CN101370511B - Activin-ActRIIa antagonist and its application in promoting bone growth - Google Patents

Abstract

In certain aspects, the present invention provides compositions and methods for promoting bone growth and increasing bone density.

Description

Activin-ActRIIa antagonist and its application in promoting bone growth

related application

This application claims Provisional Application No. 60/739,462 filed November 23, 2005, Provisional Application No. 60/783,322 filed March 17, 2006, and Provisional Application No. 60/844,855 filed September 15, 2006 The above-mentioned application is hereby incorporated by reference in its entirety.

Background technique

Skeletal disorders, 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 immobilization, exercise, and dietary changes. For the treatment of various skeletal disorders, it would be beneficial to have therapeutic agents that promote bone growth as well as increase bone density.

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

Fractures and other structural breaks in bone heal by a process that at least superficially resembles a developmental sequence of osteogenesis involving the formation of cartilage tissue followed by mineralization. The process of fracture healing can occur in two ways. Immediate or initial bone healing occurs without callus formation. Indirect or secondary bone healing occurs with a callous precursor stage. Primary healing of fractures involves the remodeling of mechanical connections across closely spaced fractures. Under suitable conditions, resorbing cells surrounding ruptured bone display a tunnel resorbing response and construct pathways for puncturing blood vessels and subsequent healing. Secondary healing of bone proceeds in the sequence of inflammatory response, soft callus formation, callus mineralization, and callus remodeling. During the inflammatory phase, hematomas and hemorrhages originate from rupture of the periosteum at the site of injury and rupture of endosteal vessels. Inflammatory cells invade the area. In the stage of soft callus formation, cells generate new blood vessels, fibroblasts, intracellular material, and supporting cells to form granulation tissue in the spaces between fracture fragments. Clinical healing across the rupture is established by fibrous or cartilaginous tissue (soft callus). Osteoblasts form and regulate the mineralization of the soft callus, which is then replaced by lamellar bone and is subject to normal remodeling processes.

In addition to fractures and other physical breakdowns in bone structure, loss of bone mineral content and bone density can be caused by a variety of different conditions and can lead to important medical problems. Changes in bone density occur in a relatively predictable manner throughout an individual's lifespan. By approximately age 30, the bones of both males and females grow to maximum mass through linear and radial growth of endochondral osteogenic growth discs. Slow bone loss occurs in both males and females around age 30 (for cancellous bone, eg, flat bones such as the vertebrae and pelvis) and 40 years (for cortical bone, eg, long bones in the limbs). In women, there is also a final stage of parenchymal bone loss, perhaps due to postmenopausal estrogen deficiency. During this period, women may lose an additional 10% of mass in the cortical bone and an additional 25% in the cancellous septum. Whether progressive bone mass loss leads to pathological conditions such as osteoporosis is critically dependent on the individual's initial bone mass and the presence or absence of exacerbating conditions.

Bone loss is sometimes characterized by an imbalance in the normal skeletal remodeling process. Healthy bones are often subject to remodeling. Remodeling begins with bone resorption by osteoclasts. The resorbed bone is then replaced by new bone tissue, characterized by collagen formation by osteoblasts and subsequent calcification. In a healthy individual, the rates of absorption and formation are balanced. Osteoporosis is a chronic, progressive condition marked by a shift toward resorption, resulting in an overall loss of bone mass as well as bone mineralization. Osteoporosis in humans precedes clinical osteopenia (bone mineral density 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 for osteoporosis.

Therefore, methods of controlling the activity of osteoclasts and osteoblasts are effective in promoting the healing of fractures and other injuries to the bone and in the treatment of diseases such as osteoporosis, which are associated with loss of bone mass and bone mineralization.

For osteoporosis, estrogen, calcitonin, osteocalcin, and vitamin K, or high doses of dietary calcium are used as interventions. Other therapeutic approaches for osteoporosis include bisphosphonates, parathyroid hormone, calcimimetics, statins, lanthanum and strontium salts, and sodium fluoride. However, these treatments are often associated with adverse side effects.

Accordingly, it is an object of the present invention to provide compositions and methods for promoting bone growth and mineralization.

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 increased bone strength. In particular, the present invention demonstrates that a soluble form of ActRIIa acts as an inhibitor of activin-ActRIIa signaling and promotes 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 (often also referred to as "catabolic agents") (eg, bisphosphonates) or as anabolic agents (eg, thyroid paraglandular hormone, PTH, when dosed appropriately), the soluble ActRIIa protein exhibits dual activity, with both catabolic and anabolic effects. Thus, 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 nevertheless demonstrates that desirable therapeutic agents can be selected based on activin-ActRIIa antagonist activity. Accordingly, in certain embodiments, the present invention provides methods of using activin-ActRIIa antagonists to treat diseases associated with low bone density or low bone strength (such as osteoporosis) or to promote Activin-ActRIIa antagonists include, for example, activin-binding ActRIIa polypeptides, anti-activin antibodies, anti-ActRIIa antibodies, activin-targeting or ActRIIa Targeted small molecules and aptamers, and nucleic acids that reduce the expression of activin and ActRIIa. Furthermore, soluble ActRIIa polypeptides promote bone growth without causing a sustained and measurable increase in muscle mass.

In certain aspects, the invention provides polypeptides comprising soluble activin-binding ActRIIa polypeptides that bind to activin. The ActRIIa polypeptide can be made into a pharmaceutical preparation 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, activin-binding ActRIIa selectively binds activin relative to GDF11 and/or GDF8, preferably, the _KD of activin is at least 10 times, 20 times or 50 times lower than the _KD of GDF11 and/or GDF8. While not wishing to be bound by a particular mechanism of action, it is expected that the selectivity of activin inhibition over GDF11/GDF8 demonstrates that selective effects on bone do not result in consistently measurable effects on muscle. In some embodiments, the dose of ActRIIa that causes less than 15%, less than 10%, or less than 5% muscle growth is selected to achieve a desirable effect on bone. Preferably, the composition is at least 95% pure, as determined by size exclusion chromatography with respect to other polypeptide components, more preferably, the composition is at least 98% pure. The activin-binding ActRIIa polypeptide used in such preparations can be any polypeptide disclosed in the present invention, such as a polypeptide having an amino acid sequence selected from SEQ ID Nos: 2, 3, 7, 12 or 13, or a polypeptide selected from SEQ ID Nos : A polypeptide having at least 80%, 85%, 90%, 95%, 97% or 99% identity to the amino acid sequence of 2, 3, 7 or 12. Activin-binding ActRIIa polypeptides may comprise native ActRIIa functional fragments, such as comprising at least 10 of the sequence selected from SEQ ID NOs: 1-3 or the sequence of SEQ ID Nos: 2 (lacking 10 to 15 amino acids at the C-terminus). 1, 20 or 30 amino acids ("tail").

A soluble, activin-binding ActRIIa polypeptide can include one or more amino acid sequence alterations (eg, in the ligand-binding domain) relative to a naturally-occurring ActRIIa polypeptide. Examples of altered ActRIIa polypeptides are provided on pages 59-60 of WO 2006/012627, which is hereby incorporated by reference. Amino acid sequence changes can be, for example, altering the glycosylation of the polypeptide when produced in mammalian, insect or other eukaryotic cells, or altering the proteolytic cleavage of the polypeptide relative to a naturally occurring ActRIIa polypeptide.

Activin-binding ActRIIa polypeptides can be fusion proteins that have one domain of an ActRIIa polypeptide (e.g., the ligand-binding portion of an ActRIIa polypeptide) and one or more additional domains that provide desirable properties (e.g., improved pharmacokinetics, easier domains for purification, targeting to specific tissues, etc.). For example, domains of the fusion protein may enhance one or more of in vivo stability, in vivo half-life, absorption/administration, tissue localization or distribution, formation of protein complexes, multimerization and/or purification of the fusion protein. Activin-binding ActRIIa fusion proteins may include immunoglobulin Fc domains (wild-type or mutant) or serum albumin or other polypeptides that provide desirable properties such as improved pharmacokinetics, improved solubility, or improved stability part. In a preferred embodiment, the ActRIIa-Fc fusion includes a relatively unstructured linker located between the Fc domain and the ActRIIa extracellular domain. This unstructured column key may correspond to an approximately 15 amino acid unstructured region of the C-terminal end ("tail") of the ActRIIa extracellular domain, or it may be 1, 2, 3, 4 or 5 An artificial sequence of amino acids, or a length between 5 and 15, 20, 30, 50 or more amino acids relatively free of secondary structure, or a mixture of both. The linker may be rich in glycine and proline residues and may, for example, contain a simple sequence of threonine/serine and glycine or a repeated sequence of threonine/serine and glycine (e.g., _TG4 or _SG4 singlet or repeat sequence). Fusion proteins can include purified subsequences such as epitope tags, FLAG tags, polyhistidine sequences, and GST fusions. Optionally, the soluble activin-binding type IIA receptor polypeptide comprises one or more modified amino acid residues selected from the group consisting of glycosylated amino acids, PEGylated amino acids, farnesylated (farnesylated) amino acids, acetylated amino acids, biotinylated amino acids, amino acids coupled to liposomal moieties, amino acids coupled to organic derivative preparations. A pharmaceutical formulation may also include one or more additional compounds such as compounds used in the treatment of bone disorders. Preferably, the pharmaceutical formulation is substantially pyrogen-free. In general, it is preferred that the ActRIIa protein is expressed in a mammalian cell line that regulates the proper native glycosylation of the ActRIIa protein to eliminate the possibility of an adverse immune response in the patient. Human cell lines as well as CHO cell lines have been used successfully, and other common mammalian expression systems are expected to be useful.

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

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

In certain aspects, the invention provides methods of making soluble activin-binding ActRIIa polypeptides. Such methods may comprise expressing any nucleic acid disclosed herein (e.g., SEQ ID NO: 4, 5 or 14) in a suitable cell, such as a Chinese Hamster Ovary (CHO) cell. Such methods may comprise: a) culturing cells under suitable conditions to express a soluble ActRIIa polypeptide, wherein said cells are transformed with an expression construct for soluble ActRIIa; and b) renaturing the expressed soluble ActRIIa polypeptide. Soluble ActRIIa polypeptides can be refolded as native, partially purified or highly purified fragments. Purification can be accomplished by a series of purification steps including, for example, one, two or three or more of the following, in any order: protein A affinity chromatography, anion exchange chromatography ( For example, Q Sepharose), hydrophobic chromatography (eg, Phenyl Sepharose), size exclusion chromatography and cation exchange chromatography.

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

In certain aspects, the invention provides methods of identifying agents that stimulate bone growth or increase bone mineralization. The method comprises: a) identifying the drug to be tested 1 that binds to the ligand binding domain of the activin or ActRIIa polypeptide; b) evaluating the effect of the drug on bone growth or mineralization.

Description of drawings

Figure 1 shows the purification of ActRIIa-hFc expressed in CHO cells. The purified protein was a single peak with good peak shape.

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

Figure 3 shows a schematic diagram of A-204 reporter gene analysis. The figure shows the reporter vector: pGL3(CAGA)12 (described in EMBO 17:3091-3100, Dennler et al., 1998). The CAGA12 motif is present in the TGF-Beta responsive gene (PAI-1 gene), so this vector is commonly used for factors that signal through the Smad 2 and 3 pathways.

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

Figure 5 shows the effect of three different ActRIIa-hFc preparations on GDF-11 signaling in the 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. Light shades show increased bone density.

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

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

Figure 9 shows quantification of the effect of ActRIIa-mFc on cancellous bone mineral density in ovariectomized (OVX) or sham-operated C57BL6 mice over 6 weeks. Treatment was divided into control (PBS) or ActRIIa-mFc (ActRIIa) at a dose of 10 mg/kg.

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

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

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

Figure 13 depicts the results of pQCT analysis of bone density in sham-operated mice during the 12-week treatment period. Treatments were divided into control (PBS, light bars) or ActRIIa-mFc at a dose of 10 mg/kg (ActRIIa, dark bars). Y-axis: mg/ccm

Figures 14A and 14B show whole body DEXA analysis and femoral ex vivo analysis (B) after a 12 week treatment period (A). Bright areas describe areas of high bone density.

Figure 15 shows ex vivo pQCT analysis of the midshaft femur after a 12 week treatment period. Treatments are 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 every four bars represents data from ovariectomized mice and the second pair represents data from sham-operated mice.

Figure 16 shows ex vivo pQCT analysis and diaphysis content of midshaft femur after a 12 week treatment period. Treatments were classified as 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 every 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 midshaft femur and femoral cortical thickness. Treatments were classified as Control (PBS, dark bars) or ActRIIa-mFc (light bars). The four bars on the left show the endosteal perimeter and the four bars on the right show the periosteal perimeter. The first pair of every four bars represents data from ovariectomized mice and the second pair represents data from sham-operated mice.

Figure 18 depicts the results of femoral mechanical testing after the 12-week treatment period. Treatments were classified as Control (PBS, dark bars) or ActRIIa-mFc (light bars). The two bars on the left represent data from ovariectomized mice and the last two bars represent data from sham-operated mice.

Figure 19 shows the effect of ActRIIa-mFc on cancellous bone volume.

Figure 20 shows the effect of ActRIIa-mFc on cancellous bone structure of the distal femur.

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

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

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

Figure 24 shows that histomorphometry indicates 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 with identical consensus sequence units and structural motifs. These proteins are known to exert biological functions on many types of cells in vertebrates and invertebrates. Members of the superfamily perform important functions in patterning during embryonic development and tissue differentiation, and can affect a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis, and epithelial cell differentiation. This family is divided into two general clades: the BMP/GDF and TGF-beta/Activin/BMP10 clades, whose members have distinct, often complementary effects. By manipulating the activity of TGF-beta family members, important physiological changes in organisms can often be caused. For example, the Piedmontese and Belgian Blue cattle breeds carry a loss-of-function mutation within the GDF8 (also called myostatin) gene, which results in a dramatic 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) abnormal 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 (A, B and AB) of activin, which are homo/heterodimers of two closely related β subunits ( _{βΑβA , βΒβB and βΑβB} ). The human genome also encodes activin C and activin E, which are initially expressed in the liver. In the TGF-beta superfamily, activins are able to stimulate hormone production in ovarian and placental cells, support neural cell survival, affect cell cycle progression positively or negatively depending on cell type, and induce mesoderm in at least one amphibian embryo Unique and multifunctional factors of differentiation (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). Furthermore, 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 releases follicle stimulating hormone (FSH), activin promotes the secretion and synthesis of FSH, while inhibin prevents the secretion and synthesis of FSH. Other proteins that can modulate activin biological activity and/or bind to activin include follistatin (FS), follistatin-related protein (FSRP), α2-macroglobulin, Cerberus, and endoglin.

TGF-β signaling is regulated by a heteromeric complex of type I and type II serine/threonine kinase receptors, which upon ligand activation phosphorylate and activate downstream Smad proteins (Massague, 2000, Nat. Rev. MoI. Cell Biol. 1:169-178). These type I and type II receptors are 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 specificity. domain composition. Type I is required 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 Sci.98:9306-9311; Yeo and Whitman, 2001, MoI. Cell 7:949-957; Oh et al., 2002, Genes Dev.16:2749-54). ALK4 is the major activin type I receptor, especially for activin A, and ALK-7 can also act as a receptor for activins, especially for activin B.

As demonstrated by the present invention, soluble ActRIIa polypeptide (sActRIIa), which shows a substantial preference for binding to activin A relative to other TGF-beta family members such as GDF8 or GDF11, is effective in promoting bone growth and increasing bone density. While not wishing to be bound by any particular mechanism, given the very strong binding (picomolar dissociation constants) of activin exhibited by the particular sActRIIa constructs employed in these studies, it is expected that the effects of sActRIIa are primarily mediated by activin antagonists. caused by the effect. Regardless of mechanism, the data presented here clearly show that ActRIIa-activin antagonists indeed increase bone density in normal mice as well as in a mouse model of osteoporosis. Note that bone is a dynamic tissue that grows or shrinks and increases or decreases in density depending on factors that produce bone and stimulate mineralization (mainly osteoblasts) versus factors that destroy and remove bone mineral (mainly osteoclasts) balance between. Growth and mineralization of bone can be increased by increasing productive factors or decreasing destructive factors or both. The terms "promoting bone growth" and "increasing bone mineralization" refer to physically visible changes in bone and are used to neutrally describe the mechanism by which changes in bone occur.

The mouse model of osteoporosis and bone growth/density used in the studies described herein are believed to be highly predictive of efficacy in humans, and thus, the present invention provides the use of ActRIIa polypeptides and other activin-ActRIIa antagonists To promote bone growth and increase bone density in the human body. Activin-ActRIIa antagonists include, for example, activin that binds to soluble ActRIIa polypeptide, antibodies that bind to activin (particularly activin A or B subunit, also referred to as βA or βB) and interrupt ActRIIa binding, antibodies that bind to ActRIIa and interrupt activin binding , selected from activin or ActRIIa binding non-antibody proteins (see for example, WO/2002/088171, WO/2006/055689, WO/2002/032925, WO/2005/037989, US 2003/0133939, and US 2005/0238646 Examples of such proteins and methods of designing and selecting the same), random peptides selected from activin or ActRIIa binding usually conjugated to the Fc domain. Two different proteins (or other moieties) having activin or ActRIIa binding activity, in particular block Type I (eg, soluble Type I activin receptor) or Type II (eg, soluble Type II activin receptor) binding sites, respectively Dotted activin binders can be linked to generate bifunctional conjugated molecules. Aptamers, small molecules and other agents that inhibit the activin-ActRIIa signaling axis. A variety of proteins have activin-ActRIIa antagonist activity, including inhibin (ie, inhibin alpha subunit) (although inhibin does not antagonize activin in all tissues), follistatins (such as follistatin 288 and follistatin Inhibin 315), Cerberus, follistatin-related protein (FSRP), endoglin, activin C, _α2 -macroglobulin, and M108A (methionine to alanine at position 108) mutations activin A. In general, alternative forms of activin, especially those with changes in the type I receptor binding domain, can bind to type II receptors and fail to form active ternary complexes, thus 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 (in particular) ActRIIa, can be used as activin-ActRIIa antagonists. Preferably, the activin-ActRIIa antagonist employed will exhibit selectivity for inhibiting activin regulated signaling 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 show significant selectivity for binding to activin relative to GDF8/11, and preliminary experiments show that this does not provide a desirable effect on bone, but can still Causes significant muscle growth. However, altered forms of ActRIIb with different binding properties have been identified (see eg WO2006/012627, pp. 55-59, incorporated herein by reference), and these proteins may have desirable effects on bone. Native or altered ActRIIb can provide additional specificity for activin by coupling a second activin selective binding moiety.

The terms used in the specification generally have their ordinary meanings in the art, both in the context of the present invention and in the specific context where 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 invention and how they should be used. The scope or meaning of any application of a term is clear in the particular context in which it is applied.

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

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

The methods of the invention may comprise the step of comparing sequences to one another, including comparing the wild-type sequence to one or more mutants (sequence variants). Such comparisons typically involve alignment of polymeric sequences, eg, using sequence alignment programs and/or algorithms known in the art (eg, BLAST, FASTA, and MEGALIGN, to name a few). Those skilled in the art will readily appreciate that in such an alignment, in a mutation containing an inserted or deleted residue, the aligned sequences will create a "gap" in the sequence of the polymer that does not contain the inserted or deleted residue. " (gap) (usually represented by a dash or "A").

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

The term "sequence similarity", in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences which may or may not have a common evolutionary origin.

However, in common usage and in immediate applications, 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 being involved.

2. ActRIIa polypeptide

In certain aspects, the invention relates to ActRIIa polypeptides. As used herein, the term "ActRIIa" refers to the family of activin type IIa receptors from any species and derived from ActRIIa protein variants by mutation or other modification. ActRIIa of the present invention can be understood as any previously identified form. Members of the ActRIIa family are generally 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 variant (including mutants, fragments, fusions, and peptidomimetic forms) that retains useful activity. For example, ActRIIa polypeptides include polypeptides derived from any known ActRIIa sequence that is at least about 80% identical to an ActRIIa polypeptide, preferably at least 85%, 90%, 95%, 97%, 99% or more identical. For example, the ActRIIa polypeptide of the present invention can bind to ActRIIa protein and/or activin and inhibit its 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:

MGAA′AKLAFAVFLISSSGA ILGRSETQECLFFNANWEKDRT

QTGVEPCYGDKDKRRHCFATWK ISGSIEIVKQGCWLDDINCY

DRTDCVEKKD SPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNP

VTPKPPYYNILLYSLVPLMLIAGIVICAFWVYRHHKMAYPPVL

VPTQDPGPPPPSPLLGLKPLQLLEVKARGRFGCVWKAQLLNEY

VAVKIFPIQDKQSWQNEYEVYSLPGMKHENILQFIGAEKRGTS

VDVDLWLITAFHEKGSLSDFLKANVVSWNELCHIAETMARGL

AYLHEDIPGLKDGHKPAISHRDIKSKNVLLKNNLTACIADFGLA

LKFEAGKSAGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDM

YAMGLVLWELASRCTAADGPVDEYMLPFEEEIGQHPSLEDMQE

VVVHKKKRPVLRDYWQKHAGMAMLCETIEECWDHDAEARL

SAGCVGERITQMQRLTNIITTEDIVTVVTMVTNVDFPPKESSL (SEQ ID NO: 1)

Signal peptides are single underlined; extracellular domains are in bold and potential N-linked glycosylation sites are double underlined.

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

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM EVTQPTSPVTPKPP (SEQ ID NO: 2)

The C-terminal "tail" of the extracellular domain is underlined. The sequence with the "tail" (Δ15 sequence) removed is as follows:

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM (SEQ ID NO: 3)

The nucleic acid sequence encoding the human ActRIIa precursor polypeptide protein is as follows (164-1705 nucleotides of Genebank accession number_001616):

ATGGGAGCTGCTGCAAAGTTGGCGTTTGCCGTCTTTTCTTATCTCCTGTTCTT

CAGGTGCTATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTCTTTAATGC

TAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGT

GACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTT

CCATTGAAATAGTGA′A′ACAAGGTTGTTGGCTGGATGAT′ATCAACTGCTATGA

CAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTTGTTGC

TGTGAGGGCAATATGTGTAATGAAA′AGTTTTCTTATTTTCCAGAGATGGAAG

TCACACAGCCCACTTCAAATCCAGTTAC'ACCTA'AGCCACCCCT'ATTACAACAT

CCTGCTCTATTCCTTGGTGCCACTTATGTTAATTGCGGGGATTGTCATTTGT

GCATTTTGGGTGTACAGGCATCACAAGATGGCCTACCCTCCTGTACTTGTTC

CAACTCAAGACCCAGGACCACCCCCACCTTCTCCATTACTAGGGTTGAAACC

ACTGCA'GTTATTAGAAGTGAAAGCAAGGGGAAGATTTGGTTGTGTCTGGAAA

GCCCAGTTGCTTAACGAATATGTGGCTGTCAAAATATTTCCAATACAGGACA

AACAGTCATGGCAAAATGAATACGAAGTCTACAGTTTGCCTGGAATGAAGCA

TGAGAACATATTACAGTTCATTGGTGCAGAAAAACGAGGCACCAGTGTTGAT

GTGGATCTTTGGCTGATCAC'AGCATTTCATGAAA'AGGGTTC'ACTATCAGACT

TTCTTAAGGCTA′ATGTGGTCTCTTGGAATGAACTGTGTCATATTGCAGAAAC

CATGGCTAGAGGATTGGCATATTTACATGAGGATATACCTGGCCTAAAAGAT

GGCCACAAACCTGCCATATCTCACAGGGACATCAAAAGTAAAAAATGTGCTGT

TGAAAAACAACCTGACAGCTTGCATTGCTGACTTTGGGTTGGCCTTAAAATT

TGAGGCTGGCAAGTCTGCAGGCGATACCCATGGACAGGTTGGTACCCGGAGG

TACATGGCTCCAGAGGTATTAGAGGGTGCTATAAACTTCCAAAGGGATGCAT

TTTTGAGGATAGATATGTATGCCATGGGATTAGTCCTATGGGAACTGGCTTC

TCGCTGTACTGCTGCAGATGGACCTGTAGATGAATACATGTTGCCATTTGAG

GAGG′AAATTGGCCAGCATCCATCTCTTGAAGACATGCAGGAAGTTGTTGTGC

ATAAAAAAAAGAGGCCTGTTTTAAGAGATTATTGGCAGAAACATGCTGGAAT

GGCAATGCTCTGTGAAACCATTGAAGAATGTTGGGATCACGACGCAGA′AGCC

AGGTTATCAGCTGGATGTGTAGGTGAAAGAATTACCCAGATGCAGAGACTAA

CAAATATTATTACCACAGAGGACATTGTAACAGTGGTCACAATGGTGACAAA

TGTTGACTTTCCTCCCAAAGAATCTAGTCTATGA (SEQ IDNO: 4)

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

ATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTTCTTTAATGCTAATTGGG

AAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGA

TAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTCTGGTTCCATTGAA

ATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTG

ATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTTTTGTTGCTGTGAGGG

CAATATGTGTAATGAAAAGTTTTCTTATTTTCCAGAGATGGAAGTCACACAG CCCACTTCAAATCCAGTTACACcTAAGCCACCC (SEQ ID NO: 5)

In a specific embodiment, the invention relates to soluble ActRIIa polypeptides. As described herein, the term "soluble ActRIIa polypeptide" generally refers to a polypeptide comprising the extracellular domain of the ActRIIa protein. As used herein, the term "soluble ActRIIa polypeptide" includes the naturally occurring extracellular domain of any ActRIIa protein and any variants (including mutants, fragments and peptidomimetic forms) thereof. An Activin-binding ActRIIa polypeptide is a polypeptide that retains the ability to bind to activin, particularly activin AA, AB or BB. Preferably, the activin-binding ActRIIa polypeptide binds to activinAA with a dissociation constant of 1 nM or less. The amino acid sequence of the human ActRIIa precursor protein is provided below. ActRIIa proteins bind to activin and are generally soluble, and are thus 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 refers to ActRIIa-hFc and is further described in the Examples. Other soluble activin-binding ActRIIa polypeptides include a signal sequence in addition to the extracellular domain of the ActRIIa protein, for example, honeybee melittin leader sequence (SEQ ID NO: 8), tissue plasminogen activator (TPA) leader (SEQ ID NO: 9) or native ActRIIa leader (SEQ ID NO: 10). The ActRIIa-hFc polypeptide illustrated in SEQ ID NO: 13 was used as a TPA leader.

Functionally active fragments of ActRIIa polypeptides can be obtained by screening polypeptides produced from corresponding nucleic acid fragments encoding ActRIIa polypeptides. Alternatively, fragments can be chemically synthesized using well known on-site 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 those peptidyl fragments that function as antagonists (inhibitors) of the ActRIIa protein or that are signaled by activin.

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

Functional variants can be generated by modifying the structure of ActRIIa polypeptides for purposes such as enhancing therapeutic efficacy or stability (e.g., ex vivo shelf life and resistance to protein degradation in vivo), such modified ActRIIa polypeptides being selected While retaining activin binding, it is considered to be a functional equivalent of the naturally occurring ActRIIa polypeptide. Modified ActRIIa polypeptides can also be produced, for example, by amino acid substitutions, deletions or additions. For example, it is reasonable to expect to substitute isoleucine or valine for leucine alone, glutamic acid for aspartic acid alone, serine for threonine alone, or a structurally related amino acid for another amino acid. (eg, conservative mutations) do not have a large effect on the biological activity of the resulting molecule. Conservative substitutions are those that occur between side chain related amino acids within a family. Whether changes in the amino acid sequence of an ActRIIa polypeptide result in functional homology is readily determined by assessing the ability of the ActRIIa polypeptide variant to generate a response in the cell in a manner similar to the wild-type ActRIIa polypeptide.

In certain embodiments, the invention encompasses specific mutations of ActRIIa polypeptides in order to alter the glycosylation of the polypeptides. Such mutations can be selected to introduce or eliminate one or more glycosylation sites, O-linked or N-linked glycosylation sites. Asparagine-linked glycosylation recognition sites typically include the tripeptide sequence, asparagine-X-threonine (or asparagine-X-serine) (where X can be any amino acid), which is suitably specifically recognized by cellular glycosylases. Engineering can also be performed by the addition or substitution of one or more serine or threonine residues to the wild-type ActRIIa polypeptide (for O-linked glycosylation sites). Various amino acid substitutions or deletions at one or both of the first or third amino acid positions of the glycosylation recognition site (and/or amino acid deletions at the second position) result in Aglycosylation of peptide sequences. Another method of increasing the number of carbohydrates on an ActRIIa polypeptide may be by chemical coupling or enzymatic coupling of glycosides to the ActRIIa polypeptide. Depending on the coupling mode employed, sugars can be appended to (a) arginine and histidine; (b) free carboxyl groups; (c) free thiol groups such as those of cysteine; (d) free hydroxyl groups such as free hydroxyl groups of serine, threonine or hydroxyproline; (e) aromatic residues such as phenylalanine, tyrosine or tryptophan; or (f) amide groups of glutamine. These methods are described in WO 87/05330 (published September 11, 1987) and Aplin and Wriston (1981) CRC Crit. Rev. Biochem., pages 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 an ActRIIa polypeptide to the compound triflate or an equivalent compound. This treatment results in the cleavage of most or all sugars except the one to which they are attached (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 cleavage of carbohydrates on ActRIIa polypeptides can be achieved by using various endoglycosidases or exoglycosidases as described by Thotakura et al. (1987) Meth. Enzymol. 138:350. The sequence of the ActRIIa polypeptide can be adjusted appropriately, which depends on the type of expression system used, for example, mammalian, yeast, insect and plant cells can introduce different glycosylation patterns, which can be affected by the amino acid sequence of the peptide. Typically, ActRIIa polypeptides for use in humans can be expressed in mammalian cell lines that provide correct glycosylation, such as HEK293 cells or CHO cell lines, although other mammalian expression cell lines, with engineered glycosylation Yeast cell lines as well as insect cell lines that base the enzyme can also be used for expression.

The invention further encompasses methods of 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 purpose of screening such combinatorial libraries is to generate, for example, variants of the ActRIIa polypeptide that act either as agonists or as antagonists, or alternatively, mutants that possess all novel activities. Various screening methods are provided below, and such methods can be used to evaluate variants. For example, variants of an ActRIIa polypeptide can be screened for the ability to bind to an ActRIIa ligand, prevent the ActRIIa ligand from binding to the ActRIIa polypeptide, or interfere with signaling by the ActRIIa ligand.

The activity of ActRIIa polypeptides or variants thereof can also be tested on a cell basis or in vivo. For example, the effect of ActRIIa polypeptide variants on the expression of genes involved in bone production or bone destruction can be assessed. If desired, this can be accomplished in the presence of one or more recombinant ActRIIa ligand proteins (e.g., activin), and cells can be transfected to produce ActRIIa polypeptides and/or variants thereof and, where appropriate, ActRIIa ligands . Likewise, an ActRIIa polypeptide can be administered to a mouse or other animal and one or more bone properties such as density or volume assessed. Fracture healing rates can also be assessed. Dual energy X-ray absorptiometry (DEXA) is a well-established, non-invasive, quantitative technique for assessing bone density in animals. The human central DEXA system can be used to assess bone density in the spine and pelvis. These are the best predictors of total bone density. The peripheral DEXA system 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.

Variants of combinatorial origin can be generated that have selective or general increased potential relative to naturally occurring ActRIIa polypeptides. Likewise, mutations can result in variants with intracellular half-lives that differ dramatically from the corresponding wild-type ActRIIa polypeptides. For example, the altered protein is either more stable or less stable to proteolytic or other cellular processes that lead to its destruction or conversely inactivation of the native ActRIIa polypeptide. Such variants, and the genes encoding them, can be used to alter the level of the ActRIIa polypeptide by modulating the half-life of the ActRIIa polypeptide. For example, a short half-life can result in more transient biological effects and can allow tighter control of the level of recombinant ActRIIa polypeptide in a patient. In Fc fusion proteins, mutations can be in the linker (if any) and/or the Fc portion to alter the half-life of the protein.

A recombinant library can be generated by means of a degenerate gene library encoding a library of polypeptides each comprising 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 a degenerate form of the underlying ActRIIa polypeptide nucleotide sequence can be expressed as a single polypeptide, or alternatively, as a larger fusion protein ( For example, phage display) expression.

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

Alternatively, other forms of mutagenesis can be used to generate combinatorial libraries. For example, variants of ActRIIa polypeptides can be generated and screened for mutations using, for example, alanine and similar (Ruf et al. (1994) Biochemistry 33:1565-1572; Wang et al. (1994) J. Biol. Chem. 269:3095 -3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al., (1993) Eur.J.Biochem.218:597-601; Nagashima et al., (1993) J.Biol.Chem.268:2888- 2892; Lowman et al., (1991) Biochemistry 30:10832-10838; and Cunningham et al., (1989) Science 244:1081-1085), mutation by linker partitioning (Gustin et al., (1993) Virology 193:653-660; Brown et al. , (1992) MoI.CellBiol.12:2644-2652; McKnight et al., (1982) Science 232:316), by saturation mutation (Meyers et al., (1986) Science 232:613), by PCR mutation (Leung et al., ( 1989) Method Cell MoI Biol 1:11-19) or by random mutation, including chemical mutation etc. (Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY; and Greener et al., ( 1994) Strategies in MoIBiol 7:32-34) were screened and isolated from the library. Linker partition mutagenesis, especially in a combinatorial setting, is an attractive approach to identify truncated (biologically active) forms of ActRIIa polypeptides.

A wide range of techniques are known in the art for screening gene products in combinatorial libraries obtained by point mutations and truncations, and, for that matter, cDNA libraries for screening gene products with certain properties. Such techniques are generally adapted for rapid screening of gene libraries generated by combinatorial mutagenesis of ActRIIa polypeptides. The technique most widely used for screening large gene libraries generally involves cloning the gene library into replicable expression vectors, transforming the obtained vector library into suitable cells, and upon detection of the desired activity allows isolation of the gene encoding the product being detected vectors for expression of combinatorial genes under relatively easy conditions. Preferred methods include activin binding assays and activin regulated cell signaling assays.

In certain embodiments, the ActRIIa polypeptides of the invention may further comprise post-translational modifications of any naturally occurring ActRIIa polypeptides. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. As a result, modified ActRIIa polypeptides may comprise non-amino acid elements such as polyethylene glycols, lipids, polysaccharides or monosaccharides, and phosphates. The effect of such non-amino acid elements on the function of an ActRIIa polypeptide can be tested as described herein for other ActRIIa polypeptide variants. When the ActRIIa polypeptide is produced intracellularly by cleavage of the nascent form of the ActRIIa polypeptide, post-translational processes are also important for proper folding and/or function of the protein. 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 actions and can be chosen to ensure correct modification and activation of ActRIIa polypeptides. process.

In certain aspects, functional variants or modified forms of ActRIIa polypeptides include fusion proteins having at least a portion of an ActRIIa polypeptide and one or more fusion domains. Well-known examples of such fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione transferase (GST), thioredoxin, protein A, protein G, immunoglobulin heavy chain invariant region (Fc), maltose binding protein (MBP) or human serum albumin. Fusion domains can be selected to confer desirable properties. For example, some fusion domains are specifically useful for isolating fusion proteins by affinity chromatography. For the purpose of affinity purification, affinity chromatography-related matrices such as glutathione, amylase, and nickel- or cobalt-conjugated resins are used. Many such matrices are available in "kit" form, such as the Pharmacia GST purification system and the QIAexpress ^™ system (Qiagen) with useful (HISg) fusion partners. As a further example, fusion domains can be selected to facilitate detection of ActRIIa polypeptides. Examples of such detection domains include various fluorescent proteins (eg GFP) as well as "epitope tags", which are usually short peptide sequences for which specific antibodies are available. Well-known epitope tags for specific monoclonal antibodies are readily available, including FLAG, influenza virus hemagglutinin (HA), and c-myc tags. In some examples, the fusion domain has a proteolytic cleavage site, such as for Factor Xa or thrombin, which allows the relevant protease to partially digest the fusion protein and thus 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 "stabilized" is meant any substance that increases serum half-life, regardless of whether this is due to reduced destruction, decreased renal clearance, or other pharmacokinetic effects. Fusions to the Fc portion of immunoglobulins are known to confer desirable pharmacokinetic properties on a wide range of proteins. Furthermore, fusion to human serum albumin may impart desirable properties. Alternative other types of fusion domains include multimerization domains (e.g., dimerization, tetramerization) as well as functional domains (which confer additional biological functions, such as further stimulation of bone growth or muscle growth, as desired ).

As a specific example, the invention provides fusion proteins comprising the 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 QKSLSLSPGK ^*

Alternatively, the Fc domain has one or more mutations in residues such as aspartate 265, lysine 322 and asparagine 434. In certain instances, a mutant Fc domain having one or more such mutations (eg, an aspartate 265 mutation) has an abridged ability to bind to an Fcγ receptor relative to a wild-type Fc domain. In other examples, mutant Fc domains having one or more such mutations (e.g., paragine 434 mutation) have increased binding to MHC class I-associated Fc receptors (FcRN) relative to wild-type Fc domains Ability.

Of course, the various elements of the fusion protein may be arranged in any manner consistent with the desired function. For example, an ActRIIa polypeptide can be placed C-terminal to a heterologous domain, or alternatively, a heterologous domain can be placed C-terminal to an 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 may include the C-terminus or N-terminus to one of the two domains or be between the two domains.

In certain embodiments, the ActRIIa polypeptides of the invention contain one or more modifications that stabilize the ActRIIa polypeptides. For example, such modifications enhance the half-life of the ActRIIa polypeptide in vivo, 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), modification of glycosylation sites (including, for example, addition of glycosylation sites to an ActRIIa polypeptide) and modification of carbohydrates (including, for example, removal of carbohydrates from an ActRIIa polypeptide). For fusion proteins, the ActRIIa polypeptide is fused to a stabilizer domain (eg, an Fc domain) such as an IgG molecule. As used in the present invention, the term "stabilizer domain" not only refers to fusion domains (eg Fc) in fusion proteins, 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, isolated or otherwise substantially free from other proteins. ActRIIa polypeptides are typically produced by expression from recombinant nucleic acids.

3. Nucleic acid encoding ActRIIa polypeptide

In certain aspects, the invention provides isolated and/or recombinant nucleic acids encoding any of the ActRIIa polypeptides (eg, soluble ActRIIa polypeptides), 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. Target nucleic acids can be single-stranded or double-stranded. Such nucleic acids may be DNA molecules or RNA molecules. These nucleic acids can be used, for example, in methods of making ActRIIa polypeptides or as direct therapeutic agents (eg, in gene therapy approaches).

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

In certain embodiments, the invention provides isolated or Recombinant nucleic acid sequence. Those of ordinary skill in the art should understand that the nucleic acid sequence complementary to SEQ ID NO: 4 or 5 and the variant complementary to SEQ ID NO: 4 or 5 can be included in the scope of the present invention. In a further embodiment, the nucleic acid sequences of the invention may be isolated, recombinant and/or fused to heterologous nucleotide sequences or in a DNA library.

In other embodiments, the nucleic acid of the present invention can also include a nucleic acid that hybridizes to the specified nucleotide sequence in SEQ ID NO: 4 or 5, the complementary sequence of SEQ ID NO: 4 or 5, or a fragment thereof under stringent conditions. nucleotide sequence. As discussed above, those of ordinary skill in the art will readily appreciate that conditions of appropriate stringency to promote DNA hybridization may vary. For example, hybridization can be performed in 6.0X sodium chloride/sodium citrate (SSC) at about 45°C, followed by a 2.0X SSC wash at 50°C. For example, the salt concentration of the washing step can be selected from a low stringency condition of 2.0×SSC, 50°C to a high stringency condition of 0.2×SSC, 50°C. Additionally, the temperature of the wash steps can range from low stringency conditions at room temperature of about 22°C to high stringency conditions of about 65°C. Both the temperature and the salt can be varied, or the temperature or the salt concentration can be kept constant while the other variable is changed. In summary of certain embodiments, the invention provides nucleic acids that hybridize under low stringency conditions of 6*SSC, room temperature, followed by washing in 2*SSC at room temperature.

Isolated nucleic acid molecules that differ from the nucleic acid in SEQ ID NO: 4 or 5 due to degeneracy of the genetic code are also included within the scope of the present invention. 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 (eg, CAU and CAC are synonyms for histidine) can result in "silent" mutations that do not affect the amino acid sequence of the protein. However, we expect that polymorphisms in the DNA sequence that do result in changes in the amino acid sequence of the protein of interest are present 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 may exist in individuals of a given species due to natural allelic variation. middle. Any or all such nucleotide variations and resulting amino acid polymorphisms are within the scope of the invention.

In certain embodiments, a recombinant nucleic acid of the invention is operably linked to one or more regulatory nucleotide sequences of an expression construct. Regulatory DNA sequences are usually suitable for expressing the cells in which they are applied. Many types of suitable expression vectors, as well as suitable regulatory sequences, are known in the art for use in various host cells. Generally, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcriptional initiation and termination sequences, translational initiation and termination sequences, and enhancing sub or activation subsequence. Constructed or induced promoters known in the art are encompassed by the present invention. The promoter is either a naturally occurring promoter or a hybrid promoter combining elements of more than one promoter. The expression construct may be present episomally within the cell, such as a plasmid, or the expression construct may be inserted chromosomally. In a preferred embodiment, the expression vector contains a selectable marker gene to allow selection of transformed hosts. Selectable marker genes are well known in the art and will vary with the host cell employed.

In certain aspects of the invention, the nucleic acid of interest is provided in an expression vector comprising a nucleotide sequence encoding an ActRIIa polypeptide, operably linked to at least one regulatory sequence. Regulatory sequences are art recognized and selected to direct expression of the ActRIIa polypeptide. Accordingly, the term regulatory sequence includes promoters, enhancers and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Techniques; Methods in Enzymology, Academic Press, San Diego, CA (1990). For example, any of a wide variety of expression control sequences that control the expression of a DNA sequence, when operably linked, can be used to express the expression of 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 adenovirus or cytomegalovirus immediate early promoter, the RSV promoter, the lactose operon system, the tryptophan system, TAC or TRC system, T7 promoter expressed by T7 RNA polymerase, the main operation and promoter region of lambda phage, the control region of fd goat protein, the promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, the promoter of acid phosphatase Promoters such as Pho5, the promoter of the yeast alpha-hybrid factor, the polyhedral promoter of the baculovirus system and other known sequences controlling the expression of prokaryotic or eukaryotic cells or their viral genes and their various recombinants. It will be appreciated that the design of the expression vector depends on factors such as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. In addition, the number of copies of the vector, the ability to control copy number and expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.

Recombinant nucleic acids of the invention can be produced by ligating cloned genes or fragments thereof into vectors suitable for expression either in prokaryotic cells or in eukaryotic cells (yeast, avian, insect or mammalian) 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 prokaryotic cells such as E. coli. plasmid.

Some mammalian expression vectors contain both prokaryotic sequences that facilitate propagation of the vector in bacteria and one or more eukaryotic transcription units for expression in eukaryotic cells. PcDNAI/amp, pcDNAI/neo, pRC/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells . Some of these vectors have been modified with sequences of bacterial plasmid origin, such as pBR322, to facilitate replication in prokaryotic and eukaryotic cells and selection for drug resistance. Alternatively, derivatives of viruses such as bovine papilloma virus (BPV-1) or eb virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found in the description of gene therapy delivery systems below. Various methods employed in the preparation of plasmids and the transformation of host organisms are well known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells and general recombination procedures, see Molecular Cloning A Laboratory Manual, Third Edition, edited by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 2001). In some instances, it is suitable to express the recombinant polypeptide using the bovine papilloma virus 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-galactoside containing pBlueBacIII enzyme).

In preferred embodiments, vectors are designed to produce the 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 the 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 a host cell transfected with a recombinant gene comprising a coding sequence (eg, SEQ ID NO: 4 or 5) encoding one or more target ActRIIa polypeptides. 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 E. coli, insect cells (eg, using the bovine papillomavirus 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 of producing a target ActRIIa polypeptide. For example, an expression vector transformed with an ActRIIa polypeptide can be cultured under suitable conditions to allow expression of the ActRIIa polypeptide. ActRIIa polypeptides can be secreted and isolated from mixtures of cells and matrices comprising ActRIIa polypeptides. Alternatively, the ActRIIa polypeptide can remain in the cytoplasm or as a membrane fraction and the cells harvested, lysed and the protein isolated. Cell cultures include host cells, matrix and other by-products. Suitable cell culture substrates are well known in the art. The target ActRIIa polypeptide can be isolated from the cell culture substrate, the host cell, or both, and the protein can be purified using techniques well known in the art, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, using methods specific for the ActRIIa polypeptide Immunoaffinity purification of antibodies to specific epitopes of the ActRIIa polypeptide and affinity purification using preparations that bind to domains fused to the ActRIIa polypeptide (eg, Protein A columns can be used to purify ActRIIa-Fc fusion proteins). In a preferred embodiment, the ActRIIa polypeptide is a fusion protein comprising 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 Agarose chromatography, size exclusion chromatography, and cation exchange chromatography. Purification can be accomplished by virus filtration and buffer exchange. As demonstrated in the present invention, the ActRIIa-hFc protein was purified to a purity of greater than 98% by size exclusion chromatography and greater than 95% by SDS PAGE. Such purity is sufficient to achieve the desired effect on mouse bone and acceptable drug safety 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, allows passage using Ni2 ⁺ metal resins. The expressed fusion protein was purified by affinity chromatography. The purified leader sequence is then removed by enterokinase treatment to provide purified ActRIIa polypeptide (see, eg, 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. Essentially, the joining of the various DNA fragments encoding the different polypeptide sequences is accomplished by corresponding conventional techniques, using blunt-ended or stagger-ended ends for ligation, restriction enzyme digestion to provide suitable ends, where appropriate Fill in sticky ends, alkaline phosphatase treatment to avoid unwanted ligation, and enzymatic ligation. In another embodiment, fusion genes can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using adapter primers that create complementary overhangs between two consecutive gene fragments that can then anneal to generate chimeric gene sequences (see, e.g., Current Protocols in Molecular Biology, pp. Ausubel et al., eds., John Wiley & Sons: 1992).

4. Selective Activin and ActRIIa antagonists

The data presented herein demonstrate that antagonists of activin-ActRIIa signaling can be used to promote bone growth and bone 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 be an indicator of a tendency to inhibit spectral molecular activity, Including, possibly, other members of the TGF-beta superfamily, and such quorum inhibition may result in the desired skeletal effect), 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 an ActRIIa polypeptide (e.g., a soluble ActRIIa polypeptide), and agents that either compete for binding to the ActRIIa polypeptide or otherwise inhibit ActRIIa-mediated signaling, can be used as antagonists of ActRIIa polypeptide activity. In addition, antibodies that specifically react with activinA polypeptides as well as antibodies that disrupt ActRIIa binding can also be used as antagonists.

Anti-protein/anti-polypeptide antisera or monoclonal antibodies can be prepared by standard methods (see, e.g., A Laboratory Manual edited by Harlow and Lane (Cold Spring Harbor Press: 1988)) using immunogens derived from ActRIIa polypeptides or activin polypeptides . Mammals such as mice, hamsters or rabbits can be immunized with immunogenic forms of ActRIIa polypeptides, antigenic fragments or fusion proteins capable of eliciting an antibody response. Techniques for conferring immunogenicity to proteins or polypeptides include conjugation to carriers or other techniques known in the art. An immunogenic portion of an ActRIIa or activin polypeptide can be administered in the presence of an adjuvant. The immunization process can be detected by detecting antibody titers in plasma or serum. Standard ELISA or other immunization methods can be used with the immunogen as antigen to assess antibody levels.

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

The term "antibody" as used herein is intended to include those fragments which also specifically react with the polypeptide of interest. 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 produce Fab fragments with fewer disulfide bridges. Antibodies of the invention are further intended to include bispecific, single-chain, chimeric, humanized, and full-length human molecules with ActRIIa or activin polypeptide affinity conferred by at least one antibody CDR region. Antibodies may further include additionally attached and detectable labels (eg, labels may be radioisotopes, fluorescent compounds, enzymes or coenzyme factors).

In certain embodiments, the antibody is a recombinant antibody, the term encompassing any antibody partially produced by molecular biology techniques, including CDR-grafted or chimeric antibodies, human or assembled from library-selected antibody domains antibodies, single chain antibodies, and single domain antibodies (eg, human V _H protein or camelid V _HH protein). In certain embodiments, the antibodies of the invention are monoclonal antibodies, and in certain embodiments, the invention enables methods of producing novel antibodies. For example, a method of producing a monoclonal antibody that specifically binds to an ActRIIa polypeptide or an activin polypeptide comprises administering to a mouse a dose of an immunogenic composition comprising an effective amount of an antigenic polypeptide that stimulates a detectable immune response, from the mouse In obtaining antibody-producing cells (eg, cells in the spleen) and fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, the antibody-producing hybridomas are tested to identify hybridomas that produce a monoclonal that specifically binds the antigen. Once obtained, the hybridomas are propagated from the cell culture medium, optionally under the culture conditions of the hybridoma-derived cells that produce cells that specifically bind the antigen. Monoclonal antibodies can be purified from cell culture media.

The adjective "specifically reacts with" herein refers to an antibody, meaning that, as generally understood in the art, the antibody reacts with the antigen of interest (e.g., an ActRIIa polypeptide) and other non-antigens of interest in a particular biological sample. There is sufficient selectivity among them. In certain methods of employing antibodies, such as therapeutic applications, a high degree of specificity of binding is necessary. Monoclonal antibodies generally have a greater tendency (compared to polyclonal antibodies) to efficiently discriminate between desired antigens and cross-reactive polypeptides. Influencing Antibodies: The specificity of an antibody interaction is characterized by the affinity of the antibody for the antigen. Although a range of different affinities is available for desired specificity, generally preferred antibodies have affinities (dissociation constants) of about 10 ^"6 , 10 ^"7 , 10 ^"8 , 10" ⁹ or less. Given the exceptionally tight binding between activin and ActRIIa, we expect neutralizing anti-activin antibodies or anti-ActRIIa antibodies to generally have dissociation constants of 10 ⁻¹⁰ or less.

In addition, the technique 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 the antigen in solution, it may be appropriate to test solution binding. A variety of different techniques can be used to test the interaction between antibodies and antigens to identify specific desired antibodies. Such techniques include ELISAs, surface plasmon resonance binding assays (e.g., the Biacore ^™ binding assay, Biacore AB, Upps ala, Sweden), sandwich methods (e.g., the paramagnetic bead system of IGEN International, Inc., Gaithersburg, Maryland), Western blotting, immunoprecipitation, and immunohistochemistry.

Examples of classes of nucleic acid compounds that are Activin or ActRIIa antagonists 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 may also include overhangs or regions of non-complementarity wherein one or the other segment is single stranded. Single-stranded compounds may include regions that are self-complementary, meaning that the compounds form structures known as "hairpins" or "stem-loops," with regions of a double-stranded helical structure. The nucleic acid compound may comprise 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 complementary to the full-length ActRIIa nucleic acid sequence or activin βA or activin βB nucleic acid sequence or a nucleotide sequence of a region of 18 nucleotides. 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 may belong to an intron, the coding sequence of the target transcript, or a non-coding sequence such as part of the coding sequence. Typically, nucleic acid compounds have a length of 8 to about 500 nucleotides or base pairs, optionally from about 14 to about 50 nucleotides in length. The nucleic acid may be DNA (especially used as antisense nucleic acid), RNA or RNA:DNA hybrid. Either strand may include a mixture of DNA and RNA, as well as modified forms that cannot easily be distinguished as DNA or RNA. In addition, the double-stranded compound can be DNA:DNA, DNA:RNA, or RNA:RNA, and either strand can include a mixture of DNA and RNA, as well as modified forms that cannot easily be distinguished as DNA or RNA. Nucleic acid compounds may include any of a variety of modifications, including one or more modifications of the backbone (the sugar-phosphate portion of natural nucleic acids, including internucleoside linkages) or the base portion (the purine or pyrimidine portion of natural nucleic acids). 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 the site where the compound might be administered, the Sites of administration are eg 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 usually RNA or a modification thereof. The other strands can be RNA, DNA or any other variant. The biphasic portion of a double-stranded or single-stranded "hairpin" RNAi construct is preferably 18 to 40 nucleotides in length, optionally about 21 to 23 nucleotides in length, so long as it functions as a Dicer substrate . Catalytic or enzymatic nucleic acids may be ribozymes or DNases, and may also include modified forms. The nucleic acid compound inhibits the expression of the target by about 50%, 75%, 90% or more when contacted with the cell under physiological conditions at a nonsense or sense control concentration that produces little or no effect. Preferred concentrations for detecting the effect of nucleic acid compounds are 1, 5 and 10 micromolar. Nucleic acid compounds can be used to test for example effects on bone growth and bone mineralization.

5. Screening method

In certain aspects, the invention relates to the use of ActRIIa polypeptides (eg, soluble ActRIIa polypeptides) and activin polypeptides to identify compounds (drugs) that can agonize or antagonize the activin-ActRIIa signaling pathway. Agents identified by this screen can be tested to assess their ability to modulate bone growth and bone mineralization in vivo, and optionally, these compounds can be further tested in animals to assess their ability to modulate tissue growth in vivo.

There are numerous avenues for screening therapeutics 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 modulating effects of activin or ActRIIa on bone. In certain embodiments, the method is used to screen and identify compounds that specifically inhibit or reduce the binding of an ActRIIa polypeptide to activin. Alternatively, the method is used to identify compounds that enhance binding of an ActRIIa polypeptide to activin. In another embodiment, a compound can be identified by its ability to interact with an activin or ActRIIa polypeptide.

Versions of various methods are sufficient and within the scope of the present disclosure, however those not expressly described herein will be appreciated by those of ordinary skill in the art. As described herein, the detection compounds (agents) of the present invention can be produced by any combination chemistry method. Alternatively, the compound of interest may be a naturally occurring biomolecule synthesized in vivo or in vitro. Compounds (agents) to be tested for their ability to act as tissue growth regulators can be produced, for example, by bacteria, yeast, plants, or other organisms (e.g., natural products), or produced chemically (e.g., small molecules, including peptidomimetics), or recombinantly to produce. Detection compounds encompassed by the present invention include non-peptidic organic molecules, peptides, polypeptides, peptidomimetics, sugars, hormones and nucleic acid molecules. In specific embodiments, the detection agent is a small organic molecule with a molecular weight of less than about 2000 Daltons.

The detection compounds of the invention may be single, discrete entities, or libraries of greater complexity, such as those prepared by combinatorial chemistry. These libraries can include, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers, and other classes of organic compounds. The detection of the presence of compounds in the detection system may be in the form of individual or in the form of a mixture of compounds, especially in the initial screening step. Compounds can optionally be derivatized with other compounds and derivatized populations facilitate isolation of the compounds, as appropriate. Examples of a non-limiting group of derivatives include biotin, fluorescein, digoxin, green fluorescent protein, isotopes, polyhistidine, magnetic beads, glutathione transferase (GST), optically activated crosslinkers (photoactivatible crosslinkers) or any combination thereof.

In many drug screening programs that test compound libraries as well as natural extracts, high-throughput methods are required in order to maximize the number of compounds tested in a given time. Such methods in a cell-free environment, such as those that can purify or semi-purify proteins anywhere, are often preferred as "primary" screening methods, allowing rapid development and relatively easy detection of changes in target molecules modulated by test compounds. Furthermore, testing compounds for cytotoxicity or bioavailability can be neglected in in vitro systems, and the approach instead focuses primarily on the effect of the drug on the target molecule, which can be manifested in changes in the binding affinity of the ActRIIa polypeptide and activin.

By way of illustration only, in an exemplary screening method of the invention, a compound of interest is contacted with an isolated and purified ActRIIa polypeptide that normally binds to activin. The mixture of compound and ActRIIa polypeptide is then added to the composition comprising the ActRIIa ligand. Detection and quantification of the ActRIIa/activin complex provides a method for determining the potency of a compound to inhibit (or potentiate) complex formation between an ActRIIa polypeptide and activin. The potency of a compound can be assessed by generating dose response curves using data obtained at various concentrations of the test compound. Additionally, control analyzes can be used to provide a baseline 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 should be understood that, in general, the order in which the reactants are mixed can vary, and can be mixed simultaneously. In addition, cell extracts and lysates can be used instead of purified proteins to provide suitable cell-free analysis systems.

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

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 of an ActRIIa polypeptide with its binding protein. Further, other detection modes, 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 compatible with many aspects of the present invention. The implementation is the same.

Furthermore, the present invention encompasses the use of the interaction trap approach (also referred to as the "two-hybrid approach") to identify agents that disrupt or enhance the interaction of an ActRIIa polypeptide with its binding protein. See, eg, U.S. Patent 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 specific embodiments, the invention encompasses the use of the reverse two-hybrid system to identify compounds (eg, small molecules or peptides) that isolate the interaction of an ActRIIa polypeptide with 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 U.S. Patents 5,525,490; 5,955,280; and 5,965,368.

In certain embodiments, a compound of interest 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 is either covalent or non-covalent. For example, such interactions can be identified at the protein level using in vivo biochemical methods,

The biochemical methods described include photocrosslinking, isotope-labeled ligand binding, and affinity chromatography (Jakoby WB et al., 1974, Methods in Enzymology 46:1). In some instances, compounds can be screened by certain mechanism-based methods such as detection of compounds that bind to activin or ActRIIa polypeptides. This can involve solid phase or liquid phase combinations. 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 screened, preferably in high-throughput screening, or with Individuals in the library were transfected together. Other mechanisms based on binding methods can be used, for example, in binding assays that detect free energy changes. Binding assays can be performed with targets immobilized to wells, beads or chips or captured by immobilized antibodies or resolved by capillary electrophoresis. Confined compounds can usually be measured colorimetrically or by fluorescence or surface plasmon resonance.

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

For example, the effect of ActRIIa or activin polypeptides or test compounds on bone or cartilage growth can be determined on a cellular basis by measuring induction of Ms×2 or differentiation of osteoblasts into osteoblasts (see, e.g., (Daluiski et al., Nat Genet.2001, 27 (1): 84-8; Hino et al., Front Biosci.2004, 9: 1520-9). Examples of analysis on the basis of other cells include the analysis of the osteogenic activity of the target ActRIIa or activin polypeptide and in the interstitial Test compounds in progenitor cells as well as osteoblasts. For example, recombinant adenoviruses expressing activin or ActRIIa polypeptides can be constructed to infect multipotent mesenchymal progenitor cells C3H10T 1/2, osteoblast precursors C2C12, and osteoblast TE- 85. Osteogenic activity is then determined by measuring alkaline phosphatase, osteocalcin, and induction of matrix mineralization (see, eg, Cheng et al., Jbone Joint Surg Am. 2003, 85-A(8):1544-52).

The invention also encompasses methods of detecting bone or cartilage growth in vivo. For example, Namkung-Matthai et al., Bone, 28:80-86 (2001 ) disclose a rat model of osteoporosis to study early bone repair after fracture. Kubo et al., Steroid Biochemistry & Molecular Biology, 68: 197-202 (1999) also disclose a rat model of osteoporosis to study late bone repair after fracture. Andersson et al., J. Endocrinol. 170: 529-537 disclose a mouse model of osteoporosis in which the mice were ovariectomized, which resulted in a loss of parenchymal bone mineral content and bone mineral density in mice, wherein the cancellous bone lost approximately 50% bone mineral density. Bone density in ovariectomized mice can be increased by administering factors such as parathyroid hormone. In certain aspects, the present invention employs methods of fracture healing known in the art. These methods include fracture techniques, histological analysis, and biomechanical analysis, which are described in, for example, U.S. Pat. invention.

6. Exemplary Therapeutic Applications

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

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 a treated sample relative to an untreated sample, or that , a compound that delays the onset or lessens the severity of one or more symptoms of a disease or condition. The term "treating" as used herein includes preventing the indicated condition or ameliorating or eliminating the condition once established. In either case, prophylaxis or treatment and the expected outcome of the administration of the therapeutic agent are for the consideration of the diagnostic physician.

The present invention provides methods involving bone and/or cartilage formation, preventing bone loss, increasing bone mineralization, or preventing bone demineralization. For example, target activin-ActRIIa antagonists have been applied in the treatment of osteoporosis and the healing of fractures and cartilage defects in humans or animals. ActRIIa or activin peptides 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 invention have medical utility in fracture healing and cartilage defects in humans and other animals. The subject methods and compositions also have prophylactic application in closed and open fracture reduction and improved fixation of artificial joints. De novo bone formation induced by osteogenic agents is helpful for the repair of congenital, trauma-induced or tumor resection-induced craniofacial deformities, and is also effective in medical aesthetics. In some instances, the targeted activin-ActRIIa antagonist provides 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 s disease), Paget's disease, hyperthyroidism, chronic dysentery or malabsorption, renal tubular acidosis, or anorexia nervosa.

Osteoporosis can be caused by or related to various factors. For women, especially postmenopausal women, low body weight and resulting sedentary behavior are risk factors for osteoporosis (loss of bone mineral density, leading to fracture risk). Individuals with any of the following characteristics are candidates for ActRIIa antagonist therapy: postmenopausal women who are 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 taking drugs known to cause bone loss, including glucocorticoids such as prednisone (Prednisone ^™ ), various anticonvulsant drugs such as phenytoin (Dilantin ^TM ) and certain barbiturates, or high doses of thyroid replacement drugs; people with type 1 diabetes, liver disease, kidney disease, or a family history of osteoporosis; people with high bone turnover (eg, in urine People with hypercollagen); people with thyroid conditions such as hyperthyroidism; people with fractures after minor trauma; people with x-ray evidence of vertebral fractures or other phenomena of osteoporosis.

As mentioned above, osteoporosis can also be a condition associated with another disease or with the use of certain drugs. Osteoporosis caused by drugs or other medical conditions is called secondary osteoporosis. In a condition called Cushing's disease, too much cortisol produced by the body leads to osteoporosis and bone fractures. The drugs most commonly 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 sustainably reduce bone mass. Bone loss can occur when high doses of antacids containing aluminum are taken by people with kidney problems, especially those on dialysis. Other agents that can cause secondary osteoporosis include phenytoin (Dilantin) and barbiturates, used to prevent epilepsy; methotrexate (Rheumatrex, Immunex, Folex PFS), a drug used in some drugs for 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 stimulator (Lupron, Zoladex), for prostate cancer and endometriosis; heparin (Calciparine, Liquaemin), an anticoagulant drug; and cholestyramine (Questran) and colestipol (Colestid), for high cholesterol. Cancer therapy-induced bone loss is widely recognized and referred to as cancer therapy-induced bone loss (CTIBL). Bone metastases can create cavities in the bone that can be treated and corrected with activin-ActRIIa antagonists.

In a preferred embodiment, the activin-ActRIIa antagonist disclosed in the present invention, especially soluble ActRIIa, can be applied to cancer patients. Patients with certain tumors (eg, prostate, breast, multiple myeloma, or any tumor that causes hyperthyroidism) who are at high risk for bone loss due to tumor-induced bone loss as well as bone metastases and therapeutic agents . Even in the absence of evidence of bone loss or bone metastases, such patients could be treated with an activin-ActRIIa antagonist. Patients can also be monitored for evidence of bone loss or bone metastases and treated with an activin-ActRIIa antagonist when indicators indicate an increased risk. Typically, changes in bone density are assessed using a DEXA (dual energy x-ray absorptiometry study) scan, and indicators of skeletal 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 bone metastasis. An increase in BSAP was detected in patients with bone metastases from prostate cancer and was present to varying degrees in bone metastases from breast cancer. High levels of bone morphogenetic protein 7 (BMP-7) are present in prostate cancers that have metastasized to bone, but not in bone metastases from bladder, skin, liver, or lung cancers. Type I carboxy-terminal peptide (ICTP) is a cross-linker found in collagen formed during bone resorption. ICTP is found throughout the body as bones are continually broken down and remodeled. However, at sites of bone metastases, levels will be significantly higher than in areas of normal bone. High levels of ICTP are found in the prostate, lung, and bone metastases from breast cancer. Another collagen cross-linker, type I amino-terminal peptide (NTx), is produced with ICTP during bone turnover. NTx levels are elevated in bone metastases from many different cancers including lung, prostate, and breast cancer. Moreover, the increase in NTx levels was accompanied by the development of bone metastases. Therefore, this marker can be used to detect bone metastases as well as measure the extent of disease. Other absorbing labels include pyridyl ethers and deoxypyridine ethers. Any increase in resorption markers as well as markers of bone metastases indicates that the patient requires treatment with an activin-ActRIIa antagonist.

Activin-ActRIIa antagonists may be administered in combination with other pharmaceutical agents. Joint administration can be accomplished by a single co-formulation, by simultaneous or spaced-apart administration. Activin-ActRIIa antagonists are of particular advantage if administered in combination with other bone activating agents. Patients may benefit from the administration of an activin-ActRIIa antagonist in combination with intake of calcium supplements, vitamin D, appropriate exercise, and/or, in some cases, other medications. Examples of other drugs include bisphosphonates (alendronate, ibandronate, and risedronate), calcitonin, estrogens, 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 stop the bone resorption portion of the bone remodeling cycle without slowing the bone formation portion of the cycle. The result is that the rate of new formation is consistently greater than the rate of resorption, and bone density continues to increase. Parathyroid hormone fragment, a form of parathyroid hormone that increases the rate of bone formation in the bone remodeling cycle. Alendronate has been approved for the prevention (5 mg daily or 35 mg weekly) and treatment (10 mg daily or 70 mg weekly) 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 in men and women as a result of long-term use of these drugs (ie, prednisone and cortisone) and for osteoporosis in men. loose treatment. Alendronate plus vitamin D is approved for the treatment of postmenopausal osteoporosis in women (70 mg once weekly plus vitamin D) and for treatment to improve bone mass in men with osteoporosis . Ibandronate sodium is approved for the prevention and treatment of postmenopausal osteoporosis. Taken once a month (150 mg), ibandronate must be taken on the same day of 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-vertebral fractures. Risedronate is also approved for use in men and women to prevent and/or treat glucocorticoid-induced osteoporosis resulting from the chronic administration of these drugs (ie, prednisone and cortisone). Calcitonin is a naturally occurring hormone involved in calcium regulation and bone metabolism. In women after 5 years of menopause, calcitonin slows bone loss, increases spinal bone density and may reduce fracture-related pain. Calcitonin reduces the risk of spinal fractures. Calcitonin is available as an injection (50-100 IU per day) or as a nasal spray (200 IU per day). Estrogen therapy (ET)/hormone therapy (HT) is approved for the prevention of osteoporosis. ET has been shown to reduce bone loss, increase spine and hip bone density, and reduce the risk of hip and spine fractures in postmenopausal women. ET administration is most commonly in the form of pills or skin patches, which deliver low doses of about 0.3 mg per day or standard doses of about 0.625 mg per day, and are effective even when started after age 70. When taken alone, estrogen can increase a woman's risk of developing cancer of the cervical wall (endometrial cancer). To eliminate this risk, healthcare providers prescribe the hormone progesterone in combination with estrogen for women with an intact uterus. ET/HT alleviates menopausal symptoms and has been shown to have beneficial effects on bone health. Side effects include vaginal bleeding, breast tenderness, mood disturbances, and gallbladder disease. Raloxifene, 60 mg daily, is approved for the prevention and treatment of postmenopausal osteoporosis. It is derived from a family of drugs called selective estrogen receptor modulators (SERMs), which were developed to provide the beneficial effects of estrogen without the potential pitfalls. Raloxifene increases estimates and reduces the risk of spinal fractures. Data are still not available to demonstrate that raloxifene reduces the risk of hip and other non-spine fractures. This drug stimulates the formation of new bone 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 was significant in the spine, but insufficient data were available to assess fracture reduction in other locations. Parathyroid hormone fragments are self-administered as injections 24 hours a day.

7. Pharmaceutical composition

In certain embodiments, an activin-ActRIIa antagonist (eg, an ActRIIa polypeptide) of the invention is formulated with a pharmaceutically acceptable carrier. For example, an ActRIIa polypeptide can be administered alone or as a component of a formulation (therapeutic composition). The compound of interest may be formulated for administration by any route suitable for human or veterinary use.

In certain embodiments, the methods of treatment of the present invention comprise administering the composition systemically or locally as an implant or device. When administered, the therapeutic combinations for use in the present invention are of course in a pyrogen-free, physiologically acceptable form. Therapeutically effective agents other than ActRIIa antagonists are optionally included in the compositions described above, which may be administered simultaneously or sequentially with the compound of interest (eg, ActRIIa polypeptide) in the methods of the invention.

Typically, the ActRIIa antagonist is administered parenterally. Pharmaceutical compositions suitable for parenteral administration include one or more ActRIIa polypeptides and one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solvents, dispersions, suspensions or emulsions, or reconstituted into Sterile powders for sterile injectable solutions or dispersions previously used in combination, which may contain antioxidants, buffers, antibacterial agents, solutes to render the formulation isotonic with the blood of the future recipient, or drugs to suspend or thicken . Examples of suitable aqueous or non-aqueous carriers for use in the 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 coating substances such as lecithin, in the case of dispersions by maintaining the required particle size, and by the use of surfactants.

Additionally, the compositions may be encapsulated or injected for delivery to a target tissue site (eg, bone). In certain embodiments, compositions of the invention include a matrix that can release one or more therapeutic compounds (e.g., ActRIIa polypeptides) to a target tissue location (e.g., bone), provide a structure to the developing tissue and, Ideally, it can be absorbed into the body. For example, the matrix can provide slow release of the ActRIIa polypeptide. Such matrices may be formed from materials already available for other implanted medical applications.

The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, appearance, and interfacial properties. The particular 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 substances are biodegradable and biologically very clear, such as bone or dermal collagen. Other matrices include pure protein or extracellular matrix components. Other potential matrices are non-biodegradable and chemically clear, such as sintered hydroxyapatite, bioglass, aluminates or other ceramics. The matrix may consist of a combination of any of the types of substances mentioned above, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. Bioceramics can be altered in composition, such as in calcium-aluminum-phosphate and in treatments that alter pore size, particle size, particle shape, and biodegradability.

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

In solid dosage forms (capsules, tablets, pills, dragees, powders, granules, etc.) for oral administration, one or more therapeutic compounds of the invention may be combined 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, alginate, gelatin, polyvinylpyrrolidone, sucrose, and/or gum arabic; (3) humectants, such as glycerin; (4) disintegrants, such as agar-agar, carbonic acid Calcium, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution slow-release agents, such as paraffin; (6) absorption enhancers, such as quaternary ammonium compounds; (7) wetting agents , such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite; (9) lubricants, such as talc, calcium stearate, magnesium stearate, and solid polyethylene glycol Alcohol, sodium lauryl sulfate and mixtures thereof; (10) Colorants. In the case of capsules, tablets and pills, the pharmaceutical composition may comprise buffering agents. Solid compositions of a similar type may also be used in the filling of soft-filled and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, syrups and elixirs. In addition to the active ingredient, liquid dosage forms contain inert diluents (such as water or other solvents), co-solvents and emulsifiers (such as alcohol, isopropanol, ethyl carbonate, ethyl acetate, phenylpropanol, benzoic acid) commonly used in the art. Fatty acids of benzyl esters, propylene glycol, 1,3-butanediol, oils (especially cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerin, THF, polyethylene glycol, and sorbitan Esters and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, perfuming, coloring, perfuming and preservative agents.

Suspensions, which contain, besides the active compound, substances such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar -Suspending agents for agar-agar and tragacanth and mixtures thereof.

The compositions of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents (eg, paragum, chlorobutanol, phenol, and the like). Isotonic agents such as sugars, sodium chloride, and the like can also be included in the compositions as necessary. In addition, prolonged absorption of the injectable drugs can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Of course, the dosing regimen is determined by the attending physician considering various factors that alter the effect of the target compound (eg, ActRIIa polypeptide) of the present invention. The various factors described include, but are not limited to, the weight of bone that needs to be formed, the extent of bone density loss, the location of the bone injury, the condition of the damaged bone, the patient's age, sex, and diet, and the severity of any disease that promotes bone loss , administration time, and other clinical factors. Dosages may vary depending on the type of matrix used in the reconstitution and the type of compound in the composition, as appropriate. Other known growth factors added to the final composition may also affect dosage. Progress can be monitored by periodic assessment of bone growth and/or repair (eg, X-rays (including DEXA), histomorphometry, and tetracycline labeling).

Experiments in mice demonstrated detectable effects of ActRIIa-Fc on bone when the compound was administered at intervals and at doses sufficient to achieve plasma concentrations of 0.2 μg/kg or greater, and serum drug levels of 1 μg /kg or 2 μg/kg or higher is necessary to obtain significant effects on bone density and strength. Treatment regimens were designed to achieve plasma concentrations between 0.2 and 15 μg/kg, optionally between 1 and 5 μg/kg, although there was no indication that higher doses of ActRIIa-Fc would be undesirable due to side effects. 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 are obtained, for example, by 0.2- 0.4 mg/kg dose, or higher doses with longer intervals between doses. For example, a dose of 1-3 mg/kg may be used on a monthly or bi-monthly Required once a month.

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 ActRIIa polynucleotide sequences into cells or tissues suffering from the diseases listed above. Delivery of ActRIIa polynucleotide sequences can be accomplished through the use of 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 that can be used for gene therapy as taught by the present invention include adenoviruses, herpesviruses, 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 foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous sarcoma virus ( RSV). A number of other retroviruses incorporate multiple genes. All of these vectors can transfer or incorporate genes for selectable markers so that transduced cells can be identified and generated. Retroviral vectors can be made target-specific by attachment of, for example, sugars, glycolipids or proteins. Preferably targeting is achieved through the use of antibodies. Those skilled in the art will appreciate that specific polynucleotide sequences can be inserted into the retroviral genome or adhered to the viral envelope protein to allow target-specific delivery of retroviral vectors comprising ActRIIa polynucleotides. In a preferred embodiment, the vector targets bone or cartilage.

Alternatively, tissue construct 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 ActRIIa polynucleotide targeted release system is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and liposome systems include water-in-oil emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal dispersion system of the present invention is liposomes. Liposomes are artificial membrane vesicles that are effective as dispersion vehicles in vitro or in vivo. RNA, DNA, and intact virus particles can be encapsulated with an internal aqueous solution and dispersed in a biologically active form (see, eg, Fraley, et al., Trends Biochem. Sci., 6:77, 1981). Methods for efficient gene transfection using liposome vectors are well known in the art, see, eg, Mannino, et al., Biotechniques, 6:682,1988. Liposome compositions are usually combinations of phospholipids, usually with steroids, especially cholesterol. Other phosphates or other lipids may also be used. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

Examples of lipids that can be used to produce liposomes include phosphatidyl compounds such as phosphatidylglycerol, lecithin, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, cerebrosides, and gangliosides. Illustrative examples of phospholipids include egg yolk lecithin, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine. Targeting of liposomes may also be based on, for example, organ specificity, cell specificity, and organelle specificity, among others known in the art.

Example

The invention is 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. invention.

Embodiment 1: ActRIIa-Fc fusion protein

Applicants constructed soluble ActRIIa fusion proteins with the extracellular domain of human ActRIIa fused to a human or mouse Fc domain with minimal linkers in between. Constructs are referred to as ActRIIa-hFc and ActRIIa-mFc, respectively.

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

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSI

EIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEV

TQPTSNPVTPKPP TGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV

TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVL

HODWLNGKEYKCKVSNKALPVPIEKTISKAKGOPREPOVYTLPPSREEMTK

NOVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWOOGNVFSCSVMHEALHNHYTOKSLSLSPGK

ActRIIa-hFc and ActRIIa-mFc proteins were expressed in CHO cell lines. Three different preamble sequences were considered:

(i) Honey bee melittin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ IDNO: 8)

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

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

The selected form employs a TPA leader and has the following unprocessed amino acid sequence:

MDAMKRGLCCVLLLCGAVFVSPGAAILGRSETQECLFFNANWEKDRTNQT

GVEPCYGDKDKRRHCFATWKNIS GSIEIVKQGCWLDDINCYDRTDCVEKKD

SPEVYFCCCEGNMCNEKF SYFPEMEVTQPT SNPVTPKPPTGGGTHTCPPCPA

PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE

VHNAKTKPREEQYNSTYIVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIE

KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG

QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 13)

The peptide is encoded by the following nucleotide sequence:

ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGT GGAG

CAGTCTTCGTTTCGCCCGGCGCCGCTATACTTGGTAGATCAGAAACTCAG

GAGTGTCTTTTTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAAC

TGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTG

CTACCTGGAAGAATATTTCTGGTTCCATTGAATAGTGAAACAAGGTTGTT

GGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAA

AGACAGCCCTGAAGTATATTTCTGTTGCTGTGAGGGCAATATGTGTAATG

AAAAGTTTTCTTATTTTCCGGAGATGGAAGTCACACAGCCACTTCAAAT

CCAGTTACACCTAAGCCACCCACCGGTGGTGGAACTCACACATGCCCAC

CGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCC

CCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT

GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTG

GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGA

GGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTG

CACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA

AAGCCTCCCAGTCCCCATCGAGAAAACCATTCTCAAAGCCAAAGGGCA

GCCCCGAGAACCACAGGTGTACACCCTGCCCCCCATCCCGGGAGG AGATG

ACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCA

GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT

ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAT

AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCT

CATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAG CCCTTCCCCTGTCTCCGGGTAAATGAGAATTC (SEQ ID NO: 14)

Both ActRIIa-hFc and ActRIIa-mFc are remarkably amenable to recombinant expression. As shown in Figure 1, the protein was purified as a single, well-defined protein peak. N-terminal sequencing revealed a unique sequence for 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 restriction chromatography and cation exchange chromatography. Purification can be accomplished by virus filtration and buffer exchange. The ActRIIa-hFc protein was purified to a purity of greater than 98% as determined by size exclusion chromatography and greater than 95% as determined by SDS PAGE.

ActRIIa-hFc and ActRIIa-mFc showed high affinity to ligands, especially activinA. GDF-11 or ActivinA ("ACA") was immobilized on a Biacore CM5 chip using standard amino acid coupling procedures. ActRIIa-hFc and ActRIIa-mFc proteins were loaded into the system and their binding was measured. ActRIIa-hFc binds to activin with a dissociation constant (K _D ) of 5×10 ^-12 , while the protein binds to GDF11 with a K _D of 9.96×10 ^-9 . See Figure 2. The situation is similar for ActRIIa-mFc.

The A-204 reporter gene assay evaluates the effect of ActRIIa-hFc protein on signaling by GDF-11 and ActivinA. Cell line: human rhabdomyosarcoma (derived from muscle). 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 gene), so this vector is commonly used for signaling factors through Smad2 and 3.

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

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

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

This was followed by a luciferase assay. Typically in this method, without the presence of any inhibitors, Activin A was shown to stimulate reporter gene expression about 10-fold with an ED50 ~ 2 ng/ml. GDF-11: 16-fold stimulation, ED50: ~1.5 ng/ml. GDF-8 showed similar effects to GDF-11.

As shown in Figure 4, ActRIIa-hFc and ActRIIa-mFc inhibit GDF-8-mediated signaling at picomolar concentrations. As shown in Figure 5, three different ActRIIa-hFc formulations inhibited GDF-11 signaling with an IC50 close to 200 pM.

ActRIIa-hFc is very stable in pharmacokinetic assays. Rats were dosed with 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 at 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 1 mg/kg, 10 mg/kg or 30mg/kg starting dose). In cynomolgus monkeys, the plasma half-life was substantially greater than 14 days and 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 show that the serum drug drug half-life is between approximately 20 and 30 days.

Example 2: ActRIIa-hFc promotes bone growth in vivo

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

In female BALB/c, 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, antagonists of ActRIIa caused increases in bone density and mass in normal female mice. As a subsequent step, the effect of ActRIIa-mFc was tested in an ovariectomized mouse model.

Andersson et al. (2001), determined that ovariectomized mice underwent considerable bone loss (approximately 50% of cancellous bone was lost at 6 weeks postoperatively), and that bone loss in these mice could be cured by candidate therapeutic agents such as parathyroid glandular hormone therapy.

Applicants employed 4-5 week old ovariectomized (OVX) female C57BL6 mice or sham-operated female mice. Eight weeks after surgery, treatment with ActRIIa-mFc (10 mg/kg, twice a week) or control (PBS) was started. Bone density is measured with a 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. The ActRIIa-mFc treatment group retained bone mineral density at the level of the sham group. ActRIIa-mFc caused comparable growth of cancellous bone in OVX mice at 6 and 12 weeks of treatment. 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%.

ActRIIa-mFc also caused a comparable increase in cancellous bone in sham-operated mice. See Figure 11. Treatment produced a 35% increase relative to control after 6 and 12 weeks. the

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) over 12 weeks. Similar to the results described above for ActRIIa-mFc, OVX mice receiving ActRIIa-mFc exhibited a 15% increase in cancellous bone density as early as 4 weeks of treatment and a 15% increase after 12 weeks of treatment (Fig. 12 ). Sham-operated mice receiving ActRIIa-mFc also showed a 25% increase in cancellous bone density as early as 4 weeks of treatment and a 32% increase after 12 weeks of treatment (Figure 13).

After 12 weeks of treatment with ActRIIa-mFc, whole-body and ex vivo femur DEXA analysis showed that treatment induced an increase in bone density in both ovariectomized and sham-operated mice (Figure 14A and Figure 14B, respectively). These results were also supported by ex vivo pQCT analysis of the midshaft femur, which demonstrated a significant increase in total bone density as well as cortical bone density after 12 weeks of ActRIIa-mFc treatment. Vehicle-treated control ovariectomized mice exhibited bone density compared 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 midshaft femur demonstrated a significant increase in total and cortical bone mass after 12 weeks of ActRIIa-mFc treatment, whereas ovariectomized mice and sham-operated vehicle control-treated mice both displayed comparable Bone mass (Figure 16). Ex vivo pQCT analysis of the mid-femur also revealed that ActRIIa-mFc-treated mice did not show changes in periosteal perimeter, whereas ActRIIa-mFc treatment resulted in a decrease in endosteal perimeter suggesting that the increase in cortical thickness is driven by the inner surface of the femur. caused by growth (Fig. 17). Mechanical testing of the femur determined that ActRIIa-mFc can increase the extrinsic properties of the bone (maximum load, stiffness, and energy to fracture), which contributes to a significant increase in the intrinsic properties of the bone (ultimate strength). Ovariectomized mice treated with ActRIIa-mFc displayed increased bone strength above sham-operated, vehicle-treated control levels, indicating complete reversal of the osteoporotic phenotype (Figure 18).

These data demonstrate that activin-ActRIIa antagonists increase bone density in female mice and, moreover, correct defects in bone density, bone content, and maximal bone strength in a mouse model of osteoporosis.

In another set of experiments, mice were ovariectomized or sham-operated at week 4, and received placebo or ActRIIa-mFc (twice a week, 10 mg/kg) at the beginning of week 12 (as shown in Figures 19-24 RAP-11) for another 12 weeks. Assess various skeletal parameters. As shown in Figure 19, ActRIIa-mFc increased the ratio of spinal cancellous bone volume to total volume (BV/TV) in OVX and sham groups. ActRIIa-mFc also improved cancellous bone structure (FIG. 20), increased periosteal thickness (FIG. 21) and improved bone strength (FIG. 22). As shown in Figure 23, ActRIIa-mFc produced satisfactory effects in the dose range from 1 mg/kg to 10 mg/kg.

Histomorphometric measurements of bones were 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 as well as 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 deletes the 15 amino acids of the C-terminal tail (last) of the ActRIIa extracellular domain. The sequence of such a construct is as follows: (Fc portion is underlined) (SEQ ID NO: 12):

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSI

EIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM TG

GGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV

KFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKEYKCK

VSNKALPVPIEKTISKAKGOPREPOVYTLPPSREEMTKNOVSLTCLVKGFYPS

DIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEALHNHYTOKSLSLSPGK

Integration of References

All publications and patents mentioned herein are herein incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

While specific examples of the subject matter have been discussed, the foregoing description is illustrative and not restrictive. Many modifications will become apparent to those skilled in the art from reading the foregoing specification and the following claims. The entire protection scope of the present invention should be determined with reference to the claims and their equivalent protection scope and the description and such changes.

Claims (40)

1. activin-is in conjunction with an ActRIIa polypeptide, and it comprises the aminoacid sequence be made up of the aminoacid sequence of SEQ ID NO:7.

2. activin-according to claim 1 is in conjunction with ActRIIa polypeptide, considers protein impurities, and the purity of wherein said polypeptide is at least 95%, and it is determined by size exclusion chromatography.

3. the activin-described in claim 1 or 2 in conjunction with ActRIIa polypeptide, wherein said polypeptide relative to GDF-11 to the selectivity dissociation constant of activin showing at least 10 times.

4. a pharmaceutical preparation, it comprises activin-any one of claim 1-3 in conjunction with ActRIIa polypeptide and the pharmaceutically acceptable adjuvant of at least one.

5. pharmaceutical preparation according to claim 4, wherein said preparation is apyrogeneity material substantially.

6. the polynucleotide be separated, it comprises the coded sequence of the activin-any one of claim 1-3 in conjunction with ActRIIa polypeptide.

7. the polynucleotide of separation according to claim 6, the polynucleotide of wherein said separation comprise the sequence of SEQ ID NO:14.

8. a recombination of polynucleotide, it comprises promoter sequence and is operably connected to polynucleotide described in claim 6 or 7.

9. a cell, it transforms with the recombination of polynucleotide any one of claim 6-8, and wherein said cell is not the plant cell of the plant variety that can develop into whole plant.

10. cell according to claim 9, wherein said cell is mammalian cell.

11. cells according to claim 10, wherein said cell is Chinese hamster ovary celI or human cell.

12. 1 kinds manufacture the method for activin-in conjunction with ActRIIa polypeptide, and the method comprises:

A) cultured cell under the condition of applicable expression ActRIIa polypeptide, wherein said cell transforms with the recombination of polynucleotide any one of claim 6-8; And

B) activin-expressed by renaturation is in conjunction with ActRIIa polypeptide.

13. 1 kinds of pharmaceutical preparatioies, it comprises:

A the activin-any one of () claims 1 to 3 is in conjunction with ActRIIa polypeptide; And

B () second bone-active agent, wherein said second bone-active agent is selected from: bisphosphonate, estrogen, selective estrogen receptor modulators, parathyroid hormone, calcitonin, calsium supplement and Vitamin D supplements.

Activin-any one of 14. claim 1-3 is in conjunction with ActRIIa polypeptide, or the pharmaceutical preparation any one of claim 4-5, and wherein said polypeptide is glycosylated.

15. 1 peptide species, it comprises SEQ ID NO:7.

16. 1 kinds of soluble polypeptides according to claim 15.

17. 1 kinds of polypeptide according to claim 16, it is bonded to activin.

18. 1 kinds of polypeptide according to claim 17, it is bonded to activin to be less than 1 micromolar dissociation constant.

19. 1 kinds of polypeptide according to claim 18, it is bonded to activin with the dissociation constant being less than 100 nanomoles.

20. 1 kinds of polypeptide according to claim 19, it is bonded to activin with the dissociation constant being less than 10 nanomoles.

21. 1 kinds of polypeptide according to claim 20, it is bonded to activin with the dissociation constant being less than 1 nanomole.

22. 1 kinds of polypeptide according to claim 15, its purity is at least 95%.

23. 1 kinds of polypeptide according to claim 22, its purity is at least 98%.

24. 1 kinds of polypeptide according to claim 15, it is acetylizad.

25. 1 kinds of polypeptide according to claim 15, it is carboxylated.

26. 1 kinds of polypeptide according to claim 15, it is glycosylated.

27. 1 kinds of polypeptide according to claim 15, it is phosphorylation.

28. 1 kinds of polypeptide according to claim 15, it is lipidization.

29. 1 kinds of polypeptide according to claim 15, it is acidylate.

30. 1 kinds of polypeptide according to claim 15, it comprises Polyethylene Glycol.

31. 1 kinds of polypeptide according to claim 15, it comprises lipid.

32. 1 kinds of polypeptide according to claim 15, it comprises phosphoric acid.

33. 1 kinds of polypeptide according to claim 15, it comprises polysaccharide.

34. 1 kinds of polypeptide according to claim 15, it comprises monosaccharide.

35. 1 kinds of polypeptide according to claim 15, it is Pegylation.

36. 1 kinds of polypeptide according to claim 15, it is farnesylation.

37. 1 kinds of polypeptide according to claim 15, it is biotinylated.

38. 1 kinds of polypeptide according to claim 15, it is coupled to organic derivative.

39. activin-according to claim 1 are in conjunction with ActRIIa polypeptide, and wherein said polypeptide is bonded to activin.

The method of 40. claim 12, wherein said activin-is soluble polypeptide in conjunction with ActRIIa polypeptide.