HK1228411B - Variants derived from actriib and uses therefor - Google Patents

Description

Variants derived from ACTRIIB and their uses

This application is a divisional application of the following application: Application date: February 4, 2008; Application number: 200880011247.6 (PCT/US2008/001506); Invention name: same as above.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No. 60/899,304, filed February 2, 2007, U.S. Provisional Application Serial No. 60/927,088, filed May 1, 2007, and U.S. Provisional Application Serial No. 60/931,880, filed May 25, 2007. All teachings of the above-cited applications are incorporated herein by reference.

Background of the Invention

The transforming growth factor-β (TGF-β) superfamily includes a variety of growth factors that share common sequence elements and structural motifs. These proteins are known to exert biological effects on a wide range of cell types in both vertebrates and invertebrates. Superfamily members perform important functions in pattern formation and tissue specification during embryonic development and can influence a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis, cardiogenesis, erythropoiesis, neurogenesis, and epithelial differentiation. The family is divided into two general branches: the BMP/GDF and the TGF-β/activin/BMP10 branches, whose members have distinct, often complementary, effects. By manipulating the activity of TGF-β family members, it is often possible to produce significant physiological changes in an organism. For example, Piedmontese and Belgian Blue cattle breeds carry disabling mutations in the GDF8 (also known as myostatin) gene, which results in a significant increase in muscle mass. Grobet et al., Nat Genet. 1997, 17(1):71-4. Furthermore, in humans, the inactive allele of GDF8 is associated with increased muscle mass and reportedly associated with abnormal strength. Schuelke et al., N Engl J Med 2004, 350:2682-8.

Changes in muscle, bone, cartilage, and other tissues can be achieved by agonizing or antagonizing signal transduction mediated by appropriate TGF-β family members. Therefore, there is a need for drugs that act as effective modulators of TGF-β signal transduction.

SUMMARY OF THE INVENTION

In certain aspects, the disclosure herein provides ActRIIB polypeptides, particularly ActRIIB variants, including amino-terminal and carboxyl-terminal truncations and sequence alterations. Such ActRIIB polypeptides can be used to treat a variety of diseases or conditions, particularly muscle and neuromuscular diseases (e.g., muscular dystrophy, amyotrophic lateral sclerosis (ALS), and muscle atrophy), adipose tissue diseases (e.g., obesity), metabolic diseases (e.g., type 2 diabetes), neurodegenerative diseases and muscle atrophy associated with aging (sarcopenia), prostate cancer treatment, and cancer cachexia. In specific embodiments, ActRIIB polypeptides (e.g., soluble ActRIIB polypeptides) can antagonize ActRIIB receptors in any process associated with ActRIIB activity. Optionally, the ActRIIB polypeptides of the present invention can be designed to preferentially antagonize one or more ligands of the ActRIIB receptor, such as GDF8 (also known as myostatin), GDF11, activin A, activin B, activin AB, Nodal, and BMP7 (also known as OP-1), and thus can be used to treat other diseases. Examples of ActRIIB polypeptides include native ActRIIB polypeptides and functional variants thereof. The disclosure also provides a group of variants derived from ActRIIB that have greatly reduced affinity for activin while retaining binding to GDF11. These variants exhibit desirable effects on muscle while reducing effects on other tissues.

In certain aspects, the disclosure provides pharmaceutical formulations containing a soluble ActRIIB polypeptide that binds to an ActRIIB ligand, such as GDF8, GDF11, activin, BMP7, or nodal, and a pharmaceutically acceptable carrier. Optionally, the soluble ActRIIB polypeptide binds to the ActRIIB ligand with a Kd of less than 10 micromolar, or less than 1 micromolar, 100, 10, or 1 nanomolar. Optionally, the soluble ActRIIB polypeptide inhibits ActRIIB signaling, such as intracellular signaling events triggered by an ActRIIB ligand. The soluble ActRIIB polypeptide used in such a formulation can be any of those disclosed herein, such as a polypeptide having an amino acid sequence selected from SEQ ID NOs: 1, 2, 5, 6, and 12, or a polypeptide having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 1, 2, 5, 6, and 12. Soluble ActRIIB polypeptides can include functional fragments of native ActRIIB polypeptides, such as fragments comprising at least 10, 20, or 30 amino acids selected from the group consisting of SEQ ID NOs: 1, 2, 5, 6, and 12, or the sequence of SEQ ID NO: 1, which lacks 1, 2, 3, 4, 5, or 10-15 amino acids at the C-terminus and lacks 1, 2, 3, 4, or 5 amino acids at the N-terminus. Preferred polypeptides will contain a truncation of 2-5 amino acids at the N-terminus and no more than 3 amino acids at the C-terminus relative to SEQ ID NO: 1. Another preferred polypeptide is the polypeptide provided as SEQ ID NO: 12. Soluble ActRIIB polypeptides can include one or more alterations in the amino acid sequence (e.g., in the ligand-binding domain) relative to native ActRIIB polypeptides. Alterations in the amino acid sequence can, for example, alter the glycosylation of the polypeptide when produced in mammalian, insect, or other eukaryotic cells, or alter proteolytic cleavage of the polypeptide relative to native ActRIIB polypeptides. A soluble ActRIIB polypeptide can be a fusion protein having an ActRIIB polypeptide (e.g., a ligand-binding domain of ActRIIB or a variant thereof) as a domain and one or more other domains that provide desired properties, such as improved pharmacokinetics, easier purification, targeting to specific tissues, etc. For example, the domains of the fusion protein can enhance one or more of the following properties: in vivo stability, in vivo half-life, absorption/administration, tissue localization or distribution, formation of protein complexes, multimeric fusion proteins, and/or purification. A soluble ActRIIB fusion protein can include an immunoglobulin Fc domain (wild type or mutant) or serum albumin. In certain embodiments, the ActRIIB-Fc fusion comprises a relatively unstructured linker located between the Fc domain and the extracellular ActRIIB domain. This unstructured linker can correspond to an unstructured region of approximately 15 amino acids at the C-terminus of the ActRIIB extracellular domain ("tail"), or it can be an artificial sequence of 5 to 15, 20, 30, 50, or more amino acids with relatively free secondary structure. The linker can be rich in glycine and proline residues and can, for example, contain repeats of threonine/serine and glycine (e.g., TG ₄ or SG ₄ repeats). The fusion protein can include a purification subsequence, such as an epitope tag, a FLAG tag, a polyhistidine sequence, and a GST fusion. Optionally, the soluble ActRIIB polypeptide includes one or more modified amino acid residues selected from the group consisting of glycosylated amino acids, PEGylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, amino acids conjugated to a lipid moiety, and amino acids conjugated to an organic derivative. The pharmaceutical formulation can also include one or more additional compounds, such as compounds for treating ActRIIB-related diseases. Preferably, the pharmaceutical formulation is substantially pyrogen-free. Generally, ActRIIB proteins expressed in mammalian cell lines are preferably mediated by suitable native glycosylation of the ActRIIB protein to reduce the possibility of adverse immune responses in patients. Human cell lines and CHO cell lines have been successfully used, and it is expected that other common mammalian expression vectors will also be useful.

In certain aspects, the disclosure herein provides packaged pharmaceuticals containing the pharmaceutical formulations described herein and labeled for use in humans to promote tissue growth or reduce or prevent tissue loss. Exemplary tissues include bone, cartilage, muscle, adipose, and neuronal tissue.

In certain aspects, the disclosure herein provides soluble ActRIIB polypeptides comprising an altered ligand-binding (e.g., GDF8-binding) domain. Such altered ligand-binding domains of the ActRIIB receptor comprise one or more mutations at amino acid residues such as E37, E39, R40, K55, R56, Y60, A64, K74, W78, L79, D80, F82, and F101 of human ActRIIB (numbered relative to SEQ ID NO: 2). Optionally, the altered ligand-binding domain can have increased selectivity for ligands such as GDF8/GDF11 relative to the wild-type ligand-binding domain of the ActRIIB receptor. For example, the following mutations have been demonstrated herein to increase the selectivity of the altered ligand-binding domain for GDF11 (and, presumably, GDF8) over activin (which provides selectivity for ActRIIB): K74Y, K74F, K74I, and D80I. The following mutations have the opposite effect, increasing the ratio of activin binding to GDF11: D54A, K55A, L79A, and F82A. The overall (GDF11 and activin) binding activity can be increased by including the "tail" region, or presumably unstructured linker region, and also by using the K74A mutation. Other mutations that overall decrease ligand binding affinity include: R40A, E37A, R56A, W78A, D80K, D80R, D80A, D80G, D80F, D80M, and D80N. Mutations can be combined to achieve the desired effect. For example, many mutations that affect the GDF11:activin binding ratio have an overall negative effect on ligand binding; therefore, these mutations can be combined with mutations that generally increase ligand binding to produce binding proteins with improved ligand selectivity.

Optionally, the altered ligand-binding domain has a ratio of activin binding _Kd to GDF8 binding _Kd that is at least 2, 5, 10, or even 100 times greater than that of the wild-type ligand-binding domain. Optionally, the altered ligand-binding domain has a ratio of activin inhibition _IC50 to GDF8/GDF11 inhibition _IC50 that is at least 2, 5, 10, or even 100 times greater than that of the wild-type ligand-binding domain. Optionally, the altered ligand-binding domain inhibits GDF8/GDF11 with an _IC50 that is at least 2, 5, 10, or even 100 times lower than _that of activin inhibition. These soluble ActRIIB polypeptides can be fusion proteins comprising an immunoglobulin Fc domain (wild-type or mutant). In some cases, the soluble ActRIIB polypeptides of the present invention are antagonists (inhibitors) of GDF8/GDF11.

Other variants of ActRIIB are contemplated, for example, as follows: A variant ActRIIB fusion protein comprising a portion of the ActRIIB sequence derived from SEQ ID NO: 2 and a second partial polypeptide, wherein the portion derived from ActRIIB corresponds to a sequence beginning with any one of amino acids 21-29 of SEQ ID NO: 2 (optionally beginning with 22-25 of SEQ ID NO: 2) and ending with any one of amino acids 109-134 of SEQ ID NO: 2, wherein the ActRIIB fusion protein inhibits signaling through activin, myostatin, and/or GDF11 in a cell-based assay. A variant ActRIIB fusion protein as above, wherein the portion derived from ActRIIB corresponds to a sequence beginning with any one of amino acids 20-29 of SEQ ID NO: 2 (optionally beginning with 22-25 of SEQ ID NO: 2) and ending with any one of amino acids 109-133 of SEQ ID NO: 2. The variant ActRIIB fusion protein above, wherein the portion derived from ActRIIB corresponds to a sequence beginning with any one of amino acids 20-24 of SEQ ID NO: 2 (optionally beginning with 22-25 of SEQ ID NO: 2) and ending with any one of amino acids 109-133 of SEQ ID NO: 2. The variant ActRIIB fusion protein above, wherein the portion derived from ActRIIB corresponds to a sequence beginning with any one of amino acids 21-24 of SEQ ID NO: 2 and ending with any one of amino acids 109-134 of SEQ ID NO: 2. The variant ActRIIB fusion protein above, wherein the portion derived from ActRIIB corresponds to a sequence beginning with any one of amino acids 20-24 of SEQ ID NO: 2 and ending with any one of amino acids 118-133 of SEQ ID NO: 2. The variant ActRIIB fusion protein above, wherein the portion derived from ActRIIB corresponds to a sequence beginning with any one of amino acids 21-24 of SEQ ID NO: 2 and ending with any one of amino acids 118-134 of SEQ ID NO: 2. The variant ActRIIB fusion protein above, wherein the portion derived from ActRIIB corresponds to a sequence beginning with any one of amino acids 20-24 of SEQ ID NO: 2 and ending with any one of amino acids 128-133 of SEQ ID NO: 2. The variant ActRIIB fusion protein above, wherein the portion derived from ActRIIB corresponds to a sequence beginning with any one of amino acids 20-24 of SEQ ID NO: 2 and ending with any one of amino acids 128-133 of SEQ ID NO: 2. The variant ActRIIB fusion protein above, wherein the portion derived from ActRIIB corresponds to a sequence beginning with any one of amino acids 21-29 of SEQ ID NO: 2 and ending with any one of amino acids 118-134 of SEQ ID NO: 2. The variant ActRIIB fusion protein above, wherein the portion derived from ActRIIB corresponds to a sequence beginning with any one of amino acids 20-29 of SEQ ID NO: 2 and ending with any one of amino acids 118-133 of SEQ ID NO: 4. The variant ActRIIB fusion protein above, wherein the portion derived from ActRIIB corresponds to a sequence beginning with any one of amino acids 21-29 of SEQ ID NO: 2 and ending with any one of amino acids 128-134 of SEQ ID NO: 2. The above variant ActRIIB fusion proteins, wherein the portion derived from ActRIIB corresponds to a sequence beginning with any one of amino acids 20-29 of SEQ ID NO: 2 and ending with any one of amino acids 128-133 of SEQ ID NO: 2. Surprisingly, constructs beginning with amino acids 22-25 of SEQ ID NO: 2 had higher activity levels than proteins containing the entire extracellular domain of human ActRIIB. Any of the above variant ActRIIB fusion proteins can be produced as homodimers. Any of the above ActRIIB fusion proteins can have a heterologous portion containing a constant region from an IgG heavy chain, such as an Fc domain.

Other variant ActRIIB proteins are contemplated, for example, as follows: a variant ActRIIB protein comprising an amino acid sequence at least 80% identical to amino acids 29-109 of SEQ ID NO:2, wherein the position corresponding to position 64 of SEQ ID NO:2 is R or K, wherein the variant ActRIIB protein inhibits signaling through activin, myostatin, and/or GDF11 in a cell-based assay; a variant ActRIIB protein above, wherein at least one alteration in the sequence of SEQ ID NO:2 is located outside the ligand binding pocket; a variant ActRIIB protein above, wherein at least one alteration in the sequence of SEQ ID NO:2 is a conservative alteration located within the ligand binding pocket; a variant ActRIIB protein above, wherein at least one alteration in the sequence of SEQ ID NO:2 is at one or more positions selected from the group consisting of K74, R40, Q53, K55, F82, and L79. The variant ActRIIB protein described above, wherein the protein comprises at least one N-X-S/T sequence, which is located outside the endogenous N-X-S/T sequence of ActRIIB and outside the ligand binding pocket.

Other variant ActRIIB proteins are contemplated, such as: an ActRIIB protein comprising an amino acid sequence that is at least 80% identical to amino acids 29-109 of SEQ ID NO:2, wherein the protein comprises at least one N-X-S/T sequence located outside the endogenous N-X-S/T sequence of ActRIIB and outside the ligand binding pocket; a variant ActRIIB protein comprising an N at a position corresponding to position 24 of SEQ ID NO:2 and an S or T at a position corresponding to position 26 of SEQ ID NO:2, wherein the variant ActRIIB protein inhibits signaling through activin, myostatin, and/or GDF11 in a cell-based assay; a variant ActRIIB protein comprising an R or K at a position corresponding to position 64 of SEQ ID NO:2; or a variant ActRIIB protein comprising at least one alteration to the sequence of SEQ ID NO:2 that is a conservative alteration located in the ligand binding pocket. A variant ActRIIB protein as described above, wherein at least one alteration to the sequence of SEQ ID NO: 2 is at one or more positions selected from the group consisting of K74, R40, Q53, K55, F82, and L79. A variant ActRIIB protein as described above, wherein the protein is a fusion protein further comprising a heterologous portion. Any of the variant ActRIIB fusion proteins as described above can be produced as a homodimer. Any of the ActRIIB fusion proteins as described above can have a heterologous portion comprising a constant region from an IgG heavy chain, such as an Fc domain.

In certain aspects, the disclosure herein provides nucleic acids encoding soluble ActRIIB polypeptides that do not encode the complete ActRIIB polypeptide. An isolated polynucleotide can comprise, for example, a coding sequence for a soluble ActRIIB polypeptide as described above. For example, an isolated nucleic acid can include a coding sequence for an extracellular domain (e.g., a ligand-binding domain) of ActRIIB and a sequence encoding part or all of the transmembrane domain and/or cytoplasmic domain of ActRIIB, wherein a stop codon is located within the transmembrane domain or cytoplasmic domain or between the extracellular domain and the transmembrane domain or cytoplasmic domain. For example, an isolated polynucleotide can comprise a full-length ActRIIB polynucleotide sequence, such as SEQ ID NO: 4, or a partially truncated form, further comprising a transcription stop codon at least 600 nucleotides prior to the 3' end, or the transcription stop codon is positioned such that translation of the polynucleotide produces the extracellular domain, optionally fused to a truncated portion of the full-length ActRIIB. The nucleic acids disclosed herein can be operably linked to a promoter for expression, and the disclosure herein provides cells transformed with such recombinant polynucleotides. Preferably, the cells are mammalian cells, such as CHO cells.

In certain aspects, the disclosure herein provides methods for producing soluble ActRIIB polypeptides. Such methods can comprise expressing any of the nucleic acids disclosed herein (e.g., SEQ ID NO: 3) in suitable cells, such as Chinese hamster ovary (CHO) cells. Such methods can comprise: a) culturing the cells under conditions suitable for expression of the soluble ActRIIB polypeptide, wherein the cells are transformed with a soluble ActRIIB expression construct; and b) recovering the soluble ActRIIB polypeptide so expressed. The soluble ActRIIB polypeptide can be recovered as a crude, partially purified, or highly purified fraction using any of the well-known techniques for obtaining proteins from cell culture.

In certain aspects, the soluble ActRIIB polypeptides disclosed herein can be used in methods for treating patients suffering from diseases associated with muscle loss or insufficient muscle growth. Such diseases include muscle atrophy, muscular dystrophy, amyotrophic lateral sclerosis (ALS), and muscle wasting disorders (e.g., cachexia, anorexia, DMD syndrome, BMD syndrome, AIDS wasting syndrome, muscular dystrophy, neuromuscular diseases, motor neuron disease, neuromuscular junction disease, and inflammatory myopathy). The methods can comprise administering an effective amount of a soluble ActRIIB polypeptide to a patient in need thereof.

In certain aspects, the soluble ActRIIB polypeptides disclosed herein can be used in methods for reducing body fat content or reducing the rate of increase in body fat content, and in methods for treating diseases associated with unwanted weight gain, such as obesity, non-insulin-dependent diabetes mellitus (NIDDM), cardiovascular disease, cancer, hypertension, osteoarthritis, stroke, respiratory problems, and gallbladder disease. These methods can comprise administering an effective amount of a soluble ActRIIB polypeptide to a patient in need thereof.

In certain specific aspects, the soluble ActRIIB polypeptides disclosed herein can be used in methods for treating disorders associated with aberrant GDF8 activity. Such disorders include metabolic disorders such as type 2 diabetes, impaired glucose tolerance, metabolic syndrome (e.g., syndrome X), and insulin resistance induced by trauma (e.g., burns or nitrogen imbalance); adipose tissue diseases (e.g., obesity); muscular dystrophy (including Duchenne muscular dystrophy); amyotrophic lateral sclerosis (ALS); muscle atrophy; organ atrophy; frailty; carpal tunnel syndrome; congestive embolic pulmonary disease; sarcopenia, cachexia, and other muscle wasting syndromes; osteoporosis; glucocorticoid-induced osteoporosis; osteopenia; osteoarthritis; osteoporosis-related fractures; low bone mass due to chronic glucocorticoid therapy, premature gonadal failure, androgen suppression, vitamin D deficiency, secondary hyperparathyroidism, malnutrition, and anorexia nervosa. The method may comprise administering to a patient in need thereof an effective amount of a soluble ActRIIB polypeptide.

In certain aspects, the disclosure herein provides methods for identifying drugs that stimulate tissue growth (e.g., bone, cartilage, muscle, and fat). The methods include: a) identifying a test drug that competes with a soluble ActRIIB polypeptide for binding to the ligand-binding domain of the ActRIIB polypeptide; and b) evaluating the effect of the drug on tissue growth.

In certain aspects, the disclosure herein provides methods for antagonizing the activity of an ActRIIB polypeptide or ActRIIB ligand (e.g., GDF8, GDF11, activin, BMP7, and Nodal) in a cell. The method comprises contacting the cell with a soluble ActRIIB polypeptide. Optionally, the activity of the ActRIIB polypeptide or ActRIIB ligand is monitored by signaling mediated by the ActRIIB/ActRIIB ligand complex, for example, by monitoring cell proliferation. Cells used in the methods include osteoblasts, chondrocytes, myocytes, adipocytes, and myocytes.

In certain aspects, the disclosure herein provides for the use of a soluble ActRIIB polypeptide in the preparation of a medicament for treating a disease or condition as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the human ActRIIB soluble (extracellular) polypeptide sequence (SEQ ID NO: 1). The C-terminal "tail" is underlined.

Figure 2 shows the sequence of the human ActRIIB precursor protein (SEQ ID NO: 2). The signal peptide is underlined; the extracellular domain is in bold (also known as SEQ ID NO: 1); potential N-linked glycosylation sites are boxed.

Figure 3 shows the nucleic acid sequence encoding the human ActRIIB soluble (extracellular) polypeptide, designated as SEQ ID NO:3.

Figure 4 shows the nucleic acid sequence encoding human ActRIIB precursor protein, designated as SEQ ID NO:4.

Figure 5 shows body weight gain in mice treated with vehicle (diamonds), ActRIIB(R64 20-134)-mFc (squares), or the long-half-life form of ActRIIB(R64 A24N 20-134) (triangles).

Figure 6 shows the weights of dissected muscles at the end of the study. Vehicle: left column of each group (light shading); ActRIIB(R64 20-134)-mFc: middle column of each group (medium shading); ActRIIB(R64 A24N 20-134): right column of each group (dark shading).

Figure 7 shows grip strength testing of SOD mice treated with PBS and murine ActRIIB(R64 K74A 20-134)-mFc (or "K74A+15 tails") (white and black bars, respectively). The figure illustrates the increased strength of the murine ActRIIB(R64 K74A 20-134)-mFc group compared to the PBS group at both early (117 days) and late (149 days) stages of the disease. *P < 0.05, two-tailed Student's t-test.

Figure 8 shows a Kaplan-Meier survival comparison of PBS- and ActRIIB(R64 K74A 20-134)-mFc-treated SOD mice (white and black lines, respectively). The ActRIIB(R64 K74A 20-134)-mFc-treated group had an increased mean survival day compared to the PBS group.

Figure 9 shows the percent change in body composition of PBS- and ActRIIB(R64 20-134)-mFc HFD-fed mice (white and black bars, respectively). Treatment with mouse ActRIIB(R64 20-134)-Fc protein significantly reduced fat mass and increased lean tissue.

Figure 10 shows muscle cross-sections (4x magnification) of the femoris muscles of aged mice (A) or aged mice treated with ActRIIB(R64 20-134)-mFc (B).

Figure 11 shows the average weight of mice in a cancer cachexia experiment using CT26 colon cancer cells. Diamonds: tumor-free, saline-treated animals; squares: tumor-free, ActRIIB(R64 20-134)-mFc-treated mice; triangles: tumor-bearing, saline-treated animals; "x": tumor-bearing, ActRIIB(R64 20-134)-mFc-treated mice (10 mg/kg); "*": tumor-bearing, ActRIIB(R64 20-134)-mFc-treated mice (30 mg/kg); circles: tumor-bearing, ActRIIB(R64 20-134)-mFc-treated mice (10 mg/kg). Treatment was initiated in a preventive manner at the time of tumor implantation.

Figure 12 shows an alignment of human ActRIIA and ActRIIB with the residues inferred herein, based on compositional analysis of multiple ActRIIB and ActRIIA crystal structures, with direct ligand contacts (ligand binding pocket) boxed.

Figure 13 shows a multiple sequence alignment of various vertebrate ActRIIB proteins and human ActRIIA.

Detailed Description of the Invention

1. Overview

In certain aspects, the present invention relates to ActRIIB polypeptides. As used herein, the term "ActRIIB" refers to a family of activin receptor type IIB (ActRIIB) proteins and ActRIIB-related proteins derived from any species. ActRIIB family members are generally all 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 specificity. The amino acid sequences of the human ActRIIA precursor protein (provided for comparison) and the ActRIIB precursor protein are shown in Figure 1 (SEQ ID NO: 1) and Figure 2 (SEQ ID NO: 2), respectively.

The term "ActRIIB polypeptide" is used to refer to polypeptides comprising any naturally occurring polypeptide of an ActRIIB family member, as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity. For example, ActRIIB polypeptides include polypeptides derived from any known ActRIIB sequence that have a sequence that is at least about 80% identical to an ActRIIB polypeptide sequence, preferably at least 85%, 90%, 95%, 97%, 99%, or more identical.

In specific embodiments, the present invention relates to soluble ActRIIB polypeptides. As described herein, the term "soluble ActRIIB polypeptide" generally refers to a polypeptide comprising the extracellular domain of an ActRIIB protein. As used herein, the term "soluble ActRIIB polypeptide" includes any native extracellular domain of an ActRIIB protein, as well as any variants thereof (including mutants, fragments, and peptidomimetic forms) that retain useful activity. For example, the extracellular domain of an ActRIIB protein binds a ligand and is generally soluble. Examples of soluble ActRIIB polypeptides include the ActRIIB soluble polypeptide shown in Figure 1 (SEQ ID NO: 1). Other examples of soluble ActRIIB polypeptides that, in addition to comprising the extracellular domain of an ActRIIB protein, also include a signal sequence are described in Example 1. The signal sequence can be the native signal sequence of ActRIIB, or it can be the signal sequence of another protein, such as the tissue plasminogen activator (TPA) signal sequence or the honey bee melatin (HBM) signal sequence.

TGF-β signaling is mediated by a heteromeric complex of type I and type II serine/threonine kinase receptors, which phosphorylate and activate downstream Smad proteins upon ligand stimulation (Massagué, 2000, Nat. Rev. Mol. Cell Biol. 1:169-178). These type I and type II receptors are all 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. Type I receptors are essential for signal transduction, while type II receptors are 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 activins (Mathews and Vale, 1991, Cell 65:973-982; Attisano et al., 1992, Cell 68:97-108). In addition to activins, ActRIIA and ActRIIB can biochemically interact with several other TGF-β family proteins, including BMP7, Nodal, GDF8, and GDF11 (Yamashita et al., 1995, J. Cell Biol. 130:217-226; Lee and McPherron, 2001, Proc. Natl. Acad. Sci. 98:9306-9311; Yeo and Whitman, 2001, Mol. Cell 7:949-957; Oh et al., 2002, Genes Dev. 16:2749-54). Applicants have discovered that soluble ActRIIA-Fc fusion proteins and ActRIIB-Fc fusion proteins have significantly different effects in vivo, with ActRIIA-Fc having a primary effect on bone and ActRIIB-Fc having a primary effect on skeletal muscle.

In certain embodiments, the present invention relates to antagonizing ActRIIB receptor ligands (also referred to as ActRIIB ligands) with target ActRIIB polypeptides (e.g., soluble ActRIIB polypeptides). Thus, the compositions and methods of the present invention can be used to treat diseases associated with aberrant activity of one or more ligands of the ActRIIB receptor. Exemplary ActRIIB receptor ligands include certain TGF-β family members, such as activin, Nodal, GDF8, GDF11, and BMP7.

Activin is a dimeric polypeptide growth factor that belongs to the TGF-β superfamily. There are three activins (A, B and AB), which are homodimers of two closely related β subunits (β _A β _A , β _B β _B and β _A β _B ). In the TGF-β superfamily, activin is a unique multifunctional factor that can stimulate hormone production in ovarian and placental cells, support nerve cell survival, affect cell cycle progression positively or negatively according to cell type, and affect mesoderm differentiation at least in amphibian embryos (DePaolo et al., 1991, Proc Soc Ep Biol Med. 198: 500-512; Dyson et al., 1997, Curr Biol. 7: 81-84; Woodruff, 1998, Biochem Pharmacol. 55: 953-963). In addition, it was found that the erythroid differentiation factor (EDF) isolated from stimulated human monocytic leukemia cells was identical to activin A (Murata et al., 1988, PNAS, 85:2434). It has been shown that activin A is used as a natural regulator of erythropoiesis in the bone marrow. In some tissues, activin signal transduction is antagonized by its related heterodimer, inhibin. For example, during the release of follicle-stimulating hormone (FSH) by the pituitary gland, activin promotes FSH secretion and synthesis, while inhibin prevents FSH secretion and synthesis. Other proteins that can regulate the biological activity of activin and/or are bound to activin include follistatin (FS), follistatin-related protein (FSRP), α ₂ -macroglobulin, Cerberus, and endoglin described below.

Nodal proteins play a role in the induction and formation of mesoderm and endoderm and the subsequent organization of axial structures (e.g., heart and stomach in early embryogenesis). It has been shown that the dorsal tissue of the developing vertebrate embryo primarily contributes to the axial structure of the notochord and prechordal plate, while recruiting surrounding cells to form non-axial embryonic structures. Nodal appears to signal through both type I and type II receptors and intracellular effectors called Smad proteins. Recent studies support the idea that ActRHA and ActRIIB serve as type II receptors for Nodal (Sakuma et al., Genes Cells. 2002, 7: 401-12). It has been shown that Nodal ligands interact with their cofactors (e.g., cripto) to activate activin type I and type II receptors that phosphorylate Smad2. Nodal proteins are involved in many key events in early vertebrate embryos, including mesoderm formation, anterior patterning, and left-right axis specialization. Experimental evidence has demonstrated that Nodal signaling activates pAR3-Lux, a luciferase reporter previously shown to be specifically responsive to activin and TGF-β. However, Nodal fails to induce pTlx2-Lux, a reporter specifically responsive to bone morphogenetic protein. Recent studies have provided direct biochemical evidence that Nodal signaling is mediated by two activin-TGF-β pathway Smads: Smad2 and Smad3. Further evidence has shown that Nodal signaling requires the extracellular Cripto protein, distinguishing it from activin or TGF-β signaling.

Growth and differentiation factor-8 (GDF8), also known as myostatin, is a negative regulator of skeletal muscle mass. GDF8 is highly expressed in developing and adult skeletal muscle. Transgenic mice harboring a GDF8 null mutation are characterized by significant hypertrophy and proliferation of skeletal muscle (McPherron et al., Nature, 1997, 387:83-90). Similar increases in skeletal muscle mass are evident in naturally occurring mutations of GDF8 in cattle (Ashmore et al., 1974, Growth, 38:501-507; Swatland and Kieffer, J. Anim. Sci, 1994, 38:752-757; McPherron and Lee, Proc. Natl. Acad. Sci. USA, 1997, 94:12457-12461; and Kambadur et al., Genome Res., 1997, 7:910-915) and are even more pronounced in humans (Schuelke et al., N Engl J Med 2004; 350:2682-8). Studies have also shown that muscle wasting associated with HIV infection in humans is accompanied by increased expression of GDF8 protein (Gonzalez-Cadavid et al., PNAS, 1998, 95:14938-43). In addition, GDF8 can regulate the production of muscle-specific enzymes (e.g., creatine kinase) and regulate myoblast proliferation (WO 00/43781). The GDF8 propeptide can non-covalently bind to the mature GDF8 domain dimer, inactivating its biological activity (Miyazono et al. (1988) J. Biol. Chem., 263:6407-6415; Wakefield et al. (1988) J. Biol. Chem., 263:7646-7654; and Brown et al. (1990) Growth Factors, 3:35-43). Other proteins that bind to GDF8 or structurally related proteins and inhibit their biological activity include follistatin, and potentially follistatin-related proteins (Gamer et al. (1999) Dev. Biol., 208:222-232).

Growth and differentiation factor-11 (GDF11), also known as BMP11, is a secreted protein (McPherron et al., 1999, Nat. Genet. 22:260-264). During mouse development, GDF11 is expressed in the tail bud, limb buds, maxillary and mandibular arches, and dorsal root ganglia (Nakashima et al., 1999, Mech. Dev. 80:185-189). GDF11 plays a unique role in patterning mesoderm and neural tissue (Gamer et al., 1999, Dev Biol., 208:222-32). GDF11 has been shown to be a negative regulator of chondrogenesis and myogenesis in the developing chick limb bud (Gamer et al., 2001, Dev Biol. 229:407-20). GDF11 expression in muscle also suggests a role in regulating muscle growth in a similar manner to GDF8. Furthermore, GDF11 expression in the brain suggests that it may also have activities related to nervous system function. Interestingly, GDF11 was found to inhibit neurogenesis in the olfactory epithelium (Wu et al., 2003, Neuron. 37:197-207). Therefore, GDF11 may be used in vitro and in vivo to treat diseases such as muscle diseases and neurodegenerative diseases (e.g., amyotrophic lateral sclerosis).

Bone morphogenetic protein (BMP7), also known as osteogenic protein-1 (OP-1), is well known for inducing cartilage and bone formation. Additionally, BMP7 regulates a range of physiological processes. For example, BMP7 may be an osteoinductive factor responsible for the phenomenon of epithelial osteogenesis. BMP7 has also been found to play a role in calcium regulation and bone homeostasis. Like activins, BMP7 binds to type II receptors ActRIIA and IIB. However, BMP7 and activins recruit different type I receptors into heteromeric receptor complexes. The predominant BMP7 type I receptor observed is ALK2, while activins specifically bind to ALK4 (ActRIIB). BMP7 and activins elicit different biological responses and activate different Smad pathways (Macias-Silva et al., 1998, J Biol Chem. 273:25628-36).

In certain aspects, the present invention relates to the use of certain ActRIIB polypeptides (e.g., soluble ActRIIB polypeptides) to antagonize signaling of ActRIIB ligands, generally in any process associated with ActRIIB activity. Optionally, the ActRIIB polypeptides of the present invention can antagonize one or more ligands of the ActRIIB receptor, such as activin, Nodal, GDF8, GDF11, and BMP7, and thus can be used to treat other diseases.

Thus, the present invention contemplates the use of ActRIIB polypeptides to treat or prevent diseases or conditions associated with aberrant activity of ActRIIB or an ActRIIB ligand. ActRIIB or an ActRIIB ligand is involved in the regulation of many key biological processes. Due to their critical functions in these processes, they may be ideal targets for therapeutic intervention. For example, ActRIIB polypeptides (e.g., soluble ActRIIB polypeptides) can be used to treat human or animal diseases or conditions. Examples of such diseases or conditions include, but are not limited to, metabolic diseases such as type 2 diabetes, impaired glucose tolerance, metabolic syndrome (e.g., syndrome X), and insulin resistance induced by trauma (e.g., burns or nitrogen imbalance); adipose tissue diseases (e.g., obesity); muscle and neuromuscular disorders such as muscular dystrophy (including Duchenne muscular dystrophy); amyotrophic lateral sclerosis (ALS); muscle atrophy; organ atrophy; weakness; carpal tunnel syndrome; congestive embolic lung disease; and sarcopenia, cachexia, and other muscle wasting syndromes. Other examples include osteoporosis, particularly in the elderly and/or postmenopausal women; glucocorticoid-induced osteoporosis; osteopenia; osteoarthritis; and osteoporosis-related fractures. Still further examples include low bone mass due to chronic glucocorticoid therapy, premature gonadal failure, androgen suppression, vitamin D deficiency, secondary hyperparathyroidism, malnutrition, and anorexia nervosa. These disorders and conditions are discussed below under "Exemplary Therapeutic Applications."

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

"About" and "approximately" generally refer to an acceptable degree of error in the amount measured given the nature or precision of the measurement. Typically, exemplary degrees of error are within 20% of a given value or range of values, preferably within 10%, and more preferably within 5%.

Additionally, especially in biological systems, the terms "about" and "approximately" can refer to values within an order of magnitude, preferably within 5 times, and more preferably within 2 times of a given value. Unless otherwise indicated, the numerical values given herein are approximate, meaning that the terms "about" or "approximately" can be inferred when not expressly specified.

The methods of the present invention may include a step of comparing sequences to each other, including comparing a wild-type sequence to one or more mutants (sequence variants). Such comparisons typically involve alignment of polymer sequences, for example, using sequence alignment programs and/or algorithms well known in the art (e.g., BLAST, FASTA, and MEGALIGN, to name a few). Those skilled in the art will readily appreciate that, in such alignments, where the mutations comprise insertions or deletions of residues, the sequence alignment will introduce "gaps" (typically indicated by a dash or "A") in the polymer sequence that do not contain the inserted or deleted residues.

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

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

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

2. ActRIIB peptide

In certain aspects, the present invention relates to ActRIIB variant polypeptides (e.g., soluble ActRIIB polypeptides). Optionally, fragments, functional variants, and modified forms have similar or identical biological activities as their corresponding wild-type ActRIIB polypeptides. For example, ActRIIB variants of the present invention can bind to and inhibit the function of an ActRIIB ligand (e.g., activin A, activin AB, activin B, Nodal, GDF8, GDF11, or BMP7). Optionally, the ActRIIB polypeptide regulates the growth of tissues such as bone, cartilage, muscle, or fat. Examples of ActRIIB polypeptides include human ActRIIB precursor polypeptide (SEQ ID NO: 2) and soluble human ActRIIB polypeptides (e.g., SEQ ID NOs: 1, 5, 6, and 12).

The disclosure herein identifies functionally active portions and variants of ActRIIB. Applicants have determined that an Fc fusion protein having the sequence disclosed by Hilden et al. (Blood. 1994 Apr 15;83(8):2163-70) containing an alanine at position (A64) corresponding to amino acid 64 of SEQ ID NO: 2 has relatively low affinity for activin and GDF-11. In contrast, the same Fc fusion protein having an arginine at position 64 (R64) has an affinity for activin and GDF-11 in the low nanomolar to high picomolar range. Therefore, the sequence containing R64 is used as the wild-type reference sequence for human ActRIIB in the disclosure herein.

Attisano et al. (Cell. 1992 Jan 10;68(1):97-108) demonstrated that deletion of the proline knot at the C-terminus of the ActRIIB extracellular domain reduced the receptor's affinity for activin. The data presented herein demonstrate that the ActRIIB-Fc fusion protein, "ActRIIB(20-119)-Fc," comprising amino acids 20-119 of SEQ ID NO: 2, has reduced binding to GDF-11 and activin relative to ActRIIB(20-134)-Fc, which contains the proline knot region and an intact juxtamembrane domain. However, the ActRIIB(20-129)-Fc protein retained similar, but slightly reduced, activity relative to the wild-type protein, even with the proline knot region destroyed. Thus, ActRIIB extracellular domains terminating at amino acids 134, 133, 132, 131, 130, and 129 are all expected to be active, but constructs terminating at 134 or 133 are likely to be most active. Similarly, mutations at any of residues 129-134 are not expected to significantly alter ligand binding affinity. Supporting this, mutations at P129 and P130 do not significantly reduce ligand binding. Thus, the ActRIIB-Fc fusion protein can terminate as early as amino acid 109 (the last cysteine), but termination at or between 109 and 119 is expected to reduce ligand binding. Amino acid 119 is poorly conserved and therefore easily altered or truncated. Termination at or after 128 retains ligand binding activity. Termination at or between 119 and 127 will have intermediate binding capacity. Any of these formats may need to be used, depending on the clinical or experimental setting.

At the N-terminus of ActRIIB, proteins starting at or before amino acid 29 are expected to retain ligand binding activity. Amino acid 29 is the first cysteine. Mutation of alanine at position 24 to asparagine introduces an N-linked glycosylation sequence without significantly affecting ligand binding. This demonstrates that mutations corresponding to amino acids 20-29 in the region between the signal cleavage peptide and the cysteine crosslinking region are fully tolerated. Specifically, constructs starting at positions 20, 21, 22, 23, and 24 will retain activity, and constructs starting at positions 25, 26, 27, 28, and 29 are also expected to retain activity. The data presented in the Examples demonstrate that constructs starting at positions 22, 23, 24, or 25 are surprisingly the most active.

In summary, the active portion of ActRIIB contains amino acids 29-109 of SEQ ID NO: 2, and the construct can, for example, begin at residues corresponding to amino acids 20-29 and end at positions corresponding to amino acids 109-134. Other examples include constructs that begin at positions 20-29 or 21-29 and end at positions 119-134, 119-133, or 129-134, 129-133. Other examples include constructs that begin at positions 20-24 (or 21-24 or 22-25) and end at positions 109-134 (or 109-133), 119-134 (or 119-133), or 129-134 (or 129-133). Variants within these ranges are also contemplated, especially those having at least 80%, 85%, 90%, 95% or 99% identity to the corresponding portion of SEQ ID NO: 2.

The disclosure herein includes analysis of the composite ActRIIB structure shown in Figure 12, which demonstrates that the ligand binding pocket is defined by residues Y31, N33, N35, L38 to T41, E47, E50, Q53 to K55, L57, H58, Y60, S62, K74, W78 to N83, Y85, R87, A92, and E94 to F101. At these positions, conservative mutations are expected to be tolerated, although K74A mutations are fully tolerated, and R40A, K55A, F82A, and mutations at position L79 are also tolerated. R40 is K in Xenopus, indicating that basic amino acids at this position will be tolerated. Q53 is R in bovine ActRIIB and K in Xenopus ActRIIB, thus amino acids including R, K, Q, N, and H will be tolerated at this position. Thus, the general formula of an active ActRIIB variant protein is that comprising amino acids 29-109, but optionally starting at a position within the range of 20-24 or 22-25 and ending at a position within the range of 129-134, and containing no more than 1, 2, 5, 10, or 15 conservative amino acid changes in the ligand binding pocket, and 0, 1, or more non-conservative changes at positions 40, 53, 55, 74, 79, and/or 82 in the ligand binding pocket. Such a protein may retain greater than 80%, 90%, 95%, or 99% sequence identity to amino acids 29-109 of SEQ ID NO: 2. Sites outside the binding pocket where variability is particularly tolerated include the amino and carboxyl termini of the extracellular domain (as indicated above) and positions 42-46 and 65-73. The asparagine to alanine change at position 65 (N65A) actually improved ligand binding in the A64 background and is therefore not expected to have a deleterious effect on ligand binding in the R64 background. This change likely eliminates glycosylation at N65 in the A64 background, demonstrating that significant changes in this region may be tolerated. While the R64A change is less tolerated, the R64K change is fully tolerated, potentially allowing for another basic residue, such as H, at position 64.

ActRIIB is highly conserved across nearly all vertebrates, with large sections of the extracellular domain being completely conserved. Many ligands that bind to ActRIIB are also highly conserved. Therefore, comparisons of ActRIIB sequences from multiple vertebrate organisms provide insights into residues that can be altered. Thus, active human ActRIIB variants can include one or more amino acids at corresponding positions in the sequence of another vertebrate ActRIIB, or can include residues that are analogous to residues in the human or other vertebrate sequence. The following examples illustrate this approach to defining active ActRIIB variants. L46 is a valine in Xenopus laevis ActRIIB, so this position can be altered and, optionally, to another hydrophobic residue, such as V, I, or F, or to a nonpolar residue, such as A. E52 is a K in Xenopus laevis, indicating that this position can tolerate a wide range of variations, including polar residues such as E, D, K, R, H, S, T, P, G, Y, and potentially A. T93 is K in Xenopus, indicating that this position tolerates a wide range of structural variation, with polar residues such as S, K, R, E, D, H, G, P, G, and Y being preferred. F108 is Y in Xenopus, so Y or other hydrophobic groups such as I, V, or L should be tolerated. E111 is K in Xenopus, indicating that charged residues, including D, R, K, and H, as well as Q and N, will be tolerated at this position. R112 is K in Xenopus, indicating that basic residues, including R and H, will be tolerated at this position. The A at position 119 is relatively poorly conserved, being expressed as P in rodents and V in Xenopus, so essentially any amino acid should be tolerated at this position.

The disclosure herein demonstrates that the addition of further N-linked glycosylation sites (N-X-S/T) increases the serum half-life of ActRIIB-Fc fusion proteins relative to the ActRIIB(R64)-Fc form. By introducing an asparagine at position 24 (A24N construct), an NXT sequence conferring a longer half-life was generated. Other NX(T/S) sequences were found at 42-44 (NQS) and 65-67 (NSS), although the latter may not be efficiently glycosylated by the R at position 64. N-X-S/T sequences can generally be introduced at positions outside the ligand-binding pocket defined in FIG12 . Particularly suitable sites for the introduction of non-endogenous N-X-S/T sequences include amino acids 20-29, 20-24, 22-25, 109-134, 120-134, or 129-134. An N-X-S/T sequence can also be introduced into the linker between the ActRIIB sequence and the Fc or other fusion component. Such sites can be introduced with minimal effort by introducing an N at the correct position relative to a pre-existing S or T, or by introducing an S or T at a position corresponding to a pre-existing N. Thus, suitable alterations that should result in N-linked glycosylation sites are: A24N, R64N, S67N (which can be combined with an N65A alteration), E106N, R112N, G120N, E123N, P129N, A132N, R112S, and R112T. Any S predicted to be glycosylated can be changed to a T without creating an immunogenic site, as glycosylation provides protection. Similarly, any T predicted to be glycosylated can be changed to an S. Thus, alterations S67T and S44T are contemplated. Similarly, in the A24N variant, an S26T alteration can be used. Thus, ActRIIB variants may include one or more additional non-endogenous N-linked glycosylation consensus sequences.

Position L79 can be altered to confer modified activin-myostatin (GDF-11) binding properties. L79A or L79P significantly reduce GDF-11 binding compared to activin binding. L79E or L79D retain GDF-11 binding. Remarkably, the L79E and L79D variants significantly reduce activin binding. In vivo experiments demonstrate that these non-activin receptors retain substantial ability to increase muscle mass but exhibit reduced effects on other tissues. These data demonstrate the desirability and feasibility of obtaining polypeptides with reduced activin effects.

The variations can be combined in a variety of ways. Furthermore, the results of the mutagenesis program described herein indicate that there are amino acid positions in ActRIIb that are often beneficial to conserve. These positions include position 64 (basic amino acid), position 80 (acidic or hydrophobic amino acid), position 78 (hydrophobic, particularly tryptophan), position 37 (acidic, particularly aspartic acid or glutamic acid), position 56 (basic amino acid), and position 60 (hydrophobic amino acid, particularly phenylalanine or tyrosine). Thus, within each variant disclosed herein, the disclosure herein provides an amino acid framework that may be conserved. Other positions that may be conserved are as follows: position 52 (acidic amino acid), position 55 (basic amino acid), position 81 (acidic), and position 98 (polar or charged, particularly E, D, R, or K).

In certain embodiments, isolated fragments of an ActRIIB polypeptide can be obtained by screening polypeptides recombinantly produced from corresponding fragments of nucleic acids encoding ActRIIB polypeptides (e.g., SEQ ID NOs: 3 and 4). Alternatively, fragments can be chemically synthesized using techniques known in the art, such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. Such fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments that are, for example, useful as antagonists (inhibitors) or agonists (activators) of an ActRIIB protein or ActRIIB ligand.

In certain embodiments, a functional variant of an ActRIIB polypeptide has an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NOs: 3, 4, and 10. In some cases, a functional variant has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 3, 4, and 10.

In certain embodiments, the present invention contemplates the creation of functional variants by modifying the structure of an ActRIIB polypeptide for purposes such as enhancing efficacy or stability (e.g., ex vivo shelf life and resistance to in vivo protease degradation). Modified ActRIIB polypeptides can also be produced, for example, by amino acid substitutions, deletions, or additions. For example, it is reasonable to expect that a single substitution of leucine with isoleucine or valine, a single substitution of aspartic acid with glutamic acid, a single substitution of threonine with serine, or a similar substitution of an amino acid with a structurally related amino acid (e.g., a conservative mutation) will not significantly affect the biological activity of the resulting molecule. Conservative substitutions are those that occur within a family of amino acids that are related in their side chains. Whether a change in the amino acid sequence of an ActRIIB polypeptide results in a functional homolog can be readily determined by assessing the ability of a variant ActRIIB polypeptide to generate a response in a cell similar to that of a wild-type ActRIIB polypeptide, or by assessing the ability of a variant ActRIIB polypeptide to bind to one or more ligands (e.g., activin, GDF-11, or myostatin) in a manner similar to that of the wild-type ActRIIB polypeptide.

In certain specific embodiments, the present invention contemplates generating mutations in the extracellular domain (also referred to as the ligand-binding domain) of an ActRIIB polypeptide such that the variant (or mutant) ActRIIB polypeptide has altered ligand-binding activity (e.g., binding affinity or binding specificity). In some cases, such variant ActRIIB polypeptides have altered (increased or decreased) binding affinity for a particular ligand. In other cases, the variant ActRIIB polypeptides have altered binding specificity for their ligand.

For example, the disclosure herein provides variant ActRIIB polypeptides that preferentially bind to GDF8/GDF11 relative to activin. The disclosure herein further establishes the desirability of such polypeptides for reducing off-target effects, although such selective variants are rarely needed to treat serious diseases where a very large increase in muscle mass may be required for therapeutic efficacy and where a certain level of off-target effects can be tolerated. For example, amino acid residues of the ActRIIB protein, such as E39, K55, Y60, K74, W78, D80, and F101, are located in the ligand-binding pocket and mediate binding to its ligands, such as activin and GDF8. Accordingly, the present invention provides altered ligand-binding domains (e.g., GDF8-binding domains) of the ActRIIB receptor, comprising one or more mutations at those amino acid residues. Optionally, the altered ligand-binding domain can have increased selectivity for a ligand, such as GDF8, relative to the wild-type ligand-binding domain of the ActRIIB receptor. For example, these mutations increase the selectivity of the altered ligand-binding domain for GDF8 over activin. Optionally, the altered ligand-binding domain has a ratio of activin binding _Kd to GDF8 binding _Kd that is at least 2-fold, 5-fold, 10-fold, or even 100-fold greater relative to the ratio of the wild-type ligand-binding domain. Optionally, the altered ligand-binding domain has a ratio of activin inhibition _IC50 to GDF8 inhibition _IC50 that is at least 2-fold, 5-fold, 10-fold, or even 100-fold greater relative to the wild-type ligand-binding domain. Optionally, the altered ligand-binding domain inhibits GDF8 with an _IC50 that is at least 2-fold, 5-fold, 10-fold, or even 100-fold lower than the _IC50 for inhibition of activin.

As a specific example, the positively charged amino acid residue Asp (D80) in the ligand-binding domain of ActRIIB can be mutated to a different amino acid residue such that the variant ActRIIB polypeptide preferentially binds to GDF8 but not activin. Preferably, the D80 residue is changed to an amino acid residue selected from the group consisting of an uncharged amino acid residue, a negatively charged amino acid residue, and a hydrophobic amino acid residue. As another specific example, the hydrophobic residue L79 can be changed to the acidic amino acids aspartic acid or glutamic acid to significantly reduce activin binding while retaining GDF11 binding. Those skilled in the art will appreciate that most of the described mutations, variants, or modifications can be prepared at the nucleic acid level or, in some cases, by post-translational modification or chemical synthesis. Such techniques are well known in the art.

In certain embodiments, the present invention contemplates specific mutations of ActRIIB polypeptides that alter the glycosylation of the polypeptide. Exemplary glycosylation sites in ActRIIB polypeptides are shown in Figure 2. Such mutations can be selected to introduce or eliminate one or more glycosylation sites, such as O-linked or N-linked glycosylation sites. Asparagine-linked glycosylation recognition sites typically comprise the tripeptide sequence asparagine-X-threonine (where X is any amino acid), which is specifically recognized by appropriate cellular glycosylation enzymes. Alterations can also be made by adding or substituting one or more serine or threonine residues to the wild-type ActRIIB polypeptide sequence (for O-linked glycosylation sites). Multiple amino acid substitutions or deletions at either or both of the first or third amino acid positions of a glycosylation recognition site (and/or amino acid deletions at the second position) result in a non-glycosylated modified tripeptide sequence. Another method for increasing the number of carbohydrate moieties on an ActRIIB polypeptide is by chemically or enzymatically coupling glycosides to the ActRIIB polypeptide. Depending on the coupling mode used, the sugar can be attached to (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups, such as those of cysteine; (d) free hydroxyl groups, such as those of serine, threonine, or hydroxyproline; (e) aromatic residues, such as those of phenylalanine, tyrosine, or tryptophan; or (f) the amide group of glutamine. These methods are described in WO 87/05330, published September 11, 1987, and in Aplin and Wriston (1981) CRC Crit. Rev. Biochem., pp. 259-306, which are incorporated herein by reference. Removal of one or more carbohydrate moieties present on an ActRIIB polypeptide can be accomplished chemically and/or enzymatically. Chemical deglycosylation can include, for example, contacting the ActRIIB polypeptide with the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the removal of most or all sugars other than the attached sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the amino acid sequence intact. Chemical deglycosylation is further described by Hakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and Edge et al. (1981) Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties on ActRIIB polypeptides can be achieved using various endoglycosidases and exoglycosidases as described by Thotakura et al. (1987) Meth. Enzymol. 138:350. The sequence of the ActRIIB polypeptide can be appropriately adjusted depending on the type of expression system employed, as mammalian, yeast, insect, and plant cells can all introduce different glycosylation patterns, which can be influenced by the amino acid sequence of the peptide. Generally, ActRIIB proteins for use in humans will be expressed in mammalian cell lines that provide correct glycosylation (such as HEK293 cells or CHO cell lines), although other mammalian expression cell lines are contemplated to be useful.

The disclosure herein further contemplates methods for generating variants, particularly panels of combinatorial variants of ActRIIB polypeptides, optionally including truncation variants; combinatorial mutant libraries are particularly useful for identifying functional variant sequences. Screening such combinatorial libraries can be aimed at generating, for example, ActRIIB polypeptide variants with altered properties, such as altered pharmacokinetics or altered ligand binding. Various screening assays are provided below that can be used to evaluate variants. For example, ActRIIB polypeptide variants can be screened for their ability to bind to an ActRIIB polypeptide to prevent binding of an ActRIIB ligand to the ActRIIB polypeptide.

The activity of an ActRIIB polypeptide or variant thereof can also be tested in cell-based or in vivo assays. For example, the effect of an ActRIIB polypeptide variant on the expression of genes involved in bone production in osteoblasts or precursors can be assessed. This can be done in the presence of one or more recombinant ActRIIB ligand proteins (e.g., BMP7), as desired, and the cells can be transfected to produce an ActRIIB polypeptide and/or variant thereof and, optionally, an ActRIIB ligand. Similarly, an ActRIIB polypeptide can be administered to mice or other animals, and one or more bone properties, such as density or volume, can be assessed. The healing rate of fractures can also be assessed. Similarly, the activity of an ActRIIB polypeptide or variant thereof can be tested in muscle cells, adipocytes, and neuronal cells, for example, by testing any effect on the growth of these cells using assays as described below. Such assays are well known and routine in the art. SMAD-responsive reporter genes can be used in such cell lines to monitor the effects of downstream signaling.

Combinatorially derived variants can be generated that have selective potency relative to native ActRIIB polypeptides. Such variant proteins, when expressed from recombinant DNA constructs, can be used in gene therapy protocols. Similarly, mutagenesis can generate variants with intracellular half-lives that differ significantly from the corresponding wild-type ActRIIB polypeptide. For example, the altered protein can be rendered more or less stable to protease degradation or other processes that result in the destruction or inactivation of the native ActRIIB polypeptide. Such variants, and the genes encoding them, can be used to alter ActRIIB polypeptide levels by modulating its half-life. For example, a short half-life can produce a more transient biological effect and, in the case of partially inducible expression systems, can allow for tighter control of recombinant ActRIIB polypeptide levels in cells.

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

In certain aspects, functional variants or modified forms of an ActRIIB polypeptide include fusion proteins comprising at least a portion of an ActRIIB polypeptide and one or more fusion domains. Well-known examples of such fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S-transferase (GST), thioredoxin, protein A, protein G, immunoglobulin heavy chain constant region (e.g., Fc), maltose binding protein (MBP), or human serum albumin. The fusion domain can be selected to confer desired properties. For example, some fusion domains are particularly useful for separating fusion proteins by affinity chromatography. For affinity purification purposes, affinity chromatography-related matrices are employed, such as resins conjugated to glutathione, amylase, and nickel or cobalt. Many such matrices are available in "kit" form, such as the Pharmacia GST purification system and the QIAexpress ^™ system (Qiagen) used with (HIS ₆ ) fusion partners. As another example, the fusion domain can be selected to facilitate detection of the ActRIIB polypeptide. Examples of such detection domains include various fluorescent proteins (e.g., GFP) and "epitope tags," which are typically short peptide sequences for which specific antibodies are available. Well-known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus hemagglutinin (HA), and c-myc tags. In some cases, the fusion domain has a protease cleavage site, such as for Factor Xa or thrombin, which allows the relevant protease to partially digest the fusion protein and thereby release the recombinant protein therefrom. The released protein can then be separated from the fusion domain by subsequent chromatographic separation. In certain preferred embodiments, the ActRIIB polypeptide is fused to a domain that stabilizes the ActRIIB polypeptide in vivo (a "stabilizer" domain). By "stabilize" is meant increasing serum half-life, whether this is due to reduced destruction, reduced renal clearance, or other pharmacokinetic effects. Fusion to the Fc portion of an immunoglobulin is known to impart desirable pharmacokinetic properties to a variety of proteins. Similarly, fusion to human serum albumin can impart desired properties. Other types of fusion domains that may be selected include multimerization (eg, dimerization, tetramerization) domains as well as functional domains (which confer additional biological functions, such as further stimulation of muscle growth).

As a specific example, the present invention provides fusion proteins that are GDF8 antagonists, comprising an extracellular (e.g., GDF8-binding) domain fused to an Fc domain (e.g., SEQ ID NO: 13).

THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV D (A)VSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC K (A)VSNKALPVPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYS KLTVDKSRWQQGNVFSCSVMHEALH N (A)HYTQKSLSLSPGK＊

Preferably, the Fc domain has one or more mutations at residues such as Asp-265, lysine 322, and Asn-434. In some cases, a mutant Fc domain having one or more of these mutations (e.g., an Asp-265 mutation) has reduced binding to Fcγ receptors relative to a wild-type Fc domain. In other cases, a mutant Fc domain having one or more of these mutations (e.g., an Asn-434 mutation) has increased binding to MHC class I-related Fc receptors (FcRNs) relative to a wild-type Fc domain.

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

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

In certain embodiments, the present invention makes available an ActRIIB polypeptide in isolated and/or purified form, separated from other proteins, or substantially free of other proteins.

In certain embodiments, ActRIIB polypeptides of the invention (unmodified or modified) can be produced by a variety of techniques known in the art. For example, ActRIIB polypeptides of the invention can be synthesized using standard protein chemistry techniques, such as those described in Bodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant G.A. (ed.), Synthetic Peptides: A User's Guide, W.H. Freeman and Company, New York (1992). In addition, automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Model 396; Milligen/Biosearch 9600). Alternatively, ActRIIB polypeptides, fragments, or variants thereof can be recombinantly produced using a variety of expression systems well known in the art (e.g., E. coli, Chinese hamster ovary cells, COS cells, baculovirus) (see also below). In further embodiments, a naturally or recombinantly produced full-length ActRIIB polypeptide can be digested using, for example, a protease (e.g., trypsin, thermolysin, chymotrypsin, pepsin, or paired basic amino acid convertase (PACE)) to produce a modified or unmodified ActRIIB polypeptide. Protease cleavage sites can be identified using computer analysis (using commercially available software, such as Mac Vector, Omega, PCGene, Molecular Simulation, Inc.). Alternatively, such ActRIIB polypeptides can be generated from naturally or recombinantly produced full-length ActRIIB polypeptides, for example, by standard techniques known in the art, such as chemical cleavage (e.g., cyanogen bromide, hydroxylamine).

3. Nucleic Acids Encoding ActRIIB Polypeptides

In certain aspects, the present invention provides isolated and/or recombinant nucleic acids encoding any ActRIIB polypeptide (e.g., a soluble ActRIIB polypeptide), including any variant disclosed herein. For example, SEQ ID NO:4 encodes a native ActRIIB precursor polypeptide, while SEQ ID NO:3 encodes a soluble ActRIIB polypeptide. Nucleic acids of the invention can be single-stranded or double-stranded. Such nucleic acids can be DNA molecules or RNA molecules. These nucleic acids can be used, for example, in methods for preparing ActRIIB polypeptides or as direct therapeutic agents (e.g., in gene therapy approaches).

In certain aspects, it will be further understood that nucleic acids of the invention encoding ActRIIB polypeptides include nucleic acids that are variants of SEQ ID NO: 3. Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions, or deletions, such as allelic variants, and will therefore include coding sequences that differ from the nucleotide sequence of the coding sequence indicated in SEQ ID NO: 4.

In certain embodiments, the present invention provides isolated or recombinant nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3. One of ordinary skill in the art will recognize that nucleic acid sequences complementary to SEQ ID NO: 3 and variants of SEQ ID NO: 3 are also encompassed by the present invention. In further embodiments, the nucleic acid sequences of the present invention can be isolated, recombinant, and/or fused to heterologous nucleotide sequences, or present in a DNA library.

In other embodiments, nucleic acids of the present invention also include nucleotide sequences that hybridize under highly stringent conditions to the nucleotide sequence specified in SEQ ID NO:3, the complement of SEQ ID NO:3, or a fragment thereof. As discussed above, one of ordinary skill in the art will readily appreciate that suitable stringency conditions for promoting DNA hybridization can vary. For example, one can hybridize with 6.0x sodium chloride/sodium citrate (SSC) at approximately 45°C, followed by a wash with 2.0x SSC at 50°C. For example, the salt concentration in the wash step can be selected from low stringency conditions of about 2.0x SSC at 50°C to high stringency conditions of about 0.2x SSC at 50°C. Alternatively, the temperature of the wash step can be increased 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 concentration can be varied, or the temperature or salt concentration can be kept constant while the other variable is varied. In one embodiment, the present invention provides nucleic acids that hybridize under low stringency conditions of 6x SSC at room temperature, followed by a wash with 2x SSC at room temperature.

Separate nucleic acids that differ from the nucleic acid shown in SEQ ID NO: 3 due to the degeneracy of the genetic code also fall within the scope of the present invention. For example, many amino acids are specified in more than one triplet. Codons or synonymous codons that specify the same amino acid (e.g., CAU and CAC are synonymous codons for histidine) can result in "silent" mutations that do not affect the amino acid sequence of the protein. However, it is expected that DNA sequence polymorphisms exist in mammalian cells that do result in changes in the amino acid sequence of the target protein of the present invention. It will be understood by those skilled in the art that these variations in one or more nucleotides (up to about 3-5% of the nucleotides) of a nucleic acid encoding a particular protein may exist between individuals of a given species due to natural allelic variation. Any such nucleotide variation and the resulting amino acid polymorphism are within the scope of the present invention.

In certain embodiments, the recombinant nucleic acid of the present invention can be operatively linked to one or more regulatory nucleotide sequences in an expression construct. The regulatory nucleotide sequence is generally suitable for the host cell being used for expression. Numerous types of suitable expression vectors and suitable regulatory sequences for various host cells are known in the art. Generally, the one or more regulatory nucleotide sequences may include, but are not limited to, a promoter sequence, a leader or signal sequence, a ribosome binding site, a transcription start and stop sequence, a translation start and stop sequence, and an enhancer or activator sequence. The present invention contemplates constitutive promoters or inducible promoters known in the art. The promoter may be a natural promoter or a hybrid promoter combining elements of more than one promoter. The expression construct may be present in an intracellular episome, such as a plasmid, or the expression construct may be inserted into a chromosome. In preferred embodiments, the expression vector comprises a selectable marker gene to allow selection of transformed host cells. Selectable marker genes are well known in the art and vary with the host cell used.

In certain aspects of the invention, a nucleic acid of the invention is provided in an expression vector comprising a nucleotide sequence encoding an ActRIIB polypeptide and operably linked to at least one regulatory sequence. Regulatory sequences are art-recognized and are selected to direct expression of an ActRIIB polypeptide. Thus, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990). For example, any of a variety of expression control sequences that control the expression of a DNA sequence, when operably linked to a DNA sequence, can be used in these vectors to express a DNA sequence encoding an ActRIIB polypeptide. Such useful expression control sequences include, for example, the early and late promoters of SV40, the tet promoter, the immediate early promoters of adenovirus or cytomegalovirus, the RSV promoter, the lac system, the trp system, the TAC or TRC system, the T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter region of lambda phage, the control region of the fd capsid protein, the promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatases such as Pho5, the promoter of yeast α-hybrid factor, the polyhedron promoter of the baculovirus system, and other sequences known to control gene expression in prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It will be understood that the design of the expression vector may depend 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 vector copy number, the ability to control copy number, and the expression of any other proteins encoded by the vector (such as antibiotic markers) should also be considered.

The recombinant nucleic acids of the present invention can be produced by ligating the cloned gene or portion thereof into a vector suitable for expression in prokaryotes, eukaryotic cells (yeast, birds, insects, or mammals), or both. Expression vectors for producing recombinant ActRIIB polypeptides include plasmids and other vectors. For example, suitable vectors include the following types of plasmids: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids for expression in prokaryotes such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences that facilitate vector propagation 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 are modified with sequences derived from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotes and eukaryotes. Alternatively, derivatives of viruses, such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived, and p205), can be used to transiently express proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found below in the description of gene therapy delivery systems. The various methods for preparing plasmids and transforming host organisms are well known in the art. About other expression systems suitable for both prokaryotic and eukaryotic cells and common recombination steps, see Molecular Cloning A Laboratory Manual, 2nd edition, edited by Sambrook, Fritsch and Maniatis, (Cold Spring Harbor Laboratory Press, 1989), Chapter 16 and Chapter 17. In some cases, it may be necessary to adopt a baculovirus expression system to express the recombinant polypeptide. The example of such a baculovirus expression system includes pVL derivative vectors (such as pVL1392, pVL1393 and pVL941), pAcUW derivative vectors (such as pAcUW1) and pBlueBac derivative vectors (such as β-gal comprising pBlueBac III).

In a preferred embodiment, vectors are designed for producing the ActRIIB polypeptides 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, Wis.). Obviously, the gene constructs of interest can be used to express the ActRIIB polypeptides of interest in cultured cells, for example, to produce proteins, including fusion proteins or variant proteins, for purification.

The present invention also relates to host cells transfected with a recombinant gene comprising one or more coding sequences for an ActRIIB polypeptide of the invention (e.g., SEQ ID NO: 4). The host cell can be any prokaryotic or eukaryotic cell. For example, the ActRIIB polypeptides of the invention can be expressed in bacterial cells such as Escherichia coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.

Therefore, the present invention further relates to methods for producing the ActRIIB polypeptides of the present invention. For example, host cells transformed with an expression vector encoding an ActRIIB polypeptide can be cultured under suitable conditions to allow expression of the ActRIIB polypeptide. The ActRIIB polypeptide can be secreted and isolated from a mixture of cells and culture medium containing the ActRIIB polypeptide. Alternatively, the ActRIIB polypeptide can remain in the cytoplasm or membrane fraction, and the cells can be harvested, lysed, and the protein isolated. Cell cultures comprise host cells, culture medium, and other byproducts. Culture media suitable for cell culture are well known in the art. The ActRIIB polypeptides of the present invention can be isolated from cell culture medium, host cells, or both using protein purification techniques known in the art, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification using antibodies specific for a particular epitope of the ActRIIB polypeptide. In preferred embodiments, the ActRIIB polypeptide is a fusion protein comprising a domain that facilitates its purification.

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

Techniques for preparing fusion genes are well known in the art. Essentially, ligation of various DNA fragments encoding different polypeptide sequences is performed using conventional techniques employing blunt or staggered ends for ligation, restriction enzyme digestion to provide suitable ends, filling in sticky ends when appropriate, alkaline phosphatase treatment to prevent undesirable ligation, and enzymatic ligation. In another embodiment, fusion genes can be synthesized using conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that create complementary overhangs between two consecutive gene fragments, which can then be annealed to generate a chimeric gene sequence (see, e.g., Current Protocols in Molecular Biology, ed. Ausubel et al., John Wiley & Sons: 1992).

4. Antibodies

Another aspect of the present invention relates to antibodies. Antibodies that specifically react with ActRIIB polypeptides (e.g., soluble ActRIIB polypeptides), as well as antibodies that competitively bind to ActRIIB polypeptides, can be used as antagonists of ActRIIB polypeptide activity. For example, anti-protein/anti-peptide antisera or monoclonal antibodies can be prepared using immunogens derived from ActRIIB polypeptides by standard methods (see, e.g., Antibodies: A Laboratory Manual, edited by Harlow and Lane (Cold Spring Harbor Press: 1988)). Mammals, such as mice, hamsters, or rabbits, can be immunized with immunogenic forms of ActRIIB polypeptides, antigenic fragments, or fusion proteins capable of eliciting an antibody response. Techniques for conferring immunogenicity on proteins or peptides include conjugation to carriers or other techniques well known in the art. Immunogenic portions of ActRIIB polypeptides can be administered in the presence of an adjuvant. The progress of immunization can be monitored by measuring antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as the antigen to assess antibody levels.

After immunizing an animal with an antigenic formulation of an ActRIIB polypeptide, antiserum can be obtained, and if necessary, polyclonal antibodies can be isolated from the serum. To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be collected from the immunized animal and fused with immortalized cells such as myeloma cells using standard somatic cell fusion procedures 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 for producing human monoclonal antibodies (Cole et al., (1985) Monoclone Antibody and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with an ActRIIB polypeptide and monoclonal antibodies isolated from cultures containing such hybridoma cells.

The term "antibody" as used herein is intended to include fragments thereof that are also capable of specifically reacting with the target ActRIIB polypeptides of the present invention. Antibodies can be fragmented using conventional techniques and the fragments can be screened for use in the same manner as described above for intact antibodies. For example, F(ab) ₂ fragments can be generated by treating the antibody with pepsin. The resulting F(ab) ₂ fragments can be treated to reduce disulfide bridges to produce Fab fragments. The antibodies of the present invention are further intended to include bispecific, single-chain, and chimeric humanized molecules having affinity for the ActRIIB polypeptide conferred by at least one CDR region of the antibody. In a preferred embodiment, the antibody further includes a label attached thereto that is capable of being detected (e.g., the label can be a radioisotope, a fluorescent compound, an enzyme, or a coenzyme factor).

In certain embodiments, the antibodies of the present invention are monoclonal antibodies, and in certain embodiments, the present invention provides methods for producing novel antibodies. For example, a method for producing a monoclonal antibody that specifically binds to an ActRIIB polypeptide can include administering to a mouse an amount of an immunogenic composition comprising an ActRIIB polypeptide effective to stimulate a detectable immune response, obtaining antibody-producing cells (e.g., spleen cells) from the mouse, fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and detecting the antibody-producing hybridomas to identify hybridomas that produce monoclonal antibodies that specifically bind to an ActRIIB polypeptide. Once obtained, the hybridomas can be propagated in cell culture, optionally under culture conditions in which the hybridoma-derived cells produce monoclonal antibodies that specifically bind to an ActRIIB polypeptide. The monoclonal antibodies can be purified from the cell culture.

As generally understood in the art, the adjective "specifically reactive with" when used in reference to an antibody means that the antibody has sufficient selectivity between the target antigen (e.g., an ActRIIB polypeptide) and other non-target antigens such that the antibody can be used, at a minimum, to detect the presence of the target antigen in a particular type of biological sample. In certain methods using antibodies, such as therapeutic applications, a higher degree of binding specificity may be desired. Monoclonal antibodies generally have a greater tendency (compared to polyclonal antibodies) to effectively distinguish between the desired antigen and cross-reacting polypeptides. One characteristic that affects the specificity of an antibody:antigen interaction is the affinity of the antibody for the antigen. Although different affinity ranges can be achieved for the desired specificity, antibodies having an affinity (dissociation constant) of about ^10-6 , ^10-7 , ^10-8 , ^10-9 or less are generally preferred.

In addition, the technique used to screen antibodies to identify the desired antibody can affect the properties of the obtained antibody. For example, if the antibody is used to bind to an antigen in solution, a test solution may be required for binding. A variety of different techniques can be used to test the interaction between the antibody and the antigen to identify a particularly desired antibody. Such techniques include ELISA, surface plasmon resonance binding assays (e.g., Biacore binding assays, Bia-core AB, Uppsala, Sweden), sandwich assays (e.g., paramagnetic bead systems, IGEN International, Inc., Gaithersburg, Maryland), Western blotting, immunoprecipitation assays, and immunohistochemistry.

In certain aspects, the disclosure herein provides antibodies that bind to soluble ActRIIB polypeptides. Such antibodies can be produced in large quantities as described above using soluble ActRIIB polypeptides or fragments thereof as antigens. Such antibodies can be used, for example, to detect ActRIIB polypeptides in biological samples and/or to monitor soluble ActRIIB polypeptide levels in individuals. In certain instances, antibodies that specifically bind to soluble ActRIIB polypeptides can be used to modulate the activity of ActRIIB polypeptides and/or ActRIIB ligands, thereby modulating (promoting or inhibiting) the growth of tissues (e.g., bone, cartilage, muscle, fat, and neurons).

5. Screening Assay

In certain aspects, the present invention relates to the use of compounds (drugs) identified as agonists or antagonists of an ActRIIB polypeptide (e.g., a soluble ActRIIB polypeptide) of the invention. Compounds identified through this screening can be tested in tissues such as bone, cartilage, muscle, fat, and/or neurons to assess their ability to regulate tissue growth in vitro. Optionally, these compounds can be further tested in animal models to assess their ability to regulate tissue growth in vivo.

There are numerous approaches to screening for therapeutic drugs that modulate tissue growth by targeting ActRIIB polypeptides. In certain embodiments, high-throughput screening of compounds can be performed to identify drugs that interfere with ActRIIB-mediated effects on bone, cartilage, muscle, fat, and/or neuronal growth. In certain embodiments, this assay is performed to screen for and identify compounds that specifically inhibit or reduce the binding of an ActRIIB polypeptide to its binding partner, such as an ActRIIB ligand (e.g., activin, Nodal, GDF8, GDF11, or BMP7). Alternatively, this assay can be used to identify compounds that enhance the binding of an ActRIIB polypeptide to its binding protein (e.g., an ActRIIB ligand). In further embodiments, compounds can be identified by their ability to interact with an ActRIIB polypeptide.

A variety of assay formats will suffice, and those not clearly described herein will be understood by those of ordinary skill in the art in light of the disclosure herein. As described herein, the test compounds (drugs) of the present invention can be manufactured by any combinatorial chemical method. Alternatively, the target compound can be a natural biomolecule synthesized in vivo or in vitro. The compound (drug) to be tested for its ability to act as a tissue growth regulator can be produced, for example, by bacteria, yeast, plants, or other organisms (e.g., natural products), chemically (e.g., small molecules, including peptidomimetics), or recombinantly. Test compounds contemplated by the present invention include non-peptidyl organic molecules, peptides, polypeptides, peptidomimetics, sugars, hormones, and nucleic acid molecules. In specific embodiments, the test drug is a small organic molecule having a molecular weight of less than about 2,000 daltons.

The test compounds of the present invention can be provided as single discrete entities or as libraries with greater complexity, such as libraries prepared using combinatorial chemistry. These libraries can include, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers, and other types of organic compounds. The test compounds can be presented in a test system in a single form or as a mixture of compounds, particularly in the initial screening step. Optionally, the compounds can be optionally derivatized with other compounds and have a derivatization group that helps separate the compounds. Non-limiting examples of derivatization groups include biotin, fluorescein, digoxin, green fluorescent protein, isotopes, polyhistidine, magnetic beads, glutathione S-transferase (GST), photosensitizable crosslinkers, or any combination thereof.

In many drug screening programs for test compound libraries and natural extracts, high-throughput assays are required to maximize the number of compounds tested in a given time period. Assays performed in cell-free systems (e.g., available with purified or semi-purified proteins) are often preferred as "primary" screens because they can be generated to allow for rapid development and relatively easy detection of changes in the molecular target mediated by the test compound. Furthermore, the effects of the cytotoxicity or bioavailability of the test compound are generally negligible in in vitro systems, and the assays instead focus primarily on the effect of the drug on the molecular target, which can manifest as changes in the binding affinity between the ActRIIB polypeptide and its binding protein (e.g., ActRIIB ligand).

By way of example only, in an exemplary screening assay of the present invention, a target compound is contacted with an isolated and purified ActRIIB polypeptide, typically capable of binding an ActRIIB ligand, as appropriate for the purpose of the assay. A composition comprising an ActRIIB ligand is then added to the mixture of compound and ActRIIB polypeptide. Detection and quantification of ActRIIB/ActRIIB ligand complexes provides a method for determining the potency of a compound to inhibit (or enhance) complex formation between an ActRIIB polypeptide and its binding protein. The potency of a compound can be assessed by generating a dose-response curve using data obtained at various test compound concentrations. In addition, control assays can be performed to provide a baseline for comparison. For example, in a control assay, an isolated and purified ActRIIB ligand is added to a composition comprising an ActRIIB polypeptide, and the formation of the ActRIIB/ActRIIB ligand complex is quantified in the absence of the test compound. It will be appreciated that, in general, the order in which the reactants are mixed can vary and can be mixed simultaneously. Furthermore, cell extracts and lysates can be used in place of purified protein to provide suitable cell-free assay systems.

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

In certain embodiments, the present invention contemplates the use of fluorescence polarization assays and fluorescence resonance energy transfer (FRET) assays to directly or indirectly measure the extent of interaction between an ActRIIB polypeptide and its binding protein. In addition, other detection modalities, such as those based on optical waveguides (PCT Publication No. WO 96/26432 and U.S. Patent No. 5,677,196), surface plasmon resonance (SPR), surface charge sensors, and surface force sensors, are consistent with many embodiments of the present invention.

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

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

In certain aspects, the present invention provides methods and medicaments for stimulating muscle growth and increasing muscle mass, for example, by antagonizing the function of ActRIIB polypeptides and/or ActRIIB ligands. Thus, any compound identified can be tested in vitro or in vivo in whole cells or tissues to confirm its ability to modulate muscle growth. A variety of methods known in the art can be used for this purpose. For example, the methods of the present invention are practiced such that signal transduction via an ActRIIB protein activated by binding to an ActRIIB ligand (e.g., GDF8) is reduced or inhibited. It will be appreciated that growth of muscle tissue in an organism should result in an increase in the organism's muscle mass compared to the muscle mass of a corresponding organism (or population of organisms) in which signal transduction via the ActRIIB protein is not so affected.

For example, the effect of an ActRIIB polypeptide or test compound on muscle cell growth/proliferation can be determined by measuring gene expression of Pax-3 and Myf-5, which are associated with proliferation of myogenic cells, and gene expression of MyoD, which is associated with muscle differentiation (e.g., Amthor et al., Dev Biol. 2002, 251:241-57). GDF8 is known to downregulate gene expression of Pax-3 and Myf-5 and to inhibit gene expression of MyoD. It is expected that an ActRIIB polypeptide or test compound antagonizes this activity of GDF8. Another example of a cell-based assay includes measuring the proliferation of myoblasts, such as C(2)C(12) myoblasts, in the presence of an ActRIIB polypeptide or test compound (e.g., Thomas et al., J Biol Chem. 2000, 275:40235-43).

The present invention also contemplates in vivo assays for detecting muscle mass and strength. For example, Whittemore et al. (Biochem Biophys Res Commun. 2003, 300:965-71) disclose methods for detecting increased skeletal muscle mass and increased grip strength in mice. Optionally, this method can be used to determine the efficacy of a test compound (e.g., an ActRIIB polypeptide) for treating muscle diseases or conditions, such as those in which muscle mass is limiting.

In certain aspects, the present invention provides methods and medicaments for modulating (stimulating or inhibiting) bone formation and increasing bone mass. Thus, any identified compounds can be tested in vitro or in vivo in intact cells or tissues to confirm their ability to modulate bone or cartilage growth. Various methods known in the art can be used for this purpose.

For example, the effect of an ActRIIB polypeptide or test compound on bone or cartilage growth can be determined by detecting Msx2 induction or differentiation of osteoprogenitor cells into osteoblasts in a cell-based assay (see, e.g., Daluiski et al., Nat Genet. 2001, 27(1):84-8; Hino et al., Front Biosci. 2004, 9:1520-9). Another example of a cell-based assay includes analyzing the osteogenic activity of the target ActRIIB polypeptide and test compound in mesenchymal progenitor cells and osteoblasts. For example, a recombinant adenovirus expressing an ActRIIB polypeptide is constructed to infect multipotent mesenchymal progenitor cells C3H10T1/2 cells, pre-osteoblast C2C12 cells, and osteoblast TE-85 cells. Osteogenic activity is then determined by measuring alkaline phosphatase, osteocalcin, and induction of matrix mineralization (see, e.g., Cheng et al., J Bone Joint Surg Am. 2003, 85-A(8):1544-52).

The present invention also contemplates in vivo assays for detecting bone or cartilage growth. For example, Namkung-Matthai et al., Bone, 28:80-86 (2001) discloses a rat osteoporosis model in which bone repair is studied during the early post-fracture period. Kubo et al., Steroid Biochemistry & Molecular Biology, 68:197-202 (1999) also discloses a rat osteoporosis model in which bone repair is studied during the later post-fracture period. The disclosures of these references regarding rat models for studying osteoporotic fractures are incorporated herein by reference in their entirety. In certain aspects, the present invention utilizes fracture healing assays known in the art. These assays include fracture scissoring, histological analysis, and biomechanical analysis, which are described, for example, in U.S. Patent No. 6,521,750, which is incorporated herein by reference in its entirety for its disclosures regarding experimental protocols for inducing and detecting fracture extent and the repair process.

In certain aspects, the present invention provides methods and medicaments for controlling weight gain and obesity. At the cellular level, adipocyte proliferation and differentiation are critical to the development of obesity, leading to the production of additional adipocytes. Therefore, any identified compounds can be tested in vitro or in vivo in intact cells or tissues by measuring adipocyte proliferation or differentiation to confirm their ability to modulate adipogenesis. A variety of methods known in the art can be used for this purpose. For example, the effect of an ActRIIB polypeptide (e.g., a soluble ActRIIB polypeptide) or a test compound on adipogenesis can be determined by measuring the differentiation of 3T3-L1 preadipocytes into mature adipocytes in a cell-based assay, for example, by observing the accumulation of triacylglycerols in Oil Red O-stained vesicles and by the presence of certain adipocyte markers such as FABP (aP2/422) and PPARγ. See, e.g., Reusch et al., 2000, Mol Cell Biol. 20:1008-20; Deng et al., 2000, Endocrinology. 141:2370-6; Bell et al., 2000, Obes Res. 8:249-54. Another example of a cell-based assay includes analyzing the effects of an ActRIIB polypeptide and a test compound on the proliferation of adipocytes or adipocyte precursor cells (e.g., 3T3-L1 cells), for example, by monitoring bromodeoxyuridine (BrdU)-positive cells. See, e.g., Pico et al., 1998, Mol Cell Biochem. 189:1-7; Masuno et al., 2003, Toxicol Sci. 75:314-20.

It should be understood that the screening assays of the present invention are applicable not only to the ActRIIB polypeptides and ActRIIB polypeptide variants of the present invention, but also to any test compound, including agonists and antagonists of ActRIIB polypeptides. In addition, these screening assays can be used for drug target identification and quality control purposes.

6. Exemplary Therapeutic Applications

In certain embodiments, the compositions of the present invention (e.g., ActRIIB polypeptides) can be used to treat or prevent diseases or conditions associated with aberrant activity of ActRIIB polypeptides and/or ActRIIB ligands (e.g., GDF8). These diseases, disorders, or conditions are generally referred to herein as "ActRIIB-related conditions." In certain embodiments, the present invention provides methods for treating or preventing a subject in need thereof by administering a therapeutically effective amount of an ActRIIB polypeptide as described above. These methods are particularly directed to therapeutic and prophylactic treatment of animals, and more specifically, humans.

As used herein, a therapeutic agent that "prevents" a disease or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disease or condition in treated subjects relative to untreated control subjects, or delays the onset of or lessens the severity of one or more symptoms of the disease or condition relative to untreated control subjects. As used herein, the term "treating" includes preventing a specified condition or ameliorating or eliminating the condition after it has occurred.

The ActRIIB/ActRIIB ligand complex plays an essential role in tissue growth and early developmental processes (e.g., the proper formation of various structures) or one or more post-developmental capabilities (including sexual development, pituitary hormone production, and bone and cartilage production). Therefore, ActRIIB-associated disorders include abnormal tissue growth and developmental defects. Additionally, ActRIIB-associated disorders include, but are not limited to, disorders of cell growth and differentiation, such as inflammation, allergies, autoimmune diseases, infectious diseases, and tumors.

Exemplary ActRIIB-associated disorders include neuromuscular diseases (e.g., muscular dystrophy and muscle atrophy), congestive obstructive pulmonary disease (and muscle wasting associated with COPD), muscle wasting syndrome, sarcopenia, cachexia, adipose tissue diseases (e.g., obesity), type 2 diabetes, and bone degenerative diseases (e.g., osteoporosis). Other exemplary ActRIIB-associated disorders include muscle degenerative diseases and neuromuscular diseases, tissue repair (e.g., wound healing), neurodegenerative diseases (e.g., amyotrophic lateral sclerosis), immune diseases (e.g., diseases associated with abnormal proliferation or function of lymphocytes), and obesity or disorders associated with abnormal proliferation of adipocytes.

In certain embodiments, compositions of the invention (e.g., soluble ActRIIB polypeptides) are used as part of a muscular dystrophy treatment. The term "muscular dystrophy" refers to a group of degenerative muscle diseases characterized by the gradual weakening and degeneration of skeletal muscle and, in some cases, cardiac and respiratory muscles. Muscular dystrophy is a genetic disease characterized by progressive muscle wasting and weakening that begins with microscopic changes in the muscle. As muscle degenerates over time, a person's muscle strength decreases. Exemplary muscular dystrophies that can be treated with regimens including the subject ActRIIB polypeptides of the invention include Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Emery-Dreifuss muscular dystrophy (EDMD), Limb-Girdle muscular dystrophy (LGMD), facioscapulohumeral muscular dystrophy (FSH or FSHD) (also known as Landouzy-Dejerine), myotonic dystrophy (MMD) (also known as Steinert's disease), oculopharyngeal muscular dystrophy (OPMD), distal muscular dystrophy (DD), and congenital muscular dystrophy (CMD).

Duchenne muscular dystrophy (DMD) was first described by French neurologist Guillaume Benjamin Amand Duchenne in the 1860s. Becker muscular dystrophy (BMD) was named after German physician Peter Emil Becker, who first described this DMD variant in the 1950s. DMD is one of the most common genetic diseases in men, affecting one in 3,500 boys. DMD occurs when the dystrophin gene, located on the short arm of the X chromosome, is damaged. Because men carry only one copy of the X chromosome, they only have one copy of the dystrophin gene. Without dystrophin, muscles are easily damaged by cycles of contraction and relaxation. Although muscles compensate through regeneration in the early stages of the disease, later on, muscle progenitor cells cannot keep up with the ongoing damage, and healthy muscle is replaced by nonfunctional fibro-fatty tissue.

BMD is caused by different mutations in the dystrophin gene. People with BMD have some dystrophin, but it's either in insufficient quantity or of poor quality. Having some dystrophin protects BMD patients from muscle degeneration to a lesser or slower degree than in patients with DMD.

For example, recent studies have demonstrated that blocking or eliminating the function of GDF8 (the ActRIIB ligand) in vivo can effectively treat at least some symptoms in patients with DMD and BMD. Therefore, target ActRIIB polypeptides can be used as GDF8 inhibitors (antagonists) and constitute an alternative approach to blocking GDF8 and/or ActRIIB function in patients with DMD and BMD. This approach is confirmed and supported by the data presented herein, which show that ActRIIB-Fc proteins increase muscle mass in a mouse model of muscular dystrophy.

Similarly, the ActRIIB polypeptides of the present invention provide an effective method for increasing muscle mass in other diseases where muscle growth is desired. For example, ALS (motor neuron disease), also known as Lou Gehrig's disease, is a chronic, incurable, and incurable CNS disease that attacks motor neurons, the components of the CNS that connect the brain to skeletal muscle. In ALS, motor neurons degenerate and eventually die. Although the brain typically remains fully functional and alert, the commands they transmit no longer reach the muscles. Most people who develop ALS are between the ages of 40 and 70. The first motor neurons to weaken are those that connect to the arms or legs. ALS patients may have difficulty walking, may drop objects, fall, slur their speech, and laugh or cry uncontrollably. Eventually, limb muscles begin to atrophy from disuse. This muscle weakening can lead to frailty, requiring a wheelchair or becoming unable to leave their bed. Most ALS patients die within 3-5 years of onset due to respiratory failure or complications of ventilator support, such as pneumonia. This approach is confirmed and supported by the data presented herein, which show that ActRIIB-Fc protein improves the appearance, muscle mass and lifespan of ALS mouse models.

The increase in muscle mass induced by ActRIIB polypeptides may also benefit patients suffering from muscle-wasting diseases. Gonzalez-Cadavid et al. (supra) reported that GDF8 expression is negatively correlated with fat-free mass in humans, and that increased GDF8 gene expression is associated with weight loss in men with AIDS wasting syndrome. By inhibiting GDF8 function in AIDS patients, at least some AIDS symptoms could be alleviated, if not completely eliminated, thereby significantly improving the quality of life of AIDS patients.

Because loss of GDF8 (ActRIIB ligand) function is also associated with fat loss in the absence of reduced nutrient intake (Zimmers et al., supra; McPherron and Lee, supra), the subject ActRIIB polypeptides can further be used as therapeutic agents to slow or prevent the development of obesity and type 2 diabetes. This approach is confirmed and supported by the data presented herein, which demonstrate that ActRIIB-Fc protein improves the metabolic status of obese mice.

Cancer anorexia-cachexia syndrome is one of the most debilitating and life-threatening complications of cancer. Progressive weight loss in cancer anorexia-cachexia syndrome is a common feature of many cancer types, leading not only to a decreased quality of life and poor response to chemotherapy, but also to shorter survival times than patients with comparable tumors who have not lost weight. Cachexia, associated with anorexia, fat and muscle wasting, mental distress, and a lower quality of life, results from a complex interaction between the cancer and the host. It is one of the most common causes of death in cancer patients, accounting for 80% of cancer deaths. It is a complex example of a metabolic disorder affecting protein, carbohydrate, and lipid metabolism. Tumors induce direct and indirect bodily abnormalities, leading to anorexia and weight loss. Currently, there are no treatments to control or reverse this process. Cancer anorexia-cachexia syndrome affects cytokine production, the release of lipid metabolism factors and proteolysis-inducing factors, and alterations in intermediary metabolism. Although anorexia is common, decreased food intake alone cannot explain the changes in body composition observed in cancer patients, and increased nutrient intake does not reverse the wasting syndrome. Cachexia is suspected in cancer patients if involuntary weight loss of greater than 5% of pre-morbid weight occurs within 6 months.

Because systemic overexpression of GDF8 in adult mice was found to induce severe muscle and fat loss similar to that observed in human cachexia (Zimmers et al., supra), the subject ActRIIB polypeptides of the present invention can be beneficially used as pharmaceutical compositions to prevent, treat, or alleviate the symptoms of cachexia in which muscle growth is desired.

In other embodiments, the present invention provides methods for inducing bone and/or cartilage formation, preventing bone loss, increasing bone mineralization, or preventing bone demineralization. For example, the subject ActRIIB polypeptides and compounds identified herein are useful for treating osteoporosis in humans and other animals, as well as healing bone fractures and cartilage defects. ActRIIB polypeptides can be used as protective tests against the development of osteoporosis and for diagnosing patients with subclinical low bone density.

In a specific embodiment, the methods and compositions of the present invention can be used medically to heal bone fractures and cartilage defects in humans and other animals. The subject methods and compositions can also have preventive uses for closed and open fracture reduction and improved artificial joint fixation. De novo bone formation induced by osteogenic agents helps repair congenital, trauma-induced, or tumor resection-induced craniofacial defects and can also be used in cosmetic plastic surgery. In addition, the methods and compositions of the present invention can be used to treat periodontal disease and other tooth repair procedures. In some cases, the subject ActRIIB polypeptide can provide an environment that attracts bone-forming cells, stimulates the growth of bone-forming cells, or induces the differentiation of bone-forming cell progenitors. The ActRIIB polypeptides of the present invention can also be used to treat osteoporosis. In addition, ActRIIB polypeptides can be used to repair cartilage defects and prevent/reverse osteoarthritis.

In another specific embodiment, the present invention provides methods and compositions for repairing bone fractures and other conditions associated with cartilage and/or bone defects or periodontal disease. The present invention further provides methods and compositions for wound healing and tissue repair. Wound types include, but are not limited to, burns, incisions, and ulcers. See, for example, PCT Publication No. WO84/01106. Such compositions comprise a therapeutically effective amount of at least one ActRIIB polypeptide of the present invention, mixed with a pharmaceutically acceptable solvent, carrier, or matrix.

In another specific embodiment, the methods and compositions of the present invention can be applied to conditions that cause bone loss, such as osteoporosis, hyperparathyroidism, Cushing's disease, thyrotoxicosis, chronic diarrheal states or malabsorption, renal tubular acidosis, or anorexia nervosa. It is well known that being female, having a low body weight, and leading a predominantly sedentary lifestyle are risk factors for osteoporosis (which decreases bone mineral density and leads to fracture risk). However, osteoporosis can also be caused by long-term use of certain medications. Osteoporosis caused by medications or another medical condition is called secondary osteoporosis. In a condition called Cushing's disease, excess cortisol produced by the body leads to bone loss and fractures. The most common medications associated with secondary osteoporosis are glucocorticoids, a class of drugs that act like cortisol, a hormone naturally 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 reduce bone mass over time. Aluminum-containing antacids can cause bone loss when taken in high doses by people with kidney problems, especially those undergoing dialysis. Other medications that can cause secondary osteoporosis include phenytoin (Dilantin) and barbiturates used to prevent epilepsy; methotrexate (Rheumatrex, Immunex, Folex PFS), a medication used for certain forms of arthritis, cancer, or immune disorders; cyclosporine (Sandimmune, Neoral), a medication used to treat some autoimmune diseases and suppress the immune system in organ transplant patients; luteinizing hormone-releasing hormone agonists (Lupron, Zoladex), used to treat prostate cancer and endometriosis; heparin (Calciparine, Liquaemin), an anticoagulant; and cholestyramine (Questran) and colestipol (Colestid), used to treat high cholesterol. Periodontal disease causes bone loss because harmful bacteria in our mouths force our bodies to defend themselves against them. Bacteria produce toxins and enzymes below the gum line, causing chronic infection.

In a further embodiment, the present invention provides methods and therapeutic agents for treating diseases or disorders associated with abnormal or unwanted bone growth. For example, patients with a disease called myositis ossificans progressiva (FOP) grow an abnormal "second skeleton" that prevents any movement. In addition, abnormal bone growth can occur after hip replacement surgery and thus undermine the surgical results. This is a more common example of pathological bone growth and a situation in which the subject methods and compositions may be therapeutically useful. The same methods and compositions can also be used to treat other forms of abnormal bone growth (e.g., pathological growth of bone after trauma, burns, or spinal cord injury) and can be used to treat or prevent adverse conditions associated with abnormal bone growth observed in metastatic prostate cancer or osteosarcoma. Examples of such therapeutic agents include, but are not limited to, ActRIIB polypeptides (e.g., BMP7) that antagonize the function of ActRIIB ligands, compounds that disrupt the interaction between ActRIIB and its ligands (e.g., BMP7), and antibodies that specifically bind to ActRIIB receptors, rendering the ActRIIB ligands (e.g., BMP7) unable to bind to the ActRIIB receptors.

In another embodiment, the present invention provides compositions and methods for regulating body fat content in animals, as well as compositions and methods for treating or preventing conditions associated with body fat content, particularly health-damaging diseases associated with body fat content. According to the present invention, regulating (controlling) body weight can refer to reducing or increasing body weight, reducing or increasing the rate of weight gain, or increasing or reducing the rate of weight loss, and also includes actively maintaining or not significantly changing body weight (e.g., resisting external or internal influences that might otherwise increase or decrease body weight). One embodiment of the present invention relates to regulating body weight by administering an ActRIIB polypeptide to an animal (e.g., a human) in need thereof.

In one specific embodiment, the present invention relates to methods and compounds for reducing weight and/or reducing weight gain in animals, and more specifically, to methods and compounds for treating or ameliorating obesity in patients at risk for or suffering from obesity. In another specific embodiment, the present invention relates to methods and compounds for treating animals that are unable to gain or maintain weight (e.g., animals suffering from wasting syndrome). Such methods are effective in increasing body weight and/or mass, or reducing weight and/or mass loss, or ameliorating conditions associated with or caused by undesirable low (e.g., unhealthy) body weight and/or mass.

Other diseases that can be treated with ActRIIB proteins are described in the Examples, including high cholesterol.

7. Pharmaceutical Compositions

In certain embodiments, the compounds of the invention (e.g., ActRIIB polypeptides) are formulated with a pharmaceutically acceptable carrier. For example, ActRIIB polypeptides can be administered alone or as a component of a pharmaceutical formulation (therapeutic composition). The subject compounds of the invention can be formulated for administration by any convenient route for human or veterinary use.

In certain embodiments, the therapeutic methods of the present invention comprise administering the compositions topically, systemically, or locally as an implant or device. When administered, the therapeutic compositions of the present invention are, of course, in a pyrogen-free, physiologically acceptable form. Furthermore, the compositions may need to be encapsulated or injected in a viscous form for delivery to target tissue sites (e.g., bone, cartilage, muscle, fat, or neurons), such as sites of tissue damage. Topical administration may be suitable for wound healing and tissue repair. Therapeutically useful agents other than ActRIIB polypeptides may also optionally be included in the compositions described above and, alternatively or additionally, may be administered simultaneously or sequentially with the compound of interest (e.g., ActRIIB polypeptide) in the methods of the present invention.

In certain embodiments, the compositions of the present invention may include a matrix capable of delivering one or more therapeutic compounds (e.g., ActRIIB polypeptides) to a target tissue location, providing a structure for developing tissues, and preferably being reabsorbed into the body. For example, the matrix may provide for a slow release of the ActRIIB polypeptide. Such matrices may be formed from existing materials used for other implantable medical applications.

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

In certain embodiments, the methods of the present invention can be administered orally in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, typically sucrose and gum arabic or tragacanth), powders, granules, solutions or suspensions in aqueous or non-aqueous liquids, oil-in-water or water-in-oil liquid emulsions, elixirs or syrups, lozenges (using an inert base, such as gelatin and glycerin, or sucrose and gum arabic), and/or mouthwashes, each containing a predetermined amount of the active ingredient. The drug can also be administered as a bolus, dry electuary, or paste.

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

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage form may also contain inert diluents commonly used in the art (such as water or other solvents), cosolvents and emulsifiers (such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol), oils (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuran methanol, polyethylene glycol, and sorbitan fatty acid esters, and mixtures thereof. In addition to the inert diluent, the oral composition may also include adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, spices, colorants, flavoring agents, and preservatives.

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

Certain compositions disclosed herein can be administered topically to the skin or mucous membranes. Topical formulations may further include one or more of a variety of substances known to be effective as skin or stratum corneum penetration enhancers. Examples of such substances are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methanol or isopropyl alcohol, dimethyl sulfoxide, and azone. Other substances may be further included to produce a cosmetically acceptable formulation. Examples of such substances are fats, waxes, oils, dyes, fragrances, preservatives, stabilizers, and surfactants. Keratin softeners, such as those known in the art, may also be included. Examples are salicylic acid and sulfur.

Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required. In addition to the target compound of the invention (e.g., ActRIIB polypeptide), ointments, pastes, creams, and gels can also contain excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

Powders and sprays may contain, in addition to the target compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants, such as chlorofluorocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

In certain embodiments, pharmaceutical compositions suitable for parenteral administration may comprise one or more ActRIIB polypeptides together with one or more pharmaceutically acceptable sterile, isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions prior to use, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Suitable fluidity can be maintained by using coating materials (e.g., lecithin), or by maintaining the desired particle size for dispersions, and by using surfactants.

The compositions of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Various antibacterial and antifungal agents (e.g., parahydroxybenzoic acid, chlorobutanol, phenol sorbate, etc.) may be included to ensure protection against microbial activity. It may also be desirable to include isotonic agents (e.g., sugars, sodium chloride, etc.) in the compositions. Furthermore, prolonged absorption of injectable pharmaceutical forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

It will be understood that the dosage regimen will be determined by the attending physician taking into account a variety of factors that alter the effects of the subject compounds of the invention (e.g., ActRIIB polypeptides). Various factors will depend on the disease being treated. In the case of muscle diseases, factors may include, but are not limited to, the amount of muscle mass to be developed, the muscle most affected by the disease, the condition of the degenerating muscle, the patient's age, sex, and diet, the timing of administration, and other clinical factors. The addition of other known growth factors to the final composition may also affect the dosage. Progress can be monitored by periodic assessment of muscle growth and/or repair, for example, by strength testing, MRI assessment of muscle size, and analysis of muscle biopsies.

In certain embodiments of the present invention, one or more ActRIIB polypeptides can be administered together (simultaneously) or at different times (sequentially or overlappingly). Furthermore, an ActRIIB polypeptide can be administered with a class of therapeutic agents, such as a chondroinductive agent, an osteoinductive agent, a myogenic agent, a lipogenic agent, or a neuroinductive agent. The two classes of compounds can be administered simultaneously or at different times. It is contemplated that the ActRIIB polypeptides of the present invention may act in conjunction with, or potentially synergistically with, another therapeutic agent.

In particular, various osteogenic, chondroinductive, and osteoinductive factors have been described, particularly bisphosphonates. See, for example, European Patent Application Nos. 148,155 and 169,016. For example, other factors that can be combined with the ActRIIB polypeptide of interest include various growth factors, such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF).

In certain embodiments, the present invention also provides gene therapy for in vivo production of ActRIIB polypeptides. Such therapy achieves its therapeutic effect by introducing an ActRIIB polynucleotide sequence into cells or tissues affected by the diseases listed above. Delivery of ActRIIB polynucleotide sequences can be accomplished using recombinant expression vectors, such as chimeric viruses, or colloidal dispersion systems. Targeted liposomes are preferred for therapeutic delivery of ActRIIB polynucleotide sequences.

The various viral vectors taught herein that can be used for gene therapy include adenovirus, herpes virus, vaccinia, or preferably RNA viruses such as retroviruses. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus. Examples of retroviral vectors into which a single exogenous gene can be inserted include, but are not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), and Rous sarcoma virus (RSV). A large number of other retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a selectable marker gene, allowing identification and generation of transduced cells. Retroviral vectors can be made target-specific by attaching, 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 attached to viral envelope proteins to allow target-specific delivery of retroviral vectors containing ActRIIB polynucleotides. In a preferred embodiment, the vector is targeted to bone, cartilage, muscle or neuronal cells/tissue.

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

Another targeted delivery system for ActRIIB polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and liposome-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system of the present invention is a liposome. Liposomes are artificial membrane vesicles that can be used as delivery vehicles in vitro and in vivo. RNA, DNA, and intact virions can be encapsulated within an aqueous interior and delivered to cells in a biologically active form (see, e.g., Fraley et al., Trends Biochem. Sci., 6:77, 1981). Methods for efficient gene transfer using liposome vectors are well known in the art, see, e.g., Mannino et al., Biotechniques, 6:682, 1988. Liposome compositions are typically combinations of phospholipids, often in combination with steroids, particularly cholesterol. Other phospholipids or other lipids can 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 for liposome production include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Exemplary phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine. Targeting of liposomes may also be based on, for example, organ specificity, cell specificity, and organelle specificity, and is known in the art.

Example

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

Example 1. Production of ActRIIb-Fc fusion protein

Applicants constructed soluble ActRIIb fusion proteins with the extracellular domain of human ActRIIb fused to either the human or mouse Fc domain, with a minimal linker (3 glycine amino acids) between the two. The constructs were termed ActRIIb-hFc and ActRIIb-mFc, respectively.

ActRIIb-hFc (SEQ ID NO: 5) purified from the CHO cell line is shown below: GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKPLHCYASWRNSSGTIELVKKGC WLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT GGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK

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

(i) Bee melittin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ ID NO: 7)

(ii) Tissue plasminogen activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 8)

(iii) Native: MGAAAKLAFAVFLISCSSGA (SEQ ID NO: 9).

The selected version utilizes a TPA leader sequence and has the following unprocessed amino acid sequence:

MDAMKRGLCCVLLLCGAVFVSPGAS GRGEAETRECIYYNANWELERTNOSGLERCEG EQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNF CNERFTHLPEAGGPEVTYEPPPTAPT GG GTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The polypeptide is encoded by the following nucleic acid sequence (SEQ ID NO: 10):

N-terminal sequencing of the CHO-cell produced material revealed the -GRGEAE primary sequence (SEQ ID NO: 11). Note that other constructs reported in the literature begin with the -SGR... sequence.

Purification can be achieved by a series of column chromatography steps, including, for example, three or more of the following steps in any order: Protein A chromatography, Q Sepharose chromatography, Phenyl Sepharose chromatography, size exclusion chromatography, and cation exchange chromatography. Purification can be accomplished by virus filtration and buffer exchange.

ActRIIb-Fc fusion proteins were also expressed in HEK293 cells and COS cells. Although material from all cell lines and appropriate culture conditions provided proteins with muscle-building activity in vivo, variations in potency were observed that may be related to cell line selection and/or culture conditions.

Example 2: Generation of ActRIIb-Fc mutants

Applicants generated a series of mutations in the extracellular domain of ActRIIB and produced these mutant proteins as soluble fusion proteins between the extracellular ActRIIB and the Fc domain. Background ActRIIB-Fc fusion has the following sequence (Fc portion is underlined) (SEQ ID NO: 12):

SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKG CWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT GGGTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Various mutations, including N- and C-terminal truncations, were introduced into the background ActRIIB-Fc protein. Based on the data provided in Example 1, it was expected that these constructs would lack the N-terminal serine if expressed with the TPA leader sequence. Mutations were generated in the ActRIIB extracellular domain by PCR mutagenesis. Following PCR, the fragments were purified using a Qiagen column, digested with SfoI and AgeI, and gel purified. These fragments were ligated into the expression vector pAID4 (see WO2006/012627) such that upon ligation, they formed a fusion chimera with human IgG1. Upon transformation into E. coli DH5α, colonies were picked and DNA isolated. For the murine construct (mFc), murine IgG2a was substituted for human IgG1. All mutants were sequence confirmed.

All mutants were generated in HEK293T cells by transient transfection. In summary, HEK293T cells were grown overnight in 250 ml of Freestyle (Invitrogen) medium at 6 × ¹⁰ cells/ml in a 500 ml spinner. The next day, the cells were treated with a DNA:PEI (1:1) complex at a final DNA concentration of 0.5 μg/ml. After 4 hours, 250 ml of culture medium was added, and the cells were cultured for 7 days. The conditioned medium was collected by centrifugation and concentrated.

The mutants were purified using a variety of techniques including, for example, a Protein A column and elution with a low pH (3.0) glycine buffer. Following neutralization, the eluate was dialyzed against PBS.

Mutants were also generated in CHO cells by a similar approach.

The mutants were tested in the binding assays and/or bioassays described below. In some cases, conditioned medium was used rather than purified protein for the assays.

Example 2. Bioassays for GDF-11 and Activin-mediated Signaling

The effect of ActRIIB-Fc protein on signaling via GDF-11 and activin A was assessed using an A-204 reporter gene assay. Cell line: human rhabdomyosarcoma (muscle-derived). Reporter vector: pGL3(CAGA)12 (described in Dennler et al., 1998, EMBO 17: 3091-3100). See Figure 5. The CAGA12 motif is present in a TGF-β-responsive gene (PAI-1 gene), so this vector is commonly used for factors that signal via Smad2 and 3.

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

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

Day 3: Add factors (diluted in culture medium + 0.1% BSA). Inhibitors should be pre-incubated with factors for 1 hour before addition to cells. After 6 hours, rinse cells with PBS and lyse.

This was followed by a luciferase assay. In the absence of any inhibitor, activin A showed 10-fold stimulation of reporter gene expression and an ED50 of approximately 2 ng/ml. GDF-11: 16-fold stimulation, ED50: approximately 1.5 ng/ml.

ActRIIB (R64,20-134) is a potent inhibitor of activin, GDF-8, and GDF-11 activity in this assay. Variants were also tested in this assay.

Example 3. N-terminally and C-terminally truncated GDF-11 inhibits

Truncations were generated at the N-terminus and C-terminus of the ActRIIB portion, ActRIIB-Fc (R64,20-134), and tested for their activity as inhibitors of GDF-11 and activin. The activities are shown below (measured in conditioned medium):

C-terminal ActRIIb-hFc truncation:

As can be seen, truncation of 3 (ending with ...PPT), 6 (ending with ...YEP) or more amino acids at the C-terminus reduces the activity of the molecule by more than 3-fold. Truncation of the final 15 amino acids of the ActRIIB portion results in an even greater loss of activity (see WO2006/012627).

The amino-terminal truncation was performed in the context of the ActRIIb-hFc (R64 20-131) protein. The activities are shown below (measured in conditioned medium):

N-terminal ActRIIb-hFc truncation:

Thus, truncations of 2, 3, or 4 amino acids at the N-terminus result in more active proteins than forms with the full-length extracellular domain. Additional experiments demonstrated that a 5-amino acid truncation of ActRIIb-hFc (R64, 25-131) exhibited activity equivalent to the untruncated form, with further deletions at the N-terminus continuing to degrade the protein. Thus, the optimal construct would have a C-terminus ending between amino acids 133-134 of SEQ ID NO: 4 and an N-terminus beginning at amino acids 22-24 of SEQ ID NO: 4. An N-terminus corresponding to amino acids 21 or 25 would produce activity similar to that of the ActRIIb-hFc (R64, 20-134) construct.

Example 4. ActRIIb-Fc variants, cell-based activity

The activity of the ActRIIB-Fc protein was tested in the cell-based assay described above. The results are summarized in Table 1 below. Several variants were tested in various C-terminal truncation constructs. As discussed above, truncation of 5 or 15 amino acids resulted in decreased activity. Notably, the L79D and L79E variants showed a significant loss of activin binding while retaining nearly wild-type GDF-11 inhibition.

Soluble ActRIIB-Fc binds GDF11 and activin A:

+ Poor activity (about 1×10 ^-6 K ₁ )

++ Moderate activity (about 1×10 ^-7 K ₁ )

+++ Good (wild-type) activity (approximately 1×10 ^-8 K ₁ )

++++ Greater than wild-type activity

The serum half-life of several variants has been evaluated in rats. ActRIIB(R64 20-134)-Fc has a serum half-life of approximately 70 hours. ActRIIB(R64 A24N 20-134)-Fc has a serum half-life of approximately 100-150 hours. The A24N variant has activity equivalent to the wild-type molecule in cell-based assays (above) and in vivo assays (below). This, combined with a longer half-life, suggests that over time, the A24N variant will produce a greater effect per unit protein than the wild-type molecule.

Strikingly, introducing an acidic amino acid (aspartic acid or glutamic acid) at position 79 selectively reduces activin binding while maintaining GDF11/GDF8 binding. As described below, the wild-type ActRIIB-Fc protein appears to have effects on tissues other than muscle, some of which may be undesirable. As disclosed herein, these effects are expected to involve a variety of different ligands bound and inhibited by ActRIIB-Fc, potentially including activin. Preliminary data suggest that in mice, the L79E and L79D variants have reduced effects on non-muscle tissues while retaining their effects on muscle. While this type of variation can be considered an ActRIIB variant, it should be noted that these proteins no longer function as activin receptors, and the name "ActRIIB" is therefore only appropriate as an indicator of the origin of these polypeptides. Although the acidic residue at position 79 reduces activin binding while maintaining GDF11 binding, other changes at this position do not have this effect. The L79A change increases activin binding relative to GDF11 binding. The L79P change reduces both activin binding and GDF11 binding.

Example 5. Binding of GDF-11 and activin A

Certain ActRIIB-Fc proteins were tested for binding to ligands using the BiaCore ^™ assay.

ActRIIB-Fc variants or wild-type proteins were captured on the system using anti-hFc antibodies. Ligand was injected and allowed to flow over the captured receptor protein. The results are summarized in the table below.

Ligand Binding Specificity IIB Variants.

These data confirm the cell-based assay data, showing that the A24N variant retains ligand binding activity similar to that of the ActRIIb-hFc(R64 20-134) molecule, and that the L79D or L79E molecules retain myostatin and GDF11 binding but exhibit significantly reduced (non-quantitative) activin A binding.

Other variants have been generated and tested as reported in WO 2006/012627 using ligands coupled to the device and flowing the receptors over the coupled ligands. The data tables for these variants are reproduced below:

Soluble ActRIIB-Fc variants that bind GDF11 and activin A (BiaCore assay)

ActRIIB ActA GDF11 WT(64A) KD＝1.8e-7M(+) KD＝2.6e-7M(+) WT(64R) na KD＝8.6e-8M(+++) +15 tails KD～2.6e-8M(+++) KD＝1.9e-8M(++++) E37A * * R40A - - D54A - * K55A ++ * R56A * * K74A KD＝4.35e-9M+++++ KD＝5.3e-9M+++++ K74Y * -- K74F * -- K74I * -- W78A * * L79A + * D80K * * D80R * * D80A * * D80F * * D80G * * D80M * * D80N * * D80I * -- F82A ++ -

* No binding observed

--＜1/5WT combination

- ～1/2WT combined

+ WT

++ ＜2x increased binding

+++ ~5x increased bonding

++++ ~10x increased binding

+++++ ~40x increased binding

Example 6: Effect of ActRIIB-Fc protein on muscle mass in wild-type mice

Applicants determined the ability of ActRIIB-Fc protein to increase muscle mass in wild-type mice.

C57B110 mice were dosed with human ActRIIB (R64 20-134) protein or human ActRIIB (K74A 20-134) intraperitoneally (i.p.) twice weekly (10 mg/kg). Mice were scanned by NMR on days 0 and 28 to determine the percentage change in whole-body lean tissue mass. Compared to the vehicle control group, mice treated with human ActRIIB (R64 20-134)-Fc showed a significant increase in lean tissue mass of 31.1%. Mice treated with human ActRIIB (K74A 20-134)-Fc protein showed a significant increase in lean tissue mass compared to the control group, although the extent was less than that of the human ActRIIB (R64 20-134)-treated group. In a similar study, mice were treated intraperitoneally with PBS, 1 mg/kg, 3 mg/kg, or 10 mg/kg of murine ActRIIB (WT, 20-134)-Fc twice a week. At the end of the study, the femoris, gastrocnemius, pectoralis, and diaphragm muscles were dissected and weighed. The results are summarized in Table 3 below.

Table 3: Tissue weights of wild-type mice treated with vehicle and murine ActRIIB (WT, 20-134)-Fc

As shown in Table 3, the murine ActRIIB(WT,20-134)-Fc fusion protein significantly increased muscle mass in wild-type mice. In mice treated with murine ActRIIB(WT,20-134)-Fc, the gastrocnemius muscle increased by 26.5%, the femoris muscle increased by 28.9%, and the pectoral muscle increased by 40.0%. We also observed changes in the diaphragm muscle, which increased by 63% compared to vehicle-treated control mice. Decreased diaphragm muscle mass is a common complication in various muscular dystrophies. Therefore, the increase in diaphragm weight observed after murine ActRIIB(WT,20-134)-Fc treatment may be clinically important.

Example 7: Effect of long half-life ActRIIB-Fc protein on muscle mass in wild-type mice

Applicants determined the ability of a long half-life ActRIIB-mFc (R64, A24N 20-134) protein variant to increase muscle mass in wild-type mice.

C57B110 mice were dosed twice weekly with human ActRIIB-mFc (R64 20-134) protein or human ActRIIB-mFc (R64, A24N 20-134) (10 mg/kg, intraperitoneally (i.p.)). NMR scans were performed at multiple locations up to day 25 to determine the percentage change in whole-body lean tissue mass. Both molecules produced comparable increases in total body weight and muscle mass, with effects on the gastrocnemius, femoris, and pectoralis muscles showing increases of 40-70%. See Figures 5 and 6.

These data demonstrate that the half-life-enhanced form of the molecule promotes muscle growth with equal potency to the wild-type molecule in short-term studies.

Example 8: Effect of ActRIIB-Fc Protein with Reduced Activin Binding on Muscle Mass in Wild-Type Mice

Applicants determined the ability of a long half-life ActRIIB-mFc(R64, L79D 20-134) protein variant to increase muscle mass in wild-type mice.

C57B110 mice were administered human ActRIIB-mFc (R64 20-134) protein or human ActRIIB-mFc (R64, L79D 20-134) twice weekly (10 mg/kg, intraperitoneally (i.p.)). NMR scans were performed at multiple locations on day 24 to determine the percentage change in whole-body lean tissue mass. The data are shown in the table below.

These data demonstrate that the L79D variant of ActRIIB (reduced activin A binding) is active in promoting muscle growth in vivo, however, the amount of muscle growth is less than that of wild-type ActRIIB. This reduced effect may be due in part to slightly reduced myostatin binding or loss of binding to another, as yet unknown, negative regulator of muscle growth. The ability to stimulate muscle growth without affecting activin A signaling is highly desirable, as activin is a widely expressed regulatory molecule known to have effects on the reproductive system, bone, liver, and many other tissues. In mice, ActRIIB-mFc(R64 20-134) produced significant effects on the reproductive system and, in some cases, increased spleen size. The effects of the ActRIIB-mFc(R64, L79D 20-134) molecule on reproductive tissues and the spleen were greatly reduced, suggesting that this molecule is particularly suitable for promoting muscle growth in reproductively active patients or those who need to minimize effects on the reproductive system.

Example 9: Effects of ActRIIB-Fc Protein on Muscle Mass and Strength in Mdx Mice

To determine the ability of murine ActRIIB (WT, 20-134)-Fc protein to increase muscle mass in diseased conditions, Applicants determined the ability of ActRIIB-Fc protein to increase muscle mass in the mdx mouse model of muscular dystrophy.

Adult Mdx mice were treated twice weekly with murine ActRIIB (WT, 20-134)-Fc protein (1, 3, or 10 mg/kg intraperitoneally) or PBS vehicle control. Forelimb grip strength was determined by measuring the force exerted by the mice while pulling on a force transducer. The average force from five pull tests was used to compare grip strength between cohorts. At the end of the study, the femoris, gastrocnemius, pectoralis, and diaphragm muscles were dissected and weighed. Grip strength testing also demonstrated significant increases. Muscle mass results are summarized in the table below.

Tissue weights of vehicle- and murine ActRIIB (WT, 20-134)-Fc-treated mdx mice

As shown in the table, ActRIIB (WT, 20-134)-Fc-treated mice demonstrated increased lean tissue mass in mdx mice compared to PBS-treated mice. ActRIIB-Fc treatment increased gastrocnemius muscle size by 25.9%, femoris muscle size by 31.8%, and pectoral muscle size by 85.4% compared to vehicle controls. Of potential clinical importance, we also observed a 34.2% increase in diaphragm muscle weight in ActRIIB (WT, 20-134)-Fc-treated mice compared to controls. These data demonstrate the efficacy of ActRIIB-Fc protein against muscular dystrophy.

Additionally, mdx mice treated with ActRIIB-Fc protein demonstrated increased grip strength compared to vehicle-treated controls. At 16 weeks, the 1, 3, and 10 mg/kg ActRIIB groups demonstrated 31.4%, 32.3%, and 64.4% increases in grip strength, respectively, compared to the vehicle control group. The improved grip strength performance of the murine ActRIIB(WT,20-134)-Fc-treated groups supports the notion that the increased muscle mass found in the treated groups is physiologically relevant. Mdx mice are susceptible to contraction-induced injury and undergo significantly more rounds of degeneration and regeneration than their wild-type counterparts. Despite these muscle phenotypes, murine ActRIIB(WT,20-134)-Fc treatment increased grip strength in mdx mice.

In Duchenne muscular dystrophy, disease onset occurs early in childhood, often as early as 5 years of age. Therefore, the data presented above for adult mice do not necessarily reflect the role that ActRIIB molecules should have in children with Duchenne muscular dystrophy. To address this issue, studies were performed using juvenile mdx mice.

ActRIIB-mFc(R64,20-134) treatment significantly increased body weight in young (4-week-old) C57BL/10 and mdx mice. Body composition analysis using in vivo NMR spectroscopy revealed that the increased lean tissue mass was accompanied by higher body weight. ActRIIB-mFc(R64,20-134)-treated C57BL/10 mice gained 35.2% more lean tissue mass, and the treated mdx group gained 48.3% more lean tissue mass than their corresponding control group. Furthermore, the effect of ActRIIB-mFc(R64,20-134) treatment on strength was evaluated. Grip strength scores in vehicle-treated mdx mice were 15.7% lower than those in the vehicle-treated C57BL/10 group, indicating muscle weakness associated with dystrophin deficiency. In contrast, ActRIIB-mFc(R64,20-134)-treated mdx mice showed improved grip strength compared to the mdx vehicle group, achieving grip strength scores that were superior to those of C57BL/10 vehicle-treated mice and approached the grip strength scores of treated C57BL/10 mice (vehicle mdx: 0.140 ± 0.01 kgF; treated mdx: 0.199 ± 0.02 kgF; vehicle C57BL/10: 0.166 ± 0.03; 0.205 ± 0.02 kgF). Remarkably, treatment restored grip strength in young mdx mice to wild-type levels. Therefore, the ActRIIB-mFc(R64,20-134) molecule has the potential to have important clinical applications for Duchenne muscular dystrophy, particularly in young patients approaching the age of onset.

Example 7: Effects of ActRIIB-Fc Protein on Strength and Survival of SOD1 Mice

To determine the ability of ActRIIB polypeptides to increase strength and survival in an ALS mouse model, Applicants tested ActRIIB-Fc protein in SOD1 mice.

B6.Cg-Tg(SOD1-G93A)1Gur/J mice, or SOD1 mice, carry a high-copy mutant allele of the human superoxide dismutase transgene. High levels of this protein in these mice result in a phenotype resembling human ALS. SOD1 mice develop ascending paralysis by 91 days of age and exhibit early symptoms of the disease, which leads to premature death between 19 and 23 weeks of age.

SOD1 mice were dosed with either vehicle control or ActRIIB-mFc (K74A 20-134) (i.p., 5 mg/kg, twice weekly) starting at 10 weeks of age. The force exerted by the mice when pulling on a force transducer was a measure of forelimb grip strength. The average force from five traction tests was used to compare grip strength between cohorts. Survival was calculated as the number of days between the date of birth and the date the mouse could no longer right itself in a supine position within 30 seconds. Figure 7 shows the grip strength test, while Figure 8 shows the survival data.

Mice at the end of the disease had difficulty grooming their fur, presumably due to the development of paralysis, and appeared matted. A cursory observation of the mice revealed that the murine ActRIIB(K74A 20-134)-Fc-treated group groomed their fur better than the PBS group, even at the late stages of the disease. This observation suggests that the treated mice were in better health and maintained a higher quality of life than the controls.

As shown in Figure 7, SOD1 mice treated with murine ActRIIB(K74A 20-134)-Fc demonstrated significantly higher grip strength compared to the PBS control group. This was observed at day 117, early in the disease, and at day 149, after the disease had progressed. Figure 8 shows that ActRIIB(K74A 20-134)-Fc-treated mice survived significantly longer than vehicle controls. This study demonstrates the utility of murine ActRIIB(K74A 20-134)-Fc in improving strength and survival in mice in an ALS mouse model.

Similar experiments were performed using SOD1 mice, but treatment was delayed until the onset of significant detectable disease (day 130) to better mimic treatment of human ALS after the onset of significant disease symptoms. On day 130, SOD1 mice were divided into a vehicle-treated group (modified TBS) or an ActRIIB(R64 20-134)-mFc (10 mg/kg)-treated group. Mice were dosed subcutaneously once a week. NMR scans were performed on the mice on day -1 and day 27 of the study (129 and 157 days of age, respectively). Grip strength was measured on days 0 and 20 of the study. At the end of the study, male controls had lost 4.3% of their day 0 body weight, while the treated group had gained 7.8% of their day 0 body weight. Female controls had lost 1.5% of their day 0 body weight, while the treated females had gained 15% of their day 0 body weight.

SOD1 grip strength test

Grip strength was measured on days 0 and 20 in male and female SOD1 mice. Superscript "a" indicates a significant difference (p < 0.05) compared to the corresponding day 0 test. Superscript "b" indicates a significant difference (p < 0.05) between the day 20 test of PBS (Group 1) and ActRIIB (R64 20-134)-mFc (Group 2).

Mice were scanned by NMR to determine changes in body composition attributable to treatment. Male control mice lost 6.0% of their lean tissue mass over the course of the study (-1 day: 18.2 g ± 1.28; 27 days: 17.1 g ± 1.10), while male treated mice gained 9.1% of their lean tissue mass at study day 0 (-1 day: 19.17 g ± 0.77; 27 days: 20.92 g ± 0.74). Female control mice lost 0.83% of their lean tissue mass at the start of the study (-1 day: 13.18 g ± 0.84; 27 days: 13.08 g ± 0.71), while female treated mice gained 10.7% of their body weight at study day 0 (-1 day: 13.66 g ± 0.83; 27 days: 15.12 g ± 1.21). Both male and female treated groups had significantly increased lean tissue mass compared to their corresponding PBS controls (p &lt; 0.001).

SOD1 muscle effects of ActRIIB(R64 20-134)-mFc

Gastrocnemius (L+R) Femoris (L+R) Chest muscles (L+R) Male control 0.18±0.03 0.12±0.03 0.20±0.04 Male ActRIIB(R64 20-134)-mFc 0.22±0.04 0.15±0.02 0.30±0.04 Female control 0.13±0.02 0.089±0.016 0.11±0.01 Female ActRIIB(R64 20-134)-mFc 0.17±0.03 0.01±0.02 0.15±0.05

These data suggest that ActRIIB-Fc therapy may be beneficial in the treatment of patients with active ALS, with improvements in both muscle function and quality of life.

Example 8: Effects of ActRIIB-Fc Protein on Obesity and Diabetes in Obese Mice

Applicants tested ActRIIB-mFc protein in mice fed a high-fat diet (HFD) to determine the ability of ActRIIB-Fc to induce weight loss in a mouse model of obesity.

Type II diabetes is a major complication of obesity and is characterized by insulin resistance. Elevated fasting insulin levels are indicative of insulin resistance and provide methods for testing whether an animal is in a state of insulin resistance. Applicants determined the effect of treatment with murine ActRIIB(R64K74A20-134)-Fc on normalizing fasting insulin levels in an obese mouse model.

HFD-fed C57BL/6 mice were maintained on a 35% fat diet and were considered obese when their body weight exceeded approximately 50% that of age-matched mice fed a standard chow diet (4.5% fat). Obese mice were administered vehicle control or human ActRIIB (R64 K74A 20-134)-Fc (10 mg/kg; i.p.) twice weekly. NMR scans of obese mice were performed to determine body composition at the start of dosing and after 3 weeks of dosing. Changes in body composition from baseline are summarized in Figure 9.

Mice were fed an HFD and were considered obese when their body weight was 50% greater than that of their standard chow-fed counterparts. HFD-fed mice were administered either vehicle control or murine ActRIIB(R64 K74A 20-134)-Fc (5 mg/kg, twice weekly; i.p.) for 35 weeks. At the end of the study, mice were fasted overnight. At the end of the fast, blood was collected and processed for serum. The serum was then used to determine fasting insulin levels in both cohorts. The results of the effect of murine ActRIIB(K74A 20-134)-Fc on fasting insulin levels in obese mice are summarized in the table below.

Fasting insulin levels in vehicle- and murine ActRIIB(K74A 20-134)-Fc-treated mice

Figure 9 shows that the murine ActRIIB(R64 K74A 20-134)-Fc group reduced adiposity compared to vehicle-treated controls. The treated mice showed a 25.9% decrease in fat mass compared to their baseline levels. Additionally, the treated group increased lean tissue mass by 10.1% above their baseline levels. The percentage changes in adipose and lean tissue mass were significantly greater in the ActRIIB(R64 K74A 20-134)-mFc group than in the PBS-treated group.

In this model, mice are maintained on a high-fat diet until they are over 50% heavier than chow-fed test mice. Based on this significant increase in weight and adiposity, it is clear that this model corresponds to humans characterized by morbid obesity. Therefore, the finding that human ActRIIB (R64 K74A 20-134)-Fc protein reduces obesity in obese mice may be clinically relevant for the treatment of morbidly obese humans.

The results summarized in Table 5 indicate that treatment with murine ActRIIB (K74A 20-134)-Fc protein significantly reduces obesity-associated elevated fasting serum insulin levels. This finding supports the potential clinical relevance of the role of ActRIIB polypeptides in the treatment of type 2 diabetes.

Further experiments were performed using ActRIIB-mFc (R64 20-134) in a HFD model of obesity and diabetes. 30-week-old HFD-fed C57BL/6 mice were divided into two groups (PBS and 10 mg/kg ActRIIB-mFc (R64 20-134)). Mice were weighed and intraperitoneally administered 2X/week for 12 weeks. Mice were evaluated by NMR on days 0 and 94 of the study.

Treated mice lost 1.9% of their body weight on day 0 of the study, while PBS-treated mice gained 6.7% of their starting weight over the course of the study. Treated mice also gained significantly more lean tissue over the course of the study than the PBS group (21.1% ± 6.28 vs. 3.7% ± 4.08). Treated mice also lost significantly more adipose tissue (-34% ± 10.95) compared to the PBS group (+10.2 ± 10.18). Individual muscle mass also increased in the ActRIIB-mFc (R64 20-134)-treated group.

In addition to the beneficial effects on fat and muscle associated with ActRIIB-Fc treatment in these mice, positive effects on serum lipids were also observed. Serum cholesterol and triglyceride levels were significantly reduced, suggesting that the ActRIIB-Fc fusion protein could be used to lower the levels of these lipids in patients.

Example 9: Effect of ActRIIB-Fc protein on muscle mass in cachectic mice

Applicants tested the ability of ActRIIB(R64 20-134)-mFc to attenuate muscle loss in a mouse model of glucocorticoid-induced muscle wasting.

Mice were subcutaneously administered PBS or dexamethasone (2 mg/kg) once daily for 13 days to induce muscle wasting. Over the same 13 days, PBS- and dexamethasone-treated groups received either vehicle or ActRIIB(R64 20-134)-mFc (10 mg/kg; i.p.; twice weekly) to provide all treatment combinations. Mice underwent NMR scanning on days 0 and 13 to determine changes in lean tissue mass between groups. The NMR results are summarized in Table 6 below.

Table 6: Lean tissue mass of vehicle and murine ActRIIB(R64 20-134)-Fc treated mice

NMR scans showed a significant 2.5% decrease in lean tissue mass in the dexamethasone:PBS group compared to the PBS:PBS group. In contrast, the dexamethasone:ActRIIB(R64 20-134)-mFc group showed a significant 13.5% increase in lean tissue mass compared to both the PBS:PBS and dexamethasone:PBS groups. Cachexia is an unwanted side effect of many therapeutic treatments, including chronic glucocorticoid therapy. Therefore, attenuation of cachexia-associated muscle wasting by treatment with human ActRIIB(R64 20-134)-mFc protein may be clinically important.

Example 10: Effects of ActRIIB-Fc on muscle mass and adiposity in aged or ovarectomized mice

Sarcopenia is a form of muscle loss associated with aging in otherwise healthy individuals. The condition is associated with a progressive loss of skeletal muscle mass and impaired strength and mobility. The cause of sarcopenia is unclear. In women, menopause accelerates muscle loss much as it accelerates bone loss. Therefore, ActRIIB(R64,20-134)-mFc was tested in very old (2-year-old) mice and in ovariectomized mice, a model of the postmenopausal state.

Eight-week-old C57BL/6 female mice were ovariectomized (OVX) or sham-operated and then allowed to reach 16 weeks of age before the study began. At the start of the study, sham-operated and OVX mice were each divided into treatment and vehicle groups. All groups were weighed and administered ActRIIB (R64,20-134)-mFc or buffer control once weekly for 11 weeks. All mice underwent NMR scanning on days 0 and 83 to determine body composition.

At the end of the study, sham-operated PBS mice had lost 4.7% of their original lean tissue mass, while the sham-treated group gained 21% of their lean tissue mass over the course of the study. OVX controls lost 12.1% of their lean tissue mass (significantly more than sham-operated vehicle), while treated OVX mice gained 12.9% by the end of the study.

These data suggest that ActRIIB-Fc fusion protein can be used to combat muscle loss commonly seen in postmenopausal women.

To evaluate the effects of ActRIIB-Fc in a naturally aging population, male C57BL/6 mice were treated starting at 70 weeks of age. Mice were divided into two groups: PBS and 10 mg/kg ActRIIB (R64,20-134)-mFc. Each group was weighed and dosed twice weekly for 10 weeks. Over the course of the study, the treated groups gained significantly more lean tissue mass than the PBS group.

% Lean Tissue Mass Change PBS 10 mg/kg Mean (% from baseline) 101.76 117.27 Standard Deviation 3.83 3.91 P-value for PBS <0.001

The treated groups also had significantly higher individual muscle weights compared to PBS mice.

The muscle integrity of the treated group also appeared to be higher than that of the PBS group, as intramuscular fat was significantly reduced and muscle structure improved (see Figure 10).

These data demonstrate that ActRIIB-Fc fusion protein can be used to treat age-related muscle wasting in men and women.

Example 11: Effect of ActRIIB-Fc on Castration-Associated Muscle Loss

Prostate cancer is commonly treated with anti-androgen therapy. Side effects of treatment include muscle loss and increased obesity. Castrated mice undergo similar changes, making them a good model for studying the potential of ActRIIB-Fc in this clinical setting.

Eight-week-old male C57BL/6 mice were castrated or sham-operated and allowed to recover for 3 weeks before the study began. The sham-operated and castrated groups were further subdivided into a PBS group and an ActRIIB (R64,20-134)-mFc (10 mg/kg) group. Mice were weighed and administered subcutaneously once weekly for 12 weeks. NMR scans were performed on days 0 and 83 of the study.

Throughout the study, sham-operated PBS mice gained an average of 9.72% ± 3.67% of their lean tissue mass on day 0, while sham-operated ActRIIB(R64,20-134)-mFc mice gained 35.79% ± 3.1% of their lean tissue mass on day 0. Castrated PBS-treated mice lost 8.1% ± 4.22% of their lean tissue mass on day 0, while treated castrated mice gained 17.77% ± 3.86%. Additionally, castration resulted in increased adiposity, but ActRIIB(R64,20-134)-mFc treatment helped to reduce the extent of fat mass gain.

The gastrocnemius and pectoral muscles of castrated vehicle-treated mice were smaller than those of sham-operated PBS mice (castrated gastrocnemius: 0.275 ± 0.03 g, castrated pectoral muscle: 0.196 ± 0.06 g; sham-operated gastrocnemius: 0.313 ± 0.02 g, sham-operated pectoral muscle: 0.254 ± 0.03 g). ActRIIB(R64,20-134)-mFc treatment significantly attenuated this castration-induced decrease in muscle weight (castrated gastrocnemius: 0.421 ± 0.03 g, castrated pectoral muscle: 0.296 ± 0.06 g).

Example 12: Effect of ActRIIB-Fc on cancer cachexia

Many tumors are associated with loss of appetite and severe muscle loss. Patients who exhibit cachexia have a worse prognosis than those who do not. The colon cancer cell line CT26 induces significant cachexia in mice. The effect of ActRIIB (R6420-134) on xenograft-induced cachexia was tested in this model.

The following 6 groups of mice were used in the experiment:

Groups 3-6 received 5×10 ⁶ tumor cells subcutaneously. Group 6 immediately began treatment with ActRIIB-Fc twice weekly. Groups 1-5 began dosing on study day 28, when tumors reached 300-500 mm ³ in size. As shown in Figure 11, ActRIIB-Fc significantly reduced CT26 tumor-associated muscle loss in mice with established tumors and when used prophylactically prior to tumor introduction.

Example 13: Effects of ActRIIB-Fc variants on muscle mass in wild-type mice

This study demonstrates the effects of the following ActRIIB-related Fc constructs on muscle mass and other tissues in 6-week-old C57BL/6 male mice. Mice were weighed and injected intraperitoneally twice weekly with PBS or ActRIIB-related Fc constructs (10 mg/kg):

ActRIIB(R64 20-134)-Fc

ActRIIB(L79D 20-134)-Fc

ActRIIB(L79E 20-134)-Fc

ActRIIB(A24N 20-134)-Fc

ActRIIB(R64K 20-134)-Fc

Mice were scanned by NMR at the beginning, middle, and end of the study. Femoral, pectoral, and gastrocnemius muscles, as well as the liver, kidneys, and spleen were weighed and preserved in formalin.

Initial data analysis indicated that ActRIIB(R6420-134)-Fc produced the greatest increases in muscle mass and lean body mass, while also having the greatest effects on other tissues. The L79D and L79E variants increased muscle mass to a lesser extent, while having little effect on other tissues. The A24N and R64K constructs had intermediate effects on muscle and other tissues. These data demonstrate that ActRIIB variants with reduced activin binding possess desirable properties, particularly selective effects on muscle tissue.

Integration of references

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

While specific embodiments of the subject invention have been discussed, the above description is illustrative and not restrictive. Numerous variations will become apparent to those skilled in the art upon reviewing this specification and the following embodiments. The full scope of the invention should be determined by reference to the embodiments, along with their full scope of equivalents, and the specification and such variations.

1. A variant ActRIIB protein comprising an amino acid sequence that is at least 80% identical to amino acids 29-109 of SEQ ID NO: 2, wherein the protein comprises at least one N-X-S/T sequence at a position other than the endogenous N-X-S/T sequence of ActRIIB and outside the ligand binding pocket.

2. The variant ActRIIB protein of embodiment 1, wherein the protein comprises an N at a position corresponding to position 24 of SEQ ID NO: 2 and an S or T at a position corresponding to position 26 of SEQ ID NO: 2, and wherein the variant ActRIIB protein inhibits signal transduction through activin, myostatin and/or GDF11 in a cell-based assay.

3. The variant ActRIIB protein of embodiment 2, wherein the protein comprises R or K at the position corresponding to position 64 of SEQ ID NO:2.

4. The variant ActRIIB protein of embodiment 2, wherein at least one change in the sequence of SEQ ID NO: 2 is a conservative change located in the ligand binding pocket.

5. The variant ActRIIB protein of embodiment 2, wherein at least one alteration of the sequence of SEQ ID NO: 2 is an alteration at one or more positions selected from the group consisting of K74, R40, Q53, K55, F82 and L79.

6. The variant ActRIIB protein of embodiment 2, wherein the protein comprises an amino acid sequence that begins at an amino acid corresponding to any one of amino acids 22-24 of SEQ ID NO:2 and ends at an amino acid corresponding to any one of amino acids 133 or 134 of SEQ ID NO:2.

7. The variant ActRIIB protein of any one of embodiments 1-6, wherein the protein is a fusion protein further containing a heterologous portion.

8. The variant ActRIIB fusion protein of embodiment 7, wherein the protein is a homodimer.

9. A variant of the ActRIIB fusion protein of embodiment 7, wherein the heterologous portion comprises the constant region of an IgG heavy chain.

10. A variant ActRIIB protein comprising an amino acid sequence that is at least 80% identical to amino acids 29-109 of SEQ ID NO:2, wherein the protein comprises an acidic amino acid at a position corresponding to position 79 of SEQ ID NO:2, and wherein the variant ActRIIB protein inhibits signal transduction through myostatin and/or GDF11 in a cell-based assay.

11. A variant of embodiment 10, wherein the protein comprises at least one N-X-S/T sequence at a position other than the endogenous N-X-S/T sequence of ActRIIB and at a position other than the ligand binding pocket.

12. The variant ActRIIB protein of embodiment 11, wherein the protein comprises an N at a position corresponding to position 24 of SEQ ID NO:2 and an S or T at a position corresponding to position 26 of SEQ ID NO:2.

13. The variant ActRIIB protein of embodiment 10, wherein the protein comprises an R or a K at a position corresponding to position 64 of SEQ ID NO:2.

14. The variant ActRIIB protein of embodiment 10, wherein at least one change in the sequence of SEQ ID NO:2 is a conservative change located in the ligand binding pocket.

15. The variant ActRIIB protein of embodiment 10, wherein at least one alteration in the sequence of SEQ ID NO: 2 is an alteration at one or more positions selected from the group consisting of K74, R40, Q53, K55, F82 and L79.

16. The variant ActRIIB protein of embodiment 10, wherein the protein comprises an amino acid sequence that begins at an amino acid corresponding to any one of amino acids 22-24 of SEQ ID NO:2 and ends at an amino acid corresponding to any one of amino acids 133 or 134 of SEQ ID NO:2.

17. The variant ActRIIB protein of any one of embodiments 10-16, wherein the protein is a fusion protein further containing a heterologous portion.

18. The variant ActRIIB fusion protein of embodiment 17, wherein the protein is a homodimer.

19. A variant of the ActRIIB fusion protein of embodiment 17, wherein the heterologous portion comprises the constant region of an IgG heavy chain.

20. A variant ActRIIB protein comprising an amino acid sequence that is at least 80% identical to amino acids 29-109 of SEQ ID NO: 2, wherein the protein comprises at least one N-X-S/T sequence at a position other than the endogenous N-X-S/T sequence of ActRIIB and at a position other than the ligand binding pocket.

21. The variant ActRIIB protein of embodiment 20, wherein the protein comprises an N at a position corresponding to position 24 of SEQ ID NO:2 and an S or T at a position corresponding to position 26 of SEQ ID NO:2, wherein the variant ActRIIB protein inhibits signal transduction through activin, myostatin and/or GDF11 in a cell-based assay.

22. The variant ActRIIB protein of embodiment 21, wherein the protein comprises an R or a K at a position corresponding to position 64 of SEQ ID NO:2.

23. The variant ActRIIB protein of embodiment 21, wherein at least one change in the sequence of SEQ ID NO:2 is a conservative change located in the ligand binding pocket.

24. The variant ActRIIB protein of embodiment 21, wherein at least one alteration of the sequence of SEQ ID NO: 2 is an alteration at one or more positions selected from the group consisting of K74, R40, Q53, K55, F82 and L79.

25. The variant ActRIIB protein of embodiment 21, wherein the protein comprises an amino acid sequence that begins at an amino acid corresponding to any one of amino acids 22-24 of SEQ ID NO:2 and ends at an amino acid corresponding to any one of amino acids 133 or 134 of SEQ ID NO:2.

26. The variant ActRIIB protein of any one of embodiments 20-25, wherein the protein is a fusion protein further containing a heterologous portion.

27. The variant ActRIIB fusion protein of embodiment 26, wherein the protein is a homodimer.

28. A variant of the ActRIIB fusion protein of embodiment 27, wherein the heterologous portion comprises the constant region of an IgG heavy chain.

29. An ActRIIB fusion protein comprising a portion of the ActRIIB sequence derived from SEQ ID NO:2 and a second partial polypeptide, wherein the portion derived from SEQ ID NO:2 corresponds to a sequence beginning at any one of amino acids 22-24 of SEQ ID NO:2 and ending at any one of amino acids 133 or 134 of SEQ ID NO:2, wherein the ActRIIB fusion protein additionally differs from the SEQ ID NO:2 sequence at no more than 5 amino acid positions, wherein the ActRIIB protein inhibits signal transduction through myostatin and/or GDF11 in a cell-based assay.

30. A variant of embodiment 29, wherein the protein comprises at least one N-X-S/T sequence at a position other than the endogenous N-X-S/T sequence of ActRIIB and at a position other than the ligand binding pocket.

31. The variant ActRIIB protein of embodiment 30, wherein the protein comprises an N at a position corresponding to position 24 of SEQ ID NO:2 and an S or T at a position corresponding to position 26 of SEQ ID NO:2.

32. The variant ActRIIB protein of embodiment 29, wherein the protein comprises an R or a K at a position corresponding to position 64 of SEQ ID NO:2.

33. The variant ActRIIB protein of embodiment 29, wherein at least one change in the sequence of SEQ ID NO:2 is a conservative change located in the ligand binding pocket.

34. The variant ActRIIB protein of embodiment 29, wherein at least one alteration in the sequence of SEQ ID NO: 2 is an alteration at one or more positions selected from the group consisting of K74, R40, Q53, K55, F82, and L79.

35. The variant ActRIIB protein of any one of embodiments 29-34, wherein the protein is a fusion protein further containing a heterologous portion.

36. The variant ActRIIB fusion protein of embodiment 35, wherein the protein is a homodimer.

37. A variant of the ActRIIB fusion protein of embodiment 35, wherein the heterologous portion comprises the constant region of an IgG heavy chain.

38. A variant ActRIIB fusion protein comprising a portion of the ActRIIB sequence derived from SEQ ID NO:2 and a second partial polypeptide, wherein the portion derived from SEQ ID NO:2 corresponds to a sequence starting at any one of amino acids 20-29 of SEQ ID NO:2 and ending at any one of amino acids 109-134 of SEQ ID NO:2, wherein the ActRIIB fusion protein has arginine (R), lysine (K) or histidine (H) at position 64 of SEQ ID NO:2, and inhibits signal transduction through activin, myostatin and/or GDF11 in a cell-based assay.

39. The variant ActRIIB fusion protein of embodiment 38, wherein the portion derived from SEQ ID NO:2 corresponds to a sequence starting at any one of amino acids 21-29 of SEQ ID NO:2 and ending at any one of amino acids 118-134 of SEQ ID NO:4.

40. The variant ActRIIB fusion protein of embodiment 38, wherein the portion derived from SEQ ID NO:2 corresponds to a sequence starting at any one of amino acids 21-29 of SEQ ID NO:2 and ending at any one of amino acids 128-134 of SEQ ID NO:2.

41. The variant ActRIIB fusion protein of embodiment 38, wherein the portion derived from SEQ ID NO:2 corresponds to a sequence starting at any one of amino acids 21-24 of SEQ ID NO:2 and ending at any one of amino acids 109-134 of SEQ ID NO:2.

42. The variant ActRIIB fusion protein of embodiment 38, wherein the portion derived from SEQ ID NO:2 corresponds to a sequence starting at any one of amino acids 21-24 of SEQ ID NO:2 and ending at any one of amino acids 118-134 of SEQ ID NO:2.

43. The variant ActRIIB fusion protein of embodiment 38, wherein the portion derived from SEQ ID NO:2 corresponds to a sequence starting at any one of amino acids 21-24 of SEQ ID NO:2 and ending at any one of amino acids 128-134 of SEQ ID NO:2.

44. The variant ActRIIB fusion protein of embodiment 38, further comprising one or more modifications relative to the sequence of SEQ ID NO: 2, wherein the modifications confer a property selected from the group consisting of: (a) greater inhibition of GDF11 or GDF8 signaling relative to inhibition of activin signaling; (b) increased serum half-life; and (c) both (a) and (b).

45. A variant ActRIIB fusion protein comprising a portion of the ActRIIB sequence derived from SEQ ID NO:2 and a second partial polypeptide, wherein the portion derived from SEQ ID NO:2 corresponds to a sequence starting at any one of amino acids 20-29 of SEQ ID NO:2 and ending at any one of amino acids 109-133 of SEQ ID NO:2, wherein the ActRIIB fusion protein has arginine (R), lysine (K) or histidine (H) at position 64 of SEQ ID NO:2, and inhibits signal transduction through activin, myostatin and/or GDF11 in a cell-based assay.

46. The variant ActRIIB fusion protein of embodiment 45, wherein the portion derived from SEQ ID NO:2 corresponds to a sequence starting at any one of amino acids 20-29 of SEQ ID NO:2 and ending at any one of amino acids 118-133 of SEQ ID NO:2.

47. The variant ActRIIB fusion protein of embodiment 45, wherein the portion derived from SEQ ID NO:2 corresponds to a sequence starting at any one of amino acids 20-29 of SEQ ID NO:2 and ending at any one of amino acids 128-133 of SEQ ID NO:2.

48. The variant ActRIIB fusion protein of embodiment 45, wherein the portion derived from SEQ ID NO:2 corresponds to a sequence starting at any one of amino acids 20-24 of SEQ ID NO:2 and ending at any one of amino acids 109-133 of SEQ ID NO:2.

49. The variant ActRIIB fusion protein of embodiment 45, wherein the portion derived from SEQ ID NO:2 corresponds to a sequence starting at any one of amino acids 20-24 of SEQ ID NO:2 and ending at any one of amino acids 118-133 of SEQ ID NO:2.

50. The variant ActRIIB fusion protein of embodiment 45, wherein the portion derived from SEQ ID NO:2 corresponds to a sequence starting at any one of amino acids 20-24 of SEQ ID NO:2 and ending at any one of amino acids 128-133 of SEQ ID NO:2.

51. The variant ActRIIB fusion protein of embodiment 45, further comprising one or more modifications relative to the sequence of SEQ ID NO: 2, wherein the modifications confer properties selected from the group consisting of: (a) greater inhibition of GDF11 or GDF8 signaling relative to inhibition of activin signaling; (b) increased serum half-life; and (c) both (a) and (b).

52. The variant ActRIIB fusion protein of any one of embodiments 38-51, wherein the protein is a homodimer.

53. A variant of the ActRIIB fusion protein of any one of embodiments 38-51, wherein the heterologous portion comprises the constant region of an IgG heavy chain.

54. The variant ActRIIB fusion protein of embodiment 53, wherein the heterologous portion is an Fc domain.

55. A variant ActRIIB protein comprising an amino acid sequence that is at least 80% identical, but not 100% identical, to amino acids 29-109 of SEQ ID NO:2, wherein the position corresponding to position 64 in SEQ ID NO:4 is K, wherein the variant ActRIIB protein inhibits signal transduction through activin, myostatin and/or GDF11 in a cell-based assay.

56. The variant ActRIIB protein of embodiment 55, wherein at least one change in the sequence of SEQ ID NO:2 is located outside the ligand binding pocket.

57. The variant ActRIIB protein of embodiment 55, wherein at least one change in the sequence of SEQ ID NO:2 is a conservative change located within the ligand binding pocket.

58. The variant ActRIIB protein of embodiment 55, wherein at least one alteration of the sequence of SEQ ID NO: 2 is an alteration at one or more positions selected from the group consisting of K74, R40, Q53, K55, F82 and L79.

59. The variant ActRIIB protein of embodiment 55, wherein the protein comprises at least one N-X-S/T sequence at a position other than the endogenous N-X-S/T sequence of SEQ ID NO:2 and at a position other than the ligand binding pocket.

60. The variant ActRIIB protein of embodiment 55, wherein the protein comprises one or more modifications relative to the sequence of SEQ ID NO:2, wherein the modifications confer a property selected from the group consisting of: (a) greater inhibition of GDF11 or GDF8 signaling relative to inhibition of activin signaling; (b) increased serum half-life; and (c) both (a) and (b).

61. A pharmaceutical formulation comprising the protein of any one of embodiments 1-60.

62. A method of treating a patient suffering from a disease associated with muscle loss or insufficient muscle growth, the method comprising administering to the patient an effective amount of the pharmaceutical formulation of embodiment 61.

63. The method of embodiment 62, wherein the patient suffers from muscle atrophy.

64. The method of embodiment 62, wherein the patient suffers from muscular dystrophy.

65. The method of embodiment 64, wherein the muscular dystrophy is Duchenne muscular dystrophy.

66. The method of embodiment 64, wherein the patient is an adolescent and treatment is initiated within 1-5 years after diagnosis of Duchenne muscular dystrophy.

67. The method of embodiment 62, wherein the muscular dystrophy is FSH muscular dystrophy.

68. The method of embodiment 62, wherein the patient has ALS.

69. The method of embodiment 68, wherein the patient receives treatment after being diagnosed with ALS.

70. The method of embodiment 62, wherein the disease is cachexia associated with cancer or cancer treatment.

71. The method of embodiment 70, wherein the disease is muscle loss associated with prostate cancer treatment.

72. The method of embodiment 70, wherein the disease is tumor-associated muscle loss.

73. The method of embodiment 72, wherein the tumor is a colon cancer tumor.

74. A method of treating a patient suffering from a disease associated with neurodegeneration, the method comprising administering to the patient an effective amount of the pharmaceutical formulation of embodiment 61.

75. The method of embodiment 74, wherein the disease is amyotrophic lateral sclerosis.

76. A method of reducing body fat content or reducing the rate of increase of body fat content in a patient, the method comprising administering to the patient an effective amount of the pharmaceutical formulation of embodiment 61.

77. A method of treating a condition associated with unwanted weight gain in a patient, the method comprising administering to the patient an effective amount of the pharmaceutical formulation of embodiment 61.

78. The method of embodiment 77, wherein the disease is selected from obesity, severe or morbid obesity, non-insulin dependent diabetes mellitus (NIDDM), cardiovascular disease, cancer, hypertension, osteoarthritis, stroke, respiratory disease and gallbladder disease.

79. A method of lowering cholesterol and/or triglycerides, said method comprising administering to a patient an effective amount of the pharmaceutical formulation of embodiment 61.

80. A method of treating sarcopenia, comprising administering to a patient an effective amount of the pharmaceutical formulation of embodiment 61.

81. An ActRIIB-Fc protein comprising the amino acid sequence of SEQ ID NO: 5.

82. The ActRIIB-Fc protein of embodiment 81, wherein the protein has an N at the position corresponding to position 5 of SEQ ID NO:5.

83. The ActRIIB-Fc protein of embodiment 81, wherein the protein has a D or E at the position corresponding to position 60 of SEQ ID NO:5.

84. The ActRIIB-Fc protein of embodiment 82, wherein the protein has a D or E at the position corresponding to position 60 of SEQ ID NO:5.

85. The ActRIIB-Fc protein of embodiment 81, wherein the protein is a dimer.

86. The ActRIIB-Fc protein of embodiment 82, wherein the protein is a dimer.

87. The ActRIIB-Fc protein of embodiment 83, wherein the protein is a dimer.

88. The ActRIIB-Fc protein of embodiment 84, wherein the protein is a dimer.

89. A pharmaceutical preparation comprising the protein of any one of embodiments 81-88.

90. A method of treating a patient suffering from a disease associated with muscle loss or insufficient muscle growth, said method comprising administering to said patient an effective amount of the pharmaceutical formulation of embodiment 89.

91. The method of embodiment 90, wherein the patient suffers from muscle atrophy.

92. The method of embodiment 90, wherein the patient suffers from muscular dystrophy.

93. The method of embodiment 92, wherein the muscular dystrophy is Duchenne muscular dystrophy.

94. The method of embodiment 93, wherein the patient is an adolescent and treatment is initiated within 1-5 years after diagnosis of Duchenne muscular dystrophy.

95. The method of embodiment 92, wherein the muscular dystrophy is FSH muscular dystrophy.

96. The method of embodiment 90, wherein the patient has ALS.

97. The method of embodiment 96, wherein the patient receives treatment after being diagnosed with ALS.

98. The method of embodiment 90, wherein the disease is cachexia associated with cancer or cancer treatment.

99. The method of embodiment 98, wherein the disease is muscle loss associated with prostate cancer treatment.

100. The method of embodiment 98, wherein the disease is tumor-associated muscle loss.

101. The method of embodiment 100, wherein the tumor is a colon cancer tumor.

102. A method of treating a patient suffering from a disease associated with neurodegeneration, the method comprising administering to the patient an effective amount of the pharmaceutical formulation of embodiment 89.

103. The method of embodiment 102, wherein the disease is amyotrophic lateral sclerosis.

104. A method of reducing body fat content or reducing the rate of increase of body fat content in a patient, the method comprising administering to the patient an effective amount of the pharmaceutical formulation of embodiment 89.

105. A method of treating a condition associated with unwanted weight gain in a patient, the method comprising administering to the patient an effective amount of the pharmaceutical formulation of embodiment 104.

106. The method of embodiment 105, wherein the disease is selected from obesity, severe or morbid obesity, non-insulin dependent diabetes mellitus (NIDDM), cardiovascular disease, cancer, hypertension, osteoarthritis, stroke, respiratory disease, and gallbladder disease.

107. A method of lowering cholesterol and/or triglycerides, said method comprising administering to a patient an effective amount of the pharmaceutical formulation of embodiment 89.

108. A method of treating sarcopenia, comprising administering to a patient an effective amount of the pharmaceutical formulation of embodiment 107.

109. Use of the pharmaceutical formulation of embodiment 61 or 89 for the preparation of a medicament for treating a disease associated with muscle loss or insufficient muscle growth.

110. The pharmaceutical formulation of embodiment 61 or 89 for use in treating a disease associated with muscle loss or insufficient muscle growth.

111. Use of the pharmaceutical formulation of embodiment 61 or 89 for the preparation of a medicament for treating a disease associated with neurodegeneration.

112. The pharmaceutical formulation of embodiment 61 or 89 for use in treating a disease associated with neurodegeneration.

113. Use of the pharmaceutical formulation of embodiment 61 or 89 in the preparation of a medicament for reducing body fat content in a patient or reducing the rate of increase in body fat content in a patient.

114. The pharmaceutical formulation of embodiment 61 or 89 for reducing body fat content or reducing the rate of increase of body fat content in a patient.

115. Use of the pharmaceutical formulation of embodiment 61 or 89 for the preparation of a medicament for treating a disease associated with unwanted weight gain in a patient.

116. The pharmaceutical formulation of embodiment 61 or 89 for use in treating a disease associated with unwanted weight gain in a patient.

117. Use of the pharmaceutical formulation of embodiment 61 or 89 for the preparation of a medicament for lowering cholesterol and/or triglycerides.

118. The pharmaceutical formulation of embodiment 61 or 89 for lowering cholesterol and/or triglycerides.

119. Use of the pharmaceutical formulation of embodiment 61 or 89 in the preparation of a medicament for treating sarcopenia.

120. The pharmaceutical formulation of embodiment 61 or 89 for use in treating sarcopenia.

Claims (30)

1. A variant ActRIIB protein, which consists of the amino acid sequence of SEQ ID NO:5. 2. The ActRIIB protein variant of claim 1, wherein the protein is a homodimer. 3. A pharmaceutical formulation comprising the variant ActRIIB protein as described in claim 1 or 2. 4. An isolated polynucleotide encoding the variant ActRIIB protein of claim 1. 5. The isolated polynucleotide of claim 4, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:10. 6. A vector comprising the polynucleotide of claim 4 or 5. 7. A cell comprising the polynucleotide of claim 4 or 5 or the vector of claim 6, wherein the cell is not in a human body. 8. A method for preparing a variant ActRIIB protein, the method comprising culturing the cells of claim 7 and expressing the variant ActRIIB protein. 9. The method of claim 8, wherein the cell is a CHO cell. 10. The method of claim 8 or 9, wherein the variant ActRIIB protein is expressed with a TPA leader sequence. 11. The method of claim 10, wherein the TPA leader sequence comprises the amino acid sequence of SEQ ID NO:8. 12. An ActRIIB-Fc fusion protein, wherein the ActRIIB domain of the fusion protein is composed of amino acids 25-131 of SEQ ID NO:2. 13. The ActRIIB-Fc fusion protein of claim 12, wherein the fusion protein binds to GDF11. 14. The ActRIIB-Fc fusion protein of claim 12 or 13, wherein the fusion protein binds to activin. 15. The ActRIIB-Fc fusion protein of claim 14, wherein the fusion protein binds activin A. 16. The ActRIIB-Fc fusion protein of claim 14, wherein the fusion protein binds activin B. 17. The ActRIIB-Fc fusion protein of any one of claims 12-13, wherein the fusion protein inhibits GDF11 signal transduction in cell morphology assays. 18. The ActRIIB-Fc fusion protein of any one of claims 12-13, wherein the fusion protein inhibits activin signal transduction in cell type assays. 19. The ActRIIB-Fc fusion protein of claim 18, wherein the fusion protein inhibits activin A signal transduction in cell type assays. 20. The ActRIIB-Fc fusion protein of claim 18, wherein the fusion protein inhibits activin B signaling in cell type assays. 21. The ActRIIB-Fc fusion protein of any one of claims 12-13, wherein the fusion protein comprises a constant region derived from the IgG heavy chain. 22. The ActRIIB-Fc fusion protein of any one of claims 12-13, wherein the fusion protein comprises a linker domain located between the ActRIIB domain and the Fc domain. 23. A pharmaceutical formulation comprising the ActRIIB-Fc fusion protein according to any one of claims 12-22. 24. An isolated polynucleotide encoding the ActRIIB-Fc fusion protein of claim 12. 25. A vector comprising the polynucleotide of claim 24. 26. A cell comprising the polynucleotide of claim 24 or the carrier of claim 25, wherein the cell is not in a human body. 27. A method for preparing ActRIIB-Fc fusion protein, the method comprising culturing the cells of claim 26 and expressing the ActRIIB-Fc fusion protein. 28. The method of claim 27, wherein the cell is a CHO cell. 29. The method of claim 27 or 28, wherein the ActRIIB-Fc fusion protein is expressed using a TPA leader sequence. 30. The method of claim 29, wherein the TPA leader sequence comprises the amino acid sequence of SEQ ID NO:8.