Bispecific agonistic antibodies to activin a receptor like type 1 (ALK1)

HK40138673APending Publication Date: 2026-10-09DIAGONAL THERAPEUTICS INC
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Patent Information

Application Number
HK62026126552
Authority / Receiving Office
HK · HK
Patent Type
Applications
Current Assignee / Owner
Priority Date
2023-11-07
Filing Date
2026-07-23
Publication Date
2026-10-09
Estimated Expiration
2044-04-04

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Abstract

Provided herein are bispecific agonistic antibodies that bind to ALK1, BMPR2, ActRIIA, and / or ActRIIB, and methods of using the same.
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Description

Abstract This article provides bispecific agonistic antibodies that bind to ALK1, BMPR2, ActRIIA and / or ActRIIB, and their methods of use.

Claims

CLAIMS1 . A multispecific binding protein comprising 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, wherein: the VH1 binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB and the VH2 binds specifically to ALK1 ; or the VH1 binds specifically to ALK1 and the VH2 binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB.

2. The multispecific binding protein of claim 1, wherein one or both of VH1 and VH2 are VH domains or VHH domains.

3. The multispecific binding protein of claim 1 or 2, further comprising 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), wherein: the VL1 binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB and the VL2 binds specifically to a ALK1 ; or the VL1 binds specifically to ALK1 and the VL2 binds specifically to a target selected from BMPRII, ActRIIA, and ActRIIB.

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

5. The multispecific binding protein of any one of claims 1-4, wherein one or both of VH1 and VH2 is truncated at the C-terminal end.

6. The multispecific binding protein of claim 5, wherein the C-terminal end is truncated by at least one residue.

7. The multispecific binding protein of claim 5 or 6, wherein the C-terminal end is truncated by at least two residues.

8. The multispecific binding protein of any one of claims 5-7, wherein the SS amino acid residues of the C-terminal end are deleted.

9. The multispecific binding protein of any one of claims 1-8, comprising 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; 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.

10. The multispecific binding protein of any one of claims 1-9, wherein the modified hinge region comprises; i) an upper hinge region of up to 7 amino acids in length or is absent; and ii) a lower hinge region.

11. The multispecific binding protein of any one of claims 1-10, wherein the modified hinge region comprises or consists of an amino acid sequence of PLAP or PAPNLLGGP.

12. The multispecific binding protein of any one of claims 1-11 , wherein: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; orB) 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; orC) 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.

13. The multispecific binding protein of any one of claims 1-12, wherein: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; orB) 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; orC) 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.

14. The multispecific binding protein of any one of claims 1-13, wherein:A) the VH binding to ALK1 comprises an amino acid sequence of EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVANINQDGSEKN YVDSMRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREFDYWGQGTLVTVSS, or an amino acid sequence with at least 90% identity thereto; and the VL binding to ALK1 comprises an amino acid sequence of QSVLAQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYGNNKRPSGVPD RFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGRVFGGGTKLTVL, or an amino acid sequence with at least 90% identity thereto; orB) the VH binding to ALK1 comprises an amino acid sequence of EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINQDGSEKY YVDSMRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREYDYWGQGTLVTVSS, or an amino acid sequence with at least 90% identity thereto; and the VL binding to ALK1 comprises an amino acid sequence ofQSVLAQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYGNNKRPSGVPD RFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGRVFGGGTKLTVL, or an amino acid sequence with at least 90% identity thereto; orC) the VH binding to ALK1 comprises an amino acid sequence ofEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKN YVDSMRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREFDFWGQGTLVTVSS, or an amino acid sequence with at least 90% identity thereto; and the VL binding to ALK1 comprises an amino acid sequence ofQSVLAQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYGNNKRPSGVPD RFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGRVFGGGTKLTVL, or an amino acid sequence with at least 90% identity thereto.

15. The multispecific binding protein of any one of claims 1-14, wherein:A) the VH binding to BMPRII comprises an amino acid sequence of 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 ofQSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYRSNQRPSGVP DRFSGSKSGTSASLAISGLRSEDEADYYCSSYAGNYNLVFGGGTKLTVL, or an amino acid sequence with at least 90% identity thereto; orB) the VH binding to BMPRII comprises an amino acid sequence ofEVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMNWIRQAPGKGLEWVSSISGGSTYYAD SVKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDFGVAGWFGQFGMDVWGQGTLVT VSS, or an amino acid sequence with at least 90% identity thereto; and the VL binding to BMPRII comprises an amino acid sequence ofQSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYRSNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCSSYAGNYNLVFGGGTKLTVL, or an amino acid sequence with at least 90% identity thereto; orC) the VH binding to BMPRII comprises an amino acid sequence of 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 ofQSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYRSNQRPSGVP DRFSGSKSGTSASLAISGLRSEDEADYYCSSYAGNYNLVFGGGTKLTVL, or an amino acid sequence with at least 90% identity thereto.

16. The multispecific binding protein of any one of claims 3-15, wherein the first polypeptide chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 136-142, and the second polypeptide chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 143-146.

17. The multispecific binding protein of any one of claims 3-15, wherein 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.

18. The multispecific binding protein of any one of claims 3-15, wherein 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.

19. The multispecific binding protein of any one of claims 3-15, wherein 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.

20. The multispecific binding protein of any one of claims 3-15, wherein 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.

21. The multispecific binding protein of any one of claims 3-15, wherein 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.

22. The multispecific binding protein of any one of claims 3-15, wherein the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 142, and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 146.

23. The multispecific binding protein of any one of claims 3-15, wherein the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 68, and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 69.

24. The multispecific binding protein of any one of claims 3-15, wherein the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 70, and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 71.

25. The multispecific binding protein of any one of claims 3-15, wherein the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 72, and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 73.

26. The multispecific binding protein of any one of claims 3-15, wherein the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 74, and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 75.

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

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

29. The multispecific binding protein of 27 or 28, wherein the scFv binding to ALK1 comprises:A):a VH domain comprising an HCDR1 amino acid sequence of SYAMS, an HCDR2 amino acid sequence of NINQDGSEKNYVDSMRG, and an HCDR3 amino acid sequence of EFDY; and a 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; orB): a VH domain comprising an an HCDR1 amino acid sequence of SYWMS, an HCDR2 amino acid sequence of NINQDGSEKYYVDSMRG, and an HCDR3 amino acid sequence of EYDY; and a VL domain comprising an an LCDR1 amino acid sequence of SGSSSNIGSNYVY, an LCDR2 amino acid sequence of GNNKRPS, and an LCDR3 amino acid sequence of AAWDDSLNGRV; orC): a VH domain comprising an HCDR1 amino acid sequence of SYWMS, an HCDR2 amino acid sequence of NIKQDGSEKNYVDSMRG, and an HCDR3 amino acid sequence of EFDF; and a VL domain comprising an an LCDR1 amino acid sequence of SGSSSNIGSNYVY, an LCDR2 amino acid sequence of GNNKRPS, and an LCDR3 amino acid sequence of AAWDDSLNGRV.

30. The multispecific binding protein of any one of claims 27-29, wherein the scFv binding to BMPRII comprises:A): a VH domain comprising an HCDR1 amino acid sequence of DYYMT, an HCDR2 amino acid sequence of SISGGSTYYADSRKG, and an HCDR3 amino acid sequence of DFGVAGWFGQYGMDV; and a VL domain comprising an LCDR1 amino acid sequence of TGSSSNIGAGYDVH, an LCDR2 amino acid sequence of RSNQRPS, and an LCDR3 amino acid sequence of SSYAGNYNLV; orB):a VH domain comprising an HCDR1 amino acid sequence of DYYMN, an HCDR2 amino acid sequence of SISGGSTYYADSVKG, and an HCDR3 amino acid sequence of DFGVAGWFGQFGMDV; and a VL domain comprising an LCDR1 amino acid sequence of TGSSSNIGAGYDVH, an LCDR2 amino acid sequence of RSNQRPS, and an LCDR3 amino acid sequence of SSYAGNYNLV; orC): a VH domain comprising an HCDR1 amino acid sequence of DYYMN, an HCDR2 amino acid sequence of SISGGSTYYADSVKG, and an HCDR3 amino acid sequence of DFGVAGWFGYYGMDV; and a VL domain comprising an LCDR1 amino acid sequence of TGSSSNIGAGYDVH, an LCDR2 amino acid sequence of RSNQRPS, and an LCDR3 amino acid sequence of SSYAGNYNLV.

31. The multispecific binding protein of any one of claims 27-30, wherein the scFv binding to ALK1 comprises: a VH domain comprising an amino acid sequence ofEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVANINQDGSEKN YVDSMRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREFDYWGQGTLVTVSS, or an amino acid sequence with at least 90% identity thereto; and a VL domain comprising an amino acid sequence ofQSVLAQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYGNNKRPSGVPD RFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGRVFGGGTKLTVL, or an amino acid sequence with at least 90% identity thereto.

32. The multispecific binding protein of any one of claims 27-31 , wherein the scFv binding to BMPRII comprises: a VH domain comprising an amino acid sequence ofEVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMTWIRQAPGKGLEWVSSISGGSTYYAD SRKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDFGVAGWFGQYGMDVWGQGTLVT VSS, or an amino acid sequence with at least 90% identity thereto; and a VL domain comprising an amino acid sequence ofQSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYRSNQRPSGVP DRFSGSKSGTSASLAISGLRSEDEADYYCSSYAGNYNLVFGGGTKLTVL, or an amino acid sequence with at least 90% identity thereto.

33. The multispecific binding protein of any one of claims 27-30, wherein the scFv binding to ALK1 comprises an amino acid sequence of SEQ ID NO: 120, or an amino acid sequence with at least 90% identity thereto.

34. The multispecific binding protein of any one of claims 27-30, wherein the scFv binding to ALK1 comprises an amino acid sequence of SEQ ID NO: 122, or an amino acid sequence with at least 90% identity thereto.

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

36. The multispecific binding protein of any one of claims 27-30, wherein the scFv binding to ALK1 comprises an amino acid sequence of SEQ ID NO: 123, or an amino acid sequence with at least 90% identity thereto.

37. The multispecific binding protein of any one of claims 27-30, wherein the first and second polypeptide chain each comprise an amino acid sequence of any one of SEQ ID Nos: 60-63.

38. The multispecific binding protein of any one of claims 1-37, wherein the multispecific binding protein is capable of inducing signaling by inducing proximity between ALK1 and BMPRII, ActRIIA, or ActRIIB.

39. The multispecific binding protein of any one of claims 1-38, wherein the multispecific binding protein has greater agonist activity compared to a multispecific binding protein that lacks at least one modified hinge region.

40. The multispecific binding protein of any one of claims 1-39, wherein the multispecific binding protein induces at least about 35% of the activity of BMP9.

41. The multispecific binding protein of claim 40, wherein the activity of BMP9 is determined by measuring phosphorylated SMAD1 (pSMADI) levels in cells incubated with the multispecific binding protein and / or in cells incubated with BMP9.

42. The multispecific binding protein of any one of claims 1-41 , wherein the melting temperature onset of unfolding (Tonset) of the multispecific binding protein is at least about 55 °C.

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

44. The multispecific binding protein of claim 42 or 43, wherein the Tonset and Tm of the multispecific binding protein is determined by differential scanning calorimetry (DSC).

45. The multispecific binding protein of any one of claims 1-44, capable of stimulating expression of ID1 in a cell.

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

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

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

49. The multispecific binding protein of claim 48, wherein the substitution at amino acid position 234 is an alanine (A).

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

51. The multispecific binding protein of claim 50, wherein the substitution at amino acid position 235 is an alanine (A).

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

53. The multispecific binding protein of claim 52, wherein the substitution at amino acid position 237 is an alanine (A).

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

55. The multispecific binding protein of claim 54, 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).

56. The multispecific binding protein 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.

57. The multispecific binding protein of claim 56, wherein the heterodimerization mutations are Knob-in-Hole (KIH) mutations.

58. The multispecific binding protein of claim 57, 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.

59. The multispecific binding protein of claim 58, 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.

60. The multispecific binding protein of claim 56, wherein the heterodimerization mutations are charge stabilization mutations.

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

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

63. The multispecific binding protein of claim 56, wherein the heterodimerization mutations comprise an engineered disulfide bond.

64. The multispecific binding protein of claim 63, 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.

65. The multispecific binding protein of claim 63 or 64, 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.

66. The multispecific binding protein of claim 65, 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.

67. The multispecific binding protein 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.

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

69. The multispecific binding protein of claim 68, 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).

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

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

72. A pharmaceutical composition comprising the multispecific binding protein of any one of the preceding claims and a pharmaceutically acceptable carrier.

73. An isolated nucleic acid molecule encoding the multispecific binding protein of any one of claims 1-71.

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

75. A host cell comprising the expression vector of claim 74.

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

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

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

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

80. The multispecific binding protein according to any one of claims 1-71 , for use as a medicament.

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

82. The method of any one of claims 76-81 , wherein the multispecific binding protein is capable of inducing signaling by inducing proximity between ALK1 and BMPRII, ActRIIA, or ActRIIB.

83. The method of any one of claims 76-82, wherein the multispecific binding protein has greater agonist activity compared to a multispecific binding protein that lacks at least one modified hinge region.

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

85. The method of claim 84, wherein the activity of BMP9 is determined by measuring phosphorylated SMAD1 (pSMADI) levels in cells incubated with the multispecific binding protein and / or in cells incubated with BMP9.