 domain.

[0295] In certain embodiments, the two Fc domains of the antigen binding construct are heterodimerized through Fab arm exchange (FAE). A human IgG 1 possessing a P228S hinge mutation may contain an F405L or K409R CH3 domain mutation. Mixing of the two antibodies with a reducing agent leads to FAE. This technology is described in US Patent 9,212,230 and Labrijn A. F. PNAS (2013) 110(13):5145-5150, which are incorporated herein by reference.

[0296] In other embodiments, the two Fc domains of the antigen binding construct are heterodimerized through electrostatic steering effects. This dimerization technique utilizes electrostatic steering to promote and strengthen Fc domain pairing in the CH3 / CH3 domain interface. The charge complementarity between two CH3 domains is altered to favor

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[0298] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC heterodimerization (opposite charge pairing) over homodimerization (same charge pairing). In this method, the electrostatic repulsive forces prevent homodimerization. Certain exemplary amino acid residue substitutions which confer electrostatic steering effects include K409D, K392D, and / or K370D in a first CH3 domain and D399K, E356K, and / or E357K in a second CH3 domain. This technology is described in US Patent Publication No. 2014 / 0154254 A1 and Gunasekaran K. JBC (2010) 285(25): 19637- 19646, which are incorporated herein by reference.

[0299] In other embodiments, the charge complementarity is formed by a first Fc domain comprising a N297K and / or a T299K mutation, and a second Fc domain comprising a N297D and / or a T299D mutation.

[0300] In an aspect of the invention, the two Fc domains of the antigen binding construct are heterodimerized through hydrophobic interaction effects. This dimerization technique utilizes hydrophobic interactions instead of electrostatic ones to promote and strengthen Fc domain pairing in the CH3 / CH3 domain interface. Exemplary amino acid residue substitution may include K409W, K360E, Q347E, Y349S, and / or S354C in a first CH3 domain and D399V, F405T, Q347R, E357W, and / or Y349C in a second CH3 domain. Preferred pairs of amino acid residue substitutions between a first CH3 domain and a second CH3 domain include K409W:D399V, K409W:F405T, K360E:Q347R, Y349S:E357W, and S354C:Y349C. This technology is described in US Patent Publication No. 2015 / 0307628 A1.

[0301] In an aspect of the invention, heterodimerization can be mediated through the use of leucine zipper fusions. Leucine zipper domains fused to the C terminus of each CH3 domain of the antibody chains force heterodimerization. This technology is described in Wranik B. JBC (2012) 287(52):43331-43339.

[0302] In an aspect of the invention, heterodimerization can be mediated through the use of a Strand Exchange Engineered Domain (SEED) body. CH3 domains derived from an IgG and IgA format force heterodimerization. This technology is described in Muda M. PEDS (2011) 24(5): 447-454.

[0303] In other embodiments, the heterodimerization motif may comprise non-native, disulfide bonds formed by engineered cysteine residues. In certain embodiments, the first set of disulfide may comprise a Y349C mutation in the first Fc domain and a S354C mutation in the second Fc domain. In other embodiment, an engineered disulfide bond may be introduced by fusion a C-terminal extension peptide with an engineered cysteine residue to the C-terminus of each of the two Fc domains. In certain embodiments, the first Fc domain may comprise the substitution of the carboxyl-terminal as “PGK” with “GEC”, and the second Fc domain may comprise the substitution of the carboxyl terminal amino acids “PGK” with “KSCDKT”.

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[0306] In yet another approach, the multispecific antibodies may employ the CrossMab principle (as reviewed in Klein et al.), which involves domain swapping between heavy and light chains so as to promote the formation of the correct pairings. Yet another approach involves engineering the interfaces between the paired VH-VL domains or paired CH1 -CL domains of the heavy and light chains so as to increase the affinity between the heavy chain and its cognate light chain (Lewis et al. Nature Biotechnology (2014) 32: 191 -198).

[0307] An alternative approach to the production of multispecific antibody preparations having the correct antigen specificity has been the development of methods that enrich for antibodies having the correct heavy chain-light chain pairings. For example, Spiess et al. (Nature Biotechnology (2013) 31 : 753-758) describe a method for the production of a MET-EGFR bispecific antibody from a co-culture of bacteria expressing two distinct half-antibodies.

[0308] Methods have also been described wherein the constant region of at least one of the heavy chains of a bispecific antibody is mutated so as to alter its binding affinity for an affinity agent, for example Protein A. This allows correctly paired heavy chain heterodimers to be isolated based on a purification technique that exploits the differential binding of the two heavy chains to an affinity agent (see US2010 / 0331527, WO2013 / 136186).

[0309] International patent application no. PCT / EP2012 / 071866 (WO2013 / 064701) addresses the problem of incorrect chain pairing using a method for multispecific antibody isolation based on the use of anti-idiotypic binding agents, in particular anti-idiotypic antibodies. The anti-idiotype binding agents are employed in a two-step selection method in which a first agent is used to capture antibodies having a VH-VL domain pairing specific for a first antigen and a second agent is subsequently used to capture antibodies also having a second VH-VL domain pairing specific for a second antigen.

[0310] In yet another embodiment, the multispecific antibody employs a first binding specificity having a conventional Fab binding region and a second binding specificity comprising a single domain antibody (VHH) binding region. The heterodimerization method employed forces the binding of the heavy chain region of the Fab and the full, heavy chain only, of the VHH. Because the VHH chain does not associate with light chains, the light chain region of the Fab portion will only associate with its corresponding heavy chain.

[0311] In certain other embodiments, the multispecific binding protein described herein further comprises a common light chain. The term “common light chain” as used herein refers to a light chain which is capable of pairing with a first heavy chain of an antibody which binds to a first antigen in order to form a binding site specifically binding to said first antigen and which is also capable of pairing with a second heavy chain of an antibody which binds to a second antigen in order to form a binding site specifically binding to said second antigen. A common

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[0313] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC light chain is a polypeptide comprising in N-terminal to C-terminal direction an antibody light chain variable domain (VL), and an antibody light chain constant domain (CL), which is herein also abbreviated as “VL-CL”. Multispecific binding proteins with a common light chain require heterodimerization of the distinct heavy chains. In certain embodiments, the heterodimerization methods listed above may be used with a common light chain. In certain exemplary embodiments, the heterodimerization motif may comprise non-native, disulfide bonds formed by engineered cysteine residues. Adding disulfide bonds, both between the heavy and light chain of an antibody has been shown to improve stability. Additionally, disulfide bonds have also been used as a solution to improve light-chain pairing within bispecific antibodies (Geddie M. L. et al, mABs (2022) 14(1)).

[0314] Unless otherwise stated, all antibody constant region numbering employed herein corresponds to the EU numbering scheme, as described in Edelman et al. (Proc. Natl. Acad. Sci. 63(1): 78-85. 1969).

[0315] Additional methods of heterodimerization of heavy and / or light chains and the generation 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 9,499,634, each of which is incorporated herein by reference.

[0316] Effector Function Mutations

[0317] As discussed above, multispecific binding proteins of the disclosure can be provided in various isotypes and with different constant regions. The Fc region of the multispecific binding primarily determines its effector function in terms of Fc binding, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement dependent cytotoxicity (CDC) activity, and antibody-dependent cell phagocytosis (ADCP) activity. These "cellular effector functions", as distinct from effector T cell function, involve the recruitment of cells bearing Fc receptors to the site of the target cells, resulting in killing of the antibody-bound cell.

[0318] An antibody according to the present invention may be one that exhibits reduced effector function. In certain embodiments, the one or more mutations reduces one or more of antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), or complement dependent cytotoxicity (CDC). In certain embodiments, an antibody according to the present invention may lack ADCC, ADCP and / or CDC activity. In either case, an antibody according to the present invention may comprise, or may optionally lack, an Fc region that binds to one or more types of Fc receptor. Use of different antibody formats, and the presence or absence of FcR binding and cellular effector functions, allow the antibody to

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[0320] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC be tailored for use in particular therapeutic purposes as discussed elsewhere herein.

[0321] In certain embodiments, the first and the second Fc domain comprise one or more mutations that reduces Fc effector function. In certain embodiments, the first Fc domain and the second Fc domain each comprise a L234A and L235A mutation. These lgG1 mutations are also known as the “LALA” mutations and are described in further detail in Xu et al. (Cell Immunol. 2000; 200:16-26). In certain embodiments the first Fc domain and the second Fc domain each comprise a L234A, L235A, G237A, and / or P329G mutation. The Fc domain amino acid positions referred to herein are based on Ell antibody numbering. Alternatively, an antibody may have a constant region which is effector null. An antibody may have a heavy chain constant region that does not bind Fey receptors, for example the constant region may comprise a L235E mutation. Another optional mutation for a heavy chain constant region is S228P, which increases stability. A heavy chain constant region may be an lgG4 comprising both the L235E mutation and the S228P mutation. This "lgG4-PE" heavy chain constant region is effector null. A disabled IgG 1 heavy chain constant region is also effector null. A disabled lgG1 heavy chain constant region may contain alanine at position 234, 235 and / or 237 (Ell index numbering), e.g., it may be an lgG1 sequence comprising the L234A, L235A and / or G237A mutations ("LALAGA"). In certain embodiments, the first Fc domain and the second Fc domain each comprise a L234A, L235A, and P329A mutation (“LALAPA).

[0322] Human I gG 1 constant regions containing specific mutations or altered glycosylation on residue Asn297 (e.g., N297Q, N297D, and N297K, Ell index numbering) have been shown to reduce binding to Fc receptors.

[0323] In some embodiments, the first Fc domain and / or the second Fc domain each comprise a L234F, L235E, and P331S mutation (FES).

[0324] In some embodiments, the first Fc domain and / or the second Fc domain each comprise a E233P, L234V, L235A, G236Del, and S267K mutation.

[0325] In other embodiments, it may be desirable to enhance the binding of the Fc region of a multispecific antibody to human Fc gamma receptor II IA (FcgRIIIA) relative to that of the Fc region of a corresponding naturally occurring antibody. In certain embodiments, a constant region may be engineered for enhanced ADCC and / or CDC and / or ADCP. The potency of Fc- mediated effects may be enhanced by engineering the Fc domain by various established techniques. Such methods increase the affinity for certain Fc-receptors, thus creating potential diverse profiles of activation enhancement. This can be achieved by modification of one or several amino acid residues. Example mutations are one or more of the residues selected from 239, 332 and 330 for human IgG 1 constant regions (or the equivalent positions in other IgG isotypes). An antibody may thus comprise a human lgG1 constant region having one or

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[0327] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC more mutations independently selected from S239D, I332E and A330L (Ell index numbering).

[0328] Increased affinity for Fc receptors can also be achieved by altering the natural glycosylation profile of the Fc domain by, for example, generating under fucosylated or de- fucosylated variants. Non-fucosylated antibodies harbor a tri-mannosyl core structure of complex-type N-glycans of Fc without fucose residue. These glycoengineered antibodies that lack core fucose residue from the Fc N-glycans may exhibit stronger ADCC than fucosylated equivalents due to enhancement of FcyRIIIA binding capacity. For example, to increase ADCC, residues in the hinge region can be altered to increase binding to FcyRIIIA. Thus, an antibody may comprise a human IgG heavy chain constant region that is a variant of a wildtype human IgG heavy chain constant region. In certain embodiments, the variant human IgG heavy chain constant region binds to human Fey receptors selected from the group consisting of FcyRIIB and FcyRIIA with higher affinity than the wild type human IgG heavy chain constant region binds to the human FcyRIIIA. The antibody may comprise a human IgG heavy chain constant region that is a variant of a wild type human IgG heavy chain constant region, wherein the variant human IgG heavy chain constant region binds to human FcyRI I B with higher affinity than the wild type human IgG heavy chain constant region binds to human FcyRIIB. The variant human IgG heavy chain constant region can be a variant human I gG 1 , a variant human lgG2, or a variant human lgG4 heavy chain constant region. In one embodiment, the variant human IgG heavy chain constant region comprises one or more amino acid mutations selected from G236D, P238D, S239D, S267E, L328F, and L328E (Ell 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 (Ell index numbering system).

[0329] The enhancement of CDC may be achieved by amino acid changes that increase affinity for C1 q, the first component of the classic complement activation cascade. Another approach is to create a chimeric Fc domain created from human lgG1 and human lgG3 segments that exploit the higher affinity of lgG3 for C1 q. Antibodies of the present invention may comprise mutated amino acids at residues 329, 331 and / or 322 to alter the 01 q binding and / or reduced or abolished CDC activity. In another embodiment, the antibodies or antibody fragments disclosed herein may contain Fc regions with modifications at residues 231 and 239, whereby the amino acids are replaced to alter the ability of the antibody to fix complement. In one embodiment, the antibody or fragment has a constant region comprising

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[0331] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC one or more mutations selected from E345K, E430G, R344D and D356R, in particular a double mutation comprising R344D and D356R (Ell index numbering system).

[0332] The functional properties of the multispecific binding proteins may be further tuned 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 binding to FcRn (also referred to herein as “FcRn variants”) may in certain situations also increase serum half-life in vivo as compared to an unmodified binding protein. As will be appreciated, any combination of Fc and FcRn variants may be used to tune clearance of the antigenantibody complex. Suitable FcRn variants that may be combined with any of the Fc variants described herein that include without limitation N434A, N434S, M428L, V308F, V259I, M428L I N434S, V259I I V308F, Y436I I M428L, Y436I I N434S, Y436V I N434S, Y436V I M428L, M252Y, M252Y I S254T I T256E, and V259I I V308F I M428L (EU index numbering system). In some embodiments, the Fc variant includes H443K / N434K. In some embodiments, the Fc variant includes M328L / N434S (EU index numbering system).

[0333] While the present disclosure has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the claims appended hereto. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.

[0334] EXAMPLES

[0335] Example 1. Bispecific antibodies to BMPR Type I and Type II receptors with optimized hinges, linkers and valencies

[0336] Bispecific antibodies targeting the BMRP Type I receptor ALK1 and BMPR Type II receptor BMPRII were designed, with sequences provided below. Some constructs include

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[0338] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC upper hinge variants: hinge 1 = no upper hinge; hinge 3 = an upper hinge sequence of PLAP (SEQ ID NO: 2); hinge 6 = an upper hinge sequence of DKTHT (SEQ ID NO: 5).

[0339] Three-dimensional structures of BMP10 in complex with ALK1 and BMPRII (PDB ID 7PPC) was used in combination with structural models from AlphaFold2 AF-P37023-F1- model_v4 (ALK1), AF-Q13873-F1-model_v4 (BMPRII) and a model from Agnew et al. (DOI: 10.1038 / s41467-021-25248-5) to construct a model of the intra and extra cellular domains of the BMPRII / ALK1 / BMP10 active tetrameric receptor complex that enables phosphorylation of the GS domain and activation of SMADs. We predicted that tetravalent format of agonistic antibodies would facilitate the predicted tetrameric receptor assembly, required for signaling of ALK1 / BMPRI I complex.

[0340] The DIAGONAL platform predicted epitopes on ALK1 and BMPRII that binders could target to engage the receptor in this tetravalent format. Those predictions were used to design CDRs of the binding modules of the DGL molecules and connecting linkers compatible with the geometrical constraints of tetravalent antibody formats. Antibodies were transiently transfected using the ExpiCHO (Thermo) system according to the manufacturer’s instructions. Cells were harvested six days post transfection and harvested using batch purification with mabSelect resin. Purity of the final product was assessed using SDS-PAGE and analytical gel filtration.

[0341] Table 1. Hinge variant and Fc domain sequences

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[0344] Table 2. Bispecific antibodies to ALK1 and BMPRII constructs

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[0379] Example 2. Screen for Agonistic Activity

[0380] The bispecific antibodies were screened for agonist activity. PathHunter U2Os ALK- 1 / BMPR-2 dimerization assay was obtained from DiscoverX Corporation (93-0962C3). These cells use Enzyme Fragment Complementation (EFC) technology using - galactosidase fragments to evaluate protein-protein interactions. Reporter cells were revived and cultured according to supplier’s recommendations. Bispecific antibodies were compared to the natural ligands, BMP-9 and BMP-10.

[0381] To perform the assay, cells were detached and removed from the flask with cell detachment reagent (DiscoverX, 92-0009). Cells were spun at 300g for four minutes and resuspended at a density of 250K / ml in assay plating media (DiscoverX 93-0563R22A). 20 ul of the suspension were plated / well of a 384 well plate and incubated at 37°C for 24 hours.

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[0384] Bispecifics were made at 5x the final concentration. 12-point titrations using a 1:10 dilution were done to generate curves. 5 ul of the bispecific was added to the 384 well plate and incubated for three hours. 25 ul of flash detection reagent (DiscoverX, 93-0247) was added / well and the plates were read on a Verilux Skan at 60 minutes. Data was analyzed using PRISM.

[0385] Table 3: Agonist activity of the bispecific antibody constructs

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[0388] Example 3: Measurement of pSMAD in HUVEC cells

[0389] HLIVEC cells from ATCC (CRL-1730) were plated at 15K cells per well of a 96 well plate in 100ul of complete HUVEC media overnight (F12K (Corning, 10-025-CV), 10% FBS (Gibco, A31605-02), ECGS (30 ug / ml, Corning, 356006), 0.1 mg / ml Heparin (Sigma, H3393), 1x Pen / Strep (Gibco, 15140-122). The following morning, cells were starved for 4 hours by replacing media with 50ul serum free / ECGS free F12K media. Cells were then treated with 50ul of serum free / ECGS free media containing 2X concentration dose curve of the bispecifics or BMP ligands. At various time points (5, 15, 30, 60 min) media was removed from cells and 50ul lysis buffer (Abeam ELISA kit, AB186037) was added per well. After lysis, buffer from four wells were pooled for a single 200ul lysed sample per condition, which was frozen and later run on ELISAs measuring either total SMAD1 (Abeam, AB186037) or pSMADI (Abeam, AB186036). As a negative control, an anti-HEL antibody with LALA-PG mutations (BioXCell, CP149) was used. Table 4. Phosphorylation of SMAD1 following treatment with bispecific antibodies.

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[0392] Example 4: Measurement of in vivo activity

[0393] Antibodies were measured for agonistic activity in a mouse model of HHT wherein circulating BMP9 / BMP10 were neutralized by anti-BMP9 / 10 antibodies (Ruiz S, et al, Scientific Reports, 2016 Nov 22: 5:37366). These mice develop vascular defects in the postnatal retina. Three animals were dosed with either DGL288 or a negative control antibody (Anti-HEL, LAI-A-PG, BioXCell, CP149) for two days, P3 and P4, at 15mg / kg / day. BMP9 / 10 antibodies were dosed on the same days. Analysis was completed on P6. Retinas were dissected and whole-mount prepared, then stained with both isolectin B4 and SMA to label retinal vasculature and detect ateriovenous malformations (AVMs). Results are in FIG. 1A. Mice dosed with DGL288 showed no formation of AVMs, whereas the negative control showed an average of 4.8 AVMs / retina.

[0394] For the second set of experiments, all animals were dosed with BMP9 / 10 antibodies on P3 and P4. DGL288, DGL292 or PBS control were dosed at 1 mg / kg / day on P4 and P5. Analysis was completed on P6 for DGL288 and the littermate negative control animals, or P7 for DGL292 and littermates dosed with the PBS control. Retinas were dissected and wholemount prepared, then stained with both isolectin B4 and SMA to detect AVMs. Mice dosed with DGL292 did not form AVMs, compared with an average of 5.7 / retina for the controls (FIG. 1 B). Mice dosed with DGL288 did not form AVMs, compared with an average of 4.5 / retina for the controls (FIG. 10). No differences in body weight were observed, suggesting that the agonists are well tolerated.

[0395] Example 5: Additional engineering of Binders

[0396] Based on structural modeling of the receptor / antibody complex, the binders were engineered to further optimize the complementary regions of the binding to the antigen. Both ALK1 and BMPRII variants were designed for improved potency and / or stability.

[0397] Table 5. Optimized ALK1 and BMPRII binders.

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[0402] Table 6. Optimized ALK1 and BMPRII bispecific antibodies.

[0403] These binders were then tested for ELISA high binding plates (Corning, 9018) were coated with either 2 ug / ml of human BMPRII protein (Sino Biological, #10551-H08H) or 2

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[0405] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC ug / ml of human ALK1 protein (Sino Biological, #10066-H08H) overnight at 4C. The plates were then washed three times with wash buffer (R&D Systems, WA126). The plates were blocked with 1% BSA in PBS for one hour at room temperature, then blocked with 1% BSA and 2 ug / ml of goat anti-human IgG (Jackson ImmunoResearch, 109-005-190) in PBS for another hour at room temperature. The plates were then washed three times with wash buffer and DGL antibodies or controls, which were diluted with PBS and 0.1% BSA. The antibodies were incubated for one hour at room temperature and then the plates were washed three times with wash buffer. The plates were then incubated with mouse antihuman IgG Fc secondary - HRP (diluted with PBS / 0.1% BSA); 100 ul per well at 2 ug / ml and incubated at room temperature for one hour. The plate was washed three times in wash buffer and then 100 ul TMB (R&D Systems, DY9998B, substrate reagent pack). After the wells turn blue, 50 ul of stop solution (R&D Systems, DY994) was added to each well and the absorbance of the plate was read at 450nm. results can be found in Table 7.

[0406] Table 7. Binding of optimized ALK1 and BMPRII bispecific antibodies.

[0407] Example 6. DiscoverX data for variants

[0408] The bispecific antibodies were screened for agonist activity as described in Example

[0409] 2. Data reported (RLU) is the average of two replicates at the highest concentration tested. Antibodies were compared to the natural ligand, BMP-9 on every plate.

[0410] Table 8. Agonist activity of exemplary bispecific antibodies

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[0413] Example 7. Engineering of scFv containing bispecific agonist antibodies with optimized hinges

[0414] Agonist activity of heteromeric antibodies with modified hinges identified by 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. Heteromeric antibodies were tested using the DiscoverX assay. DGL809 outperformed the parental DGL288, as seen in Table 9 (average values across two different experiment is shown), which shows the activity level relative to BMP9 at 1000 nM antibody concentration.

[0415] Table 9. Agonist activity of exemplary bispecific antibodies

[0416] Example 8. Engineering bispecific agonist antibodies with optimized linkers in DVD-lg format

[0417] An alternative way to rigidity agonist antibodies is to optimize the linkers between IgG and additional variable domains in the DVD-lg format. To pursue this route, the agonist activity of heteromeric antibodies with modified VH to IgG hinge linkers identified by the DIAGONAL platform was tested. Variants of DGL292, designated DGL810, DGL811, and DGL812, were designed, expressed, and purified as described above. Heteromeric antibodies were tested using the DiscoverX assay where variants outperformed the parental DGL292, as seen in Table 11 (average values across two different experiment is shown).

[0418] Table 10. Linkers used in DVD-lg format

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[0421] Table 11. Agonist activity in DVD-lg format

[0422] Example 9. ALK1 / BMPRII bispecific antibodies in models of ocular pathologies associated with vascular dysfunction

[0423] The activity of a ALK1 I BMPRII bispecific antibody in models of ocular pathologies associated with vascular dysfunction will be evaluated.

[0424] The first aim will evaluate how a ALK1 I BMPRII bispecific antibody affects choroidal neovascularization (CNV) in a clinically relevant model of laser-induced CNV. Twelve-week old C57 / BI6 mice will be subjected to laser impact, followed by three subcutaneous administrations of a ALK1 I BMPRII bispecific antibody one, three and five days after laser impact. OCT imaging will be performed to visualize CNV lesions and retinal thickness prior to sacrifice, 14 days after laser impact. Choroidal neovessels will be detected by staining choroidsclera whole-mounts with lsoB4 (blood vessels) and extent of CNV will be detected by image analysis using imaged. A group of mice receiving intraocular delivery of Eylea following laser- CNV will be used as positive control.

[0425] In the second aim, the potential effects of a ALK1 I BMPRII bispecific antibody in a model of oxygen-induced retinopathy (OIR), a pre-clinical model which reproduces some of pathological vascular features of retinopathy of prematurity and diabetic retinopathy, will be evaluated. Briefly, dams and their litters (P7) will be placed in oxygen chambers (75% oxygen) for 5 days. After return to room air (P12), pups will receive subcutaneous injections of the ALK1 I BMPRII bispecific antibody on 3 consecutive days (P12-P15), and retinas will be harvested at P17. Retinas will be stained with lsoB4 and neovascularization (vascular tufts) will be quantified using image.

[0426] Additional exemplary ALK1 I BMPRII bispecific antibodies for use in the methods described herein

[0427] The following exemplary ALK1 I BMPRII bispecific antibody sequences may be used in the method of treating or preventing an ocular disease in a subject as described herein. The

[0428] 68

[0429] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC following antibodies are further described in WO2024211796, incorporated herein by reference.

[0430] Table 12. Sequences

[0431] 69

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[0434] 81386151. v1 Attorney Docket No. 769472: DGT9-010PC

[0435] Table 13. Sequences - CDRs

[0436] 71

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[0438] Table 14. Sequences - VH / VL

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[0442] 81386151. v1

Claims

Attorney Docket No. 769472: DGT9-010PCCLAIMS1. A method of treating or preventing an ocular disease in a subject, the method comprising administering to the subject a multispecific binding protein comprising at least a first binding moiety which binds specifically to human ALK1 and at least a second binding moiety which binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB, thereby treating or preventing the ocular disease in the subject.

2. The method of claim 1 , wherein the ocular disease is selected from the group consisting of age-related macular degeneration (AMD), retinitis pigmentosa (RP), diabetic retinopathy, diabetic macular edema (DME), and hypertensive retinopathy.

3. The method of claim 2, wherein the AMD is wet AMD.

4. The method of any one of claims 1-3, wherein the multispecific binding protein reduces neovascularization in the subject.

5. The method of any one of claims 1-4, wherein the multispecific binding protein reduces vascular lesion volume.

6. The method of any one of claims 1-5, wherein the multispecific binding protein is capable of inducing signaling by inducing proximity between ALK1 and BMPRII, ActRIIA, or ActRIIB.

7. The method of any one of claims 1-6, wherein the multispecific binding protein is capable of inducing SMAD1 / 5 phosphorylation.

8. The method of any one of claims 1-7, wherein the multispecific binding protein comprises at least one modified hinge region.

9. The method of claim 8, wherein the first modified hinge region comprises: a. an upper hinge region of up to 7 amino acids in length or is absent; and b. a lower hinge region, wherein the lower hinge region is linked to the N-terminus of a first constant region.7481386151. v1Attorney Docket No. 769472: DGT9-010PC10. The method of claim 8 or 9, the multispecific binding protein further comprises a second modified hinge region linked to the N-terminus of a second constant region.

11. The method of claim 10, wherein the second modified hinge region comprises a. an upper hinge region of up to 7 amino acids in length or is absent; and b. a lower hinge region, wherein the lower hinge region is linked to the N-terminus of the second constant region.

12. The method of claim 10 or 11 , wherein the upper hinge region of the first and the second modified hinge region are the same sequence.

13. The method of claim 10 or 11 , wherein the upper hinge region of the first and the second modified hinge regions are different sequences.

14. The method of any one of claims 9-13, wherein the upper hinge region comprises an amino acid sequence derived from an upper hinge region of a human IgG antibody.

15. The method of claim 14, wherein the IgG antibody is selected from lgG1, lgG2, lgG3, and lgG4.

16. The method of claim 14 or 15, wherein the IgG antibody is I gG 1.

17. The method of any one of claims 9-16, wherein the upper hinge region comprises an amino acid sequence of SEQ ID NO: 2.

18. The method of any one of claims 9-16, wherein the upper hinge region comprises an amino acid sequence of SEQ ID NO: 5.

19. The method of claim 14 or 15, wherein the IgG antibody is lgG4.

20. The method of any one of claims 9-15, wherein the upper hinge region comprises an amino acid sequence of SEQ ID NO: 4.

21. The method of any one of claims 9-13, wherein the upper hinge is absent.7581386151. v1Attorney Docket No. 769472: DGT9-010PC22. The method of any one of claims 1-21 , wherein the binding protein is multivalent.

23. The method of any one of claims 1-22, wherein the binding protein is tetravalent.

24. The method of any one of claims 1-23, wherein the first heavy chain constant region and / or the second heavy chain constant region comprise a human lgG1, lgG2, lgG3, or lgG4.

25. The method of any one of claims 1-24, wherein the first heavy chain constant region and / or the second heavy chain constant region comprise an amino acid sequence of SEQ ID NO: 10.

26. The method of any one of claims 1-25, wherein at least one heavy chain constant region comprises a substitution at amino acid position 234, according to Ell numbering.

27. The method of claim 26, wherein the substitution at amino acid position 234 is an alanine (A).

28. The method of any one of claims 1-27, wherein at least one heavy chain constant region comprises a substitution at amino acid position 235, according to Ell numbering.

29. The method of claim 28, wherein the substitution at amino acid position 235 is an alanine (A).

30. The method of any one of claims 1-29, wherein at least one heavy chain constant region comprises a substitution at amino acid position 237 according to Ell numbering.

31. The method of claim 30, wherein the substitution at amino acid position 237 is an alanine (A).

32. The method of any one of claims 1-31 , wherein at least one heavy chain constant region comprises one or more substitutions at amino acid positions 234, 235, or 237, according to Ell numbering.

33. The method of claim 32,7681386151. v1Attorney Docket No. 769472: DGT9-010PC wherein the substitution at amino acid position 234 is an alanine (A), wherein the substitution at amino acid position 235 is an alanine (A), and wherein the substitution at amino acid position 237 is an alanine (A).

34. The method of any of the previous claims, wherein the heavy chain constant region comprises heterodimerization mutations to promote heterodimerization of the first binding moiety with the second binding moiety.

35. The method of claim 34, wherein the heterodimerization mutations are Knob-in-Hole (KIH) mutations.

36. The method of claim 35, wherein the first heavy chain constant region comprises an amino acid substitution at position 366, 368, or 407 which produced a hole, and the second heavy chain constant region comprises an amino acid substitution at position 366 which produce a knob.

37. The method of claim 36, wherein the first heavy chain constant region comprises the amino acid substitution T366S, L368A, or Y407V, and the second heavy chain constant region comprises the amino acid substitution T366W.

38. The method of claim 34, wherein the heterodimerization mutations are charge stabilization mutations.

39. The method of claim 38, 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.

40. The method of claim 38 or 39, 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.

41. The method of claim 33, wherein the heterodimerization mutations comprise an engineered disulfide bond.7781386151. v1Attorney Docket No. 769472: DGT9-010PC42. The method of claim 41, wherein the engineered disulfide bond is formed by a first heavy chain constant region comprising the amino acid substitution Y349C, and a second heavy chain constant region comprising the amino acid substitution S354C.

43. The method of claim 42, wherein the engineered disulfide bond is formed by a C- terminal extension peptide fused to the C-terminus of each of the first heavy chain constant region and the second heavy chain constant region.

44. The method of claim 43, wherein the first heavy chain constant region C-terminal extension comprises the amino acid sequence GEC, and the second heavy chain constant region C-terminal extension comprises the amino acid sequence SCDKT.

45. The method of any one of the previous claims, wherein at least one heavy chain constant region comprises one or more mutations to promote increased half-life.

46. The method of claim 45, wherein at least one heavy chain constant region comprises one or more substitutions at amino acid positions 252, 254, or 256, according to Ell numbering.

47. The method of claim 46, wherein the substitution at amino acid position 252 is a tyrosine (Y), wherein the substitution at amino acid position 254 is a threonine (T), and wherein the substitution at amino acid position 256 is a glutamic acid (E).

48. The method of claim 45, wherein at least one heavy chain constant region comprises one or more substitutions at amino acid positions 428 or 434, according to Ell numbering.

49. The method of claim 48, wherein the substitution at amino acid position 428 is a leucine (L), and wherein the substitution at amino acid position 434 is a serine (S).

50. The method of any one of claims 1-49, wherein the first binding moiety that binds specifically to human ALK1 is selected from a single chain Fv (scFv), VHH, Fab, F(ab’)2, or a single domain antibody.7881386151. v1Attorney Docket No. 769472: DGT9-010PC51. The method of any one of claims 1-50, wherein the second binding moiety that binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB is selected from a single chain Fv (scFv), VHH, Fab, F(ab’)2, or a single domain antibody.

52. The method of any one of claims 1-51 , the multispecific 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; or aiv) a first polypeptide chain comprising a first antigen binding domain, a second antigen binding domain, a first modified hinge region, and a first constant region; and biv) a second polypeptide chain comprising a third antigen binding domain, a fourth antigen binding domain, a second modified hinge region, and a second constant region.

53. The method of claim 52, wherein the first antigen binding domain, second antigen binding domain, third antigen binding domain, and fourth antigen binding domain comprise an scFv.

54. The method of any one of claims 1-53, wherein:(a) the first binding moiety comprises an VHH domain and the second moiety comprises a VHH domain;(b) the first binding moiety comprises a Fab domain and the second binding moiety comprises a VHH domain;(c) the first binding moiety comprises a VHH domain and the second binding moiety comprises a Fab domain;7981386151. v1Attorney Docket No. 769472: DGT9-010PC(d) the first binding moiety comprises a Fab domain and the second binding moiety comprises a Fab domain;(e) the first binding moiety comprises a Fab domain and the second binding moiety comprises an scFv;(f) the first binding moiety comprises a scFv and the second binding moiety comprises a Fab domain;(g) the first binding moiety comprises a scFv and the second binding moiety comprises a scFv;(h) the first binding moiety comprises a scFv and the second binding moiety comprises a VHH; or(i) the first binding moiety comprises a VHH and the second binding moiety comprises a scFv.

55. The method of any one of claims 1-54, wherein the first and / or the second antibody binding domain is truncated at the C-terminal end adjacent to the upper hinge domain.

56. The method of claim 55, wherein the C-terminal end adjacent to the upper hinge domain is truncated by at least one residue.

57. The method of claim 55 or 56, wherein the C-terminal end adjacent to the upper hinge domain is truncated by at least two residues.

58. The method of any one of claims 1-57, the multispecific binding protein comprises a first and a second polypeptide chain, wherein: said first polypeptide chain comprisesVH1-(HX1)n-VH2-C-(HX2)n, wherein: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 said second polypeptide chain comprises8081386151. v1Attorney Docket No. 769472: DGT9-010PCVL1-(LX1)n-VL2-C-(LX2)n, wherein: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 comprise an Fc region; and n is independently 0 or 1.

59. The method of claim 58, wherein VH1 binds specifically to human ALK1 and VH2 binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB.

60. The method of claim 58 or 59, wherein VL1 binds specifically to human ALK1 and VL2 binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB.

61. The method of claim 58, wherein VH1 binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB and VH2 binds specifically to human ALK1.

62. The method of claim 58, wherein VL1 binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB and VL2 binds specifically to human ALK1.

63. The method of any one of claims 58-62, wherein linker HX1 comprises an amino acid sequence of PLAP or PAPNLLGGP.

64. The method of any one of claims 58-63, wherein linker LX1 comprises an amino acid sequence of PLAP or PAPNLLGGP.

65. The method of any one of claims 58-64, wherein linker HX1 comprises an amino acid sequence of PLAP and linker LX1 comprises an amino acid sequence of PLAP or PAPNLLGGP.

66. The method of any one of claims 1-65, wherein linker HX1 comprises an amino acid sequence of PAPNLLGGP and linker LX1 comprises an amino acid sequence of PLAP.8181386151. v1Attorney Docket No. 769472: DGT9-010PC67. The method of any one of claims 1-8, wherein the multispecific binding protein comprises at least a first polypeptide chain, wherein: said 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; and the VH1 binds specifically to ALK1 and the VH2 binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB.

68. The method of claim 67, wherein one or both of VH1 and VH2 are VH domains or VHH domains.

69. The method of claim 67 or 68, wherein the multispecific binding protein further comprises a second polypeptide chain, wherein said second polypeptide chain comprises a first variable light chain domain (VL1) linked to a second variable light chain domain (VL2), and wherein VL1 binds specifically to ALK1 and the VL2 binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB.

70. The method of claim 69, wherein the VL1 is linked to the VL2 via at least one modified hinge region.

71. The method of any one of claims 67-70, wherein one or both of VH 1 and VH2 is truncated at the C-terminal end.

72. The method of claim 71 , wherein the C-terminal end is truncated by at least one residue.

73. The method of claim 71 or 72, wherein the C-terminal end is truncated by at least two residues.

74. The method of any one of claims 71-73, wherein the SS amino acid residues of the C- terminal end are deleted.

75. The method of any one of claims 67-74, wherein the multispecific binding protein comprises a first polypeptide chain of VH1-HX1-VH2-C-Fc, wherein:VH1 is a first heavy chain variable domain;VH2 is a second heavy chain variable domain;8281386151. v1Attorney Docket No. 769472: DGT9-010PCC is a heavy chain constant domain;HX1 is a modified hinge region linker; andFc is an Fc region; and a second polypeptide chain of VL1-LX1-VL2-C, wherein:VL1 is a first light chain variable domain;VL2 is a second light chain variable domain;C is a light chain constant domain; andLX1 is a modified hinge region linker.

76. The method of any one of claims 67-75, wherein the modified hinge region comprises or consists of an amino acid sequence of PLAP or PAPNLLGGP.

77. The method of any one of claims 1-76, wherein the multispecific binding protein is administered intraocularly to the subject.

78. The method of any one of claims 1-76, wherein the multispecific binding protein is administered intravenously to the subject.

79. The method of any one of claims 1-78, wherein the multispecific binding protein is administered subcutaneously to the subject.81386151. v1

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