Methods for Treating Heterotopic Ossification - Patent application

Abstract

In part, the present disclosure relates to a method for treating heterotopic ossification or one or more complications of heterotopic ossification.In particular, the present disclosure provides a method for treating heterotopic ossification by administering one or more TGFβ antagonists to a patient in need thereof, optionally in combination with one or more additional active agents or supportive care for treating heterotopic ossification.For example, in some embodiments, the present disclosure provides a method for treating heterotopic ossification by administering an effective amount of TβRII polypeptide to a patient in need thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 628,649, filed February 9, 2018, and U.S. Provisional Application No. 62 / 666,235, filed May 3, 2018. The foregoing applications are incorporated herein by reference in their entireties. [Background technology]

[0002] Heterotopic ossification (HO) is the pathological formation of extra-skeletal bone in soft tissue. Vanden Bossche and Vanderstraeten, J Rehabil Med 27, 129 (2005). This process generally occurs in patient populations with severe trauma, including large surface area burns, musculoskeletal injuries, orthopedic surgery and spinal cord injuries, as well as in patient populations with a genetic disease known as fibrodysplasia ossificans progressiva (FOP). FOP is caused by a hyperactivating mutation in the ACVR1 type I bone morphogenetic protein (BMP) receptor, and patients with FOP develop heterotopic bone lesions in the absence of any substantial trauma. The clinical sequelae of these pathological heterotopic bone formations include non-healing wounds, chronic pain and joint immobility, regardless of the circumstances of trauma or genetic mutation. In the case of FOP, progressive ossification can lead to death due to loss of rib cage extensibility. Treatment options for HO are limited because the bone often recurs after surgical resection, and some patients may have unresectable HO due to its sensate location. The risks of surgery, especially in the face of recurrence, may outweigh the benefits of resection. Thus, there is a high unmet need for effective therapies to treat HO. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Vanden Bossche and Vanderstraeten, J Rehabil Med 27, 129 (2005) Summary of the Invention [Means for solving the problem]

[0004] In part, the present disclosure relates to the discovery that TGFβ antagonists (inhibitors) can be used to treat heterotopic ossification (HO). In particular, the data presented herein show that soluble TβRII polypeptides that bind to TGFβ1 and TGFβ3 are effective in attenuating the development of HO anlagen with reduced bone deposition and formation of bone marrow cavity in a mouse model of HO. Furthermore, treatment with TβRII polypeptides significantly reduced the volume of mature HO in this animal model. Without wishing to be bound by any particular mechanism, it is expected that the effect of TβRII polypeptides is mainly caused by the antagonistic effect of TGFβ signaling, particularly the antagonistic effect of TGFβ1 and / or TGFβ3 signaling. Therefore, other TGFβ antagonists (e.g., antagonists of TβRII receptors, antagonists of one or more TβRII binding ligands (e.g., TGFβ1, TGFβ2 and TGFβ3), antagonists of one or more TβRII-related type I receptors (e.g., ALK5), antagonists of one or more TβRII-related co-receptors (betaglycan), antagonists of one or more downstream signaling components of TβRII (e.g., Smads), or combinations of such antagonists) are expected to be useful in treating HO. Such agents are collectively referred to herein as "TGFβ antagonists" or "TGFβ inhibitors".

[0005] In certain aspects, the present disclosure relates to a method for treating HO, comprising administering an effective amount of a transforming growth factor-β (TGFβ) antagonist to a patient in need thereof. In some embodiments, the method prevents or reduces the severity and / or duration of one or more complications of HO. For example, treatment with a TGFβ antagonist can prevent or reduce the severity and / or duration of one or more complications of HO selected from the group consisting of joint contracture, joint ankylosis, pain, spasticity, swelling fever, neurovascular compression syndrome and bedsore. In some embodiments, the method relates to treating HO associated with one or more disorders or conditions selected from the group consisting of spinal cord injury, trauma, brain injury, burn, fracture, muscle strain, joint arthroplasty / joint replacement, hip surgery / total hip replacement, acetabular surgery / acetabular replacement, elbow fracture, fracture of long bones of lower leg, combat-related trauma, limb amputation, neuromuscular blockade used to manage adult respiratory distress syndrome, non-traumatic myelopathy. In some embodiments, the present disclosure relates to a method for treating fibrodysplasia ossificans progressiva (FOP), comprising administering an effective amount of a TGFβ antagonist to a patient in need thereof. For example, the method prevents or reduces the severity and / or duration of one or more complications of FOP (e.g., heterotopic ossification). In some embodiments, the present disclosure relates to a method for treating fibrodysplasia ossificans progressiva (FOP), comprising administering an effective amount of a TGFβ antagonist to a patient in need thereof. For example, the method prevents or reduces the severity and / or duration of one or more complications of FOP (e.g., heterotopic ossification). In some embodiments, the present disclosure relates to a method for treating fibrodysplasia ossificans progressiva (FOP), comprising administering an effective amount of a TGFβ antagonist to a patient in need thereof. For example, the method prevents or reduces the severity and / or duration of one or more complications of fibrodysplasia (e.g., heterotopic ossification). In some embodiments, the heterotopic ossification occurs in one or more tissues selected from the group consisting of bone, skin, subcutaneous tissue, skeletal muscle, fibrotic tissue in the vicinity of a joint, blood vessel wall, and ligament. Optionally, such methods described herein further comprise administering to the patient an effective amount of one or more additional active agents or supportive care for treating HO.For example, such additional active agents or supportive therapies include isotretinoin, etidronate and oral corticosteroids, perhexiline maleate, ALK2 small molecule inhibitors, palovarotene, retinoic acid receptor gamma agonists, retinoic acid receptor alpha agonists, activin antibodies (e.g., activin A antibodies such as REGN 2477), ALK2 allele-specific RNA interference, bisphosphonates, radiation therapy, anti-inflammatory agents (e.g., indomethacin, ibuprofen and aspirin) and conservative treatments such as passive range of motion exercises or other mobilization techniques. In some embodiments, the additional active agent or supportive therapies are treatments with activin antagonists. In some embodiments, the activin antagonist is an activin antibody. In some embodiments, the activin antibody is an activin A antibody. In some embodiments, the TGFβ antagonist is a multispecific antibody that binds to one or more of TGFβ1, TGFβ2, and TGFβ3 and inhibits signal transduction mediated by one or more of these, and further binds to and inhibits activin (e.g., activin A).

[0006] In certain aspects, the TGFβ antagonist or combination of antagonists used according to the methods and uses described herein is an agent that inhibits at least TGFβ1 (e.g., a TGFβ1 antagonist). The effect on TGFβ1 inhibition can be determined using, for example, a cell-based assay (e.g., a Smad signaling assay), including the assays described herein. Thus, in some embodiments, the TGFβ antagonist or combination of antagonists disclosed herein can bind to at least TGFβ1. Ligand binding activity can be determined using, for example, a binding affinity assay, including the assays described herein. In some embodiments, the TGFβ antagonist or combination of antagonists disclosed herein can bind to at least 1×10 -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1 × 10 -10 M, at least 1 × 10 -11M, or at least 1 × 10 -12 M)K D and binds to at least TGFβ1. As described herein, various TGFβ antagonists that inhibit TGFβ1 can be used according to the methods and uses described herein, including, for example, ligand traps (e.g., TβRII polypeptides and variants thereof), antibodies, small molecules, nucleotide sequences, and combinations thereof. In certain embodiments, a TGFβ antagonist or combination of antagonists that inhibits TGFβ1 can further inhibit one or more of TGFβ2, TGFβ3, TβRII, ALK5, and betaglycan. In some embodiments, a TGFβ antagonist or combination of antagonists that inhibits TGFβ1 further inhibits TGFβ3. In some embodiments, a TGFβ antagonist or combination of antagonists that inhibits TGFβ1 does not inhibit TGFβ2 or does not substantially inhibit TGFβ2. In some embodiments, a TGFβ antagonist or combination of antagonists that inhibits TGFβ1 further inhibits TGFβ3 but does not inhibit TGFβ2 or does not substantially inhibit TGFβ2.

[0007] In certain aspects, the TGFβ antagonist or combination of antagonists used according to the methods and uses described herein is an agent that inhibits at least TGFβ2 (e.g., a TGFβ2 antagonist). The effect on TGFβ2 inhibition can be determined, for example, using a cell-based assay (e.g., a Smad signaling assay), including the assays described herein. Thus, in some embodiments, the TGFβ antagonist or combination of antagonists disclosed herein can bind to at least TGFβ2. Ligand binding activity can be determined, for example, using a binding affinity assay, including the assays described herein. In some embodiments, the TGFβ antagonist or combination of antagonists disclosed herein can bind to at least 1×10 -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1 × 10 -10M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)K D and binds to at least TGFβ2. As described herein, various TGFβ antagonists that inhibit TGFβ2 can be used according to the methods and uses described herein, including, for example, ligand traps (e.g., TβRII polypeptides and variants thereof), antibodies, small molecules, nucleotide sequences, and combinations thereof. In certain embodiments, the TGFβ antagonist or combination of antagonists that inhibit TGFβ2 can further inhibit one or more of TGFβ1, TGFβ3, TβRII, ALK5, and betaglycan.

[0008] In certain aspects, the TGFβ antagonist or combination of antagonists used according to the methods and uses described herein is an agent that inhibits at least TGFβ3 (e.g., a TGFβ3 antagonist). The effect on TGFβ3 inhibition can be determined, for example, using a cell-based assay (e.g., a Smad signaling assay), including the assays described herein. Thus, in some embodiments, the TGFβ antagonist or combination of antagonists disclosed herein can bind to at least TGFβ3. Ligand binding activity can be determined, for example, using a binding affinity assay, including the assays described herein. In some embodiments, the TGFβ antagonist or combination of antagonists disclosed herein can bind to at least 1×10 -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1 × 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)K Dand binds to at least TGFβ3. As described herein, various TGFβ antagonists that inhibit TGFβ3 can be used according to the methods and uses described herein, including, for example, ligand traps (e.g., TβRII polypeptides and variants thereof), antibodies, small molecules, nucleotide sequences, and combinations thereof. In certain embodiments, a TGFβ antagonist or combination of antagonists that inhibits TGFβ3 can further inhibit one or more of TGFβ1, TGFβ2, TβRII, ALK5, and betaglycan. In some embodiments, a TGFβ antagonist or combination of antagonists that inhibits TGFβ3 further inhibits TGFβ1. In some embodiments, a TGFβ antagonist or combination of antagonists that inhibits TGFβ3 does not inhibit TGFβ2 or does not substantially inhibit TGFβ2. In some embodiments, a TGFβ antagonist or combination of antagonists that inhibits TGFβ3 further inhibits TGFβ1 but does not inhibit TGFβ2 or does not substantially inhibit TGFβ2.

[0009] In certain aspects, the TGFβ antagonist or combination of antagonists used according to the methods and uses described herein is an agent that inhibits at least TβRII (e.g., a TβRII receptor antagonist). The effect on TβRII inhibition can be determined using, for example, a cell-based assay (e.g., a Smad signaling assay), including the assays described herein. Thus, in some embodiments, the TGFβ antagonist or combination of antagonists disclosed herein can bind to at least TβRII. Ligand binding activity can be determined using, for example, a binding affinity assay, including the assays described herein. In some embodiments, the TGFβ antagonist or combination of antagonists disclosed herein can bind to at least 1×10 -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1 × 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10-12 M)K D and binds to at least TβRII. As described herein, various TGFβ antagonists that inhibit TβRII can be used according to the methods and uses described herein, including, for example, ligand traps (e.g., TβRII polypeptides and variants thereof), antibodies, small molecules, nucleotide sequences, and combinations thereof. In certain embodiments, the TGFβ antagonist or combination of antagonists that inhibit TβRII can further inhibit one or more of TGFβ1, TGFβ2, TGFβ3, ALK5, and betaglycan. In some embodiments, the TGFβ antagonist or combination of antagonists that inhibit TβRII does not inhibit TGFβ2 or does not substantially inhibit TGFβ2.

[0010] In certain aspects, the TGFβ antagonist or combination of antagonists used according to the methods and uses described herein is an agent that inhibits at least ALK5 (e.g., an ALK5 antagonist). The effect on ALK5 inhibition can be determined, for example, using a cell-based assay (e.g., a Smad signaling assay), including the assays described herein. Thus, in some embodiments, the TGFβ antagonist or combination of antagonists disclosed herein can bind to at least ALK5. Ligand binding activity can be determined, for example, using a binding affinity assay, including the assays described herein. In some embodiments, the ALK5 antagonist or combination of antagonists disclosed herein can bind to at least 1×10 -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1 × 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)K Dand binds to at least ALK5. As described herein, various TGFβ antagonists that inhibit ALK5 can be used according to the methods and uses described herein, including, for example, ligand traps (e.g., TβRII polypeptides and variants thereof), antibodies, small molecules, nucleotide sequences, and combinations thereof. In certain embodiments, the TGFβ antagonist or combination of antagonists that inhibit ALK5 can further inhibit one or more of TGFβ1, TGFβ2, TGFβ3, TβRII, and betaglycan. In some embodiments, the TGFβ antagonist or combination of antagonists that inhibit ALK5 does not inhibit TGFβ2 or does not substantially inhibit TGFβ2.

[0011] In certain aspects, the TGFβ antagonist or combination of antagonists used according to the methods and uses described herein is an agent that inhibits at least betaglycan (e.g., a betaglycan antagonist). The effect on betaglycan inhibition can be determined, for example, using a cell-based assay (e.g., a Smad signaling assay), including the assays described herein. Thus, in some embodiments, the TGFβ antagonist or combination of antagonists disclosed herein can bind at least betaglycan. The ligand binding activity can be determined, for example, using a binding affinity assay, including the assays described herein. In some embodiments, the betaglycan antagonist or combination of antagonists disclosed herein can bind at least 1×10 -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1 × 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)K Dand binds to at least betaglycan. As described herein, various TGFβ antagonists that inhibit betaglycan can be used according to the methods and uses described herein, including, for example, ligand traps (e.g., TβRII polypeptides and variants thereof), antibodies, small molecules, nucleotide sequences, and combinations thereof. In certain embodiments, the TGFβ antagonist or combination of antagonists that inhibit betaglycan can further inhibit one or more of TGFβ1, TGFβ2, TGFβ3, TβRII, and ALK5. In some embodiments, the TGFβ antagonist or combination of antagonists that inhibit betaglycan does not inhibit TGFβ2 or does not substantially inhibit TGFβ2.

[0012] In certain aspects, the disclosure provides TβRII polypeptides and the use of such TβRII polypeptides as selective antagonists for TGFβ1 and / or TGFβ3. As described herein, polypeptides comprising part or all of the extracellular domain (ECD) of TβRII bind to and / or inhibit TGFβ1 and / or TGFβ3 with altered affinity, with or without additional mutations. Thus, in certain aspects, the disclosure provides TβRII polypeptides for use in selectively inhibiting TGFβ superfamily-related disorders.

[0013] In part, the disclosure provides fusion proteins of TβRII polypeptides and the use of such fusion proteins as selective antagonists for TGFβ1 or TGFβ3. As described herein, polypeptides comprising part or all of the extracellular domain (ECD) of TβRII bind and / or inhibit TGFβ1 or TGFβ3 with altered affinity, with or without additional mutations. In particular, TβRII polypeptides comprising a heterologous portion (e.g., an Fc immunoglobulin domain) and a linker of at least 10 amino acids in length (e.g., a linker having the amino acid sequence of SEQ ID NO:6) are associated with surprisingly superior TGFβ1 and TGFβ3 binding properties compared to TβRII polypeptides having shorter linkers. Thus, in certain aspects, the disclosure provides TβRII polypeptides for use in selectively inhibiting TGFβ superfamily-related disorders.

[0014] In some embodiments, the disclosure provides a transforming growth factor-beta receptor II (TβRII) fusion polypeptide comprising: a) an extracellular domain of a TβRII portion; b) a heterologous portion; and c) a linker portion, wherein the linker is at least 10 amino acids in length, and wherein the extracellular domain of the TβRII portion comprises an amino acid sequence at least 80% identical to i) a sequence starting at any of positions 23-35 of SEQ ID NO:1 and ending at any of positions 153-159 of SEQ ID NO:1, or ii) a sequence starting at any of positions 23-60 of SEQ ID NO:2 and ending at any of positions 178-184 of SEQ ID NO:2. In some embodiments, the extracellular domain of the TβRII portion comprises an amino acid sequence at least 80% identical to a sequence starting at any of positions 23-35 of SEQ ID NO:1 and ending at any of positions 153-159 of SEQ ID NO:1. In some embodiments, the extracellular domain of the TβRII portion comprises an amino acid sequence that is at least 90% identical to a sequence that starts at any of positions 23-35 of SEQ ID NO:1 and ends at any of positions 153-159 of SEQ ID NO:1. In some embodiments, the extracellular domain of the TβRII portion comprises an amino acid sequence that is at least 95% identical to a sequence that starts at any of positions 23-35 of SEQ ID NO:1 and ends at any of positions 153-159 of SEQ ID NO:1. In some embodiments, the extracellular domain of the TβRII portion comprises an amino acid sequence that is at least 97% identical to a sequence that starts at any of positions 23-35 of SEQ ID NO:1 and ends at any of positions 153-159 of SEQ ID NO:1. In some embodiments, the extracellular domain of the TβRII portion comprises an amino acid sequence that starts at any of positions 23-35 of SEQ ID NO:1 and ends at any of positions 153-159 of SEQ ID NO:1. In some embodiments, the extracellular domain of the TβRII portion comprises an amino acid sequence that is at least 80% identical to a sequence starting at any of positions 23-60 of SEQ ID NO:2 and ending at any of positions 178-184 of SEQ ID NO:2. In some embodiments, the extracellular domain of the TβRII portion comprises an amino acid sequence that is at least 90% identical to a sequence starting at any of positions 23-60 of SEQ ID NO:2 and ending at any of positions 178-184 of SEQ ID NO:2.In some embodiments, the extracellular domain of the TβRII portion comprises an amino acid sequence that is at least 95% identical to a sequence that starts at any of positions 23-60 of SEQ ID NO:2 and ends at any of positions 178-184 of SEQ ID NO:2. In some embodiments, the extracellular domain of the TβRII portion comprises an amino acid sequence that is at least 97% identical to a sequence that starts at any of positions 23-60 of SEQ ID NO:2 and ends at any of positions 178-184 of SEQ ID NO:2. In some embodiments, the extracellular domain of the TβRII portion comprises an amino acid sequence that starts at any of positions 23-60 of SEQ ID NO:2 and ends at any of positions 178-184 of SEQ ID NO:2. In some embodiments, the extracellular domain of the TβRII portion comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:18. In some embodiments, the extracellular domain of the TβRII portion comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:18. In some embodiments, the extracellular domain of the TβRII portion comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:18. In some embodiments, the extracellular domain of the TβRII portion comprises an amino acid sequence at least 97% identical to SEQ ID NO: 18. In some embodiments, the extracellular domain of the TβRII portion comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the extracellular domain of the TβRII portion comprises an amino acid sequence at least 80% identical to a sequence starting at any of positions 23-35 of SEQ ID NO: 1 and ending at any of positions 153-159 of SEQ ID NO: 1. In some embodiments, the extracellular domain of the TβRII portion comprises an amino acid sequence at least 90% identical to a sequence starting at any of positions 23-35 of SEQ ID NO: 1 and ending at any of positions 153-159 of SEQ ID NO: 1. In some embodiments, the extracellular domain of the TβRII portion comprises an amino acid sequence at least 95% identical to a sequence starting at any of positions 23-35 of SEQ ID NO: 1 and ending at any of positions 153-159 of SEQ ID NO: 1. In some embodiments, the extracellular domain of the TβRII portion consists of an amino acid sequence that is at least 97% identical to a sequence starting at any of positions 23-35 of SEQ ID NO:1 and ending at any of positions 153-159 of SEQ ID NO:1.In some embodiments, the extracellular domain of the TβRII portion consists of an amino acid sequence starting at any of positions 23-35 of SEQ ID NO:1 and ending at any of positions 153-159 of SEQ ID NO:1. In some embodiments, the extracellular domain of the TβRII portion consists of an amino acid sequence that is at least 80% identical to a sequence that starts at any of positions 23-60 of SEQ ID NO:2 and ends at any of positions 178-184 of SEQ ID NO:2. In some embodiments, the extracellular domain of the TβRII portion consists of an amino acid sequence that is at least 90% identical to a sequence that starts at any of positions 23-60 of SEQ ID NO:2 and ends at any of positions 178-184 of SEQ ID NO:2. In some embodiments, the extracellular domain of the TβRII portion consists of an amino acid sequence that is at least 95% identical to a sequence that starts at any of positions 23-60 of SEQ ID NO:2 and ends at any of positions 178-184 of SEQ ID NO:2. In some embodiments, the extracellular domain of the TβRII portion consists of an amino acid sequence that is at least 97% identical to a sequence that starts at any of positions 23-60 of SEQ ID NO:2 and ends at any of positions 178-184 of SEQ ID NO:2. In some embodiments, the extracellular domain of the TβRII portion consists of an amino acid sequence beginning at any one of positions 23-60 of SEQ ID NO:2 and ending at any one of positions 178-184 of SEQ ID NO:2. In some embodiments, the extracellular domain of the TβRII portion consists of an amino acid sequence that is at least 80% identical to SEQ ID NO:18. In some embodiments, the extracellular domain of the TβRII portion consists of an amino acid sequence that is at least 90% identical to SEQ ID NO:18. In some embodiments, the extracellular domain of the TβRII portion consists of an amino acid sequence that is at least 95% identical to SEQ ID NO:18. In some embodiments, the extracellular domain of the TβRII portion consists of an amino acid sequence that is at least 97% identical to SEQ ID NO:18. In some embodiments, the extracellular domain of the TβRII portion consists of the amino acid sequence of SEQ ID NO:18. In some embodiments, the polypeptide comprises an N-terminal leader sequence. In some embodiments, the N-terminal leader sequence comprises the amino acid sequence of any one of SEQ ID NOs:22-24. In some embodiments, the N-terminal leader sequence comprises the amino acid sequence of SEQ ID NO:23. In some embodiments, the heterologous moiety is an immunoglobulin Fc domain. In some embodiments, the immunoglobulin Fc domain is a human immunoglobulin Fc domain. In some embodiments, the heterologous moiety comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:49.In some embodiments, the heterologous portion comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 49. In some embodiments, the heterologous portion comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 49. In some embodiments, the heterologous portion comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 49. In some embodiments, the heterologous portion comprises the amino acid sequence of SEQ ID NO: 49. In some embodiments, the linker is less than 25 amino acids in length. In some embodiments, the linker is 10-25 amino acids in length. In some embodiments, the linker is 15-25 amino acids in length. In some embodiments, the linker is 17-22 amino acids in length. In some embodiments, the linker is 21 amino acids in length. In some embodiments, the linker comprises (GGGGS)n, where n=≧2. In some embodiments, the linker comprises (GGGGS)n, where n=≧3. In some embodiments, the linker comprises (GGGGS)n, where n=≧4. In some embodiments, the linker comprises (GGGGS)n, where n ≠ ≧ 5. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs: 4-7. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 13.In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:63. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:63. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:63. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:63. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:48. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:48. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:48. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:48. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:65. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:65. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:65. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:65. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:68. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:68. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:68. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:68. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:15. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:15. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:15. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:15.In some embodiments, a TβRII polypeptide does not include amino acids 185-592 of SEQ ID NO: 2. In some embodiments, a TβRII polypeptide does not include amino acids 1-22 of SEQ ID NO:2. In some embodiments, the polypeptide consists of, or consists essentially of, a) a TβRII polypeptide portion comprising an amino acid sequence at least 85%, 90%, 95%, 97% or 99% identical to the amino acid sequence of SEQ ID NO:18, and no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids; b) a linker portion comprising an amino acid sequence at least 85%, 90%, 95%, 97% or 99% identical to the amino acid sequence of SEQ ID NO:6, and no more than 5, 4, 3, 2 or 1 additional amino acids; c) a heterologous portion comprising an amino acid sequence at least 85%, 90%, 95%, 97% or 99% identical to the amino acid sequence of SEQ ID NO:49, and no more than 25, 20, 15, 10, 5, 4, 3, 2 or 1 additional amino acids; and d) optionally, a leader sequence (e.g., SEQ ID NO:23). In some embodiments, the polypeptide consists of, or essentially consists of, a) a TβRII polypeptide portion comprising the amino acid sequence of SEQ ID NO:18 and not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids; b) a linker portion comprising the amino acid sequence of SEQ ID NO:6 and not more than 5, 4, 3, 2 or 1 additional amino acids; c) a heterologous portion comprising the amino acid sequence of SEQ ID NO:49 and not more than 25, 20, 15, 10, 5, 4, 3, 2 or 1 additional amino acids; and d) optionally a leader sequence (e.g., SEQ ID NO:23). In some embodiments, the polypeptide comprises: a) an extracellular domain of a TβRII portion comprising an amino acid sequence that is at least 85%, 90%, 95%, 97% or 99% identical to the sequence of SEQ ID NO: 18; b) a heterologous portion comprising an amino acid sequence that is at least 85%, 90%, 95%, 97% or 99% identical to the sequence of SEQ ID NO: 49; and c) a linker portion connecting the extracellular domain and the heterologous portion, wherein the linker comprises an amino acid sequence that is at least 85%, 90%, 95%, 97% or 99% identical to the amino acid sequence of SEQ ID NO: 6.In some embodiments, the polypeptide comprises: a) an extracellular domain of the TβRII portion comprising the amino acid sequence of SEQ ID NO: 18; b) a heterologous portion comprising the amino acid sequence of SEQ ID NO: 49; c) a linker portion linking the extracellular domain and the heterologous portion, the linker comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 97% or 99% identical to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 48. In some embodiments, the polypeptide does not comprise a leader sequence or the leader sequence has been removed. In some embodiments, the polypeptide comprises one or more modified amino acid residues selected from glycosylated amino acids, pegylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, amino acids conjugated to a lipid moiety, and amino acids conjugated to an organic derivatizing agent. In some embodiments, the polypeptide is glycosylated. In some embodiments, the polypeptide has a glycosylation pattern characteristic of expression of the polypeptide in a CHO cell. In some embodiments, the polypeptide binds to human TGFβ1 with an equilibrium dissociation constant (KD) of less than 100 pM. In some embodiments, the polypeptide binds to human TGFβ1 with an equilibrium dissociation constant (KD) of less than 75 pM. In some embodiments, the polypeptide binds to human TGFβ3 with an equilibrium dissociation constant (KD) of less than 60 pM. In some embodiments, the polypeptide binds to human TGFβ3 with an equilibrium dissociation constant (KD) of less than 50 pM. In some embodiments, the polypeptide inhibits TGFβ1 with an IC50 of less than 1.0 nM, as determined using a reporter gene assay. In some embodiments, the polypeptide inhibits TGFβ1 with an IC50 of less than 0.25 nM, as determined using a reporter gene assay. In some embodiments, the polypeptide inhibits TGFβ1 with an IC50 of less than 0.1 nM, as determined using a reporter gene assay. In some embodiments, the polypeptide inhibits TGFβ1 with an IC50 of less than 0.05 nM, as determined using a reporter gene assay.In some embodiments, the polypeptide inhibits TGFβ3 with an IC50 of less than 0.3 nM, as determined using a reporter gene assay. In some embodiments, the polypeptide inhibits TGFβ3 with an IC50 of less than 0.1 nM, as determined using a reporter gene assay. In some embodiments, the polypeptide inhibits TGFβ3 with an IC50 of less than 0.05 nM, as determined using a reporter gene assay. In some embodiments, the polypeptide inhibits TGFβ3 with an IC50 of less than 0.04, as determined using a reporter gene assay. Inhibits TGFβ3 with an IC50 of sub-nM. In some embodiments, the reporter gene assay is a CAGA reporter assay.

[0015] In some embodiments, the disclosure provides a homodimer comprising any two polypeptides disclosed herein.

[0016] In some embodiments, the present disclosure provides an isolated polynucleotide comprising a coding sequence for any of the polypeptides disclosed herein. In some embodiments, the present disclosure provides a recombinant polynucleotide comprising a promoter sequence operably linked to any of the polynucleotides disclosed herein. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97% or 100% identical to any one of SEQ ID NOs: 10, 12, 14, 46 or 47.

[0017] In some embodiments, the present disclosure provides a cell transformed with any of the polynucleotides disclosed herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a CHO cell or a human cell.

[0018] In some embodiments, the present disclosure provides a pharmaceutical preparation comprising a pharma- ceutically acceptable excipient and any of the polypeptides disclosed herein or any of the homodimers disclosed herein.

[0019] In certain aspects, the TGFβ antagonist is an antibody or a combination of antibodies. In certain aspects, the antibody binds to at least TβRII. In some embodiments, the TGFβ antagonist antibody that binds to TβRII inhibits signaling of TβRII, optionally measured by a cell-based assay such as the assay described herein. In some embodiments, the TGFβ antagonist antibody that binds to TβRII inhibits one or more TGFbeta superfamily ligands, TGFβ superfamily type I receptors, or TGFβ superfamily co-receptors from binding to TβRII. In some embodiments, the TGFβ antagonist antibody that binds to TβRII inhibits one or more TGFbeta superfamily ligands selected from the group consisting of TGFβ1, TGFβ2, and TGFβ3 from binding to TβRII. In certain aspects, the antibody binds to at least ALK5. In some embodiments, the TGFβ antagonist antibody that binds to ALK5 inhibits signaling of ALK5, optionally measured by a cell-based assay such as the assay described herein. In some embodiments, the TGFβ antagonist antibody that binds to ALK5 inhibits one or more TGFbeta superfamily ligands, TGFβ superfamily type II receptors, or TGFβ superfamily co-receptors from binding to ALK5. In some embodiments, the TGFβ antagonist antibody that binds to ALK5 inhibits one or more TGFbeta superfamily ligands selected from the group consisting of TGFβ1, TGFβ2, and TGFβ3 from binding to ALK5. In certain aspects, the antibody binds to at least betaglycan. In some embodiments, the TGFβ antagonist antibody that binds to betaglycan inhibits betaglycan signaling, optionally measured in a cell-based assay such as the assay described herein. In some embodiments, the TGFβ antagonist antibody that binds to betaglycan inhibits one or more TGFbeta superfamily ligands, TGFβ superfamily type I receptors, or TGFβ superfamily type II receptors from binding to betaglycan.In some embodiments, the TGFβ antagonist antibody that binds to betaglycan inhibits one or more TGF beta superfamily ligands selected from the group consisting of TGFβ1, TGFβ2, and TGFβ3 from binding to betaglycan. In certain aspects, the TGFβ antagonist antibody binds to at least TGFβ1. In some embodiments, the TGFβ antagonist antibody that binds to TGFβ1 inhibits TβRII signaling, optionally measured in a cell-based assay such as the assay described herein. In some embodiments, the TGFβ antagonist antibody that binds to TGFβ1 inhibits TGFβ1-TβRII, TGFβ1-ALK5, and / or TGFβ1-betaglcyan binding. In certain aspects, the TGFβ antagonist antibody binds to at least TGFβ2. In some embodiments, the TGFβ antagonist antibody that binds to TGFβ2 inhibits TβRII signaling, optionally measured in a cell-based assay such as the assay described herein. In some embodiments, the TGFβ antagonist antibody that binds to TGFβ2 inhibits the binding of TGFβ2-TβRII, TGFβ1-ALK5, and / or TGFβ1-betaglycan. In certain embodiments, the TGFβ antagonist antibody binds to at least TGFβ3. In some embodiments, the TGFβ antagonist antibody that binds to TGFβ3 inhibits the signaling of TβRII, optionally measured in a cell-based assay such as the assay described herein. In some embodiments, the TGFβ antagonist antibody that binds to TGFβ3 inhibits the binding of TGFβ3-TβRII, TGFβ1-ALK5, and / or TGFβ1-betaglycan. In some embodiments, the TGFβ antagonist antibody is a multispecific antibody or combination of multispecific antibodies that inhibits the signaling in one or more cell-based assays of TGFβ1, TGFβ2, TGFβ3, TβRII, ALK5, and betaglycan. In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody.In some embodiments, the antibody is a single chain antibody, a F(ab')2 fragment, a single chain antibody, a tandem single chain Fv fragment, a tandem single chain diabody, or a fusion protein comprising a single chain diabody and at least a portion of an immunoglobulin heavy chain constant region.

[0020] In certain aspects, the TGFβ antagonist is a small molecule inhibitor or a combination of small molecule inhibitors. In some embodiments, the small molecule inhibitor of the TGFβ antagonist is at least an inhibitor of TβRII. In some embodiments, the small molecule inhibitor of the TGFβ antagonist is at least an inhibitor of ALK5. In some embodiments, the small molecule inhibitor of the TGFβ antagonist is at least an inhibitor of betaglycan. In some embodiments, the small molecule inhibitor of the TGFβ antagonist is at least an inhibitor of TGFβ1. In some embodiments, the small molecule inhibitor of the TGFβ antagonist is at least an inhibitor of TGFβ2. In some embodiments, the small molecule inhibitor of the TGFβ antagonist is at least an inhibitor of TGFβ3.

[0021] In certain aspects, the TGFβ antagonist is a nucleic acid inhibitor or a combination of nucleic acid inhibitors. In some embodiments, the nucleic acid inhibitor of the TGFβ antagonist is at least an inhibitor of TβRII. In some embodiments, the nucleic acid inhibitor of the TGFβ antagonist is at least an inhibitor of ALK5. In some embodiments, the nucleic acid inhibitor of the TGFβ antagonist is at least an inhibitor of betaglycan. In some embodiments, the nucleic acid inhibitor of the TGFβ antagonist is at least an inhibitor of TGFβ1. In some embodiments, the nucleic acid inhibitor of the TGFβ antagonist is at least an inhibitor of TGFβ2. In some embodiments, the nucleic acid inhibitor of the TGFβ antagonist is at least an inhibitor of TGFβ3. [Brief description of the drawings]

[0022] [Figure 1]Figure 1 shows the amino acid sequence of the native precursor for the B (short) isoform of human TGFβ receptor type II (hTβRII) (NP_003233.4). Solid underlining indicates the mature extracellular domain (ECD) (residues 23-159) and double underlining indicates the valine replaced in the A (long) isoform. Dotted underlining represents the leader (residues 1-22).

[0023] [Diagram 2] Figure 2 shows the amino acid sequence of the native precursor for the A (long) isoform of human TβRII (NP_001020018.1). Solid underlining indicates the mature ECD (residues 23-184), double underlining indicates the isoleucine substitution that occurs in the splice, and dotted underlining represents the leader (residues 1-22).

[0024] [Diagram 3] FIG. 3 shows a comparison of the linker sequences of five different TβRII constructs.

[0025] [Figure 4-1] 4A and 4B show in tabular form the binding affinity between TGFβ1 and TGFβ3 and one of several different TβRII-Fc fusion protein constructs. [Figure 4-2] Same as above.

[0026] [Figure 5-1] Figures 5A and 5C graphically represent the results from a reporter gene assay testing the affinity of TGFβ1 for one of several different TβRII-Fc fusion protein constructs. Figures 5B and 5D graphically represent the results from a reporter gene assay testing the affinity of TGFβ3 for one of several different TβRII-Fc fusion protein constructs. Figures 5E and 5F present the IC50 data from these same experiments in tabular format. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 5-4] Same as above. [Figure 5-5] Same as above. [Figure 5-6] Same as above.

[0027] [Figure 6] Figures 6A, 6B and 6C show the effects of mTβRII-mFc in an in vitro inflammation model and an animal model of heterotopic ossification. Figure 6A shows that mTβRII-mFc can suppress TGFβ1 secretion by regenerating M2 macrophages in an in vitro inflammatory environment. Figure 6B shows that treatment with mTβRII-mFc reduces bone deposition and formation of bone marrow cavities in an animal model of traumatic heterotopic ossification. Figure 6C shows that treatment with mTβRII-mFc significantly reduces the volume of mature HO in an animal model of traumatic heterotopic ossification.

[0028] [Figure 7] Figure 7 shows a multiple sequence alignment of Fc domains from human IgG isotypes using Clustal 2.1. The hinge region is indicated by a dotted underline. Double underlines indicate examples of positions designed in IgG1 Fc to promote asymmetric chain pairing, as well as corresponding positions for the other isotypes IgG2, IgG3 and IgG4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] 1. Overview Proteins described herein are in human form unless otherwise specified. The NCBI reference for the protein is as follows: human TβRII isoform A (hTβRII long ), NP_001020018.1 and human TβRII isoform B (hTβRII short ), (NP_003233.4). The sequence of a native TβRII protein from human is set forth in Figures 1 and 2. In some embodiments, the TβRII protein is from a non-human animal, such as mouse, rat, cow or monkey.

[0030] The TGFβ superfamily contains a variety of growth factors that share common sequence elements and structural motifs. These proteins are known to exert biological effects on a wide variety of cell types in both vertebrates and invertebrates. Members of the superfamily play important functions in pattern formation and tissue specification during embryogenesis and can affect a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis, and epithelial cell differentiation. By manipulating the activity of members of the TGFβ family, it is often possible to produce significant physiological changes in the organism. For example, the bovine breeds Piedmont Blue and Belgian Blue carry a loss-of-function mutation in the GDF8 (also called myostatin) gene that causes a marked increase in muscle mass. Grobet et al., Nat Genet. 1997, 17(1):71-4. Similarly, in humans, an inactive allele of GDF8 is associated with increased muscle mass and reportedly exceptional strength. Schuelke et al., N Engl J Med 2004, 350:2682-8.

[0031] TGFβ signals are mediated by heterogeneous complexes of type I (e.g., TβRI) and type II (e.g., TβRII) serine / threonine kinase receptors, which phosphorylate and activate downstream SMAD proteins upon ligand stimulation (Massague, 2000, Nat. Rev. Mol. Cell Biol. 1:169-178). These type I and type II receptors are transmembrane proteins composed of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine specificity. Type I receptors are essential for signal transduction, while type II receptors are required for ligand binding and for expression of type I receptors. Type I and type II receptors form stable complexes after ligand binding, resulting in phosphorylation of type I receptors by type II receptors. TGFβ has three mammalian isoforms, TGFβ1, TGFβ2, and TGFβ3, each with distinct functions in vivo. Binding of TGFβ to TβRII is a critical step in initiating activation of the TGFβ signaling pathway, directing the phosphorylation of SMAD2, and translocating the activated SMAD2 / SMAD4 complex to the nucleus to regulate gene expression.

[0032] Thus, in certain embodiments, the present disclosure provides TβRII polypeptides as antagonists of TGFβ1 or TGFβ3 for use in treating various TGFβ1 or TGFβ3-related disorders. Without wishing to be bound by any particular mechanism of action, such polypeptides are expected to act by binding to TGFβ1 or TGFβ3 and inhibiting the ability of these ligands to form the three-way signaling complex.

[0033] The present disclosure provides a fusion protein comprising a TβRII polypeptide and a heterologous moiety (e.g., an Fc moiety). In certain embodiments, the TβRII moiety and the heterologous moiety are fused by a linker. As described in more detail below, the present disclosure demonstrates that a TβRII-Fc fusion protein comprising a linker of a certain length (e.g., a linker having 21 amino acids) was surprisingly able to bind to TGFβ-1 and TGFβ-3 with stronger affinity than a TβRII-Fc fusion protein having a linker of only 4 amino acids.

[0034] As demonstrated herein, soluble TβRII polypeptides that bind TGFβ1 and TGFβ3 are effective in significantly attenuating the development of ectopic ossification (HO) primordia with reduced bone deposition and formation of bone marrow cavity in a mouse model of HO. Furthermore, treatment with TβRII polypeptides significantly reduced the volume of mature HO in this animal model. Without wishing to be bound by any particular mechanism, it is expected that the effect of TβRII polypeptides is mainly caused by the antagonistic effect of TGFβ signaling, particularly the antagonistic effect of TGFβ1 and / or TGFβ3 signaling. Regardless of the mechanism, it is clear from the data presented herein that TGFβ signaling antagonists reduce the severity of bone deposition in HO. It should be noted that ectopic ossification is dynamic and changes depending on the balance of factors that increase HO and factors that decrease HO. HO can be decreased by increasing factors that decrease HO, decreasing factors that increase HO, or both. Thus, the term decreasing (reducing) HO refers to an observable physical change in bone deposition and is intended to be neutral with respect to the mechanism by which the change occurs.

[0035] The animal model for HO used in the studies described herein is believed to be descriptive of efficacy in humans, and thus the present disclosure provides methods for using TGFβ signaling antagonists to treat HO, particularly for preventing or reducing the severity or duration of one or more HO complications in humans.As disclosed herein, the term TGFβ antagonist refers to various agents that can be used to antagonize TGFβ signaling, including, for example, antagonists that inhibit one or more TGFβ ligands [e.g., TGFβ1, TGFβ2 and / or TGFβ3]; antagonists that inhibit one or more TGFβ-interacting type I, type II or co-receptors (e.g., TβRII, ALK5 and betaglycan); and antagonists that inhibit one or more downstream signaling components (e.g., Smad proteins such as Smad2 and 3). TGFβ antagonists used in accordance with the disclosed methods and uses include various forms, such as ligand traps (e.g., soluble TβRII polypeptides and betaglycan polypeptides), antibody antagonists (e.g., antibodies that inhibit one or more of TGFβ1, TGFβ2, TGFβ3, TβRII and betaglycan), small molecule antagonists (e.g., small molecules that inhibit one or more of TGFβ1, TGFβ2, TGFβ3, TβRII, betaglycan, and one or more Smad proteins (e.g., Smad2 and 3)), and nucleotide antagonists (e.g., nucleotide sequences that inhibit one or more of TGFβ1, TGFβ2, TGFβ3, TβRII, betaglycan, and one or more Smad proteins (e.g., Smad2 and 3)).

[0036] The terms used herein generally have their usual meaning in the art, within the context of the present invention and in the specific context in which each term is used.Certain terms are discussed below or elsewhere in this specification to provide additional guidance to the skilled artisan in the description of the compositions and methods of the present invention, and the methods of making and using them.The scope or meaning of any use of a term will be clear from the specific context in which it is used.

[0037] "Homologous," in all its grammatical forms and spelling variations, refers to the relationship between two proteins that have a "common evolutionary origin," and includes proteins from superfamilies in the same species, and homologous proteins from different species. Such proteins (and their encoding nucleic acids) have sequence homology that reflects their sequence similarity, regardless of percent identity terms, or that reflects the presence of certain residues or motifs and conserved positions.

[0038] The term "sequence similarity," in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin. However, in common usage and in this application, the term "homologous," when modified with an adverb such as "high," may refer to sequence similarity, which may or may not be related to a common evolutionary origin.

[0039] "Percent sequence identity" or "percent identity" with respect to a reference polypeptide (or nucleotide) sequence is defined as the percentage of amino acid residues (or nucleic acids) in a candidate sequence that are identical to the amino acid residues (or nucleic acids) in a reference polypeptide (nucleotide) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. For purposes herein, however, amino acid (nucleic acid) sequence identity percent values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been submitted with user documentation to the U.S. Copyright Office, Washington DC, 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program must be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and are not altered.

[0040] "Agonize," in all its grammatical forms, refers to the process of activating a protein and / or gene (e.g., by activating or amplifying gene expression of a protein or by inducing an inactive protein to become active) or increasing the activity of a protein and / or gene.

[0041] "Antagonize," in all its grammatical forms, refers to the process of inhibiting a protein and / or gene (e.g., by inhibiting or reducing gene expression of a protein or by inducing an active protein to become inactive) or decreasing the activity of a protein and / or gene.

[0042] The terms "about" and "approximately" as used in connection with numerical values ​​throughout this specification and claims refer to intervals of accuracy well known and accepted by those of skill in the art.

[0043] The numerical ranges disclosed herein are inclusive of the numbers defining the range.

[0044] The terms "a" and "an" include plural referents unless the context in which the term is used clearly dictates otherwise. The terms "a" (or "an"), as well as "one or more" and "at least one" can be used interchangeably herein. Furthermore, "and / or" as used herein should be a specific disclosure of each of two or more specific features or components, with or without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (single) and "B" (single). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to encompass each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0045] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to include the meaning of the inclusion of a reference integer or group of integers, but not the exclusion of any other integer or group of integers. As used herein, the term "comprises" also encompasses use of the narrower terms "consisting of" and "consisting essentially of."

[0046] The term "consisting essentially of" is limited to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the invention disclosed herein.

[0047] As used herein, the term "appreciable affinity" refers to an affinity that has a dissociation constant (K D) means bonding at

[0048] The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein to refer to a chain of amino acids of any length. The chain may be linear or branched, and may contain modified amino acids and / or may be interrupted by non-amino acids. The term also encompasses amino acid chains that are natural or modified by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within this definition are polypeptides that contain, for example, one or more analogs of amino acids (including, for example, non-natural amino acids, etc.) and other modifications known in the art. It is understood that polypeptides can exist as single chains or associated chains. 2. TGFβ antagonists

[0049] As disclosed herein, the term TGFβ antagonist refers to a variety of agents that can be used to antagonize TGFβ-mediated signal transduction, including, for example, antagonists that inhibit one or more TGFβ ligands [e.g., TGFβ1, TGFβ2 and / or TGFβ3]; antagonists that inhibit one or more type I, type II or co-receptors that interact with TGFβ (e.g., TβRII, ALK5 and betaglycan); and antagonists that inhibit one or more downstream signaling components (e.g., Smad proteins such as Smad2 and 3). TGFβ antagonists used in accordance with the disclosed methods and uses include various forms, such as ligand traps (e.g., soluble TβRII polypeptides and betaglycan polypeptides), antibody antagonists (e.g., antibodies that inhibit one or more of TGFβ1, TGFβ2, TGFβ3, TβRII and betaglycan), small molecule antagonists (e.g., small molecules that inhibit one or more of TGFβ1, TGFβ2, TGFβ3, TβRII, betaglycan, and one or more Smad proteins (e.g., Smad2 and 3)), and nucleotide antagonists (e.g., nucleotide sequences that inhibit one or more of TGFβ1, TGFβ2, TGFβ3, TβRII, betaglycan, and one or more Smad proteins (e.g., Smad2 and 3)). A. TβRII Polypeptides and Betaglycan Polypeptides

[0050] Naturally occurring TβRII proteins are transmembrane proteins with a portion of the protein located outside the cell (extracellular portion) and a portion of the protein located inside the cell (intracellular portion). Embodiments of the present disclosure encompass variant TβRII polypeptides that include mutations within the extracellular domain of TβRII and / or truncated portions of the extracellular domain of TβRII. As described above, human TβRII naturally exists in at least two isoforms, A (long) and B (short), resulting from alternative splicing in the extracellular domain (ECD) (FIGS. 1 and 2, SEQ ID NOs: 1 and 2). SEQ ID NO: 27, corresponding to residues 23-159 of SEQ ID NO: 1, represents the short isoform of the natural full-length extracellular domain of TβRII. SEQ ID NO: 18, corresponding to residues 23-184 of SEQ ID NO: 2, represents the long isoform of the natural full-length extracellular domain of TβRII. Unless otherwise stated, the numbering of amino acid positions for variants based on the short and long isoforms of TβRII refer to the corresponding positions in the naturally occurring precursors SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0051] In certain embodiments, the present disclosure provides variant TβRII polypeptides. The TβRII polypeptides of the present disclosure can bind to and inhibit the function of members of the TGFβ superfamily, such as, but not limited to, TGFβ1 or TGFβ3. The TβRII polypeptides can include polypeptides consisting of or including an amino acid sequence at least 70% identical, optionally at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical, to the truncated ECD domain of a naturally occurring TβRII polypeptide, the C-terminus of which is located at any of amino acids 153-159 of SEQ ID NO:1. A TβRII polypeptide can include a polypeptide consisting of or comprising an amino acid sequence at least 70% identical, and optionally at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical, to the truncated ECD domain of a naturally occurring TβRII polypeptide, the C-terminus of which is located at any of amino acids 178-184 of SEQ ID NO: 2. In certain embodiments, a TβRII polypeptide comprises an amino acid sequence at least 70% identical, and optionally at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical, to the amino acid sequence of SEQ ID NO: 18. Optionally, the TβRII polypeptide does not include more than 5 contiguous amino acids, or 10, 20, 30, 40, 50, 52, 60, 70, 80, 90, 100, 150, or 200 or more contiguous amino acids from the sequence consisting of amino acids 160-567 of SEQ ID NO:1, or from the sequence consisting of amino acids 185-592 of SEQ ID NO:2. In some embodiments, the TβRII polypeptide does not include amino acids 160-567 of SEQ ID NO:1. In some embodiments, the TβRII polypeptide does not include amino acids 1-22 of SEQ ID NO:1. In some embodiments, the TβRII polypeptide does not include amino acids 1-22 and 160-567 of SEQ ID NO:1.In some embodiments, a TβRII polypeptide does not include amino acids 185-592 of SEQ ID NO:2. In some embodiments, a TβRII polypeptide does not include amino acids 1-22 of SEQ ID NO:2. In some embodiments, a TβRII polypeptide does not include amino acids 1-22 and 185-592 of SEQ ID NO:2. An unprocessed TβRII polypeptide can either include or omit any signal sequence, and any N-terminal to signal sequence sequence. As detailed herein, the N-terminus of a mature (processed) TβRII polypeptide can be at either amino acids 23-35 of SEQ ID NO:1 or amino acids 23-60 of SEQ ID NO:2. Examples of mature TβRII polypeptides include, but are not limited to, amino acids 23 to 159 of SEQ ID NO:1 (described in SEQ ID NO:27), amino acids 29 to 159 of SEQ ID NO:1 (described in SEQ ID NO:28), amino acids 35 to 159 of SEQ ID NO:1 (described in SEQ ID NO:29), amino acids 23 to 153 of SEQ ID NO:1 (described in SEQ ID NO:30), amino acids 29 to 153 of SEQ ID NO:1 (described in SEQ ID NO:31), amino acids 35 to 153 of SEQ ID NO:1 (described in SEQ ID NO:32), amino acids 23 to 184 of SEQ ID NO:2 (described in SEQ ID NO:18), amino acids 29 to 184 of SEQ ID NO:2 (described in SEQ ID NO:33), amino acids 60 to 184 of SEQ ID NO:2 (described in SEQ ID NO:29), amino acids 23 to 178 of SEQ ID NO:2 (described in SEQ ID NO:34), amino acids 29 to 178 of SEQ ID NO:2 (described in SEQ ID NO:35), and amino acids 60 to 178 of SEQ ID NO:2 (described in SEQ ID NO:32). It will be understood by those skilled in the art that the corresponding variant based on the long isoform of TβRII contains a nucleotide sequence encoding a 25 amino acid insertion with a conservative Val-Ile substitution at the C-terminal position adjacent to the insertion.Thus, a TβRII polypeptide may include the extracellular portion of an isolated TβRII polypeptide, including both short and long isoforms, variants thereof (including, for example, variants containing no more than 2, 3, 4, 5, 10, 15, 20, 25, 30 or 35 amino acid substitutions in the sequence corresponding to amino acids 23-159 of SEQ ID NO:1 or amino acids 23-184 of SEQ ID NO:2), fragments thereof, and fusion proteins comprising any of the foregoing, although in each case, preferably, any of the foregoing TβRII polypeptides maintain substantial affinity for at least one or both of TGFβ1 or TGFβ3. Generally, TβRII polypeptides are designed to be soluble in aqueous solutions at biologically relevant temperatures, pH levels, and osmolality.

[0052] In some embodiments, variant TβRII polypeptides of the present disclosure contain one or more mutations in the extracellular domain that confer an altered ligand binding profile. TβRII polypeptides may contain one, two, five or more changes in amino acid sequence relative to the corresponding portion of a naturally occurring TβRII polypeptide. In some embodiments, the mutation results in a substitution, insertion or deletion at a position corresponding to position 70 of SEQ ID NO:1. In some embodiments, the mutation results in a substitution, insertion or deletion at a position corresponding to position 110 of SEQ ID NO:1. Examples include, but are not limited to, an N to D or a D to K substitution at positions corresponding to positions 70 and 110 of SEQ ID NO:1, respectively. Examples of such variant TβRII polypeptides include, but are not limited to, the sequences set forth in SEQ ID NOs:36-39. A TβRII polypeptide may comprise a polypeptide or portion thereof encoded by any one of SEQ ID NO:8, 10, 12, 14, 16, 46 or 47, or silent variants thereof, or nucleic acids that hybridize to their complements under stringent hybridization conditions. In certain embodiments, a TβRII polypeptide may comprise a polypeptide or portion thereof encoded by any one of SEQ ID NO:12, or silent variants thereof, or nucleic acids that hybridize to their complements under stringent hybridization conditions.

[0053] In some embodiments, the variant TβRII polypeptides of the present disclosure further comprise an insertion of 36 amino acids (SEQ ID NO:41) between the pair of glutamic acid residues located near the C-terminus of the human TβRII ECD (positions 151 and 152 of SEQ ID NO:1, or positions 176 and 177 of SEQ ID NO:2), as naturally occurring in human TβRII isoform C (Konrad et al., BMC Genomics 8:318, 2007).

[0054] It has been demonstrated that TβRII polypeptides can be modified to selectively antagonize TβRII ligands. The N70 residue represents a potential glycosylation site. In some embodiments, the TβRII polypeptide is not glycosylated. In some embodiments, the TβRII polypeptide is not glycosylated or has reduced glycosylation at Asn157. In some embodiments, the TβRII polypeptide is not glycosylated or has reduced glycosylation at Asn73.

[0055] In certain embodiments, the TβRII polypeptide binds to TGFβ1, and the TβRII polypeptide does not exhibit substantial binding to TGFβ3. In certain embodiments, the TβRII polypeptide binds to TGFβ3, and the TβRII polypeptide does not exhibit substantial binding to TGFβ1. Binding can be assessed using purified proteins in solution or in a surface plasmon resonance system, such as a Biacore™ system.

[0056] In certain embodiments, TβRII polypeptide inhibits TGFβ1 intracellular signal transduction, and TβRII polypeptide has moderate or limited inhibitory effect on TGFβ3 signal transduction.In certain embodiments, TβRII polypeptide inhibits TGFβ3 intracellular signal transduction, and TβRII polypeptide has moderate or limited inhibitory effect on TGFβ1 signal transduction.The inhibitory effect on cell signal transduction can be assayed by methods known in the art.

[0057] Based on these, the active portion of the TβRII polypeptide may include the amino acid sequence 23-153, 23-154, 23-155, 23-156, 23-157 or 23-158 of SEQ ID NO: 1, and variants of these sequences starting with any of amino acids 24-35 of SEQ ID NO: 1. Similarly, the active portion of the TβRII polypeptide may include the amino acid sequence 23-178, 23-179, 23-180, 23-181, 23-182 or 23-183 of SEQ ID NO: 2, and variants of these sequences starting with any of amino acids 24-60 of SEQ ID NO: 2. Exemplary TβRII polypeptides include amino acid sequences 29-159, 35-159, 23-153, 29-153, and 35-153 of SEQ ID NO: 1, or amino acid sequences 29-184, 60-184, 23-178, 29-178, and 60-178 of SEQ ID NO: 2. Variants within these ranges are also contemplated, particularly those having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 or the corresponding portion of SEQ ID NO: 2. TβRII polypeptides may be selected that do not include a sequence consisting of amino acids 160-567 of SEQ ID NO: 1 or amino acids 185-592 of SEQ ID NO: 2. In certain embodiments, the TβRII polypeptide comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:18, optionally at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical.

[0058] The term "betaglycan polypeptide" includes polypeptides comprising any naturally occurring betaglycan protein (encoded by TGFBR3, or one of its non-human orthologs), and any variants thereof (including mutants, fragments, fusions and peptidomimetics) that maintain useful activity.

[0059] The precursor protein sequence of human betaglycan isoform A (NCBI Reference Sequence NP_003234.2) is as follows: [ka]

[0060] The signal peptide is indicated by a single underline, the extracellular domain is indicated in bold font, and the transmembrane domain is indicated by a dotted underline. This isoform differs from isoform B of betaglycan by the insertion of a single alanine, indicated above by a double underline.

[0061] The polypeptide sequence of processed betaglycan isoform A is as follows: [ka] [ka]

[0062] The nucleic acid sequence encoding the unprocessed precursor protein of human betaglycan isoform A is shown below (SEQ ID NO: 52), which corresponds to nucleotides 516 to 3068 of the NCBI reference sequence NM_003243.4. The signal sequence is indicated by a solid underline and the transmembrane region is indicated by a dotted underline. [ka] [ka]

[0063] The nucleic acid sequence encoding the extracellular domain of processed betaglycan isoform A is shown below (SEQ ID NO:53). [ka]

[0064] The precursor protein sequence of human betaglycan isoform B (NCBI Reference Sequence NP_001182612.1) is as follows: [ka]

[0065] The signal peptide is indicated by a single underline, the extracellular domain is indicated in bold font, and the transmembrane domain is indicated by a dotted underline.

[0066] The polypeptide sequence of processed betaglycan isoform B is as follows: [ka]

[0067] The nucleic acid sequence encoding the unprocessed precursor protein of human betaglycan isoform B is shown below (SEQ ID NO: 56) and corresponds to nucleotides 516 to 3065 of the NCBI reference sequence NM_001195683.1. The signal sequence is indicated by a solid underline and the transmembrane region is indicated by a dotted underline. [ka]

[0068] The nucleic acid sequence encoding the extracellular domain of processed betaglycan isoform B is shown below (SEQ ID NO:57). [ka] [ka]

[0069] In certain embodiments, the present disclosure relates to betaglycan polypeptides, including fragments, functional variants, and modified forms thereof. Preferably, the betaglycan polypeptides for use with the disclosed invention are soluble (e.g., include the extracellular domain of betaglycan). In other preferred embodiments, the betaglycan polypeptides for use with the disclosed invention bind to and / or inhibit (antagonize) one or more TGF-beta superfamily ligands and / or their activity (e.g., Smad signaling). In some embodiments, the betaglycan polypeptides comprise an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 50, 51, 54 or 55. In some embodiments, the betaglycan polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a polypeptide beginning with any one of amino acids 21-28 of SEQ ID NO: 50 and ending with any one of amino acids 381-787 of SEQ ID NO: 50. In some embodiments, the polypeptides of the present disclosure comprise at least one betaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 21-381 of SEQ ID NO: 50. In some embodiments, the polypeptides of the present disclosure comprise at least one betaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 21-787 of SEQ ID NO: 50. In some embodiments, the polypeptides of the present disclosure comprise at least one betaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 28-381 of SEQ ID NO: 50.In some embodiments, the polypeptides of the present disclosure comprise at least one betaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 28-787 of SEQ ID NO: 50. In some embodiments, the polypeptides of the present disclosure are comprised of at least one betaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 21-781 of SEQ ID NO: 50. In some embodiments, the polypeptides of the present disclosure are comprised of at least one betaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 28-781 of SEQ ID NO: 50. In some embodiments, the polypeptides of the present disclosure comprise at least one betaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a polypeptide beginning with any one of amino acids 21-28 of SEQ ID NO: 54 and ending with any one of amino acids 380-786 of SEQ ID NO: 54. In some embodiments, the polypeptides of the present disclosure are comprised of at least one betaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 21-380 of SEQ ID NO: 54. In some embodiments, the polypeptides of the present disclosure are comprised of at least one betaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 21-786 of SEQ ID NO: 54.In some embodiments, the polypeptides of the present disclosure are comprised of at least one betaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 28-380 of SEQ ID NO: 54. In some embodiments, the polypeptides of the present disclosure are comprised of at least one betaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 28-786 of SEQ ID NO: 54. In some embodiments, the polypeptides of the present disclosure are comprised of at least one betaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 21-780 of SEQ ID NO: 54. In some embodiments, the polypeptides of the present disclosure are comprised of at least one betaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 28-780 of SEQ ID NO: 54. In some embodiments, the polypeptides of the present disclosure comprise at least one betaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a polypeptide beginning with any one of amino acids 21-28 of SEQ ID NO: 50 and ending with any one of amino acids 730-787 of SEQ ID NO: 50. In some embodiments, the polypeptides of the present disclosure are comprised of at least one betaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 21-787 of SEQ ID NO: 50.In some embodiments, the polypeptides of the present disclosure are comprised of at least one betaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 28-730 of SEQ ID NO: 50. In some embodiments, the polypeptides of the present disclosure comprise at least one betaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a polypeptide beginning with any one of amino acids 21-28 of SEQ ID NO: 50 and ending with any one of amino acids 730-787 of SEQ ID NO: 50. In some embodiments, the polypeptides of the present disclosure are comprised of at least one betaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 21-787 of SEQ ID NO: 50. In some embodiments, the polypeptides of the present disclosure are comprised of at least one betaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 28-730 of SEQ ID NO: 50. In some embodiments, the polypeptides of the present disclosure comprise at least one betaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a polypeptide beginning with any one of amino acids 21-28 (e.g., amino acid residues of SEQ ID NO:54) and ending with any one of amino acids 730-787 of SEQ ID NO:54. In some embodiments, the polypeptides of the present disclosure are comprised of at least one betaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 21-786 of SEQ ID NO:54.In some embodiments, the polypeptides of the present disclosure are comprised of at least one betaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 28-729 of SEQ ID NO:54.

[0070] In certain aspects, the present disclosure relates to heteromultimers comprising ALK5 polypeptide.As used herein, the term "ALK5" refers to the family of activin receptor-like kinase-5 proteins from any species, and variants derived from such ALK4 proteins by mutagenesis or other modifications.Reference to ALK5 herein is understood to refer to any one of the currently identified forms.Members of the ALK5 family are generally transmembrane proteins that are composed of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine kinase activity.

[0071] The term "ALK5 polypeptide" includes polypeptides including any naturally occurring member of the ALK5 polypeptide family, and any variants thereof (including mutants, fragments, fusions and peptidomimetic forms) that retain useful activity.

[0072] The sequence of human ALK5 precursor protein isoform 1 (NCBI reference sequence NP_004603.1) is as follows: [ka]

[0073] The signal peptide is indicated by a single underline and the extracellular domain is indicated in bold font.

[0074] The processed extracellular ALK5 polypeptide sequence is as follows: [ka]

[0075] The nucleic acid sequence encoding the ALK5 precursor protein is shown below (SEQ ID NO: 32) and corresponds to nucleotides 77 to 1585 of the Genbank reference sequence NM_004612.2.

[0076] [ka] [ka]

[0077] The nucleic acid sequence encoding the extracellular human ALK5 polypeptide is as follows: [ka]

[0078] The alternative isoform of isoform 2 of the precursor protein sequence of human ALK5 (NCBI reference sequence XP_005252207.1) is as follows: [ka]

[0079] The signal peptide is indicated by a single underline and the extracellular domain is indicated in bold font.

[0080] The processed extracellular ALK5 polypeptide sequence (isoform 2) is as follows: [ka]

[0081] The nucleic acid sequence encoding the human ALK5 precursor protein (isoform 2) is shown below (SEQ ID NO: 75) and corresponds to nucleotides 77-1597 of the Genbank reference sequence XM_005252150.1. The signal sequence is underlined and the extracellular domain is shown in bold font. [ka]

[0082] The nucleic acid sequence encoding the processed extracellular ALK5 polypeptide is as follows: [ka]

[0083] In certain embodiments, the present disclosure relates to heteromultimers comprising at least one ALK5 polypeptide, including fragments, functional variants, and modified forms thereof.Preferably, the ALK5 polypeptide for use according to the present disclosure (e.g., heteromultimers comprising ALK5 polypeptides and their uses) is soluble (e.g., the extracellular domain of ALK5).In other preferred embodiments, the ALK5 polypeptide for use according to the present disclosure binds to one or more TGF beta superfamily ligands and / or inhibits (antagonizes) their activity (e.g., Smad signaling).In some embodiments, the heteromultimer of the present disclosure comprises at least one ALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:69, 70, 73 or 74. In some embodiments, a heteromultimer of the present disclosure begins at any one of amino acids 25-36 of SEQ ID NO:69 (e.g., amino acid residues 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36) and begins at any one of amino acids 101-126 of SEQ ID NO:69 (e.g., amino acid residues 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 120, 122, 124, 126, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 0, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, or 126). In some embodiments, the heteromultimers of the disclosure comprise at least one ALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 25-101 of SEQ ID NO:69.In some embodiments, heteromultimers of the disclosure comprise at least one ALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 25-126 of SEQ ID NO: 69. In some embodiments, heteromultimers of the disclosure comprise at least one ALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 36-101 of SEQ ID NO: 69. In some embodiments, heteromultimers of the disclosure comprise at least one ALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 36-126 of SEQ ID NO: 69. In some embodiments, heteromultimers of the disclosure comprise at least one ALK5 polypeptide that begins at any one of amino acids 25-36 of SEQ ID NO: 73 (e.g., amino acid residues 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36) and begins at any one of amino acids 101-130 of SEQ ID NO: 73 (e.g., amino acid residues 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 330, 34, 35 or 36). 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130). In some embodiments, heteromultimers of the present disclosure comprise at least one ALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 25-101 of SEQ ID NO:73.In some embodiments, heteromultimers of the disclosure comprise at least one ALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 25-130 of SEQ ID NO: 73. In some embodiments, heteromultimers of the disclosure comprise at least one ALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 36-101 of SEQ ID NO: 73. In some embodiments, heteromultimers of the present disclosure comprise at least one ALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 36-130 of SEQ ID NO:73.

[0084] As described above, the present disclosure provides TβRII polypeptides or betaglycan polypeptides that share a certain degree of sequence identity or similarity with naturally occurring TβRII polypeptides or betaglycan polypeptides. To determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). The amino acid residues at corresponding amino acid positions are then compared. If a position in the first sequence is occupied by the same amino acid residue at the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, amino acid "identity" is equivalent to amino acid "homology"). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.

[0085] Comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using mathematical algorithms (Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).

[0086] In one embodiment, the percent identity between two amino acid sequences is determined using the algorithm of Needleman and Wunsch (J Mol. Biol. (48):444-453 (1970)) incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com). In a detailed embodiment, the following parameters are used in the GAP program: either Blosum 62 matrix or PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6 or 4, and length weights of 1, 2, 3, 4, 5 or 6. In yet another embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984)) (available at http: / / www.gcg.com). Exemplary parameters include using a NWSgapdna.CMP matrix, with a gap weight of 40, 50, 60, 70 or 80, and a length weight of 1, 2, 3, 4, 5 or 6. Unless otherwise specified, percent identity between two amino acid sequences should be determined using the GAP program using a Blosum 62 matrix, a GAP weight of 10 and a length weight of 3; if such an algorithm is not capable of computing the desired percent identity, then a suitable alternative disclosed herein should be selected.

[0087] In another embodiment, the percent identity between two amino acid sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) as incorporated into the ALIGN program (version 2.0), using a PAM120 weight residual table, a gap length penalty of 12, and a gap penalty of 4.

[0088] Another embodiment for determining the best overall alignment between two amino acid sequences can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci., 6:237-245 (1990)). In sequence alignment, both the query and subject sequences are amino acid sequences. The results of the global sequence alignment are presented in terms of percent identity. In one embodiment, amino acid sequence identity is performed using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci., 6:237-245 (1990)). In a detailed embodiment, the parameters used to calculate the identity and similarity percentage of amino acid alignment include matrix=PAM 150, k-tuple=2, mismatch penalty=1, linkage penalty=20, randomization group length=0, cutoff score=1, gap penalty=5 and gap size penalty=0.05.

[0089] The polypeptides of the present disclosure (e.g., TβRII or betaglycan polypeptides) may additionally include any of a variety of leader sequences at the N-terminus. Such sequences would allow the peptides to be expressed in eukaryotic systems and targeted to the secretory pathway. See, for example, Ernst et al., U.S. Pat. No. 5,082,783 (1992). Alternatively, a native signal sequence (e.g., a native TβRII or betaglycan signal sequence) may be used to effect release from the cell. Possible leader sequences include the native leaders, tissue plasminogen activator (TPA) and honeybee melittin (SEQ ID NOs: 22-24, respectively). Examples of TβRII-Fc fusion proteins incorporating the TPA leader sequence include SEQ ID NOs: 11, 13, 15 and 17. Signal peptide processing may vary depending on the leader sequence selected, the cell type and culture conditions used, among other variables, and therefore the actual N-terminal start site for the mature polypeptide may shift by up to 1, 2, 3, 4 or 5 amino acids in either the N- or C-terminal direction. Examples of TβRII-Fc fusion proteins include SEQ ID NOs: 11, 13, 15 and 17. It will be understood by those skilled in the art that the corresponding variant based on the long isoform of TβRII contains a 25 amino acid insertion with a conservative Val-Ile substitution at the C-terminal position adjacent to the insertion.

[0090] In some embodiments, any TβRII polypeptide disclosed herein is at least 80%, 85%, 90%, 92%, 94%, 95%, 97%, 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 18, 20, 27, 30, 34, 36, 37, 38, 39 or 48, but is missing one or more N-terminal amino acids compared to the amino acid sequence of SEQ ID NOs: 18, 20, 27, 30, 34, 36, 37, 38, 39 or 48. In some embodiments, a TβRII polypeptide is missing an amino acid corresponding to the first amino acid (threonine) of any one of SEQ ID NOs: 18, 20, 27, 30, 34, 36, 37, 38, 39 or 48. In some embodiments, the TβRII polypeptide lacks amino acids corresponding to the first and second amino acids (threonine and isoleucine, respectively) of any one of SEQ ID NOs: 18, 20, 27, 30, 34, 36, 37, 38, 39, or 48. In some embodiments, the TβRII polypeptide lacks amino acids corresponding to the first, second, and third amino acids (threonine, isoleucine, and proline, respectively) of any one of SEQ ID NOs: 18, 20, 27, 30, 34, 36, 37, 38, 39, or 48. In some embodiments, the TβRII polypeptide lacks amino acids corresponding to the first, second, third, and fourth amino acids (threonine, isoleucine, proline, proline, respectively) of any one of SEQ ID NOs: 18, 20, 27, 30, 34, 36, 37, 38, 39, or 48.

[0091] In some embodiments, any TβRII polypeptide disclosed herein is at least 80%, 85%, 90%, 92%, 94%, 95%, 97%, 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 18 or 48, but lacks one or more N-terminal amino acids compared to the amino acid sequence of SEQ ID NOs: 18 or 48. In some embodiments, the TβRII polypeptide lacks amino acids corresponding to the first and second amino acids (threonine and isoleucine, respectively) of any one of SEQ ID NOs: 18 or 48. In some embodiments, the TβRII polypeptide lacks amino acids corresponding to the first, second and third amino acids (threonine, isoleucine and proline, respectively) of SEQ ID NOs: 18 or 48. In some embodiments, the TβRII polypeptide lacks amino acids corresponding to the first, second, third and fourth amino acids (threonine, isoleucine, proline, proline, respectively) of SEQ ID NOs: 18 or 48.

[0092] In some embodiments, the disclosure provides a composition comprising a mixture of TβRII polypeptides, wherein the TβRII polypeptides in the composition comprise an amino acid sequence that is at least 80%, 85%, 90%, 92%, 94%, 95%, 97%, 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 18, 20, 27, 30, 34, 36, 37, 38, 39 or 48, respectively, but wherein at least a portion (e.g., at least 1%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%) of the TβRII polypeptides in the composition are identical to the amino acid sequence of any one of SEQ ID NOs: 18, 20, 27, 30, 34, 36, 37, 38, 39 or 48, respectively. 48; and wherein at least a portion (e.g., at least 1%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%) of the TβRII polypeptides in the composition are lacking one or more of the amino acids corresponding to the first, second, third and fourth amino acids (threonine, isoleucine, proline and proline, respectively) of any one of SEQ ID NOs: 18, 20, 27, 30, 34, 36, 37, 38, 39 or 48. In some embodiments, the disclosure provides a composition comprising a mixture of TβRII polypeptides, wherein the TβRII polypeptides are at least 80%, 85%, 90%, 92%, 94%, 95%, 97%, 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 18 or 48, but at least 30%-80% of the TβRII polypeptides in the composition lack an amino acid corresponding to the first amino acid (threonine) of SEQ ID NOs: 18 or 48. In some embodiments, the TβRII polypeptides are at least 80%, 85%, 90%, 92%, 94%, 95%, 97%, 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 63-68. In some embodiments, the TβRII polypeptides are at least 80%, 85%, 90%, 92%, 94%, 95%, 97%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 68.

[0093] In certain embodiments, the present disclosure contemplates specific mutations of a polypeptide (e.g., a TβRII polypeptide or a betaglycan polypeptide) to alter the glycosylation of the polypeptide. Such mutations may be selected to introduce or remove one or more glycosylation sites, such as O-linked or N-linked glycosylation sites. An asparagine-linked glycosylation recognition site generally comprises the tripeptide sequence asparagine-X-threonine (or asparagine-X-serine), where "X" is any amino acid, which is specifically recognized by the appropriate intracellular glycosylation enzyme. Alterations may also be made by the addition or substitution of one or more serine or threonine residues into the sequence of the wild-type polypeptide (for O-linked glycosylation sites). Various amino acid substitutions or deletions at one or both of the first or third amino acid positions (and / or amino acid deletions at the second position) of the glycosylation recognition site result in non-glycosylation of the modified tripeptide sequence. Another means of increasing the number of carbohydrate moieties in a polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Depending on the mode of coupling used, sugars can be attached to (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups such as those of cysteine; (d) free hydroxyl groups such as those of serine, threonine or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine or tryptophan; or (f) the amide group of glutamine. These methods are described in WO 87 / 05330 published September 11, 1987 and Aplin and Wriston (1981) CRC Crit. Rev. Biochem., pp. 259-306, which are incorporated herein by reference. Removal of one or more carbohydrate moieties present in a polypeptide can be accomplished chemically and / or enzymatically. Chemical deglycosylation can involve, for example, exposure of the polypeptide to the compound trifluoromethanesulfonic acid, or an equivalent compound.This treatment results in the cleavage of most or all sugars except the linked sugar (N-acetylglucosamine or N-acetylgalactosamine), but leaves the amino acid sequence intact. Chemical deglycosylation is further described by Hakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and Edge et al. (1981) Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties in polypeptides can be achieved by the use of various endoglycosidases and exoglycosidases as described by Thotakura et al. (1987) Meth. Enzymol. 138:350. The sequence of the polypeptide may be adjusted, if necessary, depending on the type of expression system used, since mammalian cells, yeast cells, insect cells and plant cells can all introduce different glycosylation patterns that may affect the amino acid sequence of the peptide. Generally, polypeptides for use in humans (e.g., TβRII polypeptides or betaglycan polypeptides) are expressed in mammalian cell lines that provide appropriate glycosylation, such as HEK293 or CHO cell lines, although other mammalian expression cell lines, yeast cell lines and insect cells with engineered glycosylation enzymes are expected to be useful as well.

[0094] The present disclosure further contemplates a method for generating mutants, particularly sets of combinatorial mutants of polypeptides (e.g., TβRII polypeptides or betaglycan polypeptides), as well as truncation mutants, and the pool of combinatorial mutants is particularly useful for identifying functional variant sequences. The purpose of screening such combinatorial libraries can be, for example, to generate polypeptide variants that can act as either agonists or antagonists, or all together have novel activities. Various screening assays are provided below, and such assays can be used to evaluate variants. For example, TβRII polypeptide variants may be screened for their ability to bind to TβRII ligands, prevent TβRII ligands from binding to TβRII polypeptides, or interfere with signal transduction caused by TβRII ligands. The activity of TβRII polypeptides or variants thereof can also be tested in cell-based or in vivo assays, particularly any of the assays disclosed in the Examples.

[0095] Combinatorially derived variants can be generated that have selectivity or generally increased potency for polypeptides that include the extracellular domain of a naturally occurring polypeptide (e.g., a TβRII polypeptide or a betaglycan polypeptide). Similarly, mutagenesis can result in variants that have dramatically different serum half-lives than the corresponding wild-type polypeptide. For example, the altered protein can be made either more stable or less stable to proteolysis or other processes that result in the destruction, or otherwise elimination or inactivation, of the native TβRII polypeptide. Such variants, and the genes that encode them, can be utilized to alter the levels of TβRII polypeptides by modulating the half-life of the TβRII polypeptide. For example, a shorter half-life can result in a more transient biological effect and may allow for tighter control of the levels of the recombinant polypeptide in the patient. In an Fc fusion protein, mutations can be made in the linker (if any) and / or the Fc portion to alter the half-life of the protein.

[0096] Combinatorial libraries may be generated by means of a degenerate library of genes encoding a library of polypeptides, each of which contains at least a portion of a potential polypeptide (e.g., a TβRII polypeptide or a betaglycan polypeptide) sequence. For example, a mixture of synthetic oligonucleotides can be enzymatically ligated to a gene sequence such that a degenerate set of potential polypeptide nucleotide sequences can be expressed as individual polypeptides or as a set of larger fusion proteins (e.g., for phage display).

[0097] There are many ways in which a library of potential polypeptide (e.g., TβRII or betaglycan polypeptide) variants can be generated from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene can then be ligated into an appropriate vector for expression. The synthesis of degenerate oligonucleotides is well known in the art (see, e.g., Narang, SA (1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 11:477). Such techniques have been used in the directed evolution of other proteins (see, e.g., Scott et al., (1990) Science 249:386-390; Roberts et al., (1992) PNAS USA 89:2429-2433; Devlin et al., (1990) Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; and U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

[0098] Alternatively, other forms of mutagenesis can be used to generate combinatorial libraries. For example, polypeptide (e.g., TβRII polypeptide or betaglycan polypeptide) variants can be constructed using, for example, alanine scanning mutagenesis, etc. (Ruf et al., (1994) Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al., (1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol. Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838; and Cunningham et al., (1989) Science 244:1081-1085); by linker scanning mutagenesis (Gustin et al., (1993) Virology 193:653-660; Brown et al., (1992) Mol. Cell Biol. 12:2644-2652; McKnight et al., (1982) Science 232:316); by saturation mutagenesis (Meyers et al., (1986) Science 232:613); by PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol 1:11-19); or by random mutagenesis, including chemical mutagenesis (Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY; and Greener et al., (1994) Strategies in Mol Biol 7:32-34), can be produced and isolated from libraries.Linker scanning mutagenesis is an attractive method for identifying truncated (bioactive) forms of a polypeptide, especially in combinatorial conditions.

[0099] A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutation and truncation, and even for screening cDNA libraries for gene products with certain properties. Such techniques are generally applicable to the rapid screening of gene libraries made by combinatorial mutagenesis of polypeptides (e.g., TβRII polypeptides or betaglycan polypeptides). The most widely used techniques for screening large gene libraries typically involve cloning the gene library into a replicable expression vector, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions where detection of the desired activity facilitates the relatively easy isolation of the vectors encoding the genes whose products are detected. Preferred assays include ligand binding assays and ligand-mediated cell signaling assays.

[0100] In certain embodiments, the polypeptides of the present disclosure (e.g., TβRII or betaglycan polypeptides) may further comprise post-translational modifications in addition to any that are naturally present in the native polypeptide. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, pegylation (polyethylene glycol), and acylation. As a result, modified polypeptides may contain non-amino acid elements such as polyethylene glycol, lipids, mono- or polysaccharides, and phosphates. The effect of such non-amino acid elements on the functionality of the polypeptide may be tested as described herein for other polypeptide variants. When a polypeptide is produced in a cell by cleaving a nascent form of the polypeptide, post-translational processing may also be important for the proper folding and / or function of the protein. Different cells (such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293) have specific cellular machinery and characteristic mechanisms for such post-translational activities and can be selected to ensure proper modification and processing of the polypeptide.

[0101] In certain aspects, the present disclosure provides a fusion protein (e.g., a TβRII fusion protein or a betaglycan fusion protein), in which in some embodiments, a first portion (e.g., a TβRII polypeptide portion or a betaglycan polypeptide portion) is connected to a heterologous portion (e.g., an Fc portion) by a linker. In some embodiments, the linker is a glycine and serine-rich linker. Other near-neutral amino acids, such as, but not limited to, Thr, Asn, Pro and Ala, may also be used in the linker sequence. In some embodiments, the linker comprises various permutations of amino acid sequences containing Gly and Ser. In some embodiments, the linker is greater than 10 amino acids in length. In further embodiments, the linker has a length of at least 12, 15, 20, 21, 25, 30, 35, 40, 45 or 50 amino acids. In some embodiments, the linker is less than 40, 35, 30, 25, 22 or 20 amino acids. In some embodiments, the linker is 10-50, 10-40, 10-30, 10-25, 10-21, 10-15, 10, 15-25, 17-22, 20 or 21 amino acids long. In some preferred embodiments, the linker comprises the amino acid sequence GlyGlyGlyGlySer(GGGGS) (SEQ ID NO: 19), or repeats thereof (GGGGS)n, where n≧2. In certain embodiments, n≧3 or n=3-10. The present application teaches the surprising finding that proteins comprising a TβRII portion and a heterologous portion fused together by a (GGGGS)4 linker associate with stronger affinity to TGFβ1 and TGFβ3 compared to TβRII fusion proteins where n<4. Thus, in preferred embodiments, n≧4 or n=4-10. The present application also teaches that proteins containing (GGGGS)n linkers where n>4 had similar inhibitory properties as proteins with (GGGGS)4 linkers. Thus, in some embodiments, n is not greater than 4 in the (GGGGS)n linker. In some embodiments, n=4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, or 5-6. In some embodiments, n=3, 4, 5, 6, or 7. In certain embodiments, n=4.In some embodiments, (GGGGS). n The linker comprising the sequence also comprises an N-terminal threonine. In some embodiments, the linker is any one of the following: [ka]

[0102] In some embodiments, the linker comprises the amino acid sequence of TGGGPKSCDK (SEQ ID NO: 7). In some embodiments, the linker is any one of SEQ ID NOs: 21, 4-7, 25-26, or 40 lacking the N-terminal threonine. In some embodiments, the linker does not comprise the amino acid sequence of SEQ ID NO: 26 or 40.

[0103] In certain embodiments, functional variants or modified forms of TβRII (or betaglycan) polypeptides include fusion proteins having at least a portion of a TβRII (or betaglycan) polypeptide and one or more heterologous moieties. Well-known examples of such heterologous moieties include, but are not limited to, polyhistidine, Glu-Glu, glutathione S-transferase (GST), thioredoxin, protein A, protein G, immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP) or human serum albumin. The heterologous moiety may be selected to confer desired properties. For example, some heterologous moieties are particularly useful for the isolation of fusion proteins by affinity chromatography. For the purpose of affinity purification, relevant matrices for affinity chromatography are used, such as glutathione-bound resins, amylase-bound resins, and nickel- or cobalt-bound resins. Many such matrices are available in the form of "kits," such as the useful QIAexpress™ system (Qiagen) using the Pharmacia GST purification system and (HIS6) fusion partners. As another example, the heterologous moiety may be selected to facilitate detection of the TβRII (or betaglycan) polypeptide. Examples of such detection domains include various fluorescent proteins (e.g., GFP) and "epitope tags," which are usually short peptide sequences for which specific antibodies are available. Well-known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus hemagglutinin (HA) and c-myc tags. In some cases, the heterologous moiety has a protease cleavage site, such as for factor Xa or thrombin, which allows the relevant protease to partially digest the fusion protein, thereby liberating the recombinant protein from the fusion protein. The liberated protein can then be isolated from the heterologous moiety by subsequent chromatographic separation.In certain preferred embodiments, the TβRII (or betaglycan) polypeptide is fused to a domain that stabilizes the TβRII polypeptide in vivo (a "stabilizer" domain). By "stabilize" is meant something that increases serum half-life, whether this is through reduced destruction, reduced clearance by the kidney, or other pharmacokinetic effects. Fusions with the Fc portion of immunoglobulins are known to confer desirable pharmacokinetic properties on a wide range of proteins. Similarly, fusions with human serum albumin can confer desirable properties. Other types of heterologous moieties that can be selected include multimerization (e.g., dimerization, tetramerization) domains and functional domains.

[0104] As a detailed example, the present disclosure provides a fusion protein comprising a variant of a TβRII polypeptide fused to an Fc domain sequence of SEQ ID NO: 49. Optionally, the Fc domain has one or more mutations at residues such as Asp-265, Lys-322 and Asn-434 (numbering according to the corresponding full-length IgG). In certain cases, the mutant Fc domain with one or more of these mutations (e.g., Asp-265 mutation) has a reduced ability to bind to Fcγ receptors relative to the wild-type Fc domain. In other cases, the mutant Fc domain with one or more of these mutations (e.g., Asn-434 mutation) has an increased ability to bind to MHC class I-associated Fc receptors (FcRN) relative to the wild-type Fc domain.

[0105] It is understood that the different elements of the fusion protein may be arranged in any manner consistent with the desired functionality. For example, the TβRII (or betaglycan) polypeptide may be placed C-terminal to the heterologous domain, or the heterologous domain may be placed C-terminal to the TβRII (or betaglycan) polypeptide. The TβRII (or betaglycan) polypeptide domain and the heterologous domain do not need to be adjacent in the fusion protein, and additional domains or amino acid sequences may be included at the C-terminus or N-terminus of either domain, or between the domains.

[0106] As used herein, the term "immunoglobulin Fc domain" or simply "Fc" is understood to mean the carboxyl terminal portion of the constant region of an immunoglobulin chain, preferably an immunoglobulin heavy chain constant region or a portion thereof. For example, an immunoglobulin Fc region may include 1) a CH1 domain, a CH2 domain and a CH3 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, or 5) a combination of two or more domains and an immunoglobulin hinge region. In a preferred embodiment, the immunoglobulin Fc region includes at least an immunoglobulin hinge region, a CH2 domain and a CH3 domain, and preferably lacks a CH1 domain. In some embodiments, the immunoglobulin Fc region is a human immunoglobulin Fc region.

[0107] In one embodiment, the immunoglobulin class from which the heavy chain constant region is derived is IgG (Igγ) (γ subclasses 1, 2, 3, or 4).

[0108] An example of a natural amino acid sequence that can be used for the Fc portion of human IgG1 (G1Fc) is shown below (SEQ ID NO:58). The dotted underline indicates the hinge region and the solid underline indicates the position with a naturally occurring variant. In part, the disclosure provides a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence having 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:58. Naturally occurring variants in G1Fc would include E134D and M136L according to the numbering system used in SEQ ID NO:58 (see Uniprot P01857). [ka]

[0109] Optionally, the IgG1 Fc domain has one or more mutations at residues such as Asp-265, lysine 322 and Asn-434. In certain cases, the mutant IgG1 Fc domain with one or more of these mutations (e.g., Asp-265 mutation) has a reduced ability to bind to Fcγ receptors compared to the wild-type Fc domain. In other cases, the mutant Fc domain with one or more of these mutations (e.g., Asn-434 mutation) has an increased ability to bind to MHC class I-associated Fc receptors (FcRN) compared to the wild-type IgG1 Fc domain.

[0110] An example of a native amino acid sequence that may be used for the Fc portion of human IgG2 (G2Fc) is shown below (SEQ ID NO:59). The dotted underline indicates the hinge region and the double underline indicates the position in the sequence where there is a database conflict (from UniProt P01859). In part, the disclosure provides polypeptides comprising, consisting essentially of, or consisting of an amino acid sequence having 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:59. [ka]

[0111] Two examples of amino acid sequences that can be used for the Fc portion of human IgG3 (G3Fc) are shown below. The hinge region in G3Fc can be up to four times as long as other Fc chains and contains three identical 15-residue segments preceding a similar 17-residue segment. The first G3Fc sequence shown below (SEQ ID NO: 60) contains a short hinge region consisting of a single 15-residue segment, while the second G3Fc sequence (SEQ ID NO: 61) contains a full-length hinge region. In each case, the dotted underline indicates the hinge region and the solid underline indicates the position with a naturally occurring variant according to UniProt P01859. In part, the disclosure provides polypeptides comprising, consisting essentially of, or consisting of an amino acid sequence having 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 60 or 61. [ka]

[0112] Naturally occurring variants in G3Fc (see, for example, Uniprot P01860) include E68Q, P76L, E79Q, Y81F, D97N, N100D, T124A, S169N, S169del, F221Y when converted to the numbering system used in SEQ ID NO:60, and the present disclosure provides fusion proteins that include a G3Fc domain that contains one or more of these variations. In addition, the human immunoglobulin IgG3 gene (IGHG3) shows structural polymorphisms characterized by different hinge lengths (see, Uniprot P01859). In particular, variant WIS lacks most of the V region and all of the CH1 region. It has an extra interchain disulfide bond at position 7 in addition to the 11 normally present in the hinge region. Variant ZUC lacks most of the V region, all of the CH1 region, and a portion of the hinge. The variant OMM may represent an allelic form or another gamma chain subclass. The present disclosure provides additional fusion proteins that include a G3Fc domain that contains one or more of these variants.

[0113] An example of a native amino acid sequence that may be used for the Fc portion of human IgG4 (G4Fc) is shown below (SEQ ID NO:62). The dotted underline indicates the hinge region. In part, the disclosure provides polypeptides comprising, consisting essentially of, or consisting of an amino acid sequence having 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:62. [ka]

[0114] Various engineered mutations in the Fc domain are presented herein with respect to the G1Fc sequence (SEQ ID NO: 58), and similar mutations in G2Fc, G3Fc and G4Fc can be derived from their alignment with G1Fc in Figure 7. Due to the heterogeneous hinge lengths, similar Fc positions based on the isotype alignment (Figure 7) have different amino acid numbers in SEQ ID NOs: 58, 59, 60, 61 and 62. Hinge, C H 2 and C H A given amino acid position in a three-region immunoglobulin sequence (e.g., SEQ ID NOs: 58, 59, 60, 61, and 62) is numbered according to the total IgG1 heavy chain constant domain (C H 1, hinge, C H 2 and C H It will also be apparent that when the term encompasses a sequence comprising a plurality of regions (e.g., a sequence of a nucleic acid molecule, a nucleic acid ...

[0115] Other classes of immunoglobulins, IgA (Igα), IgD (Igδ), IgE (Igε) and IgM (Igμ), may also be used. Selection of appropriate immunoglobulin heavy chain constant regions is discussed in detail in U.S. Pat. Nos. 5,541,087 and 5,726,044. Selection of a particular immunoglobulin heavy chain constant region sequence from a particular immunoglobulin class and subclass to achieve a particular result is considered to be within the level of skill in the art. The portion of the DNA construct encoding the immunoglobulin Fc region includes at least a portion of the hinge domain, and preferably includes at least a portion of the CH3 domain of Fc gamma or a homologous domain in any of IgA, IgD, IgE or IgM.

[0116] Furthermore, it is contemplated that substitutions or deletions of amino acids within the immunoglobulin heavy chain constant region may be useful in the practice of the methods and compositions disclosed herein. One example would be to introduce amino acid substitutions in the upper portion of the CH2 region to create an Fc variant with reduced affinity for the Fc receptor (Cole et al. (1997) J. Immunol. 159:3613).

[0117] In some embodiments, the disclosure provides a TβRII polypeptide fusion protein comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 11, 13, 15 and 17, or a biologically active fragment thereof. In some embodiments, the TβRII polypeptide fusion protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 11, 13 and 15, or a biologically active fragment thereof. In some embodiments, a TβRII polypeptide fusion protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 13, or a biologically active fragment thereof. In some embodiments, a TβRII polypeptide fusion protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 63, or a biologically active fragment thereof. In some embodiments, a TβRII polypeptide fusion protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 48, or a biologically active fragment thereof. In some embodiments, a TβRII polypeptide fusion protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 64, or a biologically active fragment thereof. In some embodiments, a TβRII polypeptide fusion protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 65, or a biologically active fragment thereof.In some embodiments, a TβRII polypeptide fusion protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 66, or a biologically active fragment thereof. In some embodiments, a TβRII polypeptide fusion protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 67, or a biologically active fragment thereof. In some embodiments, a TβRII polypeptide fusion protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 68, or a biologically active fragment thereof. In some embodiments, the TβRII polypeptide fusion protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:49, or a biologically active fragment thereof.

[0118] In some embodiments, the fusion proteins described herein have improved binding affinity for TGFβ1 and TGFβ3. In some embodiments, a fusion protein comprising a linker of at least 10 amino acids in length (e.g., a fusion protein having an amino acid sequence of any one of SEQ ID NOs: 11, 13, 15, 48, and 63-68) has improved binding affinity for TGFβ1 and TGFβ3 compared to a reference fusion protein (e.g., a fusion protein having an amino acid sequence of SEQ ID NO: 9). In some embodiments, the fusion protein has a K of less than 200 pM, less than 150 pM, less than 100 pM, less than 75 pM, less than 50 pM, or less than 25 pM. D In some embodiments, the fusion protein binds to TGFβ1 with a K of less than 75 pM, less than 70 pM, less than 60 pM, less than 50 pM, less than 40 pM, less than 35 pM, less than 25 pM, less than 15 pM, less than 10 pM, or less than 5 pM. D It binds to TGFβ3.

[0119] In some embodiments, any of the polypeptides disclosed herein inhibit TGFβ1 and / or TGFβ3 in a measurable assay. In some embodiments, the polypeptide has an IC of less than 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.08, 0.09, 0.07, 0.06, 0.05, 0.04, 0.03, or 0.02 nM as determined using a reporter gene assay. 50 In some embodiments, the polypeptide has an IC of less than 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03 or 0.02 nM as determined using a reporter gene assay. 50 In some embodiments, the reporter gene assay is a CAGA reporter assay. In some embodiments, the CAGA assay is based on a human lung cancer cell line transfected with pGL3(CAGA)12 reporter plasmid (Dennler et al, 1998, EMBO 17: 3091-3100) and Renilla reporter plasmid (pRLCMV) to control for transfection efficiency. CAGA motifs are present in the promoters of TGFβ-responsive genes (e.g., PAI-1), so this vector is commonly used for factors that signal through SMAD2 and SMAD3. See, for example, Example 2.

[0120] In some embodiments, the present disclosure provides a TβRII (or betaglycan)-containing fusion polypeptide. The fusion polypeptide can be prepared by any of the methods disclosed herein or known in the art.

[0121] In some embodiments, any fusion polypeptide disclosed herein comprises the following components: a) any TβRII (or betaglycan) polypeptide disclosed herein ("A"), b) any linker disclosed herein ("B"), c) any heterologous moiety disclosed herein ("C"), and optionally a linker ("X"). In such embodiments, the fusion polypeptide may be arranged in the following manner (N-terminus to C-terminus): ABC or CBA. In such embodiments, the fusion polypeptide may be arranged in the following manner (N-terminus to C-terminus): XABC or XCBA. In some embodiments, the fusion polypeptide comprises each of A, B, and C (and optionally a leader sequence, such as the amino acid sequence of SEQ ID NO: 23), and comprises no more than 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 additional amino acids (but may comprise further post-translational modifications, such as pegylation).

[0122] In some embodiments, the fusion polypeptide comprises a leader sequence (e.g., SEQ ID NO: 23) located in the following manner (N-terminus to C-terminus): XABC, where the fusion polypeptide comprises 1, 2, 3, 4 or 5 amino acids between X and A. In some embodiments, the fusion polypeptide comprises a leader sequence (e.g., SEQ ID NO: 23) located in the following manner (N-terminus to C-terminus): XCBA, where the fusion polypeptide comprises 1, 2, 3, 4 or 5 amino acids between X and C. In some embodiments, the fusion polypeptide comprises a leader sequence (e.g., SEQ ID NO: 23) located in the following manner (N-terminus to C-terminus): XABC, where the fusion polypeptide comprises an alanine between X and A. In some embodiments, the fusion polypeptide comprises a leader sequence (e.g., SEQ ID NO: 23) located in the following manner (N-terminus to C-terminus): XCBA, where the fusion polypeptide comprises an alanine between X and C. In some embodiments, the fusion polypeptide comprises a leader sequence (e.g., SEQ ID NO: 23) located in the following manner (N-terminus to C-terminus): XABC, where the fusion polypeptide comprises a glycine and an alanine between X and A. In some embodiments, a fusion polypeptide comprises a leader sequence (e.g., SEQ ID NO:23) located in the following manner (N-terminus to C-terminus): XCBA, where the fusion polypeptide comprises a glycine and an alanine between X and C. In some embodiments, a fusion polypeptide comprises a leader sequence (e.g., SEQ ID NO:23) located in the following manner (N-terminus to C-terminus): XABC, where the fusion polypeptide comprises a threonine between X and A. In some embodiments, a fusion polypeptide comprises a leader sequence (e.g., SEQ ID NO:23) located in the following manner (N-terminus to C-terminus): XCBA, where the fusion polypeptide comprises a threonine between X and C.

[0123] In some embodiments, the fusion polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of the amino acid sequences of the TβRII polypeptides disclosed herein (e.g., SEQ ID NO: 18), and the TβRII polypeptide portion of the fusion polypeptide comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids (but may include further post-translational modifications such as pegylation). In some embodiments, the fusion polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of the linker sequences disclosed herein (e.g., SEQ ID NO: 6), and the linker portion of the fusion polypeptide comprises no more than 5, 4, 3, 2 or 1 additional amino acids (but may include further post-translational modifications such as pegylation). In some embodiments, a fusion polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of the sequences of the heterologous moieties disclosed herein (e.g., SEQ ID NO: 49), and the heterologous moiety of the fusion polypeptide comprises no more than 25, 20, 15, 10, 5, 4, 3, 2 or 1 additional amino acid (but may include further post-translational modifications such as pegylation). In some embodiments, a fusion polypeptide comprises any of the amino acid sequences of the TβRII polypeptides disclosed herein (e.g., SEQ ID NO: 18), and the TβRII polypeptide portion of the fusion polypeptide comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acid (but may include further post-translational modifications such as pegylation).In some embodiments, a fusion polypeptide comprises any of the linker sequences disclosed herein (e.g., SEQ ID NO: 6), where the linker portion of the fusion polypeptide comprises no more than 5, 4, 3, 2, or 1 additional amino acids (but may comprise further post-translational modifications such as pegylation). In some embodiments, a fusion polypeptide comprises any of the heterologous portion sequences disclosed herein (e.g., SEQ ID NO: 49), where the heterologous portion of the fusion polypeptide comprises no more than 25, 20, 15, 10, 5, 4, 3, 2, or 1 additional amino acids (but may comprise further post-translational modifications such as pegylation).

[0124] In some embodiments, the disclosure provides a fusion polypeptide, the fusion polypeptide comprising: a) an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of the amino acid sequences of the TβRII polypeptides disclosed herein (e.g., SEQ ID NO: 18), where the TβRII polypeptide portion of the fusion polypeptide includes no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids (but may include further post-translational modifications such as pegylation); b) an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of the linker sequences disclosed herein (e.g., SEQ ID NO: 6). and c) an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of the sequences of the heterologous moieties disclosed herein (e.g., SEQ ID NO:49), where the heterologous portion of the fusion polypeptide comprises no more than 25, 20, 15, 10, 5, 4, 3, 2 or 1 additional amino acids (but may comprise further post-translational modifications such as pegylation); and d) optionally consisting of or consisting essentially of (not necessarily in this order) a leader sequence (e.g., SEQ ID NO:23).In some embodiments, the disclosure provides a fusion polypeptide, the fusion polypeptide comprising: a) any of the amino acid sequences of a TβRII polypeptide disclosed herein (e.g., SEQ ID NO: 18), where the TβRII polypeptide portion of the fusion polypeptide comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 additional amino acids (but may comprise further post-translational modifications such as pegylation); b) any of the linker sequences disclosed herein (e.g., SEQ ID NO: 6), where the heterologous portion of the fusion polypeptide comprises no more than 25, 20, 15, 10, 5, 6, 7, 8, 9, 10, 15 ... , 4, 3, 2 or 1 or less additional amino acids (but may include further post-translational modifications such as pegylation); and c) any of the heterologous moiety sequences disclosed herein (e.g., SEQ ID NO:49), where the heterologous moiety of the fusion polypeptide includes no more than 25, 20, 15, 10, 5, 4, 3, 2 or 1 or less additional amino acids (but may include further post-translational modifications such as pegylation); and d) optionally consisting of or consisting essentially of (not necessarily in this order) a leader sequence (e.g., SEQ ID NO:23).

[0125] In some embodiments, the disclosure provides a TβRII polypeptide portion consisting of: a) an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18, and up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids (but may include further post-translational modifications such as pegylation); or b) an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 6, and up to 5, 4, 3, 2 or 1 additional amino acids. and c) a heterologous portion consisting of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:49 and no more than 25, 20, 15, 10, 5, 4, 3, 2 or 1 additional amino acid sequence (but which may include further post-translational modifications such as pegylation); and d) optionally a fusion polypeptide consisting of or consisting essentially of (not necessarily in this order) a leader sequence (e.g., SEQ ID NO:23). In some embodiments, the disclosure provides fusion polypeptides consisting of or consisting essentially of (not necessarily in this order): a) a TβRII polypeptide portion consisting of the amino acid sequence of SEQ ID NO: 18 and not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids (but which may include further post-translational modifications such as pegylation); b) a linker portion consisting of the amino acid sequence of SEQ ID NO: 6 and not more than 5, 4, 3, 2 or 1 additional amino acids (but which may include further post-translational modifications such as pegylation); and c) a heterologous portion consisting of the amino acid sequence of SEQ ID NO: 49 and not more than 25, 20, 15, 10, 5, 4, 3, 2 or 1 additional amino acids (but which may include further post-translational modifications such as pegylation); and d) optionally a leader sequence (e.g., SEQ ID NO: 23).

[0126] In some embodiments, the fusion protein does not include a leader sequence. In some embodiments, the fusion protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:48. [ka]

[0127] In some embodiments, the disclosure provides a TβRII fusion polypeptide, wherein the polypeptide does not comprise an antibody or an antigen-binding portion thereof. In some embodiments, the polypeptide does not bind with appreciable affinity to a cytokine other than a ligand of the transforming growth factor beta superfamily (e.g., TGFβ1, TGFβ2, and / or TGFβ3). In some embodiments, the polypeptide does not bind with appreciable affinity to a cytokine other than TGFβ1, TGFβ2, and / or TGFβ3. In some embodiments, the polypeptide does not bind with appreciable affinity to a cytokine other than TGFβ1 and / or TGFβ3. In some embodiments, the polypeptide is CD4, CD8, CD25, CTLA-4, IL-10, TGFβ receptor, PD-1, PD-L1, PD-L2, RANK, RANKL, HER2 / neu, EGFR1, CD20, VEGF, TNF-α, TNFR2, FoxP3, CD80, CD86, IFN-α, IFN-β, IFN-γ, GITR, 4-1BB, OX-40, TLR1-10, ErbB-1, HER1, ErbB-3 / HER3, ErbB-4 / HER4, IGFR, IGFBP, IGF-1R, PDGFR, FGFR, VEGFR, HGFR, TRK receptor, ephrin receptor, AXL receptor, LTK receptor, TIE receptor , angiopoietin 1, 2, ROR receptor, DDR receptor, RET receptor, KLG receptor, RYK receptor, MuSK receptor, ILβR, IlαR, TNTRSF, TRAIL receptor, ARTC1, alpha-actinin-4, Bcr-abl, B-RAF, caspases, beta-catenin, fibronectin, GPNMB, GDP-L, LDLR, HLA-A2, MLA-A11, HSP70, KIAA205, MART2, MUM-1, 2, 3, PAP, neo-PAP, NFYC, OGT, OS-9, pml-RAR alpha fusion protein, PRDX5, PTPRK, KRAS2, NRAS, HRAS, RBAF600, SIRT2.SNRPD1, SYT-SSX1 or -SSX2 fusion protein, triosephosphate isomerase, BAGE, BAGE-1, BAGE-2, 3, 4, 5, GAGE-1, 2, 3, 4, 5, 6, 7, 8, GnT-V, HERV-K MEL, KK-LC, KM-HN-1, LAGE, LAGE-1, CAMEL, MAGE-1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-AS, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-3 , MAGE-B1, MAGE-B2, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2, mucin 1 (MUC1), MART-1 / Melan-A (MLANA), gp100, gp100 / Pme117 (S1LV), tyrosinase (TYR), TRP-1, HAGE, NA-88 , NY-ESO-1, NY-ESO-1 / LAGE-2, SAGE, Sp17.SSX-1, 2, 3, 4, TRP2-1NT2, carcinoembryonic antigen (CEA), Kallikfein 4, mammaglobm-A, OA1, prostate-specific antigen (PSA), pre Standing specific membrane antigen, TRP-1 / , 75.TRP-2, AIM-2.BING-4, CPSF, cyclin D1, Ep-CAM, EpbA3, FGF-5, gp250, iCE, AFP, M-CSF, mdm-2, MUCI, p53 (TP53), PBF, FRAME, PSMA, RAGE-1.RNF43, RU2AS, SOX10, STEAP1, Survivin (BIRCS), hTERT, Telomerase, WT1, SYCP1, BRDT, SPANX, XAGE, ADAM2, PAGE-5, LIP1, CTAGE-1, CSAGE, MMA1, CAGE, BORIS, HOM-TES-85, AF15q14, HCA66I, LDHC, MORC, SGY-1, SPO11, TPX1, NY-SAR-35, FTHLI7, NXF2 TDRD1, TEX 15, FATE, TPTE, Estrogen Receptor (ER), Androgen Receptor (AR), CD40, CD30, CD20, CD19, CD33, CD4, CD25, CD3, CA 72-4, CA 15-3, CA 27-29, CA 125, CA It does not bind with any appreciable affinity to 19-9, beta-human chorionic gonadotropin, 1-2 microglobulin, squamous cell carcinoma antigen, neuron-specific enoJase, heat shock protein gp96, GM2, sargramostim, CTLA-4, 707-AP, ART-4, CAP-1, CLCA2, Cyp-B, HST-2, HPV proteins, EBV proteins, Hepatitis B or C virus proteins, and / or HIV proteins.

[0128] In some embodiments, the disclosure provides a TβRII fusion polypeptide, where the polypeptide does not include an additional ligand binding domain in addition to the TβRII domain. In some embodiments, the polypeptide includes a linear amino acid sequence including a TβRII domain and a heterologous portion (e.g., an Fc portion), but the linear amino acid sequence does not include any additional ligand binding domain. In some embodiments, the polypeptide includes a linear amino acid sequence including a TβRII domain and an Fc portion, but the linear amino acid sequence does not include any additional ligand binding domain. In some embodiments, the disclosure provides a TβRII fusion polypeptide, where the polypeptide does not include multiple ligand binding domains in a single linear amino acid sequence. In some embodiments, the disclosure provides a TβRII fusion polypeptide, where the polypeptide does not include two or more consecutive linker sequences in a single linear amino acid sequence. In some embodiments, the polypeptide does not include multiple consecutive glycine and / or serine linkers (e.g., a linker including (GGGGS)n, where n=>4) in a single linear amino acid sequence. In some embodiments, the disclosure provides a TβRII fusion polypeptide in which the heterologous moiety is an Fc domain and the only contiguous linker is covalently linked to the Fc domain. In some embodiments, the only contiguous linker comprises or consists of a (GGGGS)n linker, where n=>4.

[0129] The present application further provides Fc fusion proteins with engineered or variant Fc regions. Such antibodies and Fc fusion proteins may be useful in modulating effector functions, such as, for example, antigen-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In addition, modifications may improve the stability of antibodies and Fc fusion proteins. Amino acid sequence variants of antibodies and Fc fusion proteins are prepared by introducing appropriate nucleotide changes into DNA or by peptide synthesis. Such variants include, for example, deletions from and / or insertions into and / or substitutions of residues within the amino acid sequences of the antibodies and Fc fusion proteins disclosed herein. Any combination of deletions, insertions and substitutions may be made to arrive at the final construct, provided that the final construct has the desired properties. Amino acid changes, such as changes in the number or position of glycosylation sites, may also alter post-translational processing of antibodies and Fc fusion proteins.

[0130] Antibodies and Fc fusion proteins with reduced effector function can be generated by introducing changes in the amino acid sequence, including, but not limited to, Ala-Ala mutations as described by Bluestone et al. (see WO 94 / 28027 and WO 98 / 47531; see also Xu et al. 2000 Cell Immunol 200; 16-26). Thus, in certain embodiments, Fc fusion proteins of the present disclosure with mutations in the constant region, including Ala-Ala mutations, can be used to reduce or abolish effector function. According to these embodiments, the antibodies and Fc fusion proteins may include a mutation at position 234 to alanine, or a mutation at position 235 to alanine, or a combination thereof. In one embodiment, the antibody or Fc fusion protein comprises an IgG4 framework, and the Ala-Ala mutation describes a mutation from phenylalanine to alanine at position 234 and / or a mutation from leucine to alanine at position 235. In another embodiment, the antibody or Fc fusion protein comprises an IgG1 framework and the Ala-Ala mutation describes a leucine to alanine mutation at position 234 and / or a leucine to alanine mutation at position 235. The antibody or Fc fusion protein may alternatively or additionally carry other mutations, including the point mutation K322A in the CH2 domain (Hezareh et al. 2001 J Virol. 75: 12161-8).

[0131] In certain embodiments, the antibody or Fc fusion protein may be modified to either enhance or inhibit complement-dependent cytotoxicity (CDC). Modulated CDC activity may be achieved by introducing one or more amino acid substitutions, insertions or deletions in the Fc region (see, for example, U.S. Pat. No. 6,194,551). Alternatively or in addition, cysteine ​​residues may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus produced may have improved or reduced internalization capability and / or increased or reduced complement-mediated cell killing. See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, BJ Immunol. 148:2918-2922 (1992), WO 99 / 51642, Duncan & Winter Nature 322: 738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351. B. Nucleic Acids and Methods of Production

[0132] In certain embodiments, the present disclosure makes available isolated and / or purified forms of polypeptides (e.g., TβRII or betaglycan polypeptides) that are isolated from or otherwise substantially free (e.g., at least 80%, 90%, 95%, 96%, 97%, 98% or 99% free) of other proteins and / or other polypeptide species. Polypeptides are generally produced by expression from recombinant nucleic acids.

[0133] In certain embodiments, the present disclosure includes a nucleic acid encoding a soluble polypeptide (e.g., a TβRII polypeptide or a betaglycan polypeptide) that includes a coding sequence for an extracellular portion of a protein (e.g., a TβRII protein or a betaglycan protein). In further embodiments, the present disclosure also relates to a host cell that includes such a nucleic acid. The host cell can be any prokaryotic or eukaryotic cell. For example, the polypeptide of the present disclosure can be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast or mammalian cells. Other suitable host cells are known to those of skill in the art. Thus, some embodiments of the present disclosure further relate to methods of producing a polypeptide.

[0134] In certain aspects, the present disclosure provides isolated and / or recombinant nucleic acids encoding any of the polypeptides (e.g., TβRII or betaglycan polypeptides), including fragments, functional variants, and fusion proteins disclosed herein. SEQ ID NOs: 8, 10, 12, 14, 16, 46, or 47 encode variants of the TβRII extracellular domain fused with an IgG Fc domain. The subject nucleic acids can be single-stranded or double-stranded. Such nucleic acids can be DNA or RNA molecules. These nucleic acids can be used, for example, in methods for making polypeptides or as direct therapeutic agents (e.g., antisense, RNAi, or gene therapy approaches).

[0135] It is further understood that in certain embodiments, a subject nucleic acid encoding a polypeptide comprises a nucleic acid that is a variant of SEQ ID NO: 8, 10, 12, 14, 16, 46, or 47. Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions, or deletions, such as allelic variants.

[0136] In certain embodiments, the disclosure provides isolated or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 8, 10, 12, 14, 16, 46 or 47. In certain embodiments, the disclosure provides isolated or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 12 or a fragment thereof. It will be apparent to one of skill in the art that nucleic acid sequences complementary to SEQ ID NO: 8, 10, 12, 14, 16, 46 or 47, as well as variants of SEQ ID NO: 8, 10, 12, 14, 16, 46 or 47, are also within the scope of the present disclosure. In further embodiments, the nucleic acid sequences of the present disclosure may be isolated, recombinant and / or fused with a heterologous nucleotide sequence, or may be nucleic acid sequences in a DNA library.

[0137] In other embodiments, the nucleic acid of the present disclosure also includes a nucleotide sequence that hybridizes under highly stringent conditions to the nucleotide sequence designated in SEQ ID NO: 8, 10, 12, 14, 16, 46 or 47, the complement sequence of SEQ ID NO: 8, 10, 12, 14, 16, 46 or 47, or a fragment thereof. As discussed above, those skilled in the art will readily appreciate that suitable stringency conditions that promote DNA hybridization can vary. For example, hybridization can be performed at about 45°C with 6.0x sodium chloride / sodium citrate (SSC), followed by washing at 50°C with 2.0x SSC. For example, the salt concentration during the washing step can be selected from a low stringency of about 2.0x SSC, 50°C to a high stringency of about 0.2x SSC, 50°C. In addition, the temperature in the wash step can be increased from low stringency conditions of about 22° C. at room temperature to high stringency conditions of about 65° C. Both temperature and salt can be varied, or temperature or salt concentration can be held constant while the other variable is changed. In some embodiments, the disclosure provides nucleic acids hybridized under low stringency conditions of 6×SSC at room temperature, followed by washing at 2×SSC at room temperature.

[0138] Isolated nucleic acids that differ from the nucleic acids described in SEQ ID NO: 8, 10, 12, 14, 16, 46 or 47 are also within the scope of this disclosure due to degeneracy in the genetic code. For example, some amino acids are designated by two or more triplet bases. Codons that specify the same amino acid, or synonyms (e.g., CAU and CAC are synonyms for histidine), may result in "silent" mutations that do not affect the amino acid sequence of the protein. However, DNA sequence polymorphisms that result in changes in the amino acid sequence of a protein of interest are expected to exist among mammalian cells. It is clear to one of skill in the art that these variations in one or more nucleotides (up to about 3-5% of the nucleotides) of a nucleic acid encoding a particular protein may exist among individuals of a given species due to natural allelic variations. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this disclosure.

[0139] It will be apparent to one skilled in the art that the corresponding variant based on the long isoform of TβRII contains a nucleotide sequence encoding a 25 amino acid insertion with a conservative Val-Ile substitution at the C-terminal position adjacent to the insertion. It will also be apparent that the corresponding variant based on either the long (A) or short (B) isoform of TβRII contains a variant nucleotide sequence (SEQ ID NO: 41) that contains a 108 nucleotide insertion and encodes a 36 amino acid insertion at the same location as described for the naturally occurring isoform C of TβRII.

[0140] In certain embodiments, the recombinant nucleic acid of the present disclosure may be operably linked to one or more regulatory nucleotide sequences in an expression construct. The regulatory nucleotide sequence is generally appropriate for the host cell used for expression. Numerous types of suitable expression vectors and suitable regulatory sequences are known in the art for various host cells. Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription start and end sequences, translation start and end sequences, and enhancer or activator sequences. Constitutive or inducible promoters known in the art are contemplated by the present disclosure. The promoter may be either a naturally occurring promoter or a hybrid promoter that combines elements of two or more promoters. The expression construct may be present in the cell in an episome, such as a plasmid, or the expression construct may be inserted into a chromosome. In a preferred embodiment, the expression vector contains a selection marker gene that allows for the selection of transformed host cells. Selection marker genes are well known in the art and vary with the host cell used.

[0141] In certain embodiments disclosed herein, the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding a polypeptide (e.g., a TβRII polypeptide or a betaglycan polypeptide) and operably linked to at least one regulatory sequence. Regulatory sequences are art-recognized and selected to direct the expression of a T polypeptide. Thus, the term regulatory sequence includes promoters, enhancers and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990). For example, any of a wide range of expression control sequences that control the expression of a DNA sequence when operably linked can be used in these vectors to express a DNA sequence encoding a polypeptide (e.g., a TβRII polypeptide or a betaglycan polypeptide). Such useful expression control sequences include, for example, the SV40 early and late promoters, the tet promoter, the adenovirus or cytomegalovirus immediate early promoters, the RSV promoter, the lac system, the trp system, the TAC or TRC system, the T7 promoter whose expression is directed by the T7 RNA polymerase, the major operator and promoter region of the phage lambda, the control region for the fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoter of acid phosphatase, e.g., Pho5, the promoter of yeast α-mating factor, the polyhedron promoter of the baculovirus system, and other sequences known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and / or the type of protein desired to be expressed. Also to be considered are the copy number of the vector, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers.

[0142] The recombinant nucleic acid included in the present disclosure can be produced by ligating the cloned gene or a portion thereof into a suitable vector for expression in either or both prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian cells). Expression vehicles for the production of recombinant polypeptides (e.g., TβRII polypeptides or betaglycan polypeptides) include plasmids and other vectors. For example, suitable vectors include the following types of plasmids: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells such as E. coli.

[0143] Some mammalian expression vectors contain both prokaryotic sequences to facilitate propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. Vectors derived from pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg are examples of suitable mammalian expression vectors for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids such as pBR322 to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found below in the discussion of gene therapy delivery systems. Various methods used in preparing plasmids and transforming host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, please refer to Molecular Cloning A Laboratory Manual, 3rd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 2001). In some cases, it may be desirable to express recombinant polypeptides by using baculovirus expression systems. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as pBlueBacIII containing β-gal).

[0144] In certain embodiments, vectors such as Pcmv-Script vector (Stratagene, La Jolla, Calif.), pcDN4 vector (Invitrogen, Carlsbad, Calif.) and pCI-neo vector (Promega, Madison, Wisc.) are designed for the production of a polypeptide of interest (e.g., a TβRII polypeptide or a betaglycan polypeptide) in CHO cells. In a preferred embodiment, the vector is designed for the production of a polypeptide of interest in HEK-293 cells. As will be apparent, the genetic construct of interest can be used to cause expression of a polypeptide of interest in cells grown in culture, for example, to produce a protein, including a fusion protein or a variant protein, for purification.

[0145] The present disclosure also relates to host cells transfected with a recombinant gene comprising a coding sequence (e.g., SEQ ID NO: 8, 10, 12, 14, 16, 46 or 47) for one or more of the polypeptides of interest (e.g., TβRII polypeptide or betaglycan polypeptide). The host cell can be any prokaryotic or eukaryotic cell. For example, the TβRII polypeptides disclosed herein may be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast or mammalian cells. Other suitable host cells are known to those of skill in the art.

[0146] Thus, the present disclosure further relates to a method for producing a polypeptide of interest (e.g., a TβRII polypeptide or a betaglycan polypeptide). For example, a host cell transfected with an expression vector encoding a polypeptide can be cultured under appropriate conditions to result in expression of the polypeptide. The polypeptide can be secreted and isolated from a mixture of cells and medium containing the polypeptide. Alternatively, the polypeptide may be maintained in the cytoplasm or membrane fraction, the cells are harvested, lysed, and the protein isolated. The cell culture comprises host cells and medium. Suitable media for cell culture are well known in the art. The polypeptide of interest can be isolated from the cell culture medium, the host cells, or both using techniques known in the art for purifying proteins, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, immunoaffinity purification using an antibody specific for a particular epitope of the polypeptide, and affinity purification using an agent that binds to a domain fused to the polypeptide (e.g., a protein A column can be used to purify an Fc fusion). In a preferred embodiment, the polypeptide is a fusion protein that contains a domain that facilitates its purification. By way of example, purification can be accomplished by a series of column chromatography including, for example, three or more of the following in any order: Protein A chromatography, Q Sepharose chromatography, phenyl sepharose chromatography, size exclusion chromatography and cation exchange chromatography. Purification can be completed using viral filtration and buffer exchange.

[0147] In another embodiment, a fusion gene encoding a purification leader sequence, such as a poly-(His) / enterokinase cleavage site sequence, at the N-terminus of a desired portion of a recombinant polypeptide (e.g., a TβRII polypeptide or a betaglycan polypeptide) is 2+Affinity chromatography using metal resins may allow purification of the expressed fusion protein. The purification leader sequence can then be subsequently removed by treatment with enterokinase to provide the purified polypeptide (see, e.g., Hochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al., PNAS USA 88:8972).

[0148] The technique for making fusion genes is well known. Essentially, the linking of various DNA fragments encoding different polypeptide sequences is carried out according to conventional techniques, using blunt or alternate ends for ligation, restriction enzyme digestion to provide suitable ends, filling cohesive ends as necessary, alkaline phosphatase treatment to avoid undesired ligation, and enzymatic ligation. In another embodiment, fusion genes can be synthesized by conventional techniques, including automatic DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that generate complementary overhangs between two consecutive gene fragments, which can then be annealed to create chimeric gene sequences (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992). C.Antibodies

[0149] In certain aspects, the TGFβ antagonist used according to the methods and uses disclosed herein is an antibody (TGFβ antagonist antibody) or antibody combination.TGFβ antagonist antibody or antibody combination can, for example, inhibit and / or bind to one or more ligands (e.g., TGFβ1, TGFβ2 and TGFβ3), TβRII receptor, TβRII-related type I receptor (e.g., ALK5) and / or TβRII co-receptor (e.g., betaglycan).In some embodiments, the ability of TGFβ antagonist antibody or antibody combination to inhibit signal transduction (e.g., Smad signal transduction) and / or bind to target is determined in an in vitro assay or cell-based assay, including, for example, the assay described herein. As described herein, a TGFβ antagonist antibody or combination of antagonist antibodies may be used alone or in combination with one or more additional supportive therapies or active agents for treating heterotopic ossification, preferably preventing or reducing the severity or duration of one or more complications of heterotopic ossification.

[0150] In certain embodiments, the TGFβ antagonist antibody or antibody combination is an antibody that inhibits at least TGFβ1. Thus, in some embodiments, the TGFβ antagonist antibody or antibody combination binds at least TGFβ1. As used herein, TGFβ1 antibody (anti-TGFβ1 antibody) generally refers to an antibody that is capable of binding to TGFβ1 with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent in targeting TGFβ1. In certain embodiments, the degree of binding of the anti-TGFβ1 antibody to unrelated non-TGFβ1 proteins is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the antibody's binding to TGFβ1, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the anti-TGFβ1 antibody binds to an epitope of TGFβ1 that is conserved among TGFβ1 from different species. In certain preferred embodiments, anti-TGFβ1 antibodies bind to human TGFβ1. In some embodiments, TGFβ1 antibodies can inhibit TGFβ1 from binding to type I, type II and / or co-receptors (e.g., TβRII, ALK5 and / or betaglycan), and thus inhibit TGFβ1 signaling (e.g., Smad signaling). It should be noted that TGFβ1 shares some sequence homology with TGFβ2 and TGFβ3. Thus, in some embodiments, antibodies that bind to TGFβ1 can also bind to TGFβ2 and / or TGFβ3. In some embodiments, the disclosure relates to multispecific antibodies (e.g., bispecific antibodies) and uses thereof that bind to TGFβ1 and further bind, for example, to one or more additional ligands (e.g., TGFβ2, TGFβ3, or TGFβ2 and TGFβ3), one or more type I and / or type II receptors (e.g., TβRII and ALK5), and / or one or more co-receptors (e.g., betaglycan). In some embodiments, a multispecific antibody that binds to TGFβ1 further binds to TGFβ3 but does not bind or does not substantially bind to TGFβ2 (e.g., 1×10 -7 K exceeds M Dor binds to TGFβ2 at, for example, about 1×10 -8 M or approx. 1×10 -9 M). In some embodiments, the present disclosure relates to antibody combinations and uses thereof, where the antibody combination comprises a TGFβ1 antibody and one or more additional antibodies that bind, for example, to one or more additional TβRII ligands (e.g., TGFβ2, TGFβ3, or TGFβ2 and TGFβ3), one or more type I and / or type II receptors (e.g., TβRII and ALK5), and / or one or more co-receptors (e.g., betaglycan). In some embodiments, the antibody combination comprising a TGFβ1 antibody further comprises a TGFβ3 antibody but does not comprise a TGFβ2 antibody.

[0151] In certain embodiments, the TGFβ antagonist antibody or antibody combination is an antibody that inhibits at least TGFβ2. Thus, in some embodiments, the Tβ TGFβ RII antagonist antibody or antibody combination binds at least TGFβ2. As used herein, TGFβ2 antibody (anti-TGFβ2 antibody) generally refers to an antibody that is capable of binding to TGFβ2 with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent in targeting TGFβ2. In certain embodiments, the degree of binding of the anti-TGFβ2 antibody to unrelated non-TGFβ2 proteins is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the antibody's binding to TGFβ2, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the anti-TGFβ2 antibody binds to an epitope of TGFβ2 that is conserved among TGFβ2 from different species. In certain preferred embodiments, anti-TGFβ2 antibodies bind to human TGFβ2. In some embodiments, TGFβ2 antibodies can inhibit TGFβ2 from binding to type I, type II and / or co-receptors (e.g., TβRII, ALK5 and / or betaglycan), and thus inhibit TGFβ2 signaling (e.g., Smad signaling). It should be noted that TGFβ2 shares some sequence homology with TGFβ1 and TGFβ3. Thus, in some embodiments, antibodies that bind to TGFβ2 can also bind to TGFβ1 and / or TGFβ3. In some embodiments, the disclosure relates to multispecific antibodies (e.g., bispecific antibodies) and uses thereof that bind to TGFβ2 and further bind, e.g., to one or more additional ligands (e.g., TGFβ1, TGFβ3, or TGFβ1 and TGFβ3), one or more type I and / or type II receptors (e.g., TβRII and ALK5), and / or one or more co-receptors (e.g., betaglycan).In some embodiments, the disclosure relates to antibody combinations and uses thereof, wherein the antibody combination comprises a TGFβ2 antibody and one or more additional antibodies that bind, for example, to one or more additional ligands (e.g., TGFβ1, TGFβ3, or TGFβ1 and TGFβ3), one or more type I and / or type II receptors (e.g., TβRII and ALK5), and / or one or more co-receptors (e.g., betaglycan).

[0152] In certain embodiments, the TGFβ antagonist antibody or antibody combination is an antibody that inhibits at least TGFβ3. Thus, in some embodiments, the TGFβ antagonist antibody or antibody combination binds at least TGFβ3. As used herein, TGFβ3 antibody (anti-TGFβ3 antibody) generally refers to an antibody that is capable of binding to TGFβ3 with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent in targeting TGFβ3. In certain embodiments, the degree of binding of the anti-TGFβ3 antibody to unrelated non-TGFβ3 proteins is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the antibody's binding to TGFβ3, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the anti-TGFβ3 antibody binds to an epitope of TGFβ3 that is conserved among TGFβ3 from different species. In certain preferred embodiments, anti-TGFβ3 antibody binds to human TGFβ3. In some embodiments, TGFβ3 antibody can inhibit TGFβ3 from binding to type I, type II and / or co-receptors (e.g., TβRII, ALK5 and / or betaglycan), and thus inhibit TGFβ3 signaling (e.g., Smad signaling). It should be noted that TGFβ3 shares some sequence homology with TGFβ2 and TGFβ1. Thus, in some embodiments, the antibody that binds to TGFβ3 can also bind to TGFβ2 and / or TGFβ1. In some embodiments, the disclosure relates to multispecific antibodies (e.g., bispecific antibodies) and uses thereof that bind TGFβ3 and further bind, for example, to one or more additional TβRII ligands (e.g., TGFβ2, TGFβ1, or TGFβ2 and TGFβ1), one or more type I and / or type II receptors (e.g., TβRII and ALK5), and / or one or more co-receptors (e.g., betaglycan). In some embodiments, a multispecific antibody that binds to TGFβ3 does not bind or does not substantially bind TGFβ2 (e.g., 1×10 -7 K exceeds M D or binds to TGFβ2 at, for example, about 1×10-8 M or approx. 1×10 -9 In some embodiments, a multispecific antibody that binds to TGFβ3 further binds to TGFβ1, but does not bind or does not substantially bind to TGFβ2 (e.g., 1×10 -7 K exceeds M D or binds to TGFβ2 at, for example, about 1×10 -8 M or approx. 1×10 -9 M). In some embodiments, the present disclosure relates to antibody combinations and uses thereof, where the antibody combination comprises a TGFβ3 antibody and one or more additional antibodies that bind, for example, to one or more additional ligands (e.g., TGFβ2, TGFβ1, or TGFβ2 and TGFβ1), one or more type I and / or type II receptors (e.g., TβRII and ALK5), and / or one or more co-receptors (e.g., betaglycan). In some embodiments, the antibody combination comprising a TGFβ3 antibody further comprises a TGFβ1 antibody but does not comprise a TGFβ2 antibody.

[0153] In some embodiments, the TGFβ antagonist antibody binds to all three TGFβ isoforms, namely TGFβ1, TGFβ2 and TGFβ3. In some embodiments, the TGFβ antagonist antibody is fresolimumab.

[0154] In certain aspects, the TGFβ antagonist antibody or antibody combination is an antibody that inhibits at least TβRII. Thus, in some embodiments, the TGFβ antagonist antibody or antibody combination binds at least TβRII. As used herein, TβRII antibody (anti-TβRII antibody) generally refers to an antibody that binds to TβRII with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent in targeting TβRII. In certain embodiments, the degree of binding of the anti-TβRII antibody to unrelated non-TβRII proteins is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of the antibody's binding to TβRII, as measured, for example, by radioimmunoassay (RIA), Biacore, or other protein-protein interaction assay or binding affinity assay. In certain embodiments, the anti-TβRII antibody binds to an epitope of TβRII that is conserved among TβRII from different species. In certain preferred embodiments, anti-TβRII antibody binds to human TβRII. In some embodiments, anti-TβRII antibody can inhibit one or more ligands [e.g., TGFβ1; TGFβ2; TGFβ3; TGFβ1 and TGFβ3; TGFβ1 and TGFβ2; TGFβ2 and TGFβ3; or TGFβ1, TGFβ2 and TGFβ3] from binding to TβRII. In some embodiments, anti-TβRII antibody is a multispecific antibody (e.g., bispecific antibody) that binds to TβRII and one or more ligands [e.g., TGFβ1, TGFβ2 and TGFβ3], type I receptor (e.g., ALK5) and / or co-receptor (e.g., betaglycan). In some embodiments, the present disclosure relates to antibody combinations and uses thereof, wherein the antibody combination comprises an anti-TβRII antibody and one or more additional antibodies that bind, for example, to one or more ligands [e.g., TGFβ1, TGFβ2 and TGFβ3], type I receptors (e.g., ALK5), and / or co-receptors (e.g., betaglycan).

[0155] In certain aspects, the TGFβ antagonist antibody or antibody combination is an antibody that inhibits at least ALK5.Thus, in some embodiments, the TGFβ antagonist antibody or antibody combination binds at least ALK5.As used herein, ALK5 antibody (anti-ALK5 antibody) generally refers to an antibody that binds to ALK5 with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent in targeting ALK5.In certain embodiments, the degree of binding of anti-ALK5 antibody to unrelated non-ALK5 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of the binding of the antibody to ALK5, as measured, for example, by radioimmunoassay (RIA), Biacore, or other protein-protein interaction assay or binding affinity assay.In certain embodiments, the anti-ALK5 antibody binds to an epitope of ALK5 that is conserved among ALK5 from different species. In certain preferred embodiments, anti-ALK5 antibody binds to human ALK5.In some embodiments, anti-ALK5 antibody can inhibit one or more ligands [e.g., TGFβ1; TGFβ2; TGFβ3; TGFβ1 and TGFβ3; TGFβ1 and TGFβ2; TGFβ2 and TGFβ3; or TGFβ1, TGFβ2 and TGFβ3] from binding to ALK5.In some embodiments, anti-ALK5 antibody is a multispecific antibody (e.g., bispecific antibody) that binds to ALK5 and one or more ligands [e.g., TGFβ1, TGFβ2 and TGFβ3], type II receptor (e.g., TβRII) and / or co-receptor (e.g., betaglycan). In some embodiments, the disclosure relates to antibody combinations and uses thereof, wherein the antibody combination comprises an anti-ALK5 antibody and one or more additional antibodies that bind, for example, to one or more ligands [e.g., TGFβ1, TGFβ2 and TGFβ3], type II receptors (e.g., TβRII), and / or co-receptors (e.g., betaglycan).

[0156] In certain aspects, the TGFβ antagonist antibody or antibody combination is an antibody that inhibits at least betaglycan. Thus, in some embodiments, the TGFβ antagonist antibody or antibody combination binds at least betaglycan. As used herein, betaglycan antibody (anti-betaglycan antibody) generally refers to an antibody that binds to betaglycan with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent in targeting betaglycan. In certain embodiments, the degree of binding of the anti-betaglycan antibody to unrelated non-betaglycan proteins is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of the antibody's binding to betaglycan, as measured, for example, by radioimmunoassay (RIA), Biacore, or other protein-protein interaction assay or binding affinity assay. In certain embodiments, the anti-betaglycan antibody binds to an epitope of betaglycan that is conserved among betaglycans from different species. In certain preferred embodiments, anti-betaglycan antibody binds to human betaglycan.In some embodiments, anti-betaglycan antibody can inhibit one or more ligands [e.g., TGFβ1; TGFβ2; TGFβ3; TGFβ1 and TGFβ3; TGFβ1 and TGFβ2; TGFβ2 and TGFβ3; or TGFβ1, TGFβ2 and TGFβ3] from binding to betaglycan.In some embodiments, anti-betaglycan antibody is a multispecific antibody (e.g., bispecific antibody) that binds to betaglycan and one or more ligands [e.g., TGFβ1, TGFβ2 and TGFβ3], type I receptor (e.g., ALK5) and / or type II receptor (e.g., TβRII). In some embodiments, the present disclosure relates to antibody combinations and uses thereof, wherein the antibody combination comprises an anti-betaglycan antibody and one or more additional antibodies that bind, for example, to one or more ligands [e.g., TGFβ1, TGFβ2 and TGFβ3], type I receptors (e.g., ALK5), and / or type II receptors (e.g., TβRII).

[0157] The term antibody is used herein in the broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. An antibody fragment refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. See, for example, Hudson et al. (2003) Nat. Med. 9:129-134; Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); WO 93 / 16185; and U.S. Patent Nos. 5,571,894, 5,587,458, and 5,869,046. The antibodies disclosed herein can be polyclonal or monoclonal antibodies. In certain embodiments, the antibodies of the present disclosure include a detectable label attached thereto (e.g., the label can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor). In a preferred embodiment, the antibodies of the present disclosure are isolated antibodies. Diabodies are antibody fragments that have two antigen-binding sites, and can be bivalent or bispecific.See, for example, EP404,097; WO 1993 / 01161; Hudson et al. (2003) Nat. Med. 9:129-134 (2003); and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448.Triabodies and tetrabodies are also described in Hudson et al. (2003) Nat. Med. 9:129-134.A single domain antibody is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody. See, for example, U.S. Patent No. 6,248,516. Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage) as described herein.

[0158] The antibody herein may be of any class. The class of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major antibody classes: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu.

[0159] In general, an antibody for use in the methods disclosed herein will specifically bind to its target antigen, preferably with high binding affinity. Affinity is measured by the K D The binding affinity may be expressed as a value, which reflects the intrinsic binding affinity (e.g., with minimal avidity effects). Typically, the binding affinity is measured in vitro, whether in a cell-free or cell-associated context. Any of a number of assays known in the art, including the assays disclosed herein, can be used to obtain the binding affinity measurements, including, for example, surface plasmon resonance (Biacore™ assay), radiolabeled antigen binding assay (RIA), and ELISA. In some embodiments, the antibodies of the present disclosure have a binding affinity of at least 1×10 -7 Or stronger, 1×10 -8 Or stronger, 1×10 -9 Or stronger, 1×10 -10 Or stronger, 1×10-11 Or stronger, 1×10 -12 Or stronger, 1×10 -13 or stronger, or 1×10 -14 Or even stronger K D and bind to their target antigens (e.g., TGFβ1, TGFβ2, ALK5, betaglycan, and TβRII).

[0160] In certain embodiments, K D is measured by an RIA performed with the Fab version of the antibody of interest and its target antigen, as described by the following assay: The solution binding affinity of the Fab for the antigen is determined by binding to the lowest concentration of radiolabeled antigen (e.g., 125I-labeled) and then capture the bound antigen using an anti-Fab antibody-coated plate [see, e.g., Chen et al. (1999) J. Mol. Biol. 293:865-881]. To establish conditions for the assay, a multi-well plate (e.g., MICROTITER®, Thermo Scientific) is coated (e.g., overnight) with a capture anti-Fab antibody (e.g., Cappel Labs), and then blocked, preferably, with bovine serum albumin at room temperature (approximately 23° C.). In a non-adsorbing plate, radiolabeled antigen is mixed with a serial dilution of the Fab of interest [e.g., consistent with the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599]. The Fab of interest is then incubated, preferably overnight, although incubation may continue for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation, preferably at room temperature, for about 1 hour. The solution is then removed and the plate is washed several times, preferably with a mixture of polysorbate 20 and PBS. Once the plate is dry, a scintillation agent (e.g., MICROSCINT®, Packard) is added and the plate is counted in a gamma counter (e.g., TOPCOUNT®, Packard).

[0161] According to another embodiment, K Dis measured using a surface plasmon resonance assay, for example, using a BIACORE® 2000 or BIACORE® 3000 (Biacore, Inc., Piscataway, NJ) with an immobilized antigen CM5 chip at about 10 response units (RU). Briefly, a carboxymethylated dextran biosensor chip (CM5, Biacore, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. For example, the antigen can be diluted to 5 μg / mL (about 0.2 μM) in 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of coupled protein. After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN®-20) surfactant (PBST) at a flow rate of approximately 25 μl / min. on ) and dissociation rate (k off The equilibrium dissociation constant (K) is calculated, for example, by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). D ) to compare k off / k on [See, e.g., Chen et al., (1999) J. Mol. Biol. 293:865-881]. The on-rate, e.g., 10 6 M -1 seconds -1If it exceeds 100%, then the on-rate can be determined using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (e.g., excitation=295 nm; emission=340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab type) in PBS in the presence of increasing concentrations of antigen when measured using a stirred cuvette in a spectrometer such as a spectrophotometer equipped with stopped-flow (Aviv Instruments) or an 8000 series SLM-AMINCO® spectrophotometer (ThermoSpectronic).

[0162] The nucleic acid and amino acid sequences of TβRII, ALK5, betaglycan, TGFβ1, TGFβ2 and TGFβ3, particularly the human sequences, are well known in the art, and thus antibody antagonists for use according to the present disclosure can be routinely generated by one of ordinary skill in the art based on knowledge in the art and the teachings provided herein.

[0163] In certain embodiments, the antibody provided herein is a chimeric antibody. A chimeric antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, but the remaining portion of the heavy and / or light chain is derived from a different source or species. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855. In some embodiments, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate, such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In some embodiments, a chimeric antibody is a "class-switched" antibody, in which the class or subclass is changed from that of the parent antibody. In general, a chimeric antibody comprises an antigen-binding fragment thereof.

[0164] In certain embodiments, the chimeric antibody provided herein is a humanized antibody. A humanized antibody refers to a chimeric antibody that comprises amino acid residues from a non-human hypervariable region (HVR) and amino acid residues from a human framework region (FR). In certain embodiments, a humanized antibody comprises substantially all of at least one, typically two, variable domains, with all or substantially all of the HVRs (e.g., CDRs) corresponding to those of a non-human antibody and all or substantially all of the FRs corresponding to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been subjected to humanization.

[0165] Humanized antibodies and methods for making them are reviewed, for example, in Almagro and Fransson (2008) Front. Biosci. 13:1619-1633, and further described, for example, in Riechmann et al., (1988) Nature 332:323-329; Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., (2005) Methods 36:25-34 [describing SDR (a-CDR) grafting]; Padlan, Mol. Immunol. (1991) 28:489-498 (describing "resurfacing"); Dall'Acqua et al. (2005) Methods 36:43-60 (describing "FR shuffling"); Osbourn et al. (2005) Methods 36:61-68; and Klimka et al. Br. J. Cancer (2000) 83:252-260 (describing a "guided selection" approach to FR shuffling).

[0166] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method [see, e.g., Sims et al. (1993) J. Immunol. 151:2296]; framework regions derived from the constant sequences of human antibodies of a particular subgroup of light or heavy chain variable regions [see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; and Presta et al. (1993) J. Immunol., 151:2623]; human mature (somatically mutated) framework regions or human germline framework regions [see, e.g., Almagro and Fransson (2008) Front. Biosci. 13:1619-1633]; and framework regions derived from screening of FR libraries [see, e.g., Baca et al., (1997) J. Biol. Chem. 272:10678-10684; and Rosok et al., (1996) J. Biol. Chem. 271:22611-22618].

[0167] In certain embodiments, the antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel (2001) Curr. Opin. Pharmacol. 5: 368-74 and Lonberg (2008) Curr. Opin. Immunol. 20:450-459.

[0168] Human antibodies can be prepared by administering immunogens (e.g., TβRII, ALK5, betaglycan, TGFβ1, TGFβ2 or TGFβ3 polypeptides) to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or a portion of the human immunoglobulin loci that replace endogenous immunoglobulin loci or that are extrachromosomal or randomly integrated into the animal's chromosomes. In such transgenic animals, endogenous immunoglobulin loci are generally inactivated. For reviews of methods for obtaining human antibodies from transgenic animals, see, e.g., Lonberg (2005) Nat. Biotechnol. 23:1117-1125; U.S. Pat. Nos. 6,075,181 and 6,150,584 (describing XENOMOUSE™ technology); U.S. Pat. No. 5,770,429 (describing HuMab® technology); U.S. Pat. No. 7,041,870 (describing KM MOUSE® technology); and U.S. Patent Application Publication No. 2007 / 0061900 (describing VelociMouse® technology). The human variable regions from intact antibodies made by such animals can be further modified, for example, by combining with different human constant regions.

[0169] The human antibody provided herein can also be produced by hybridoma-based methods.Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, for example, Kozbor J. Immunol., (1984) 133: 3001;Brodeur et al. (1987) Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York;and Boerner et al. (1991) J. Immunol., 147: 86).Human antibodies produced by human B-cell hybridoma technology are also described in Li et al., (2006) Proc. Natl. Acad. Sci. USA, 103:3557-3562. Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue (2006) 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein (2005) Histol. Histopathol., 20(3):927-937 (2005) and Vollmers and Brandlein (2005) Methods Find Exp. Clin. Pharmacol., 27(3):185-91.

[0170] The human antibodies provided herein may also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described herein.

[0171] For example, antibodies of the present disclosure can be isolated by screening combinatorial libraries for antibodies having the desired activity(ies). A variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al. (2001) in Methods in Molecular Biology 178:1-37, O'Brien et al., ed., Human Press, Totowa, NJ, and in, for example, McCafferty et al. (1991) Nature 348:552-554; Clackson et al., (1991) Nature 352: 624-628; Marks et al. (1992) J. Mol. Biol. 222:581-597; Marks and Bradbury (2003) in Methods in Molecular Biology 248:161-175, Lo, ed., Human Press, Totowa, NJ; Sidhu et al. (2004) J. Mol. Biol. 338(2):299-310; Lee et al. (2004) J. Mol. Biol. 340(5):1073-1093; Fellouse (2004) Proc. Natl. Acad. Sci. USA 101(34):12467-12472; and Lee et al. (2004) J. Immunol. Methods 284(1-2): 119-132.

[0172] In one particular phage display method, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for antigen-binding phages as described in Winter et al. (1994) Ann. Rev. Immunol., 12: 433-455. Phages typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies against the immunogen (e.g., TβRII, TGFβ1, TGFβ2 or TGFβ3 polypeptides) without the requirement of constructing hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a broad range of non-self antigens, as well as against self antigens, without any immunization, as described in Griffiths et al. (1993) EMBO J, 12: 725-734. Finally, naive libraries can be synthetically generated by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 regions and achieve in vitro rearrangement, as described in Hoogenboom and Winter (1992) J. Mol. Biol., 227: 381-388. Patent publications describing human antibody phage libraries include, for example, U.S. Pat. No. 5,750,373, as well as U.S. Patent Application Publication No. 2005 / 0079574, U.S. Patent Application Publication No. 2005 / 0119455, U.S. Patent Application Publication No. 2005 / 0266000, U.S. Patent Application Publication No. 2007 / 0117126, U.S. Patent Application Publication No. 2007 / 0160598, U.S. Patent Application Publication No. 2007 / 0237764, U.S. Patent Application Publication No. 2007 / 0292936, and U.S. Patent Application Publication No. 2009 / 0002360.

[0173] In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies (typically monoclonal antibodies) have binding specificities for at least two different epitopes (e.g., 2, 3, 4, 5, or 6 or more) on one or more (e.g., 2, 3, 4, 5, 6 or more) antigens.

[0174] Also included herein are engineered antibodies with three or more functional antigen binding sites, including "Octopus antibodies" (see, e.g., U.S. Patent Application Publication No. 2006 / 0025576).

[0175] In certain embodiments, the antibody disclosed herein is a monoclonal antibody. A monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up this population are identical and / or bind to the same epitope, except for possible variant antibodies that contain naturally occurring mutations or arise during the production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies that are made against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is made against a single epitope on an antigen. Thus, the modifier "monoclonal" refers to the characteristics of an antibody obtained from a population of substantially homogeneous antibodies, and should not be interpreted as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present methods may be made by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals that contain all or a portion of the human immunoglobulin loci; such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0176] For example, by using immunogens derived from TβRII, anti-protein / anti-peptide antisera or monoclonal antibodies can be made by standard protocols [see, for example, Antibodies: A Laboratory Manual (1988) ed. by Harlow and Lane, Cold Spring Harbor Press]. Mammals such as mice, hamsters or rabbits can be immunized with immunogenic forms of TβRII polypeptides, antigenic fragments capable of eliciting an antibody response, or fusion proteins. Techniques for conferring immunogenicity to proteins or peptides include conjugation to carriers or other techniques well known in the art. Immunogenic portions of TβRII polypeptides can be administered in the presence of adjuvants. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the level of antibody production and / or the level of binding affinity.

[0177] After immunization of an animal with an antigenic preparation of TβRII, antisera can be obtained, and polyclonal antibodies can be isolated from the serum, if desired. To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be collected from the immunized animal and fused with immortalized cells, such as myeloma cells, by standard somatic cell fusion procedures to obtain hybridoma cells. Such techniques are well known in the art, and include, for example, hybridoma techniques [see, for example, Kohler and Milstein (1975) Nature, 256: 495-497], human B-cell hybridoma techniques [see, for example, Kozbar et al. (1983) Immunology Today, 4:72], and EBV hybridoma techniques for producing human monoclonal antibodies [Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96]. Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with TβRII polypeptides and monoclonal antibodies can be isolated from cultures containing such hybridoma cells.

[0178] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., substitution, deletion, and / or addition) at one or more amino acid positions.

[0179] For example, the present disclosure contemplates antibody variants with some, but not all, effector functions that make them desirable candidates for applications in which the half-life of the antibody in vivo is important, and yet certain effector functions [e.g., complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC)] are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / lack of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus may lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is reviewed, for example, in Ravetch and Kinet (1991) Annu. Rev. Immunol. 9:457-492. Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom, I. et al. (1986) Proc. Nat'l Acad. Sci. USA 83:7059-7063; Hellstrom, I. et al. (1985) Proc. Nat'l Acad. Sci. USA 82:1499-1502; U.S. Patent No. 5,821,337; and Bruggemann, M. et al. (1987) J. Exp. Med. 166:1351-1361. Alternatively, non-radioactive assay methods may be used (e.g., ACTI™, a non-radioactive cytotoxicity assay for flow cytometry; CellTechnology, Inc., Mountain View, Calif.; and CytoTox 96®, a non-radioactive cytotoxicity assay, Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. (1998) Proc. Nat'l Acad. Sci. USA 95:652-656. C1q binding assays may also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity [see, e.g., C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402]. To assess complement activation, CDC assays may be performed (see, e.g., Gazzano-Santoro et al. (1996) J. Immunol. Methods 202:163; Cragg, MS et al. (2003) Blood 101:1045-1052; and Cragg, M. S, and MJ Glennie (2004) Blood 103:2738-2743). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al. (2006) Int. Immunol. 18(12):1759-1769).

[0180] Antibodies of the present disclosure with reduced effector function include antibodies with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0181] In certain embodiments, it may be desirable to create cysteine ​​engineered antibodies, e.g., "thioMAbs" in which one or more residues of an antibody are replaced with cysteine ​​residues. In certain embodiments, the replaced residues are located at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are thereby placed at accessible sites of the antibody, which can be used to conjugate the antibody with other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be replaced with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibodies may be made, for example, as described in U.S. Pat. No. 7,521,541.

[0182] In addition, the technique used to screen antibodies to identify desirable antibodies may affect the properties of the resulting antibodies. For example, if an antibody is used to bind an antigen in solution, it may be desirable to test the binding in solution. A variety of different techniques are available to test the interaction between antibodies and antigens to identify specific desirable antibodies. Various techniques include ELISA, surface plasmon resonance binding assays (e.g., Biacore™ binding assay, BiacoreAB, Uppsala, Sweden), sandwich assays (e.g., paramagnetic bead system from IGEN International, Inc., Gaithersburg, Maryland), Western blots, immunoprecipitation assays, and immunohistochemistry.

[0183] In certain embodiments, amino acid sequence variants of the antibodies and / or binding polypeptides provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibodies and / or binding polypeptides. Amino acid sequence variants of the antibodies and / or binding polypeptides can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibodies and / or binding polypeptides or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions into and / or substitutions of residues within the amino acid sequence of the antibodies and / or binding polypeptides. Any combination of deletions, insertions and substitutions can be made to arrive at the final construct, provided that the final construct has the desired properties, e.g., target binding (TβRII, ALK5, betaglycan, TGFβ1, TGFβ2 and / or TGFβ3).

[0184] Alterations (e.g., substitutions) may be made in HVRs, for example, to improve antibody affinity. Such alterations can be made in HVR "hot spots", i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury (2008) Methods Mol. Biol. 207:179-196 (2008)), and / or in SDRs (a-CDRs), and the resulting variants VH or VL are tested for binding affinity. Affinity maturation by constructing secondary libraries and reselecting from the secondary libraries has been described in the art [see, e.g., Hoogenboom et al., in Methods in Molecular Biology 178:1-37, O'Brien et al., ed., Human Press, Totowa, NJ, (2001)]. In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity includes the HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0185] In certain embodiments, substitution, insertion or deletion can be made in one or more HVRs, as long as such changes do not substantially reduce the ability of antibody to bind to antigen.For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in HVRs.Such changes can be outside HVR "hot spots" or SDRs.In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unaltered or contains no more than 1, 2 or 3 amino acid substitutions.

[0186] A useful method for identifying residues or regions of an antibody and / or binding polypeptide that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or groups of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody or binding polypeptide with the antigen is affected. Further substitutions may be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody complex can be used to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues may be targeted or excluded as candidates for substitution. The variants may be screened to determine whether they contain the desired properties.

[0187] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intersequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertion variants of antibody molecules include the fusion of the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0188] In certain embodiments, the antibodies and / or binding polypeptides provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Suitable moieties for derivatization of antibodies and / or binding polypeptides include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages during manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody and / or binding polypeptide may vary, and when two or more polymers are attached, they may be the same or different molecules. In general, the number and / or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the specific properties or functions of the antibody and / or binding polypeptide to be improved, regardless of whether the antibody derivative and / or binding polypeptide derivative is used in treatment under a defined condition. D. Small molecules

[0189] In certain aspects, the TGFβ antagonist used according to the methods and uses disclosed herein is a small molecule (TGFβ antagonist small molecule) or a combination of small molecules.The TGFβ antagonist small molecule or combination of small molecules can, for example, inhibit one or more ligands (e.g., TGFβ1, TGFβ2 and TGFβ3), TβRII receptor, TβRII-related type I receptor (e.g., ALK5), TβRII-related co-receptor (e.g., betaglycan) and / or downstream signaling components (e.g., Smad).In some embodiments, the ability of the TGFβ antagonist small molecule or combination of small molecules to inhibit signaling (e.g., Smad signaling) is determined in a cell-based assay, including, for example, the assay described herein. The TGFβ antagonist small molecule or combination of small molecules may be used alone or in combination with one or more additional supportive therapies or active agents for treating heterotopic ossification, preferably preventing or reducing the severity or duration of one or more complications of heterotopic ossification.

[0190] In certain aspects, the TGFβ antagonist small molecule or combination of small molecules inhibits at least TGFβ1 (e.g., inhibits Smad signaling). Thus, in some embodiments, the small molecule inhibitor of TGFβ1 binds to TGFβ1. In some embodiments, the small molecule inhibitor of TGFβ1 inhibits the expression (e.g., transcription, translation, secretion, or a combination thereof) of TGFβ1. In some embodiments, the small molecule inhibitor of TGFβ1 further inhibits one or more of TGFβ2, TGFβ3, TβRII, ALK5, and betaglycan. In some embodiments, the small molecule inhibitor of TGFβ1 does not inhibit or does not substantially inhibit TGFβ2. In some embodiments, the small molecule inhibitor of TGFβ1 further inhibits TGFβ3, but does not inhibit or does not substantially inhibit TGFβ2. In certain aspects, the TGFβ antagonist small molecule or combination of small molecules inhibits at least TGFβ2 (e.g., inhibits Smad signaling). Thus, in some embodiments, the small molecule inhibitor of TGFβ2 binds to TGFβ2. In some embodiments, the small molecule inhibitor of TGFβ2 inhibits the expression (e.g., transcription, translation, secretion, or a combination thereof) of TGFβ2. In some embodiments, the small molecule inhibitor of TGFβ2 further inhibits one or more of TGFβ3, TGFβ1, TβRII, ALK5, and betaglycan. In certain aspects, the TGFβ antagonist small molecule or combination of small molecules inhibits at least TGFβ3 (e.g., inhibits Smad signaling). Thus, in some embodiments, the small molecule inhibitor of TGFβ3 binds to TGFβ3. In some embodiments, the small molecule inhibitor of TGFβ3 inhibits the expression (e.g., transcription, translation, secretion, or a combination thereof) of TGFβ3. In some embodiments, the small molecule inhibitor of TGFβ3 further inhibits one or more of TGFβ2, TGFβ1, TβRII, ALK5, and betaglycan. In some embodiments, the small molecule inhibitor of TGFβ3 does not inhibit or does not substantially inhibit TGFβ2. In some embodiments, the small molecule inhibitor of TGFβ3 further inhibits TGFβ1, but does not inhibit or does not substantially inhibit TGFβ2.In certain aspects, the TGFβ antagonist small molecule or small molecule combination inhibits at least TβRII (e.g., inhibits Smad signaling). Thus, in some embodiments, the small molecule inhibitor of TβRII binds to TβRII. In some embodiments, the small molecule inhibitor of TβRII inhibits expression (e.g., transcription, translation, secretion, or a combination thereof) of TβRII. In some embodiments, the small molecule inhibitor of TβRII further inhibits one or more of TGFβ1, TGFβ2, TGFβ3, ALK5, and betaglycan. In certain aspects, the TGFβ antagonist small molecule or small molecule combination inhibits TGFβ1 binding to TβRII. In certain aspects, the TGFβ antagonist small molecule or small molecule combination inhibits TGFβ2 binding to TβRII. In certain aspects, the TGFβ antagonist small molecule or small molecule combination inhibits TGFβ3 binding to TβRII. In certain embodiments, the TGFβ antagonist small molecule or small molecule combination inhibits TGFβ1 and TGFβ3 from binding to TβRII. In certain embodiments, the TGFβ antagonist small molecule or small molecule combination inhibits TGFβ1, TGFβ2 and TGFβ3 from binding to TβRII. In certain embodiments, the TGFβ antagonist small molecule or small molecule combination inhibits TGFβ1 from binding to TβRII but does not inhibit or does not substantially inhibit TGFβ2 from binding to TβRII. In certain embodiments, the TGFβ antagonist small molecule or small molecule combination inhibits TGFβ3 from binding to TβRII but does not inhibit or does not substantially inhibit TGFβ2 from binding to TβRII. In certain aspects, TGFβ antagonist small molecule or small molecule combination inhibits TGFβ1 and TGFβ3 from binding to TβRII, but does not inhibit or does not substantially inhibit TGFβ2 from binding to TβRII.In certain aspects, Tβ TGFβ RII antagonist small molecule or small molecule combination inhibits at least ALK5 (e.g., inhibits Smad signaling).Thus, in some embodiments, the small molecule inhibitor of ALK5 binds to ALK5.In some embodiments, the small molecule inhibitor of ALK5 inhibits the expression (e.g., transcription, translation, secretion, or a combination thereof) of ALK5. In some embodiments, the small molecule inhibitor of ALK5 further inhibits one or more of TGFβ1, TGFβ2, TGFβ3, TβRII, and betaglycan. In certain aspects, the TGFβ antagonist small molecule or combination of small molecules inhibits TGFβ1 binding to ALK5. In certain aspects, the TGFβ antagonist small molecule or combination of small molecules inhibits TGFβ2 binding to ALK5. In certain aspects, the TGFβ antagonist small molecule or combination of small molecules inhibits TGFβ3 binding to ALK5. In certain aspects, the TGFβ antagonist small molecule or combination of small molecules inhibits TGFβ1 and TGFβ3 binding to ALK5. In certain embodiments, the TGFβ antagonist small molecule or small molecule combination inhibits TGFβ1, TGFβ2 and TGFβ3 from binding to ALK5. In certain embodiments, the TGFβ antagonist small molecule or small molecule combination inhibits TGFβ1 from binding to ALK5, but does not or does not substantially inhibit TGFβ2 from binding to ALK5. In certain embodiments, the TGFβ antagonist small molecule or small molecule combination inhibits TGFβ3 from binding to ALK5, but does not or does not substantially inhibit TGFβ2 from binding to ALK5. In certain embodiments, the TGFβ antagonist small molecule or small molecule combination inhibits TGFβ1 and TGFβ3 from binding to ALK5, but does not or does not substantially inhibit TGFβ2 from binding to ALK5. In certain aspects, the TGFβ antagonist small molecule or combination of small molecules inhibits at least betaglycan (e.g., inhibits Smad signaling). Thus, in some embodiments, the small molecule inhibitor of betaglycan binds to betaglycan. In some embodiments, the small molecule inhibitor of betaglycan inhibits the expression (e.g., transcription, translation, secretion, or a combination thereof) of betaglycan. In some embodiments, the small molecule inhibitor of betaglycan further inhibits one or more of TGFβ1, TGFβ2, TGFβ3, TβRII, and ALK5.In certain embodiments, the TGFβ antagonist small molecule or small molecule combination inhibits TGFβ1 from binding to betaglycan. In certain embodiments, the TGFβ antagonist small molecule or small molecule combination inhibits TGFβ2 from binding to betaglycan. In certain embodiments, the TGFβ antagonist small molecule or small molecule combination inhibits TGFβ3 from binding to betaglycan. In certain embodiments, the TGFβ antagonist small molecule or small molecule combination inhibits TGFβ1 and TGFβ3 from binding to betaglycan. In certain embodiments, the TGFβ antagonist small molecule or small molecule combination inhibits TGFβ1, TGFβ2 and TGFβ3 from binding to betaglycan. In certain embodiments, the TGFβ antagonist small molecule or small molecule combination inhibits TGFβ1 from binding to betaglycan but does not inhibit or does not substantially inhibit TGFβ2 from binding to betaglycan. In certain embodiments, a TGFβ antagonist small molecule or combination of small molecules inhibits TGFβ3 from binding to betaglycan, but does not inhibit or does not substantially inhibit TGFβ2 from binding to betaglycan. In certain embodiments, a TGFβ antagonist small molecule or combination of small molecules inhibits TGFβ1 and TGFβ3 from binding to betaglycan, but does not inhibit or does not substantially inhibit TGFβ2 from binding to betaglycan.

[0191] A TGFβ antagonist small molecule can be a direct inhibitor or an indirect inhibitor. For example, a TGFβ antagonist small molecule or a combination of small molecules can inhibit the expression (e.g., transcription, translation, cell secretion, or a combination thereof) of at least one or more of TβRII, ALK5, betaglycan, TGFβ1, TGFβ2, TGFβ3, and / or one or more downstream signaling factors (Smads). Alternatively, a direct TGFβ antagonist small molecule or a combination of small molecules can directly bind, for example, to one or more of TβRII, ALK5, betaglycan, TGFβ1, TGFβ2, and TGFβ3, or one or more downstream signaling factors. A combination of one or more indirect TGFβ antagonist small molecules and one or more direct TGFβ antagonist small molecules can be used according to the methods disclosed herein.

[0192] The binding small organic molecule antagonist of the present disclosure can be identified and chemically synthesized using known methodology (see, for example, PCT Publications WO 00 / 00823 and WO 00 / 39585).In general, the small molecule antagonist of the present disclosure is usually less than about 2000 daltons in size, or less than about 1500, 750, 500, 250 or 200 daltons in size, and such small organic molecules are preferably capable of specifically binding to the polypeptides described herein (e.g., TβRII, ALK5, betaglycan, TGFβ1, TGFβ2 and TGFβ3).Such small molecule antagonists can be identified without undue experimentation using well-known techniques. In this regard, it is noted that techniques for screening small organic molecule libraries for molecules capable of binding to polypeptide targets are well known in the art (see, e.g., International Application Publication Nos. WO 00 / 00823 and WO 00 / 39585).

[0193] The conjugated organic small molecules of the present disclosure can be, for example, aldehydes, ketones, oximes, hydrazones, semicarbazones, carbazides, primary amines, secondary amines, tertiary amines, N-substituted hydrazines, hydrazides, alcohols, ethers, thiols, thioethers, disulfides, carboxylic acids, esters, amides, ureas, carbamates, carbonates, ketals, thioketals, acetals, thioacetals, aryl halides, aryl sulfonates, alkyl halides, alkyl sulfonates, aromatic compounds, heterocyclic compounds, anilines, alkenes, alkynes, diols, amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolines, enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonyl chlorides, diazo compounds, and acid chlorides. E. Nucleic acid

[0194] In certain aspects, the TGFβ antagonist used according to the methods and uses disclosed herein is a polynucleotide (TGFβ antagonist polynucleotide) or a combination of polynucleotides.TGFβ antagonist polynucleotide or a combination of polynucleotides can, for example, inhibit one or more ligands (e.g., TGFβ1, TGFβ2 and TGFβ3), TβRII receptor, TβRII-related type I receptor (e.g., ALK5), TβRII-related co-receptor (e.g., betaglycan) and / or downstream signaling components (e.g., Smad).In some embodiments, the ability of TGFβ antagonist polynucleotide or a combination of polynucleotides to inhibit signaling (e.g., Smad signaling) is determined in a cell-based assay, including, for example, the assay described herein. The TGFβ antagonist polynucleotide or combination of polynucleotides may be used alone or in combination with one or more additional supportive therapies or active agents for treating heterotopic ossification, preferably preventing or reducing the severity or duration of one or more complications of heterotopic ossification.

[0195] In certain aspects, the TGFβ antagonist polynucleotide or combination of polynucleotides inhibits at least TGFβ1 (e.g., inhibits Smad signaling). Thus, in some embodiments, the polynucleotide inhibitor of TGFβ1 binds to TGFβ1. In some embodiments, the polynucleotide inhibitor of TGFβ1 inhibits the expression (e.g., transcription, translation, secretion, or a combination thereof) of TGFβ1. In some embodiments, the polynucleotide inhibitor of TGFβ1 further inhibits one or more of TGFβ2, TGFβ3, TβRII, ALK5, and betaglycan. In some embodiments, the polynucleotide inhibitor of TGFβ1 does not inhibit or does not substantially inhibit TGFβ2. In some embodiments, the polynucleotide inhibitor of TGFβ1 further inhibits TGFβ3, but does not inhibit or does not substantially inhibit TGFβ2. In certain aspects, the TGFβ antagonist polynucleotide or combination of polynucleotides inhibits at least TGFβ2 (e.g., inhibits Smad signaling). Thus, in some embodiments, the polynucleotide inhibitor of TGFβ2 binds to TGFβ2. In some embodiments, the polynucleotide inhibitor of TGFβ2 inhibits the expression (e.g., transcription, translation, secretion, or a combination thereof) of TGFβ2. In some embodiments, the polynucleotide inhibitor of TGFβ2 further inhibits one or more of TGFβ3, TGFβ1, TβRII, ALK5, and betaglycan. In certain aspects, the TGFβ antagonist polynucleotide or combination of polynucleotides inhibits at least TGFβ3 (e.g., inhibits Smad signaling). Thus, in some embodiments, the polynucleotide inhibitor of TGFβ3 binds to TGFβ3. In some embodiments, the polynucleotide inhibitor of TGFβ3 inhibits the expression (e.g., transcription, translation, secretion, or a combination thereof) of TGFβ3. In some embodiments, the polynucleotide inhibitor of TGFβ3 further inhibits one or more of TGFβ2, TGFβ1, TβRII, ALK5, and betaglycan.In some embodiments, the polynucleotide inhibitor of TGFβ3 does not inhibit or does not substantially inhibit TGFβ2. In some embodiments, the polynucleotide inhibitor of TGFβ3 further inhibits TGFβ1, but does not inhibit or does not substantially inhibit TGFβ2. In certain aspects, the TGFβ antagonist polynucleotide or combination of polynucleotides inhibits at least TβRII (e.g., inhibits Smad signaling). Thus, in some embodiments, the polynucleotide inhibitor of TβRII binds to TβRII. In some embodiments, the polynucleotide inhibitor of TβRII inhibits expression (e.g., transcription, translation, secretion, or combinations thereof) of TβRII. In some embodiments, the polynucleotide inhibitor of TβRII further inhibits one or more of TGFβ1, TGFβ2, TGFβ3, ALK5, and betaglycan. In some embodiments, the TGFβ antagonist polynucleotide or combination of polynucleotides inhibits TGFβ1 binding to TβRII. In some embodiments, the TGFβ antagonist polynucleotide or polynucleotide combination inhibits TGFβ2 from binding to TβRII. In some embodiments, the TGFβ antagonist polynucleotide or polynucleotide combination inhibits TGFβ3 from binding to TβRII. In some embodiments, the TGFβ antagonist polynucleotide or polynucleotide combination inhibits TGFβ1 and TGFβ3 from binding to TβRII. In some embodiments, the TGFβ antagonist polynucleotide or polynucleotide combination inhibits TGFβ1, TGFβ2 and TGFβ3 from binding to TβRII. In some embodiments, the TGFβ antagonist polynucleotide or polynucleotide combination inhibits TGFβ1 from binding to TβRII but does not inhibit or does not substantially inhibit TGFβ2 from binding to TβRII. In some embodiments, the TGFβ antagonist polynucleotide or combination of polynucleotides inhibits TGFβ3 binding to TβRII but does not inhibit, or does not substantially inhibit, TGFβ2 binding to TβRII.In some embodiments, the TGFβ antagonist polynucleotide or polynucleotide combination inhibits TGFβ1 and TGFβ3 from binding to TβRII, but does not inhibit or does not substantially inhibit TGFβ2 from binding to TβRII. In certain aspects, the TGFβ antagonist polynucleotide or polynucleotide combination inhibits at least ALK5 (e.g., inhibits Smad signaling). Thus, in some embodiments, the ALK5 polynucleotide inhibitor binds to ALK5. In some embodiments, the ALK5 polynucleotide inhibitor inhibits the expression (e.g., transcription, translation, secretion, or combinations thereof) of ALK5. In some embodiments, the ALK5 polynucleotide inhibitor further inhibits one or more of TGFβ1, TGFβ2, TGFβ3, TβRII, and betaglycan. In some embodiments, the TGFβ antagonist polynucleotide or polynucleotide combination inhibits TGFβ1 from binding to ALK5. In some embodiments, the TGFβ antagonist polynucleotide or polynucleotide combination inhibits TGFβ2 from binding to ALK5. In some embodiments, the TGFβ antagonist polynucleotide or polynucleotide combination inhibits TGFβ3 from binding to ALK5. In some embodiments, the TGFβ antagonist polynucleotide or polynucleotide combination inhibits TGFβ1 and TGFβ3 from binding to ALK5. In some embodiments, the TGFβ antagonist polynucleotide or polynucleotide combination inhibits TGFβ1, TGFβ2 and TGFβ3 from binding to ALK5. In some embodiments, the TGFβ antagonist polynucleotide or polynucleotide combination inhibits TGFβ1 from binding to ALK5, but does not inhibit or does not substantially inhibit TGFβ2 from binding to ALK5. In some embodiments, the TGFβ antagonist polynucleotide or combination of polynucleotides inhibits TGFβ3 binding to ALK5 but does not inhibit, or does not substantially inhibit, TGFβ2 binding to ALK5.In some embodiments, the TGFβ antagonist polynucleotide or polynucleotide combination inhibits TGFβ1 and TGFβ3 from binding to ALK5, but does not inhibit or does not substantially inhibit TGFβ2 from binding to ALK5. In certain aspects, the TGFβ antagonist polynucleotide or polynucleotide combination inhibits at least betaglycan (e.g., inhibits Smad signaling). Thus, in some embodiments, the betaglycan polynucleotide inhibitor binds to betaglycan. In some embodiments, the betaglycan polynucleotide inhibitor inhibits the expression (e.g., transcription, translation, secretion, or combinations thereof) of betaglycan. In some embodiments, the betaglycan polynucleotide inhibitor further inhibits one or more of TGFβ1, TGFβ2, TGFβ3, TβRII, and ALK5. In some embodiments, the TGFβ antagonist polynucleotide or polynucleotide combination inhibits TGFβ1 from binding to betaglycan. In some embodiments, the TGFβ antagonist polynucleotide or polynucleotide combination inhibits TGFβ2 from binding to betaglycan. In some embodiments, the TGFβ antagonist polynucleotide or polynucleotide combination inhibits TGFβ3 from binding to betaglycan. In some embodiments, the TGFβ antagonist polynucleotide or polynucleotide combination inhibits TGFβ1 and TGFβ3 from binding to betaglycan. In some embodiments, the TGFβ antagonist polynucleotide or polynucleotide combination inhibits TGFβ1, TGFβ2 and TGFβ3 from binding to betaglycan. In some embodiments, the TGFβ antagonist polynucleotide or polynucleotide combination inhibits TGFβ1 from binding to betaglycan, but does not inhibit or does not substantially inhibit TGFβ2 from binding to betaglycan. In some embodiments, a TGFβ antagonist polynucleotide or combination of polynucleotides inhibits TGFβ3 binding to betaglycan but does not inhibit or does not substantially inhibit TGFβ2 binding to betaglycan.In some embodiments, a TGFβ antagonist polynucleotide or combination of polynucleotides inhibits TGFβ1 and TGFβ3 from binding to betaglycan, but does not inhibit or does not substantially inhibit TGFβ2 from binding to betaglycan.

[0196] The polynucleotide antagonist of the present disclosure can be an antisense nucleic acid, an RNAi molecule (e.g., small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA)), an aptamer and / or a ribozyme.The nucleic acid and amino acid sequences of human TβRII, ALK5, betaglycan, TGFβ1, TGFβ2 and TGFβ3 are known in the art, and therefore, the polynucleotide antagonist for use in the method of the present disclosure can be routinely made by those skilled in the art based on the knowledge in the art and the teachings provided herein.

[0197] For example, antisense technology can be used to control gene expression by antisense DNA or RNA or by triple helix formation.Antisense technology is discussed, for example, in Okano (1991) J. Neurochem. 56:560;Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).Triple helix formation is discussed, for example, in Cooney et al. (1988) Science 241:456;and Dervan et al., (1991) Science 251:1300.These methods are based on the binding of polynucleotides to complementary DNA or RNA.In some embodiments, antisense nucleic acid comprises single-stranded RNA or DNA sequence that is complementary to at least a portion of the RNA transcript of desired gene.However, absolute complementarity, although preferred, is not necessary.

[0198] The sequence "complementary to at least a portion of an RNA" referred to herein means a sequence with sufficient complementarity to be able to hybridize with the RNA and form a stable duplex; in the case of the double-stranded antisense nucleic acid of the gene disclosed herein, therefore, one strand of the double-stranded DNA can be tested or triplex formation can be assayed. The ability to hybridize depends on both the degree of complementarity and the length of the antisense nucleic acid. In general, the larger the hybridizing nucleic acid, the more base mismatches it can contain with the RNA and still form a stable duplex (or triplex, as the case may be). Those skilled in the art can ascertain the degree of mismatch that can be tolerated by using standard procedures to determine the melting temperature of the hybridized complex.

[0199] Polynucleotides that are complementary to the 5' end of the message, for example, the 5' untranslated sequence up to and including the AUG start codon, should work most efficiently in inhibiting translation. However, sequences complementary to the 3' untranslated sequence of mRNA have been shown to be effective in inhibiting translation of mRNA as well [see, e.g., Wagner, R., (1994) Nature 372:333-335]. Thus, oligonucleotides complementary to either the 5' or 3' untranslated non-coding regions of the genes of the present disclosure could be used in an antisense approach to inhibit the translation of endogenous mRNA. Polynucleotides complementary to the 5' untranslated region of mRNA should include the complement of the AUG start codon. Antisense polynucleotides complementary to mRNA coding regions are less effective inhibitors of translation, but could be used according to the methods of the present disclosure. Whether designed to hybridize with the 5'-untranslated region, 3'-untranslated region, or coding region of an mRNA of the present disclosure, the antisense nucleic acid should be at least six nucleotides in length, and is preferably an oligonucleotide ranging from 6 to about 50 nucleotides in length. In detailed embodiments, the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides, or at least 50 nucleotides.

[0200] In one embodiment, the antisense nucleic acid of the present disclosure is generated intracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof is transcribed to generate an antisense nucleic acid (RNA) of the gene of the present disclosure. Such a vector will contain a sequence that codes for the desired antisense nucleic acid. Such a vector can remain episomal or be integrated into a chromosome, so long as it can be transcribed to generate the desired antisense RNA. Such a vector can be constructed by recombinant DNA technology methods standard in the art. The vector can be a plasmid, a virus, or other known in the art, used for replication and expression in vertebrate cells. Expression of the sequence that codes for the desired gene of the present disclosure, or a fragment thereof, can be by any promoter known in the art that acts in vertebrate cells, preferably human cells. Such a promoter can be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter region [see, e.g., Benoist and Chambon (1981) Nature 29:304-310], the promoter contained in the 3' terminal repeat of Rous sarcoma virus [see, e.g., Yamamoto et al. (1980) Cell 22:787-797], the herpes thymidine promoter [see, e.g., Wagner et al. (1981) Proc. Natl. Acad. Sci. USA 78:1441-1445], and the regulatory sequences of metallothionein genes [see, e.g., Brinster, et al. (1982) Nature 296:39-42].

[0201] In some embodiments, the polynucleotide antagonist is an interfering RNA or RNAi molecule that targets the expression of one or more genes. RNAi refers to the expression of an RNA that interferes with the expression of a targeted mRNA. In particular, RNAi silences a targeted gene through interaction with a specific mRNA by an siRNA (small interfering RNA). The dsRNA complex is then targeted for degradation by the cell. siRNA molecules are double-stranded RNA duplexes of 10-50 nucleotides in length that interfere with the expression of a target gene that is sufficiently complementary (e.g., at least 80% identical to that gene). In some embodiments, the siRNA molecule comprises a nucleotide sequence that is at least 85, 90, 95, 96, 97, 98, 99, or 100% identical to the nucleotide sequence of the target gene.

[0202] Additional RNAi molecules include short hairpin RNAs (shRNAs), also short interfering hairpins and microRNAs (miRNAs). shRNA molecules contain sense and antisense sequences from a target gene linked by a loop. shRNAs are transported from the nucleus to the cytoplasm and degraded along with the mRNA. Pol III or U6 promoters can be used to express RNA for RNAi. Paddison et al. [Genes & Dev. (2002) 16:948-958, 2002] have used small RNA molecules that fold into hairpins as a means to affect RNAi. Thus, such short hairpin RNA (shRNA) molecules are also advantageously used in the methods described herein. The length of the stem and loop of a functional shRNA can vary, with the stem length ranging anywhere from about 25 to about 30 nt, and the size of the loop can be within the range of 4 to about 25 nt without affecting silencing activity. Without wishing to be bound by any particular theory, these shRNAs have in common with the double-stranded RNA (dsRNA) products of DICER RNase and have the same ability to inhibit the expression of specific genes in any event. shRNAs can be expressed from lentiviral vectors. miRNAs are single-stranded RNAs of about 10-70 nucleotides long that are initially transcribed as pre-miRNAs characterized by a "stem-loop" structure, and then processed into mature miRNAs after further processing by RISC.

[0203] Molecules that mediate RNAi, including but not limited to siRNAs, can be generated in vitro by chemical synthesis (Hohjoh, FEBS Lett 521:195-199, 2002), by hydrolysis of dsRNA (Yang et al., Proc Natl Acad Sci USA 99:9942-9947, 2002), by in vitro transcription with T7 RNA polymerase (Donzeet et al., Nucleic Acids Res 30:e46, 2002; Yu et al., Proc Natl Acad Sci USA 99:6047-6052, 2002), and by hydrolysis of double-stranded RNA using nucleases such as E. coli RNase III (Yang et al., Proc Natl Acad Sci USA 99:9942-9947, 2002).

[0204] According to another aspect, the present disclosure provides polynucleotide antagonists, including, but not limited to, decoy DNA, double-stranded DNA, single-stranded DNA, complexed DNA, encapsulated DNA, viral DNA, plasmid DNA, naked RNA, encapsulated RNA, viral RNA, double-stranded RNA, molecules capable of causing RNA interference, or combinations thereof.

[0205] In some embodiments, the polynucleotide antagonist of the present disclosure is an aptamer. Aptamers are nucleic acid molecules, including double-stranded DNA molecules and single-stranded RNA molecules, that combine to form a tertiary structure that specifically binds to target molecules, such as TβRII, TGFβ1, TGFβ2 and TGFβ3 polypeptides. The creation and therapeutic use of aptamers is well established in the art. For example, see U.S. Patent No. 5,475,096. Additional information on aptamers can be found in U.S. Patent Application Publication No. 20060148748. Nucleic acid aptamers are selected using methods known in the art, for example, through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. SELEX is a method for in vitro evolution of nucleic acid molecules with highly specific binding to target molecules, as described, for example, in U.S. Pat. No. 5,475,096, U.S. Pat. No. 5,580,737, U.S. Pat. No. 5,567,588, U.S. Pat. No. 5,707,796, U.S. Pat. No. 5,763,177, U.S. Pat. No. 6,011,577 and U.S. Pat. No. 6,699,843. Another screening method for identifying aptamers is described in U.S. Pat. No. 5,270,163. The SELEX process is based on the ability of nucleic acids to form a variety of two- and three-dimensional structures, and the chemical diversity available within nucleotide monomers to function as ligands (form specific binding pairs) with virtually any chemical compound, including other nucleic acid molecules and polypeptides, whether monomeric or polymeric. Molecules of any size or composition can serve as targets. The SELEX method involves selection from a mixture of candidate oligonucleotides, and stepwise repetition of binding, partitioning and amplification using the same general selection scheme to achieve a desired binding affinity and selectivity.Starting with a mixture of nucleic acids, which may contain segments of randomized sequences, the SELEX method includes the steps of contacting the mixture with the target under conditions favorable for binding; partitioning unbound nucleic acids from those nucleic acids that are specifically bound to the target molecule; dissociating the nucleic acid-target complexes; and amplifying the nucleic acids dissociated from the nucleic acid-target complexes to obtain a ligand-enriched mixture of nucleic acids. The steps of binding, partitioning, dissociation and amplification are optionally repeated for many cycles to provide highly specific, high affinity nucleic acid ligands for the target molecule.

[0206] Typically, such binding molecules are administered separately to the animal [see, e.g., O'Connor (1991) J. Neurochem. 56:560], but such binding molecules can also be expressed in vivo from a polynucleotide which is taken up by a host cell and expressed in vivo [see, e.g., Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)]. 3. Screening Assays

[0207] In certain embodiments, the present invention relates to the use of TβRII polypeptides (e.g., soluble TβRII polypeptides) to identify compounds (drugs) that are agonists or antagonists of the signal transduction pathways of TGFβ1, TGFβ3 and TβRII. Compounds identified by this screening can be tested in vitro to assess their ability to modulate the signal transduction activity of TGFβ1 and TGFβ3. If necessary, these compounds can be further tested in animal models to assess their ability to modulate tissue growth in vivo.

[0208] There are many approaches to screening for therapeutic agents for regulating tissue growth by targeting TGFβ1, TGFβ3 and TβRII polypeptide.In certain embodiments, high-throughput screening of compounds can be performed to identify agents that disrupt TGFβ1, TGFβ3 or TβRII-mediated cell signaling.In certain embodiments, assays are performed to screen and identify compounds that specifically inhibit or reduce the binding of TβRII polypeptide to TGFβ1 or TGFβ3.Alternatively, assays can be used to identify compounds that enhance the binding of TβRII polypeptide to TGFβ1 or TGFβ3.In further embodiments, compounds can be identified by their ability to interact with TGFβ1, TGFβ3 or TβRII polypeptide.

[0209] A variety of assay formats are sufficient in view of the present disclosure, and those not explicitly described herein will nevertheless be understood by those skilled in the art. As described herein, the test compounds (agents) of the present invention can be produced by any combinatorial chemical method. Alternatively, the compounds of interest can be naturally occurring biomolecules that are synthesized in vivo or in vitro. The compounds (agents) that are tested for their ability to act as modulators of tissue growth can be produced, for example, by bacteria, yeast, plants or other organisms (e.g., natural products), can be produced chemically (e.g., small molecules, including peptidomimetics), or can be produced recombinantly. Test compounds contemplated by the present invention include non-peptidyl organic molecules, peptides, polypeptides, peptidomimetics, sugars, hormones, and nucleic acid molecules. In a detailed embodiment, the test agent is a small organic molecule having a molecular weight of less than about 2,000 daltons.

[0210] The test compounds of the present invention can be provided as single separate entities or in libraries of higher complexity, such as those created by combinatorial chemistry. These libraries can include, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers and other classes of organic compounds. Presentation of test compounds to the test system can be either in isolated form or as a mixture of compounds, especially in the initial screening step. If necessary, the compounds can be derivatized with other compounds or have derivatization groups that facilitate the isolation of the compounds. Non-limiting examples of derivatization groups include biotin, fluorescein, digoxygenin, green fluorescent protein, isotopes, polyhistidine, magnetic beads, glutathione S-transferase (GST), photoactivatable crosslinkers, or any combination thereof.

[0211] In many drug screening programs that test libraries of compounds and natural extracts, high-throughput assays are desirable to maximize the number of compounds that are investigated in a given period of time.Assays that are performed in cell-free systems, such as those that can be derived from purified or semi-purified protein, are often preferred as "primary" screening, in that they can be made to allow rapid development and relatively easy detection of the changes in molecular targets mediated by test compounds.In addition, the effects of cytotoxicity or bioavailability of test compounds can generally be ignored in in vitro systems, and the assay instead focuses mainly on the effects of drugs on molecular targets, which can be expressed in the change of binding affinity between TβRII polypeptide and TGFβ1 or TGFβ3.

[0212] For illustrative purposes only, in an exemplary screening assay of the present invention, a compound of interest is contacted with an isolated and purified TβRII polypeptide that is normally capable of binding to TGFβ1 or TGFβ3. A composition containing a TβRII ligand is then added to the mixture of compound and TβRII polypeptide. Detection and quantification of TβRII / TGFβ1 complex or TβRII / TGFβ3 complex provides a means for determining the effectiveness of a compound that inhibits (or enhances) the formation of a complex between a TβRII polypeptide and TGFβ1 or TGFβ3. The effectiveness of a compound can be evaluated by generating a dose-response curve from data obtained using various concentrations of a test compound. A control assay can also be performed to provide a baseline for comparison. For example, in a control assay, isolated and purified TGFβ1 or TGFβ3 is added to a composition containing a TβRII polypeptide, and the formation of a TβRII / TGFβ1 complex or TβRII / TGFβ3 complex is quantified in the absence of a test compound. In general, it will be understood that the order in which reactants may be mixed can be varied and can be mixed simultaneously. Also, instead of purified proteins, cell extracts and lysates may be used making them suitable cell-free assay systems.

[0213] Complex formation between a TβRII polypeptide and TGFβ1 or TGFβ3 can be detected by a variety of techniques. For example, modulation of the formation of a complex can be detected by, for example, radioactive labeling (e.g., 32 P, 35 S, 14 C or 3 H), fluorescently labeled (e.g., FITC), or enzyme-labeled TβRII polypeptide or TGFβ1 or TGFβ3 can be used to quantitate by immunoassay or by chromatographic detection.

[0214] In certain embodiments, the present invention contemplates the use of fluorescence polarization assays and fluorescence resonance energy transfer (FRET) assays in measuring, either directly or indirectly, the degree of interaction between a TβRII polypeptide and its binding protein. Additionally, other detection modalities, such as those based on optical waveguides (PCT Publication No. WO 96 / 26432 and U.S. Patent No. 5,677,196), surface plasmon resonance (SPR), surface charge sensors, and surface force sensors, are compatible with many embodiments of the present invention.

[0215] The present invention also contemplates the use of interaction trap assays, also known as "two-hybrid assays", to identify agents that disrupt or enhance the interaction between TβRII polypeptides and their binding proteins.See, for example, U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene 8:1693-1696.In a specific embodiment, the present invention contemplates the use of reverse two-hybrid systems to identify compounds (e.g., small molecules or peptides) that dissociate the interaction between TβRII polypeptides and their binding proteins. See, e.g., Vidal and Legrain, (1999) Nucleic Acids Res 27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81; and U.S. Patent Nos. 5,525,490; 5,955,280; and 5,965,368.

[0216] In certain embodiments, compounds of interest are identified by their ability to interact with the TβRII or TGFβ1 or TGFβ3 polypeptide of the present invention. The interaction between a compound and a TβRII or TGFβ1 or TGFβ3 polypeptide can be covalent or non-covalent. For example, such interactions can be identified at the protein level using in vitro biochemical methods, including photocrosslinking, radiolabeled ligand binding and affinity chromatography (Jakoby WB et al., 1974, Methods in Enzymology 46: 1). In certain cases, compounds may be screened with mechanism-based assays, such as assays for detecting compounds that bind to TGFβ1 or TGFβ3 or TβRII polypeptide. This may include solid-phase or fluid-phase binding events. Alternatively, genes encoding TGFβ1 or TGFβ3 or TβRII polypeptides can be transfected into cells with a reporter system (e.g., β-galactosidase, luciferase or green fluorescent protein) and screened against the library or with individual members of the library, preferably by high-throughput screening. Binding assays based on other mechanisms can also be used, such as binding assays that detect changes in free energy. Binding assays can be performed with targets immobilized on wells, beads or chips, captured by immobilized antibodies, or resolved by capillary electrophoresis. Bound compounds can usually be detected using colorimetry or fluorescence, or surface plasmon resonance.

[0217] In certain embodiments, the present invention provides methods and agents for regulating (stimulating or inhibiting) TGFβ1 or TGFβ3 mediated cell signaling. Thus, any compounds identified can be tested in whole cells or tissues, in vitro or in vivo, to confirm their ability to regulate TGFβ1 or TGFβ3 signaling. Various methods known in the art can be utilized for this purpose. 4. Exemplary Therapeutic Uses

[0218] As used herein, a therapeutic agent that "prevents" a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in a treated sample compared to an untreated control sample, or delays the occurrence of or reduces the severity of one or more symptoms of the disorder or condition compared to an untreated control sample.

[0219] The terms "treatment", "treating", "alleviating" and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect, and may also be used to refer to improving, alleviating, and / or reducing the severity of one or more symptoms of the condition being treated. The effect may be preventative in terms of delaying, completely or partially, the onset or recurrence of the disease, condition, or its symptoms, and / or may be therapeutic in terms of partially or completely curing the disease or condition, and / or the deleterious effects caused by the disease or condition. As used herein, "treatment" encompasses any treatment of a mammalian, particularly a human, disease or condition, including (a) preventing the disease or condition from occurring in a subject who may be susceptible to the disease or condition, but has not yet been diagnosed as having it; (b) inhibiting the disease or condition (e.g., preventing its occurrence); or (c) relieving the disease or condition (e.g., causing regression of the disease or condition, providing improvement in one or more symptoms).

[0220] The terms "patient", "subject" or "individual" are used interchangeably herein and refer to either human or non-human animals. These terms include mammals, such as humans, non-human primates, laboratory animals, livestock animals (including cows, pigs, camels, etc.), companion animals (e.g., dogs, cats, other domestic animals, etc.), and rodents (e.g., mice and rats). In certain embodiments, the patient, subject or individual is a human.

[0221] As used herein, "combination," "in combination with," "co-administration," and the like refer to any form of administration such that the second therapy remains effective in the body (e.g., two compounds are effective in the patient simultaneously, which may include synergistic effects of the two compounds). Efficacy may not be related to measurable concentrations of the agents in blood, serum, or plasma. For example, different therapeutic compounds can be administered on different schedules, either simultaneously or sequentially, either in the same formulation or in separate formulations. Thus, an individual undergoing such treatment can benefit from the combined effects of the different therapies. One or more TGFβ antagonists of the present disclosure can be administered simultaneously with, before, or after one or more other additional agents or supportive therapies. In general, each therapeutic agent is administered at a dose and / or time schedule determined for the particular agent. The particular combination used in the regimen takes into account the compatibility of the antagonists of the present disclosure with the therapy and / or the desired therapeutic effect to be achieved.

[0222] In part, the present disclosure provides a method for treating ectopic ossification by administering an effective amount of a TGFβ antagonist to a patient in need thereof. In some embodiments, the method relates to preventing or reducing the severity and / or duration of ectopic ossification in a patient in need thereof with an effective amount of a TGFβ antagonist. In some embodiments, the present disclosure provides a method for treating a disease or condition associated with ectopic ossification by administering an effective amount of a TGFβ antagonist to a patient in need thereof. In some embodiments, the method relates to preventing or reducing the severity and / or duration of a disease or condition associated with ectopic ossification in a patient in need thereof with an effective amount of a TGFβ antagonist. Optionally, such a method further comprises administering a TGFβ antagonist in combination with one or more additional active agents or supportive therapies for treating ectopic ossification.

[0223] In general, heterotopic ossification (HO) is the result of osteoid formation of mature lamellar bone in soft tissue sites outside the periosteum (skeletal system) of the skeleton. In many cases, there is a precipitating event prior to the initiation of HO, usually an episode of trauma that may result in a hematoma. In general, the progression of HO requires the supply of multipotent (multi-potent) mesenchymal cells that can differentiate into osteoblasts or chondroblasts. However, the mechanism of action behind other HO development is largely unknown. Osteoid formation is often associated with an inflammatory phase characterized by local swelling, pain, erythema, and sometimes fever. This pathological process can occur in sites such as the skin, subcutaneous tissue, skeletal muscles, and fibrous tissue adjacent to joints. Bone can also form in blood vessel walls and ligaments. Lesions range from small clinically insignificant foci to numerous deposits that extend throughout the body. HO can occur in joint contractures and ankylosis, pain, spasticity, swelling fever, neurovascular compression syndromes, pressure ulcers, and significant disability. In some embodiments, the present disclosure relates to methods of treating one or more complications associated with heterotopic ossification (e.g., bone deposition, formation of bone marrow cavities, joint contractures, joint ankylosis, pain, spasticity, swelling, fever, neurovascular compression syndromes, and bedsores) by administering to a subject in need thereof an effective amount of a TGFβ antagonist, optionally in combination with one or more additional active agents or supportive therapies for treating heterotopic ossification.

[0224] HO is most commonly associated with spinal cord injury, trauma, brain injury, burns, bone fractures, muscle strains, and joint arthroplasty. In some embodiments, the present disclosure relates to a method for treating heterotopic ossification associated with one or more disorders or conditions selected from the group consisting of spinal cord injury, trauma, brain injury, burns, bone fractures, muscle strains, and joint arthroplasty, by administering to a subject in need thereof an effective amount of a TGFβ antagonist, optionally in combination with one or more additional active agents or supportive care for treating heterotopic ossification.

[0225] HO is a serious complication of hip, acetabulum and elbow fracture surgeries. Approximately one in three patients with total hip arthroplasty (joint replacement) or severe fractures of the long bones of the lower leg develop HO, but it is rarely symptomatic. 50% to 90% of patients who develop HO after a previous hip arthroplasty also develop heterotopic ossification. In some embodiments, the present disclosure relates to a method of treating heterotopic ossification associated with one or more of joint, hip, acetabulum and elbow fracture surgeries (e.g., replacement surgery) by administering to a subject in need thereof an effective amount of a TGFβ antagonist, optionally in combination with one or more additional active agents or supportive care for treating heterotopic ossification.

[0226] HO often occurs in patients with trauma. In particular, HO is associated with brain and spinal cord injuries, which may be the primary cause of the clinical observation that traumatic brain injury causes accelerated fracture healing. Morley et al. (2005) Injury 36(3): 363. HO is also associated with severe burns, combat-related trauma, and amputation. In traumatic heterotopic ossification, patients may complain of warmth, tenderness, hard swelling in the muscles, and reduced range of motion in the joints innervated by the involved muscles. There is often a history of a blow or other trauma to the area several weeks to months prior. Patients with traumatic nerve injury, severe nerve damage, or severe burns who have developed heterotopic ossification experience limited movement in the affected area. In some embodiments, the present disclosure relates to methods of treating heterotopic ossification associated with one or more of trauma, brain or spinal cord injury, burns, combat-related trauma, and amputation by administering to a patient in need thereof an effective amount of a TGFβ antagonist, optionally in combination with one or more additional active agents or supportive care for treating heterotopic ossification.

[0227] There are also rare genetic disorders that cause heterotopic ossification, such as fibrodysplasia ossificans progressiva (FOP). FOP is an extremely rare connective tissue disease. The disease is caused by a mutation in the body's repair mechanism, which causes ossification in fibrous tissues (including muscles, tendons, and ligaments), either spontaneously or when injured. In many cases, the injury can cause the joint to become permanently stiff in place. For unknown reasons, the bones of children with FOP may have a deformed big toe, possibly a joint defect, or simply show noticeable lumps in minor joints. The first "flare-up" that results in the formation of FOP bones usually occurs before the age of 10. Bone growth proceeds from top to bottom, just as bones grow in a fetus. Children with FOP typically develop bones starting in the neck, then the shoulders, arms, chest region, and finally the feet. In particular, ossification is typically first seen in the dorsal, axial, cranial, and basal regions of the body. The disease then progresses to the abdominal, limb, caudal, and distal regions of the body. However, it does not always occur in that order due to the damage that causes the flare-ups. In many cases, the tumor-like lumps that characterize the disease suddenly appear. The condition causes loss of motor function affecting the joints, including an inability to open the mouth completely, limiting speech and eating. Extra bone formation around the rib cage limits the expansion of the lungs and diaphragm causing respiratory complications. Because the disease is so rare, the symptoms are often misdiagnosed as cancer or fibrosis. This leads the physician to order a biopsy, which may exacerbate the growth of these lumps. The median age of survival is 40 years, with proper management. However, if the diagnosis is delayed, trauma and infection may reduce the life expectancy. Generally, FOP is caused by an autosomal dominant allele at chromosome 2q23-24. The allele has variable expressivity but complete penetrance. Most cases are caused by spontaneous mutations in gametes. A similar, but less intractable, disease is fibrous dysplasia, which is caused by postzygotic mutations. Mutations in the ACVR1 gene (also known as activin-like kinase 2 (ALK2)) are responsible for this disease.ACVR1 encodes activin receptor type 1, a BMP type 1 receptor. The mutation causes an arginine to histidine substitution at codon 206 in the ACVR1 protein. This substitution causes the abnormal activation of ACVR1, resulting in the conversion of connective tissue and muscle tissue into the secondary skeleton. This causes endothelial cells to convert into mesenchymal stem cells and then into bone. In some embodiments, the present disclosure relates to a method for treating ectopic ossification associated with fibrodysplasia ossificans progressiva by administering to a subject in need thereof an effective amount of a TGFβ antagonist, optionally in combination with one or more additional active agents or supportive therapies for treating ectopic ossification and / or fibrodysplasia ossificans progressiva. In some embodiments, the present disclosure relates to a method for treating ectopic ossification associated with fibrodysplasia ossificans progressiva by administering to a subject in need thereof an effective amount of a TGFβ antagonist, optionally in combination with one or more additional active agents or supportive therapies for treating ectopic ossification and / or fibrodysplasia ossificans progressiva.

[0228] There is no cure or approved treatment for FOP. Attempts to surgically remove the bone result in explosive bone growth. The variability of the disease's course and low prevalence induce uncertainty, although clinical trials of isotretinoin, etidronate and oral corticosteroids, and perhexiline maleate have failed to demonstrate efficacy. A handful of pharmaceutical companies looking at rare diseases are currently in various stages of exploring different treatment approaches for FOP. In August 2015, the U.S. Food and Drug Administration's Office of Orphan Product Development granted La Jolla Pharmaceuticals orphan drug designation for two novel compounds for FOP. The compounds are small molecule kinase inhibitors designed to selectively block ACVR1 (ALK2). In August 2015, Clementia Pharmaceuticals also began enrolling children into a Phase II clinical trial examining palovarotene for the treatment of FOP. Preclinical studies have demonstrated that palovarotene, a retinoic acid receptor gamma agonist, blocked abnormal bone formation in animal models through inhibition of second messenger systems in the BMP pathway. In September 2015, Regeneron published new insights into the mechanism of disease involving activation of the ACVR1 receptor by activin A. In 2016, the company began a phase 1 study of REGN 2477, an activin A antibody, in healthy volunteers. Another potential therapeutic approach involves allele-specific RNA interference, which targets mutated mRNA for degradation while preserving normal ACVR1 gene expression. JW Lowery et al. (2012) Gene Therapy 19(701-702): 701-702. A mouse model of FOP expressing a potent constitutively active ALK2 R206H mutant has been found to be useful in identifying selective agonists for nuclear retinoic acid receptor-α (RAR-α) in mesenchymal cells. RAR-α agonists were found to partially inhibit HO, whereas agonists for RAR-γ were found to be potent inhibitors of intramuscular and subcutaneous HO in the FOP model.Shimono et al., 2011, Nature Medicine 17:454-60). In some embodiments, the disclosure relates to a method of treating heterotopic ossification associated with FOP by administering to a subject in need thereof an effective amount of a TGFβ antagonist in combination with one or more additional active agents or supportive care for treating FOP, wherein the additional active agents or supportive care are selected from the group consisting of isotretinoin, etidronate and oral corticosteroids, perhexiline maleate, ALK2 small molecule inhibitors, palovarotene, retinoic acid receptor gamma agonists, retinoic acid receptor alpha agonists, activin antibodies (e.g., activin A antibodies such as REGN 2477), and allele-specific RNA interference of ALK2.

[0229] Another rare genetic disorder causing heterotopic ossification is progressive osseous dysplasia (POH), a condition characterized by cutaneous or subcutaneous bone formation. POH is a condition characterized by the alpha subunit of the stimulatory guanine nucleotide-binding protein Gα, which acts downstream of many G protein-coupled receptors in activating adenylyl cyclase. sIt is associated with inactivating mutations in the GNAS gene, which encodes for POH. Kaplan, et al. 1994, J Bone Joint Surg Am 76, 425-436;Shore, et al., 2002, N Engl J Med 346, 99-106;and Eddy, et al., 2000, J Bone Miner Res 15, 2074-2083. Clinically, POH presents with cutaneous and subcutaneous ossifications in infancy, progressing to skeletal muscle and deep connective tissues (e.g., tendons, ligaments, fascia) during childhood. Over time, these ossifications result in joint stiffness, bone and joint fusion, and growth retardation of the affected limb. Currently, patients with POH undergo extensive surgical resection of the heterotopic bone to arrest the spread of the disease. This often results in partial or complete limb amputation, and the lesion frequently recurs (Kaplan, et al., 2000, J Bone Miner Res 15, 2084-94; and Shehab, et al., 2003, J Nucl Med 43, 346-353), which clearly indicates the importance of developing improved therapeutic interventions. Observations made in patients with POH suggest that mesenchymal stem cells present in soft tissues inappropriately differentiate into osteoblasts and begin depositing into bone. Lacking both in vitro and in vivo animal models, the pathogenesis of POH remains unknown, and as with all forms of HO, suitable treatments are lacking. In some embodiments, the present disclosure relates to a method of treating heterotopic ossification associated with progressive skeletal dysplasia by administering to a subject in need thereof an effective amount of a TGFβ antagonist, optionally in combination with one or more additional active agents or supportive care for treating heterotopic ossification and / or progressive skeletal dysplasia.

[0230] HO may also occur in patients who are neuromuscularly blocked to manage adult respiratory distress syndrome and in patients with non-traumatic myelopathy.In some embodiments, the present disclosure relates to a method for treating the heterotopic ossification associated with neuromuscular blockade used to manage adult respiratory distress syndrome by administering to a subject in need thereof an effective amount of a TGFβ antagonist, optionally in combination with one or more additional active agents or supportive care for treating heterotopic ossification.In some embodiments, the present disclosure relates to a method for treating the heterotopic ossification associated with non-traumatic myelopathy by administering to a subject in need thereof an effective amount of a TGFβ antagonist, optionally in combination with one or more additional active agents or supportive care for treating heterotopic ossification.

[0231] No well-defined form of treatment exists for HO. Initially, bisphosphonates were hoped to be of value after hip surgery, but despite their prophylactic use, there was no convincing evidence of benefit. Prophylactic radiation therapy for the prevention of heterotopic ossification has also been used since the 1970s. Various doses and techniques have been used. In general, radiation therapy should be delivered as close as possible to the time of surgery. Doses of 7 to 8 Gray to a single compartment within 24 to 48 hours of surgery have been used successfully. Treatment volumes include periarticular areas and can be used for the hip, knee, elbow, shoulder, jaw, or in patients after spinal cord injury. Single-dose radiation therapy has been found to be well tolerated, with no increase in bleeding, infection, or interference with wound healing, and is cost-effective. Certain anti-inflammatory agents, such as indomethacin, ibuprofen, and aspirin, have shown some efficacy in preventing the recurrence of heterotopic ossification after total hip arthroplasty. Conservative treatments such as passive range of motion exercises or other mobilization techniques provided by a physical or occupational therapist may also aid in the prevention of HO. In some embodiments, the present disclosure relates to a method of treating heterotopic ossification by administering to a subject in need thereof an effective amount of a TGFβ antagonist in combination with one or more additional active agents or supportive therapies for treating heterotopic ossification, wherein the one or more additional active agents or supportive therapies are selected from the group consisting of bisphosphonates, radiation therapy, anti-inflammatory agents (e.g., indomethacin, ibuprofen, and aspirin), and conservative treatments such as passive range of motion exercises or other mobilization techniques. 5. Pharmaceutical Compositions

[0232] The therapeutic agent (e.g., TβRII fusion polypeptide) described herein can be formulated into pharmaceutical compositions.The pharmaceutical compositions for use according to the present disclosure can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients.Such formulations are generally substantially pyrogen-free, which meets most regulatory requirements.

[0233] In certain embodiments, the therapeutic method of the present disclosure comprises administering the composition systemically or locally as an implant or device.When administered, the therapeutic composition for use in the present disclosure is in a pyrogen-free, physiologically acceptable form.The therapeutically useful agent other than TGFβ signaling antagonist, which may be optionally included in the composition described above, can be administered simultaneously or sequentially with the compound of interest (e.g., TβRII polypeptide) in the method disclosed herein.

[0234] Typically, the protein therapeutic agent disclosed herein is administered parenterally, particularly intravenously or subcutaneously. Pharmaceutical compositions suitable for parenteral administration may contain one or more TGFβ antagonists in combination with one or more pharma- ceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted immediately before use into sterile injectable solutions or dispersions, which may contain antioxidants, buffers, bacteriostatic agents, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0235] The compositions and formulations may be optionally provided in a pack or dispenser device that may contain one or more unit dosage forms containing the active ingredient.The pack may, for example, comprise a metal or plastic foil, such as a blister pack.The pack or dispenser device may be accompanied by instructions for administration.

[0236] Furthermore, the composition may be encapsulated or injected in a form for delivery to the target tissue site.In certain embodiments, the composition of the present invention may comprise a matrix capable of delivering one or more therapeutic compounds (e.g., TGFβ antagonist) to the target tissue site, providing a structure for tissue growth, and optimally capable of being resorbed in the body.For example, the matrix may provide sustained release of the TGFβ antagonist.Such a matrix may be formed of materials currently used for other implantation medical applications.

[0237] The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the composition of interest will define the appropriate formulation. Potential matrices for the composition may be biodegradable, chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid and polyanhydrides. Other potential materials are biodegradable, biologically well defined, such as bone or dermal collagen. Further matrices are composed of pure proteins or extracellular matrix components. Other potential matrices are non-biodegradable, chemically defined, such as sintered hydroxyapatite, bioglass, aluminates or other ceramics. Matrices may be composed of any combination of the above mentioned types of materials, such as polylactic acid and hydroxyapatite, or collagen and tricalcium phosphate. Bioceramics may be modified in the composition, such as in calcium-aluminate-phosphate, and processed to modify pore size, particle size, particle shape and biodegradability.

[0238] In certain embodiments, the methods of the invention can be administered orally, for example, in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powders, granules, each containing a predetermined amount of the agent as the active ingredient, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a troche (using an inert base such as gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, etc. The agent may also be administered as a bolus, electuary, or paste.

[0239] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), one or more therapeutic compounds of the present invention may be mixed with one or more pharma- ceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol, ... (4) disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) dissolution retardants such as paraffin; (6) absorption enhancers such as quaternary ammonium compounds; (7) wetting agents such as cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be used as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols, and the like.

[0240] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.In addition to active ingredients, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.Other than inert diluents, oral compositions may also contain auxiliary agents such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings, coloring agents, flavors and preservatives.

[0241] Suspensions may contain, in addition to the active compound, suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0242] The composition of the present invention may also contain auxiliary agents, such as preservatives, wetting agents, emulsifiers and dispersants.Prevention of microbial action can be ensured by including various antibacterial and antifungal agents, such as paraben, chlorobutanol, phenol sorbic acid, etc.It may also be desirable to include isotonic agents, such as sugar, sodium chloride, etc. in the composition.In addition, sustained absorption of injectable pharmaceutical forms can be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0243] It is understood that the dosage regimen is determined by the attending physician, taking into consideration various factors that change the action of the compound of the present invention (e.g., TGFβ antagonist) in the subject. Various factors include, but are not limited to, the age, sex and diet of the patient, the severity of the disease, the administration time, and other clinical factors. If necessary, the dosage can be changed according to the type of matrix used in the reconstruction and the type of compound in the composition. The addition of other known growth factors to the final composition can also affect the dosage. Progress can be monitored by regular evaluation of bone growth and / or repair, for example, X-ray (including DEXA), histomorphometric determination, and tetracycline labeling.

[0244] In certain embodiments, the present invention also provides gene therapy for the in vivo production of TGFβ antagonists. Such therapy will achieve its therapeutic effect by introducing TGFβ antagonist polynucleotide sequences into cells or tissues with disorders such as those listed above. Delivery of TGFβ antagonist polynucleotide sequences can be achieved using recombinant expression vectors such as chimeric viruses, or colloidal dispersion systems. The use of targeted liposomes is preferred for therapeutic delivery of TGFβ antagonist polynucleotide sequences.

[0245] Various viral vectors that can be utilized for gene therapy as taught herein include adenovirus, herpes virus, vaccinia, or preferably RNA viruses such as retrovirus. Preferably, the retroviral vector is a derivative of mouse or avian retrovirus. Examples of retroviral vectors into which a single foreign gene can be inserted include, but are not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV) and Rous sarcoma virus (RSV). Some additional retroviral vectors can incorporate multiple genes. All these vectors can transfer or incorporate genes for selection markers so that transduced cells can be identified and generated. Retroviral vectors can be made target specific, for example, by attaching sugars, glycolipids or proteins. Preferred targeting is achieved by using antibodies. Those skilled in the art will recognize that specific polynucleotide sequences can be inserted into the retroviral genome or attached to the viral envelope to enable target-specific delivery of retroviral vectors containing TGFβ antagonist polynucleotides. In a preferred embodiment, the vector is targeted to bone or cartilage.

[0246] Alternatively, tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env by conventional calcium phosphate transfection. These cells are then transfected with a vector plasmid containing the gene of interest. The resulting cells release the retroviral vector into the culture medium.

[0247] Another targeted delivery system for TGFβ antagonist polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of the present invention is a liposome. Liposomes are artificial membrane vesicles that are useful as delivery vehicles in vitro and in vivo. RNA, DNA, and intact virions can be encapsulated in the aqueous interior and delivered to cells in a biologically active form (see, for example, Fraley, et al., Trends Biochem. Sci., 6:77, 1981). Methods for efficient gene transfer using liposomal vehicles are known in the art, see, for example, Mannino, et al., Biotechniques, 6:682, 1988. Liposome compositions are usually a combination of phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used.The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0248] Examples of lipids useful in the manufacture of liposome include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides and gangliosides.Exemplary phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.Targeting of liposome can also be based on, for example, organ specificity, cell specificity and organelle specificity, and is known in the art.

[0249] The disclosure provides formulations that can be modified, including acids and bases to adjust the pH; and buffers to keep the pH within narrow ranges. Example

[0250] The present invention will now be generally described and will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain particular embodiments of the invention and are not intended to limit the invention. EXAMPLES

[0251] Example 1 Generation of receptor fusion protein variants TβRII ECD variants TβRII fusion proteins were generated that comprised the soluble extracellular portion of human TβRII and a human Fc portion. For each fusion protein, the TβRII amino acid sequence having the amino acid sequence of SEQ ID NO: 18 was fused by one of several different linkers to an IgG Fc portion having the amino acid sequence of SEQ ID NO: 49. Each fusion protein also contained a TPA leader sequence having the amino acid sequence of SEQ ID NO: 23 (below).

[0252] Tissue plasminogen activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 23)

[0253] An illustrative overview of some of the constructs designed is provided as Figure 3. A table detailing the sequences for the different constructs tested in the exemplary section is provided below. [Table 1]

[0254] The amino acid sequences for the components of the constructs and their respective constructs are presented below, along with the nucleic acid sequences used to express these constructs. [ka] [ka]

[0255] For the animal studies described below, a mouse version of SEQ ID NO:9 was generated by fusing the mouse extracellular domain of TβRII, corresponding to isoform A of human TβRII, to mouse G1Fc via a linker domain (TGGG). This fusion protein is referred to herein as mTβRII-mFc.

[0256] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0257] The various constructs were successfully expressed in CHO cells and purified to high purity as determined by analytical size exclusion chromatography and SDS-PAGE. The hTβRII(G4S)2-hFc, hTβRII(G4S)3-hFc, hTβRII(G4S)4-hFc, hTβRII(G4S)5-hFc and hTβRII(G4S)6-hFc proteins showed similar strong stability as determined by SDS-PAGE analysis when kept in PBS at 37°C for 13 days. The hTβRII(G4S)2-hFc, hTβRII(G4S)3-hFc, hTβRII(G4S)4-hFc proteins were also kept in rat, mouse or human serum, showing similar strong stability. TβRII ECD variants

[0258] In addition to the TβRII domain contained in the fusion protein described above (e.g., SEQ ID NO: 18), the present disclosure also contemplates fusion proteins that contain alternative TβRII domains. For example, the fusion protein may contain the wild-type hTβRII domain shown below. short (23-159) sequence (SEQ ID NO:27), or any other TβRII polypeptide disclosed below. [ka]

[0259] (1) hTβRII shown below short (23~159 / D110K) amino acid sequence (SEQ ID NO:36), where the substituted residues are underlined. [ka]

[0260] (2) N-terminal truncated hTβRII as shown below short (29-159) Amino acid sequence (sequence number 28). [ka]

[0261] (3) N-terminally truncated hTβRII as shown below short (35-159) Amino acid sequence (sequence number 29). [ka]

[0262] (4) C-terminal truncated hTβRII as shown below short (23-153) Amino acid sequence (sequence number 30). [ka]

[0263] (5) C-terminal truncated hTβRII as shown below short(23~153 / N70D) amino acid sequence (SEQ ID NO:38), where the substituted residues are underlined. [ka]

[0264] Applicant also provides wild-type hTβRII as described above and below. long Based on the (23-184) sequence (SEQ ID NO: 20), five corresponding variants (SEQ ID NOs: 37, 33, 34, 39) are also envisioned, where the 25 amino acid insertion is underlined. Note that splicing results in a conservative amino acid substitution (Val→Ile) at the C-terminal position adjacent to the insertion. [ka]

[0265] (1) hTβRII shown below long (23~184 / D135K) amino acid sequence (SEQ ID NO:37), where the substituted residues are double underlined. [ka]

[0266] (2) N-terminal truncated hTβRII as shown below long (29-184) Amino acid sequence (sequence number 33). [ka]

[0267] (3) N-terminally truncated hTβRII as shown below long (60-184) Amino acid sequence (same as SEQ ID NO:29). [ka]

[0268] (4) C-terminal truncated hTβRII as shown below long(23-178) Amino acid sequence (sequence number 34). [ka]

[0269] (5) C-terminal truncated hTβRII as shown below long (23~178 / N95D) amino acid sequence (SEQ ID NO:39), where the substituted residues are double underlined. [ka] [ka]

[0270] Additional TβRII ECD variants include: (A) N- and C-terminal truncations of hTβRII as shown below. short (35-153) or hTβRII long (60-178) Amino acid sequence (sequence number 32). [ka]

[0271] (B) hTβRII truncated at the N- and C-terminus as shown below. short (29-153) Amino acid sequence (sequence number 31). [ka]

[0272] (C) hTβRII truncated at the N- and C-terminus as shown below. long (29-178) Amino acid sequence (sequence number 35). [ka]

[0273] Any of the above variants (SEQ ID NOs: 36, 28, 29, 30, 38, 37, 33, 34, 39, 32, 31 and 35) could incorporate an insertion of 36 amino acids (SEQ ID NO: 41) between a pair of glutamic acid residues located near the C-terminus of the hTβRII ECD (positions 151 and 152 of SEQ ID NO: 1, or positions 176 and 177 of SEQ ID NO: 2), as naturally occurring in hTβRII isoform C (Konrad et al., BMC Genomics 8:318, 2007). [ka]

[0274] As an example, the pair of glutamic acid residues adjacent to any insertion site can be expressed as short The (29-159) variant (SEQ ID NO: 28) is shown below (underlined). [ka] Fc domain variants

[0275] Although the constructs described above were made with an Fc domain having the amino acid sequence of SEQ ID NO: 49, the present disclosure contemplates hTβRII-hFc fusion proteins containing alternative Fc domains, including a human IgG2 Fc domain (SEQ ID NO: 42, below) or a full-length human IgG1 Fc (hG1Fc) (SEQ ID NO: 43, below). If desired, a polypeptide unrelated to the Fc domain could be attached in place of the Fc domain. [ka] Leader sequence variants

[0276] The constructs made as described above contained the TPA leader sequence, however alternative leader sequences may be used, such as the native leader sequence (SEQ ID NO:22, below) or the leader sequence of honeybee melittin (SEQ ID NO:24, below). [ka] Example 2 Differentiation Ligand Inhibition by Receptor Fusion Protein Variants in Cell-Based Assays

[0277] The affinity of TGFβ1, TGFβ2 and TGFβ3 for hTβRII(G4S)2-hFc, hTβRII(G4S)3-hFc, hTβRII(G4S)4-hFc, hTβRII-hFc and hTβRII extended hinge-hFc proteins was evaluated in vitro by a Biacore™ instrument, and the results are summarized in Figures 4A and 4B. Each fusion protein was capable of binding to TGFβ1 and TGFβ3 with high affinity, but constructs with linkers longer than (G4S)4 or the same length as (G4S)4 were surprisingly capable of binding to both TGFβ1 and TGFβ3 with higher affinity than constructs with linkers shorter than (G4S)4. Binding between TGFβ2 and either construct was low or transient. Deglycosylation of the constructs did not alter binding.

[0278] A reporter gene assay in A549 cells was used to determine the ability of hTβRII-hFc variants to inhibit the activity of TGFβ1, TGFβ2 and TGFβ3. The assay is based on a human lung cancer cell line transfected with the pGL3(CAGA)12 reporter plasmid (Dennler et al, 1998, EMBO 17: 3091-3100) and the Renilla reporter plasmid (pRLCMV) to control for transfection efficiency. The CAGA motif is present in the promoter of TGFβ-responsive genes (e.g., PAI-1), so this vector is commonly used for factors that signal through SMAD2 and SMAD3.

[0279] On the first day of the assay, A549 cells (ATCC®: CCL-185™) were distributed into 48-well plates. On the second day, a solution containing pGL3(CAGA)12, pRLCMV, X-tremeGENE 9 (Roche Applied Science) and OptiMEM (Invitrogen) was preincubated and then added to Eagle's Minimum Essential Medium (EMEM, ATCC®) supplemented with 0.1% BSA, which was applied to the plated cells for incubation overnight at 37° C., 5% CO2. On the third day, the medium was removed and the cells were incubated overnight at 37° C., 5% CO2 with a mixture of ligands and inhibitors prepared as described below.

[0280] Serial dilutions of test substances were performed in 48-well plates in assay buffer (EMEM + 0.1% BSA). An equal volume of assay buffer containing test ligand was added to obtain a final ligand concentration equal to the previously determined EC50. Human TGFβ1, human TGFβ2 and human TGFβ3 were obtained from PeproTech. Test solutions were incubated at 37°C for 30 minutes, and then a portion of the mixture was added to all wells. After overnight incubation with test solutions, cells were rinsed with phosphate-buffered saline, then lysed with passive lysis buffer (Promega E1941) and stored overnight at -70°C. On the fourth and final day, plates were warmed to room temperature with gentle shaking. Cell lysates were transferred in duplicate to chemiluminescence plates (96 wells) and analyzed in a luminometer using reagents from the Dual-Luciferase Reporter assay system (Promega E1980) to determine normalized luciferase activity.

[0281] As shown in Figures 5A-5F, hTβRII(G4S)2-hFc, hTβRII(G4S)3-hFc, hTβRII(G4S)4-hFc, hTβRII(G4S)5-hFc, hTβRII(G4S)6-hFc, hTβRII-hFc and hTβRII extended hinge-hFc proteins were all capable of inhibiting both TGFβ1 and TGFβ3. Interestingly, there was a correlation between improved TGFβ1 and TGFβ3 inhibition and linker length for the hTβRII(G4S)2-hFc, hTβRII(G4S)3-hFc, and hTβRII(G4S)4-hFc constructs (Figure 5E), but this trend for improvement appeared to plateau for the hTβRII(G4S)5-hFc and hTβRII(G4S)6-hFc constructs (Figure 5F). Example 3 Effect of TβRII-Fc in an animal model of heterotopic bone formation

[0282] Heterotopic ossification (HO) can commonly occur after severe trauma, burns, and is a debilitating consequence of the congenital disease fibrodysplasia ossificans progressiva (FOP). The pathogenesis remains poorly understood, however inflammation is presumed to play a key role with several inflammatory cell types being recruited to the site of HO development. The effect of the above-described mTβRII-mFc on the progression of HO was investigated using an HO animal model.

[0283] Recruited macrophages are a known source of cytokines that influence the inflammatory microenvironment. Therefore, we first assessed the secretion of TGFβ1 in cultured macrophages. Bone marrow-derived macrophages were isolated and polarized in vitro to the M2 phenotype. Secreted TGFβ was measured in the conditioned medium using ELISA. Interestingly, while M0 macrophages secrete only minimal TGFβ1, regenerating M2 macrophages, known to be recruited to inflammatory sites such as those occurring at the sites of bone deposition in HO, were observed to have a 500-fold increase in TGFβ1 secretion. Furthermore, the increase in TGFβ1 secretion was completely abolished when mTβRII-mFc (5.44 μg / ml) was present in the culture medium (Figure 6A).

[0284] Next, the effect of systemically administered mTβRII-mFc was observed in a model of traumatic heterotopic ossification, including a 30% dorsal burn and Achilles tenotomy. Agarwal et al. (2016) Proc Natl Acad Sci USA 113(3): E338-347. Six-week-old male C57BL / 6 mice were induced for HO and then randomized into one of two treatment groups: 1) treatment with mTβRII-mFc (10 mg / kg) (n=10) or 2) PBS control (n=10). This was administered subcutaneously twice weekly for three weeks. At week 3, histology samples were collected, decalcified, and stained with Safranin O to assess the formation of HO primordia. The volume of mature HO was quantified at week 9 using micro-CT analysis and image reconstruction. After 3 weeks of treatment, substantially attenuated development of HO primordia in mice treated with mTβRII-mFc was observed during histological examination, along with reduced bone deposition and formation of bone marrow cavities (Figure 6B). Furthermore, the volume of mature HO was significantly reduced in the mTβRII-mFc-treated group (Figure 6C).

[0285] In this study, we demonstrated that TGFβ1 signaling plays a key role in HO formation and that treatment with mTβRII-mFc is effective in attenuating ectopic bone deposition, thus resulting in a reduction in the volume of HO. Thus, these data suggest that TβRII polypeptides and other TGFβ antagonists can be used to treat HO, revealing a new avenue for the treatment of this condition in human patients. Incorporation by Reference

[0286] All publications and patents mentioned in this specification are herein incorporated by reference in their entirety, to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference herein.

[0287] While specific embodiments of the subject matter have been described, the above specification is illustrative and not restrictive. Many modifications will become apparent to those skilled in the art upon review of this specification and the claims that follow. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, together with such variations. The present invention provides, for example, the following items. (Item 1) A method for treating heterotopic ossification (HO), comprising administering to a patient in need thereof an effective amount of a transforming growth factor-β (TGFβ) antagonist. (Item 2) The method according to item 1, wherein said heterotopic ossification is associated with one or more disorders or conditions selected from the group consisting of spinal cord injury, trauma, brain injury, burns, bone fractures, muscle strains, arthroplasty / joint replacement, hip surgery / total hip replacement, acetabular surgery / replacement, elbow fractures, fractures of the long bones of the lower leg, combat-related trauma, limb amputation, neuromuscular blockade used to manage adult respiratory distress syndrome, and non-traumatic myelopathy. (Item 3) A method for treating fibrodysplasia ossificans progressiva (FOP), comprising administering to a patient in need thereof an effective amount of a TGFβ antagonist. (Item 4) A method for treating progressive osseous dysplasia (POH), comprising administering to a patient in need thereof an effective amount of a TGFβ antagonist. (Item 5) A method for treating fibrous dysplasia comprising administering to a patient in need thereof an effective amount of a TGFβ antagonist. (Item 6) The method of any of the preceding items, wherein the method prevents or reduces the severity or duration of one or more complications of HO selected from the group consisting of joint contractures, joint ankylosis, pain, spasticity, swelling fever, neurovascular compression syndromes, and bedsores. (Item 7) The method of any of the preceding items, wherein the severity or duration of heterotopic ossification in said patient is prevented or reduced. (Item 8) The method of any of the preceding items, wherein the ectopic ossification occurs in one or more tissues selected from the group consisting of bone, skin, subcutaneous tissue, skeletal muscle, fibrotic tissue in the vicinity of joints, blood vessel walls and ligaments. (Item 9) The method of any of the preceding items, further comprising administration of an additional active agent or supportive care for treating heterotopic ossification, FOP, POH or fibrous dysplasia. (Item 10) The method of item 10, wherein the additional active agent or supportive therapy for treating heterotopic ossification, FOP, POH or fibrous dysplasia is selected from the group consisting of isotretinoin, etidronate and oral corticosteroids, perhexiline maleate, ALK2 small molecule inhibitors, palovarotene, retinoic acid receptor gamma agonists, retinoic acid receptor alpha agonists, activin antibodies (e.g., activin A antibodies such as REGN 2477), allele-specific RNA interference of ALK2, bisphosphonates, radiation therapy, anti-inflammatory agents (e.g., indomethacin, ibuprofen and aspirin) and conservative treatments such as passive range of motion exercises or other mobilization techniques. (Item 11) The method of item 10, wherein the additional active agent or supportive therapy is treatment with an activin antagonist. (Item 12) The method of claim 11, wherein the activin antagonist is an activin antibody. (Item 13) The method of claim 12, wherein the activin antibody is an activin A antibody. (Item 14) The method of any of the preceding items, wherein the TGFβ antagonist is a transforming growth factor-β receptor II (TβRII) polypeptide. (Item 15) the TβRII polypeptide a. an amino acid sequence that is at least 75% identical to a sequence beginning at any one of positions 23-35 of SEQ ID NO:1 and ending at any one of positions 153-159 of SEQ ID NO:1; and b. an amino acid sequence that is at least 75% identical to a sequence that begins at any one of positions 23 to 60 of SEQ ID NO:2 and ends at any one of positions 178 to 184 of SEQ ID NO:2 15. The method of claim 14, comprising an amino acid sequence selected from the group consisting of: (Item 16) Item 15. The method according to item 14, wherein the TβRII polypeptide comprises an amino acid sequence that is at least 90% identical to a sequence starting at any one of positions 23 to 35 of SEQ ID NO:1 and ending at any one of positions 153 to 159 of SEQ ID NO:1. (Item 17) Item 15. The method according to item 14, wherein the TβRII polypeptide comprises an amino acid sequence that is at least 95% identical to a sequence starting at any one of positions 23 to 35 of SEQ ID NO:1 and ending at any one of positions 153 to 159 of SEQ ID NO:1. (Item 18) Item 15. The method according to item 14, wherein the TβRII polypeptide comprises an amino acid sequence that is at least 99% identical to a sequence starting at any one of positions 23 to 35 of SEQ ID NO:1 and ending at any one of positions 153 to 159 of SEQ ID NO:1. (Item 19) Item 15. The method according to item 14, wherein the TβRII polypeptide comprises an amino acid sequence starting at any one of positions 23 to 35 of SEQ ID NO:1 and ending at any one of positions 153 to 159 of SEQ ID NO:1. (Item 20) Item 15. The method according to item 14, wherein the TβRII polypeptide comprises an amino acid sequence that is at least 90% identical to a sequence starting at any one of positions 23 to 60 of SEQ ID NO:2 and ending at any one of positions 178 to 184 of SEQ ID NO:2. (Item 21) Item 15. The method according to item 14, wherein the TβRII polypeptide comprises an amino acid sequence that is at least 95% identical to a sequence starting at any one of positions 23 to 60 of SEQ ID NO:2 and ending at any one of positions 178 to 184 of SEQ ID NO:2. (Item 22) Item 15. The method according to item 14, wherein the TβRII polypeptide comprises an amino acid sequence that is at least 99% identical to a sequence starting at any one of positions 23 to 60 of SEQ ID NO:2 and ending at any one of positions 178 to 184 of SEQ ID NO:2. (Item 23) Item 15. The method according to item 14, wherein the TβRII polypeptide comprises an amino acid sequence starting at any one of positions 23 to 60 of SEQ ID NO:2 and ending at any one of positions 178 to 184 of SEQ ID NO:2. (Item 24) 15. The method of claim 14, wherein the TβRII polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:18. (Item 25) 15. The method of claim 14, wherein the TβRII polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:18. (Item 26) The method of claim 14, wherein the TβRII polypeptide comprises the amino acid sequence of SEQ ID NO:18. (Item 27) the TβRII polypeptide a. the extracellular domain of the TβRII portion; and b. Different parts a fusion polypeptide comprising the extracellular domain of the TβRII portion being i) a sequence starting at any of positions 23 to 35 of SEQ ID NO:1 and ending at any of positions 153 to 159 of SEQ ID NO:1, or ii) a sequence starting at any one of positions 23 to 60 of SEQ ID NO:2 and ending at any one of positions 178 to 184 of SEQ ID NO:2 comprising an amino acid sequence at least 80% identical to Item 15. The method according to item 14. (Item 28) 28. The method of claim 27, wherein the TβRII polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:18. (Item 29) 28. The method according to item 27, wherein the extracellular domain of the TβRII portion consists of an amino acid sequence that is at least 80% identical to a sequence starting at any one of positions 23 to 35 of SEQ ID NO: 1 and ending at any one of positions 153 to 159 of SEQ ID NO: 1. (Item 30) 28. The method according to item 27, wherein the extracellular domain of the TβRII portion consists of an amino acid sequence that is at least 80% identical to a sequence starting at any one of positions 23 to 60 of SEQ ID NO: 2 and ending at any one of positions 178 to 184 of SEQ ID NO: 2. (Item 31) 28. The method of claim 27, wherein the extracellular domain of the TβRII portion consists of an amino acid sequence that is at least 80% identical to SEQ ID NO:18. (Item 32) 32. The method of any one of items 27 to 31, wherein the heterologous moiety is an immunoglobulin Fc domain. (Item 33) 33. The method of claim 32, wherein the immunoglobulin Fc domain is a human immunoglobulin Fc domain. (Item 34) 33. The method of claim 32, wherein the heterologous portion comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:49. (Item 35) 33. The method of claim 32, wherein the heterologous portion comprises the amino acid sequence of SEQ ID NO:49. (Item 36) 36. The method of any one of items 27 to 35, wherein the fusion protein further comprises a linker domain. (Item 37) 37. The method of claim 36, wherein the linker is between 10 and 25 amino acids in length. (Item 38) The linker is a.(GGGGS) n (In the formula, n=≧2); b.(GGGGS) n (In the formula, n=≧3); c.(GGGGS) n (wherein n=≧4); and d. The method according to item 36, comprising an amino acid sequence selected from the amino acid sequences of any one of SEQ ID NOs: 4 to 7, 19, 21, 25, 26 and 40. (Item 39) The linker is (GGGGS) n 37. The method according to claim 36, comprising: (Item 40) 15. The method of claim 14, wherein the TβRII polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:11. (Item 41) 15. The method of claim 14, wherein the TβRII polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 13, 65 or 68. (Item 42) 15. The method of claim 14, wherein the TβRII polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:15. (Item 43) 43. The method of any one of items 14 to 42, wherein the TβRII polypeptide or fusion polypeptide does not contain amino acids 185 to 592 of SEQ ID NO:2. (Item 44) 44. The method of any one of i...

Claims

1. 1. A composition for treating heterotopic ossification (HO) comprising a transforming growth factor-β (TGFβ) antagonist, The TGFβ antagonist is a transforming growth factor-β receptor II (TβRII) fusion polypeptide comprising: a. a portion of the TβRII extracellular domain, i) a sequence starting at any of positions 23 to 35 of SEQ ID NO:1 and ending at any of positions 153 to 159 of SEQ ID NO:1; or ii) a sequence starting at any one of positions 23 to 60 of SEQ ID NO:2 and ending at any one of positions 178 to 184 of SEQ ID NO:2 comprising an amino acid sequence at least 90% identical to b. a heterologous moiety comprising an immunoglobulin Fc domain, and c. a linker domain having an amino acid sequence of any one of SEQ ID NOs: 4-6, 25, and 26; composition.

2. 2. The composition of claim 1, wherein the heterotopic ossification is associated with one or more disorders or conditions selected from the group consisting of spinal cord injury, trauma, brain injury, burns, bone fractures, muscle strains, arthroplasty / joint replacement, hip surgery / total hip replacement, acetabular surgery / replacement, elbow fractures, fractures of long bones of the lower leg, combat-related trauma, limb amputations, neuromuscular blockade used to manage adult respiratory distress syndrome, and non-traumatic myelopathy.

3. The composition of claim 1 or 2, wherein the ectopic ossification occurs in one or more tissues selected from the group consisting of bone, skin, subcutaneous tissue, skeletal muscle, fibrotic tissue in the vicinity of joints, blood vessel walls and ligaments.

4. Administered in combination with an additional active agent or supportive therapy for treating heterotopic ossification, fibrodysplasia ossificans progressiva (FOP), progressive osseous dysplasia (POH) or fibrous dysplasia; the additional active agent or supportive care is selected from the group consisting of isotretinoin, etidronate and oral corticosteroids, perhexiline maleate, palovarotene, activin antibodies (e.g., activin A antibodies such as REGN 2477), allele-specific RNA interference of ALK2, bisphosphonates, radiation therapy, anti-inflammatory agents (e.g., indomethacin, ibuprofen, and aspirin), and conservative treatments such as passive range of motion exercises or other mobilization techniques; A composition according to any one of claims 1 to 3.

5. wherein the additional active agent or supportive therapy is treatment with an activin antibody. The composition of claim 4.

6. The composition of claim 5 , wherein the activin antibody is an activin A antibody.

7. 7. The composition of any one of claims 1 to 6, wherein the fusion polypeptide comprises an amino acid sequence that is at least 90% identical to a sequence starting at any one of positions 23 to 35 of SEQ ID NO:1 and ending at any one of positions 153 to 159 of SEQ ID NO:

1.

8. 8. The composition of claim 7, wherein the fusion polypeptide comprises an amino acid sequence that is at least 95% identical to a sequence starting at any one of positions 23 to 35 of SEQ ID NO:1 and ending at any one of positions 153 to 159 of SEQ ID NO:

1.

9. 8. The composition of claim 7, wherein the fusion polypeptide comprises an amino acid sequence that is at least 99% identical to a sequence starting at any one of positions 23 to 35 of SEQ ID NO:1 and ending at any one of positions 153 to 159 of SEQ ID NO:

1.

10. 8. The composition of claim 7, wherein the fusion polypeptide comprises an amino acid sequence starting at any one of positions 23 to 35 of SEQ ID NO:1 and ending at any one of positions 153 to 159 of SEQ ID NO:

1.

11. 7. The composition of any one of claims 1 to 6, wherein the fusion polypeptide comprises an amino acid sequence that is at least 90% identical to a sequence starting at any one of positions 23 to 60 of SEQ ID NO:2 and ending at any one of positions 178 to 184 of SEQ ID NO:

2.

12. 12. The composition of claim 11, wherein the fusion polypeptide comprises an amino acid sequence that is at least 95% identical to a sequence starting at any one of positions 23 to 60 of SEQ ID NO:2 and ending at any one of positions 178 to 184 of SEQ ID NO:

2.

13. 12. The composition of claim 11, wherein the fusion polypeptide comprises an amino acid sequence that is at least 99% identical to a sequence starting at any one of positions 23 to 60 of SEQ ID NO:2 and ending at any one of positions 178 to 184 of SEQ ID NO:

2.

14. 12. The composition of claim 11, wherein the fusion polypeptide comprises an amino acid sequence starting at any one of positions 23 to 60 of SEQ ID NO:2 and ending at any one of positions 178 to 184 of SEQ ID NO:

2.

15. The composition of any one of claims 1 to 6, wherein the fusion polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

18.

16. The composition of claim 15, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:

18.

17. The composition of any of claims 1 to 6, wherein the TβRII extracellular domain portion consists of an amino acid sequence that is at least 90% identical to a sequence starting at any of positions 23 to 35 of SEQ ID NO:1 and ending at any of positions 153 to 159 of SEQ ID NO:

1.

18. The composition of any of claims 1 to 6, wherein the TβRII extracellular domain portion consists of an amino acid sequence that is at least 90% identical to a sequence starting at any of positions 23 to 60 of SEQ ID NO:2 and ending at any of positions 178 to 184 of SEQ ID NO:

2.

19. The composition of any one of claims 1 to 6, wherein the TβRII extracellular domain portion consists of an amino acid sequence that is at least 90% identical to SEQ ID NO:

18.

20. 20. The composition of any one of claims 1 to 19, wherein the immunoglobulin Fc domain is a human immunoglobulin Fc domain.

21. 21. The composition of any of claims 1 to 20, wherein the heterologous moiety comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

49.

22. The composition of claim 21 , wherein the heterologous moiety comprises the amino acid sequence of SEQ ID NO:

49.

23. 23. The composition of any preceding claim, wherein the linker domain is between 10 and 25 amino acids in length.

24. The linker domain is (GGGGS) ｎ 24. The composition of claim 23, comprising: wherein n ≠ ≧ 5.

25. The composition of any one of claims 1 to 6, wherein the fusion polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

11.

26. The composition of any one of claims 1 to 6, wherein the fusion polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13, 65 or 68.

27. The composition of any one of claims 1 to 6, wherein the fusion polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

15.

28. 28. The composition of any one of claims 1 to 27, wherein the fusion polypeptide does not include amino acids 185 to 592 of SEQ ID NO:

2.

29. The composition of any one of claims 1 to 28, wherein the fusion polypeptide does not include amino acids 1 to 22 of SEQ ID NO:

2.

30. The fusion polypeptide comprises: a) the extracellular domain of the TβRII portion comprising the amino acid sequence of SEQ ID NO: 18; b) a heterologous portion comprising the amino acid sequence of SEQ ID NO:49; c) a linker domain linking the extracellular domain and the heterologous moiety, the linker domain comprising the amino acid sequence of SEQ ID NO:6; 7. The composition according to any one of claims 1 to 6.

31. The composition of any one of claims 1 to 6, wherein the fusion polypeptide comprises an amino acid sequence that is at least 90%, 95%, 97% or 99% identical to the amino acid sequence of SEQ ID NO:

48.

32. The composition of any one of claims 1 to 6, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:

48.

33. 33. The composition of any one of claims 1 to 32, wherein the fusion polypeptide does not include a leader sequence or the leader sequence has been removed.

34. 34. The composition of any one of claims 1 to 33, wherein the fusion polypeptide comprises one or more modified amino acid residues selected from glycosylated amino acids, pegylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, and amino acids conjugated to a lipid moiety.

35. The composition of claim 34, wherein the fusion polypeptide is glycosylated.

36. 36. The composition of claim 35, wherein the fusion polypeptide has a glycosylation pattern characteristic of expression of a polypeptide in a CHO cell.

37. 37. The composition of any one of claims 1 to 36, wherein the fusion polypeptide binds to TGFβ1 and TGFβ3.

38. 38. The composition of any one of claims 1 to 37, wherein the fusion polypeptide inhibits TGFβ1 and TGFβ3 signaling as determined using a reporter gene assay.

39. 39. The composition of any one of claims 1 to 38, wherein the fusion polypeptide is a homodimer.

40. 1. A combination for treating heterotopic ossification (HO) comprising a transforming growth factor-β (TGFβ) antagonist and an activin antagonist, The TGFβ antagonist is a transforming growth factor-β receptor II (TβRII) fusion polypeptide comprising: a. a portion of the TβRII extracellular domain, i) a sequence starting at any of positions 23 to 35 of SEQ ID NO:1 and ending at any of positions 153 to 159 of SEQ ID NO:1; or ii) a sequence starting at any one of positions 23 to 60 of SEQ ID NO:2 and ending at any one of positions 178 to 184 of SEQ ID NO:2 and b. a heterologous moiety comprising an immunoglobulin Fc domain; wherein the fusion polypeptide comprises a linker domain comprising the amino acid sequence of any one of SEQ ID NOs: 4-6, 25 and 26; The activin antagonist is an activin antibody. Combination.

41. 41. A combination according to claim 40, wherein the TGFβ antagonist corresponds to any one of the TGFβ antagonists defined in claims 7 to 39.

42. The combination of claim 40 or 41, wherein the activin antibody inhibits activin A.

43. 43. The combination of any one of claims 40 to 42, wherein the activin antibody binds to activin A.

44. A composition according to any one of claims 1 to 39 or a combination according to any one of claims 40 to 43, wherein the TGFβ antagonist is a heteromultimer comprising a TβRII polypeptide and an ALK5 polypeptide.

45. A composition described in any one of claims 1 to 39 or a combination described in any one of claims 40 to 44, wherein the TGFβ antagonist is a heteromultimer comprising a TβRII polypeptide and a betaglycan polypeptide.

46. A composition described in any one of claims 1 to 39 or a combination described in any one of claims 40 to 45, wherein the TGFβ antagonist is a heteromultimer comprising an ALK5 polypeptide and a betaglycan polypeptide.

47. The composition or combination of claim 45 or 46, wherein the betaglycan polypeptide comprises an amino acid sequence that is at least 90%, 91%, 92%, 94%, 95%, 97%, 99% or 100% identical to SEQ ID NO: 51 or 55, or a biologically active fragment thereof.

48. The composition or combination of claim 44 or 46, wherein the ALK5 polypeptide comprises an amino acid sequence that is at least 90%, 91%, 92%, 94%, 95%, 97%, 99% or 100% identical to SEQ ID NO: 70 or 74, or a biologically active fragment thereof.

49. 47. The composition or combination of any one of claims 44 to 46, wherein the heteromultimer is a heterodimer.

50. A composition for treating heterotopic ossification (HO) comprising a transforming growth factor-β (TGFβ) antagonist, wherein the composition is administered in combination with an activin antagonist; The TGFβ antagonist is a transforming growth factor-β receptor II (TβRII) fusion polypeptide comprising: a. a portion of the TβRII extracellular domain, i) a sequence starting at any of positions 23 to 35 of SEQ ID NO:1 and ending at any of positions 153 to 159 of SEQ ID NO:1; or ii) a sequence starting at any one of positions 23 to 60 of SEQ ID NO:2 and ending at any one of positions 178 to 184 of SEQ ID NO:2 and b. a heterologous moiety comprising an immunoglobulin Fc domain; wherein the fusion polypeptide comprises a linker domain comprising the amino acid sequence of any one of SEQ ID NOs: 4-6, 25 and 26; The activin antagonist is an activin antibody. composition.

51. A composition for treating heterotopic ossification (HO) comprising an activin antagonist, wherein the composition is administered in combination with a transforming growth factor-β (TGFβ) antagonist; The TGFβ antagonist is a transforming growth factor-β receptor II (TβRII) fusion polypeptide comprising: a. a portion of the TβRII extracellular domain, i) a sequence starting at any of positions 23 to 35 of SEQ ID NO:1 and ending at any of positions 153 to 159 of SEQ ID NO:1; or ii) comprises an amino acid sequence that is at least 90% identical to a sequence beginning at any of positions 23-60 of SEQ ID NO:2 and ending at any of positions 178-184 of SEQ ID NO:2; and b. a heterologous moiety comprising an immunoglobulin Fc domain; wherein the fusion polypeptide comprises a linker domain comprising the amino acid sequence of any one of SEQ ID NOs: 4-6, 25 and 26; The activin antagonist is an activin antibody. composition.

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