Treatment of fibrodysplasia ossificans progressiva

Abstract

To provide treatment of fibrodysplasia ossificans progressiva.SOLUTION: Methods for treating fibrodysplasia ossificans progressiva (FOP) in a human subject are provided. Such methods involve administering to a subject having FOP a therapeutically effective amount of an activin A antagonist, such as an antibody to activin A. The present disclosure provides methods of treating fibrodysplasia ossificans progressiva (FOP) comprising administering to a subject having FOP a therapeutically effective amount of an activin A antagonist. In particular, the inventors of the present application have surprisingly discovered only after conducting a Phase II clinical trial in humans that treating FOP subjects with an activin A antagonist dramatically reduces and / or prevents the progression of new heterotopic ossification (HO) bone growth and reduces the average rate of lesion growth and mineralization.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 958,448, filed January 8, 2020, U.S. Provisional Application No. 63 / 076,691, filed September 10, 2020, and U.S. Provisional Application No. 63 / 081,428, filed September 22, 2020, the entire contents of each of which are expressly incorporated herein by reference in their entirety. [Background technology]

[0002] Fibrodysplasia ossificans progressiva (FOP) is a condition characterized by Munchmeyer's disease. FOP, also known as heterotopic ossification disease, is an autosomal dominant disorder characterized by early-onset, recurrent, and progressive ossification of skeletal muscle and associated connective tissues. In FOP subjects, bone forms in soft tissues outside of the normal skeleton, a process known as heterotopic ossification (HO), which can lead to the development of secondary skeletal structures and gradually limit the patient's mobility. Elimination of new bone formation has been shown to be ineffective and leads to the progression of further new bone growth.

[0003] FOP is caused by mutations in the intracellular domain of ACVR1 (ALK2), which result in a change of most arginine 206 to histidine (R206H) (Pignolo, RJ et al. 2011, Orphanet J. Rare Dis. 6:80). ACVR1 is a type I receptor for bone morphogenetic proteins (BMPs). The R206H mutation is thought to specifically increase the receptor's sensitivity to activation and further enhance its resistance to silencing. Certain types of medications have been used to relieve the pain and swelling associated with FOP during flare-ups, but there is currently no known effective medical treatment for FOP. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Pignolo,RJet al.2011,Orphanet J.Rare Dis.6:80 Summary of the Invention [Means for solving the problem]

[0005] The present disclosure provides a method for treating fibrodysplasia ossificans progressiva (FOP), comprising administering a therapeutically effective amount of an activin A antagonist to a subject with FOP. In particular, the inventors of the present application surprisingly discovered, only after conducting a Phase II clinical trial in humans, that treating FOP subjects with an activin A antagonist dramatically reduces and / or prevents the progression of new heterotopic ossification (HO) bone growth and reduces the average rate of lesion growth and mineralization.

[0006] In one aspect, the present disclosure provides a method of treating fibrodysplasia ossificans progressiva (FOP), the method comprising administering to a human subject having FOP a therapeutically effective amount of an activin A antagonist, thereby treating FOP.

[0007] In some embodiments, the activin A antagonist is an anti-activin A antibody, or an antigen-binding fragment thereof. In some embodiments, the antibody competes for binding with an antibody comprising the heavy chain variable region and the light chain variable region of the antibody designated H4H10446P, H4H10430P, or A1. In some embodiments, the antibody comprises the heavy chain variable region and the light chain variable region of the antibody designated H4H10446P, H4H10430P, or A1. In some embodiments, the antibody is a chimeric antibody, a veneered antibody, a humanized antibody, or a human antibody. In some embodiments, the antibody is an intact antibody. In some embodiments, the antibody is a human kappa IgG1 antibody. In some embodiments, the antibody is administered in combination with an ACVR1, ACVR2A, or ACVR2B extracellular domain-Fc fusion protein.

[0008] The present disclosure further provides an activin A antagonist for use in a method for treating fibrodysplasia ossificans progressiva (FOP), the method comprising administering a therapeutically effective amount of the activin A antagonist to a subject with FOP. Optionally, the activin A antagonist is an anti-activin A antibody, or an antigen-binding fragment thereof. Optionally, the disclosure provides use of an anti-activin A antibody, or an antigen-binding fragment thereof, in the manufacture of a medicament for treating FOP. Optionally, the antibody is a chimeric, veneered, humanized, or human antibody. Optionally, the antibody is an intact antibody. Optionally, the antibody is a human kappa IgG1 antibody. Optionally, the antibody is administered in combination therapy with an ACVR1, ACVR2A, or ACVR2B extracellular domain-Fc fusion protein.

[0009] In one aspect, the present disclosure provides a method for reducing the formation of new heterotopic ossification lesions in a human subject having FOP, the method comprising administering to the human subject a therapeutically effective amount of an activin A antagonist, thereby reducing the formation of new heterotopic ossification lesions in the human subject.

[0010] In one embodiment, the formation of new heterotopic ossification lesions is prevented in a human subject.

[0011] In one embodiment, the human subject exhibits at least a 5%, at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, at least a 5% to 90%, at least a 10% to 90%, at least a 20% to 90%, at least a 30% to 90%, at least a 40% to 90%, at least a 50% to 90%, at least a 60% to 90%, at least a 70% to 90%, at least a 80% to 90%, at least a 5% to 80%, at least a 5% to 70%, at least a 5% to 60%, at least a 5% to 50%, at least a 5% to 40%, at least a 5% to 30%, at least a 5% to 20%, or at least a 5% to 10% reduction in the number of new heterotopic ossification lesions compared to a control.

[0012] In one embodiment, the human subject exhibits at least a 5%, at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 40%, at least a 50%, at least a 5% to 50%, at least a 10% to 50%, at least a 20% to 50%, at least a 30% to 50%, at least a 40% to 50%, at least a 5% to 40%, at least a 5% to 30%, at least a 5% to 20%, or at least a 5% to 10% reduction in the volume of new heterotopic ossification lesions compared to a control.

[0013] In one embodiment, the human subject exhibits at least a 5%, at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 5% to 50%, at least a 10% to 50%, at least a 20% to 50%, at least a 30% to 50%, at least a 40% to 50%, at least a 5% to 40%, at least a 5% to 30%, at least a 5% to 20%, or at least a 5% to 10% decrease in the rate of growth and mineralization of new heterotopic ossification lesions compared to a control.

[0014] In one embodiment, the human subject exhibits at least a 5%, at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 5% to 50%, at least a 10% to 50%, at least a 20% to 50%, at least a 30% to 50%, at least a 40% to 50%, at least a 5% to 40%, at least a 5% to 30%, at least a 5% to 20%, or at least a 5% to 10% decrease in the intensity of new heterotopic ossification lesions compared to a control.

[0015] In one embodiment, the human subject exhibits at least a 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 5% to 80%, at least 10% to 80%, at least 20% to 80%, at least 30% to 80%, at least 40% to 80%, at least 50% to 80%, at least 60% to 80%, at least 70% to 80%, at least 5% to 70%, at least 5% to 60%, at least 5% to 50%, at least 5% to 40%, at least 5% to 30%, at least 5% to 20%, or at least 5% to 10% reduction in total lesion activity (TLA) of heterotopic ossification lesions compared to a control.

[0016] In one embodiment, the human subject exhibits about a 0.2-fold, 0.5-fold, 1-fold, 1.5-fold, 2-fold, 3-fold, 0.2-3-fold, 0.5-3-fold, 1-3-fold, 1.5-3-fold, 2-3-fold, 2.5-3-fold, 0.2-2.5-fold, 0.2-2-fold, 0.2-1.5-fold, 0.2-1-fold, or 0.2-0.5-fold decrease in average daily Pain-NRS compared to a control.

[0017] In one embodiment, the control is a mean measurement or value collected from a population of human subjects with FOP who have not received an activin A antagonist.

[0018] In one embodiment, a therapeutically effective amount of an activin A antagonist reduces the occurrence of painful flare-ups in a human subject compared to a control.

[0019] In one embodiment, the new heterotopic ossification lesions are analyzed by positron emission tomography (PET) scan, computed tomography (CT) scan, or a combination thereof. In one embodiment, the PET scan analysis is performed using a radiolabeled human subject. 18 Sodium fluoride ( 18 This is done by administering NaF (F-NaF).

[0020] In one embodiment, a therapeutically effective amount of an activin A antagonist is administered to a human subject for at least 8 weeks.

[0021] In one embodiment, the method further comprises selecting a subject with FOP who would benefit from reducing the formation of new heterotopic ossification lesions. In one embodiment, the subject who would benefit from reducing the formation of new heterotopic ossification lesions is about to undergo surgery.

[0022] In one embodiment, the human subject is about to undergo therapeutic treatment for FOP.

[0023] In one embodiment, the activin A antagonist does not reduce the number, volume, or size of any pre-existing lesions in a human subject.

[0024] In one embodiment, the activin A antagonist is a protein or a small molecule.

[0025] In one embodiment, the activin A antagonist is an anti-activin A antibody, or antigen-binding fragment thereof. In one embodiment, the anti-activin A antibody, or antigen-binding fragment thereof, is a chimeric, veneered, humanized, or human antibody, or antigen-binding fragment thereof.

[0026] In one embodiment, the anti-activin A antibody, or antigen-binding fragment thereof, is a human kappa IgG1 antibody.

[0027] In one embodiment, the anti-activin A antibody, or antigen-binding fragment thereof, comprises the following six CDR sequences: (a) an HCDR1 having at least about 80% identity to the sequence GGSFSSHF, (b) an HCDR2 having at least about 80% identity to the sequence ILYTGGT, (c) an HCDR3 having at least about 80% identity to the sequence ARARSGITFTGIIVPGSFDI, (d) an LCDR1 having at least about 80% identity to the sequence QSVSSSY, (e) an LCDR2 having at least about 80% identity to the sequence GAS, and (f) an LCDR3 having at least about 80% identity to the sequence QQYGSSPWT.

[0028] In one embodiment, the anti-activin A antibody, or antigen-binding fragment thereof, comprises the following six CDR sequences: (a) HCDR1 having the sequence GGSFSSHF, (b) HCDR2 having the sequence ILYTGGT, (c) HCDR3 having the sequence ARARSGITFTGIIVPGSFDI, (d) LCDR1 having the sequence QSVSSSY, (e) LCDR2 having the sequence GAS, and (f) LCDR3 having the sequence QQYGSSPWT.

[0029] In one embodiment, the anti-activin A antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having at least 90% identity to SEQ ID NO: 1 and a light chain variable region having at least 90% identity to SEQ ID NO: 5.

[0030] In one embodiment, the anti-activin A antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having at least 95% identity to SEQ ID NO: 1 and a light chain variable region having at least 95% identity to SEQ ID NO: 5.

[0031] In one embodiment, the anti-activin A antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region comprising SEQ ID NO:1 and a light chain variable region comprising SEQ ID NO:5.

[0032] In one embodiment, the anti-activin A antibody, or antigen-binding fragment thereof, comprises a heavy chain comprising SEQ ID NO:25 and a light chain comprising SEQ ID NO:26.

[0033] In one embodiment, the anti-activin A antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having at least 90% identity to SEQ ID NO: 1 and a light chain variable region having at least 90% identity to SEQ ID NO: 5.

[0034] In one embodiment, the anti-activin A antibody, or antigen-binding fragment thereof, competes for binding with an antibody comprising the following six CDR sequences: (a) HCDR1 having the sequence GGSFSSHF, (b) HCDR2 having the sequence ILYTGGT, (c) HCDR3 having the sequence ARARSGITFTGIIVPGSFDI, (d) LCDR1 having the sequence QSVSSSY, (e) LCDR2 having the sequence GAS, and (f) LCDR3 having the sequence QQYGSSPWT.

[0035] In one embodiment, the activin A antagonist is administered in combination with a second therapy.

[0036] In another aspect, the disclosure provides a method for preventing the formation of new heterotopic ossification lesions in a human subject having FOP, the method comprising administering to the human subject a therapeutically effective amount of an activin A antagonist, thereby preventing the formation of new heterotopic ossification lesions in the human subject.

[0037] In one embodiment, the human subject exhibits at least a 5%, at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, at least a 5% to 90%, at least a 10% to 90%, at least a 20% to 90%, at least a 30% to 90%, at least a 40% to 90%, at least a 50% to 90%, at least a 60% to 90%, at least a 70% to 90%, at least a 80% to 90%, at least a 5% to 80%, at least a 5% to 70%, at least a 5% to 60%, at least a 5% to 50%, at least a 5% to 40%, at least a 5% to 30%, at least a 5% to 20%, or at least a 5% to 10% reduction in the number of new heterotopic ossification lesions compared to a control.

[0038] In one embodiment, the human subject exhibits at least a 5%, at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 40%, at least a 50%, at least a 5% to 50%, at least a 10% to 50%, at least a 20% to 50%, at least a 30% to 50%, at least a 40% to 50%, at least a 5% to 40%, at least a 5% to 30%, at least a 5% to 20%, or at least a 5% to 10% reduction in the volume of new heterotopic ossification lesions compared to a control.

[0039] In one embodiment, the human subject exhibits at least a 5%, at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 5% to 50%, at least a 10% to 50%, at least a 20% to 50%, at least a 30% to 50%, at least a 40% to 50%, at least a 5% to 40%, at least a 5% to 30%, at least a 5% to 20%, or at least a 5% to 10% decrease in the rate of growth and mineralization of new heterotopic ossification lesions compared to a control.

[0040] In one embodiment, the human subject exhibits at least a 5%, at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 5% to 50%, at least a 10% to 50%, at least a 20% to 50%, at least a 30% to 50%, at least a 40% to 50%, at least a 5% to 40%, at least a 5% to 30%, at least a 5% to 20%, or at least a 5% to 10% decrease in the intensity of new heterotopic ossification lesions compared to a control.

[0041] In one embodiment, the human subject exhibits at least a 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 5% to 80%, at least 10% to 80%, at least 20% to 80%, at least 30% to 80%, at least 40% to 80%, at least 50% to 80%, at least 60% to 80%, at least 70% to 80%, at least 5% to 70%, at least 5% to 60%, at least 5% to 50%, at least 5% to 40%, at least 5% to 30%, at least 5% to 20%, or at least 5% to 10% reduction in total lesion activity (TLA) of heterotopic ossification lesions compared to a control.

[0042] In one embodiment, the human subject exhibits about a 0.2-fold, 0.5-fold, 1-fold, 1.5-fold, 2-fold, 3-fold, 0.2-3-fold, 0.5-3-fold, 1-3-fold, 1.5-3-fold, 2-3-fold, 2.5-3-fold, 0.2-2.5-fold, 0.2-2-fold, 0.2-1.5-fold, 0.2-1-fold, or 0.2-0.5-fold decrease in average daily Pain-NRS compared to a control.

[0043] In one embodiment, the control is a mean measurement or value collected from a population of human subjects with FOP who have not received an activin A antagonist.

[0044] In one embodiment, a therapeutically effective amount of an activin A antagonist reduces the occurrence of painful flare-ups in a human subject compared to a control.

[0045] In one embodiment, the new heterotopic ossification lesions are analyzed by positron emission tomography (PET) scan, computed tomography (CT) scan, or a combination thereof. In one embodiment, the PET scan analysis is performed using a radiolabeled human subject. 18 Sodium fluoride ( 18 This is done by administering NaF (F-NaF).

[0046] In one embodiment, a therapeutically effective amount of an activin A antagonist is administered to a human subject for at least 8 weeks.

[0047] In one embodiment, the method further comprises selecting a subject with FOP who would benefit from reducing the formation of new heterotopic ossification lesions. In one embodiment, the subject who would benefit from reducing the formation of new heterotopic ossification lesions is about to undergo surgery.

[0048] In one embodiment, the human subject is about to undergo therapeutic treatment for FOP.

[0049] In one embodiment, the activin A antagonist does not reduce the number, volume, or size of any pre-existing lesions in a human subject.

[0050] In one embodiment, the activin A antagonist is a protein or a small molecule.

[0051] In one embodiment, the activin A antagonist is an anti-activin A antibody, or antigen-binding fragment thereof. In one embodiment, the anti-activin A antibody, or antigen-binding fragment thereof, is a chimeric, veneered, humanized, or human antibody, or antigen-binding fragment thereof.

[0052] In one embodiment, the anti-activin A antibody, or antigen-binding fragment thereof, is a human kappa IgG1 antibody.

[0053] In one embodiment, the anti-activin A antibody, or antigen-binding fragment thereof, comprises the following six CDR sequences: (a) an HCDR1 having at least about 80% identity to the sequence GGSFSSHF, (b) an HCDR2 having at least about 80% identity to the sequence ILYTGGT, (c) an HCDR3 having at least about 80% identity to the sequence ARARSGITFTGIIVPGSFDI, (d) an LCDR1 having at least about 80% identity to the sequence QSVSSSY, (e) an LCDR2 having at least about 80% identity to the sequence GAS, and (f) an LCDR3 having at least about 80% identity to the sequence QQYGSSPWT.

[0054] In one embodiment, the anti-activin A antibody, or antigen-binding fragment thereof, comprises the following six CDR sequences: (a) HCDR1 having the sequence GGSFSSHF, (b) HCDR2 having the sequence ILYTGGT, (c) HCDR3 having the sequence ARARSGITFTGIIVPGSFDI, (d) LCDR1 having the sequence QSVSSSY, (e) LCDR2 having the sequence GAS, and (f) LCDR3 having the sequence QQYGSSPWT.

[0055] In one embodiment, the anti-activin A antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having at least 90% identity to SEQ ID NO: 1 and a light chain variable region having at least 90% identity to SEQ ID NO: 5.

[0056] In one embodiment, the anti-activin A antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having at least 95% identity to SEQ ID NO: 1 and a light chain variable region having at least 95% identity to SEQ ID NO: 5.

[0057] In one embodiment, the anti-activin A antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region comprising SEQ ID NO:1 and a light chain variable region comprising SEQ ID NO:5.

[0058] In one embodiment, the anti-activin A antibody, or antigen-binding fragment thereof, comprises a heavy chain comprising SEQ ID NO:25 and a light chain comprising SEQ ID NO:26.

[0059] In one embodiment, the anti-activin A antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having at least 90% identity to SEQ ID NO: 1 and a light chain variable region having at least 90% identity to SEQ ID NO: 5.

[0060] In one embodiment, the anti-activin A antibody, or antigen-binding fragment thereof, competes for binding with an antibody comprising the following six CDR sequences: (a) HCDR1 having the sequence GGSFSSHF, (b) HCDR2 having the sequence ILYTGGT, (c) HCDR3 having the sequence ARARSGITFTGIIVPGSFDI, (d) LCDR1 having the sequence QSVSSSY, (e) LCDR2 having the sequence GAS, and (f) LCDR3 having the sequence QQYGSSPWT.

[0061] In one embodiment, the activin A antagonist is administered in combination with a second therapy. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. A method for reducing the formation of new heterotopic ossification lesions in a human subject having FOP, said method comprising administering to said human subject a therapeutically effective amount of an activin A antagonist, thereby reducing the formation of said new heterotopic ossification lesions in said human subject. (Item 2) 2. The method of claim 1, wherein the formation of new heterotopic ossification lesions is prevented in the human subject. (Item 3) 1. A method for preventing the formation of new heterotopic ossification lesions in a human subject having FOP, said method comprising administering to said human subject a therapeutically effective amount of an activin A antagonist, thereby preventing the formation of said new heterotopic ossification lesions in said human subject. (Item 4) The human subject has a decrease in the number of new heterotopic ossification lesions compared to a control, at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 5% to 90%, at least 10% to 90%, at least 20% % to 90%, at least 30% to 90%, at least 40% to 90%, at least 50% to 90%, at least 60% to 90%, at least 70% to 90%, at least 80% to 90%, at least 5% to 80%, at least 5% to 70%, at least 5% to 60%, at least 5% to 50%, at least 5% to 40%, at least 5% to 30%, at least 5% to 20%, or at least 5% to 10% reduction. (Item 5) 10. The method of any one of the preceding items, wherein the human subject exhibits at least a 5%, at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 40%, at least a 50%, at least a 5% to 50%, at least a 10% to 50%, at least a 20% to 50%, at least a 30% to 50%, at least a 40% to 50%, at least a 5% to 40%, at least a 5% to 30%, at least a 5% to 20%, or at least a 5% to 10% reduction in the volume of new heterotopic ossification lesions compared to a control. (Item 6) 10. The method of any one of the preceding items, wherein the human subject exhibits at least a 5%, at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 5% to 50%, at least a 10% to 50%, at least a 20% to 50%, at least a 30% to 50%, at least a 40% to 50%, at least a 5% to 40%, at least a 5% to 30%, at least a 5% to 20%, or at least a 5% to 10% decrease in the rate of growth and mineralization of new heterotopic ossification lesions compared to a control. (Item 7) 10. The method of any one of the preceding items, wherein the human subject exhibits at least a 5%, at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 5% to 50%, at least a 10% to 50%, at least a 20% to 50%, at least a 30% to 50%, at least a 40% to 50%, at least a 5% to 40%, at least a 5% to 30%, at least a 5% to 20%, or at least a 5% to 10% decrease in the intensity of new heterotopic ossification lesions compared to a control. (Item 8) 10. The method of any one of the preceding items, wherein the human subject exhibits a decrease in total lesion activity (TLA) of the heterotopic ossification lesions of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 5% to 80%, at least 10% to 80%, at least 20% to 80%, at least 30% to 80%, at least 40% to 80%, at least 50% to 80%, at least 60% to 80%, at least 70% to 80%, at least 5% to 70%, at least 5% to 60%, at least 5% to 50%, at least 5% to 40%, at least 5% to 30%, at least 5% to 20%, or at least 5% to 10% compared to a control. (Item 9) 10. The method of any one of the preceding items, wherein the human subject exhibits about a 0.2-fold, 0.5-fold, 1-fold, 1.5-fold, 2-fold, 3-fold, 0.2-3-fold, 0.5-3-fold, 1-3-fold, 1.5-3-fold, 2-3-fold, 2.5-3-fold, 0.2-2.5-fold, 0.2-2-fold, 0.2-1.5-fold, 0.2-1-fold, or 0.2-0.5-fold decrease in average daily Pain-NRS compared to a control. (Item 10) 10. The method of any one of items 4 to 9, wherein the control is a mean measurement or value collected from a population of human subjects with FOP who have not been administered the activin A antagonist. (Item 11) Any of the preceding items, wherein the therapeutically effective amount of the activin A antagonist reduces the occurrence of painful flare-ups in the human subject compared to a control. 1. The method according to claim 1. (Item 12) The method of any one of the preceding items, wherein the new heterotopic ossification lesions are analyzed by positron emission tomography (PET) scan, computed tomography (CT) scan, or a combination thereof. (Item 13) The PET scan analysis comprises radiolabeling the human subject. 18 Sodium fluoride ( 18 13. The method of claim 12, wherein the method is carried out by administering F-NaF. (Item 14) The method of any one of the preceding items, wherein the therapeutically effective amount of the activin A antagonist is administered to the human subject for at least 8 weeks. (Item 15) The method of any one of the preceding items, further comprising selecting a subject with FOP that would benefit from reduced formation of new heterotopic ossification lesions. (Item 16) 16. The method of item 15, wherein the subject who may benefit from reduced formation of new heterotopic ossification lesions is about to undergo surgery. (Item 17) The method of any one of the preceding items, wherein the human subject is about to undergo therapeutic treatment for FOP. (Item 18) The method of any one of the preceding items, wherein said activin A antagonist does not reduce the number, volume, or size of any pre-existing lesions in said human subject. (Item 19) The method of any one of the preceding items, wherein the activin A antagonist is a protein or a small molecule. (Item 20) 19. The method of any one of items 1 to 18, wherein the activin A antagonist is an anti-activin A antibody, or an antigen-binding fragment thereof. (Item 21) 21. The method of claim 20, wherein the anti-activin A antibody, or antigen-binding fragment thereof, is a chimeric, veneered, humanized, or human antibody, or antigen-binding fragment thereof. (Item 22) 22. The method of claim 20 or 21, wherein the anti-activin A antibody, or antigen-binding fragment thereof, is a human kappa IgG1 antibody. (Item 23) The anti-activin A antibody, or antigen-binding fragment thereof, has the following six CDR sequences: (a) an HCDR1 having at least about 80% identity to the sequence GGSFSSHF; (b) an HCDR2 having at least about 80% identity to the sequence ILYTGGT; (c) an HCDR3 having at least about 80% identity to the sequence ARARSGITFTGIIVPGSFDI; (d) an LCDR1 having at least about 80% identity to the sequence QSVSSSY; (e) an LCDR2 having at least about 80% identity to the sequence GAS; and (f) the method of item 20 or 21, comprising an LCDR3 having at least about 80% identity to the sequence QQYGSSPWT; (Item 24) The anti-activin A antibody, or antigen-binding fragment thereof, has the following six CDR sequences: (a) HCDR1 with the sequence GGSFSSHF; (b) HCDR2 with the sequence ILYTGGT; (c) HCDR3 with the sequence ARARSGITFTGIIVPGSFDI; (d) LCDR1 with the sequence QSVSSSY; (e) LCDR2 with the sequence GAS, and (f) The method of item 23, comprising LCDR3 having the sequence QQYGSSPWT. (Item 25) 25. The method of claim 24, wherein the anti-activin A antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having at least 90% identity to SEQ ID NO: 1 and a light chain variable region having at least 90% identity to SEQ ID NO: 5. (Item 26) 26. The method of claim 25, wherein the anti-activin A antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having at least 95% identity to SEQ ID NO: 1 and a light chain variable region having at least 95% identity to SEQ ID NO: 5. (Item 27) 27. The method of claim 26, wherein the anti-activin A antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO: 5. (Item 28) 28. The method of claim 27, wherein the anti-activin A antibody, or antigen-binding fragment thereof, comprises a heavy chain comprising SEQ ID NO: 25 and a light chain comprising SEQ ID NO: 26. (Item 29) The method of item 20 or 21, wherein the anti-activin A antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having at least 90% identity to SEQ ID NO: 1 and a light chain variable region having at least 90% identity to SEQ ID NO: 5. (Item 30) The anti-activin A antibody, or antigen-binding fragment thereof, has the following six CDR sequences: (a) HCDR1 with the sequence GGSFSSHF; (b) HCDR2 with the sequence ILYTGGT; (c) HCDR3 with the sequence ARARSGITFTGIIVPGSFDI; (d) LCDR1 with the sequence QSVSSSY; (e) LCDR2 with the sequence GAS, and 22. The method of claim 20 or 21, wherein (f) the antibody competes for binding with an antibody comprising an LCDR3 having the sequence QQYGSSPWT. (Item 31) The method of any one of the preceding items, wherein the activin A antagonist is administered in combination with a second therapy. [Brief explanation of the drawings]

[0062] [Figure 1] FIG. 1 shows a schematic of a double-blind study to evaluate the effect of an anti-activin A antagonist on total lesion activity and ectopic bone volume. [Figure 2A] PET imaging showing FOP disease progression (FIG. 2A) and the effect of an activin A antagonist on heterotopic ossification (HO) lesions (FIG. 2B) are shown. [Figure 2B] PET imaging showing FOP disease progression (FIG. 2A) and the effect of an activin A antagonist on heterotopic ossification (HO) lesions (FIG. 2B) are shown. [Figure 3A] Treatment with an activin A antagonist reduced total lesion activity by approximately 25% as analyzed by PET imaging (p=0.074). A similar reduction was observed by CT analysis. [Figure 3B-1] 1 shows results from a PET / CT imaging analysis showing that the effect of activin A antagonists is more apparent when pre-existing ("target") lesions are examined separately from new lesions. [Figure 3B-2] 1 shows results from a PET / CT imaging analysis showing that the effect of activin A antagonists is more apparent when pre-existing ("target") lesions are examined separately from new lesions. [Figure 4] Figure 1 shows the percent change from baseline in total lesion activity by 18F-NaF PET in the active HO analysis set (AHO). [Figure 5] Figure 1 shows the percent change from baseline in average weekly pain by Numeric Rating Scale (NRS) in the Active HO Analysis Set (AHO). [Figure 6]Figure 1 shows total lesion activity of new lesions by 18F-NaF PET at week 28 in patients with new lesions during the double-blind period in the active HO analysis set (AHO). [Figure 7] Figure 1 shows the rate of patients with new HO lesions by 18F-NaF PET in the active HO analysis set (AHO). [Figure 8] Figure 1 shows the total volume of new lesions by CT at week 28 in patients with new lesions during the double-blind period in the active HO analysis set (AHO). [Figure 9] The rate of patients with new HO lesions by CT in the active HO analysis set (AHO) is shown. [Figure 10] 1 shows the amino acid sequences of the heavy and light chains of an exemplary anti-activin A monoclonal antibody (SEQ ID NOs: 25 and 26, respectively). DETAILED DESCRIPTION OF THE INVENTION

[0063] definition Antagonists are typically provided in isolated form. This means that the antagonist is typically at least 50% w / w pure from interfering proteins and other contaminants resulting from its production or purification, but does not exclude the possibility that the antagonist may be combined with an excess of pharmaceutically acceptable carriers or other vehicles intended to facilitate its use. In some cases, the antagonist is at least 60, 70, 80, 90, 95, or 99% w / w pure from interfering proteins and contaminants resulting from its production or purification.

[0064] For purposes of classifying amino acid substitutions as conservative or non-conservative, amino acids are grouped as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues that influence chain direction): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids within the same class. Non-conservative substitutions constitute exchanging a member of one of these classes for a member of another class.

[0065] Percent sequence identity is determined using maximally aligned antibody sequences using Kabat numbering for variable regions or EU numbering for constant regions. For other proteins, sequence identity is calculated using the Wisconsin Genetics Software Package Release 7.0, Genetics Sequence identity can be determined by aligning sequences using algorithms such as BESTFIT, FASTA, and TFASTA using default gap parameters, or by inspection and best alignment, in the Molecular Biology Group (Molecular Biology, 575 Science Dr., Madison, WI). After alignment, when a region of a subject antibody (e.g., the entire mature variable region of a heavy or light chain) is compared with the same region of a reference antibody, the percentage of sequence identity between the subject and reference antibody regions is calculated by dividing the number of positions occupied by the same amino acid in both the subject and reference antibody regions by the total number of aligned positions in the two regions (gaps not counted), and multiplying by 100 to convert to a percentage.

[0066] A composition or method "comprising" one or more recited elements may include other elements not specifically recited. For example, a composition comprising an antibody may contain the antibody alone or in combination with other components.

[0067] Humanized antibodies are genetically engineered antibodies in which CDRs from a non-human "donor" antibody are grafted onto human "acceptor" antibody sequences (see, e.g., Queen, US Pat. Nos. 5,530,101 and 5,585,089; Winter, US Pat. No. 5,225,539; Carter, US Pat. No. 6,407,213; Adair, US Pat. Nos. 5,859,205 and 6,881,557; Foote, US Pat. No. 6,881,557). The acceptor antibody sequences can be, for example, mature human antibody sequences, a composite of such sequences, a consensus sequence of human antibody sequences, or germline region sequences. Thus, a humanized antibody is an antibody possessing all or substantially some or all of the CDRs from the donor antibody and variable region framework sequences and constant regions, if present, completely or substantially from human antibody sequences. Similarly, a humanized heavy chain has at least one, two, and usually all three CDRs fully or substantially from the donor antibody heavy chain and the heavy chain variable region framework sequence and heavy chain constant region (if present, substantially from human heavy chain variable region framework and constant region sequences). Similarly, a humanized light chain has at least one, two, and usually all three CDRs fully or substantially from the donor antibody light chain and the light chain variable region framework sequence and light chain constant region (if present, substantially from human light chain variable region framework and constant region sequences). Humanized antibodies other than nanobodies and dAbs comprise a humanized heavy chain and a humanized light chain. A CDR in a humanized antibody is substantially different from the corresponding CDR in a non-human antibody if at least 85%, 90%, 95%, or 100% of the corresponding residues (as defined by Kabat) are identical between the respective CDRs. The variable region framework sequence of an antibody chain or the constant region of an antibody chain is substantially derived from a human variable region framework sequence or human constant region, respectively, if at least 85, 90, 95, or 100% of the corresponding residues as defined by Kabat are identical.

[0068] Humanized antibodies often incorporate all six CDRs (preferably as defined by Kabat) from a murine antibody, although they can also be made with fewer than all CDRs (e.g., at least three, four, or five CDRs from a murine antibody) (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320:415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al., Journal of Immunology, 164:1432-1441, 2000).

[0069] Chimeric antibodies are antibodies in which the mature variable regions of the light and heavy chains of a non-human antibody (e.g., murine) are combined with human light and heavy chain constant regions. Such antibodies substantially or completely retain the binding specificity of the murine antibody and are approximately two-thirds human in sequence.

[0070] A veneered antibody is a type of humanized antibody that retains some, and usually all, of the CDRs of a non-human antibody, as well as some of the non-human variable region framework residues, but replaces other variable region framework residues that may contribute to B-cell or T-cell epitopes, e.g., exposed residues, with residues from the corresponding positions in a human antibody sequence (Padlan, Mol. Immunol. 28:489, 1991). The result is an antibody in which the CDRs are entirely or substantially derived from a non-human antibody and the variable region framework of the non-human antibody is made more human-like by the substitutions.

[0071] Human antibodies may be isolated from humans or may otherwise result from the expression of human immunoglobulin genes (e.g., in transgenic mice, in vitro, or by phage display). Methods for producing human antibodies include the trioma method of Oestberg et al., Cys muoma 2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664; and Engleman et al., U.S. Pat. No. 4,634,666. Monoclonal antibodies may also be produced by transgenic mice carrying human immune system genes, e.g., Regeneron. VelocImmune® mice from Biosciences Pharmaceuticals, Inc. (Murphy, PNAS 111 no. 14, 5153-5158 (2014); Xenomouse, Jakobovits, Nature Biotechnology 25, 1134-1143 (2007)), or HuMAb mice from Medarex, Inc. (Lonberg, Handbook Exp. Pharmacol. 181, 69-97 (2008); Lonberg et al. al., WO93 / 12227(1993), US5,877,397, US5,874,299, US5,814,318, US5,789,650, US5,770,429, US5,661,016, US5,633,425, US5,625,126, US5,569,825, US5,545,806, Nature 148,1547-1553(1994), Nature Biotechnology 14,826(1996), Kucherlapati, WO91 / 10741(1991). Human antibodies can also be produced by phage display methods (e.g., Dower et al., WO91 / 17271 and McCafferty et al., WO91 / 17271). al., WO92 / 01047, US 5,877,218, US 5,871,907, US 5,858,657, US 5,837,242, US 5,733,743, and US 5,565,332).

[0072] When an antagonist is said to retain the properties of the parent antibody from which it is derived, that retention may be complete or partial. Complete retention of activity means that the activity of the antagonist is the same, within experimental error, as or greater than the activity of the molecule from which it is derived. Partial retention of activity means activity that is significantly above background levels of a negative control (i.e., beyond experimental error), preferably at least 50% of the corresponding activity of the molecule from which it is derived.

[0073] Two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other.

[0074] Competition between antibodies is determined by an assay in which the antibody under test inhibits the specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). A test antibody competes with a reference antibody if an excess amount of the test antibody (e.g., at least 2-fold, 5-fold, 10-fold, 20-fold, or 100-fold) inhibits the binding of the reference antibody by at least 50%, but preferably 75%, 90%, or 99%, as measured in a competitive binding assay. Antibodies identified by competitive assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to adjacent epitopes that are sufficiently close to the epitope bound by the reference antibody to create steric hindrance.

[0075] I. Overview Activin A antibodies have previously been tested in mouse models of fibrodysplasia ossificans progressiva (FOP) (see, e.g., EP3191512B1, filed September 14, 2015, the entire contents of which are expressly incorporated herein by reference). However, Acvr1 [R206H]COIN / + ;Gt(ROSA26)SorCreERT2 / + Mouse models of FOP, such as the mouse model, are all conditional knockouts because constitutive mutants of the disease have been found to be nonviable. Thus, in FOP mouse models, the disease can be "turned on" with tamoxifen, and these currently available FOP mouse models lack the ability to express mutant proteins (e.g., ALK2 R206H ) is not an optimal model for studying human diseases in which expression is constitutive.

[0076] The present disclosure is based, in part, on the surprising discovery that treating FOP human subjects with an activin A antagonist dramatically reduces and / or prevents the progression of new heterotopic ossification (HO) bone growth and reduces the average rate of lesion growth and mineralization. However, treatment with the activin A antagonist surprisingly has no effect on pre-existing bone lesions.

[0077] Accordingly, disclosed herein are methods for treating fibrodysplasia ossificans progressiva (FOP), also known as Muenchmeyer's disease, in humans, provided herein, which involve administering to a human subject with FOP a therapeutically effective amount of an activin A antagonist.

[0078] II. Activin A The transforming growth factor beta (TGFβ) superfamily of ligands includes, for example, bone morphogenetic proteins (BMPs) and growth differentiation factors (GDFs). The receptors for these ligands are heteromeric receptor complexes composed of type I and type II transmembrane serine / threonine kinase receptors. Examples of type I receptors include activin receptor type IA (ACTRIA, ACVR1, or ALK2), BMP receptor type IA, and BMP receptor type IB. Examples of type II receptors include activin receptor type IIA and type IIB (ACTRIIA or ACVR2A and ACTRIIB or ACVR2B) and BMP receptor type II. Ligands of the TGFβ superfamily each have different affinities for different type I and type II receptors.

[0079] In humans, activin A can exist as a homodimeric or heterodimeric protein. Homodimeric proteins contain a homodimeric beta A subunit pair. Heterodimeric proteins contain a beta subunit and a beta B, beta C, or beta E subunit (i.e., beta A beta B, beta A beta C, or beta A beta E). Each subunit is expressed as a precursor polypeptide containing a signal peptide, a propeptide, and a mature polypeptide. An exemplary form of the human beta A subunit precursor is a 426 amino acid polypeptide designated Swiss Prot P08476, of which residues 1-20 are the signal peptide, residues 21-310 are the propeptide, and residues 311-426 are the mature polypeptide. An exemplary form of the beta B subunit precursor polypeptide is designated Swiss Prot P09529, in which residues 1-28 are the signal peptide, residues 29-292 are the propeptide, and residues 293-407 are the mature polypeptide. An exemplary form of the beta C subunit is designated Swiss Prot P55103, in which residues 1-18 are the signal peptide, residues 19-236 are the propeptide, and residues 237-352 are the mature polypeptide. An exemplary form of the beta E subunit precursor is designated Swiss Prot P58166, in which residues 1-19 are the signal peptide, residues 20-236 are the propeptide, and residues 237-350 are the mature polypeptide. Several variants of these sequences are designated Swiss Prot P09529. Activin A is known, as described in the database. Reference to activin A includes any of the beta A homodimer, beta A beta B, beta A beta C, and beta A beta E heterodimer forms, and their subunits, as well as their precursors connected to propeptides and / or signal peptides defined by the exemplary Swiss Prot sequences provided or other naturally occurring human forms of these sequences. Activin A signals via binding to ACVR2A or ACVR2B, but is not known to be a ligand for ACVR1. Activin A abnormally signals through mutant ACVRs, transducing osteogenic signals and inducing ectopic bone formation.

[0080] Both type I and type II receptors have an extracellular ligand-binding domain (ECD) and an intracellular serine / threonine kinase domain. In addition, type I receptors have a glycine / serine-rich region (GS-box) preceding the kinase domain and an L45 loop within the kinase domain. Both receptors cooperate with ligands to activate downstream signaling pathways, such as Smad and non-Smad signaling pathways. Activation involves ligand binding, ligand-receptor oligomerization, and transphosphorylation of the GS-box of the type I receptor by type II receptor kinase. Type II receptor kinase is constitutively active and plays a role in ligand binding and activation of type I receptors.

[0081] ACVR1, also known as activin receptor type I, ACVR1A, ACVRLK2, and ALK2, is a type I receptor for the TGFβ superfamily of ligands. ACVR1 possesses serine / threonine kinase activity, phosphorylating Smad proteins and activating downstream signaling pathways. ACVR1 is found in many tissues of the body, including skeletal muscle and cartilage, where it helps regulate bone and muscle growth and development. As described elsewhere herein, certain mutations in the ACVR1 gene cause FOP. Examples of ACVR1 activity include its ability to bind to ligands, form complexes with type II receptors, or activate downstream signaling pathways (e.g., the Smad pathway).

[0082] ACVR2, also known as activin receptor type II, is a type II receptor for the TGFβ superfamily of ligands.There are at least two ACVR2 receptors, for example, activin receptor type IIA (ACVR2A or ACTRIIA) and activin receptor type IIB (ACVR2B or ACTRIIB).Reference to ACVR2 includes either or both ACVR2A and ACVR2B.ACVR2A and ACVR2B can be expressed in multiple tissues, including skeletal muscle, stomach, heart, endometrium, testis, prostate, ovary, and nervous tissue.

[0083] Upon ligand binding, the ACVR2 receptor forms a complex with type I receptors, such as ACVR1, and enhances the kinase activity of the type I receptor by phosphorylating the GS box of the type I receptor. Examples of ACVR2A and ACVR2B activities include the ability to bind ligand, form a complex with the type I receptor, or phosphorylate the type I receptor.

[0084] An exemplary form of human ACVR2A has Swiss Prot accession number P27037. Residues 1-19 are a signal peptide, residues 20-135 are an extracellular domain, residues 59-116 are an activin type I and type II receptor domain, residues 136-161 are a transmembrane domain, and residues 162-513 are a cytoplasmic domain. An exemplary form of human ACVR2B is assigned Swiss Prot number Q13705. Residues 1-18 are a signal sequence, residues 19-137 are an extracellular domain, residues 27-117 are an activin type I and type II receptor domain, residues 138-158 are a transmembrane domain, and residues 159-512 are a cytoplasmic domain. An exemplary form of human ACVR1 has Swiss Prot accession number Q04771. Residues 1-20 are the signal sequence, residues 21-123 are the extracellular domain, residues 33-104 are the activin type I and type II receptor domains, residues 124-146 are the transmembrane domain, and residues 147-509 are the cytoplasmic domain. Reference to any of ACVR1, ACVR2A, and ACVR2B includes these exemplary forms, known isoforms and polymorphisms thereof, e.g., those listed in the SwissProt database, cognate forms from other species, and other variants having at least 90, 95, 96, 97, 98, or 99% sequence identity to the exemplary forms.

[0085] Residues in forms of ACVR2A, ACVR2B, and ACVR1 other than the exemplified sequences defined above are numbered by maximum alignment with the corresponding exemplified sequences, with the aligned residues assigned the same number. Substitutions from the exemplified sequences may be conservative or non-conservative. Reference to ACVR1, ACVR2A, or ACVR2B also includes intact extracellular domains (e.g., residues 20-135, 19-137, or 21-123 of ACVR2A, ACVR2B, and ACVR1, respectively), or portions thereof that do not include or are substantially free of transmembrane and cytoplasmic portions. A portion of the extracellular domain retains sufficient residues of the intact extracellular domain to bind to at least one ligand or counterreceptor that binds to the intact extracellular domain and thereby antagonizes the associated receptor (e.g., residues 59-116, 27-117, or 33-104 of ACVR2A, ACVR2B, and ACVR1, respectively).

[0086] III. Activin A antagonists A.Antibodies The term "antibody" encompasses intact antibodies having two pairs of heavy and light chains, antibody fragments capable of binding to antigens (e.g., Fab, F(ab')2, Fv, single-chain antibodies, diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, humanized antibodies, etc.), and recombinant peptides containing the foregoing.

[0087] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, F(ab'), and Fv fragments, diabodies, linear antibodies (Zapata et al. (1995) Protein Eng. 10:1057-1062), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0088] Antibodies can be monoclonal or polyclonal. A "monoclonal antibody" is an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are often highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is typically directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population, such as that produced by a clonal population of B cells, and does not require production of the antibody by any particular method.

[0089] Monoclonal antibodies used in accordance with the methods provided herein can be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or a modification thereof. Typically, an animal such as a mouse is immunized with a solution containing an antigen (e.g., activin A, ACVR1, ACVR2A and / or ACVR2B polypeptide, or particularly the extracellular domain (intra-receptor) or a portion thereof).

[0090] Immunization can be performed by mixing or emulsifying an antigen-containing solution in saline, preferably in an adjuvant such as Freund's complete adjuvant, and parenterally injecting the mixture or emulsion. After immunization of the animal, the spleen (and optionally several large lymph nodes) is removed and dissociated into single cells. The spleen cells can be screened by applying a cell suspension to a plate or well coated with the antigen of interest. B cells expressing membrane-bound immunoglobulin specific for the antigen bind to the plate and are not washed away. The resulting B cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas and cultured in a selective medium. The resulting cells are plated by serial dilution and assayed for the production of antibodies that specifically bind to the antigen of interest (and do not bind to unrelated antigens). Selected monoclonal antibody (mAb)-secreting hybridomas are then cultured in vitro (e.g., in tissue culture bottles or hollow fiber reactors) or in vivo (as ascites in mice).

[0091] Alternatively, monoclonal antibodies can be produced by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al. (1991) Nature 352:624-628, Marks et al. (1991) J. Mol. Biol. 222:581-597, and U.S. Patent No. 5,514,548.

[0092] "Antibody" includes chimeric, veneered, humanized and human monoclonal antibodies, as defined above, directed against any of activin A, ACVR1, ACVR2A and ACVR2B.

[0093] Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the various classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known.

[0094] The monoclonal antibody or Fc fusion protein of the present invention can be any of various antibody classes. In one embodiment, the monoclonal antibody is an IgG class antibody. In other embodiments, the monoclonal antibody can be an IgM, IgE, IgD, or IgA class antibody. In a specific embodiment, the antibody is an IgG isotype, for example, IgG1, IgG2, IgG3, or IgG4, particularly human IgG1, IgG2, IgG3, or IgG4.

[0095] One or more amino acids at the amino or carboxy termini of the light and / or heavy chains, such as the C-terminal lysine of the heavy chain, may be deleted or derivatized in some or all of the molecules. Substitutions can be made within the constant region to reduce or increase effector functions such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No. 5,834,597; and Lazar et al., proc. Natl. Acad. Sci. USA 103:4005, 2006) or to extend half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). Exemplary substitutions include Gln at position 250 and / or Leu at position 428 (EU numbering) to increase the half-life of the antibody. Substitutions at any of positions 234, 235, 236, and / or 237 reduce affinity for Fcγ receptors, particularly FcγRI receptors (see, e.g., U.S. Pat. No. 6,624,821). Optionally, positions 234, 236, and / or 237 in human IgG2 are substituted with alanine, and position 235 is substituted with glutamine (see, e.g., U.S. Pat. No. 5,624,821). Effector function can also be reduced by substituting EFLG at positions 232-236 with PVA (see WO 14 / 121087). Optionally, S at position 428 is replaced with P, particularly in human IgG4, to reduce exchange between endogenous and exogenous immunoglobulins. Other mutations can add or remove post-translational modification sites, such as N-linked glycosylation at N-XS / T motifs. Mutations can also include the introduction of knobs (i.e., replacing one or more amino acids with larger ones) or holes (i.e., replacing one or more amino acids with smaller ones) to facilitate heterodimer formation between different heavy chains for the production of bispecific antibodies.Exemplary substitutions for forming knob and hole pairs are T336Y and Y407T, respectively (Ridgeway et al., Protein Engineering vol. 9 no. 7 pp. 617-621, 1996). Mutations may also include mutations that reduce Protein A interactions in the EU numbering system (e.g., H435R and Y436F). Bispecific antibodies in which one heavy chain has such mutations and the other heavy chain does not can be separated from their parent antibodies by Protein A affinity chromatography.

[0096] Antibodies may also include antibodies that specifically bind to activin A. Such antibodies may specifically bind to any or all of the betaAbetaA, betaAbetaB, betaAbetaC, and betaAbetaE forms of activin A. Some antibodies specifically bind to only one of these forms (i.e., betaAbetaA, betaAbetaB, betaAbetaC, or betaAbetaE). Specificity for the betaAbetaB, betaAbetaC, and betaAbetaE forms can be conferred by epitopes within the betaB, betaC, or betaE subunits, respectively, or by epitopes contributed by both components of a heterodimer. Specificity for betaAbeta can be conferred by epitopes contributed by both molecules within a homodimer (e.g., at the subunit interface). Some antibodies specifically bind to all of these forms of activin A, in which case the epitope is typically on the betaA subunit. Antibodies typically have epitopes within the mature polypeptide components of the precursor protein. Some antibodies exist in the form of alpha (Swiss Prot P05111) beta A or alpha beta B heterodimers and specifically bind to any or all forms of activin A without binding to human inhibin. Some antibodies specifically bind to any or all forms of activin A and bind to either or both forms of human inhibin. Such antibodies are believed to inhibit activin A signaling through one or more of its counterreceptors, ACVR2A and / or ACVR2B and / or BMPR2, although an understanding of the mechanism is not required for the use of such antibodies in methods of treating FOP.

[0097] A considerable number of antibodies against activin A have been reported. For example, U.S. Patent No. 9,718,881 discloses human antibodies designated H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P2, H4H10447P2, H4H10447P2, H4H10448P2, and H4H10452P2. U.S. Patent No. 8,309,082 discloses human antibodies A1-A14. Mouse antibodies against activin A are available from several commercial suppliers, e.g., MAB3381 from R&D Systems or 9H16 or MM0074-7L18 (ab89307) from Novus Biologicals, AbCam.

[0098] Preferred antibodies have a nucleotide sequence of at least 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 , or 10 13 M -1 (measured at 25°C as in Example 3 of US2015 / 00373339). Some antibodies have an affinity for activin A of 10 9 ~10 12 M -1 Preferred antibodies inhibit activin A signaling with an IC50 of less than 4 nM, preferably less than 400 pM or less than 40 pM. Some antibodies inhibit signaling with an IC50 in the range of 4 nM to 10 pM or 3.5 nM to 35 pM.

[0099] Signal transduction inhibition can be measured similarly to Example 6 of U.S. Pat. No. 9,718,881, which is summarized as follows: The human A204 rhabdomyosarcoma cell line was treated with Smad The A204 / CAGAx12-Luc cell line was produced by transfection with a 2 / 3-luciferase reporter plasmid. A204 / CAGAx12-Luc cells were maintained in McCoy's 5A medium supplemented with 10% fetal bovine serum, penicillin / streptomycin / glutamine, and 250 μg / mL G418. For bioassays, A204 / CAGAx12-Luc cells were seeded at 10,000 cells / well onto 96-well assay plates in low-serum medium, 0.5% FBS, and OPTIMEM and incubated overnight at 37°C and 5% CO2. Activin A was serially diluted 1:3 from 100 to 0.002 nM and added to the cells, starting with a control containing no activin. Antibodies are serially diluted 1:3 starting from 100-0.002 nM, 1000-0.02 nM, or 300-0.005 nM, including control samples containing either the appropriate isotype control antibody or no antibody, and added to cells with a constant concentration of activin A at 100 pM.

[0100] Some antibodies inhibit the binding of activin A to ACVR2A and / or ACVR2B and / or BMPR2 by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% when measured when the receptor is expressed from cells or when the extracellular domain is fused to an Fc domain as a fusion protein and the fusion protein is immobilized on a support (e.g., a Biacore sensor chip). In such measurements, the antibody and activin A should be present in equimolar amounts, and the receptor or extracellular domain should be in excess.

[0101] Some antibodies bind to epitopes within residues 321-343 or 391-421 of full-length activin A, which correspond to C11-S33 and C81-E111 of the mature protein.

[0102] The exemplary antibody used in the examples of the present invention is referred to as H4H10446P in U.S. Patent No. 9,718,881. Its heavy chain variable region and heavy chain CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 162, 164, 166, and 168, respectively, in U.S. Patent No. 9,718,881 (represented by SEQ ID NOs: 1, 2, 3, and 4 of the present invention). Its light chain variable region and light chain CDRs, CDRL1, CDRL2, and CDRL3, have the amino acid sequences of SEQ ID NOs: 146, 148, 150, and 152, respectively, in U.S. Patent No. 9,718,881 (represented by SEQ ID NOs: 5, 6, 7, and 8 of the present invention). H4H10446P inhibits activin A-mediated signal transduction through ACVR2A and / or ACVRIIB, but does not strongly inhibit the binding of activin A to ACRIIA or ACVR2B, even if it does. Also included are other antibodies that compete with H4H10446P for binding to human activin A or to the same epitope on human activin A as H4H10446P, and share its inhibition of signaling.

[0103] Another exemplary antibody for use in the methods of the present invention is designated H4H10430P in U.S. Patent No. 9,718,881. Its heavy chain variable region and heavy chain CDRs, CDRH1, CDRH2, and CDRH3, have the amino acid sequences of SEQ ID NOs: 66, 68, 70, and 72, respectively, in U.S. Patent No. 9,718,881 (SEQ ID NOs: 9, 10, 11, and 12, respectively, in the present invention). Its light chain variable region and light chain CDRs, CDRL1, CDRL2, and CDRL3, have the amino acid sequences of SEQ ID NOs: 74, 76, 78, and 80, respectively, in U.S. Patent No. 9,718,881 (SEQ ID NOs: 13, 14, 15, and 16, respectively, in the present invention). This antibody inhibits the binding of activin A to ACRV2A and / or ACVR2B, inhibiting signal transduction through one or both of these receptors. Also included are other antibodies that compete with H4H10430P for binding to activin A or to the same epitope on activin A as H4H10430P and share its properties of inhibiting activin A binding thereto and signaling through ACVR2A and ACVR2B.

[0104] An exemplary antibody for use in the methods of the present invention is garetsumab. The recombinant monoclonal antibody garetsumab is a covalent heterotetramer consisting of two disulfide-linked human heavy chains (IgG4 isotype), each covalently linked via a disulfide bond to a human kappa light chain. Based on the primary sequence, the glycan-free antibody has a predicted molecular weight of 145,235.3 Da, assuming the formation of 16 canonical disulfide bonds and the removal of Lys453 from the C-terminus of each heavy chain. Each heavy chain contains a serine-to-proline mutation at amino acid Pro234 in the hinge region of the Fc domain to reduce the tendency of IgG4 isotype antibodies to form half-antibodies in solution. One N-linked glycosylation site (Asn303) is present on each heavy chain and is located within the constant region of the Fc domain of the molecule. The complementarity-determining regions (CDRs) within the galetusumab heavy and light chain variable domains together form the binding site for its targets: activin A, activin AB, and activin AC. The heavy and light chain amino acid sequences, the location of the CDRs within each polypeptide chain, the location of the heavy chain N-linked glycosylation sites, and the predicted disulfide bond structure of the galetusumab monoclonal antibody are shown in Figure 10.

[0105] Another exemplary antibody for use in the present methods is the antibody designated A1 in U.S. Patent No. 8,309,082, which is characterized by light and heavy chain variable regions having the sequences of SEQ ID NOs: 9 and 10 in U.S. Patent No. 8,309,082 (SEQ ID NOs: 17 and 18 of the present invention, respectively). Its light chain CDRs, CDRL1, CDRL2, and CDRL3, have the sequences of SEQ ID NOs: 11, 12, and 13, respectively, in U.S. Patent No. 8,309,082 (SEQ ID NOs: 19, 20, and 21 of the present invention, respectively), and its heavy chain CDRs, CDRH1, CDRH2, and CDRH3, have the sequences of SEQ ID NOs: 62, 63, and 64, respectively, in U.S. Patent No. 8,309,082 (SEQ ID NOs: 22, 23, and 24 of the present invention, respectively). Also included are other antibodies that compete with H4H10430P for binding to activin A or to the same epitope on activin A as H4H10430P, inhibit activin A binding thereto, and share its property of transducing signals through ACVR2A and / or ACVR2B.

[0106] Other antibodies can be obtained by mutagenesis of the cDNA encoding the heavy and light chains of any of the above-mentioned antibodies. Also included in the present disclosure are monoclonal antibodies that are at least 90%, 95%, or 99% identical in amino acid sequence to any of the above-mentioned antibodies in the mature heavy and / or light chain variable regions, retain their functional properties, and / or differ from the respective antibodies by a small number of functionally insignificant amino acid substitutions (e.g., conservative substitutions), deletions, or insertions. Also included are monoclonal antibodies with at least one, two, three, four, five, and preferably all six CDRs that are 90%, 95%, 99%, or 100% identical to the corresponding CDRs of any of the exemplified antibodies. CDRs are preferably defined as defined by Kabat, but can be defined by any conventional alternative definition, such as the Chothia, combined Kabat-Chothia, contact, or AbM definitions (see the World Wide Web at bioinf.org.uk / abs).

[0107] B. Protein / Peptide Inhibitors The activin A antagonist useful in the method of the present disclosure includes various molecules, such as peptide inhibitors of activin A, and various inhibitory fragments, derivatives, and analogs thereof.The present disclosure also includes peptide inhibitors of activin A that can function as competitive inhibitors of activin A signal transduction, and various inhibitory fragments, derivatives, and analogs thereof.In some embodiments, the peptide inhibitor is follistatin (see, for example, PCT Publication No. WO2014 / 064292), or a derivative or analog thereof, which inhibits the signal transduction pathway between activin A and any of its receptors disclosed herein.Signal transduction inhibition can be measured as previously disclosed herein.

[0108] Peptide inhibitors of activin A can be recombinantly produced from the corresponding fragments of nucleic acid using various expression systems well known in the art, and various host systems are suitable for production, including bacteria (e.g., E. coli), yeast (e.g., Saccharomyces cerevisiae), insects (e.g., Sf9), and mammalian cells (e.g., CHO, COS-7). Many expression vectors have been developed and are available for each of these hosts. Vectors and procedures for cloning and expression are discussed, for example, in Sambrook et al. (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1987)) and Ausubel et al., 1995. Standard expression vectors useful in the present disclosure are well known in the art and include, but are not limited to, plasmids, cosmids, phage vectors, viral vectors, and yeast artificial chromosomes. The vector sequence may contain an origin of replication for propagation in Escherichia coli (E. coli), an SV40 origin of replication, an ampicillin, neomycin, or puromycin resistance gene for selection in the host cell, and / or a dominant selectable marker and a gene that amplifies the gene of interest (e.g., a dihydrofolate reductase gene).

[0109] Alternatively, peptide inhibitor of activin A can be chemically synthesized using techniques known in the art, such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.For peptide synthesis method, also refer to Bodansky, "Principles of Peptide Synthesis," (Springer Verlag, Berlin (1993)) and Grant (ed.), "Synthetic Peptides: A User's Guide," (WH Freeman and Company, New York (1992)).In addition, automatic peptide synthesizers are commercially available (for example, Advanced ChemTech Model 396, Milligen / Biosearch 9600).

[0110] In certain embodiments, useful activin A antagonists are small molecules such as peptides and peptidomimetics.As used herein, the term "peptidomimetic" includes chemically modified peptides and peptide-like molecules that contain non-naturally occurring amino acids, peptoids, etc.Peptidomimetics offer various advantages over peptides, including enhanced stability when administered to a subject.Methods for identifying peptidomimetics are well known in the art and include screening databases containing libraries of potential peptidomimetics.For example, the Cambridge Structural Database contains a collection of more than 300,000 compounds with known crystal structures (Allen et al., Acta Crystallogr.Section B 35:2331(1979)).If the crystal structure of the target molecule is not available, the structure can be generated, for example, using the program CONCORD (Rusinko et al., J.Chem.Inf.Comput.Sci.29:251(1989)). Another database, the Available Chemicals Directory (Molecular Design Limited, Information Systems; San Leandro Calif.), contains approximately 100,000 commercially available compounds and can also be searched to identify potential peptidomimetics.

[0111] In certain embodiments, the peptide inhibitor of activin A may further comprise post-translational modifications.Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.As a result, the modified soluble polypeptide may contain non-amino acid elements such as polyethylene glycol, lipids, polysaccharides or monosaccharides, and phosphates.The effect of such non-amino acid elements on the functionality of polypeptides can be tested using the functional assays described herein.

[0112] C. Small Molecule Inhibitors The present disclosure also encompasses small molecule inhibitors of activin A. Small molecules are a diverse group of synthetic and natural substances that generally have low molecular weights (preferably less than about 2000 daltons, less than about 1000 daltons, or less than about 500 daltons). Small molecules may be, but are not limited to, nucleic acids, peptides, polypeptides, peptide nucleic acids, peptidomimetics, carbohydrates, lipids, or other organic (carbon-containing) or inorganic molecules, and may be synthetic or naturally occurring, or optionally derivatized. Such small molecules may be therapeutically deliverable substances or may be further derivatized to facilitate delivery or targeting. Such small molecules may be isolated from natural sources (e.g., plants, fungi, microorganisms, etc.), isolated from random or combinatorial chemical libraries of synthetic or natural compounds, or synthesized. See Werner et al., (2006) Brief Funct. Genomic Proteomic 5(1):32-6. Many available random or combinatorial chemical libraries are known in the art. Numerous methods are currently used for the random and directed synthesis of sugar, peptide, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, Conn.). Rare chemical libraries are available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or easily produced, for example, from Pan Laboratories (Bothell, Wash.) or MycoSearch (NC). Additionally, natural and synthetically produced libraries and compounds are easily modified through conventional chemical, physical, and biochemical means (Blondelle et al., (1996) Tib Tech 14:60).

[0113] The identification and screening of activin A antagonists (e.g., small molecule inhibitors) can be further facilitated by determining the structural characteristics of the proteins involved, for example, using X-ray crystallography, neutron diffraction, nuclear magnetic resonance spectroscopy, and other techniques for structure determination. These techniques provide for the rational design or identification of activin A antagonists.

[0114] D. Compounds that affect activin A expression or downstream molecular events in activin A signaling The present disclosure also encompasses inhibitors of activin A that inhibit the expression of activin A or prevent activin A from engaging in its downstream signaling pathways. Non-limiting examples of useful expression inhibitors include, for example, interfering RNA (e.g., siRNA), dsRNA, RNA polymerase III-transcribed DNA, ribozymes, and antisense nucleic acids.

[0115] Antisense oligonucleotide, including antisense DNA, RNA and DNA / RNA molecules, can directly block the translation of mRNA by binding to target mRNA, and prevent protein translation.For example, the antisense oligonucleotide of at least about 15 bases complementary to the unique region of target DNA sequence can be synthesized by, for example, conventional phosphodiester technology (Dallas et al., (2006) Med.Sci.Monit.12(4):RA67-74; Kalota et al., (2006) Handb.Exp.Pharmacol.173:173-96; Lutzelburger et al., (2006) Handb.Exp.Pharmacol.173:243-59).

[0116] siRNAs typically contain 15 to 50 base pairs, preferably 21 to 25 base pairs, and comprise a double-stranded structure with a nucleotide sequence identical or nearly identical to a target gene or RNA expressed in a cell. Antisense polynucleotides include, but are not limited to, morpholinos, 2'-O-methyl polynucleotides, DNA, RNA, and the like. Examples of siRNAs that inhibit activin A expression include, but are not limited to, the anti-activin A siRNA disclosed in Hoda et al., Br J Cancer. 2012 Dec 4;107(12):1978-86.

[0117] RNA polymerase III-transcribed DNA contains a promoter such as the U6 promoter. These DNAs can be transcribed to produce small intracellular hairpin RNAs that can function as siRNAs or linear RNAs that can function as antisense RNAs. The inhibitors can be polymerized in vitro, be recombinant RNAs, contain chimeric sequences, or be derivatives of these groups. The inhibitors can contain ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination to inhibit target RNAs and / or genes. In addition, these forms of nucleic acids can be single-stranded, double-stranded, triple-stranded, or quadruple-stranded. (See, e.g., Bass (2001) Nature, 411, 428 429; Elbashir et al., (2001) Nature, 411, 494 498; and PCT Publication Nos. WO00 / 44895, WO01 / 36646, WO99 / 32619, WO00 / 01846, WO01 / 29058, WO99 / 07409, WO00 / 44914).

[0118] Ribozymes are enzymatic RNA molecules that can catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of mRNA sequences are also within the scope of this disclosure. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of approximately 15-20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be assessed by testing their accessibility to hybridization with complementary oligonucleotides, for example, using ribonuclease protection assays.

[0119] The expression inhibitors of the present disclosure can be prepared by known methods. These include techniques for chemical synthesis, such as solid-phase phosphoamite chemical synthesis. Alternatively, antisense RNA molecules can be produced by in vitro or in vivo transcription of DNA sequences encoding the RNA molecules. Such DNA sequences can be incorporated into a wide variety of vectors incorporating suitable RNA polymerase promoters, such as T7 or SP6 polymerase promoters. See, for example, Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews—Trends See Genetics, Vol. 1(1) 1986.

[0120] Various modifications can be introduced into the oligonucleotides of the present disclosure as a means of increasing intracellular stability and half-life.Possible modifications include, but are not limited to, adding a flanking sequence of ribonucleotides or deoxyribonucleotides to the 5'-end and / or 3'-end of the molecule, or using phosphorothioate or 2'-O-methyl instead of phosphodiesterase linkages in the oligonucleotide backbone.

[0121] The aptamer nucleic acid sequence that binds to a wide variety of target molecules can be easily produced.The aptamer nucleic acid sequence of the present disclosure can be composed entirely of RNA or partly of RNA, or entirely or partly of DNA and / or other nucleotide analogs.Aptamer is typically developed to bind to specific ligand by using known in vivo or in vitro (most typically in vitro) selection technology known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Methods for producing aptamers are described, for example, in Ellington and Szostak (1990) Nature 346:818, Tuerk and Gold (1990) Science 249:505, U.S. Patent No. 5,582,981, PCT Publication No. WO00 / 20040, U.S. Patent No. 5,270,163, Lorsch and Szostak (1994) Biochem. 33:973, Mannironi et al., (1997) Biochem. 36:9726, Blind (1999) Proc. Nat'l. Acad. Sci. USA 96:3606-3610, Huizenga and Szostak (1995) Biochem. 34:656-665, PCT Publication Nos. WO 99 / 54506, WO 99 / 27133, and WO 97 / 42317, and U.S. Patent No. 5,756,291.

[0122] IV. ACVR1, ACVR2A, and ACVR2B antagonists Antagonists of type I receptor ACVR1 and type II receptor ACVR2 proteins (e.g., ACVR2A and / or ACVR2B) are provided for treating FOP. Such antagonists can antagonize the receptors by, among other mechanisms, directly binding to the receptor (such as antibodies against ACVR1, ACVR2A, or ACVR2B), or indirectly binding to a ligand or counter-receptor and inhibiting the binding of the ligand or counter-receptor to ACVR1, ACVR2A, or ACVR2B (such as fusion proteins of ACVR1, ACVR2A, or ACVR2B). Antagonists of ACVR2A and ACVR2B can also bind to activin A.

[0123] The ACVR1, ACVR2A or ACVR2B antagonists provided herein can inhibit or reduce ACVR1, ACVR2A and / or ACVR2B activity by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more compared to a control cell or animal model that did not receive the antagonist.

[0124] Any antagonist of activin A can be used alone or in combination with one or more antagonists of ACVR1, ACVR2A, or ACVR2B in methods for treating FOP. Antagonists can include, for example, activin A, ACVR1, ACVR2A, or ACVR2B polypeptides, such as extracellular domains, antagonist antibodies, or small molecule inhibitors.

[0125] A. Extracellular domains of ACVR1, ACVR2A, and ACVR2B polypeptides Antagonists include ACVR1, ACVR2A, and ACVR2B proteins, as well as fragments thereof that are effective in inhibiting at least one activity of ACVR1, ACVR2A, and ACVR2B, respectively. Such antagonists typically include the extracellular domain of ACVR1, ACVR2A, or ACVR2B, or a portion thereof. Preferably, such extracellular domains are completely or substantially free of the transmembrane and cytoplasmic regions (i.e., any remaining residues from these regions do not significantly affect the function of the extracellular domain). In other words, the ACVR2A, ACVR2B, or ACVR1 component of such antagonists consists of or essentially consists of the entire or a portion of the extracellular domain of ACVR2A, ACVR2B, or ACVR1, as defined above. Such antagonists may or may not contain other components distinct from ACVR2A, ACVR2B, or ACVR1, as further described below. Such extracellular domains are soluble and do not contain or are substantially free of transmembrane and cytoplasmic domains. Such extracellular domains can function as antagonists by binding to soluble ligands or counter-receptors and effectively competing with ligands or counter-receptors bound to the ACVR1, ACVR2A, or ACVR2B cell surface receptors, thereby modulating (reducing) the availability of the ligands or counter-receptors in vivo.

[0126] The soluble extracellular domain can be initially expressed with a signal sequence that is cleaved during expression. The signal sequence can be the native signal sequence of ACVR1, ACVR2A, or ACVR2B, such as that described in U.S. Patent No. 7,709,605, the entire contents of which are incorporated herein by reference, or it can be a signal sequence from a different protein, such as honeybee melittin (HBM) or tissue plasminogen activator (TPA). Alternatively, the soluble extracellular ACVR1, ACVR2A, or ACVR2B polypeptide can be synthesized or expressed without a signal sequence.

[0127] The ECD or ligand binding domain of ACVR1, ACVR2A and ACVR2B is highly conserved among species, including mice and humans. ECD contains a cysteine-rich region and a C-terminal tail region. The ECD of ACVR1, ACVR2A and ACVR2B binds to a diverse group of TGFβ family ligands, including, for example, activin A, myostatin (GDF-8), GDF-11 and BMP. For example, see Souza et al. (2008) Molecular Endocrinology 22(12):2689-2702.

[0128] Examples of ACVR2A and ACVR2B polypeptides and soluble ACVR2A and ACVR2B polypeptides include those disclosed in U.S. Pat. No. 7,842,633, U.S. Pat. No. 7,960,343, and U.S. Pat. No. 7,709,605, each of which is incorporated by reference in its entirety.

[0129] The ECD of an ACVR1, ACVR2A, or ACVR2B polypeptide can be mutated so that the variant polypeptide has altered ligand binding properties (e.g., binding specificity or affinity). Some variant ACVR1, ACVR2A, or ACVR2B polypeptides have altered binding affinity (e.g., increased or decreased) for a particular ligand. Variants have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the naturally occurring ACVR1, ACVR2A, or ACVR2B sequence, retain biological activity, and thus have the ACVR1, ACVR2A, or ACVR2B activity described elsewhere herein. Active variants and fragments of ACVR2A and ACVR2B are described, for example, in U.S. Patent Nos. 7,842,633, 7,960,343, and 7,709,605, each of which is incorporated herein by reference in its entirety.

[0130] Assays for measuring ACVR1, ACVR2A or ACVR2B activity are disclosed, for example, in U.S. Patent Nos. 7,842,633, 7,960,343, and 7,709,605. For example, ACVR1, ACVR2A or ACVR2B polypeptide variants can be screened for the ability to bind to a ligand or to prevent the binding of a ligand to the ACVR1, ACVR2A or ACVR2B receptor protein.

[0131] B. Fusion Proteins The ACVR1, ACVR2A and ACVR2B polypeptides described above can be expressed as fusion proteins comprising the ACVR1, ACVR2A and / or ACVR2B polypeptide and at least a portion of one or more fusion domains.

[0132] Fusion domains include immunoglobulin heavy chain constant region (Fc), human serum albumin (HSA), glutathione S-transferase (GST), protein A, protein G, or any fusion domain that may be useful for stabilizing, solubilizing, isolating, or multimerizing the fusion protein.

[0133] The Fc domain of an immunoglobulin heavy chain is a preferred domain for fusion proteins. Fusion with the Fc portion of an immunoglobulin confers desirable pharmacokinetic properties to a wide range of proteins (e.g., increasing the protein's stability and / or serum half-life). Thus, the present disclosure provides fusion proteins comprising at least one ECD of ACVR1, ACVR2A, and / or ACVR2B fused to the Fc domain of an immunoglobulin.

[0134] The Fc domain for use in the methods of the present invention can be derived from any immunoglobulin. Any of the various classes of immunoglobulins can be used, including IgG, IgA, IgM, IgD, and IgE. Within the IgG class, there are different subclasses or isotypes, including, for example, IgG1, IgG2, IgG3, and IgG4. In one embodiment, the Fc fusion protein comprises the Fc domain of an IgG molecule. In a further embodiment, the Fc domain is derived from an IgG1 molecule. The immunoglobulin molecule may be of any animal type, including, for example, a mammal, a rodent, a human, a mouse, a rat, a hamster, or a rabbit. In one embodiment, the immunoglobulin Fc domain is derived from a mammal. In another embodiment, the Fc domain is derived from a human. In yet another embodiment, the Fc domain is derived from a rodent, such as a mouse or a rat. In a specific embodiment, the Fc domain of the fusion protein is derived from human IgG1.

[0135] The Fc fusion proteins provided herein can be prepared by any method known in the art. The Fc fusion proteins can include at least the CH2 and CH3 regions, typically at least a portion of the hinge region. The CH1 region can be present, but is typically omitted in the fusion protein.

[0136] Fusion can occur at any site within the Fc portion of an immunoglobulin constant domain. Fusion can occur to the C-terminus of the Fc portion of the constant domain or immediately N-terminal to the CH1 region of the heavy chain. A particular site can be selected to optimize the biological activity, secretion, or binding characteristics of the Fc fusion protein.

[0137] In some cases, the nucleic acid encoding the ECD of ACVR1, ACVR2A, and / or ACVR2B is fused to the C-terminus of the nucleic acid encoding the N-terminus of the immunoglobulin constant domain sequence. In other cases, N-terminal fusions are also possible. The ECD of ACVR1, ACVR2A, and / or ACVR2B can also be fused to both the N-terminus and C-terminus of the immunoglobulin constant domain sequence.

[0138] For the production of immunoglobulin fusions, see also U.S. Patent No. 5,428,130, U.S. Patent No. 5,843,725, U.S. Patent No. 6,018,026, and WO2005 / 070966, each of which is incorporated by reference in its entirety.

[0139] Fusion proteins can be produced, for example, by recombinant expression of a nucleic acid encoding the fusion protein. For example, a fusion protein can be created by fusing a nucleic acid encoding the ECD of ACVR1, ACVR2A, and / or ACVR2B to a nucleic acid encoding the Fc domain. The ECD nucleic acid can be fused to the N-terminus of the nucleic acid encoding the Fc domain or to the C-terminus of the gene encoding the Fc domain. Alternatively, the ECD can be fused at any position within the Fc domain.

[0140] The ECD fusion protein may also contain a linker. In the case of an Fc fusion protein, the linker is located between ACVR1, ACVR2A, or ACVR2BECD and the Fc domain, and can optionally replace part or all of the hinge region. The linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50, or more amino acids that are relatively free of secondary structure. The linker may be rich in glycine and proline residues, and can contain, for example, a repeat sequence of threonine / serine and glycine (e.g., TG4 or SG4 repeat).

[0141] Two or more ECD-Fc fusion proteins can be connected together by a linker. In such cases, the linker can be placed between the ECDs, or the linker can be placed between the Fc domains to connect the fusion proteins together. For example, one, two, three, four, or more ACVR1, ACVR2A, and / or ACVR2B Fc fusion proteins can be linked together.

[0142] Examples of ACVR2A and / or ACVR2B ECD fusion proteins are described in U.S. Pat. No. 7,842,633, U.S. Pat. No. 7,960,343, and U.S. Pat. No. 7,709,605, each of which is incorporated by reference in its entirety.

[0143] One example of an ACVR2A antagonist is known as sotatercept (also called ACE-011), which contains the ECD of ACVR2A fused to a human IgG1 Fc domain and is described in detail in Carrancio et al., (2014) British J Haematology. 165(6):870-872, the entire contents of which are incorporated herein by reference.

[0144] One example of an ACVR2B antagonist is known as ACE-031, which contains the ECD of ACVR2B fused to a human IgG1 Fc domain and is described in detail in Sako et al., (2010) J. Biol. Chem. 285(27):21037-21048, the entire contents of which are incorporated herein by reference.

[0145] Examples of ACVR1 ECD fusion proteins are known, such as those disclosed in Berasi, et al., (2011) Growth Factors, 29(4):128-139, which is incorporated herein by reference in its entirety.

[0146] C. Hybrid ECD fusion protein Hybrid or multispecific ECD fusion protein antagonists are also provided. Hybrid ECD fusion proteins can contain a combination of two or more ACVR1, ACVR2A, and / or ACVR2B ECDs. For example, the fusion protein can contain one, two, three, four, or more molecules of ACVR1, ACVR2A, and / or ACVR2B ECDs. In one embodiment, the antagonist comprises an ACVR2A ECD linked to an ACVR2B ECD. In a further embodiment, the antagonist further comprises an Fc domain.

[0147] In one embodiment, a fusion protein may comprise one or more molecules of ACVR2A ECD and one or more molecules of ACVR2B ECD. In another embodiment, a fusion protein may comprise one or more molecules of ACVR1 ECD and one or more molecules of ACVR2A ECD. In another embodiment, a fusion protein may comprise one or more molecules of ACVR1 ECD and one or more molecules of ACVR2B ECD.

[0148] In one embodiment, the fusion protein comprises one or more ACVR2A ECD-Fc fusion proteins and one or more ACVR2B ECD-Fc fusion proteins complexed together. In another embodiment, the fusion protein comprises one or more ACVR1 ECD-Fc fusion proteins and one or more ACVR2A ECD-Fc fusion proteins complexed together. In another embodiment, the fusion protein comprises one or more ACVR1 ECD-Fc fusion proteins and one or more ACVR2B ECD-Fc fusion proteins complexed together. In such cases, the fusion proteins may be connected together via their Fc domains, for example, by at least one disulfide bond or by a linker sequence. Alternatively, the ECD portions of the fusion proteins may be connected together by a linker sequence.

[0149] In one embodiment, the antagonist comprises an ACVR2A ECD fused to a first Fc domain and an ACVR2B ECD fused to a second Fc domain. In such a case, the Fc domains can be complexed to each other. In another embodiment, the antagonist comprises a linker between the ACVR2A and ACVR2B ECDs, each fused to an Fc domain.

[0150] Fusion proteins can be constructed to produce ACVR1, ACVR2A, and / or ACVR2B antagonists in tandem. In one embodiment, the fusion protein contains two or more ECDs from ACVR1, ACVR2A, and / or ACVR2B in tandem, followed by an Fc domain. In some cases, the tandemly arranged ECDs are separated by a linker sequence. Such tandem fusion proteins can contain one, two, three, four, or more ACVR1, ACVR2A, and / or ACVR2B ECDs.

[0151] D. Antibody Antagonists ACVR1, ACVR2A, or ACVR2B antagonists include antibodies against (i.e., that specifically bind to) any of these receptors, preferably antibodies having an epitope within the extracellular domain. Specific binding of an antibody or fusion protein to its target antigen is at least 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 M -1 Specific binding refers to the affinity of an antibody to a target. Specific binding is detectably large and distinguishable from non-specific binding that occurs to at least one unrelated target. Methods for preparing antibodies are known in the art. For example, see Kohler & Milstein (1975) Nature 256:495-497, and Harlow & Lane (1988) Antibodies: a Laboratory Manual, Cold Spring Harbor Lab., Cold Spring Harbor, NY.

[0152] Any antibody (e.g., antagonist antibody) that inhibits or reduces the activity of ACVR1, ACVR2A, and / or ACVR2B can be used. Such ACVR2A and ACVR2B antibodies include, for example, those disclosed in U.S. Patent No. 8,486,403, U.S. Patent No. 8,128,933, WO2009 / 137075, and Lach-Trifilieff, et al. (2014) Mol. Cell Biol. 34(4):606-618, each of which is incorporated herein by reference in its entirety. Humanized, chimeric, and veneered forms of any of these antibodies are included, as are antibodies that compete for binding with them.

[0153] In one embodiment, the antibody is an anti-ACVR2A antibody. In another embodiment, the antibody is an anti-ACVR2B antibody. In other embodiments, the antibody may be a bispecific antibody against both ACVR2A and ACVR2B. In another embodiment, the antibody is an anti-ACVR1 antibody. In other embodiments, the antibody may be a bispecific antibody against both ACVR1 and ACVR2A, or against both ACVR1 and ACVR2B.

[0154] E. Small Molecule Antagonists The antagonist of activin A, ACVR1, ACVR2A and ACVR2B can also be a small molecule agonist. Such small molecule antagonist can inhibit the activity of activin A, ACVR1, ACVR2A or ACVR2B. The small molecule antagonist of ACVR1 includes, for example, LDN-212854, described in Mohedas et al., (2013) ACS Chem.Biol.8:1291-1302, the entirety of which is incorporated herein by reference.

[0155] V. Screening Assays The activity of the various activin A, ACVR1, ACVR2A and / or ACVR2B antagonists provided herein, and variants or fragments thereof, can be screened in various assays. For example, ACVR1, ACVR2A and / or ACVR2B antagonists, and variants thereof, can be screened for their ability to bind to a ligand or to the ACVR1, ACVR2A or ACVR2B receptor, for their ability to inhibit the binding of a ligand to the ACVR1 and / or ACVR2 polypeptide and / or to inhibit the activity of the ACVR1 or ACVR2 receptor.

[0156] The activity of ACVR1 or ACVR2 antagonist or its variant or fragment can be tested in vitro or cell-based assay.In vitro binding assay and assay for measuring the inhibition of receptor activity are well known.Various assays for measuring the activity of ACVR1, ACVR2A or ACVR2B antagonist are described in detail in, for example, U.S. Patent No. 7,842,663, the entire contents of which are incorporated herein by reference.

[0157] The ability of an antagonist to modulate complex formation between an ACVR1 or ACVR2 polypeptide and its binding protein can be detected by various techniques. For example, modulation of complex formation can be detected by, for example, immunoassay or chromatographic detection using a radiolabeled (e.g., 32 P, 35 S, 14 C, or 3 H), can be quantified using a detectably labeled protein, such as a fluorescently labeled (e.g., FITC), or enzymatically labeled ACVR1 or ACVR2 polypeptide or its binding protein.

[0158] The ability of ACVR1 or ACVR2 antagonists to inhibit ACVR1 or ACVR2 receptor-mediated signaling can be monitored. For example, the effects of downstream signaling, such as Smad activation, can be monitored using Smad-responsive reporter genes.

[0159] ACVR1 and / or ACVR2 antagonists and their variants or fragments can also be screened for activity in in vivo assays.For example, ACVR1 or ACVR2 antagonists or their variants can be screened for the ability to treat FOP in FOP mouse models (for example, the ability to reduce heterotopic bone formation).Transgenic knock-in mice carrying a conditional allele encoding Acvr1[R206H] have been developed.These Acvr1 [R206H]COIN / + The mice are described in US 14 / 207,320 and PCT / US2014 / 026582, the entire contents of which are incorporated herein by reference. This allele expresses the R206H variant only after activation by Cre recombinase. This allows Cre-dependent activation of Acvr1[R206H] expression in specific tissues at specific times by using different Cre driver lines. In this way, the resulting mice also avoid the perinatal lethality observed with the non-regulated Acvr1[R206H] knock-in allele. Activation of Acvr1[R206H] expression in juvenile or adult mice leads to ectopic bone formation. For example, Acvr1[R206H] has been introduced into the Gt(ROSA26)Sor locus and is therefore constitutively and globally expressed. [R206H]COIN / + ;Gt(ROSA26)Sor CreERt2 / +Mice (CreERt2 is a tamoxifen-regulated recombinase) (see Feil et al. (1997) Biochem Biophys Res Commun. 237(3):752-7) develop FOP after exposure to tamoxifen. Briefly, in the absence of tamoxifen, CreERt2 is inactive. Tamoxifen activates expression of Cre, which then translocates Acvr1. [R206H]COIN / + Acting on this, Acvr1 [R206H] / + and thereby converting the genotype of the mice to reflect the genotype of FOP patients, who are ACVR1[R206H]. [R206H] The allele expresses Acvr1[R206H], which is [R206H] / + ;Gt(ROSA26)Sor CreERt2 / + This is sufficient to cause the progression of FOP in mice. [R206H] Knock-in mouse Acvr1 tm1Emsh Following tamoxifen treatment, ACVR1, ACVR2A, and / or ACVR2B antagonists or controls inhibited ACVR1. [R206H]COIN / + ;Gt(ROSA26)Sor CreERt2 / + It can be administered to mice and animals monitored for ectopic bone formation. Chakkalakal SA, et al. (20120) An Acvr1 R206H knock-in mouse has fibrodysplasia ossificans See progressiva. J Bone Miner Res. 27(8):1746-56. This assay is described in detail in the Examples below.

[0160] VI. Fibrodysplasia ossificans progressiva (FOP) FOP is a rare genetic disorder in which heterotopic ossification forms histologically and biomechanically "normal" bone in extraskeletal sites (e.g., connective tissue). The disorder is recurrent, but cumulative, causing permanent disability of increasing severity. FOP is a severe, progressive, and extremely rare genetic disorder in which muscles, tendons, and ligaments are gradually replaced by bone (a process known as heterotopic ossification (HO)). HO in the jaw, spine, and rib cage can make speaking, eating, or breathing difficult and cause weight loss, exacerbating loss of mobility and skeletal deformity. People with FOP also experience recurrent, localized inflammation known as "flare-ups," although HO can occur both asymptomatically and in association with symptoms. Most people with FOP are wheelchair-bound by age 30, and the median survival age is approximately 40 years. Death often results from complications of HO, such as pneumonia, heart failure, and aspiration, as well as loss of mobility of the chest, neck, and jaw.

[0161] The global prevalence of FOP is approximately 1 in 2,000,000. Approximately 800-1,000 people worldwide are diagnosed with FOP, with many others believed to remain undiagnosed or misdiagnosed. FOP does not have an ethnic, racial, gender, or geographic predilection. Not only is FOP a highly disabling disease, it is also a condition associated with a significantly shortened lifespan.

[0162] FOP is characterized by flare-ups characterized by painful soft tissue swelling of the head, neck, and / or back, accompanied by congenital malformations of the thumb, inflammation, and progressive formation of heterotopic bone via endochondral ossification.

[0163] FOP can be clinically suspected based on the presence of thumb deformities. Diagnostic tests such as x-rays or bone scans can document the thumb abnormalities and confirm the presence of heterotopic ossification. A diagnosis of FOP can also be confirmed by genetic testing, for example, by detecting the 617G-A (R206H) mutation in the ACVR1 gene.

[0164] FOP is commonly misdiagnosed as a number of other disorders, including other conditions of heterotopic ossification. FOP should be distinguished by differential diagnosis from disorders including isolated congenital malformations, lymphedema, soft tissue sarcomas, desmoid tumors, aggressive juvenile fibromatosis, juvenile bunions, isolated brachydactyly, progressive bone dysplasia, and heterotopic ossification. The presence of congenital malformations and painful soft tissue flare-ups of the thumb can be used to differentiate FOP from other disorders.

[0165] Patients with FOP have congenital malformations of the thumb but otherwise appear normal at birth. FOP-related flare-ups begin in the first decade of life. Flare-ups can be triggered, for example, by soft tissue injury, falls, fatigue, viral infections, or intramuscular injections. The result of a flare-up is the transformation of soft tissues such as ligaments, skeletal muscles, or tendons into heterotopic bone.

[0166] There was no previous therapeutic treatment for FOP or for preventing or reversing FOP-associated HO. FOP was managed through improved safety and preventative measures, including strategies to minimize injury, avoidance of intramuscular injections, and caution when undergoing dental treatment. High-dose corticosteroid treatment initiated within the first 24 hours of a flare-up can help reduce inflammation and edema associated with flare-ups. Surgical strategies to remove heterotopic bone are not recommended because they are counterproductive and can cause new trauma-induced heterotopic ossification.

[0167] "New ectopic ossification," "new ectopic ossification lesion," "new bone lesion," or "new lesion," when used interchangeably herein, refers to ectopic ossification that is not pre-existing in a subject, for example, before or at the time of administration of an activin A antagonist. In one embodiment, new ectopic ossification may be prevented or its volume may be reduced after administration of an activin A antagonist. In one embodiment, new ectopic ossification may develop in a subject after undergoing surgery to remove pre-existing ectopic ossification (and administration of an activin A antagonist may prevent such occurrence). The occurrence of new ectopic ossification lesions can be measured / determined using standard techniques in the art. For example, lesions can be determined, for example, by using positron emission tomography (PET), as discussed in more detail herein.

[0168] The "intensity" and "severity" of new heterotopic ossification lesions refer to any one or more adverse phenotypes used to analyze the formation of new heterotopic ossification lesions, for example, in terms of their activity, intensity, volume, average daily pain, growth and mineralization rate, occurrence of painful flare-ups, and / or number of new heterotopic ossification lesions. The intensity, severity, or activity of new heterotopic ossification lesions can be determined, for example, by the use of positron emission tomography (PET) using 18F-NaF PET. For example, Botman et al., 2019 (Bone. 2019 Jul;124:1-6, incorporated herein by reference in its entirety) describes the use of 18F-NaF PET as a predictor of HO growth in FOP, serving as the basis for assessing active HO lesions specifically associated with high-intensity 18F-NaF PET signals. In one embodiment, lesion activity (LA) = total 18F-NaF signal in the lesion.

[0169] The volume of a single continuous target or new heterotopic ossification lesion can be measured by one or more known methods in the art. In one embodiment, volumetric computed tomography (CT) can be used to measure changes in heterotopic bone formation and determine the volume of new heterotopic ossification lesions.

[0170] Total lesion activity (TLA), as used herein, is defined as the patient-level sum of lesion activity of all targets plus new lesions in the patient at a given time point, and is a measure of the growth and calcification of the HO burden.

[0171] "Bone growth and mineralization activity rate" refers to changes in ectopic bone formation. In one embodiment, 18F-NaF PET can be used to provide a highly sensitive and specific whole-body quantitative measurement of bone growth and mineralization activity. In another embodiment, positron emission tomography (PET) and computed tomography (CT) using 18F-NaF PET can be used to measure changes in bone growth and mineralization activity.

[0172] "Average Daily Pain-NRS" refers to the use of a 0-10 Numerical Rating Scale (NRS) for pain management, averaging pain on one day each week. Pain intensity assessment is considered one of the core outcome domains in clinical pain research (Dworkin et al., 2005, incorporated herein by reference in its entirety). The Numerical Rating Scale (NRS) is considered one of the best single-item methods available for estimating pain intensity (Jensen et al., 1999; Breivik et al., 2000, expressly incorporated herein by reference in their entirety). The NRS assesses pain intensity using a 0-10 ranking scale, with 0 representing "no pain" and 10 representing "unbearable pain" or comparable outcomes, in relation to all lesion activity measures disclosed herein.

[0173] "Flash-up" refers to painful and / or edematous swelling that may precede or accompany heterotopic ossification or new heterotopic ossification. Notably, heterotopic ossification and chronic disease progression have been reported even in the absence of flare-ups. Flare-ups, whether associated with HO or not, are a significant burden for patients with FOP. The reduction in flare-up frequency and intensity after administration of an activin A antagonist indicates that flare-ups in FOP are activin A-related.

[0174] "Existing lesions" refer to lesions that are pre-existing in a subject before and / or at the time of administration, and are not new heterotopic ossification lesions, for example. In one embodiment, administering a therapeutically effective amount of an activin A antagonist to a human subject does not affect the subject's existing lesions. In one embodiment, administering an effective amount of an activin A antagonist to a human subject does not affect the number of existing lesions in the subject compared to the number of existing lesions in the subject before administration. In one embodiment, administering an effective amount of an activin A antagonist to a human subject does not affect the volume of existing lesions in the subject compared to the volume of existing lesions in the subject before administration. In another embodiment, administering a therapeutically effective amount of an activin A antagonist does not reduce the intensity or severity of existing lesions compared to the intensity or severity of existing lesions in the subject before administration. In one embodiment, administering a therapeutically effective amount of an activin A antagonist to a human subject does not reduce the rate of growth of heterotopic ossification lesions of existing lesions and / or the mineralization of existing lesions compared to control subjects. In one embodiment, administering a therapeutically effective amount of an activin A antagonist to a human subject does not reduce the number of existing lesions compared to a control subject. In one embodiment, administering a therapeutically effective amount of an activin A antagonist to a human subject does not reduce the intensity or activity of existing lesions compared to a control subject.

[0175] VII. Treatment Methods Provided herein are methods for treating FOP, comprising administering a therapeutically effective amount of an activin A, ACVR1, ACVR2A, and / or ACVR2B antagonist to a subject with FOP. In one embodiment, FOP is treated by administering a therapeutically effective amount of an activin A antagonist. In one embodiment, FOP is treated by administering a therapeutically effective amount of an antibody against activin A. In one embodiment, therapeutically effective amounts of an ACVR2A antagonist and an ACVR2B antagonist are administered. In a further embodiment, the ACVR2A antagonist is an Fc fusion protein and the ACVR2B antagonist is an Fc fusion protein.

[0176] In one aspect, the present disclosure provides a method for reducing the intensity or severity of new heterotopic ossification lesions in a human subject with fibrodysplasia ossificans progressiva (FOP), the method comprising administering a therapeutically effective amount of an activin A antagonist to the subject with FOP. In one embodiment, the intensity or severity of new heterotopic ossification lesions in the human subject is reduced by at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% compared to a human subject not administered the activin A antagonist.

[0177] "Treating" a subject with FOP means administering a therapeutically effective amount of an antibody to activin A to a subject with FOP for the purpose of curing, curing, alleviating, mitigating, altering, correcting, ameliorating, improving or affecting the condition of one or more symptoms of FOP.

[0178] A "subject" is any animal (i.e., mammal) in which one desires to treat FOP, such as humans, primates, rodents, e.g., mice and rats, agricultural and domesticated animals, e.g., dogs, cats, cows, horses, pigs, sheep, etc. In any of the present methods, the subject may be a mammal, preferably a human.

[0179] "Control" refers to a sample, measurement, or value that serves as a reference for comparison with a subject's sample, measurement, or value. For example, a control can be taken or measured from a subject before administration of an activin A antagonist. In another embodiment, a control can be measured or taken from a subject at the time of administration of an activin A antagonist. A control can also represent an average measurement or value collected from a population of similar individuals. In another embodiment, a control can be an average or median value or measurement collected from a population of individuals with a disease or condition (e.g., FOP). In another embodiment, a control can be an average or median value or measurement collected from a healthy population, e.g., a population without FOP. Those skilled in the art will recognize that controls can be designed for evaluation of any number of parameters disclosed herein, such as HO lesion volume, new HO lesion count, etc.

[0180] A therapeutically effective amount of an activin A, ACVR1, ACVR2A, and / or ACVR2B antagonist refers to a combination of dosage, frequency, and route of administration of the antagonist that produces a positive response in at least one symptom or symptom of FOP. A positive response can include reducing, eliminating, ameliorating, inhibiting, or delaying the worsening of at least one symptom or symptom of FOP. Symptoms or symptoms of FOP that may be subject to a positive response include, for example, ectopic or heterotopic bone formation, FOP flare-ups, or pain and swelling associated with flare-ups. A therapeutically effective amount can be assessed in a single patient by comparing the symptoms and symptoms before and after treatment. If at least one symptom and symptom produces a positive response after treatment, the amount is considered effective. Alternatively or additionally, a therapeutically effective amount can be assessed by comparing the symptoms and symptoms of a population of subjects treated with the antagonist or antagonists of the present disclosure with a control population of untreated subjects. The subjects for such comparison may be animal models, or human subjects in clinical trials (e.g., Phase I, Phase II, Phase IIa, Phase IIb, or Phase III). An amount is considered effective if there is a statistically significant positive response among the population in at least one symptom and condition.

[0181] In one aspect, the present disclosure provides a method of preventing the formation of new heterotopic ossification lesions in a human subject having FOP, the method comprising administering to the human subject a therapeutically effective amount of an activin A antagonist, thereby preventing the formation of new heterotopic ossification lesions in the human subject.

[0182] In another aspect, the disclosure provides a method of reducing the intensity or severity of new heterotopic ossification lesions in a human subject having FOP, the method comprising administering to the human subject a therapeutically effective amount of an activin A antagonist, thereby reducing the intensity or severity of new heterotopic ossification lesions in the human subject.

[0183] In one embodiment, the human subject exhibits at least a 5%, at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 5-80%, at least a 10-80%, at least a 20-80%, at least a 30-80%, at least a 40-80%, at least a 50-80%, at least a 60-80%, at least a 70-80%, at least a 5-70%, at least a 5-60%, at least a 5-50%, at least a 5-40%, at least a 5-30%, at least a 5-20%, or at least a 5-10% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 10% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 20% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 30% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 40% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 50% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 60% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 70% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least an 80% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-80% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 10-80% reduction in the intensity or severity of new heterotopic ossification lesions compared to a control subject.In one embodiment, the human subject exhibits at least a 20-80% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 30-80% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 40-80% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 50-80% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 60-80% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 70-80% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-70% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-60% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-50% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-40% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-30% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-20% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-10% decrease in the intensity or severity of new heterotopic ossification lesions compared to a control subject.

[0184] In one embodiment, the human subject exhibits at least a 5%, at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 5-80%, at least a 10-80%, at least a 20-80%, at least a 30-80%, at least a 40-80%, at least a 50-80%, at least a 60-80%, at least a 70-80%, at least a 5-70%, at least a 5-60%, at least a 5-50%, at least a 5-40%, at least a 5-30%, at least a 5-20%, or at least a 5-10% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 10% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 15% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 20% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 25% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 30% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 40% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 50% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 60% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 70% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least an 80% reduction in total lesion activity of heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-80% reduction in total lesion activity of heterotopic ossification lesions compared to a control subject.In one embodiment, the human subject exhibits at least a 10-80% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 20-80% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 30-80% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 40-80% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 50-80% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 60-80% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 70-80% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-70% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-60% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-50% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-40% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-30% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-20% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-10% decrease in total lesion activity of the heterotopic ossification lesions compared to a control subject.

[0185] In one embodiment, the human subject exhibits about a 0.2-fold, 0.5-fold, 1-fold, 1.5-fold, 2-fold, 3-fold, 0.2-3-fold, 0.5-3-fold, 1-3-fold, 1.5-3-fold, 2-3-fold, 2.5-3-fold, 0.2-2.5-fold, 0.2-2-fold, 0.2-1.5-fold, 0.2-1-fold, or 0.2-0.5-fold decrease in the average daily pain-NRS compared to a control subject. In one embodiment, the human subject exhibits about a 0.2-fold decrease in the average daily pain-NRS compared to a control subject. In one embodiment, the human subject exhibits about a 0.5-fold decrease in the average daily pain-NRS compared to a control subject. In one embodiment, the human subject exhibits about a 1-fold decrease in the average daily pain-NRS compared to a control subject. In one embodiment, the human subject exhibits about a 1.5-fold decrease in the average daily pain-NRS compared to a control subject. In one embodiment, the human subject exhibits about a 2-fold decrease in the average daily pain-NRS compared to the control subject. In one embodiment, the human subject exhibits about a 2.5-fold decrease in the average daily pain-NRS compared to the control subject. In one embodiment, the human subject exhibits about a 3-fold decrease in the average daily pain-NRS compared to the control subject. In one embodiment, the human subject exhibits about a 0.2-3-fold decrease in the average daily pain-NRS compared to the control subject. In one embodiment, the human subject exhibits about a 0.5-3-fold decrease in the average daily pain-NRS compared to the control subject. In one embodiment, the human subject exhibits about a 1-3-fold decrease in the average daily pain-NRS compared to the control subject. In one embodiment, the human subject exhibits about a 1.5-3-fold decrease in the average daily pain-NRS compared to the control subject. In one embodiment, the human subject exhibits about a 2-3-fold decrease in the average daily pain-NRS compared to the control subject. In one embodiment, the human subject exhibits about a 2.5- to 3-fold decrease in the average daily pain-NRS compared to a control subject. In one embodiment, the human subject exhibits about a 0.2- to 2.5-fold decrease in the average daily pain-NRS compared to a control subject. In one embodiment, the human subject exhibits about a 0.2- to 2-fold decrease in the average daily pain-NRS compared to a control subject. In one embodiment, the human subject exhibits about a 0.2- to 1.5-fold decrease in the average daily pain-NRS compared to a control subject. In one embodiment, the human subject exhibits about a 0.2- to 1-fold decrease in the average daily pain-NRS compared to a control subject.In one embodiment, the human subject exhibits about a 0.2-0.5 fold decrease in average daily pain-NRS compared to a control subject.

[0186] In one embodiment, the human subject exhibits at least a 5%, at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 40%, at least a 50%, at least a 5-50%, at least a 10-50%, at least a 20-50%, at least a 30-50%, at least a 40-50%, at least a 5-40%, at least a 5-30%, at least a 5-20%, or at least a 5-10% reduction in the volume of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5% reduction in the volume of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 10% reduction in the volume of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 15% reduction in the volume of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 20% reduction in the volume of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 25% reduction in the volume of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 30% reduction in the volume of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 40% reduction in the volume of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 50% reduction in the volume of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-50% reduction in the volume of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 10-50% reduction in the volume of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 20-50% reduction in the volume of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 30-50% reduction in the volume of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 40-50% reduction in the volume of new heterotopic ossification lesions compared to a control subject, hi one embodiment, the human subject exhibits at least a 5-40% reduction in the volume of new heterotopic ossification lesions compared to a control subject.In one embodiment, the human subject exhibits at least a 5-30% reduction in the volume of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-20% reduction in the volume of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-10% reduction in the volume of new heterotopic ossification lesions compared to a control subject.

[0187] In one embodiment, the human subject exhibits at least a 5%, at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 5-50%, at least a 10-50%, at least a 20-50%, at least a 30-50%, at least a 40-50%, at least a 5-40%, at least a 5-30%, at least a 5-20%, or at least a 5-10% decrease in the rate of growth and mineralization of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5% decrease in the rate of growth and mineralization of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 10% decrease in the rate of growth and mineralization of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 20% decrease in the rate of growth and mineralization of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 30% decrease in the rate of growth and mineralization of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 40% decrease in the rate of growth and mineralization of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 50% decrease in the rate of growth and mineralization of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-50% decrease in the rate of growth and mineralization of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 10-50% decrease in the rate of growth and mineralization of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 20-50% decrease in the rate of growth and mineralization of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 30-50% decrease in the rate of growth and mineralization of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 40-50% reduction in the rate of growth and mineralization of new heterotopic ossification lesions compared to control subjects, hi one embodiment, the human subject exhibits at least a 5-40% reduction in the rate of growth and mineralization of new heterotopic ossification lesions compared to control subjects.In one embodiment, the human subject exhibits at least a 5-30% decrease in the rate of growth and mineralization of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-20% decrease in the rate of growth and mineralization of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-10% decrease in the rate of growth and mineralization of new heterotopic ossification lesions compared to a control subject.

[0188] In one embodiment, the human subject exhibits at least a 5%, at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 5-50%, at least a 10-50%, at least a 20-50%, at least a 30-50%, at least a 40-50%, at least a 5-40%, at least a 5-30%, at least a 5-20%, or at least a 5-10% decrease in the intensity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5% decrease in the intensity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 10% decrease in the intensity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 20% decrease in the intensity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 30% decrease in the intensity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 40% decrease in the intensity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 50% decrease in the intensity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-50% decrease in the intensity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 10-50% decrease in the intensity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 20-50% decrease in the intensity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 30-50% decrease in the intensity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 40-50% decrease in the intensity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-40% decrease in the intensity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-30% decrease in the intensity of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-20% decrease in the intensity of new heterotopic ossification lesions compared to a control subject.In one embodiment, the human subject exhibits at least a 5-10% decrease in the intensity of new heterotopic ossification lesions compared to control subjects.

[0189] In one embodiment, the human subject exhibits at least a 5%, at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, at least a 5-90%, at least a 10-90%, at least a 20-90%, at least a 30-90%, at least a 40-90%, at least a 50-90%, at least a 60-90%, at least a 70-90%, at least a 80-90%, at least a 5-80%, at least a 5-70%, at least a 5-60%, at least a 5-50%, at least a 5-40%, at least a 5-30%, at least a 5-20%, or at least a 5-10% reduction in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5% reduction in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 10% decrease in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 20% decrease in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 30% decrease in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 40% decrease in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 50% decrease in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 60% decrease in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 70% decrease in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least an 80% decrease in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 90% decrease in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-90% reduction in the number of new heterotopic ossification lesions compared to a control subject, hi one embodiment, the human subject exhibits at least a 10-90% reduction in the number of new heterotopic ossification lesions compared to a control subject.In one embodiment, the human subject exhibits at least a 20-90% reduction in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 30-90% reduction in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 40-90% reduction in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 50-90% reduction in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 60-90% reduction in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 70-90% reduction in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least an 80-90% reduction in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 50-80% reduction in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-70% decrease in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-60% decrease in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-50% decrease in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-40% decrease in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-30% decrease in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-20% decrease in the number of new heterotopic ossification lesions compared to a control subject. In one embodiment, the human subject exhibits at least a 5-10% decrease in the number of new heterotopic ossification lesions compared to a control subject.

[0190] In some methods for treating FOP, the subject does not have or is not at risk for other conditions treatable with an antagonist to activin A, ACVR1, ACVR2A and / or ACVR2B. For example, the subject may be free of any or all of type II diabetes, muscular dystrophy, amyotrophic lateral sclerosis (ALS), and osteoporosis.

[0191] A. Method of Administration Activin A, ACVR1, ACVR2A and / or ACVR2B antagonists are usually administered directly as proteins or small molecules, but in the case of proteins, they can also be administered as nucleic acids encoding such proteins. Such antagonists can be administered by various methods, such as cell transfection, gene therapy, direct administration by a delivery vehicle or a pharmaceutically acceptable carrier, or indirect delivery by providing recombinant cells containing a nucleic acid encoding an activin A, ACVR1, ACVR2A and / or ACVR2B antagonist, or an antibody against activin A.

[0192] A variety of delivery systems can be used to administer the activin A, ACVR1, ACVR2A and / or ACVR2B antagonists, or antibodies to activin A provided herein, such as liposomes, microparticles, microcapsules, encapsulation in recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retrovirus or other vector, and the like.

[0193] The method of administration may be enteral or parenteral, including intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, pulmonary, intranasal, intraocular, epidural, and oral routes. The compound may be administered by any convenient route, for example, by infusion or bolus injection, or by absorption through epithelial or mucocutaneous linings (e.g., oral, rectal, and intestinal mucosa), and may be administered together with other biologically active agents. Administration may be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the present disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection, which may be facilitated, for example, by an intraventricular catheter connected to a reservoir, such as an Omcana reservoir. Pulmonary administration may also be employed, for example, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

[0194] The pharmaceutical compositions of the present disclosure can be administered locally to the area in need of treatment, which can be achieved, for example, by local infusion during surgery, topical application, e.g., by injection, by catheter, or by implants, which are porous, non-porous, or gel-like materials, including membranes such as sialastic membranes, fibers, or commercially available skin substitutes.

[0195] In another embodiment, the active agent can be delivered in a vesicle, particularly a liposome (see Langer (1990) Science 249:1527-1533). In another embodiment, the active agent can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer (1990) supra). In another embodiment, a polymeric material can be used (see Howard et al. (1989) J. Neurosurg. 71:105). In another embodiment in which the active agent of the present disclosure is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, for example, by using a retroviral vector (see, e.g., U.S. Pat. No. 4,980,286), or by direct injection, or by using microparticle bombardment, or by coating with lipids or cell surface receptors or transfection agents, or by administering it bound to a homeobox-like peptide known to enter the nucleus (e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc. Alternatively, the nucleic acid can be introduced intracellularly for expression and integrated into the host cell DNA by homologous recombination.

[0196] B. Combination Therapy The activin A, ACVR1, ACVR2A and ACVR2B antagonists, or antibodies against activin A provided herein can be administered in combination with each other or with other treatments. In one embodiment, a method for treating FOP involves the combined administration of an ACVR2A antagonist and an ACVR2B antagonist. In another embodiment, a method for treating FOP involves the combined administration of an ACVR1, ACVR2A, and ACVR2B antagonist. In other embodiments, an ACVR1 antagonist can be administered in combination with an ACVR2A and / or ACVR2B antagonist. The ACVR1, ACVR2A, and ACVR2B antagonists can be administered as separate pharmaceutical compositions, or as a single pharmaceutical composition containing a combination of these agents. The ACVR1, ACVR2A, and / or ACVR2B antagonists, or antibodies against activin A, can be administered alone or in combination with one or more additional therapeutic compounds. The combination therapy may involve simultaneous or alternating administration. In addition, the combination may involve acute or chronic administration.

[0197] C. Pharmaceutical Compositions The present disclosure also provides pharmaceutical compositions comprising an activin A, ACVR1, ACVR2A and / or ACVR2B antagonist, or an antibody to activin A, provided herein, and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0198] These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions can be formulated as suppositories with traditional binders and carriers such as triglycerides. Oral formulations can include standard pharmaceutical-grade carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0199] In one embodiment, the composition is formulated according to standard procedures as a pharmaceutical composition suitable for intravenous administration to humans.If necessary, the composition can also contain a solubilizing agent and a local anesthetic such as lidocaine to alleviate pain at the injection site.When the composition is administered by injection, it can be dispensed in an infusion bottle containing sterile pharmaceutical-grade water or saline.When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided, allowing the components to be mixed before administration.

[0200] The activin A, ACVR1, ACVR2A, and / or ACVR2B antagonists or antibodies to activin A provided herein can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with free carboxyl groups, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0201] The amount and frequency of administration of an activin A, ACVR1, ACVR2A and / or ACVR2B antagonist, or an antibody to activin A administered by a particular route effective for treating FOP (e.g., a therapeutically effective amount) can be determined by standard clinical techniques based on the present disclosure. Additionally, in vitro assays can be used to help identify optimal dosage ranges. The precise dose employed in a formulation will also depend on the route of administration and the severity of the condition, and should be determined according to the judgment of the practitioner and each subject's circumstances. However, suitable dosage ranges for parenteral administration, preferably intravenous or subcutaneous administration, generally range from about 20 to 50,000 micrograms of active compound per kilogram of body weight. For antibodies to activin A, suitable dosage ranges include 1 to 25 mg / kg, 2 to 20 mg / kg, 5 to 15 mg / kg, 8 to 12 mg / kg, and 10 mg / kg. Suitable dosage ranges for intranasal administration are generally from about 0.01 pg / kg to 1 mg / kg of body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0202] The frequency of administration also varies depending on, among other factors, the severity of the condition and the half-life of the drug, but is typically between daily and quarterly, including, for example, twice weekly, weekly, biweekly, monthly, or every two months. The drug can also be administered at irregular intervals in response to, among other factors, the patient's condition or a decline in serum levels of the drug below a threshold.

[0203] All patent applications, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference herein in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be incorporated by reference. Where different versions of a sequence are associated with an accession number at different times, the version associated with that accession number as of the effective filing date of this application is meant. Effective filing date means the earlier of the actual filing date for the accession number or the filing date of the priority application, if applicable. Similarly, where different versions of a publication, website, etc. are published at different times, the version published closest to the effective filing date of this application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the present disclosure can be used in combination with any other, unless otherwise specified. [Example]

[0204] Example 1: Reducing the formation of new bone lesion activity in patients with fibrodysplasia ossificans progressiva (FOP) In subjects with FOP, bone forms in soft tissues outside of the normal skeleton, a process known as ectopic (misplaced) ossification (HO). A pivotal, double-blind, placebo-controlled trial was conducted to evaluate an activin A antagonist (e.g., galetumab) in patients with FOP. The primary endpoint was a 25% reduction from baseline in new HO bone growth, as measured by CT scans, and a 25% reduction in the mean rate of lesion growth and mineralization over 28 weeks, as measured by more sensitive PET bone scans. Galetumab demonstrated a statistically significant 50% reduction in PET bone scan results only at 28 weeks (post-hoc analysis), primarily driven by a significant reduction in the incidence and intensity of new lesions. Galetumab reduced the incidence of new bone lesions by approximately 90% and halved the incidence of patient-reported flare-ups, as measured by both PET and CT scans.

[0205] The results of this placebo-controlled trial demonstrate an effective treatment that can nearly eliminate new bone formation and flare-ups, reducing the rate of new bone formation by nearly 90%, a groundbreaking result for people with FOP. These data also significantly improve our understanding of the disease by showing that untreated people with FOP experience much more frequent and widespread lesions than previously thought, as seen with PET imaging.

[0206] This study enrolled 44 adult patients (aged 18-60 years) from the United States and Europe with a clinical diagnosis of FOP and documented ACVR1 gene mutations. This study used 18F-NaF PET imaging and CT scans to investigate the effect of garetsumab on changes in HO in patients with FOP. 18F-NaF is a highly sensitive, widely approved, and widely used bone-seeking PET tracer for detecting abnormal bone growth, turnover, and mineralization in several bone-related diseases, such as Paget's disease and bone-involving cancers. The study had a three-period design consisting of a randomized, double-blind, placebo-controlled treatment period (6 months), an open-label treatment period (during which placebo-treated patients were crossed over to garetsumab treatment) (6 months), and an open-label follow-up treatment period. The primary analysis was recorded at week 28.

[0207] The major clinical outcomes from this study are summarized below in Table 1. All endpoints were at 28 weeks and were assessed by blinded independent central review. [Table 1-1] [Table 1-2] [Table 1-3]

[0208] During the 28-week treatment period, garetumab was generally well tolerated. Any serious adverse events (SAEs) were considered related to the severity of the underlying disease. Treatment-emergent adverse events (TEAEs) occurred in 100% of patients in both the placebo and treatment groups. The majority were mild to moderate in severity. Notable imbalances in TEAEs included nosebleeds (50.0% vs. 16.7%) and skin events (sleeping [eyebrow hair loss, 25.0% vs. 0%], acne (30.0% vs. 8.3%), and a mix of skin infections, including abscesses, furuncles, folliculitis, and furuncles). Two treated patients in the open-label portion of the study developed severe abscesses requiring hospitalization for drainage, which reportedly resolved while continuing garetumab treatment. One patient died in the open-label portion of the study from trauma unrelated to treatment.

[0209] Compared to placebo, garetusumab reduced the percent change from baseline in total lesion activity at the primary endpoints of weeks 8 and 28 (-24.6, p < 0.07), with no reduction at the week 8 endpoint and a 49% reduction at the week 28 endpoint (end of DB period) (nominal p = 0.043). This was primarily driven by a dramatic and significant reduction in the incidence, intensity, and lesion activity of new lesions. Total lesion activity in new lesions (measured by PET at week 28) decreased by 97% (p = 0.009), while total volume of new lesions (measured by CT at week 28) decreased by 90% (p = 0.017). There was a significant reduction in patient-reported flare-ups (51%) and investigator-reported AE flare-ups (76%). Treatment with garetusumab also demonstrated an acceptable safety profile.

[0210] Blockade of activin A with activin A antagonists (e.g., galetusumab) can significantly reduce new abnormal bone formation and the occurrence of painful flare-ups. These data strongly support the hypothesis that activin A is a key trigger of both flare-ups and new bone lesions in patients with FOP.

[0211] Example 2: Activin A antagonists prevent new heterotopic bone formation (HO) A 28-week randomized, double-blind, placebo-controlled study (Period 1) was conducted to test whether an activin A antagonist (e.g., galetusumab) would stop activin A-dependent signaling for ectopic bone formation in human subjects with FOP.

[0212] Positron emission tomography (PET) and volumetric computed tomography (CT) using 18F-NaF PET were used to measure changes in ectopic bone formation. 18F-NaF PET has been shown in several other disease settings to provide a sensitive and specific whole-body quantitative measurement of bone mineralization activity. Data published by Botman, 2019 (incorporated herein by reference in its entirety) support the use of 18F-NaF PET as a predictor of HO growth in FOP and serve as the basis for the evaluation of active HO lesions specifically associated with high-intensity 18F-NaF PET signals. Furthermore, volumetric assessment of HO lesions by CT over a 6-month period also allowed for the assessment of HO lesion growth and the transition from PET-detectable lesions to CT-detectable mature HO lesions. Garetosumab 18 It was observed to rapidly reduce F-NaF uptake (by PET) and prevent new ectopic bone formation (by CT).

[0213] The study demonstrated a 57% reduction in mean total lesion activity (PET) at 28 weeks, 18The study was designed with 80% statistical power to detect a 40% reduction in F-NaF SUV maximum and a 60% difference in total volume by CT. 100% of the 44 patients enrolled had active HO at baseline, as detected by PET. The results indicate that garetumab inhibited the appearance of new lesions but did not halt the progression of established lesions (Table 2). Garetumab was also observed to be well tolerated. SAEs occurred more frequently in garetumab-treated patients, without a trend, likely reflecting disease severity. Notable imbalances in AEs included epistaxis (50.0% vs. 16.7%), and a mix of skin events—scabbing (25.0% vs. 0%), acne (30.0% vs. 8.3%), and skin infections (abscesses, furuncles, folliculitis, and furuncles).

[0214] As shown in the schematic diagram of the study design (Figure 1), this study consisted of a screening / baseline period (Day -28 to Day -1), two 6-month treatment periods, and a follow-up treatment period (Period 3). The three treatment periods were: Period 1: a 6-month randomized, double-blind, placebo-controlled treatment period; Period 2: a 6-month open-label galetusumab treatment period; and Period 3: a follow-up treatment period with galetusumab that continued until patients completed the 76-week visit. All data were collected and validated over the time period until the last patient randomized within this study completed the 28-week visit (Period 1), and the results of the primary safety and efficacy analyses were available to the sponsor.

[0215] Emerging data (Eekhoff et al., JBMR 2017 & Bone 2017, Upadhyay et al., JBMR 2017, each of which is incorporated herein by reference in its entirety) suggested that PET scans could identify "active" bone lesions. As a surrogate for preventing new lesions, we studied the effect of garetumab on these "active" bone lesions. Therefore, the primary endpoint assessed "total lesion activity" (TLA) measured by PET in both existing and potentially new bone lesions, as assessed by size and intensity (evaluated uptake of the tracer). Multiple other endpoints captured total and new lesions, as assessed by both PET and CT, as well as flare-ups reported by patients and investigators. The primary analysis (TLA by PET), as well as multiple other analyses of bone lesions (including by CT), showed that treatment with garetumab was associated with an overall reduction of approximately 25% compared with placebo. These reductions were almost entirely driven by an approximately 90% reduction in new lesions assessed by PET or CT at week 28. This, surprisingly, contributed to the higher frequency of new lesions in FOP patients (11 / 24 placebo patients had an average of 2.7 new lesions over 28 weeks, compared with 3 / 20 galetusumab patients, who each had one lesion). There was also a significant reduction in patient-reported flare-ups (50%) and investigator-reported AE flare-ups (76%). These data indicate that activin A is required for the formation of new bone lesions in FOP patients, but does not appear to play a major role in pre-existing PET-positive and CT-positive bone lesions. [Table 2]

[0216] Imaging diagnosis Radiolabeled 18F sodium fluoride was injected into the patients, and combined PET / CT images were acquired for each patient. Details of the PET / CT imaging analysis are shown below.

[0217] PET: Standardized uptake value (SUV) is a measure of radioactivity from the scan. SUV average = average concentration of 18F-NaF in a region of interest (ROI), such as heterotopic ossification (HO), on average—how "hot" the ROI is. SUV maximum = maximum ("hottest") pixel within the ROI. Lesion activity (LA) = total signal of 18F-NaF in the lesion. Average SUV across the total metabolic volume (MV) of the lesion on PET. Total lesion activity (TLA) = patient-level sum of LA for all targets and new lesions in the patient at a given time point—a measure of HO burden growth and calcification. Time-weighted average (TWA) of TLA % change = average over 28 weeks of TLA % change used to assess changes in HO growth and calcification burden across multiple time points. PET imaging clearly demonstrated disease progression (Figure 2A) and the effect of galetusumab on HO lesions (Figure 2B).

[0218] CT: HO volume = volume of a single contiguous target or new HO lesion. Total HO volume = sum of patient-level target and HO lesion volumes at a given time point—measurements formed the HO burden.

[0219] Results from imaging analysis indicate that the effect of garetosumab is more evident when pre-existing ("target") lesions are examined separately from new lesions (Figures 3A-B).

[0220] Effectiveness Garetusumab was observed to reduce total lesion activity (both new and pre-existing) from baseline (-25%, p=0.074) when averaged across the 8-week and 28-week time points. At the 8-week endpoint, there was no reduction (p=0.592), whereas at the 28-week endpoint (end of the DB period), there was a 49% reduction (nominal p=0.043). This was primarily driven by a dramatic reduction in the incidence, activity (PET) and volume (CT) of new lesions. Total lesion activity in new lesions (as measured by PET at 28 weeks) decreased by 97% (p=0.009), while total volume of new lesions (as measured by CT at 28 weeks) decreased by 90% (p=0.017). All endpoints were prespecified (except for the number of patients with new HO lesions by CT) and assessed by blinded independent central review. Lesions were assessed by PET / CT bone scan. A 100% correlation of new lesions was determined by PET scan measurements with simultaneous CT scan evaluation. There was also a significant reduction in patient-reported flare-ups (51%) and investigator-reported AE flare-ups (76%). These data strongly support the notion that activin A is a necessary ligand for the formation of new lesions in patients with FOP. Activin A does not appear to play a significant role in the progression of existing PET / CT lesions. Therefore, blocking activin A offers an opportunity to alter disease outcomes in these long-term patients.

[0221] safety During the 28-week treatment period, treatment-emergent adverse events (TEAEs) occurred in 100% of patients in both the placebo and treatment groups. The majority of TEAEs were mild to moderate in severity. Notable imbalances in TEAEs included nosebleeds (50.0% vs. 16.7%) and skin events (sleeping [eyebrow hair loss, 25.0% vs. 0%], acne [30.0% vs. 8.3%], and a mix of skin infections, including abscesses, furuncles, folliculitis, and furuncles).

[0222] Example 3: Study Design and Interpretation How the study design differs from other studies A phase 2, randomized, double-blind, placebo-controlled trial was conducted to evaluate the safety, tolerability, and effect of intravenous garetusumab (10 mg / kg administered every 4 weeks) on heterotopic bone formation in adults with FOP, eliminating potential imbalances and biases associated with external historical controls and open-label evaluations.

[0223] What does heterotopic ossification (HO) volume tell us about the disease? Progressive HO is a defining feature of FOP. HO volume can reflect disease activity and the likelihood of joint immobilization. The location of HO can be important to patients and clinically meaningful. While bone volume is related to directional movement, the number and location of lesions are important measures of disease activity. In this study, the primary endpoint focuses on total lesion activity versus placebo. For example, a small amount of bone from a new lesion in a joint, such as the elbow, can lock the joint for life, but in areas with less critical movement or pre-existing HO, significant amounts of bone may be added, and the effect may be much less pronounced. Additionally, interpreting changes in "mean volume" can be challenging when analyzing datasets containing one or two outliers with small or large volume gains.

[0224] Rationale for diagnostic imaging Imaging analysis Lesions were identified as HO by their density and morphology, which were consistent with abnormal bone, and their location, which was consistent with skeletal muscle, tendons, or ligaments as identified on CT scans. They were identified by two independent readers blinded to the specimens and confirmed by an independent reviewer, also blinded. All of them were board-certified radiologists.

[0225] Criteria for lesion selection and rationale for study design and imaging analysis The criteria for lesion selection were clearly defined in the Diagnostic Imaging Charter and approved by the FDA. Additional post-hoc imaging analysis was performed to better understand the natural history of FOP and the effect of garetosumab on HO formation. The "maximum of seven lesions" was specified because many lesions were not expected and exceeding this number was not practical for the reader. Lesions were selected based on having three times the intensity on PET compared with normotopic bone remodeling.

[0226] Exclude false positives due to inflammatory osteochondroma or osteoarthritis The Diagnostic Imaging Charter instructed the two independent readers and independent reviewers, who were board-certified radiologists, to carefully avoid areas on the PET associated with non-HO lesions in terms of their location (e.g., joints) or their characteristics from the CT scan, whenever consistent with degenerative disease, osteochondroma, etc. All readers were also trained on the Diagnostic Imaging Charter.

[0227] for detecting osteoblast activity 18 Bone scintigraphy compared with FNaF PET / CT Technetium-99m methylenediphosphonate ( 99m TcMDP) bone scintigraphy is widely used to detect osteoblast activity. PET / CT is a molecular imaging technique that combines cross-sectional functional and anatomical imaging for diagnosis. Fluorine-18 fluoride ( 18 F-fluoride) is a sensitive bone-seeking PET tracer used to detect skeletal abnormalities. 18 The mechanism of F-fluoride uptake is 99m The uptake mechanism is similar to that of TcMDP. However, 18 F-fluoride exhibits superior pharmacokinetic characteristics, including more rapid blood clearance and a twofold higher uptake in bone. 18F-fluoride uptake reflects blood flow and bone remodeling. The use of a new hybrid PET / CT system is advantageous because the CT component of this study allows for morphological characterization of functional lesions and allows for more accurate differentiation between benign lesions and metastases. 18 F-fluoride imaging significantly improved its specificity.

[0228] Fluorine-18 sodium fluoride positron emission tomography-computed tomography ( 18 FNaF PET / CT) imaging diagnosis is 99m Compared with TcMDP bone scintigraphy, it has higher sensitivity and higher spatial resolution. 18 FNaF PET / CT images are of better quality and can be more accurately quantified due to lower plasma protein binding and higher bone uptake.

[0229] 18 FNaF PET is a more sensitive technique commonly used in research. 18 FNaF PET was used as the benchmark in this study to assess whether CT imaging would produce comparable results. 18 It can monitor disease progression with a sensitivity comparable to FNaF PET, thus confirming its ability to be used in clinical practice.

[0230] Clinical Data The trial is based on the hypothesis that blocking activin A will prevent new lesion formation, and the initial 28-week results overall are encouraging.

[0231] A longer-term 56-week study will be conducted, with placebo patients crossing over to the garetumab arm to further understand and contextualize these results. The primary effect of garetumab is its effect on preventing new lesions. The primary endpoint data elegantly demonstrate that TLA was clearly reduced after 8 weeks of treatment, when more new lesions appeared in the placebo group compared to the garetumab group. On average, the placebo group experienced approximately 10 cm reduction over the 28-week study period compared to the garetumab group. 3 Achieved a greater new HO (mean change from baseline 16.7 cm 3 6.5cm 3 ).

[0232] Because a high percentage of patients with FOP die as a result of chest failure, it is of interest to evaluate the impact of treatment with garetumab on lung function. The data show that garetumab preserves lung function compared with placebo by preventing new HO at 28 weeks.

[0233] Impact of Garetosumab on FOP The inhibitory level of galetusumab must be consistent over a long period of time to prevent the progression of established HO lesions or to prevent new HO lesions. FOP is a chronic disease with ongoing HO, some of which is asymptomatic and not associated with flare-ups. Therefore, to control it, medication must be long-term. The drug level in the patient's serum must always exceed the activin A saturation level to prevent the progression or development of new HO.

[0234] Pediatric trials A 56-week Phase 2 trial will be conducted to evaluate both pediatric and adult patients.

[0235] Unlike palovarotene, preclinical data with galetusumab did not show an effect on the growth plate. This data is consistent with the data in normal bone of adult FOP patients. 18This shows that there was no change in FNaF uptake.

[0236] A dose level of 10 mg / kg administered IV every 4 weeks (Q4W) has been tested in adult patients with FOP. The adverse effects reported in this study (e.g., headache, epistaxis, skin and soft tissue infections) do not appear to be related to drug exposure (Cmax, Cmin, or AUC) among patients. The reason for including a dose confirmation cohort (Cohort A) in the pediatric study is not related to safety concerns but rather to selecting a dose regimen for each weight group (i.e., patients <30 kg or >30 kg) that achieves an exposure in pediatric patients similar to that associated with efficacy in adult patients in this study.

[0237] The following dosing regimens have been proposed for pediatric trials in patients with FOP: 15 mg / kg IV Q4W for patients weighing less than 30 kg and 10 mg / kg IV Q4W for patients weighing 30 kg or more. In an earlier study, 10 mg / kg IV Q4W, the dose tested in adult patients with FOP, demonstrated beneficial efficacy responses and an acceptable safety profile. In this study, the median (range) steady-state trough concentration after administration of 10 mg / kg IV Q4W was 120.7 (68.1–199.0) mg / L in adult patients with FOP. Based on galetusumab and total activin A concentration data generated in early studies and in two first-in-human (FIH) studies, maintaining galetusumab concentrations above approximately 50 mg / L is associated with saturation of target-mediated elimination pathways, as indicated by constant levels of total activin A of approximately 0.05-0.06 mg / L. The exposure observed in adult patients using the 10 mg / kg IV Q4W dose appears to provide a significant efficacy benefit by maintaining saturation of target-mediated elimination pathways with maximal target engagement in the majority of patients.

[0238] In pediatric patients with FOP, simulations using a population PK model based on data from healthy adult subjects and adult patients with FOP suggest that the proposed dose for Cohort A (15 mg / kg IV Q4W for patients weighing less than 30 kg and 10 mg / kg IV Q4W for patients weighing 30 kg or more) is expected to match exposure in adult patients at 10 mg / kg IV Q4W, as studied in earlier studies. Based on the results of analyses from the eight patients who received active drug in Cohort A, doses may be adjusted in one or more weight groups to ensure drug exposure comparable to adults in earlier studies. Cohort B patients will begin receiving study drug only after dose confirmation based on data from Days 1 to 85 in patients enrolled in Cohort A. Different doses will be explored in FOP because the goal is target saturation to ensure efficacy. Pediatric modeling leads to doses up to 15 mg / mL to meet target saturation in younger children. There are no data from toxicology studies suggesting increased safety risks in children.

[0239] Overview of the PET and CT Scan Imaging Procedures The PET / CT scans were processed in a pre-specified, independent, blinded manner. After Week 28, patients in the placebo arm were crossed over to galetusumab, and all study participants received the active drug thereafter. To conduct the primary analysis of this study, study treatment assignment during the double-blind, placebo-controlled period (Period 1) was unblinded. However, it is important to note that individual treatment assignments during Period 1 were not disclosed to the investigator / site staff, nor to the independent imaging reader or imaging reviewer. The sponsor was also blinded to the imaging results and did not have access to the scans until database lock at Week 56. This allowed the imaging reader to perform a blinded analysis of the PET / CT scans at Week 56. Furthermore, between Weeks 28 and 56, and beyond (for most patients, beyond Week 76), the investigators continued clinical evaluation of study patients without knowledge of what they received during the placebo-controlled period.

[0240] Example 4: Activin A antagonists prevent new heterotopic bone formation (HO) research design A phase 2, randomized, double-blind, placebo-controlled study was designed to evaluate the safety, tolerability, pharmacokinetics, and efficacy of 10 mg / kg garetusumab administered every 4 weeks (Q4W) in adult patients with fibrodysplasia ossificans progressiva (FOP). Efficacy was assessed by comparing the efficacy of garetusumab with that of ectopic (HO) bone formation. 18 F-NaF positron emission tomography (PET) and low-dose X-ray computed tomography (CT) imaging analyses. Compared to placebo, this study was expected to demonstrate that a) garetumab is well tolerated, b) garetumab reduces HO by demonstrating a reduction in PET signal and inhibition of HO growth by volumetric CT, and c) garetumab inhibits the progression of new HO lesions as assessed by PET and CT.

[0241] The study consisted of a 4-week screening / baseline period, a 6-month randomized, double-blind, placebo-controlled treatment period (Period 1), a 6-month open-label galetusumab treatment period (Period 2), and a follow-up treatment period with open-label galetusumab (Period 3). The primary analysis was performed when all patients completed double-blind treatment (Period 1). All primary and secondary 28-week efficacy endpoints were analyzed for the active HO analysis set (AHO, n=44), which included patients with any FOP-associated ACVR1 mutation, and for the active HO ACVR1[R206H] mutation set (AHOC, n=42). The imaging charter for this study defined active patients as those with a pelvic floor injury (PEA) of at least 3x the normal reference bone, as assessed by central review. 18 Patients were defined as having at least one heterotopic ossification lesion demonstrating F-NaF PET uptake. Similarly, the criteria for identifying new HO lesions by PET and CT were prespecified in the Diagnostic Imaging Charter. Safety analyses were performed in the safety analysis set and included all data available as of the data cut-off date.

[0242] Patient breakdown A total of 44 patients were randomized (20 patients in the galetusumab arm and 24 patients in the placebo arm). 43 (98%) patients completed the double-blind treatment period. One patient from the galetusumab group discontinued the study during the double-blind period due to a fever adverse event (see below for details). All 43 patients who completed the double-blind period entered the open-label second period. As of the data cutoff date, 12 patients entered the third period of follow-up treatment. One patient in the third period died due to a severe head injury (unrelated to treatment) after a fall. Demographic and baseline disease characteristics of the AHO, AHOC, and SAF populations were balanced between the two treatment groups.

[0243] Efficacy Results Results of all primary and key secondary endpoints tested using statistical strata are shown in Table 3 (in order of statistical strata). Garetusumab significantly reduced mean total lesion activity ( 18F-NaF PET (based on PET) reduced total lesion activity by approximately 25% (LS mean difference) (p=0.0741). Post-hoc analysis indicated that reductions in total lesion activity may not occur at a consistent rate. The percent change in total lesion activity from baseline to week 8 was similar in both treatment groups, and the percent change in total lesion activity from baseline to week 28 was lower in the galetusumab group compared with placebo (post-hoc, p=0.043, Figure 4). Regarding other endpoints, in AHO, an approximately 25% reduction from baseline (LS mean difference) was also observed in total HO lesion volume (by CT) at week 28 compared with placebo (p=0.3726). Similar results were observed in the AHOC population. In AHO, there was a favorable trend for reduction in average daily pain in the galetusumab arm compared with placebo (Figure 5). Similar results were observed in AHOC. The reduction in total lesion activity by PET in the garetumab arm compared with placebo, and the reduction in total HO lesion volume by CT, was primarily driven by the efficacy of garetumab in reducing new lesion growth. [Table 3] AHO = patients with at least one active HO lesion, AHOC = patients with at least one active HO lesion and the classic ACVR1R206H mutation, ANCOVA = analysis of covariance, MMRM = mixed model with repeated measures. Assessments provided by the selected reader who was reviewed at week 28 were used for the primary analysis (if no review was performed at week 28, reader 1 was selected).

[0244] Analysis of inhibition of new lesions by garetosumab The following analysis of the key prespecified secondary exploratory post-hoc analyses in the AHO population (Table 4) shows that the reductions in total lesion activity on PET and total volume of HO lesions on CT in the garetumab arm compared with placebo were primarily driven by a nearly 90% reduction in multiple endpoints related to new HO lesion growth, including the incidence, rate, lesion activity (PET), and volume (CT) of new HO lesions. The number of new PET lesions over 28 weeks was substantially reduced in the garetumab arm compared with placebo (87.4% reduction in the rate of new lesions). The number of new PET lesions was significantly lower in the garetumab arm compared with placebo (3 vs. 29). The mean total lesion activity per patient related to new lesions was lower in the garetumab arm compared with placebo (5.22 / pt vs. 205.99 / pt). Total lesion activity associated with new lesions in patients who developed new PET lesions in the garetumab group was also substantially lower compared to the placebo group (Figure 6). The rate of patients who developed new PET lesions over 28 weeks was also lower in the garetumab arm compared to placebo (15% vs. 46%, Figure 7). The number of new CT lesions over 28 weeks was lower in the garetumab arm compared to placebo (86.7% reduction in the rate of new lesions). There were 27 new CT lesions in the placebo group compared to 3 new CT lesions in the garetumab group. The mean total CT volume per patient associated with new lesions was lower in the garetumab group compared to placebo (1.06 cm). 3 / pt vs 10.21cm 3 / pt). Total CT volume associated with new lesions in patients who developed new CT lesions was also lower in the galetumab arm compared to the placebo group (Figure 8). The rate of patients who developed new CT lesions over 28 weeks was also lower in the galetumab arm compared to placebo (15% vs. 46%, Figure 9). Additionally, the proportion of patients with flare-ups, as assessed by patient diary, was favorable, as investigator-reported adverse events of flare-ups were lower in the galetumab arm compared to placebo. Similar results were observed in the AHOC population. Table 4

[0245] Security Results During the 28-week double-blind treatment period, treatment-emergent adverse events (TEAEs) occurred in 100% of patients in both the garetumab and placebo groups. The majority of TEAEs were mild to moderate in severity. Notable imbalances in TEAEs between the garetumab and placebo groups included nosebleeds (50.0% vs. 16.7%) and skin events (sleeping [eyebrow loss, 25.0% vs. 0%], acne [30.0% vs. 8.3%], and a mix of skin infections, including abscesses, furuncles, folliculitis, and furuncles). When patients in the placebo group were crossed over to garetumab during the ongoing open-label period (Period 2), the frequency of nosebleeds and the above-mentioned skin events increased. The frequency of acute infusion reactions during the 28-week double-blind period was balanced between the placebo and galetumab groups (placebo: 6 / 24 (25%), galetumab: 5 / 20 (25%)). During the second period, four patients (9.3%) experienced acute infusion reactions while receiving galetumab. Serious adverse events (SAEs) were reported in 20% and 8.3% of the galetumab- and placebo-treated groups, respectively, during the first period. One SAE (nosebleed) in a patient assigned to the galetumab group that occurred during the first period resulted in hospitalization due to nasal tamponade. This event was characterized as a suspected unexpected serious adverse reaction (SUSAR). The patient made a full recovery and continued in the study. During the open-label period, Three serious adverse events (SAEs) have been reported to date. Two patients developed an abscess SAE during Period 2 requiring hospitalization for incision and drainage (subcutaneous and cystic abscesses were reported by the investigator as study drug-related and unrelated, respectively). The abscesses resolved, and the patient continued receiving garetumab after a temporary treatment interruption. A third patient had an SAE of severe head injury after a fall and subsequently died. The head injury occurred during Period 3 after the patient had received 16 garetumab infusions and was assessed by the investigator as unrelated to study drug. Nineteen of the 20 garetumab-treated patients and 24 of the 24 placebo-treated patients completed Period 1.The patient who discontinued garetusumab during the 28-week treatment period had an adverse event of fever, which the investigator assessed as mild in severity and unrelated to the study drug. The recurrence of fever after the patient had two SAEs (hospitalizations) for pneumonia, one of which was complicated by sepsis, led the investigator to discontinue the patient from the study. Other clinical features of the patient's medical history included severe restrictive lung disease, bronchiectasis, and skeletal deformities. One patient died during the open-label follow-up treatment period (Period 3) due to a head injury unrelated to treatment.

[0246] conclusion Garetusumab reduced total lesion activity (new and pre-existing) from baseline over 28 weeks (time-weighted mean difference of approximately 25%, p=0.0741). This was primarily driven by a roughly 90% reduction in the incidence, rate, and activity of new lesions (PET) and volume (CT). In a very rare condition like FOP, where it is not possible to conduct a large enough study to ensure sufficient statistical power, it is common to accept a 10% type I error rate to avoid a type II error. In this light, the overall efficacy results are compelling and strongly support the use of garetusumab to reduce the formation of new HO in patients with FOP. These results also support the interpretation that activin A does not appear to play a significant role in the progression of pre-existing HO lesions as detected by PET / CT at baseline. Treatment with garetusumab demonstrated an acceptable safety profile, with a low incidence of acute infusion reactions and serious adverse events (SAEs). The majority of TEAEs were mild to moderate in severity. Notable imbalances in TEAEs included epistaxis, acne, and alopecia, as well as a mix of skin infections, including abscesses, cysts, folliculitis, and furuncles. In this ongoing study, all patients have progressed to the open-label period (Periods 2 and 3). Crossover of placebo patients to garetumab allows confirmation at week 56 that garetumab treatment leads to a significant reduction in new bone lesion formation compared with control ("control" being measurements from these same patients during the prior placebo treatment period). The 56-week data also provide information on the durability of treatment effects in patients who continue garetumab treatment. Collectively, the safety and efficacy data from this study demonstrate a positive benefit-risk profile for garetumab in the treatment of adult patients with FOP. Blockade of activin A with garetumab offers an opportunity to alter the course of disease in long-standing FOP patients.

[0247] Test Purpose Main purpose The primary safety objective of this study was to evaluate the safety and tolerability of garetusumab in patients with FOP. The primary efficacy objective of this study was to evaluate the efficacy and safety of garetusumab in patients with FOP. 18 The objective of this study was to evaluate the effect of garetosumab versus placebo on the change from baseline in HO in patients with FOP, as determined by F-NaF uptake and total volume of HO lesions by CT.

[0248] Secondary Objectives The secondary objectives of this study were: To compare the effect of garetosumab versus placebo on pain due to FOP, as measured by the area under the curve (AUC) of pain based on daily pain NRS scores. ● 18 To evaluate the effect of garetosumab versus placebo on the change from baseline in HO as determined by the number of new HO lesions identified by F-NaF PET or CT. ● PET analysis of individual active HO sites 18 To evaluate the effect of garetosumab versus placebo on the change from baseline in F-NaF standardized maximum uptake value (SUV max). In patients who switched from placebo to garetosumab at week 28, for the same patient between baseline and week 28: 18 To evaluate the effect of garetosumab between weeks 28 and 56 on the number, activity, and volume of HO lesions identified by F-NaF PET or CT. To assess the effect of garetosumab versus placebo on change from baseline in biochemical markers of bone formation. • To characterize total activin A concentrations at baseline and over time after the first dose of study drug. To characterize the concentration-time (pharmacokinetic [PK]) profile of garetosumab in patients with FOP. • To evaluate the immunogenicity of garetosumab.

[0249] research design This was a phase 2, randomized, double-blind, placebo-controlled study designed to evaluate the safety, tolerability, PK, and effect on heterotopic bone formation of repeated doses of 10 mg / kg IV garetusumab Q4W in adult patients with FOP. As shown in the schematic diagram of the study design (Figure 1), the study consisted of a screening / baseline period (days -28 to -1), two 6-month treatment periods, and a follow-up treatment period (period 3).

[0250] During the screening / baseline period, all patients underwent an informed consent process and screening / baseline procedures. In the double-blind treatment period (Period 1), patients were randomized to receive garetusumab at a dose of 10 mg / kg or matching placebo, administered IC Q4W for a total of seven doses through Week 24. Patients were randomized according to gender, classic ACVR1 [R206H] mutation / alternative ACVR1 mutation, and 18 Randomization was stratified by the presence or absence of baseline active HO lesions as determined by F-NaF-PET / CT. During the open-label treatment period (Period 2), all patients who completed the double-blind treatment period received garetumab administered intravenously at a dose of 10 mg / kg Q4W for a total of seven doses through Week 52. Patients who completed Period 2 continued to receive garetumab until Week 76 or later in Period 3. Imaging procedures were performed at baseline, Weeks 8, 28, 56, and 76.

[0251] statistical methods Analysis population Following guidance from the International Conference of Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guideline ICH E9 Statistical Principles for Clinical Trials (1998), the following analysis populations were used for all statistical analyses:

[0252] Baseline-active HO analysis set (AHO): The baseline-active HO analysis set (AHO) included all randomized patients who had at least one active HO lesion at baseline, based on the treatment they were assigned (randomized).

[0253] Baseline-Active HO Classical ACVR1 [R206H] Mutation Analysis Set (AHOC): The Baseline-Active HO Classical ACVR1 [R206H] Mutation Analysis Set (AHOC) included all randomized patients who had a classic ACVR1 [R206H] mutation and had at least one active HO lesion at baseline, as defined by a positive 18F-NaF PET scan, based on their assigned treatment (randomization).

[0254] Safety Analysis Set (SAF): The Safety Analysis Set (SAF) included all randomized patients who received any study medication. The actual treatment received was used for safety analyses.

[0255] Analysis of efficacy variables Study efficacy variables consisted of assessments from imaging procedures, clinical endpoints, and biomarkers of bone formation.

[0256] Multiplicity considerations: This study had four primary efficacy objectives and two secondary efficacy objectives. To control the type I error rate, a hierarchical testing procedure was applied with a two-sided 5% significance level, and the primary secondary efficacy endpoints were tested only when statistical significance was established for all primary endpoints. The order of the testing sequence for the primary and secondary efficacy endpoints was as follows: [Table 5]

[0257] Primary efficacy endpoint:

[0258] Over 28 weeks in AHO 18 Time-weighted mean percent change from baseline (standardized AUC) in total lesion activity by F-NaF PET The AUC of the percent change from baseline in total lesion activity by 18F-Naf PET over 28 weeks was calculated for each patient. Nominal times (i.e., weeks 8 and 28) were used to calculate AUC, rather than actual times. If the week 8 imaging scan was missing, linear interpolation of the percent change between baseline and week 28 was used to calculate AUC. If the week 28 imaging scan was missing, the percent change at week 8 was carried forward to week 28 to calculate AUC. The time-weighted average (standardized AUC) was calculated over 28 weeks. 18 The mean change in LS mean change from baseline was derived by dividing the AUC of the percent change from baseline in total lesion activity by F-Naf PET by 28. The time-weighted mean of percent change over 28 weeks was analyzed using an analysis of covariance (ANCOVA) model. The model included treatment, sex, and baseline total lesion activity as covariates. Because all but two patients had classic ACVR1 mutations, ACVR1 mutations were excluded from the model. The difference in LS mean change from baseline, the corresponding 95% CI, and p-value were provided from the ANCOVA model for the comparison of the galetosumab group to the placebo group.

[0259] Percent change from baseline in total HO lesion volume assessed by CT at 28 weeks in AHO The percent change from baseline in total HO lesion volume assessed by CT at weeks 8 and 28 in AHO was analyzed in the AHO analysis set using the MMRM model. The model included interactions between treatment, sex, ACVR1 variant type (classic, non-classic), visit (weeks 8 and 28), baseline total volume, and treatment by visit. Unstructured covariance was used to account for within-patient correlation across time points. The difference in LS mean change from baseline, corresponding 95% CI, and p-value were provided by the MMRM model for the comparison of the garetosumab group to the placebo group.

[0260] AHOC, over 28 weeks 18 Time-weighted mean percent change from baseline (standardized AUC) in total lesion activity by F-NaF PET AHOC, over 28 weeks 18 The time-weighted mean percent change from baseline in total lesion activity by F-NaF PET (standardized AUC) was analyzed using the same method as for the first primary endpoint in AHO. The model included the independent variables of treatment, sex, visit (weeks 8 and 28), treatment-by-visit interaction, and baseline total lesion activity.

[0261] Percent change from baseline in total HO lesion volume assessed by CT at week 28 in AHOC The percent change from baseline in total HO lesion volume assessed by CT at week 28 in AHO was analyzed in the same manner as the second primary endpoint in AHO. The model included treatment, sex, visit (weeks 8 and 28), baseline total volume, and the interaction of treatment by visit.

[0262] Key secondary efficacy endpoints: Time-weighted mean (standardized AUC) change from baseline in daily pain attributable to FOP measured using a daily NRS over 28 weeks in AHO The time-weighted mean change from baseline in daily pain scores (average of current pain, worst pain, and least pain) was calculated for each patient. If pain scores for intermediate days were missing, the time-weighted mean was calculated using linear interpolation of the change between two adjacent measurements. If monotonically missing, the missing value was imputed using the last observed value after baseline (last observation carried forward (LOCF)). In AHO, an ANCOVA model was used to analyze the time-weighted mean change in daily pain over 28 weeks. The model included treatment, sex, ACVR1 variant type (classic, non-classic), and baseline daily pain score.

[0263] Time-weighted mean (standardized AUC) change from baseline in daily pain attributable to FOP measured using a daily NRS over 28 weeks in AHOC The time-weighted mean (standardized AUC) change from baseline in daily pain attributable to FOP, measured using a daily NRS over 28 weeks, in AHOC was analyzed in the same way as the previous key secondary endpoint (in AHO). The model included treatment, sex, and baseline daily pain score.

[0264] Safety Data Analysis A summary of safety and tolerability was performed for all patients in the SAF. Safety analyses were based on reported AEs, clinical laboratory assessments, and vital signs. Thresholds for treatment-emergent potentially clinically significant values ​​(PCSV) for laboratory variables and vital signs were defined in the SAP. The baseline for determining treatment-emergent PCSV represents the baseline for the current study.

[0265] 28-week trial extension Study participants who received garetumumab continued to receive it through week 56 in the study extension. Additionally, study participants who received placebo received garetumumab during the extension period. Placebo patients who received garetumumab during the extension period experienced reductions in the number of new lesions, lesion volume, and pain scores similar to those seen in the treatment group during the blinded period (i.e., through week 28).

[0266] Unofficial sequence listing SEQ ID NO: 1 [ka] SEQ ID NO: 2 GGSFSSHF SEQ ID NO: 3 ILYTGGT SEQ ID NO:4 ARARSGITFTGIIVPGSFDI SEQ ID NO:5 [ka] SEQ ID NO:6 QSVSSSY SEQ ID NO:7 GAS SEQ ID NO:8 QQYGSSPWT SEQ ID NO:9 EVQLVESGGGLVQPGRSLRLSCKASGFAFDDFAMHWVRQAPGKGLEWVSGIVWNSGDIGY ADSVKGRFTISRDNAKNSLYLQLNSLRTEDTALYFCVKDMVRGLMGNYYGMDVWGQGTT VTVSS SEQ ID NO: 10 GFAFDDFA SEQ ID NO: 11 IVWNSGDI SEQ ID NO: 12 VKDMVRGLMGFNYYGMDV SEQ ID NO: 13 EIVLTQSPATLSLSPGERATLSCRASQTISTYLVWYRQRPGQAPSLLIYDASNRATDIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK SEQ ID NO: 14 QTISTY SEQ ID NO: 15 DAS SEQ ID NO: 16 QQRSNWPIT SEQ ID NO: 17 SYEVTQAPSVSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPVLVIYQDSKRPSGIPER FSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTAVFGGGTKLTVL SEQ ID NO: 18 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGLSWVRQAPGQGLEWMGWIIPYNGNTNS AQKLQGRVTMTTDTSTSSTAYMELRSLRSDDTAVYFCARDRDYGVNYDAFDIWGQGTMVTV SS SEQ ID NO: 19 SGDKLGDKYAC SEQ ID NO: 20 QDSKRPS SEQ ID NO: 21 QAWDSSTAV SEQ ID NO: 22 GYTFTSYGLS SEQ ID NO: 23 WIIPYNGNTNSAQKLQG SEQ ID NO: 24 DRDYGVNYDAFDI

Claims

[Claim 1] The invention described in the specification.

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