HK1193035B - Activin-actriia antagonists and uses thereof for promoting bone growth - Google Patents

Description

Activin-ActRIIa antagonists and their use in promoting bone growth

This application is a divisional application of the invention patent application with application number 200680051538.9 and application date of November 22, 2006.

Related applications

This application claims the benefit of priority to Provisional Application No. 60/739,462 filed on November 23, 2005, Provisional Application No. 60/783,322 filed on March 17, 2006, and Provisional Application No. 60/844,855 filed on September 15, 2006, all of which are incorporated herein by reference in their entireties.

Background Art

Bone diseases, ranging from osteoporosis to fractures, represent a group of pathological conditions for which few effective pharmaceutical agents are available. Instead, treatment focuses on physical and behavioral interventions, including strengthening, exercise, and dietary changes. For the treatment of various bone diseases, therapeutic agents that promote bone growth and increase bone density would be beneficial.

Bone growth and mineralization depend on the activity of two cell types, osteoblasts and osteoclasts, although chondrocytes and vascular cells also participate in key aspects of these processes. Developmentally, bone formation occurs through two mechanisms, endochondral ossification, which is responsible for longitudinal bone formation, and intramembranous ossification, which is responsible for the formation of topologically flat bones such as the bones of the skull. Endochondral ossification requires the sequential formation and degradation of cartilage structures within the growth plate, which serves as a template for the formation of osteoblasts, osteoclasts, vascular cells, and subsequent mineralization. During intramembranous ossification, bone is formed directly in the connective tissue. Both processes require the infiltration of osteoblasts and subsequent matrix deposition.

Fractures and other structural skeletal disruptions heal through a developmental sequence that at least superficially resembles osteogenesis, which involves the formation of cartilage tissue and subsequent mineralization. The fracture healing process can occur in two ways. Direct or primary bone healing occurs without callus formation. Indirect or secondary bone healing occurs with a callus precursor phase. Primary fracture healing involves the remodeling of a mechanical connection across the closely spaced rupture. Under appropriate conditions, resorbing cells surrounding the ruptured bone exhibit a tunneling resorption response and establish pathways for vascularization and subsequent healing. Secondary bone healing proceeds in a sequence of inflammation, soft callus formation, callus mineralization, and callus remodeling. During the inflammatory phase, hematoma and hemorrhage form due to the disruption of periosteum and endosteal blood vessels at the site of injury. Inflammatory cells invade the area. During the soft callus formation phase, cells generate new blood vessels, fibroblasts, intracellular material, and supporting cells, forming granulation tissue in the gap between the fracture fragments. Clinical healing across the rupture is established by the formation of fibrous or cartilaginous tissue (soft callus). Osteoblasts form and regulate the mineralization of the soft callus, which is then replaced by lamellar bone and subject to normal remodeling processes.

In addition to fractures and other physical disruptions to the skeletal structure, loss of bone mineral content and bone density can be caused by a variety of different conditions and can lead to significant medical problems. Changes in bone density occur in a relatively predictable manner throughout an individual's lifespan. By approximately age 30, the skeleton of both men and women reaches its maximum mass through linear and radial growth of endochondral bone growth discs. Slow bone loss occurs in both men and women around age 30 (for cancellous bone, e.g., flat bones such as the vertebrae and pelvis) and age 40 (for cortical bone, e.g., long bones in the limbs). In women, there is also a final phase of substantial bone loss, perhaps due to a lack of estrogen after menopause. During this period, women may lose an additional 10% of mass in cortical bone and an additional 25% of mass in cancellous septa. Whether this progressive loss of bone mass leads to a pathological condition such as osteoporosis depends heavily on the individual's initial bone mass and whether there are aggravating conditions.

Bone loss is sometimes characterized by an imbalance in the normal bone remodeling process. Healthy bones are often limited by remodeling. Remodeling begins with the absorption of bone by osteoclasts. The absorbed bone is then replaced by new bone tissue, which is characterized by the formation of collagen by osteoblasts and subsequent calcification. In healthy individuals, the rates of absorption and formation are balanced. Osteoporosis is a chronic, progressive condition marked by a shift towards absorption, resulting in an overall reduction in bone mass and bone mineralization. Osteoporosis in the human body precedes clinical osteopenia (bone mineral density is greater than one standard deviation but less than two standard deviations below the mean for young adults). Worldwide, approximately 750 million people are at risk of osteoporosis.

Therefore, methods of controlling osteoclast and osteoblast activity are useful for promoting the healing of fractures and other injuries to the skeleton, as well as for the treatment of diseases such as osteoporosis, which is associated with loss of bone mass and mineralization.

Regarding osteoporosis, estrogen, calcitonin, osteocalcin and vitamin K, or high-dose dietary calcium are used as interventional treatments. Other osteoporosis treatment approaches include bisphosphonates, parathyroid hormone, calcimimetic agents, statins, salts of lanthanum and strontium, and sodium fluoride. However, these treatments are often associated with adverse side effects.

It is therefore an object of the present invention to provide compositions and methods for promoting bone growth and mineralization.

Summary of the Invention

SUMMARY OF THE INVENTION

In part, the present invention demonstrates that molecules with activin or activin type IIA receptor (ActRIIa) antagonist activity ("activin antagonists" and "ActRIIa antagonists") can be used to increase bone density, promote bone growth, and/or increase bone strength. In particular, the present invention demonstrates that soluble forms of ActRIIa act as inhibitors of activin-ActRIIa signaling and promote increased bone density, bone growth, and bone strength in vivo. However, the vast majority of pharmaceutical agents that promote bone growth or inhibit bone loss act either as anti-catabolic agents (also often referred to as "catabolic agents") (e.g., bisphosphonates) or as anabolic agents (e.g., parathyroid hormone, PTH, when dosed appropriately), and soluble ActRIIa proteins exhibit dual activity, having both catabolic and anabolic effects. Therefore, the present invention establishes that antagonists of the activin-ActRIIa signaling pathway can be used to increase bone density and promote bone growth. While soluble ActRIIa may affect bone through mechanisms other than activin antagonism, the present invention nonetheless demonstrates that desirable therapeutic agents can be selected based on the activity of activin-ActRIIa antagonists. Thus, in certain embodiments, the present invention provides methods for treating diseases associated with low bone density or low bone strength (e.g., osteoporosis) or promoting bone growth in patients in need thereof (e.g., in patients with fractures) using activin-ActRIIa antagonists, including, for example, activin-binding ActRIIa polypeptides, anti-activin antibodies, anti-ActRIIa antibodies, activin-targeting or ActRIIa-targeting small molecules and nucleic acid aptamers, and nucleic acids that reduce activin and ActRIIa expression. Furthermore, soluble ActRIIa polypeptides promote bone growth without causing a sustained, measurable increase in muscle mass.

In certain aspects, the present invention provides polypeptides comprising a soluble activin-binding ActRIIa polypeptide that binds to activin. The ActRIIa polypeptide can be formulated into a pharmaceutical formulation comprising activin-binding ActRIIa and a pharmaceutically acceptable carrier. Preferably, the activin-binding ActRIIa polypeptide binds to activin with a _KD of less than 1 micromolar, or less than 100, 10, or 1 nanomolar. Optionally, the activin-binding ActRIIa selectively binds to activin relative to GDF11 and/or GDF8, preferably with a _KD of at least 10-fold, 20-fold, or 50-fold lower than _that of GDF11 and/or GDF8. While not wishing to be bound by a particular mechanism of action, it is anticipated that the selectivity of activin inhibition over GDF11/GDF8 indicates that selective effects on bone do not result in sustained, measurable effects on muscle. In some embodiments, a dose of ActRIIa that induces less than 15%, less than 10%, or less than 5% muscle growth is selected to achieve the desired effect on bone. Preferably, the composition is at least 95% pure, as determined by size exclusion chromatography with respect to the other polypeptide components, and more preferably, the composition is at least 98% pure. The activin-binding ActRIIa polypeptide used in such a formulation can be any polypeptide disclosed herein, such as a polypeptide having an amino acid sequence selected from SEQ ID Nos: 2, 3, 7, 12, or 13, or a polypeptide having at least 80%, 85%, 90%, 95%, 97%, or 99% identity to an amino acid sequence selected from SEQ ID Nos: 2, 3, 7, or 12. The activin-binding ActRIIa polypeptide can include a functional fragment of native ActRIIa, such as a fragment comprising at least 10, 20, or 30 amino acids ("tail") of a sequence selected from SEQ ID Nos: 1-3 or SEQ ID Nos: 2 (lacking the C-terminal 10 to 15 amino acids).

Soluble, activin-binding ActRIIa polypeptides can include one or more amino acid sequence alterations (e.g., in the ligand binding domain) relative to a naturally occurring ActRIIa polypeptide. Examples of altered ActRIIa polypeptides are provided at pages 59-60 of WO2006/012627, incorporated herein by reference. Amino acid sequence alterations can, for example, alter the glycosylation of the polypeptide when produced in mammalian, insect, or other eukaryotic cells, or alter proteolytic cleavage of the polypeptide relative to a naturally occurring ActRIIa polypeptide.

Activin-binding ActRIIa polypeptides can be fusion proteins comprising one domain of an ActRIIa polypeptide (e.g., a ligand-binding portion of an ActRIIa polypeptide) and one or more additional domains that provide desirable properties (e.g., improved pharmacokinetics, easier purification, targeting to specific tissues, etc.). For example, the domains of the fusion protein can enhance one or more of in vivo stability, in vivo half-life, absorption/administration, tissue localization or distribution, formation of protein complexes, multimerization of the fusion protein, and/or purification. Activin-binding ActRIIa fusion proteins can include an immunoglobulin Fc domain (wild-type or mutant) or serum albumin or other polypeptide portion that provides desirable properties (e.g., improved pharmacokinetics, improved solubility, or improved stability). In a preferred embodiment, the ActRIIa-Fc fusion includes a relatively unstructured linker positioned between the Fc domain and the ActRIIa extracellular domain. This unstructured sequence keyword can correspond to the approximately 15 amino acid unstructured region at the C-terminal end ("tail") of the ActRIIa extracellular domain, or it can be an artificial sequence of 1, 2, 3, 4, or 5 amino acids, or a length of 5 to 15, 20, 30, 50, or more amino acids that is relatively free of secondary structure, or a mixture of both. The linker can be rich in glycine and proline residues and can, for example, contain a simple sequence of threonine/serine and glycine or a repeating sequence of threonine/serine and glycine (e.g., TG ₄ or SG ₄ singlet or repeat sequences). Fusion proteins can include purification subsequences such as epitope tags, FLAG tags, polyhistidine sequences, and GST fusions. Optionally, the soluble activin-binding type IIA receptor polypeptide includes one or more modified amino acid residues selected from the group consisting of a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid coupled to a liposomal moiety, and an amino acid coupled to an organic derivative. The pharmaceutical formulation may also include one or more additional compounds, such as compounds for treating bone disorders. Preferably, the pharmaceutical formulation is substantially non-pyrogenic. Generally, it is preferred that the ActRIIa protein be expressed in a mammalian cell line that modulates the native glycosylation of the ActRIIa protein appropriately to eliminate the possibility of an adverse immune response in the patient. Human cell lines and CHO cell lines have been successfully used, and it is contemplated that other common mammalian expression systems will also be useful.

As described herein, the ActRIIa protein, designated ActRIIa-Fc, possesses desirable properties, including selective binding to activin relative to GDF8 and/or GDF11, high affinity ligand binding, and a serum half-life of greater than two weeks in animal models. In certain embodiments, the present invention provides ActRIIa-Fc polypeptides and pharmaceutical formulations comprising such polypeptides and a pharmaceutically acceptable excipient.

In certain aspects, the present invention provides nucleic acids encoding soluble activin-binding ActRIIa polypeptides. The isolated polynucleotides may comprise a coding sequence for a soluble activin-binding ActRIIa polypeptide such as described above. For example, the isolated nucleic acid may comprise a coding sequence for the extracellular domain of ActRIIa (e.g., a ligand binding domain), and a sequence encoding part or all of the transmembrane domain and/or the cytoplasmic domain of ActRIIa, but not a stop codon located in the transmembrane domain or in the cytoplasmic domain or between the extracellular domain and the transmembrane domain or the cytoplasmic domain. For example, the isolated polynucleotide may comprise a full-length ActRIIa polynucleotide sequence such as SEQ ID NO: 4 or 5, or a partially truncated sequence, the polynucleotide further comprising a transcription stop codon at least 600 nucleotides prior to the 3' end or other position such that translation of the polynucleotide results in random fusion of the extracellular domain to the partially truncated full-length ActRIIa. The preferred nucleic acid sequence is SEQ ID NO: 14. The nucleic acid disclosed in the present invention is operably linked to a promoter for expression, and the present invention provides cells transformed with such recombinant polynucleotides. Preferably, the cells are mammalian cells, such as CHO cells.

In certain aspects, the present invention provides methods for producing soluble activin-binding ActRIIa polypeptides. Such methods can include expressing any of the nucleic acids disclosed herein (e.g., SEQ ID NOs: 4, 5, or 14) in suitable cells, such as Chinese hamster ovary (CHO) cells. Such methods can include: a) culturing the cells under suitable conditions to express the soluble ActRIIa polypeptide, wherein the cells are transformed with an expression construct for soluble ActRIIa; and b) renaturing the expressed soluble ActRIIa polypeptide. The soluble ActRIIa polypeptide can be renatured as a native, partially purified, or highly purified fragment. Purification can be achieved by a series of purification steps, including, for example, one, two, three, or more of the following, in any order: protein A chromatography, anion exchange chromatography (e.g., Q Sepharose), hydrophobic chromatography (e.g., phenyl Sepharose), size exclusion chromatography, and cation exchange chromatography.

In certain aspects, the disclosed activin-ActRIIa antagonists, such as soluble activin-binding ActRIIa polypeptides, can be used in a method for promoting bone growth or increasing bone density. In certain embodiments, the present invention provides methods for treating diseases associated with low bone density or promoting bone growth in patients in need thereof. The methods can comprise administering an effective dose of an activin-ActRIIa antagonist to a subject in need thereof. In certain aspects, the present invention provides methods for preparing medicaments using the activin-ActRIIa antagonists for treating the diseases or conditions described above.

In certain aspects, the present invention provides methods for identifying a drug that stimulates bone growth or increases bone mineralization, comprising: a) identifying a test drug that binds to the ligand binding domain of an activin or ActRIIa polypeptide; and b) evaluating the effect of the drug on bone growth or mineralization.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the purification of ActRIIa-hFc expressed in CHO cells. The purified protein is a single peak with a well-shaped peak.

Figure 2 shows ActRIIa-hFc binding to activin and GDF11, detected by BiaCore analysis.

Figure 3 shows a schematic diagram of the A-204 reporter gene assay. The figure shows the reporter vector: pGL3(CAGA)12 (described in EMBO 17:3091-3100, Dennler et al., 1998). The CAGA12 motif appears in the TGF-Beta responsive gene (PAI-1 gene), so this vector is commonly used for factors that signal through the Smad2 and 3 pathways.

Figure 4 shows the effects of ActRIIa-hFc (diamonds) and ActRIIa-mFc (squares) on GDF-8 signaling in an A-204 reporter gene assay. Both proteins demonstrated near-complete inhibition of GDF-8-mediated signaling at picomolar concentrations.

Figure 5 shows the effect of three different ActRIIa-hFc formulations on GDF-11 signaling in an A-204 reporter gene assay.

Figure 6 shows examples of DEXA (dual-energy X-ray absorptiometry) images of control and ActRIIa-mFc-treated BALB/c mice before (top) and after (bottom) a 12-week treatment period. The light-colored areas show increased bone density.

Figure 7 shows quantification of the effect of ActRIIa-mFc on bone mineral density in BALB/c mice during 12 weeks of treatment. Treatments were divided into control (diamonds), 2 mg/kg ActRIIa-mFc (squares), 6 mg/kg ActRIIa-mFc (triangles), and 10 mg/kg ActRIIa-mFc (circles).

Figure 8 shows quantification of the effect of ActRIIa-mFc on bone mineral content in BALB/c mice during 12 weeks of treatment. Treatments were divided into control (diamonds), 2 mg/kg ActRIIa-mFc (squares), 6 mg/kg ActRIIa-mFc (triangles), and 10 mg/kg ActRIIa-mFc (circles).

Figure 9 shows the quantification of the effect of ActRIIa-mFc on cancellous bone mineral density in C57BL6 mice that were ovariectomized (OVX) or sham-operated for more than 6 weeks. Treatments were either control (PBS) or a 10 mg/kg dose of ActRIIa-mFc (ActRIIa).

Figure 10 shows quantification of the effect of ActRIIa-mFc on cancellous bone during 12 weeks of treatment in ovariectomized (OVX) C57BL6 mice. Treatments were either control (PBS, light bars) or 10 mg/kg dose of ActRIIa-mFc (ActRIIa, dark bars).

Figure 11 shows quantification of the effect of ActRIIa-mFc on cancellous bone in sham-operated C57BL6 mice after a 6- or 12-week treatment period. Treatments were either control (PBS, light bars) or 10 mg/kg dose of ActRIIa-mFc (ActRIIa, dark bars).

Figure 12 shows the results of pQCT analysis of bone density in ovariectomized (OVX) mice during 12 weeks of treatment. Treatment was divided into control (PBS, light bars) or 10 mg/kg dose of ActRIIa-mFc (ActRIIa, dark bars). Y axis: mg/ccm

Figure 13 depicts pQCT analysis of bone density in sham-operated mice during 12 weeks of treatment. Treatment was divided into control (PBS, light bars) or 10 mg/kg dose of ActRIIa-mFc (ActRIIa, dark bars). Y axis: mg/ccm

Figures 14A and 14B show whole body DEXA analysis (A) and femur ex vivo analysis (B) after a 12-week treatment period. The bright areas depict areas of high bone density.

Figure 15 shows ex vivo pQCT analysis of the mid-femur after a 12-week treatment period. Treatments were divided into control (PBS, dark bars) and ActRIIa-mFc (light bars). The four bars on the left show total bone density and the four bars on the right show cortical bone density. The first pair of each four bars represents data from ovariectomized mice and the second pair represents data from sham-operated mice.

Figure 16 shows ex vivo pQCT analysis of mid-femurs and bone content after a 12-week treatment period. Treatments were divided into vehicle control (PBS, dark bars) or ActRIIa-mFc (light bars). The four bars on the left show total bone content and the four bars on the right show cortical bone content. The first pair of each four bars represents data from ovariectomized mice and the second pair represents data from sham-operated mice.

Figure 17 shows ex vivo pQCT analysis of the mid-femur and femoral cortical thickness. Treatments were divided into control (PBS, dark bars) or ActRIIa-mFc (light bars). The four bars on the left show the endosteal circumference and the four bars on the right show the periosteal circumference. The first pair of each four bars represents data from ovariectomized mice and the second pair represents data from sham-operated mice.

Figure 18 depicts femoral mechanical testing results after a 12-week treatment period. Treatments were either control (PBS, dark bars) or ActRIIa-mFc (light bars). The left two bars represent data from ovariectomized mice, while the bottom two bars represent data from sham-operated mice.

FIG19 shows the effect of ActRIIa-mFc on cancellous bone volume.

FIG20 shows the effect of ActRIIa-mFc on cancellous bone structure in the distal femur.

Figure 21 shows the effects of ActRIIa-mFc on cortical bone.

Figure 22 shows the effect of ActRIIa-mFc on bone mechanical strength.

Figure 23 shows the effects of different doses of ActRIIa-mFc on bone properties at three different doses.

Figure 24 shows histomorphometry demonstrating that ActRIIa-mFc has dual anabolic and antiresorptive activities.

Detailed Description of the Invention

1. Overview

The transforming growth factor beta (TGF-beta) superfamily comprises a variety of growth factors that share common sequence units and structural motifs. These proteins are known to exert biological functions on many cell types in both vertebrates and invertebrates. Members of the superfamily perform important functions in pattern formation and tissue differentiation during embryonic development and can influence a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis, and epithelial differentiation. This family is divided into two general branches: the BMP/GDF and the TGF-beta/Activin/BMP10 branches, whose members have different, often complementary effects. Manipulation of the activity of TGF-beta family members can often lead to important physiological changes in organisms. For example, Piedmontese and Belgian Blue cattle breeds carry loss-of-function mutations in the GDF8 (also called myostatin) gene, which results in a significant increase in muscle mass. Grobet et al., Nat Genet. 1997, 17(1): 71-4. Furthermore, in humans, inactivation of the GDF8 allele is associated with increased muscle mass and (allegedly) exceptional strength. Schuelke et al., N Engl J Med 2004, 350: 2682-8.

Activin is a dimeric polypeptide growth factor belonging to the TGF-beta superfamily. There are three basic forms of activin (A, B, and AB), which are homodimers of two closely related _β subunits _{( βAβA} , _βBβB , _{and βAβB} ). The human genome also encodes activin C and activin E, which are primarily expressed in the liver. Within the TGF-beta superfamily, activins are unique and multifunctional factors that can stimulate hormone production in ovarian and placental cells, support neural cell survival, positively or negatively influence cell cycle progression depending on the cell type, and induce mesodermal differentiation, at least in amphibian embryos (DePaolo et al., 1991, Proc Soc Ep Biol Med. 198:500-512; Dyson et al., 1997, Curr Biol. 7:81-84; Woodruff, 1998, Biochem Pharmacol. 55:953-963). In addition, erythroid differentiation factor (EDF) isolated from stimulated human monocytic leukemia cells was found to be identical to activin A (Murata et al., 1988, PNAS, 85:2434). Activin A has been shown to act as a natural positive regulator of erythropoiesis in the bone marrow cavity. In some tissues, activin signaling is antagonized by its related heterodimer, inhibin. For example, when the pituitary gland releases follicle-stimulating hormone (FSH), activin promotes FSH secretion and synthesis, while inhibin blocks FSH secretion and synthesis. Other proteins that can regulate the biological activity of activin and/or bind to activin include follistatin (FS), follistatin-related protein (FSRP), _α2 -macroglobulin, Cerberus, and endoglin.

TGF-β signaling is mediated by a heteromeric complex of type I and type II serine/threonine kinase receptors, which phosphorylate and activate downstream Smad proteins upon ligand activation (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 is essential for signaling; type II is required for ligand binding and expression of type I receptors. Type I and type II activin receptors form a stable complex upon ligand binding, resulting in phosphorylation of type I receptors by type II receptors.

Two related type II receptors, ActRIIa and ActRIIb, have been identified as type II receptors for activin (Mathews and Vale, 1991, Cell 65:973-982; Attisano et al., 1992, Cell 68:97-108). In addition to activin, ActRIIa and ActRIIb can biochemically interact with several other TGF-β family proteins, including BMP7, Nodal, GDF8, and GDF11 (Yamashita et al., 1995, J. Cell Biol. 130:217-226; Lee and McPherron, 2001, Proc. Natl. Acad. Sci. 98:9306-9311; Yeo and Whitman, 2001, Mol. Cell 7:949-957; Oh et al., 2002, Genes Dev. 16:2749-54). ALK4 is the major activin type I receptor, particularly for activin A, and ALK-7 can also serve as a receptor for activins, particularly for activin B.

As demonstrated herein, soluble ActRIIa polypeptides (sActRIIa), which exhibit substantial preferential binding to activin A relative to other TGF-beta family members such as GDF8 or GDF11, effectively promote bone growth and increase bone density in vivo. While not wishing to be bound by any particular mechanism, given the very strong binding to activin exhibited by the specific sActRIIa constructs used in these studies (picomolar dissociation constant), it is expected that the effects of sActRIIa are primarily due to the effects of an activin antagonist. Regardless of mechanism, the data presented here clearly demonstrate that ActRIIa-activin antagonists do increase bone density in normal mice and in mouse models of osteoporosis. It is important to note that bone is a dynamic tissue, and its growth or shrinkage, as well as its increase or decrease in density, depends on a balance between factors that produce bone and stimulate mineralization (primarily osteoblasts) and factors that destroy and remove bone mineral (primarily osteoclasts). Bone growth and mineralization can be increased by increasing productive factors, decreasing destructive factors, or both. The terms "promote bone growth" and "increase bone mineralization" refer to physically observable changes in bone and are used to neutrally describe the mechanism by which the changes in bone occur.

The mouse models of osteoporosis and bone growth/density used in the studies described herein are believed to be highly predictive of efficacy in humans. Thus, the present invention provides methods for promoting bone growth and increasing bone density in humans using ActRIIa polypeptides and other activin-ActRIIa antagonists. Activin-ActRIIa antagonists include, for example, activin-binding soluble ActRIIa polypeptides, antibodies that bind to activin (particularly activin A or B subunits, also referred to as βA or βB) and disrupt ActRIIa binding, antibodies that bind to ActRIIa and disrupt activin binding, non-antibody proteins selected from those that bind to activin or ActRIIa (see, e.g., WO/2002/088171, WO/2006/055689, WO/2002/032925, WO/2005/037989, US2003/0133939, and US2005/0238646 for examples of such proteins and methods for designing and selecting the same), random peptides selected from those that bind to activin or ActRIIa, typically conjugated to an Fc domain. Two different proteins (or other moieties) with activin or ActRIIa binding activity, particularly activin binders that block type I (e.g., soluble type I activin receptor) or type II (e.g., soluble type II activin receptor) binding sites, respectively, can be linked to produce bifunctional binding molecules. Aptamers, small molecules, and other agents inhibit the activin-ActRIIa signaling axis. A variety of proteins have activin-ActRIIa antagonist activity, including inhibin (i.e., inhibin α subunit) (although inhibin does not antagonize activin in all tissues), follistatin (e.g., follistatin 288 and follistatin 315), Cerberus, follistatin-related protein (FSRP), endoglin, activin C, _α2 -macroglobulin, and the M108A (methionine to alanine at position 108) mutant activin A. Generally, alternative forms of activin, particularly those with variations in the type I receptor binding domain, can bind to the type II receptor and fail to form an active ternary complex, thereby acting as antagonists. In addition, nucleic acids, such as antisense molecules, siRNAs or ribozymes that inhibit the expression of activin A, B, C or E or (particularly) ActRIIa, can be used as activin-ActRIIa antagonists. Preferably, the activin-ActRIIa antagonist employed will exhibit selectivity for inhibiting activin-mediated signals relative to other members of the TGF-beta family (relative to GDF8 and GDF11). Soluble ActRIIb binds to activin, however, the wild-type protein does not exhibit significant selectivity for binding to activin relative to GDF8/11, and preliminary experiments have shown that this does not provide the desired effect on bone, but can still induce significant muscle growth. However, altered forms of ActRIIb with different binding properties have been identified (see, e.g., WO 2006/012627, pp. 55-59, incorporated herein by reference), and these proteins may have desirable effects on bone. Native or altered ActRIIb may provide additional specificity for activin by coupling a second activin-selective binding component.

The terms used in this specification generally have their ordinary meanings in the art within the context of the present invention and the specific context in which each term is used. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to the practitioner in describing the compositions and methods of the present invention and how to use them. The scope or meaning of any use of a term will be clear from the specific context in which it is used.

"About" and "approximately" generally mean an acceptable degree of error in physical measurements, taking into account the nature and precision of the measurement method. Typically, exemplary degrees of error are within 20% of a given value or range of values, preferably within 10%, and more preferably within 5%.

Alternatively, particularly in biological systems, the terms "about" and "approximately" may mean values within a certain order of magnitude, preferably within 5 times, and more preferably within 2 times of a given value. Unless otherwise indicated, the values given herein are approximate, meaning that the terms "about" or "approximately" can be inferred when not expressly specified.

The methods of the present invention may include the step of comparing sequences to each other, including comparing a wild-type sequence to one or more mutants (sequence variants). Such comparisons typically include alignment of polymer sequences, for example, using sequence alignment programs and/or algorithms known in the art (e.g., BLAST, FASTA, and MEGALIGN, to name a few). One skilled in the art will readily appreciate that in such an alignment, in a mutation comprising an insertion or deletion of a residue, a "gap" (typically indicated by a dash or "A") will be generated in the polymer sequence that does not contain the inserted or deleted residue in the aligned sequences.

"Homologous," in all its grammatical forms and spelling variations, refers to the relationship between two proteins that have a "common evolutionary origin," including proteins from superfamilies of the same organism, as well as homologous proteins from organisms of different species. Such proteins (and their encoding nucleic acids) have homologous sequences, as reflected by their sequence similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions.

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 have a common evolutionary origin.

However, in common usage and instant application, the term "homologous", when modified by an adverb such as "highly", may refer to sequence similarity and may or may not refer to a common evolutionary origin.

2. ActRIIa peptide

In certain aspects, the present invention relates to ActRIIa polypeptides. As used herein, the term "ActRIIa" refers to the activin type IIa receptor family from any species, as well as ActRIIa protein variants derived through mutation or other modification. The ActRIIa of the present invention is understood to include any currently identified form. Members of the ActRIIa family are typically transmembrane proteins consisting of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine kinase activity.

The term "ActRIIa polypeptide" includes any naturally occurring ActRIIa family member and any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain useful activity. For example, ActRIIa polypeptides include polypeptides derived from any known ActRIIa sequence that has at least about 80% identity to an ActRIIa polypeptide, preferably at least 85%, 90%, 95%, 97%, 99% or more identity. For example, the ActRIIa polypeptides of the present invention can bind to ActRIIa protein and/or activin and inhibit their function. Preferably, the ActRIIa polypeptide promotes bone growth and bone mineralization. Examples of ActRIIa polypeptides include human ActRIIa precursor polypeptide (SEQ ID NO: 1) and soluble human ActRIIa polypeptides (e.g., SEQ ID NOs: 2, 3, 7, and 12).

The human ActRIIa precursor polypeptide sequence is as follows:

MGAAAKLAFAVFLISCSSGA ILGRSETQECLFFNANWEKDRTQTGVEPCYGDKDKRRHCFATWKISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPYYNILLYSLVPLMLIAGIVICAFWVY RHHKMAYPPVLVPTQDPGPPPPSPLLGLKPLQLLEVKARGRFGCVWKAQLLNEYVAVKIFPIQDKQSWQNEYEVYSLPGMKHENILQFIGAEKRGTSVDVDLWLITAFHEKGSLSDFLKANVVSWNELCHIAETMARGLA YLHEDIPGLKDGHKPAISHRDIKSKNVLLKNNLTACIADFGLALKFEAGKSAGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDM

YAMGLVLWELASRCTAADGPVDEYMLPFEEEIGQHPSLEDMQE

VVVHKKKRPVLRDYWQKHAGMAMLCETIEEECWDHDAEARLSAG CVGERITQMQRLTNIITTEDIVTVVTMVTNVDFPPKESSL(SEQ ID NO: 1)

The signal peptide is single underlined; the extracellular domain is in bold, and potential N-linked glycosylation sites are double underlined.

The soluble (extracellular), processed polypeptide sequence of human ActRIIa is as follows:

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM EVTQPTSNPVTPKPP (SEQ ID NO: 2)

The C-terminal "tail" of the extracellular domain is underlined. The sequence without the "tail" (Δ15 sequence) is as follows:

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM(SEQ ID NO: 3)

The nucleic acid sequence encoding the human ActRIIa precursor polypeptide protein is as follows (nucleotides 164-1705 of Genebank accession number 001616):

ATGGGAGCTGCTGCAAAGTTGGCGTTTGCCGTCTTTCTTATCTCCTGTTCTT

CAGGTGCTATACTTGGTAGATCAGAAAACTCAGGAGTGTCTTTTCTTTAATGC

TAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGT

GACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTT

CCATTGAAATAGTGA′A′ACAAGGTTGTTGGCTGGATGAT′ATCAACTGCTATGA

CAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTTTGTTGC

TGTGAGGGCAATATGTGTAATGAAA′AGTTTTTCTTATTTTCCAGAGATGGAAG

TCACACAGCCCACTTCAAATCCAGTTAC′ACCTA′AGCCACCCT′ATTACAACAT

CCTGCTCTATTCCTTGGTGCCACTTATGTTAATTGCGGGGATTGTCATTTGT

GCATTTTGGGTGTACAGGCATCACAAGATGGCCTACCCTCCTGTACTTGTTC

CAACTCAAGACCCAGGACCACCCCCACCTTCTCCATTACTAGGGTTGAAACC

ACTGC′AGTTATTAGAAGTGAAAGCAAGGGGAAGATTTGGTTGTGTCTGGAAA

GCCCAGTTGCTTAACGAATATGTGGCTGTCAAAATATTTCCAATACAGGACA

AACAGTCATGGCAAAATGAATACGAAGTCTACAGTTTGCCTGGAATGAAGCA

TGAGAACATATTACAGTTCATTGGTGCAGAAAAACGAGGCACCAGTGTTGAT

GTGGATCTTTGGCTGATCAC′AGCATTTCATGAAAAGGGTTCACTATCAGACT

TTCTTAAGGCTAATGTGGTCTCTTGGAATGAACTGTGTCATATTGCAGAAAC

CATGGCTAGAGGATTGGCATATTTACATGAGGATATACCTGGCCTAAAAGAT

GGCCACAAACCTGCCATATCTCACAGGGACATCAAAAGTAAAAAATGTGCTGT

TGAAAAACAACCTGACAGCTTGCATTGCTGACTTTGGGTTGGCCTTAAAATT

TGAGGCTGGCAAGTTCTGCAGGCGATACCCCATGGACAGGTTGGTACCCGGAGG

TACATGGCTCCAGAGGTATTAGAGGGTGCTATAAACTTCCAAAGGGATGCAT

TTTTGAGGATAGATATGTATGCCATGGGATTAGTCCTATGGGAACTGGCTTC

TCGCTGTACTGCTGCAGATGGACCTGTAGATGAATACATGTTGCCATTTGAG

GAGGAAATTGGCCAGCATCCATCTCTTGAAGACATGCAGGAAGTTGTTGTGC

ATAAAAAAAAGAGGCCTGTTTTAAGAGATTATTGGCAGAAACATGCTGGAAT

GGCAATGCTCTGTGAAACCATTGAAGAATGTTGGGATCACGACGCAGA′AGCC

AGGTTATCAGCTGGATGTGTAGGTGAAAGAATTACCCAGATGCAGAGACTAA

CAAATATTATTACCACAGAGGACATTGTAACAGTGGTCACAATGGTGACAAA

TGTTGACTTTCCTCCCAAAGAATCTAGTCTATGA (SEQ ID NO: 4)

The nucleic acid sequence encoding the human ActRIIa soluble (extracellular) polypeptide is as follows:

ATACTTGGTAGATCAGAAAACTCAGGAGGTGTCTTTTCTTTAATGCTAATTGGG

AAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGA

TAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATTGAA

ATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTG

ATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTTTGTTGCTGTGAGGG

CAATATGTGTAATGAAAAGTTTTCTTATTTTCCAGAGATGGAAGTCACACAG CCCACTTCAAATCCAGTTACACcTAAGCCACCC (SEQ ID NO: 5)

In certain embodiments, the present invention relates to soluble ActRIIa polypeptides. As described herein, the term "soluble ActRIIa polypeptide" generally refers to a polypeptide comprising the extracellular domain of an ActRIIa protein. As used herein, the term "soluble ActRIIa polypeptide" includes any naturally occurring extracellular domain of an ActRIIa protein and any variants thereof (including mutants, fragments, and peptidomimetics). Activin-binding ActRIIa polypeptides are polypeptides that retain the ability to bind to activin, particularly activin AA, AB, or BB. Preferably, the activin-binding ActRIIa polypeptide binds to activin AA with a dissociation constant of 1 nM or less. The amino acid sequence of the human ActRIIa precursor protein is provided below. ActRIIa proteins that bind to activin and are generally soluble are therefore referred to as soluble activin-binding ActRIIa polypeptides. Examples of soluble activin-binding ActRIIa polypeptides include the soluble polypeptides set forth in SEQ ID NOs: 2, 3, 7, 12, and 13. SEQ ID NO:7 designates ActRIIa-hFc and is further described in the Examples. Other soluble activin-binding ActRIIa polypeptides include the extracellular domain of the ActRIIa protein plus a signal sequence, such as the honey bee melittin leader (SEQ ID NO:8), the tissue plasminogen activator (TPA) leader (SEQ ID NO:9), or the native ActRIIa leader (SEQ ID NO:10). The ActRIIa-hFc polypeptide depicted in SEQ ID NO:13 serves as the TPA leader.

Functionally active fragments of ActRIIa polypeptides can be obtained by screening polypeptides produced from nucleic acid fragments encoding ActRIIa polypeptides. Alternatively, fragments can be chemically synthesized using well-known techniques such as traditional Merrifield solid-phase f-Moc or t-Boc chemistry. Fragments can be produced (recombinantly or chemically synthesized) and tested to identify peptidyl fragments that function as antagonists (inhibitors) of the ActRIIa protein or that are regulated by activin signaling.

Functionally active variants of ActRIIa polypeptides can be obtained by screening libraries of modified recombinant polypeptides generated from corresponding mutagenized nucleic acids encoding ActRIIa polypeptides. Variants can be generated and tested to identify those that function as ActRIIa protein antagonists or that are regulated by activin signaling. In certain embodiments, functional variants of ActRIIa polypeptides comprise an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NOs: 2 or 3. In certain examples, functional variants comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 2 or 3.

Functional variants can be generated by modifying the structure of an ActRIIa polypeptide for purposes such as enhancing therapeutic efficacy or stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo). Such modified ActRIIa polypeptides, when selected to retain activin binding, are considered functional equivalents of naturally occurring ActRIIa polypeptides. Modified ActRIIa polypeptides can also be produced, for example, by amino acid substitutions, deletions, or additions. For example, it is reasonable to expect that the substitution of a leucine with isoleucine or valine alone, an aspartic acid with glutamic acid alone, a threonine with serine alone, or a structurally related amino acid for another amino acid (e.g., a conservative mutation) will not significantly affect the biological activity of the resulting molecule. Conservative substitutions are substitutions between amino acids whose side chains are related within a family. Whether a change in the amino acid sequence of an ActRIIa polypeptide results in a functional homology can be readily determined by evaluating the ability of the ActRIIa polypeptide variant to elicit a response in cells in a manner similar to that of the wild-type ActRIIa polypeptide.

In certain embodiments, the present invention encompasses specific mutations of ActRIIa polypeptides to alter the glycosylation of the polypeptide. Such mutations can be selected to introduce or eliminate one or more glycosylation sites, either O-linked or N-linked. Asparagine-linked glycosylation recognition sites typically comprise a tripeptide sequence, asparagine-X-threonine (or asparagine-X-serine) (where X can be any amino acid), which is specifically recognized by appropriate cellular glycosylation enzymes. Modifications can also be made by adding or substituting one or more serine or threonine residues into a wild-type ActRIIa polypeptide (for O-linked glycosylation sites). Various amino acid substitutions or deletions at either or both of the first or third amino acid positions of a glycosylation recognition site (and/or amino acid deletions at the second position) result in aglycosylation of the modified tripeptide sequence. Another method for increasing the number of carbohydrates on an ActRIIa polypeptide can be by chemically or enzymatically coupling glycosides to the ActRIIa polypeptide. Depending on the coupling mode employed, 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 carbohydrates from an ActRIIa polypeptide can be accomplished chemically and/or enzymatically. Chemical deglycosylation can involve, for example, exposure of the ActRIIa polypeptide to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the removal of most or all sugars except the attached sugar (N-acetylglucose or N-acetylgalactosamine), while leaving the amino acid sequence intact. Chemical deglycosylation is further described in Hakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and Edge et al. (1981) Anal. Biochem. 118:131. Enzymatic removal of carbohydrates from ActRIIa polypeptides can be achieved using various endo- or exo-glycosidases as described by Thotakura et al. (1987) Meth. Enzymol. 138:350. The sequence of the ActRIIa polypeptide can be appropriately adjusted, depending on the type of expression system used, as mammalian, yeast, insect, and plant cells can all introduce different glycosylation patterns, which can be influenced by the amino acid sequence of the peptide. Typically, ActRIIb polypeptides for human use are expressed in mammalian cell lines that provide correct glycosylation, such as HEK293 cells or CHO cell lines, although other mammalian expression cell lines, yeast cell lines with engineered glycosylation enzymes, and insect cell lines are also contemplated for expression.

The present invention further encompasses methods for generating mutants, particularly sets of combinatorial mutants and truncation mutants of ActRIIa polypeptides; libraries of combinatorial mutants are particularly useful for identifying functional variant sequences. The goal of screening such combinatorial libraries is to generate, for example, variants of ActRIIa polypeptides that act as either agonists or antagonists, or alternatively, mutants with all new activities. Various screening methods are provided below, and such methods can be used to evaluate variants. For example, variants of ActRIIa polypeptides can be screened for their ability to bind to an ActRIIa ligand and prevent the ActRIIa ligand from binding to the ActRIIa polypeptide or to interfere with signaling induced by the ActRIIa ligand.

The activity of an ActRIIa polypeptide or variant thereof can also be tested on a cell-based basis or in vivo. For example, the effect of an ActRIIa polypeptide variant on the expression of a gene involved in bone production or bone destruction can be evaluated. If desired, this can be accomplished in the presence of one or more recombinant ActRIIa ligand proteins (e.g., activin), and the cells can be transfected to produce the ActRIIa polypeptide and/or variant thereof and, as appropriate, the ActRIIa ligand. Similarly, an ActRIIa polypeptide can be administered to mice or other animals, and one or more bone properties such as density or volume assessed. The healing rate of fractures can also be assessed. Dual-energy X-ray absorptiometry (DEXA) is a well-established, non-invasive, quantitative technique for assessing bone density in animals. Human central DEXA systems can be used to assess bone density of the spine and pelvis. These are the best predictors of total bone density. Peripheral DEXA systems can be used to assess the density of peripheral bones, including, for example, the bones of the hands, wrists, ankles, and feet. Conventional x-ray imaging systems, including CAT scans, can be used to assess bone growth and fracture healing. The mechanical strength of the bone can also be assessed.

Combinatorially derived variants can be generated that have selective or generally increased potency relative to naturally occurring ActRIIa polypeptides. Similarly, mutations can result in variants having intracellular half-lives that differ dramatically from those of the corresponding wild-type ActRIIa polypeptide. For example, the altered protein may be more or less stable to proteolysis or other cellular processes that lead to its destruction, or conversely, to inactivation of the native ActRIIa polypeptide. Such variants, and the genes encoding them, can be used to alter the levels of the ActRIIa polypeptide by modulating its half-life. For example, a shorter half-life can result in more transient biological effects and can allow for tighter control of the levels of the recombinant ActRIIa polypeptide in a patient. In Fc fusion proteins, mutations can be in the linker (if present) and/or the Fc portion to alter the half-life of the protein.

Recombinant libraries can be generated by means of a degenerate gene library encoding a library of polypeptides, each of which comprises at least a portion of a potential ActRIIa polypeptide sequence. For example, a mixture of synthetic oligonucleotides can be enzymatically ligated into a gene sequence such that degenerate forms of potential ActRIIa polypeptide nucleotide sequences can be expressed as individual polypeptides, or alternatively, as larger fusion proteins (e.g., via phage display).

There are many methods for generating libraries of potential homologs from degenerate oligonucleotide sequences. Chemical synthesis of degenerate gene sequences can be performed in an automated DNA synthesizer, and the synthesized gene is then ligated into a suitable vector for expression. The synthesis of degenerate oligonucleotides is well known in the art (see, for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. A G Walton, Amsterdam: Elsevier pp. 273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acids Res. 11:477). These techniques have been applied to 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. Patents 5,223,409, 5,198,346, and 5,096,815).

Alternatively, other forms of mutation can be used to generate combinatorial libraries. For example, variants of the ActRIIa polypeptide can be generated by using, for example, alanine screening mutations and the like (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; Na Gashima 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). Linker-scanning mutagenesis, particularly in a combinatorial setting, is an attractive approach for identifying truncated (bioactive) forms of ActRIIa polypeptides.

A wide range of techniques are known in the art for screening combinatorial libraries of gene products generated by point mutations and truncations, and for this purpose, cDNA libraries for gene products with certain properties. Such techniques are generally suitable for rapidly screening gene libraries generated by combinatorial mutagenesis of ActRIIa polypeptides. The most widely used techniques for screening large gene libraries generally involve cloning the gene library into a replicable expression vector, transforming the resulting vector library into suitable cells, and expressing the combinatorial genes under conditions that allow for detection of the desired activity and relatively easy isolation of the vector encoding the gene encoding the detected product. Preferred methods include activin binding assays and activin-regulated cell signaling assays.

In certain embodiments, the ActRIIa polypeptides of the present invention may further include post-translational modifications, any naturally occurring in ActRIIa polypeptides. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Consequently, modified ActRIIa polypeptides may include non-amino acid elements, such as polyethylene glycol, lipids, polysaccharides or monosaccharides, and phosphates. The effects of such non-amino acid elements on the function of the ActRIIa polypeptide can be tested as described for other ActRIIa polypeptide variants herein. When ActRIIa polypeptides are produced intracellularly by cleaving the nascent form of the ActRIIa polypeptide, post-translational processes are also important for proper protein folding and/or function. Different cell lines (such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3, or HEK293) have specific cellular machinery and unique mechanisms for such post-translational events and can be selected to ensure proper modification and processing of the ActRIIa polypeptide.

In certain aspects, functional variants or modified forms of an ActRIIa polypeptide include fusion proteins comprising at least a portion of an ActRIIa polypeptide and one or more fusion domains. Examples of such fusion domains are well known, but are not limited to, polyhistidine, Glu-Glu, glutathione transferase (GST), thioredoxin, protein A, protein G, immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), or human serum albumin. Fusion domains can be selected to impart desirable properties. For example, some fusion domains are specifically designed for isolation of fusion proteins by affinity chromatography. For affinity purification, affinity chromatography-related matrices such as glutathione, amylase, and nickel- or cobalt-conjugated resins are employed. Many such matrices are available in "kit" form, such as the Pharmacia GST purification system and the QIAexpress ^™ system (Qiagen) with a useful (HISg) fusion partner. As another example, fusion domains can be selected to facilitate detection of an ActRIIa polypeptide. Examples of such detection domains include various fluorescent proteins (e.g., GFP) and "epitope tags," which are typically short peptide sequences for which specific antibodies are available. Well-known epitope tags for specific monoclonal antibodies are readily available and include FLAG, influenza virus hemagglutinin (HA), and c-myc tags. In some examples, the fusion domain has a protease cleavage site, such as for Factor Xa or thrombin, which allows the relevant protease to partially digest the fusion protein and thereby release the recombinant protein therefrom. The released protein is then separated from the fusion domain by subsequent chromatographic separation. In certain preferred embodiments, the ActRIIa polypeptide is fused to a domain that stabilizes the ActRIIa polypeptide in vivo (a "stabilizer" domain). By "stabilize" is meant any substance that increases serum half-life, regardless of whether this is due to reduced destruction, reduced renal clearance, or other pharmacokinetic effects. Fusion to the Fc portion of an immunoglobulin is known to impart desirable pharmacokinetic properties to a wide range of proteins. In addition, fusion to human serum albumin can impart desirable properties. Other types of fusion domains that may be selected include multimerization domains (e.g., dimerization, tetramerization) as well as functional domains (which impart additional biological functions, such as further stimulation of bone growth or muscle growth, as desired).

As a specific example, the present invention provides a fusion protein comprising a soluble extracellular domain of ActRIIa fused to an Fc domain (eg, SEQ ID NO: 6).

THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV D (A)VSHEDPEVKF

NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC K (A)VSNKAL

PVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN

GQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALH N (A)HYT OKSLSLSPGK*

Alternatively, the Fc domain has one or more mutations in residues such as aspartic acid 265, lysine 322, and asparagine 434. In certain examples, a mutant Fc domain having one or more such mutations (e.g., aspartic acid 265 mutation) has reduced ability to bind to Fcγ receptors relative to a wild-type Fc domain. In other examples, a mutant Fc domain having one or more such mutations (e.g., asparagine 434 mutation) has increased ability to bind to MHC class I-associated Fc receptors (FcRN) relative to a wild-type Fc domain.

Of course, the various elements of the fusion protein can be arranged in any manner consistent with the desired function. For example, the ActRIIa polypeptide can be placed C-terminally to the heterologous domain, or alternatively, the heterologous domain can be placed C-terminally to the ActRIIa polypeptide. The ActRIIa polypeptide domain and the heterologous domain need not be immediately adjacent in the fusion protein, and additional domains or amino acid sequences can be included C-terminally or N-terminally to either domain or between the two domains.

In certain embodiments, the ActRIIa polypeptides of the present invention contain one or more modifications that stabilize the ActRIIa polypeptide. For example, such modifications enhance the in vivo half-life of the ActRIIa polypeptide, enhance the circulating half-life of the ActRIIa polypeptide, or reduce proteolytic degradation of the ActRIIa polypeptide. Such stabilizing modifications include, but are not limited to, fusion proteins (including, for example, fusion proteins comprising an ActRIIa polypeptide and a stabilizer domain), modifications to glycosylation sites (including, for example, appending glycosylation sites to an ActRIIa polypeptide), and modifications to carbohydrates (including, for example, removing carbohydrates from an ActRIIa polypeptide). In the case of fusion proteins, the ActRIIa polypeptide is fused to a stabilizer domain (e.g., an Fc domain) such as an IgG molecule. As used herein, the term "stabilizer domain" refers not only to the fusion domain (e.g., Fc) in a fusion protein, but also includes non-protein modifications such as carbohydrates or non-protein polymers such as polyethylene glycol.

In certain embodiments, the present invention makes available an ActRIIa polypeptide in isolated and/or purified form, which is isolated from or otherwise substantially free from other proteins. ActRIIa polypeptides are typically produced by expression from a recombinant nucleic acid.

3. Nucleic Acids Encoding ActRIIa Polypeptides

In certain aspects, the present invention provides isolated and/or recombinant nucleic acids encoding any ActRIIa polypeptide (e.g., a soluble ActRIIa polypeptide), including fragments, functional variants, and fusion proteins disclosed herein. For example, SEQ ID NO: 4 encodes a naturally occurring human ActRIIa precursor polypeptide, while SEQ ID NO: 5 encodes the processed extracellular domain of ActRIIa. The target nucleic acid can be single-stranded or double-stranded. Such nucleic acids can be DNA molecules or RNA molecules. These nucleic acids can be used, for example, in methods for producing ActRIIa polypeptides or as direct therapeutic agents (e.g., in gene therapy approaches).

In certain aspects, it is further understood that nucleic acids of interest encoding ActRIIa polypeptides include variants of SEQ ID NO: 4 or 5. Variant nucleotide sequences include different sequences having one or more nucleotide substitutions, additions, or deletions, such as allelic variants.

In certain embodiments, the present invention provides isolated or recombinant nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4 or 5. It will be appreciated by those skilled in the art that nucleic acid sequences complementary to SEQ ID NO: 4 or 5, as well as variants of SEQ ID NO: 4 or 5, are encompassed by the present invention. In further embodiments, the nucleic acid sequences of the present invention may be isolated, recombinant, and/or fused to heterologous nucleotide sequences or in a DNA library.

In other embodiments, the nucleic acids of the present invention may also include nucleotide sequences that hybridize under stringent conditions to the nucleotide sequence specified in SEQ ID NO: 4 or 5, the complement of SEQ ID NO: 4 or 5, or a fragment thereof. As discussed above, one of ordinary skill in the art will readily appreciate that suitable stringent conditions for promoting DNA hybridization can vary. For example, hybridization can be performed in 6.0x sodium chloride/sodium citrate (SSC) at approximately 45°C, followed by a wash in 2.0x SSC at 50°C. For example, the salt concentration of the wash step can be selected from low stringency conditions of 2.0x SSC at 50°C to high stringency conditions of 0.2x SSC at 50°C. Additionally, the temperature of the wash step can be varied from low stringency conditions at room temperature of approximately 22°C to high stringency conditions of approximately 65°C. Both the temperature and the salt concentration can be varied, or the temperature or salt concentration can be held constant while the other variable is changed. In certain embodiments, the present invention provides nucleic acids that hybridize under low stringency conditions of 6x SSC at room temperature, followed by a wash in 2x SSC at room temperature.

Also included within the scope of the present invention are isolated nucleic acid molecules that differ from the nucleic acid in SEQ ID NO: 4 or 5 due to the degeneracy of the genetic code. For example, a large number of amino acids are specified by more than one triplet codon. Codons or synonymous codons that point to the same amino acid (e.g., CAU and CAC are synonymous codons for histidine) can result in "silent" mutations that do not affect the amino acid sequence of the protein. However, we expect that polymorphisms in DNA sequences that do result in changes in the amino acid sequence of the target protein exist in mammalian cells. Those skilled in the art will appreciate that variations in one or more nucleotides (up to 3-5% of the nucleotides) of a nucleic acid encoding a particular protein can exist in individuals of a given species due to natural allelic variation. Any or all such nucleotide variations and the resulting amino acid polymorphisms are within the scope of the present invention.

In certain embodiments, the recombinant nucleic acid of the present invention is operably connected to one or more regulatory nucleotide sequences of expression constructs. The regulatory nucleotide sequence is generally suitable for expressing the applied host cell. Many types of suitable expression vectors and suitable regulatory sequences for various host cells are well known in the art. Generally, 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 stop sequences, translation start and stop sequences and enhancer or activator sequences. Constructed or induced promoters well known in the art are encompassed by the present invention. The promoter is either a naturally occurring promoter or a hybrid promoter of more than one promoter combination element. The expression construct may be present in an intracellular episome, such as a plasmid, or the expression construct may be inserted into the chromosome. In a preferred embodiment, the expression vector comprises a selectable marker gene to allow selection of a transformed host. Selectable marker genes are well known in the art and vary with the host cell employed.

In certain aspects of the present invention, a nucleic acid of interest is provided within an expression vector comprising a nucleotide sequence encoding an ActRIIa polypeptide and operably linked to at least one regulatory sequence. Regulatory sequences are art-recognized and selected to direct expression of an ActRIIa polypeptide. Accordingly, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Enzymatic Methods, 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 to express a DNA sequence encoding an ActRIIa polypeptide. Such useful expression control sequences include, for example, the early or late promoter of SV40, the tet promoter, the immediate early promoter of adenovirus or cytomegalovirus, the RSV promoter, the lactose operator system, the tryptophan system, the TAC or TRC system, the T7 promoter for directing expression by T7 RNA polymerase, the major operator and initiation region of lambda phage, the control region of the fd goat protein, the promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, the promoter of acid phosphatase such as Pho5, the promoter of yeast α-hybrid factor, the polyhedron promoter of the baculovirus system, and other known sequences for controlling the expression of prokaryotic or eukaryotic cells or viral genes thereof and their various recombinant forms. It should be understood that the design of the expression vector depends on factors such as the choice of host cell to be transformed and/or the type of protein to be expressed. In addition, the number of vector copies, the ability to control the copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.

The recombinant nucleic acids of the present invention can be produced by ligating the cloned gene or fragment thereof into a vector suitable for expression in either prokaryotes or eukaryotic cells (yeast, birds, insects, or mammals), or both. Expression vectors for the production of recombinant ActRIIa 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 prokaryotes such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences that facilitate bacterial propagation and one or more eukaryotic transcription units for expression 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 mammalian expression vectors suitable for eukaryotic cell transfection. Some of these vectors, such as pBR322, are modified with sequences derived from bacterial plasmids to facilitate replication in both prokaryotes and eukaryotic cells and selection for drug resistance. Alternatively, viral derivatives such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP, and p205) can be used for transient protein expression in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found in the description of gene therapy delivery systems below. Various methods for preparing plasmids and transforming host organisms are well known in the art. Other expression systems suitable for both prokaryotes and eukaryotes, as well as general recombination procedures, are described in Molecular Cloning: A Laboratory Manual, 3rd edition, edited by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press, 2001). In some instances, it is suitable to express recombinant polypeptides using a bovine papillomavirus expression system. Examples of such bovine papillomavirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as β-galactosidase containing pBlueBacIII).

In a preferred embodiment, a vector is designed to produce an ActRIIa polypeptide of interest in CHO cells, such as the pcmv-Script vector (Stratagene, La Jolla, Calif.), the pcDNA4 vector (Invitrogen, Carlsbad, Calif.), and the pCI-neo vector (Promega, Madison, Wisc.). Obviously, the gene construct of interest can be used to cause expression of an ActRIIa polypeptide of interest in cells propagated in culture, for example, to produce proteins, including fusion proteins or mutant proteins, for purification.

The present invention also relates to host cells transfected with a recombinant gene comprising a coding sequence encoding one or more ActRIIa polypeptides of interest (e.g., SEQ ID NO: 4 or 5). The host cell can be any prokaryotic or eukaryotic cell. For example, the ActRIIa polypeptides of the present invention can be expressed in bacteria, such as Escherichia coli, insect cells (e.g., using a bovine papilloma virus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.

Accordingly, the present invention further relates to methods for producing an ActRIIa polypeptide of interest. For example, cells transformed with an expression vector encoding an ActRIIa polypeptide can be cultured under suitable conditions to allow expression of the ActRIIa polypeptide. The ActRIIa polypeptide can be secreted from a mixture of cells and a matrix containing the ActRIIa polypeptide and isolated. Alternatively, the ActRIIa polypeptide can remain in the cytoplasm or as a membrane fraction, and the cells can be harvested, solubilized, and the protein isolated. The cell culture comprises host cells, matrix, and other by-products. Suitable cell culture matrices are well known in the art. The ActRIIa polypeptide of interest can be isolated from the cell culture matrix, the host cells, or both, and the protein purified using techniques well known in the art, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, immunoaffinity purification using antibodies specific for a particular epitope of the ActRIIa polypeptide, and affinity purification using a preparation that binds to a domain fused to the ActRIIa polypeptide (e.g., a Protein A column can be used to purify an ActRIIa-Fc fusion protein). In a preferred embodiment, the ActRIIa polypeptide is a fusion protein containing a domain that facilitates purification. In a preferred embodiment, purification is achieved by a series of column chromatography steps, including, for example, three or more of the following steps, in any order: Protein A chromatography, Q Sepharose chromatography, Phenyl Sepharose chromatography, size exclusion chromatography, and cation exchange chromatography. Purification can be accomplished by viral filtration and buffer exchange. As demonstrated herein, ActRIIa-hFc protein was purified to a purity greater than 98% as determined by size exclusion chromatography and greater than 95% as determined by SDS PAGE. Such purity is sufficient to achieve the desired effect in mouse bone and an acceptable safety profile in mice, rats, and non-human primates.

In another embodiment, a fusion gene encoding a purification leader sequence, such as a poly-(histidine)/enterokinase cleavage site sequence at the N-terminal position of the desired portion of the recombinant ActRIIa polypeptide, can allow purification of the expressed fusion protein by affinity chromatography using a Ni ²⁺ metal resin. The purification leader sequence is then removed by enterokinase treatment to provide purified ActRIIa polypeptide (e.g., see Hochuli et al., (1987) J Chromatography 411:177; and Janknecht et al., PNAS USA 88:8972).

Techniques for preparing fusion proteins are well known in the art. In essence, the connection of various DNA fragments encoding different polypeptide sequences is accomplished by corresponding conventional techniques, using blunt-ended or staggered ends for connection, restriction enzyme digestion to provide suitable ends, filling of sticky ends where appropriate, alkaline phosphatase treatment to avoid unwanted connection, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automatic DNA synthesizers. Alternatively, PCR amplification of the gene fragments can be performed using linker primers that provide complementary overhangs between two consecutive gene fragments that can subsequently anneal to produce a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, edited by Ausubel et al., John Wiley & Sons: 1992).

4. Selective Activin and ActRIIa antagonists

The data provided herein demonstrate that antagonists of activin-ActRIIa signaling can be used to promote bone growth and mineralization. Although soluble ActRIIa polypeptides, particularly ActRIIa-Fc, are preferred antagonists, and although such antagonists may affect bone through mechanisms other than activin antagonism (e.g., activin inhibition may have an effect on the activity of a broad range of molecules, including, potentially, other members of the TGF-beta superfamily, and such population inhibition may result in the desired skeletal effects), other types of activin-ActRIIa antagonists are expected to be effective, including anti-activin (e.g., A, B, C, or E) antibodies, anti-ActRIIa antibodies, antisense, RNAi or ribozyme nucleic acids that inhibit ActRIIa production, and other inhibitors of activin or ActRIIa, particularly those that disrupt activin-ActRIIa binding.

Antibodies that specifically react with ActRIIa polypeptides (e.g., soluble ActRIIa polypeptides) and substances that either competitively bind to and bind to ActRIIa polypeptides or conversely inhibit ActRIIa-mediated signaling can be used as antagonists of ActRIIa polypeptide activity. Furthermore, antibodies that specifically react with activin A polypeptides and antibodies that disrupt ActRIIa binding can also be used as antagonists.

Anti-protein/anti-polypeptide antisera or monoclonal antibodies can be prepared using immunogens derived from ActRIIa polypeptides or activin polypeptides by standard methods (see, for example, A Laboratory Manual, edited by Harlow and Lane (Cold Spring Harbor Press: 1988)). Mammals, such as mice, hamsters, or rabbits, can be immunized with an immunogenic form of an ActRIIa polypeptide, an antigenic fragment, or a fusion protein capable of eliciting an antibody response. Techniques for conferring immunogenicity on proteins or polypeptides include conjugation to carriers or other techniques known in the art. Immunogenic portions of ActRIIa or activin polypeptides can be administered in the presence of an adjuvant. Immunization can be monitored by measuring antibody titers in plasma or serum. Standard ELISA or other immunological methods can be used with the immunogen as an antigen to assess antibody levels.

After immunizing an animal with an antigenic preparation of an ActRIIa polypeptide, antiserum is obtained, and if desired, polyclonal antibodies can be isolated from the serum. To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be collected from the immunized animal and fused with immortalized cells such as myeloma cells via standard somatic cell fusion procedures to produce hybridoma cells. Such techniques are well known in the art and include, for example, the hybridoma technique (originally developed by Kohler and Milstein (1975) Nature, 256:495-497), the human B cell hybridoma technique (Kozbar et al. (1983) Immunology Today, 4:72), and the EBV-hybridoma technique 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 that specifically react with an ActRIIa polypeptide, and monoclonal antibodies can be isolated from culture medium containing such hybridoma cells.

As used herein, the term "antibody" is intended to include fragments that are also capable of specifically reacting with a target polypeptide. Antibodies can be fragmented using conventional techniques, and the fragments screened using the same methods described above for whole antibodies. For example, F(ab) ₂ fragments can be generated by treatment with pepsin. The resulting F(ab) ₂ fragments can be processed to reduce disulfide bridges to produce Fab fragments. Antibodies of the present invention are further intended to include bispecific, single-chain, chimeric, humanized, and full-length human molecules with affinity for ActRIIa or activin polypeptides conferred by at least one antibody CDR region. Antibodies may further include an additional, detectable label (e.g., a radioisotope, fluorescent compound, enzyme, or cofactor).

In certain embodiments, the antibody is a recombinant antibody, which term includes any antibody partially produced by molecular biology techniques, including CDR-grafted antibodies or chimeric antibodies, antibodies that are human or assembled from antibody domains selected from a library, single-chain antibodies, and single-domain antibodies (e.g., human _VH proteins or camelid _VHH proteins). In certain embodiments, the antibodies of the present invention are monoclonal antibodies, and in certain embodiments, the present invention enables methods for producing new antibodies. For example, a method for producing monoclonal antibodies that specifically bind to an ActRIIa polypeptide or an activin polypeptide comprises administering to a mouse a dose of an immunogen composition comprising an effective amount of an antigenic polypeptide that stimulates a detectable immune response, obtaining antibody-producing cells (e.g., cells from the spleen) from the mouse, and fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and detecting the antibody-producing hybridomas to identify hybridomas that can produce monoclonal antibodies that specifically bind to the antigen. Once obtained, the hybridomas are propagated from the cell culture medium, optionally under culture conditions for the hybridoma-derived cells that produce cells that specifically bind to the antigen. The monoclonal antibodies can be purified from the cell culture medium.

The adjective "specifically reactive with" as used herein in reference to an antibody means, as is generally understood in the art, that the antibody is sufficiently selective between an antigen of interest (e.g., an ActRIIa polypeptide) and other, non-antigens of interest in a particular biological sample. In certain methods employing antibodies, such as therapeutic applications, a high degree of specificity of binding is essential. Monoclonal antibodies generally have a greater tendency (compared to polyclonal antibodies) to effectively discriminate between the desired antigen and cross-reacting polypeptides. A characteristic influencing the specificity of an antibody:antibody interaction is the affinity of the antibody for the antigen. Although a range of different affinities can be achieved with the desired specificity, antibodies having an affinity (dissociation constant) of about 10 ^{^-6}, 10^{^-7} , 10^{^-8}, or less are generally preferred. Given the exceptionally tight binding between activin and ActRIIa, we expect that neutralizing anti-activin or anti-ActRIIa antibodies will generally have a dissociation constant of 10 ^{^-10} or less.

In addition, the technology used to screen antibodies to identify desirable antibodies can affect the properties of the antibodies obtained. For example, if the antibody is used to bind to the antigen in solution, test solution binding may be appropriate. Various different techniques can be used to test the interaction between the antibody and the antigen to identify the specific desired antibody. Such techniques include ELISAs, surface plasmon resonance binding analysis (e.g., the Biacore ^™ binding assay, Biacore AB, Uppsala, Sweden), sandwich methods (e.g., the paramagnetic bead system of IGEN International, Inc., Gaithersburg, Maryland), western immunoblotting, immunoprecipitation, and immunohistochemistry.

Examples of nucleic acid compounds that are antagonists of Activin or ActRIIa include antisense nucleic acids, RNAi constructs, and catalytic nucleic acid constructs. Nucleic acid compounds can be single-stranded or double-stranded. Double-stranded nucleic acid compounds can also include overhanging or non-complementary regions, where one or the other segment is single-stranded. Single-stranded compounds can include self-complementary regions, meaning that the compound forms a region with a double-stranded helical structure known as a "hairpin" or "stem-loop" structure. Nucleic acid compounds can include a nucleotide sequence that is complementary to a region of no more than 1000, no more than 500, no more than 250, no more than 100, or no more than 50, 35, 30, 25, 22, 20, or 18 nucleotides comprising the full-length ActRIIa nucleic acid sequence or the activin βA or activin βB nucleic acid sequence. Preferred complementary regions are at least 8 nucleotides, optionally at least 10 or at least 15 nucleotides, and optionally between 15 and 25 nucleotides. The complementary region can be within an intron, a coding sequence, or a non-coding sequence such as a portion of a coding sequence of the target transcript. Typically, nucleic acid compounds have a length of 8 to about 500 nucleotides or base pairs, and optionally can be about 14 to about 50 nucleotides in length. The nucleic acid can be DNA (particularly useful as an antisense nucleic acid), RNA, or an RNA:DNA hybrid. Any one strand can include a mixture of DNA and RNA, as well as modified forms that are not readily distinguishable as DNA or RNA. In addition, double-stranded compounds can be DNA:DNA, DNA:RNA, or RNA:RNA, and any one strand can include a mixture of DNA and RNA, as well as modified forms that are not readily distinguishable as DNA or RNA. Nucleic acid compounds can include any of a variety of modifications, including one or more modifications to the backbone (sugar-phosphate portion of a natural nucleic acid, including internucleoside linkages) or the base portion (purine or pyrimidine portion of a natural nucleic acid). Antisense nucleic acid compounds are preferably about 15 to about 30 nucleotides in length and typically contain one or more modifications to improve properties such as stability in serum, in cells, or at potential sites of administration of the compound, such as the stomach (in the case of oral administration) and the lungs (for inhaled compounds). In the case of RNAi constructs, the strand complementary to the target transcript is typically RNA or a modification thereof. The other chain can be RNA, DNA or any other variant. The duplex portion of the double-stranded or single-stranded "hairpin" RNAi construct is preferably 18 to 40 nucleotides in length, optionally about 21 to 23 nucleotides in length, as long as it can function as a Dicer substrate. The catalytic or enzymatic nucleic acid can be a ribozyme or DNA enzyme, and can also include modified forms. Under physiological conditions, the nucleic acid compound, in a concentration that produces essentially no or no effect with nonsense or sense control, can inhibit the expression of the target by about 50%, 75%, 90% or more when contacted with the cell. The preferred concentration for detecting the effect of the nucleic acid compound is 1, 5 and 10 micromolar. The nucleic acid compound can be used to test, for example, the effect on bone growth and bone mineralization.

5. Screening Method

In certain aspects, the present invention relates to the use of ActRIIa polypeptides (e.g., soluble ActRIIa polypeptides) and activin polypeptides to identify compounds (drugs) that can agonize or antagonize the activin-ActRIIa signaling pathway. Agents identified through such screening can be tested to assess their ability to regulate bone growth and bone mineralization in vivo. Optionally, these compounds can be further tested in animals to assess their ability to regulate tissue growth in vivo.

Numerous approaches exist for screening therapeutic agents that modulate tissue growth by targeting activin and ActRIIa polypeptides. In certain embodiments, high-throughput screening of compounds can be used to identify agents that interfere with the regulatory effects of activin or ActRIIa on bone. In certain embodiments, the methods are used to screen and identify compounds that specifically inhibit or reduce the binding of an ActRIIa polypeptide to activin. Alternatively, the methods are used to identify compounds that enhance the binding of an ActRIIa polypeptide to activin. In another embodiment, compounds can be identified by their ability to interact with activin or ActRIIa polypeptides.

Versions of various methods are sufficient and within the scope of the present disclosure, however, those not clearly described herein can be understood by those of ordinary skill in the art. As described herein, the detection compounds (agents) of the present invention can be manufactured by any combinatorial chemical method. Alternatively, the target compound can be a naturally occurring biomolecule synthesized in vivo or in vitro. The compound (agent) to be tested for its ability as a tissue growth regulator can be produced by, for example, bacteria, yeast, plants or other organisms (e.g., natural products), or chemically produced (e.g., small molecules, including peptoids), or recombinantly produced. The detection compounds encompassed by the present invention include non-peptide organic molecules, peptides, polypeptides, peptoids, sugars, hormones and nucleic acid molecules. In a specific embodiment, the detection agent is a small organic molecule with a molecular weight of less than about 2000 Daltons.

The test compounds of the present invention can be single, discrete entities, or libraries of greater complexity, such as libraries prepared by combinatorial chemistry. These libraries can include, for example, ethanol, alkyl halides, amines, amides, esters, aldehydes, ethers, and other classes of organic compounds. The test compounds can be present in the detection system alone or in the form of a mixture of compounds, particularly in the initial screening step. Optionally, the compounds can be derivatized with other compounds and the derivative population can assist in compound separation. Non-limiting examples of derivative populations include biotin, fluorescein, digoxigenin, green fluorescent protein, isotopes, polyhistidine, magnetic beads, glutathione transferase (GST), photoactivatable crosslinkers, or any combination thereof.

In many drug screening programs involving test compound libraries and natural extracts, high-throughput methods are required to maximize the number of compounds tested in a given timeframe. Such methods, in cell-free environments, such as those that utilize purified or semi-purified proteins, are often preferred as "primary" screening methods, allowing for rapid development and relatively easy detection of changes in target molecules modulated by test compounds. Furthermore, the cytotoxicity or bioavailability of test compounds can be disregarded in in vitro systems; instead, the method focuses primarily on the drug's effect on the target molecule, which can be manifested as changes in the binding affinity between the ActRIIa polypeptide and activin.

By way of example only, in an exemplary screening method of the present invention, a compound of interest is contacted with an isolated and purified ActRIIa polypeptide that typically binds to activin. A composition comprising an ActRIIa ligand is then added to the mixture of compound and ActRIIa polypeptide. Detection and quantification of the ActRIIa/activin complex provides a method for determining the effectiveness of a compound in inhibiting (or enhancing) complex formation between the ActRIIa polypeptide and activin. The efficacy of a compound can be assessed by generating a dose-response curve using data obtained at various concentrations of the test compound. Furthermore, control assays can be used to provide a benchmark for comparison. For example, in a control assay, isolated and purified activin is added to a composition comprising an ActRIIa polypeptide, and the ActRIIa/activin complex is quantified in the absence of the test compound. It will be appreciated that, in general, the order in which the reactants are mixed can vary, and they can be mixed simultaneously. Furthermore, cell extracts and lysates can be used in place of purified protein to provide suitable cell-free assay systems.

The formation of a complex between an ActRIIa polypeptide and activin can be detected by various techniques. For example, modulation of complex formation can be quantified by immunoassay or chromatographic detection using, for example, a detectably labeled protein such as a radiolabeled (e.g., ³² P, ³⁵ S, ¹⁴ C, or ³ H), a fluorescein-labeled (e.g., FITC), or an enzyme-labeled ActRIIa polypeptide or activin.

In certain embodiments, the present invention encompasses the use of fluorescence polarization and fluorescence resonance energy transfer (FRET) to directly or indirectly measure the interaction between an ActRIIa polypeptide and its binding protein. Furthermore, other detection modalities, such as optical waveguide-based (PCT Publication WO 96/26432 and U.S. Patent 5,677,196), surface plasmon resonance (SPR), surface charge sensing, and surface force sensing, are consistent with many embodiments of the present invention.

In addition, the present invention encompasses the use of an interaction trap approach (also known as a "two-hybrid approach") to identify agents that disrupt or enhance the interaction between an ActRIIa polypeptide and its binding protein. 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 encompasses the use of a reverse two-hybrid system to identify compounds (e.g., small molecules or peptides) that disrupt the interaction between an ActRIIa polypeptide and its binding protein. See, eg, Vidal and Legrain, (1999) Nucleic Acids Res 27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81; and US Patents 5,525,490; 5,955,280; and 5,965,368.

In certain embodiments, a target compound is identified by its ability to interact with an ActRIIa or activin polypeptide of the invention. The interaction between the compound and the ActRIIa or activin polypeptide can be covalent or non-covalent. For example, such an interaction can be identified at the protein level using in vivo biochemical methods.

Such biochemical methods include photocrosslinking, isotopically labeled ligand binding, and affinity chromatography (Jakoby WB et al., 1974, Methods in Enzymology 46:1). In some instances, compounds can be screened using mechanisms based on the detection of compounds bound to activin or ActRIIa polypeptides. This can include solid-phase or liquid-phase binding. Alternatively, a gene encoding an activin or ActRIIa polypeptide can be co-transfected into cells with a reporter system (e.g., β-galactosidase, luciferase, or green fluorescent protein) and the library can be screened, preferably using high-throughput screening, or co-transfected with individuals from the library. Other binding-based mechanisms can be used, for example, in binding assays that detect changes in free energy. Binding assays can be performed with targets immobilized to wells, beads, or chips, captured by immobilized antibodies, or resolved by capillary electrophoresis. The trapped compounds can typically be measured colorimetrically, or by fluorescence or surface plasmon resonance.

In some aspects, the present invention provides methods and agents for regulating (stimulating or inhibiting) bone formation and increasing bone mass. Therefore, any identified compound can be tested in whole cells or tissues, in vitro or in vivo, to determine its ability to regulate bone growth or bone mineralization. Various methods known in the art can be used for this purpose.

For example, the effect of an ActRIIa or activin polypeptide or a test compound on bone or cartilage growth can be determined by measuring the induction of Msx2 or differentiation of osteoblasts into osteoblasts in a cell-based assay (see, e.g., (Daluiski et al., Nat Genet. 2001, 27(1):84-8; Hino et al., Front Biosci. 2004, 9:1520-9). Other examples of cell-based assays include assaying the osteogenic activity of the ActRIIa or activin polypeptide of interest and testing the compound in mesenchymal progenitor cells and osteoblasts. For example, a recombinant adenovirus expressing an activin or ActRIIa polypeptide can be constructed and used to infect multipotent mesenchymal progenitor cells C3H10T1/2, osteoblast precursors C2C12, and osteoblasts TE-85. Osteogenic activity is then determined by measuring the induction of alkaline phosphatase, osteocalcin, and matrix mineralization (see, e.g., Cheng et al., J Bone Joint Biol. 2003, 14(1):1520-9). SurgAm. 2003, 85-A(8):1544-52).

The present invention also encompasses methods for measuring bone or cartilage growth in vivo. For example, Namkung-Matthai et al., Bone, 28:80-86 (2001) discloses a rat osteoporosis model that studies bone repair in the early stages after a fracture. Kubo et al., Steroid Biochemistry & Molecular Biology, 68:197-202 (1999) also discloses a rat osteoporosis model that studies bone repair in the late stages after a fracture. Andersson et al., J. Endocrinol. 170:529-537 discloses an osteoporosis model in mice in which the ovariectomized mice lose both parenchymal bone mineral content and bone mineral density, with cancellous bone losing approximately 50% of its bone mineral density. Bone density in ovariectomized mice can be increased by administering factors such as parathyroid hormone. In certain aspects, the present invention utilizes methods known in the art for healing fractures. These methods include fracture techniques, histological analysis, and biomechanical analysis, which are described, for example, in US Patent No. 6,521,750, which is incorporated herein by reference in its entirety for its disclosure of experimental methods for establishing and measuring the extent of fractures and the progress of repair.

6. Demonstration of therapeutic applications

In certain embodiments, the activin-ActRIIa antagonists (e.g., ActRIIa polypeptides) of the present invention can be used to treat or prevent diseases or conditions associated with bone damage, whether, for example, by fracture, loss, or demineralization. In certain embodiments, the present invention provides methods for treating or preventing bone damage in an individual in need thereof by administering to the individual a therapeutically effective dose of an activin-ActRIIa antagonist (particularly an ActRIIa polypeptide). In certain embodiments, the present invention provides methods for promoting bone growth or mineralization in an individual in need thereof by administering to the individual a therapeutically effective dose of an activin-ActRIIa antagonist (particularly an ActRIIa polypeptide). These methods are preferably directed to therapeutic and prophylactic treatment in animals, and more preferably, to humans. In certain embodiments, the present invention provides uses of activin-ActRIIa antagonists (particularly soluble ActRIIa polypeptides and neutralizing antibodies targeting activin or ActRIIa) for treating diseases associated with low bone density or reduced bone strength.

As used herein, a therapeutic agent that "prevents" a disease or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disease or condition in treated subjects relative to untreated subjects, or delays the onset of or lessens the severity of one or more symptoms of the disease or condition relative to untreated subjects. As used herein, the term "treating" encompasses both preventing a specified condition and ameliorating or eliminating such a condition once established. In either case, the consideration of prevention or treatment, as well as the expected outcome of administering the therapeutic agent, is left to the discretion of the diagnosing physician.

The present invention provides methods for increasing bone and/or cartilage formation, preventing bone loss, increasing bone mineralization, or preventing bone demineralization. For example, target activin-ActRIIa antagonists have been used to treat osteoporosis and heal bone fractures and cartilage defects in humans or animals. ActRIIa or activin polypeptides are effective in patients diagnosed with subclinical low bone density as a protective measure against the development of osteoporosis.

In a specific embodiment, the methods and compositions of the present invention have medical utility in fracture healing and cartilage defects in humans and other animals. The subject methods and compositions also have preventive applications in closed and open fracture reduction and improved fixation of artificial joints. De novo bone formation induced by osteogenic agents helps repair craniofacial deformities caused by congenital, traumatic, or tumor resection, and is also effective in medical cosmetology. In certain examples, the subject activin-ActRIIa antagonists can provide an environment that attracts bone-forming cells, stimulates the growth of bone-forming cells, or induces the differentiation of bone-forming precursor cells. The activin-ActRIIa antagonists of the present invention are also effective in the treatment of osteoporosis.

The methods and compositions of the present invention can be applied to conditions characterized by or resulting in bone loss, such as osteoporosis (including secondary osteoporosis), hyperparathyroidism, Cushing's disease, Paget's disease, hyperthyroidism, chronic diarrhea or malabsorption, renal tubular acidosis, or anorexia nervosa.

Osteoporosis can be caused by or associated with various factors. For women, especially those after menopause, low body weight and the resulting sedentary behavior are risk factors for osteoporosis (loss of bone mineral density, leading to the risk of fractures). People with any of the following characteristics may be candidates for treatment with an ActRIIa antagonist: women who are postmenopausal and not taking estrogen or other hormone replacement therapy; tall (over 5 feet 7 inches) or thin (less than 125 pounds) postmenopausal women; men with clinical conditions associated with bone loss; people who use medications known to cause bone loss, including glucocorticoids such as prednisone (Prednisone ^™ ), various anticonvulsants such as phenytoin (Dilantin ^™ ), and certain barbiturates, or high-dose thyroid replacement medications; people with type 1 diabetes, liver disease, kidney disease, or a family history of osteoporosis; people with high bone turnover (e.g., excess collagen in urine); people with thyroid conditions such as hyperthyroidism; people with fractures after minor trauma; and people with x-ray evidence of vertebral fractures or other signs of osteoporosis.

As mentioned above, osteoporosis can also be a condition associated with another disease or the use of certain medications. Osteoporosis caused by medications or other medical conditions is called secondary osteoporosis. In a condition called Cushing's disease, the body produces too much cortisol, which leads to osteoporosis and fractures. The most common medications associated with secondary osteoporosis are glucocorticoids, a class of drugs that act like cortisol, a natural hormone produced by the adrenal glands. Although adequate levels of thyroid hormone (which is produced by the thyroid gland) are necessary for bone development, excess thyroid hormone can permanently reduce bone mass. Bone loss can occur when people with kidney problems, especially those on dialysis, take high doses of antacids containing aluminum. Other medications that can cause secondary osteoporosis include phenytoin (Dilantin) and barbiturates, used to prevent epilepsy; methotrexate (Rheumatrex, Immunex, Folex PFS), a drug used for some types of arthritis, cancer, or immune disorders; cyclosporine (Sandimmune, Neoral), a drug used to treat some autoimmune diseases and to suppress the immune system in organ transplant patients; luteinizing hormone-releasing hormone agonists (Lupron, Zoladex), used to treat prostate cancer and endometriosis; heparin (Calciparine, Liquaemin), an anticoagulant; and cholestyramine (Questran) and colestipol (Colestid), used to treat high cholesterol. Bone loss caused by cancer treatment is widely recognized and is referred to as cancer therapy-induced bone loss (CTIBL). Bone metastases can cause holes in the bones, which can be treated and corrected with activin-ActRIIa antagonists.

In a preferred embodiment, the disclosed activin-ActRIIa antagonists, particularly soluble ActRIIa, can be used in cancer patients. Patients with certain tumors (e.g., prostate, breast, multiple myeloma, or any tumor that causes hyperthyroidism) are at high risk for bone loss due to tumor-induced bone loss, bone metastases, and therapeutic agents. Even in the absence of evidence of bone loss or bone metastases, such patients can be treated with activin-ActRIIa antagonists. Patients can also be monitored for evidence of bone loss or bone metastases and treated with activin-ActRIIa antagonists when indicators indicate an increased risk. Typically, changes in bone density are assessed using DEXA (dual-energy X-ray absorptiometry) scans, and indicators of bone remodeling can be used to assess the likelihood of bone metastases. Serum markers can be monitored. Bone-specific alkaline phosphatase (BSAP) is an enzyme present in osteoblasts. Blood levels of BSAP are elevated in patients with bone metastases and other conditions that lead to increased bone remodeling. Osteocalcin and procollagen peptides are also associated with bone formation and metastasis. Elevated levels of BSAP have been detected in patients with bone metastases from prostate cancer and, to varying degrees, in bone metastases from breast cancer. High levels of bone morphogenetic protein-7 (BMP-7) are present in prostate cancer that has metastasized to the bone, but not in bone metastases from bladder, skin, liver, or lung cancer. Type I carboxyl-terminal peptide (ICTP) is a crosslinker found in collagen formed during bone resorption. Because bone is constantly broken down and remodeled, ICTP is found throughout the body. However, levels are significantly higher in bone metastases than in areas of normal bone. High levels of ICTP are found in bone metastases from prostate, lung, and breast cancer. Another collagen crosslinker, type I amino-terminal peptide (NTx), is produced along with ICTP during bone turnover. NTx levels are elevated in bone metastases from a variety of cancers, including lung, prostate, and breast cancer. Furthermore, rising NTx levels accompany the progression of bone metastases. Therefore, this marker can be used to detect bone metastases and measure the extent of disease. Other uptake markers include pyridinium ether and deoxypyridinium ether. Any increase in uptake markers and bone metastasis markers indicates that the patient needs treatment with an activin-ActRIIa antagonist.

Activin-ActRIIa antagonists can be administered in combination with other pharmaceutical agents. Co-administration can be accomplished through a single combined formulation, simultaneous administration, or intermittent administration. Activin-ActRIIa antagonists may have particular advantages if administered together with other bone activators. Patients may benefit from administering activin-ActRIIa antagonists in combination with calcium supplements, vitamin D, appropriate exercise, and/or (in some cases) other medications. Examples of other medications include bisphosphonates (alendronate, ibandronate, and risedronate), calcitonin, estrogen, parathyroid hormone, and raloxifene. Bisphosphonates (alendronate, ibandronate, and risedronate), calcitonin, estrogen, parathyroid hormone, and raloxifene affect the bone remodeling cycle and are classified as antiresorptive drugs. Bone remodeling consists of two distinct phases: bone resorption and bone formation. Antiresorptive drugs slow or halt the bone resorption portion of the bone remodeling cycle but do not slow the bone formation portion of the cycle. As a result, the rate of new formation continues to be greater than the rate of absorption, and bone density continues to increase. Parathyroid hormone fragment, a form of parathyroid hormone, increases the rate of bone formation in the bone remodeling cycle. Alendronate has been approved for the prevention (5 mg per day or 35 mg once a week) and treatment (10 mg per day or 70 mg once a week) of postmenopausal osteoporosis. Alendronate reduces bone loss, increases bone density, and reduces the risk of fractures of the spine, wrist, and hip. Alendronate is also approved for the treatment of glucocorticoid-induced osteoporosis caused by long-term use of these drugs (i.e., prednisone and cortisone) in men and women, as well as for the treatment of osteoporosis in men. Alendronate plus vitamin D is approved for the treatment of postmenopausal osteoporosis in women (70 mg once a week, plus vitamin D), and for treatment to improve bone mass in men with osteoporosis. Ibandronate is approved for the prevention and treatment of postmenopausal osteoporosis. Taken once monthly (150 mg), ibandronate must be taken on the same day each month. Ibandronate reduces bone loss, increases bone density, and reduces the risk of spinal fractures. Risedronate is approved for the prevention and treatment of postmenopausal osteoporosis. Taken daily (5 mg dose) or weekly (35 mg dose or 35 mg dose plus calcium), risedronate slows bone loss, increases bone density, and reduces the risk of spinal and non-spinal fractures. Risedronate is also approved for use in men and women to prevent and/or treat glucocorticoid-induced osteoporosis caused by long-term use of these medications (i.e., prednisone and cortisone). Calcitonin is a naturally occurring hormone involved in calcium regulation and bone metabolism. In women five years after menopause, calcitonin slows bone loss, increases spinal bone density, and may reduce pain associated with fractures. Calcitonin reduces the risk of spinal fractures. Calcitonin is available as an injection (50-100 IU per day) or nasal spray (200 IU per day). Estrogen therapy (ET)/hormonal therapy (HT) is approved for the prevention of osteoporosis. ET has been shown to reduce bone loss, increase bone density in the spine and hip, and reduce the risk of hip and spine fractures in postmenopausal women. ET is most commonly administered in the form of a pill or skin patch, which releases a low dose of about 0.3 mg per day or a standard dose of about 0.625 mg per day, and remains effective even after the age of 70. When taken alone, estrogen can increase the risk of developing cervical cancer (endometrial cancer) in women. To eliminate this risk, healthcare providers prescribe a combination of progesterone hormone and estrogen for women with an intact uterus. ET/HT alleviates menopausal symptoms and has been shown to have a beneficial effect on bone health. Side effects include vaginal bleeding, breast tenderness, mood swings, and gallbladder disease. Raloxifene, 60 mg per day, is approved for the prevention and treatment of postmenopausal osteoporosis. It is derived from a series of drugs called selective estrogen receptor modulators (SERMs), which are developed to provide the beneficial effects of estrogen without potential defects. Raloxifene increases the risk of estimated and reduced spinal fractures. Still no data are available to prove that raloxifene can reduce the risk of hip and non-other non-spinal fractures. This drug stimulates the formation of new bones and significantly increases bone mineral density. In postmenopausal women, fracture reduction of the spine, hip, foot, ribs and wrist is significant. In men, fracture reduction in the spine is significant, but there are not enough data to assess fracture reduction in other positions. Parathyroid hormone fragments are self-administered, as injected 24 hours a day.

7. Pharmaceutical Compositions

In certain embodiments, the activin-ActRIIa antagonists of the present invention (e.g., ActRIIa polypeptides) are formulated with a pharmaceutically acceptable carrier. For example, the ActRIIa polypeptides can be administered alone or as a component of a formulation (therapeutic composition). The compounds can be administered by any route suitable for human or veterinary use.

In certain embodiments, the therapeutic methods of the present invention include systemic administration of the composition or local administration as an implant or device. When administered, the therapeutic compositions used in the present invention are, of course, in a pyrogen-free, physiologically acceptable form. Therapeutically effective agents other than ActRIIa antagonists may optionally be included in the compositions described above and may be administered simultaneously or sequentially with the compound of interest (e.g., ActRIIa polypeptide) in the methods of the present invention.

Typically, ActRIIa antagonists are administered parenterally. Pharmaceutical compositions suitable for parenteral administration include one or more ActRIIa polypeptides in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solvents, dispersants, suspensions, or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions as previously described, which may contain antioxidants, buffers, antimicrobials, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening drugs. Examples of suitable aqueous or non-aqueous carriers for use in pharmaceutical compositions of the present invention include water, ethanol, polyols (such as 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 a coating material (such as lecithin), by maintaining the desired particle size in the case of dispersions, and by the use of surfactants.

Furthermore, the compositions can be encapsulated or injected for release to a target tissue site (e.g., bone). In certain embodiments, the compositions of the present invention include a matrix that can release one or more therapeutic compounds (e.g., ActRIIa polypeptides) to a target tissue site (e.g., bone), providing a structure for developing tissues and, ideally, allowing for absorption into the body. For example, the matrix can provide a slow release of the ActRIIa polypeptide. Such matrices can be formed from existing materials used for other implantable medical applications.

The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, appearance, and interfacial properties. The specific application of the target composition will determine the appropriate formulation. Potential matrices for the composition are biodegradable and chemically clear calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, and polyanhydrides. Other potential materials are biodegradable and biologically very clear, such as bone or dermal collagen. Other matrices include pure proteins or extracellular matrix components. Other potential matrices are non-biodegradable and chemically clear, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. The matrix can be composed of a combination of any of the above-mentioned types of materials (such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate). Bioceramics can be varied in the composition, such as in calcium-aluminum-phosphate and in treatments that change pore size, particle size, particle shape, and biodegradability.

In certain embodiments, the methods of the present invention can be administered orally, for example, in the form of capsules, cathet, pills, tablets, lozenges (using a flavored base, typically sucrose and gum arabic or tragacanth), powders, granules, or as a solution or suspension in water or a non-aqueous solvent, or as a water-in-oil or oil-in-water liquid emulsion, or as an elixir or syrup, or as a lozenge (using an inert base, such as gelatin and glycerin, or sucrose and gum arabic) and/or a mouthwash, etc., each containing a predetermined amount of the active ingredient. The agent can also be administered in the form of a pill, a syrup, or a paste.

In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), one or more therapeutic compounds of the invention may be mixed with one or more pharmaceutically acceptable carriers (such as sodium citrate or dicalcium phosphate), and/or any of the following: (1) fillers or diluents, such as starch, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, methylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or gum arabic; (3) preservatives. Wetting agents such as glycerol; (4) disintegrants such as agar-agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution-slow-release agents such as paraffin; (6) absorption promoters such as quaternary ammonium compounds; (7) wetting agents such as, for example, cetyl alcohol and glyceryl monostearate; (8) absorbents such as kaolin and bentonite; (9) lubricants such as talc, calcium stearate, magnesium oxychloride and solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof; (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical composition may contain a buffering agent. Similar types of solid compositions can also be used as fillers for soft-filled and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, syrups and elixirs. In addition to the active ingredient, the liquid dosage form comprises an inert diluent (such as water or other solvents), a solubilizing agent and an emulsifier (such as alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, phenylpropanol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oil (especially cottonseed, peanut, corn, germ, olive, castor and sesame oil), glycerol, tetrahydrofuran methanol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof, which are commonly used in the art. In addition to the inert diluent, oral compositions also include adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, spices, coloring agents, flavoring and preservatives.

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

The compositions of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of microbial activity can be ensured by including various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, etc.). If necessary, isotonic agents such as sugars and sodium chloride may also be included in the composition. Furthermore, prolonged absorption of injectable drugs can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

Of course, the dosage regimen will be determined by the attending physician, taking into account various factors that may alter the effects of the subject compounds of the invention (e.g., ActRIIa polypeptides). These factors include, but are not limited to, the amount of bone to be formed, the extent of bone density loss, the location of the bone lesion, the condition of the damaged bone, the patient's age, sex, and diet, the severity of any conditions that promote bone loss, the timing of administration, and other clinical factors. The dosage may vary depending on the type of matrix used in the reconstruction and the type of compound in the composition. Other known growth factors added to the final composition may also affect the dosage. Progress can be monitored by periodic assessment of bone growth and/or repair (e.g., X-rays (including DEXA), histomorphometry, and tetracycline labeling).

Studies in mice have demonstrated that the effects of ActRIIa-Fc on bone are detectable when the compound is administered at intervals sufficient to achieve blood concentrations of 0.2 μg/kg or higher, and that serum drug levels of 1 μg/kg or 2 μg/kg or higher are necessary to achieve significant effects on bone density and strength. Although there is no indication that higher doses of ActRIIa-Fc are undesirable due to side effects, treatment regimens are designed to achieve blood concentrations between 0.2 and 15 μg/kg, and optionally between 1 and 5 μg/kg. In humans, serum drug levels of 0.2 μg/kg can be achieved with a single dose of 0.1 mg/kg or higher, and serum drug levels of 1 μg/kg can be achieved with a single dose of 0.3 mg/kg or higher. The observed serum half-life of the molecule is between about 20 and 30 days, substantially longer than most Fc fusion proteins, and thus long-lasting effective serum drug levels can be achieved, for example, by dosing of 0.2-0.4 mg/kg on a weekly or biweekly basis, or higher doses with longer intervals between doses. For example, a dose of 1-3 mg/kg can be used on a monthly or bimonthly basis, and the effect on the skeleton is sufficiently long-lasting that such a dose is only necessary once every 3, 4, 5, 6, 9, 12 or more months.

In certain embodiments, the present invention also provides gene therapy for in vivo production of ActRIIa polypeptides. Such therapy can achieve its therapeutic effect by introducing an ActRIIa polynucleotide sequence into cells or tissues suffering from the diseases listed above. Delivery of ActRIIa polynucleotide sequences can be accomplished using recombinant expression vectors such as chimeric viruses or colloidal dispersion systems. Preferred for therapeutic delivery of ActRIIa polynucleotide sequences is the use of targeted liposomes.

The various viral vectors taught herein that can be utilized for gene therapy include adenovirus, herpes virus, vaccinia, or preferably, RNA viruses such as retroviruses. Preferably, the retroviral vector is a derivative of a mouse or avian retrovirus. Examples of retroviruses into which a single exogenous 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). A number of other retroviruses can incorporate multiple genes. All of these vectors can transfer or incorporate genes with selectable markers so that transduced cells can be identified and generated. Retroviral vectors can be made target-specific by attaching, for example, sugars, glycolipids, or proteins. Preferably, targeting is achieved using antibodies. One skilled in the art will appreciate that specific polynucleotide sequences can be inserted into the retroviral genome or attached to viral envelope proteins to allow target-specific delivery of a retroviral vector containing an ActRIIa polynucleotide. In a preferred embodiment, the vector is targeted to bone or cartilage.

Alternatively, tissue structural 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.

Another targeted delivery system for ActRIIa polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and liposomal systems, including water-in-oil emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal dispersion system of the present invention is a liposome. Liposomes are artificial membrane vesicles that can be effectively used as delivery vehicles in vitro or in vivo. RNA, DNA, and intact viral particles can be encapsulated with the internal aqueous solution and dispersed into cells in a biologically active form (see, e.g., Fraley et al., Trends Biochem. Sci., 6:77, 1981). Methods for efficient gene transfection using liposome vectors are well known in the art, see, e.g., Mannino et al., Biotechniques, 6:682, 1988. Liposome compositions are typically a combination of phospholipids, often in combination with a steroid, particularly cholesterol. Other phosphate esters or other lipids can also be used. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

The example of lipid that can be used for producing liposome comprises phosphatidyl compound, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, cerebroside and ganglioside.The example of exemplary phospholipid comprises egg yolk phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.The targeting of liposome also may be based on, for example, organ specificity, cell specificity and organelle specificity and other known in the art.

DETAILED DESCRIPTION

Example

The present invention has been generally described herein and will be more readily understood by reference to the following examples, which are included merely to illustrate certain embodiments and certain embodiments of the invention and are not intended to limit the invention.

Example 1: ActRIIa-Fc fusion protein

Applicants have constructed soluble ActRIIa fusion proteins comprising the human ActRIIa extracellular domain fused to either a human or mouse Fc domain with a minimal linker between the two. The constructs are referred to as ActRIIa-hFc and ActRIIa-mFc, respectively.

ActRIIa-hFc (SEQ ID NO: 7) purified from a CHO cell line is as follows:

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSI

EIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEV

TOPTSNPVTPKPP TGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV

TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVL

HODWLNGKEYKCKVSNKALPVPIEKTISKAKGOPREPOVYTLPPSREEMTK

NOVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWOOGNVFSCSV MHEALHNHYTOKSLSLSPGK

ActRIIa-hFc and ActRIIa-mFc proteins were expressed in a CHO cell line. Three different leader sequences were considered:

(i) Bee melittin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ ID NO: 8)

(ii) Tissue plasminogen activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 9)

(iii) Natural: MGAAAKL AFAVFLISCS SGA (SEQ ID NO: 10)

The selected version used a TPA leader and had the following unprocessed amino acid sequence:

MDAMKRGLCCVLLLCGAVFVSPGAAILGRSETQECLFFNANWEKDRTNQT

GVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKD

SPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPPTPKPPTGGGTHTCPPCPA

PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE

VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIE

KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG

QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK(SEQID NO: 13)

The peptide is encoded by the following nucleotide sequence:

ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAG

CAGTCTTCGTTTCGCCCGGCGCCGCTATACTTGGTAGATCAGAAAACTCAG

GAGTGTCTTTTTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAAC

TGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTG

CTACCTGGAAGAATATTTCTGGTTCCATGAATAGTGAAACAAGGTTGTT

GGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAA

AGACAGCCCTGAAGTATATTTCTGTTGCTGTGAGGGCAATATGTGTAATG

AAAAGTTTCTTATTTTCCGGAGATGGAAGTCACACAGCCCACTTCAAAT

CCAGTTACACCTAAGCCACCCACCGGTGGTGGAACTCACACATGCCCAC

CGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCC

CCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT

GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTG

GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGA

GGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCCTCACCGTCCTG

CACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA

AAGCCCTCCCAGTCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCA

GCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATG

ACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCA

GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT

ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCCTCTAT

AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCT

CATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGGTAAATGAGAATTC (SEQ ID NO: 14)

Both ActRIIa-hFc and ActRIIa-mFc were remarkably amenable to recombinant expression. As shown in Figure 1, the protein purified to a single, well-defined protein peak. N-terminal sequencing revealed a single sequence, ILGRSETQE (SEQ ID NO: 11). Purification can be achieved by a series of column chromatography steps, including, for example, three or more of the following steps, in any order: Protein A chromatography, Q Sepharose chromatography, Phenyl Sepharose chromatography, size exclusion chromatography, and cation exchange chromatography. Purification can be accomplished by viral filtration and buffer exchange. The ActRIIa-hFc protein was purified to a purity greater than 98% as determined by size exclusion chromatography and greater than 95% by SDS PAGE.

ActRIIa-hFc and ActRIIa-mFc display high affinity for ligands, particularly activin A. GDF-11 or Activin A ("ACA") was immobilized on a Biacore CM5 chip using a standard amino acid coupling procedure. ActRIIa-hFc and ActRIIa-mFc proteins were loaded onto the system, and their binding was measured. ActRIIa-hFc bound to activin with a dissociation constant ( _KD ) of 5× ^10⁻¹² , while the protein bound to GDF11 with a _KD of 9.96× ^10⁻¹⁺ . See Figure 2. The results for ActRIIa-mFc were similar.

The A-204 reporter gene assay evaluates the effect of ActRIIa-hFc protein on signaling via GDF-11 and Activin A. Cell line: Human rhabdomyosarcoma (muscle origin). Reporter vector: pGL3(CAGA)12 (Described in Dennler et al., 1998, EMBO 17:3091-3100). See Figure 3. The CAGA12 motif is present in the TGF-beta-responsive gene (PAI-1), so this vector is commonly used for signaling factors through Smad2 and 3.

Day 1: Split A-204 cells into 48-well plates.

The next day: A-204 cells were transfected with 10 μg of pGL3(CAGA)12 or pGL3(CAGA)12 (10 μg) + pRLCMV (1 μg) and Fugene.

Day 3: Factors were added (diluted in 0.1% BSA matrix). Inhibitors were pre-incubated with factors for 1 hour before addition to cells. After 6 hours, cells were washed with PBS and lysed.

This was followed by a luciferase assay. Typically, in this assay, without any inhibitors, Activin A stimulated reporter gene expression approximately 10-fold with an ED50 of ~2 ng/mL. GDF-11 stimulated the expression of the reporter gene 16-fold with an ED50 of ~1.5 ng/mL. GDF-8 exhibited similar effects to GDF-11.

As shown in Figure 4, ActRIIa-hFc and ActRIIa-mFc inhibited GDF-8-mediated signaling at picomolar concentrations. As shown in Figure 5, three different ActRIIa-hFc formulations inhibited GDF-11 signaling with IC50s approaching 200 pM.

ActRIIa-hFc is very stable in pharmacokinetic analysis. Rats were administered 1 mg/kg, 3 mg/kg, or 10 mg/kg of ActRIIa-hFc polypeptide, and plasma drug levels of the protein were measured at 24, 48, 72, 144, and 168 hours. In a separate study, rats were dosed with 1 mg/kg, 10 mg/kg, or 30 mg/kg. In rats, ActRIIa-hFc has a serum half-life of 11-14 days and circulating levels of the drug are very high after 2 weeks (11 μg/ml, 110 μg/ml, or 304 μg/ml, for a starting dose of 1 mg/kg, 10 mg/kg, or 30 mg/kg, respectively). In cynomolgus monkeys, the plasma half-life was substantially greater than 14 days and the circulating levels of the drug were 25 μg/ml, 304 μg/ml, or 1440 μg/ml (corresponding to starting concentrations of 1 mg/kg, 10 mg/kg, or 30 mg/kg, respectively). Preliminary results in humans showed that the serum drug half-life was between approximately 20 and 30 days.

Example 2: ActRIIa-hFc promotes bone growth in vivo

Normal female mice (BALB/c) were administered ActRIIa-mFc at 1 mg/kg/dose, 3 mg/kg/dose, or 10 mg/kg/dose twice a week, and bone mineral density and content were determined by DEXA, see Figure 6 .

In female BALB/c mice, DEXA scans showed a significant increase (>20%) in bone mineral density and content as a result of ActRIIa-mFc treatment. See Figures 7 and 8.

Thus, an ActRIIa antagonist induces an increase in bone density and bone mass in normal female mice. As a follow-up step, the effect of ActRIIa-mFc was tested in an ovariectomized mouse model.

Andersson et al. (2001) determined that ovariectomized mice undergo considerable bone loss (approximately 50% of cancellous bone lost 6 weeks after surgery) and that bone loss in these mice can be treated by candidate therapeutic agents such as parathyroid hormone.

Applicants used 4-5 week old ovariectomized (OVX) female C57BL6 mice or sham-operated female mice. Eight weeks after surgery, they began treatment with ActRIIa-mFc (10 mg/kg, twice weekly) or control (PBS). Bone density was measured by CT scan.

As shown in Figure 9, after 6 weeks, untreated, ovariectomized mice showed comparable loss of cancellous bone density relative to sham-operated mice. ActRIIa-mFc-treated mice retained sham-level bone density. At both 6 and 12 weeks of treatment, ActRIIa-mFc induced comparable increases in cancellous bone in OVX mice. See Figure 10. After 6 weeks of treatment, bone density increased by 24% relative to the PBS control group. After 12 weeks, the increase was 27%.

In sham-operated mice, ActRIIa-mFc also caused a comparable increase in cancellous bone. See Figure 11. After 6 and 12 weeks, treatment resulted in a 35% increase relative to controls.

In another set of experiments, ovariectomized (OVX) mice or sham-operated mice as described above were treated with ActRIIa-mFc (10 mg/kg twice a week) or control (PBS) for more than 12 weeks. Similar to the results described above for ActRIIa-mFc, OVX mice receiving ActRIIa-mFc showed a 15% increase in cancellous bone density as early as 4 weeks of treatment and an increase of 15% after 12 weeks of treatment (Figure 12). Sham-operated mice receiving ActRIIa-mFc also showed a 25% increase in cancellous bone density as early as 4 weeks of treatment and an increase of 32% after 12 weeks of treatment (Figure 13).

After 12 weeks of ActRIIa-mFc treatment, whole-body and ex vivo femoral DEXA analysis showed that treatment induced an increase in bone density in both ovariectomized and sham-operated mice (Figures 14A and 14B, respectively). These results were supported by ex vivo pQCT analysis of the mid-femur, which demonstrated a significant increase in both total and cortical bone density after 12 weeks of ActRIIa-mFc treatment. Vehicle-treated control ovariectomized mice exhibited bone density comparable to vehicle-treated control sham-operated mice (Figure 15). In addition to bone density, bone mass also increased with ActRIIa-mFc treatment. Ex vivo pQCT analysis of the mid-femur demonstrated a significant increase in both total and cortical bone mass after 12 weeks of ActRIIa-mFc treatment, while ovariectomized mice and sham-operated vehicle-treated mice exhibited comparable bone mass (Figure 16). Ex vivo pQCT analysis of the femur midshaft also showed that ActRIIa-mFc-treated mice showed no changes in periosteal circumference, while ActRIIa-mFc treatment resulted in a decrease in endosteal circumference, suggesting that the increase in cortical thickness was caused by growth on the inner surface of the femur (Figure 17). Mechanical testing of the femur confirmed that ActRIIa-mFc can increase extrinsic properties of bone (maximum load, stiffness, and fracture energy), which contributes to a significant increase in intrinsic bone properties (ultimate strength). Ovariectomized mice treated with ActRIIa-mFc demonstrated increased bone strength above sham-operated, vehicle-treated control levels, indicating complete reversal of the osteoporotic phenotype (Figure 18).

These data demonstrate that an activin-ActRIIa antagonist increases bone density in female mice and, further, corrects defects in bone density, bone mass, and maximum bone strength in a mouse model of osteoporosis.

In another set of experiments, mice were ovariectomized or sham-operated at week 4 and received either placebo or ActRIIa-mFc (10 mg/kg twice weekly) (e.g., RAP-11 in Figures 19-24) starting at week 12 for an additional 12 weeks. Various bone parameters were assessed. As shown in Figure 19, ActRIIa-mFc increased the ratio of spinal cancellous bone volume to total volume (BV/TV) in both the OVX and sham-operated groups. ActRIIa-mFc also improved cancellous bone structure (Figure 20), increased periosteal thickness (Figure 21), and improved bone strength (Figure 22). As shown in Figure 23, ActRIIa-mFc produced a satisfactory effect across a dose range of 1 mg/kg to 10 mg/kg.

Bone histomorphometry was performed at the 2-week time point in sham-operated mice. These data, presented in Figure 24, demonstrate that ActRIIa-mFc has a dual effect, inhibiting bone resorption and promoting bone growth. Thus, ActRIIa-mFc promotes bone growth (anabolic effect) and inhibits bone resorption (anti-catabolic effect).

Example 4: Alternative ActRIIa-Fc Proteins

An alternative construct can delete the last 15 amino acids of the C-terminal tail of the ActRIIa extracellular domain. The sequence of such a construct is as follows: (the Fc portion is underlined) (SEQ ID NO: 12):

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSI

EIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM TG

GGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV

KFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKEYKCK

VSNKALVPIEKTISKAKGOPREPOVYTLPPSREEMTKNOVSLTCLVKGFYPS

DIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCS VMHEALHNHYTOKSLSLSPGK

Integration of references

All publications and patents mentioned herein are hereby 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.

While specific embodiments of the subject matter have been discussed, the above description is illustrative and non-limiting. Numerous variations will be apparent to those skilled in the art upon reading the above description and the following claims. The full scope of the present invention should be determined by reference to the claims, their equivalents, the description, and such variations.

Sequence Listing

<110> Acceleron Pharma Inc.

<120> Activin-ActRIIa antagonists and their use in promoting bone growth

<140> WO 2007/062188

<141> 2006-11-22

<150> US 60/739,462

<151> 2005-11-23

<150> US 60/783,322

<151> 2006-03-17

<150> US 60/844,855

<151> 2006-09-15

<160> 14

<170> FastSEQ for Windows Version 4.0

<210> 1

<211> 513

<212> PRT

<213> Homo sapiens

<400> 1

Met Gly Ala Ala Ala Lys Leu Ala Phe Ala Val Phe Leu Ile Ser Cys

1 5 10 15

Ser Ser Gly Ala Ile Leu Gly Arg Ser Glu Thr Gln Glu Cys Leu Phe

20 25 30

Phe Asn Ala Asn Trp Glu Lys Asp Arg Thr Asn Gln Thr Gly Val Glu

35 40 45

Pro Cys Tyr Gly Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp

50 55 60

Lys Asn Ile Ser Gly Ser Ile Glu Ile Val Lys Gln Gly Cys Trp Leu

65 70 75 80

Asp Asp Ile Asn Cys Tyr Asp Arg Thr Asp Cys Val Glu Lys Lys Asp

85 90 95

Ser Pro Glu Val Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys Asn Glu

100 105 110

Lys Phe Ser Tyr Phe Pro Glu Met Glu Val Thr Gln Pro Thr Ser Asn

115 120 125

Pro Val Thr Pro Lys Pro Pro Tyr Tyr Asn Ile Leu Leu Tyr Ser Leu

130 135 140

Val Pro Leu Met Leu Ile Ala Gly Ile Val Ile Cys Ala Phe Trp Val

145 150 155 160

Tyr Arg His His Lys Met Ala Tyr Pro Pro Val Leu Val Pro Thr Gln

165 170 175

Asp Pro Gly Pro Pro Pro Pro Ser Pro Leu Leu Gly Leu Lys Pro Leu

180 185 190

Gln Leu Leu Glu Val Lys Ala Arg Gly Arg Phe Gly Cys Val Trp Lys

195 200 205

Ala Gln Leu Leu Asn Glu Tyr Val Ala Val Lys Ile Phe Pro Ile Gln

210 215 220

Asp Lys Gln Ser Trp Gln Asn Glu Tyr Glu Val Tyr Ser Leu Pro Gly

225 230 235 240

Met Lys His Glu Asn Ile Leu Gln Phe Ile Gly Ala Glu Lys Arg Gly

245 250 255

Thr Ser Val Asp Val Asp Leu Trp Leu Ile Thr Ala Phe His Glu Lys

260 265 270

Gly Ser Leu Ser Asp Phe Leu Lys Ala Asn Val Val Ser Trp Asn Glu

275 280 285

Leu Cys His Ile Ala Glu Thr Met Ala Arg Gly Leu Ala Tyr Leu His

290 295 300

Glu Asp Ile Pro Gly Leu Lys Asp Gly His Lys Pro Ala Ile Ser His

305 310 315 320

Arg Asp Ile Lys Ser Lys Asn Val Leu Leu Lys Asn Asn Leu Thr Ala

325 330 335

Cys Ile Ala Asp Phe Gly Leu Ala Leu Lys Phe Glu Ala Gly Lys Ser

340 345 350

Ala Gly Asp Thr His Gly Gln Val Gly Thr Arg Arg Tyr Met Ala Pro

355 360 365

Glu Val Leu Glu Gly Ala Ile Asn Phe Gln Arg Asp Ala Phe Leu Arg

370 375 380

Ile Asp Met Tyr Ala Met Gly Leu Val Leu Trp Glu Leu Ala Ser Arg

385 390 395 400

Cys Thr Ala Ala Asp Gly Pro Val Asp Glu Tyr Met Leu Pro Phe Glu

405 410 415

Glu Glu Ile Gly Gln His Pro Ser Leu Glu Asp Met Gln Glu Val Val

420 425 430

Val His Lys Lys Lys Arg Pro Val Leu Arg Asp Tyr Trp Gln Lys His

435 440 445

Ala Gly Met Ala Met Leu Cys Glu Thr Ile Glu Glu Cys Trp Asp His

450 455 460

Asp Ala Glu Ala Arg Leu Ser Ala Gly Cys Val Gly Glu Arg Ile Thr

465 470 475 480

Gln Met Gln Arg Leu Thr Asn Ile Ile Thr Thr Glu Asp Ile Val Thr

485 490 495

Val Val Thr Met Val Thr Asn Val Asp Phe Pro Pro Lys Glu Ser Ser

500 505 510

Leu

<210> 2

<211> 115

<212> PRT

<213> Homo sapiens

<400> 2

Ile Leu Gly Arg Ser Glu Thr Gln Glu Cys Leu Phe Phe Asn Ala Asn

1 5 10 15

Trp Glu Lys Asp Arg Thr Asn Gln Thr Gly Val Glu Pro Cys Tyr Gly

20 25 30

Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp Lys Asn Ile Ser

35 40 45

Gly Ser Ile Glu Ile Val Lys Gln Gly Cys Trp Leu Asp Asp Ile Asn

50 55 60

Cys Tyr Asp Arg Thr Asp Cys Val Glu Lys Lys Asp Ser Pro Glu Val

65 70 75 80

Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys Asn Glu Lys Phe Ser Tyr

85 90 95

Phe Pro Glu Met Glu Val Thr Gln Pro Thr Ser Asn Pro Val Thr Pro

100 105 110

Lys Pro Pro

115

<210> 3

<211> 100

<212> PRT

<213> Homo sapiens

<400> 3

Ile Leu Gly Arg Ser Glu Thr Gln Glu Cys Leu Phe Phe Asn Ala Asn

1 5 10 15

Trp Glu Lys Asp Arg Thr Asn Gln Thr Gly Val Glu Pro Cys Tyr Gly

20 25 30

Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp Lys Asn Ile Ser

35 40 45

Gly Ser Ile Glu Ile Val Lys Gln Gly Cys Trp Leu Asp Asp Ile Asn

50 55 60

Cys Tyr Asp Arg Thr Asp Cys Val Glu Lys Lys Asp Ser Pro Glu Val

65 70 75 80

Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys Asn Glu Lys Phe Ser Tyr

85 90 95

Phe Pro Glu Met

100

<210> 4

<211> 1542

<212> DNA

<213> Homo sapiens

<400> 4

atgggagctg ctgcaaagtt ggcgtttgcc gtctttctta tctcctgttc ttcaggtgct 60

atacttggta gatcagaaac tcaggagtgt cttttcttta atgctaattg ggaaaaagac 120

agaaccaatc aaactggtgt tgaaccgtgt tatggtgaca aagataaacg gcggcattgt 180

tttgctacct ggaagaatat ttctggttcc attgaaatag tgaaacaagg ttgttggctg 240

gatgatatca actgctatga caggactgat tgtgtagaaa aaaaagacag ccctgaagta 300

tatttttgtt gctgtgaggg caatatgtgt aatgaaaagt tttcttattt tccagagatg 360

gaagtcacac agcccacttc aaatccagtt acacctaagc caccctatta caacatcctg 420

ctctattcct tggtgccact tatgttaatt gcggggattg tcatttgtgc attttgggtg 480

tacaggcatc acaagatggc ctaccctcct gtacttgttc caactcaaga cccaggacca 540

cccccacctt ctccattact agggttgaaa ccactgcagt tattagaagt gaaagcaagg 600

ggaagatttg gttgtgtctg gaaagcccag ttgcttaacg aatatgtggc tgtcaaaata 660

tttccaatac aggacaaaca gtcatggcaa aatgaatacg aagtctacag tttgcctgga 720

atgaagcatg agaacatatt acagttcatt ggtgcagaaa aacgaggcac cagtgttgat 780

gtggatcttt ggctgatcac agcatttcat gaaaagggtt cactatcaga ctttcttaag 840

gctaatgtgg tctcttggaa tgaactgtgt catattgcag aaaccatggc tagaggattg 900

gcatatttac atgaggatat acctggccta aaagatggcc acaaacctgc catatctcac 960

agggacatca aaagtaaaaa tgtgctgttg aaaaacaacc tgacagcttg cattgctgac 1020

tttgggttgg ccttaaaatt tgaggctggc aagtctgcag gcgataccca tggacaggtt 1080

ggtacccgga ggtacatggc tccagaggta ttagagggtg ctataaactt ccaaagggat 1140

gcatttttga ggatagatat gtatgccatg ggattagtcc tatgggaact ggcttctcgc 1200

tgtactgctg cagatggacc tgtagatgaa tacatgttgc catttgagga ggaaattggc 1260

cagcatccat ctcttgaaga catgcaggaa gttgttgtgc ataaaaaaaa gaggcctgtt 1320

ttaagagatt attggcagaa acatgctgga atggcaatgc tctgtgaaac cattgaagaa 1380

tgttgggatc acgacgcaga agccaggtta tcagctggat gtgtaggtga aagaattacc 1440

cagatgcaga gactaacaaa tattattacc acagaggaca ttgtaacagt ggtcacaatg 1500

gtgacaaatg ttgactttcc tcccaaagaa tctagtctat ga 1542

<210> 5

<211> 345

<212> DNA

<213> Homo sapiens

<400> 5

atacttggta gatcagaaac tcaggagtgt cttttcttta atgctaattg ggaaaaagac 60

agaaccaatc aaactggtgt tgaaccgtgt tatggtgaca aagataaacg gcggcattgt 120

tttgctacct ggaagaatat ttctggttcc attgaaatag tgaaacaagg ttgttggctg 180

gatgatatca actgctatga caggactgat tgtgtagaaa aaaaagacag ccctgaagta 240

tatttttgtt gctgtgaggg caatatgtgt aatgaaaagt tttcttattt tccagagatg 300

gaagtcacac agcccacttc aaatccagtt acacctaagc caccc 345

<210> 6

<211> 225

<212> PRT

<213> Homo sapiens

<220>

<221> Variants

<222> 43

<223> Xaa can represent Asp or Ala

<220>

<221> Variants

<222> 100

<223> Xaa can represent Lys or Ala

<220>

<221> Variants

<222> 212

<223> Xaa can represent Asn or Ala

<400> 6

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

1 5 10 15

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

20 25 30

Arg Thr Pro Glu Val Thr Cys Val Val Val Xaa Val Ser His Glu Asp

35 40 45

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

50 55 60

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

65 70 75 80

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

85 90 95

Tyr Lys Cys Xaa Val Ser Asn Lys Ala Leu Pro Val Pro Ile Glu Lys

100 105 110

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

115 120 125

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

130 135 140

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

145 150 155 160

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

165 170 175

Asp Ser Asp Gly Pro Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

180 185 190

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

195 200 205

Ala Leu His Xaa His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

210 215 220

Lys

225

<210> 7

<211> 344

<212> PRT

<213> Homo sapiens

<400> 7

Ile Leu Gly Arg Ser Glu Thr Gln Glu Cys Leu Phe Phe Asn Ala Asn

1 5 10 15

Trp Glu Lys Asp Arg Thr Asn Gln Thr Gly Val Glu Pro Cys Tyr Gly

20 25 30

Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp Lys Asn Ile Ser

35 40 45

Gly Ser Ile Glu Ile Val Lys Gln Gly Cys Trp Leu Asp Asp Ile Asn

50 55 60

Cys Tyr Asp Arg Thr Asp Cys Val Glu Lys Lys Asp Ser Pro Glu Val

65 70 75 80

Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys Asn Glu Lys Phe Ser Tyr

85 90 95

Phe Pro Glu Met Glu Val Thr Gln Pro Thr Ser Asn Pro Val Thr Pro

100 105 110

Lys Pro Pro Thr Gly Gly Gly Thr His Thr Cys Pro Pro Cys Pro Ala

115 120 125

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

130 135 140

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

145 150 155 160

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

165 170 175

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

180 185 190

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

195 200 205

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

210 215 220

Leu Pro Val Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

225 230 235 240

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

245 250 255

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

260 265 270

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

275 280 285

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

290 295 300

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

305 310 315 320

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

325 330 335

Ser Leu Ser Leu Ser Pro Gly Lys

340

<210> 8

<211> 21

<212> PRT

<213> Bee

<400> 8

Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile

1 5 10 15

Ser Tyr Ile Tyr Ala

20

<210> 9

<211> 22

<212> PRT

<213> Homo sapiens

<400> 9

Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

1 5 10 15

Ala Val Phe Val Ser Pro

20

<210> 10

<211> 20

<212> PRT

<213> Homo sapiens

<400> 10

Met Gly Ala Ala Ala Lys Leu Ala Phe Ala Val Phe Leu Ile Ser Cys

1 5 10 15

Ser Ser Gly Ala

20

<210> 11

<211> 9

<212> PRT

<213> Homo sapiens

<400> 11

Ile Leu Gly Arg Ser Glu Thr Gln Glu

1 5

<210> 12

<211> 329

<212> PRT

<213> Homo sapiens

<400> 12

Ile Leu Gly Arg Ser Glu Thr Gln Glu Cys Leu Phe Phe Asn Ala Asn

1 5 10 15

Trp Glu Lys Asp Arg Thr Asn Gln Thr Gly Val Glu Pro Cys Tyr Gly

20 25 30

Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp Lys Asn Ile Ser

35 40 45

Gly Ser Ile Glu Ile Val Lys Gln Gly Cys Trp Leu Asp Asp Ile Asn

50 55 60

Cys Tyr Asp Arg Thr Asp Cys Val Glu Lys Lys Asp Ser Pro Glu Val

65 70 75 80

Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys Asn Glu Lys Phe Ser Tyr

85 90 95

Phe Pro Glu Met Thr Gly Gly Gly Thr His Thr Cys Pro Pro Cys Pro

100 105 110

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

115 120 125

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

130 135 140

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

145 150 155 160

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

165 170 175

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

180 185 190

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

195 200 205

Ala Leu Pro Val Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

210 215 220

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

225 230 235 240

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

245 250 255

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

260 265 270

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

275 280 285

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

290 295 300

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

305 310 315 320

Lys Ser Leu Ser Leu Ser Pro Gly Lys

325

<210> 13

<211> 369

<212> PRT

<213> Homo sapiens

<400> 13

Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

1 5 10 15

Ala Val Phe Val Ser Pro Gly Ala Ala Ile Leu Gly Arg Ser Glu Thr

20 25 30

Gln Glu Cys Leu Phe Phe Asn Ala Asn Trp Glu Lys Asp Arg Thr Asn

35 40 45

Gln Thr Gly Val Glu Pro Cys Tyr Gly Asp Lys Asp Lys Arg Arg His

50 55 60

Cys Phe Ala Thr Trp Lys Asn Ile Ser Gly Ser Ile Glu Ile Val Lys

65 70 75 80

Gln Gly Cys Trp Leu Asp Asp Ile Asn Cys Tyr Asp Arg Thr Asp Cys

85 90 95

Val Glu Lys Lys Asp Ser Pro Glu Val Tyr Phe Cys Cys Cys Glu Gly

100 105 110

Asn Met Cys Asn Glu Lys Phe Ser Tyr Phe Pro Glu Met Glu Val Thr

115 120 125

Gln Pro Thr Ser Asn Pro Val Thr Pro Lys Pro Pro Thr Gly Gly Gly

130 135 140

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

145 150 155 160

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

165 170 175

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

180 185 190

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

195 200 205

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

210 215 220

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

225 230 235 240

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Val Pro Ile Glu Lys

245 250 255

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

260 265 270

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

275 280 285

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

290 295 300

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

305 310 315 320

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

325 330 335

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

340 345 350

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

355 360 365

Lys

<210> 14

<211> 1114

<212> DNA

<213> Homo sapiens

<400> 14

atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60

tcgcccggcg ccgctatact tggtagatca gaaactcagg agtgtctttt tttaatgcta 120

attgggaaaa agacagaacc aatcaaactg gtgttgaacc gtgttatggt gacaaagata 180

aacggcggca ttgttttgct acctggaaga atatttctgg ttccattgaa tagtgaaaca 240

aggttgttgg ctggatgata tcaactgcta tgacaggact gattgtgtag aaaaaaaaga 300

cagccctgaa gtatatttct gttgctgtga gggcaatatg tgtaatgaaa agttttctta 360

ttttccggag atggaagtca cacagcccac ttcaaatcca gttacaccta agccacccac 420

cggtggtgga actcacacat gcccaccgtg cccagcacct gaactcctgg ggggacccgtc 480

agtcttcctc ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt 540

cacatgcgtg gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt 600

ggacggcgtg gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac 660

gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta 720

caagtgcaag gtctccaaca aagccctccc agtccccatc gagaaaacca tctccaaagc 780

caaagggcag ccccgagaac cacaggtgta caccctgccc ccatcccggg aggagatgac 840

caagaaccag gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt 900

ggagtgggag agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga 960

ctccgacggc tccttcttcc tctatagcaa gctcaccgtg gacaagagca ggtggcagca 1020

ggggacgtc ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa 1080

gagcctctcc ctgtctccgg gtaaatgaga attc 1114

Claims (42)

1. Use of the ActRIIa-Fc fusion protein in the preparation of a medicament for promoting bone growth, increasing bone density, inhibiting bone resorption, or increasing bone strength in subjects with such needs, wherein the fusion protein is selected from: a) A polypeptide containing the amino acid sequence of SEQ ID NO: 7; and b) wherein the ActRIIa portion of the fusion protein is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2; And the fusion protein therein binds to activin and/or GDF11. 2. The use of claim 1, wherein the ActRIIa portion of the fusion protein consists of the amino acid sequence of SEQ ID NO: 2. 3. The use of claim 1, wherein the fusion protein comprises one or more polypeptide domains in addition to the ActRIIa polypeptide domain, said polypeptide domains enhancing in vivo stability, in vivo half-life, uptake, tissue localization or distribution, protein complex formation, and purification of one or more of the following. 4. The use of claim 1, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 7. 5. The use of claim 1, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 7. 6. The use of claim 1, wherein the fusion protein has one or more of the following characteristics: i) bind to the ActRIIa ligand with at least ^10⁻⁷ M _K₂D ; and ii) Inhibit ActRIIa signaling in cells. 7. The use of claim 1, wherein the fusion protein comprises one or more modified amino acid residues selected from the group consisting of glycosylated amino acids, polyethylene glycol-modified amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, and amino acids coupled to a lipid moiety. 8. Use according to any one of claims 1-7, wherein the fusion protein is used to prepare a medicament for promoting bone growth in subjects with such needs. 9. Use according to any one of claims 1-7, wherein the fusion protein is used to prepare a medicament for increasing bone density in subjects with such need. 10. Use according to any one of claims 1-7, wherein the fusion protein is used to prepare a medicament for inhibiting bone resorption in a subject with such need. 11. Use according to any one of claims 1-7, wherein the fusion protein is used to prepare a medicament for increasing bone strength in subjects with such need. 12. Use according to any one of claims 1-7, wherein the fusion protein is bound to activin. 13. The use of claim 12, wherein the fusion protein is bound to activin A. 14. The use of claim 12, wherein the fusion protein is bound to activin B. 15. The use of claim 13 or 14, wherein the fusion protein is bound to GDF11. 16. Use according to any one of claims 1-7, wherein the fusion protein binds to activin and GDF11. 17. Use of the ActRIIa fusion protein in the preparation of a medicament for the treatment or prevention of bone-related diseases in subjects with such need, wherein the fusion protein is selected from: a) A polypeptide containing the amino acid sequence of SEQ ID NO: 7; and b) wherein the ActRIIa portion of the fusion protein is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2; And the fusion protein therein binds to activin and/or GDF11. 18. The use of claim 17, wherein the ActRIIa portion of the fusion protein consists of the amino acid sequence of SEQ ID NO: 2. 19. The use of claim 17, wherein the fusion protein comprises one or more polypeptide domains in addition to the ActRIIa polypeptide domain, said polypeptide domains enhancing in vivo stability, in vivo half-life, uptake, tissue localization or distribution, protein complex formation, and purification of one or more of the following. 20. The use of claim 17, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 7. 21. The use of claim 17, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 7. 22. The use of claim 17, wherein the fusion protein has one or more of the following properties: i) bind to the ActRIIa ligand with at least ^10⁻⁷ M _K₂D ; and ii) Inhibit ActRIIa signaling in cells. 23. The use of claim 17, wherein the fusion protein comprises one or more modified amino acid residues selected from the group consisting of glycosylated amino acids, PEGylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, and amino acids coupled to a lipid moiety. 24. Use according to any one of claims 17-23, wherein the bone-related disease is selected from the group consisting of primary osteoporosis and secondary osteoporosis. 25. Use according to any one of claims 17-23, wherein the bone-related disease is selected from the group consisting of: postmenopausal osteoporosis, hypogonadism-related bone loss, tumor-induced bone loss, cancer treatment-induced bone loss, bone metastases, multiple myeloma, and Plantar dysplasia. 26. Use according to any one of claims 17-23, wherein said object has cancer associated with bone metastases. 27. Use according to any one of claims 17-23, wherein the object is positive for indicators of bone density loss, bone resorption, or bone metastases. 28. Use according to any one of claims 17-23, wherein the object is a recipient of a cancer treatment regimen associated with bone loss. 29. Use according to any one of claims 17-23, wherein said object has cancer associated with bone loss. 30. Use according to any one of claims 17-23, wherein said object has kidney disease. 31. Use according to any one of claims 17-23, wherein the drug is used in combination with a second bone activator. 32. The use of claim 31, wherein the bone activator is selected from the group consisting of bisphosphonates, estrogens, selective estrogen receptor modulators, parathyroid hormone, calcitonin, calcium supplements, and vitamin D supplements. 33. The use of any one of claims 17-23, wherein the drug is administered such that the blood concentration of the fusion protein in the subject reaches at least 0.2 μg/kg. 34. Use according to any one of claims 17-23, wherein the fusion protein has a serum half-life between 15 and 30 days. 35. Use according to any one of claims 17-23, wherein the drug is administered to the subject at a frequency not exceeding once a week. 36. The use of any one of claims 17-23, wherein the drug is administered to the subject at a frequency not exceeding once a month. 37. Use according to any one of claims 17-23, wherein the fusion protein binds to activin. 38. The use of claim 37, wherein the fusion protein is bound to activin A. 39. The use of claim 37, wherein the fusion protein therein binds to activin B. 40. The use of claim 38 or 39, wherein the fusion protein is bound to GDF11. 41. Use according to any one of claims 17-23, wherein the fusion protein is bound to activin and GDF11. 42. Use according to any one of claims 17-23, wherein the skeletal-related disease is associated with bone loss, low bone density, and/or low bone strength.