HK1226958A1

HK1226958A1 - Activin-actrii antagonists and uses for increasing red blood cell levels

Info

Publication number: HK1226958A1
Application number: HK17100601.2A
Authority: HK
Inventors: L. Sherman Matthew
Original assignee: Acceleron Pharma Inc.
Priority date: 2006-12-18
Filing date: 2017-01-18
Publication date: 2017-10-13

Description

activin-ACTRII antagonists and uses in increasing red blood cell levels

The application is a divisional application, the application date of the original application is 12-18 th 2007, the application number is 200780051416.4(PCT/US2007/025868), and the invention name is 'activin-ACTRII antagonist and the application in increasing the level of red blood cells'.

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patent application 60/875,682 filed 2006, 12, 18, which is hereby incorporated by reference in its entirety.

Background

Mature red blood cells (or erythrocytes) are responsible for the transport of oxygen in the vertebrate circulatory system. The red blood cells carry high concentration of hemoglobin, a proteinAt a relatively high oxygen partial pressure (pO)₂) Binds oxygen in the lung and delivers oxygen to the lung with a relatively low pO₂Of the body part of (a).

Mature red blood cells can be produced from pluripotent hematopoietic stem cells in a process known as erythropoiesis. In postnatal individuals, erythropoiesis occurs primarily in the bone marrow and the red marrow of the spleen. The synergistic effects of various signal transduction pathways control the balance of cell proliferation, differentiation, survival and death. Under normal conditions, red blood cells are produced at a rate that maintains a constant amount of red blood cells in the body, and their production can be increased or decreased in response to various stimuli, including increased or decreased oxygen tension or tissue demand. The process of erythropoiesis begins with lineage-directed precursor cells and proceeds through a series of different precursor cell types. The final stage of reticulocyte erythropoiesis occurs as reticulocytes are released into the blood stream and lose their mitochondria and ribosomes, while assuming the morphology of mature erythrocytes. Rise in reticulocyte level in blood or reticulocyte: an increase in the proportion of erythrocytes indicates an increase in the rate of erythropoiesis.

Erythropoietin (Epo) is widely recognized as the most important positive regulator of erythropoiesis in post-natal vertebrates. Epo can regulate compensatory erythropoiesis in response to decreased tissue oxygen tension (hypoxia) and low red blood cell levels or low hemoglobin levels. In humans, elevated Epo levels can promote erythropoiesis by stimulating erythroid progenitor cell production in the bone marrow and spleen. In mice, Epo predominantly increases erythropoiesis in the spleen.

Physicians have employed various forms of recombinant Epo to increase red blood cell levels in various clinical settings, particularly for the treatment of anemia. Anemia is a well-defined condition characterized by lower than normal levels of hemoglobin or red blood cells in the blood. In some cases, anemia is caused by a primary disease of erythropoiesis or survival. More often, anemia is secondary to other systemic diseases (Weatherall & Provan (2000) Lancet355, 1169-1175). Anemia can be caused by a decrease in the rate of erythropoiesis or an increase in the rate of destruction, or by the loss of red blood cells due to bleeding. Anemia can be caused by a variety of diseases including, for example, chronic renal failure, myelodysplastic syndrome, rheumatoid arthritis, and bone marrow transplantation.

Treatment with Epo typically results in an approximately 1-3g/dL hemoglobin rise in healthy humans over a period of more than one week. When administered to anemic individuals, this treatment regimen generally provides a substantial increase in hemoglobin and red blood cell levels, and results in improved quality of life and prolonged survival. Epo is not uniformly effective and many individuals are not as effective even at high doses (Horl et al (2000) NephrollDial Transplant15, 43-50). More than 50% of cancer patients respond poorly to Epo, approximately 10% of end stage renal disease patients have low responsiveness (Glaspy et al (1997) JClinOncol15, 1218-. Many factors, including inflammation, iron and vitamin deficiency, inadequate dialysis, aluminum toxicity, and hyperparathyroidism can lead to poor therapeutic response, and the molecular mechanisms of tolerance to Epo remain unclear.

Accordingly, it is an object of the present disclosure to provide alternative compositions and methods for increasing red blood cell levels in a patient.

Summary of the invention

In part, the disclosure shows that activin antagonists as well as ActRIIa and ActRIIb antagonists can be used to increase red blood cell and hemoglobin levels. In particular, the disclosure shows that soluble forms of ActRIIa act as inhibitors of activin and increase red blood cell levels in the blood when administered in vivo. A milder effect was observed for soluble forms of ActRIIb, which binds to activin a with lower affinity than soluble ActRIIa. Although soluble ActRIIa and ActRIIb may affect red blood cell levels by mechanisms other than activin antagonism, the present disclosure shows that a desired therapeutic agent may be selected based on either activin antagonism or ActRII antagonism or both. This agent is collectively referred to as an activin-ActRII antagonist. Thus, in certain embodiments, the present disclosure provides methods of increasing the level of red blood cells and hemoglobin in a patient in need thereof and treating diseases associated with low red blood cells or hemoglobin levels using activin-ActRII antagonists including, for example, activin-binding ActRIIa polypeptides, activin-binding ActRIIb polypeptides, anti-activin antibodies, anti-ActRIIa antibodies, anti-ActRIIb antibodies, small molecules and aptamers targeting activin, ActRIIb, or ActRIIa and nucleic acids that decrease expression of activin, ActRIIb, or ActRIIa. activin-ActRIIa antagonists are useful for promoting bone growth and increasing bone density as described in U.S. patent application 11/603,485 (incorporated herein by reference). As described herein, the effect of such antagonists on the level of erythrocytes is more rapid and occurs at lower doses than the effect of such antagonists on bone. Thus, in certain embodiments, the present disclosure provides methods of increasing red blood cell or hemoglobin levels using activin-ActRIIa antagonists without causing a significant increase in bone density. For example, one approach may result in less than a 3%, 5%, 10%, or 15% increase in bone density. This selective effect can be achieved by using, for example, lower doses of activin-ActRIIa antagonist, lower dosing frequency, or by using an activin-ActRIIa antagonist with a shorter serum half-life at a dose and frequency calculated to provide lower serum concentrations.

In certain aspects, the disclosure provides polypeptides comprising a soluble, activin-binding ActRII polypeptide that binds to activin. The activin-binding polypeptide can be an ActRIIa polypeptide or an ActRIIb polypeptide. ActRII polypeptides may be formulated as a pharmaceutical preparation that includes the activin-binding ActRII polypeptide and a pharmaceutically acceptable carrier. The activin-binding ActRII polypeptide can be less than 1 micromolar or less than 100, 10, or 1 nanomolar K_DBinding to activin. Optionally, the activin-binding ActRII polypeptide selectively binds activin over GDF11 and/or GDF8, and optionally at least 10-fold, 20-fold less than GDF11 and/or GDF8Or 50 times K_DBinding activin. While not wishing to be bound by a particular mechanism of action, it is expected that the degree of selectivity of activin inhibition over GDF11/GDF8 inhibition explains the effect on bone or erythropoiesis, and is not consistently measurable on muscle. In many embodiments, an ActRII polypeptide will be selected to cause less than a 15%, less than a 10%, or less than a 5% increase in muscle at a dose that achieves a desired effect on red blood cell levels. The composition may be at least 95% pure, optionally at least 98% pure, relative to the other polypeptide components as estimated by size exclusion chromatography. The activin-binding ActRIIa polypeptide used in this preparation can be any such polypeptide described herein, e.g., a polypeptide having an amino acid sequence selected from seq id No. 2, 3, 7, or 12, or an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, or 99% identical to an amino acid sequence selected from seq id No. 2, 3, 7, 12, or 13. An activin-binding ActRIIa polypeptide can comprise a functional fragment of a native ActRIIa polypeptide, e.g., a functional fragment having at least 10, 20, or 30 amino acids selected from the group consisting of sequences seq id nos 1-3 or seq id No. 2, with the C-terminal 10 to 15 amino acids ("tail") deleted. The activin-binding ActRIIb polypeptide used in this preparation can be any such polypeptide described herein, e.g., a polypeptide having an amino acid sequence selected from seq id No. 16, 17, 20, or 21, or an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, or 99% identical to an amino acid sequence selected from seq id No. 16, 17, 20, or 21. An activin-binding ActRIIb polypeptide can include a functional fragment of a native ActRIIb polypeptide, e.g., a functional fragment having a sequence selected from at least 10, 20, or 30 amino acids or a deletion of the C-terminal 10 to 15 amino acids ("tails") of seq id No. 15-17, e.g., seq id No. 17.

A soluble, activin-binding ActRII polypeptide can include one or more substitutions in the amino acid sequence (e.g., in the ligand-binding domain) relative to a naturally-occurring ActRII polypeptide. Examples of altered ActRIIa and ActRIIb polypeptides are provided, for example, at pages 59-60 and pages 55-58 (incorporated herein by reference) in WO2006/012627, respectively. Changes in the amino acid sequence can, for example, alter glycosylation of the polypeptide as it is produced in a mammalian, insect, or other eukaryotic cell, or alter proteolytic cleavage of the polypeptide relative to a naturally occurring ActRII polypeptide.

An activin-binding ActRII polypeptide may be a fusion protein having an ActRII polypeptide as one domain (e.g., a ligand-binding portion of ActRIIa or ActRIIb) and one or more other domains that provide desired properties (e.g., improved pharmacokinetics, simpler purification, targeting to a particular tissue, etc.). For example, a domain of a fusion protein may enhance one or more of in vivo stability, in vivo half-life, uptake/administration, tissue localization or distribution, formation of a protein complex, multimerization and/or purification of the fusion protein. An activin-binding ActRII fusion protein may include an immunoglobulin Fc domain (wild-type or mutated) or serum albumin or other polypeptide portion that provides a desired property (e.g., improved pharmacokinetics, increased solubility, or increased stability). In preferred embodiments, the ActRII-Fc fusion comprises a relatively unstructured linker between the Fc domain and the extracellular ActRII domain. The unstructured linker may correspond to an unstructured region of about 15 amino acids ("tail") at the C-terminus of the extracellular domain of ActRII, or it may be an artificial sequence of 1, 2, 3,4, or 5 amino acids or an artificial sequence of 5 and 15, 20, 30, 50, or more amino acids in length, or a mixture of both, that is relatively free of secondary structure. The linker may be rich in glycine or proline residues and may, for example, comprise a single sequence of threonine/serine and glycine or a repeating sequence of threonine/serine and glycine (e.g., TG)₄(SEQ ID NO:22) or SG₄(SEQ ID NO:23) singlet or repeat). The fusion protein may comprise a purificant sequence, such as an epitope tag, a FLAG tag, a polyhistidine sequence, and a GST fusion. Alternatively, the soluble ActRII polypeptides comprise 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 with a lipid moiety and methods of making and using the sameAn organic derivatizing agent conjugated amino acid. The pharmaceutical formulations may also contain one or more additional compounds, such as compounds useful in the treatment of bone disorders. Preferably, the pharmaceutical composition is substantially pyrogen free. In general, it is preferred to express an ActRII protein in a mammalian cell line that suitably mediates native glycosylation of the ActRII protein, thereby reducing the likelihood of an adverse immune response in the patient. Human and CHO cell lines have been used successfully, and other common mammalian expression systems are also contemplated.

The ActRIIa proteins described herein are particularly directed to ActRIIa-Fc (a form with minimal linker between the ActRIIa portion and the Fc portion) having desirable properties, including selective binding to activin, high affinity ligand binding, and a serum half-life greater than two weeks relative to GDF8/GDF11 in an animal model. In certain embodiments, the present invention provides ActRII-Fc polypeptides and pharmaceutical formulations comprising the polypeptides and a pharmaceutically acceptable excipient.

In certain aspects, the disclosure provides nucleic acids encoding soluble activin-binding ActRII polypeptides (e.g., ActRIIa or ActRIIb polypeptides). An isolated polynucleotide may comprise a coding sequence for a soluble, activin-binding ActRII polypeptide, such as described above. For example, an isolated nucleic acid may include a sequence encoding an extracellular domain (e.g., ligand-binding domain) of an ActRII and a sequence encoding part or all of a transmembrane domain and/or a cytoplasmic domain of the ActRII except for a stop codon located in the transmembrane domain or the cytoplasmic domain, or between the extracellular domain and the transmembrane domain or the cytoplasmic domain. For example, an isolated polynucleotide may comprise a full-length ActRIIa polynucleotide sequence (e.g., seq id no:4 or 5) or a full-length ActRIIb polynucleotide sequence (e.g., seq id no:18), or a partially truncated form of ActRIIa or ActRIIb, the isolated polynucleotide further comprising a transcriptional termination codon at least six hundred nucleotides before the 3' -terminus or elsewhere, such that translation of the polynucleotide forms an extracellular domain that is optionally fused to a truncated portion of the full-length actriii. A preferred nucleic acid sequence for ActRIIa is SEQ ID NO. 14. The nucleic acids disclosed herein are operably linked to an expression promoter, and the disclosure provides cells transformed with such recombinant polynucleotides. The cell is preferably a mammalian cell, such as a CHO cell.

In certain aspects, the disclosure provides methods of making soluble, activin-binding ActRII polypeptides. The methods can include expressing any of the nucleic acids disclosed herein (e.g., SEQ ID NOs:4, 5, 14, 18, or 19) in a suitable cell, such as a Chinese Hamster Ovary (CHO) cell. Such a method may include: a) culturing a cell under conditions suitable for expression of the soluble ActRII polypeptide, wherein the cell is transformed with a soluble ActRII expression construct; and b) recovering the soluble ActRII polypeptide so expressed. Soluble ActRII polypeptides may be recovered as a crude, partially purified, or highly purified fraction. Purification may be achieved by a series of purification steps including, for example, one, two or three or more of the following steps in any order: protein a chromatography, anion exchange chromatography (e.g., Q sepharose), hydrophobic interaction chromatography (e.g., phenyl sepharose), size exclusion chromatography, and cation exchange chromatography.

In certain aspects, an activin-ActRII antagonist disclosed herein, e.g., a soluble, activin-binding ActRIIa polypeptide or a soluble, activin-binding ActRIIb polypeptide, is useful in a method of promoting erythropoiesis or increasing red blood cell levels in a subject. In certain embodiments, the present disclosure provides methods of treating a disease associated with low red blood cell count or low hemoglobin levels (e.g., anemia) in a patient in need thereof. The method may comprise administering to a subject in need thereof an effective amount of an activin-ActRII antagonist. In certain aspects, the disclosure provides for the use of an activin-ActRII antagonist in the manufacture of a medicament for treating a disease or disorder disclosed herein.

In certain aspects, the disclosure provides methods of identifying agents that stimulate erythropoiesis. The method comprises the following steps: a) identifying a test agent that binds to an activin or ligand-binding domain of an ActRII polypeptide; and b) assessing the effect of the agent on the level of red blood cells, hemoglobin, and/or red blood cell precursors (e.g., reticulocyte level).

Drawings

FIG. 1 shows purification of ActRIIa-hFc expressed in CHO cells. The protein was purified as separate, well-defined peaks shown by exclusion columns (upper panel) and as Coomassie stained SDS-PAGE (lower panel) (left lane: molecular weight standards; right lane: ActRIIa-hFc).

FIG. 2 shows the passage of BiaCore^TMAssayed binding of ActRIIa-hFc to activin and GDF-11.

FIG. 3 shows the effect of ActRIIa-hFc on red blood cell number in female non-human primates. Female cynomolgus monkeys (four groups of five monkeys) were treated with placebo or 1mg/kg, 10mg/kg, or 30mg/kg of ActRIIa-hFc at days 0, 7, 14, and 21. Fig. 3A shows Red Blood Cell (RBC) number. Fig. 3B shows hemoglobin levels. Statistical significance is relative to baseline for each treatment group. Two monkeys were kept in each group on day 57.

FIG. 4 shows the effect of ActRIIa-hFc on red blood cell number in male non-human primates. Male cynomolgus monkeys (four groups of five monkeys) were treated with placebo or 1mg/kg, 10mg/kg, or 30mg/kg of ActRIIa-hFc on days 0, 7, 14, and 21. Fig. 4A shows Red Blood Cell (RBC) number. Fig. 4B shows hemoglobin levels. Statistical significance is relative to baseline for each treatment group. Two monkeys were kept in each group on day 57.

FIG. 5 shows the effect of ActRIIa-hFc on reticulocyte number in female non-human primates. Cynomolgus monkeys (four groups of five monkeys) were treated with placebo or 1mg/kg, 10mg/kg, or 30mg/kg of ActRIIa-hFc on days 0, 7, 14, and 21. Fig. 5A shows the absolute reticulocyte count. Fig. 5B shows the percentage of reticulocytes relative to RBCs. Statistical significance is relative to the baseline of each group. Two monkeys were kept in each group on day 57.

FIG. 6 shows the effect of ActRIIa-hFc on reticulocyte number in male non-human primates. Cynomolgus monkeys (four groups of five monkeys) were treated with placebo or 1mg/kg, 10mg/kg, or 30mg/kg of ActRIIa-hFc on days 0, 7, 14, and 21. Fig. 6A shows the absolute reticulocyte count. Fig. 6B shows the percentage of reticulocytes relative to RBCs. Statistical significance is relative to the baseline of each group. Two monkeys were kept in each group on day 57.

FIG. 7 shows the results of the human clinical trial in example 5, in which the area under the curve (AUC) and the dose of ActRIIa-hFc administered have a linear relationship, regardless of whether the ActRIIa-hFc was administered Intravenously (IV) or Subcutaneously (SC).

FIG. 8 shows a comparison of ActRIIa-hFc serum levels in patients with IV or SC administration.

FIG. 9 shows Bone Alkaline Phosphatase (BAP) levels in response to various dose levels of ActRIIa-hFc. BAP is a marker of anabolic bone growth.

FIG. 10 shows the median change from baseline in hematocrit levels from the human clinical trial described in example 5. ActRIIa-hFc was administered Intravenously (IV) at the doses indicated.

Figure 11 shows the median change from baseline in hemoglobin levels from the human clinical trial described in example 5. ActRIIa-hFc was administered Intravenously (IV) at the doses indicated.

Figure 12 shows the median change from baseline in RBC (red blood cell) numbers from the human clinical trial described in example 5. ActRIIa-hFc was administered Intravenously (IV) at the doses indicated.

FIG. 13 shows the median change from baseline in reticulocyte number in the human clinical trial described in example 5. ActRIIa-hFc was administered Intravenously (IV) at the doses indicated.

Detailed Description

1.Overview

The transforming growth factor-beta (TGF- β) superfamily comprises a variety of growth factors having common sequence elements and structural motifs. These proteins are known to exert biological effects on a wide variety of cell types in both vertebrates and invertebrates. Members of this superfamily have important functions in patterning and tissue specificity during embryonic development and can influence a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis, and epithelial cell differentiation. The family is divided into two major branches: the BMP/GDF and TGF- β/activin/BMP 10 branches, the members of which have different, usually complementary, roles. By modulating the activity of TGF-. beta.family members, it is often possible to cause significant physiological changes in an organism. For example, pimont and belgium blue cattle breed harbor loss-of-function mutations in the GDF8 (also known as myostatin) gene, which results in a significant increase in muscle mass. Grobet et al, NatGenet.1997, 17(1): 71-4. Furthermore, in humans, inactivated GDF8 alleles are associated with increased muscle mass and are reported to be associated with abnormal strength. Schuelke et al, NEnglJMed2004, 350: 2682-8.

Activins are dimeric polypeptide growth factors that belong to the TGF- β superfamily there are three major forms of activins (A, B and AB) that are homo/heterodimers of two closely related β subunits (β, respectively)_Aβ_A，β_Bβ_BAnd β_Aβ_B)。

The human genome also encodes activin C and activin E, which are expressed predominantly in the liver, and are also known to include heterodimeric forms of β C or β E. Within the TGF-. beta.superfamily, activins are unique and multifunctional factors that stimulate hormone production in ovarian and placental cells, support neuronal cell survival, positively or negatively influence cell cycle progression depending on cell type, and induce mesodermal differentiation at least in amphibian embryos (DePaolo et al, 1991, ProcSocEpbiol Med.198: 500-. Furthermore, it has been found that the Erythrocyte Differentiation Factor (EDF) isolated from stimulated human monocytic leukemia cells is identical to activin A (Murata et al, 1988, PNAS, 85: 2434). Activin a has been shown to promote erythropoiesis in the bone marrow. In various tissues, activin signaling is antagonized by its associated heterodimeric inhibin. For example, during the release of Follicle Stimulating Hormone (FSH) from the pituitary, activin promotes FSH secretion and synthesis, while inhibin prevents FSH secretion and synthesis. Other proteins that modulate the biological activity of activin and/or bind activin include Follistatin (FS), follistatin-related protein (FSRP), and alpha 2-macroglobulin.

TGF-. beta.signaling is mediated by heteropeptide complexes of serine/threonine kinase receptors type I and II, which phosphorylate and activate downstream Smad proteins when stimulated by ligands (Massague, 2000, Nat. Rev. mol. CellBiol.1: 169-178). These type I and type II receptors are transmembrane proteins, consisting of a ligand-binding extracellular domain of a cysteine-rich region, a transmembrane domain, a cytoplasmic domain with a predetermined serine/threonine specificity. Type I receptors are required for signaling; type II receptors are required in the binding of ligands and expression of type I receptors. The type I and type II activin receptors form a stable complex upon ligand binding, resulting in phosphorylation of the type I receptor by the type II receptor.

Two related type II receptors (ActRII), ActRIIa and ActRIIb, have been identified as type II receptors for activins (Mathews and Vale, 1991, Cell65: 973-982; Attisano et al, 1992, Cell68: 97-108). In addition to activins, ActRIIa and ActRIIb are able to biochemically interact with a variety of other TGF- β family proteins, including BMP7, Nodal, GDF8, and GDF11 (Yamashita et al, 1995, J.CellBiol.130: 217-226; Lee and McPherron, 2001, Proc.Natl.Acad.Sci.98: 9306-9311; Yeo and Whitman, 2001, mol.Cell7: 949-957; Oh et al, 2002, GenesDev.16: 2749-54). ALK4 is the primary type I receptor for activins, particularly activin A, while ALK-7 also acts as a receptor for activins, particularly activin B.

As demonstrated herein, soluble ActRIIa polypeptides (sActRIIa) that bind significantly preferentially to activin a over other TGF- β family members (e.g., GDF8 or GDF11) are effective in increasing red blood cell levels in vivo. Without wishing to be bound by any particular mechanism, however, given the very strong activin binding (picomolar dissociation constant) exhibited by the particular sActRIIa constructs used in these studies, the effect of sActRIIa is expected to be caused primarily by activin antagonist effects. Regardless of mechanism, it is apparent from this disclosure that: ActRIIa-activin antagonists increased red blood cell levels in rodents, monkeys, and humans. It should be noted that: hematopoiesis is a complex process that is regulated by a number of factors, including the homeostasis of erythropoietin, G-CSF and iron. The terms "increasing red blood cell levels" and "promoting red blood cell formation" refer to clinically observed measures such as hematocrit, red blood cell count, and hemoglobin measurement, which are intended to be neutral to the mechanisms by which such changes occur.

Also as shown herein, soluble ActRIIb polypeptide (sActRIIb) effectively increases in vivo levels of erythrocytes, an effect that is expected to cause increased hematocrit levels over a longer period of time.

The data reported herein for non-human primates can be repeated in mice, rats, and humans, and thus, the present disclosure provides methods for promoting erythropoiesis and increasing red blood cell levels in mammals from rodents to humans using ActRII polypeptides and other activin-ActRII antagonists. activin-ActRII antagonists include, for example, an activin-binding soluble ActRIIa polypeptide, an activin-binding soluble ActRIIb polypeptide, an antibody that binds to activin (particularly activin a and B subunits, also known as β a and β B) and disrupts ActRIIa and/or ActRIIb binding, an antibody that binds to ActRIIa and disrupts activin binding, an antibody that binds to ActRIIb and disrupts activin binding, a non-antibody protein selected for activin, ActRIIb, or ActRIIa binding (see, e.g., WO/2002/088171, WO/2006/055689, and WO/2002/032925 for this protein and methods of its design and selection), a randomized peptide selected for activin, ActRIIb, or ActRIIb binding, typically attached to an Fc domain. Two different proteins (or other moieties) with activin, ActRIIb, or ActRIIa binding activity, particularly activin binding agents that block type I (e.g., soluble type I activin receptor) and type II (e.g., soluble type II activin receptor) binding sites, respectively, can be linked together to create a bifunctional binding molecule. Nucleic acid aptamers, small molecules, and other agents that inhibit the activin-ActRII signaling axis are also included in activin-ActRII antagonists. Various proteins have activin-ActRII antagonist activity, including inhibin (i.e., inhibin alpha subunit) (although inhibin does not universally antagonize activin in all tissues), follistatin (e.g., follistatin-288 and follistatin-315), FSRP, activin C, alpha (2) -macroglobulin, and M108A (mutated from methionine to alanine at position 108) mutated activin a. In general, alternative forms of activin, particularly in the type I receptor binding domain, are capable of binding to type II receptors and are incapable of forming active ternary complexes, and thus act as antagonists. In addition, nucleic acids, such as antisense molecules, sirnas or ribozymes, capable of inhibiting activin A, B, C or E, or specifically, ActRIIa or ActRIIb expression, can be used as activin-ActRII antagonists. The activin-ActRII antagonists to be used may exhibit inhibitory selectivity for activin-mediated signaling over other TGF- β family members, particularly over GDF8 and GDF 11.

The terms used in this specification generally have their ordinary meaning in the art, both in the context of the present invention and in the specific context in which each term is used. Certain terms are discussed below, as well as in other portions of the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the invention and how to make and use them. Any range of use and meaning of a term will be apparent from the specific context in which that term is used.

"about" and "approximately" generally refer to the degree of acceptable error in measuring a value given the nature and accuracy of the measurement. Exemplary degrees of error are typically within 20%, preferably within 10%, and more preferably within 5% of a given value or range of values.

Alternatively, particularly in biological systems, the terms "about" and "approximately" may refer to an average value that is within an order of magnitude, preferably within 5-fold, more preferably within 2-fold of a given value. Unless otherwise indicated, the quantities given herein are approximations that may suggest the term "about" or "approximately" when not expressly stated.

The methods of the invention may comprise the step of comparing sequences to each other, including comparing the 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 examples). The skilled person can readily understand that: in such an alignment, when a mutation comprises a residue insertion or deletion, the sequence alignment will introduce a "gap" (generally represented by a dash or "a") in the polymer sequence that does not comprise the insertion or deletion residue.

"homology" and all grammatical and spelling variants thereof refer to the relationship between two proteins having "common evolutionary origin" (including proteins from a superfamily of the same biological species, as well as homologous proteins from different biological species). Such proteins (and their encoding nucleic acids) have sequence homology, which is reflected in their sequence similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions.

The term "sequence similarity" and all grammatical forms thereof refer to the identity or correspondence between nucleic acid or amino acid sequences with or without a common evolutionary origin.

However, in general use and in the present application, when the term "homology" is modified by adverbs such as "highly", it may indicate sequence similarity and may or may not relate to common evolutionary origin.

2.ActRII polypeptides

In certain aspects, the invention relates to ActRII polypeptides. The term "ActRII" as used herein refers to the activin type II receptor family. This family includes both type IIa and type IIb activin receptors.

In certain aspects, the invention relates to ActRIIa polypeptides. The term "ActRIIa" as used herein refers to a family of activin type IIa receptor (ActRIIa) proteins from any species and variants derived from the ActRIIa proteins by mutation or other modification. Where reference is made herein to ActRIIa, it is understood that reference is made to any of the presently identified forms. The ActRIIa family members are typically transmembrane proteins, composed of a ligand-binding extracellular domain rich in cysteine regions, a transmembrane domain, a cytoplasmic domain with predetermined serine/threonine kinase activity.

The term "ActRIIa polypeptide" includes polypeptides comprising naturally occurring ActRIIa family member polypeptides and any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain useful activity. See, for example, WO/2006/012627. For example, an ActRIIa polypeptide includes a polypeptide derived from the sequence of any known ActRIIa, wherein the known ActRIIa has a sequence at least 80% identical, optionally at least 85%, 90%, 95%, 97%, 99% or more identical to the ActRIIa polypeptide sequence. For example, an ActRIIa polypeptide of the present invention may bind to and inhibit the function of an ActRIIa protein and/or activin. ActRIIa polypeptides may be selected for activity in promoting erythropoiesis in vivo. Examples of ActRIIa polypeptides include the human ActRIIa precursor polypeptide (SEQ ID NO:1) and soluble human ActRIIa polypeptides (e.g., SEQ ID NO:2, 3, 7, and 12).

The sequence of a human ActRIIa precursor polypeptide is as follows:

MGAAAKLAFAVFLISCSSGAILGRSETQECLFFNANWEKDRTQTGVEPCYGDKDKRRHCFATWKISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPYYNILLYSLVPLMLIAGIVICAFWVYRHHKMAYPPVLVPTQDPGPPPPSPLLGLKPLQLLEVKARGRFGCVWKAQLLNEYVAVKIFPIQDKQSWQNEYEVYSLPGMKHENILQFIGAEKRGTSVDVDLWLITAFHEKGSLSDFLKANVVSWNELCHIAETMARGLAYLHEDIPGLKDGHKPAISHRDIKSKNVLLKNNLTACIADFGLALKFEAGKSAGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELASRCTAADGPVDEYMLPFEEEIGQHPSLEDMQEVVVHKKKRPVLRDYWQKHAGMAMLCETIEECWDHDAEARLSAGCVGERITQMQRLTNIITTEDIVTVVTMVTNVDFPPKESSL(SEQIDNO:1)

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

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

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPP(SEQIDNO:2)

the C-terminal "tail" of the extracellular domain is underlined. The sequence in which the "tail" is deleted (Δ 15 sequence) is represented as follows:

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM(SEQIDNO:3)

the nucleic acid sequence encoding the human ActRIIa precursor protein is as follows:

(nucleotide 164-1705 of Genbank entry NM-001616):

ATGGGAGCTGCTGCAAAGTTGGCGTTTGCCGTCTTTCTTATCTCCTGTTCTTCAGGTGCTATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTCTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATTGAAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTTTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCAGAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCCTATTACAACATCCTGCTCTATTCCTTGGTGCCACTTATGTTAATTGCGGGGATTGTCATTTGTGCATTTTGGGTGTACAGGCATCACAAGATGGCCTACCCTCCTGTACTTGTTCCAACTCAAGACCCAGGACCACCCCCACCTTCTCCATTACTAGGGTTGAAACCACTGCAGTTATTAGAAGTGAAAGCAAGGGGAAGATTTGGTTGTGTCTGGAAAGCCCAGTTGCTTAACGAATATGTGGCTGTCAAAATATTTCCAATACAGGACAAACAGTCATGGCAAAATGAATACGAAGTCTACAGTTTGCCTGGAATGAAGCATGAGAACATATTACAGTTCATTGGTGCAGAAAAACGAGGCACCAGTGTTGATGTGGATCTTTGGCTGATCACAGCATTTCATGAAAAGGGTTCACTATCAGACTTTCTTAAGGCTAATGTGGTCTCTTGGAATGAACTGTGTCATATTGCAGAAACCATGGCTAGAGGATTGGCATATTTACATGAGGATATACCTGGCCTAAAAGATGGCCACAAACCTGCCATATCTCACAGGGACATCAAAAGTAAAAATGTGCTGTTGAAAAACAACCTGACAGCTTGCATTGCTGACTTTGGGTTGGCCTTAAAATTTGAGGCTGGCAAGTCTGCAGGCGATACCCATGGACAGGTTGGTACCCGGAGGTACATGGCTCCAGAGGTATTAGAGGGTGCTATAAACTTCCAAAGGGATGCATTTTTGAGGATAGATATGTATGCCATGGGATTAGTCCTATGGGAACTGGCTTCTCGCTGTACTGCTGCAGATGGACCTGTAGATGAATACATGTTGCCATTTGAGGAGGAAATTGGCCAGCATCCATCTCTTGAAGACATGCAGGAAGTTGTTGTGCATAAAAAAAAGAGGCCTGTTTTAAGAGATTATTGGCAGAAACATGCTGGAATGGCAATGCTCTGTGAAACCATTGAAGAATGTTGGGATCACGACGCAGAAGCCAGGTTATCAGCTGGATGTGTAGGTGAAAGAATTACCCAGATGCAGAGACTAACAAATATTATTACCACAGAGGACATTGTAACAGTGGTCACAATGGTGACAAATGTTGACTTTCCTCCCAAAGAATCTAGTCTATGA(SEQIDNO:4)

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

ATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTCTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATTGAAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTTTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCAGAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCC(SEQIDNO:5)

in certain aspects, the invention relates to ActRIIb polypeptides. The term "ActRIIb" as used herein refers to a family of type IIb activin receptor (ActRIIb) proteins from any species and variants derived from the ActRIIb proteins by mutation or other modification. Where reference is made herein to ActRIIb, it is to be understood that reference is made to any one of the presently identified forms. Members of the ActRIIb family are typically transmembrane proteins, composed of a cysteine-rich region of the ligand-binding extracellular domain, a transmembrane domain, a cytoplasmic domain with predetermined serine/threonine kinase activity.

The term "ActRIIb polypeptide" includes polypeptides comprising naturally occurring ActRIIb family member polypeptides and any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain useful activity. See, for example, WO/2006/012627. For example, ActRIIb polypeptides include polypeptides derived from the sequence of any known ActRIIb having a sequence at least 80% identical, optionally at least 85%, 90%, 95%, 97%, 99% or more identical to the sequence of an ActRIIb polypeptide. For example, ActRIIb polypeptides of the invention may bind to and inhibit the function of an ActRIIb protein and/or activin. ActRIIb polypeptides may be selected for their activity in promoting erythropoiesis in vivo. Examples of ActRIIB polypeptides include the human ActRIIB precursor polypeptide (SEQ ID NO:15) and soluble human ActRIIB polypeptides (e.g., SEQ ID NOS: 16, 17, 20, and 21). The sequence of the human ActRIIb precursor polypeptide is as follows:

MTAPWVALALLWGSLWPGSGRGEAETRECIYYNANWELERTQSGLERCEGEQDKRLHCYASWASSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTLLTVLAYSLLPIGGLSLIVLLAFWMYRHRKPPYGHVDIHEDPGPPPPSPLVGLKPLQLLEIKARGRFGCVWKAQLMNDFVAVKIFPLQDKQSWQSEREIFSTPGMKHENLLQFIAAEKRGSNLEVELWLITAFHDKGSLTDYLKGNIITWNELCHVAETMSRGLSYLHEDVPWCRGEGHKPSIAHRDFKSKNVLLKSDLTAVLADFGLAVRFEPGKPPGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELVSRCKAADGPVDEYMLPFEEEIGQHPSLEELQEVVVHKKMRPTIKDHWLKHPGLAQLCVTIEECWDHDAEARLSAGCVEERVSLIRRSVNGTTSDCLVSLVTSVTNVDLPPKESSI(SEQIDNO:15)

signal peptide is single underlined; the extracellular domain is shown in bold, while potential N-linked glycosylation sites are shown in boxes.

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

SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT(SEQIDNO:16)

SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA(SEQIDNO:17)

the nucleic acid sequence encoding the human ActRIIb precursor protein is as follows: (Genbank entry NM-001616 nucleotides 5-1543)

ATGACGGCGCCCTGGGTGGCCCTCGCCCTCCTCTGGGGATCGCTGTGGCCCGGCTCTGGGCGTGGGGAGGCTGAGACACGGGAGTGCATCTACTACAACGCCAACTGGGAGCTGGAGCGCACCAACCAGAGCGGCCTGGAGCGCTGCGAAGGCGAGCAGGACAAGCGGCTGCACTGCTACGCCTCCTGGGCCAACAGCTCTGGCACCATCGAGCTCGTGAAGAAGGGCTGCTGGCTAGATGACTTCAACTGCTACGATAGGCAGGAGTGTGTGGCCACTGAGGAGAACCCCCAGGTGTACTTCTGCTGCTGTGAAGGCAACTTCTGCAACGAGCGCTTCACTCATTTGCCAGAGGCTGGGGGCCCGGAAGTCACGTACGAGCCACCCCCGACAGCCCCCACCCTGCTCACGGTGCTGGCCTACTCACTGCTGCCCATCGGGGGCCTTTCCCTCATCGTCCTGCTGGCCTTTTGGATGTACCGGCATCGCAAGCCCCCCTACGGTCATGTGGACATCCATGAGGACCCTGGGCCTCCACCACCATCCCCTCTGGTGGGCCTGAAGCCACTGCAGCTGCTGGAGATCAAGGCTCGGGGGCGCTTTGGCTGTGTCTGGAAGGCCCAGCTCATGAATGACTTTGTAGCTGTCAAGATCTTCCCACTCCAGGACAAGCAGTCGTGGCAGAGTGAACGGGAGATCTTCAGCACACCTGGCATGAAGCACGAGAACCTGCTACAGTTCATTGCTGCCGAGAAGCGAGGCTCCAACCTCGAAGTAGAGCTGTGGCTCATCACGGCCTTCCATGACAAGGGCTCCCTCACGGATTACCTCAAGGGGAACATCATCACATGGAACGAACTGTGTCATGTAGCAGAGACGATGTCACGAGGCCTCTCATACCTGCATGAGGATGTGCCCTGGTGCCGTGGCGAGGGCCACAAGCCGTCTATTGCCCACAGGGACTTTAAAAGTAAGAATGTATTGCTGAAGAGCGACCTCACAGCCGTGCTGGCTGACTTTGGCTTGGCTGTTCGATTTGAGCCAGGGAAACCTCCAGGGGACACCCACGGACAGGTAGGCACGAGACGGTACATGGCTCCTGAGGTGCTCGAGGGAGCCATCAACTTCCAGAGAGATGCCTTCCTGCGCATTGACATGTATGCCATGGGGTTGGTGCTGTGGGAGCTTGTGTCTCGCTGCAAGGCTGCAGACGGACCCGTGGATGAGTACATGCTGCCCTTTGAGGAAGAGATTGGCCAGCACCCTTCGTTGGAGGAGCTGCAGGAGGTGGTGGTGCACAAGAAGATGAGGCCCACCATTAAAGATCACTGGTTGAAACACCCGGGCCTGGCCCAGCTTTGTGTGACCATCGAGGAGTGCTGGGACCATGATGCAGAGGCTCGCTTGTCCGCGGGCTGTGTGGAGGAGCGGGTGTCCCTGATTCGGAGGTCGGTCAACGGCACTACCTCGGACTGTCTCGTTTCCCTGGTGACCTCTGTCACCAATGTGGACCTGCCCCCTAAAGAGTCAAGCATCTAA(SEQIDNO:18)

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

TCTGGGCGTGGGGAGGCTGAGACACGGGAGTGCATCTACTACAACGCCAACTGGGAGCTGGAGCGCACCAACCAGAGCGGCCTGGAGCGCTGCGAAGGCGAGCAGGACAAGCGGCTGCACTGCTACGCCTCCTGGGCCAACAGCTCTGGCACCATCGAGCTCGTGAAGAAGGGCTGCTGGCTAGATGACTTCAACTGCTACGATAGGCAGGAGTGTGTGGCCACTGAGGAGAACCCCCAGGTGTACTTCTGCTGCTGTGAAGGCAACTTCTGCAACGAGCGCTTCACTCATTTGCCAGAGGCTGGGGGCCCGGAAGTCACGTACGAGCCACCCCCGACAGCCCCCACC(SEQIDNO:19)

in particular embodiments, the present invention relates to soluble ActRII polypeptides. The term "soluble ActRII polypeptide" as described herein generally refers to a polypeptide that comprises the extracellular domain of an ActRIIa or ActRIIb protein. The term "soluble ActRII polypeptide" as used herein includes the extracellular domain of any naturally occurring ActRIIa or ActRIIb protein and any variants thereof (including mutants, fragments, and peptidomimetic forms). An activin-binding ActRII polypeptide is a polypeptide that retains the ability to bind to activin (including, for example, activin AA, AB, BB or forms that include C or E subunits). An activin-binding ActRII polypeptide will bind to activin AA, optionally with a dissociation constant of 1nM or less. The extracellular domain of an ActRII protein may bind to activin and is generally soluble, and thus may be referred to as a soluble, activin-binding ActRII polypeptide. Examples of soluble, activin-binding ActRII polypeptides include the soluble polypeptides shown in seq id nos 2, 3, 7, 12, and 13. SEQ ID NO 7 is designated ActRIIa-hFc and is further described in the examples. Other examples of soluble, activin-binding ActRII polypeptides include signal sequences outside of the extracellular domain of the ActRIIa protein, such as the melittin leader sequence (SEQ ID NO:8), the Tissue Plasminogen Activator (TPA) leader sequence (SEQ ID NO:9), or the native ActRIIa leader sequence (SEQ ID NO: 10). The ActRIIa-hFc polypeptide shown in SEQ ID NO. 13 employs a TPA leader sequence. Examples of soluble, activin-binding ActRIIb polypeptides include the soluble polypeptides shown in seq id nos 16, 17, 20. Activin-binding ActRIIB polypeptides may also include signal sequences outside of the extracellular domain of the ActRIIB protein, such as the melittin leader sequence (SEQ ID NO:8) or the Tissue Plasminogen Activator (TPA) leader sequence (SEQ ID NO: 9).

Functionally active fragments of ActRII polypeptides may be obtained by screening polypeptides recombinantly produced from a corresponding fragment of a nucleic acid encoding an ActRII polypeptide. Furthermore, fragments can be chemically synthesized using techniques known in the art, such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. The fragments may be generated (by recombinant or chemical synthesis) and tested to identify peptidyl fragments that are capable of acting as antagonists (inhibitors) of ActRII protein or activin-mediated signaling.

Functionally active variants of ActRII polypeptides may be obtained by screening libraries of modified polypeptides recombinantly produced from a corresponding mutant nucleic acid encoding an ActRII polypeptide. The variants can be generated and tested to identify variants that are capable of acting as antagonists (inhibitors) of ActRII protein or activin-mediated signaling. In certain embodiments, a functional variant of an ActRIIa polypeptide comprises an amino acid sequence having at least 75% identity to an amino acid sequence selected from seq id nos. 2 or 3. In certain instances, the functional variant has 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 No. 2 or 3. In certain embodiments, a functional variant of an ActRIIb polypeptide comprises an amino acid sequence having at least 75% identity to an amino acid sequence selected from seq id nos 16 or 17. In certain instances, the 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 No. 17 or 18.

Functional variants may be generated by modifying the structure of an ActRII polypeptide for purposes such as enhancing therapeutic efficacy or stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo). Such modified ActRII polypeptides are considered functional equivalents of the naturally occurring ActRII polypeptides in selecting for retention of activin binding. Modified ActRII polypeptides may also be generated, for example, by amino acid substitution, deletion, or addition. For example, it is reasonable to expect that a substitution of leucine alone with isoleucine or valine, asparagine with glutamine, threonine with serine, or a similar amino acid substitution with a structurally related amino acid (e.g., a conservative mutation) will not have a major effect on the biological activity of the resulting molecule. Conservative substitutions are those that occur within a family of side-chain related amino acids. Whether a change in the amino acid sequence of an ActRII polypeptide results in a functional homolog can be readily determined by assessing the ability of the ActRII polypeptide variant to generate a response in a cell in a manner similar to a wild-type ActRII polypeptide.

In certain embodiments, the present disclosure contemplates specific mutations of an ActRII polypeptide to alter glycosylation of the polypeptide. Such mutations may be selected to introduce or remove one or more glycosylation sites, such as O-linked or N-linked glycosylation sites. Asparagine-linked glycosylation recognition sites typically comprise a tripeptide sequence, asparagine-X-threonine or asparagine-X-serine (where "X" is any amino acid), which is specifically recognized by the appropriate cellular glycosylation enzyme. Such alterations may also be made by the addition or substitution of one or more serine or threonine residues (to the O-linked glycosylation site) to the wild-type ActRII polypeptide sequence. Various amino acid substitutions or deletions at one or both of the first or third amino acid positions of the glycosylation recognition site (and/or amino acid deletion at the second position) can result in non-glycosylation at the modified tripeptide sequence. Another way to increase the amount of sugar moieties on an ActRII polypeptide is by chemically or enzymatically coupling glycosides to the ActRII polypeptide. Depending on the coupling mode used, the sugar may be linked to (a) arginine and histidine; (b) a free carboxylic acid group; (c) free sulfhydryl groups, such as the sulfhydryl group of cysteine; (d) free hydroxyl groups, such as the hydroxyl groups of serine, threonine, or hydroxyproline; (e) aromatic residues, such as that of phenylalanine, tyrosine or tryptophan; or (f) an amide group of glutamine. Removal of one or more sugar moieties on an ActRII polypeptide may be accomplished by chemical and/or enzymatic methods. Chemical deglycosylation may involve, for example, exposing the ActRII polypeptide to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the cleavage of most or all of the sugars, except the linked sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the amino acid sequence intact. Enzymatic cleavage of the sugar moiety on an ActRII polypeptide can be achieved by using various endo-and exo-glycosidases as described by Thotakura et al (1987) meth.Enzymol.138: 350. The sequence of an ActRII polypeptide can be adjusted as appropriate to the type of expression system used, as mammalian, yeast, insect, and plant cells can introduce different types of glycosylation, which can be affected by the amino acid sequence of the peptide. Generally, ActRII proteins for use in humans may be expressed in mammalian cell lines that provide appropriate glycosylation, such as HEK293 or CHO cell lines, although other mammalian expression cell lines are also contemplated to be useful.

The present disclosure further contemplates methods of generating mutants, particularly combinatorial mutants of ActRII polypeptides, as well as sets of truncation mutants; the combinatorial mutant collection is particularly useful for identifying functional variant sequences. The purpose of screening such combinatorial libraries may be to generate ActRII polypeptide variants that bind, for example, activin or other ligands. Provided below are a variety of screening assays that can be used to evaluate variants. For example, ActRII polypeptide variants having the ability to bind an ActRII ligand may be screened to prevent ActRII ligand binding to the ActRII polypeptide or to interfere with signaling by the ActRII ligand.

The activity of an ActRII polypeptide or variant thereof may also be tested in a cell-based assay or in an in vivo assay. For example, ActRII polypeptide variants can be evaluated for their effect on expression of genes involved in hematopoiesis. This may be performed in the presence of one or more recombinant ActRII ligand proteins (e.g., activins), as desired, and the cells may be transfected to produce an ActRII polypeptide and/or variant thereof, and optionally an ActRII ligand. Likewise, an ActRII polypeptide can be administered to a mouse or other animal, and one or more blood measurements can be made, such as RBC count, hemoglobin, or reticulocyte count.

Combinatorially-derived variants may be produced that have selective or generally higher potency relative to a naturally-occurring ActRII polypeptide. Likewise, mutations can result in variants that have an intracellular half-life that is significantly different from a corresponding wild-type ActRII polypeptide. For example, such altered proteins may become more or less stable to proteolytic degradation or other cellular processes that result in the disruption or inactivation of the native ActRII polypeptide. Such variants, as well as the genes encoding them, may be used to alter ActRII polypeptide levels by modulating the half-life of the ActRII polypeptide. For example, a shorter half-life may produce a more transient biological effect and may allow greater control of recombinant ActRII polypeptide levels in the cell for a portion of the inducible expression system. In an Fc fusion protein, mutations can be made at the linker (if any) and/or Fc portion to alter the half-life of the protein.

Combinatorial libraries may be derived from a degenerate library of genes encoding a library of polypeptides, wherein each polypeptide in the library of polypeptides comprises at least a portion of a potential ActRII polypeptide sequence. For example, a mixture of synthetic oligonucleotides may be enzymatically linked to a gene sequence such that a degenerate set of potential ActRII polypeptide nucleotide sequences may be expressed as individual polypeptides, or alternatively, as a larger set of fusion proteins (e.g., for phage display).

Libraries of potential homologs can be generated from degenerate oligonucleotide sequences in a variety of ways. Chemical synthesis of a degenerate gene sequence can be carried out in an automated DNA synthesizer, and the synthetic gene can be ligated into an appropriate vector for expression. The synthesis of degenerate oligonucleotides is well known in the art (see for example Narang, SA (1983) Tetrahedron39: 3; Itakura et al, (1981) recombined DNA, Proc.3rd Cleveland Sympos. macromolecules, ed. AGWalton, Amsterdam: Elsevierpp 273-289; Itakura et al, (1984) Annu. Rev. biochem.53: 323; Itakura et al, (1984) Science198: 1056; Ike et al, (1983) NucleicacidRes.11: 477). This technique has been used in the direct evolution of other proteins (see, for example, Scott et al, (1990) Science249: 386-.

Alternatively, other mutated forms may be employed to generate combinatorial libraries. ActRII polypeptide variants can be generated and isolated from libraries, for example, by alanine scanning mutagenesis et al (Ruf et al, (1994) Biochemistry33: 1565. sup. 1572; Wang et al, (1994) J.biol. chem.269: 3095. sup. 3099; Balint et al, (1993) Gene137: 109. sup. 118; Grodberg et al, (1993) Eur.J.biochem.218: 597. sup. 601; Nagashima et al, (1993) J.biol.chem.268: 2888. sup. 2892; Lowman et al, (1991) Biochemistry30: 10832. sup. 10838; and Cunningham et al, (1989) Science: 1081. sup. 1085), linker scanning mutations (1993) Viburnin et al, (1993) Virology193: 653: 198660; Brown et al, (19811) molecular dynamics 19811. sup. 19811) Meldrum et al, (1986. sup. 19811) molecular mutation (Meldrum) 18. sup. 19812; Meldrum. sup. 19812) and molecular dynamics); or random mutations, including chemical mutations and the like (Miller et al, (1992) AshortCorseiseinBacterialgenetics, CSHLPress, ColdSpringHarbor, NY; and Greener et al, (1994) Strategiensis nMulBiol 7: 32-34). Linker scanning mutagenesis, particularly in combinatorial setting, is an attractive method for identifying truncated (biologically active) forms of ActRII polypeptides.

Various techniques for screening gene products from combinatorial libraries generated by point mutations and truncations are known in the art, and thus cDNA library screening techniques for gene products with certain properties are known. These techniques would be generally applicable to the rapid screening of gene banks generated by ActRII polypeptide combinatorial mutations. Screening large gene libraries for widespread use generally involves cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting vector library, and expressing the combined genes under conditions in which detection of the desired activity facilitates relatively easy isolation of the vector encoding the gene whose product is being measured. Preferred assays include activin binding assays and activin-mediated cell signaling assays.

In certain embodiments, ActRII polypeptides of the disclosure may further include post-translational modifications other than any modifications naturally occurring in the ActRII polypeptide. These modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. As a result, the modified ActRII polypeptides may include non-amino acid elements, such as polyethylene glycol, lipids, poly-or monosaccharides, and phosphate esters. The effect of such non-amino acid elements on ActRII polypeptide function may be tested by methods described herein for other ActRII polypeptide variants. Post-translational processing may also be important for proper folding and/or function of the ActRII polypeptide when it is produced by cleaving a nascent form of the ActRII polypeptide in a cell. Different cells (e.g., CHO, HeLa, MDCK, 293, WI38, NIH-3T3, or HEK293) have specific cellular structures and characteristic mechanisms for this post-translational activity and are selected to ensure proper modification and processing of the ActRII polypeptide.

In certain aspects, functional variants or modified forms of the ActRII polypeptides include fusion proteins having at least a portion of the ActRII polypeptide and one or more fusion domains. Examples of such fusion domains that are well known include, but are not limited to, polyhistidine, Glu-Glu, Glutathione S Transferase (GST), thioredoxin, protein A, protein G, immunoglobulin heavy chain constant region (Fc), Maltose Binding Protein (MBP), or human serum albumin. The fusion domain may be selected to confer a desired attribute. For example, a partial fusion domain is particularly useful for isolation of the fusion protein by affinity chromatography. For the purpose of performing affinity purification, relevant matrices for affinity chromatography, such as glutathione-, amylase-, and nickel-or cobalt-bound resins, can be used. Many of these matrices can be provided in the form of "kits", such as pharmaciGST purification systems and QIAexpress, which are useful (HIS6) as fusion partners^TMSystem (Qiagen). As another example, a fusion domain may be selected to facilitate detection of the ActRII polypeptide. The detection knotExamples of domains include various fluorescent proteins (e.g., GFP) and "epitope tags," which are typically short peptide sequences from which specific antibodies can be obtained. Well known epitope tags from which specific monoclonal antibodies are readily available include FLAG, influenza Hemagglutinin (HA) and c-myc tags. In some cases, the fusion domain has protease cleavage sites, such as those of factor Xa and thrombin, which allow the relevant protease to partially digest the fusion protein, thereby releasing the recombinant protein therefrom. The released protein can then be separated from the fusion domain by subsequent chromatographic separation. In certain preferred embodiments, an ActRII polypeptide is fused to a domain that stabilizes the ActRII polypeptide in vivo (a "stabilizer" domain). "stabilization" refers to any condition that increases serum half-life, whether it be caused by decreased destruction, decreased renal clearance, or other pharmacokinetic effects. Fusions to the Fc region of immunoglobulins are known to confer desirable pharmacokinetic properties on a variety of proteins. Likewise, fusions to human serum albumin can confer desirable properties. Alternative fusion domain types include multimerization (e.g., dimerization, tetramerization) domains and functional domains (which confer additional biological functions such as further stimulation of muscle growth).

As a specific example, the invention provides a fusion protein comprising a lysable ectodomain of ActRIIa fused to an Fc domain (e.g., SEQ ID NO: 6).

THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD(A)VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK(A)VSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN(A)HYTQKSLSLSPGK*

As another specific example, the present disclosure provides a fusion protein comprising a lysable ectodomain of ActRIIB fused to an Fc domain (e.g., SEQ ID NO: 21).

SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Optionally, the Fc domain has one or more mutations at residues such as Asp-265, lysine 322, and Asn-434. In certain instances, a mutant Fc domain having one or more of these mutations (e.g., Asp-265 mutations) has reduced binding to an fey receptor relative to a wild-type Fc domain. In other cases, the mutant Fc domain having one or more of these mutations (e.g., the Asn-434 mutation) has increased binding to MHC class I-associated Fc-receptors (fcrns) relative to the wild-type Fc domain.

It is understood that the various elements of the fusion protein may be arranged in any manner consistent with the desired function. For example, an ActRII polypeptide may be placed C-terminal to the heterologous domain, or alternatively, the heterologous domain may be placed C-terminal to the ActRII polypeptide. The ActRII polypeptide domain and the heterologous domain need not be in proximity to each other in the fusion protein, and additional domains or amino acid sequences may be included at the C-or N-terminus of either domain or between domains.

In certain embodiments, an ActRII polypeptide of the disclosure includes one or more modifications that are capable of stabilizing the ActRII polypeptide. For example, the modifications increase the in vitro half-life of the ActRII polypeptide, increase the circulating half-life of the ActRII polypeptide, or decrease proteolytic degradation of the ActRII polypeptide. Such stabilizing modifications include, but are not limited to, fusion proteins (including, for example, fusion proteins comprising an ActRII polypeptide and a stabilizer domain), modifications of glycosylation sites (including, for example, the addition of glycosylation sites to the ActRII polypeptide), and modifications of sugar moieties (including, for example, the removal of sugar moieties from the ActRII polypeptide). The term "stabilizer domain" as used herein refers not only to the fusion domain (e.g., Fc) in the case of a fusion protein, but also includes non-proteinaceous modifications, such as sugar moieties, or non-proteinaceous moieties, such as polyethylene glycol.

In certain embodiments, the present disclosure provides isolated and/or purified forms of ActRII polypeptides that are isolated from, or substantially free of, other proteins. ActRII polypeptides may generally be produced by expression from a recombinant nucleic acid.

3.Nucleic acids encoding ActRII polypeptides

In certain aspects, the invention provides isolated and/or recombinant nucleic acids encoding any of the ActRII polypeptides (e.g., soluble ActRIIa polypeptides and soluble ActRIIb polypeptides), including fragments, functional variants, and fusion proteins disclosed herein. For example, SEQ ID NO. 4 encodes the naturally occurring human ActRIIa precursor polypeptide, while SEQ ID NO. 5 encodes the processed extracellular domain of ActRIIa. For example, SEQ ID NO. 18 encodes the naturally occurring human ActRIIB precursor polypeptide, while SEQ ID NO. 19 encodes the processed extracellular domain of ActRIIB. The subject nucleic acids may be single-stranded or double-stranded. Such nucleic acids may be DNA or RNA molecules. These nucleic acids may be used, for example, in methods of making ActRII polypeptides, or directly as therapeutic agents (e.g., in gene therapy methods).

In certain aspects, the subject nucleic acids encoding ActRIIa polypeptides are to be further understood as including nucleic acids that are variants of seq id nos. 4 or 5. In certain aspects, the subject nucleic acids encoding ActRIIb polypeptides should be further understood to include nucleic acids that are variants of seq id nos 18 or 19. Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions, e.g., allelic variants.

In certain embodiments, the invention provides isolated or recombinant nucleic acid sequences having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to seq id nos:4, 5, 18, or 19. One of ordinary skill in the art will understand that: nucleic acid sequences complementary to SEQ ID NOs:4, 5, 18 or 19, as well as variants of SEQ ID NOs:4, 5, 18 or 19 are also within the scope of the present invention. In a further embodiment, the nucleic acid sequence of the invention may be isolated, recombined and/or fused with a heterologous nucleotide sequence, or in a DNA pool.

In other embodiments, the nucleic acids of the invention further include nucleotide sequences that hybridize under highly stringent conditions to the nucleotide sequences set forth in SEQ ID NOs:4, 5, 18, or 19, the complement of SEQ ID NOs:4, 5, 18, or 19, or a fragment thereof. As discussed above, one of ordinary skill in the art will readily understand that: suitable stringency conditions for promoting DNA hybridization can vary. One of ordinary skill in the art will readily understand that: suitable stringency conditions for promoting DNA hybridization can vary. For example, hybridization can be performed at 6.0 XSSC/sodium citrate (SSC) and about 45 ℃ followed by washing at 2.0XSSC and 50 ℃. For example, the salt concentration in the washing step can be selected from about 2.0XSSC at 50 ℃ of low stringency to about 0.2XSSC at 50 ℃ of high stringency. In addition, the temperature in this washing step can be raised from room temperature at about 22 ℃ for low stringency to about 65 ℃ under high stringency conditions. Both temperature and salt can be varied, or the temperature or salt concentration can be kept constant while other variables are varied. In one embodiment, the invention provides nucleic acids that hybridize under low stringency conditions at 6XSSC and room temperature, and then are washed at 2XSSC and room temperature.

Isolated nucleic acids which differ from the nucleic acids shown in SEQ ID NOs:4, 5, 18 or 19 by virtue of the degeneracy of the genetic code are likewise within the scope of the present invention. For example, more than one triplet (triplet) represents a series of nucleic acids. Codons representing the same amino acid, or synonymous codons (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 that lead to changes in the amino acid sequence of the subject protein (objectprotein) are present in mammalian cells. Those skilled in the art will understand that: these changes at one or more nucleotides (up to about 3-5% of the nucleotides) of a nucleic acid encoding a particular protein may occur in an individual of a given species due to natural allelic variation. Any or all of these nucleotide changes, as well as the resulting amino acid polymorphisms, are within the scope of the invention.

In certain embodiments, a recombinant nucleic acid of the invention may be operably linked to one or more regulatory nucleotide sequences in an expression construct. Regulatory nucleotide sequences are generally suitable for use in the host cell for expression. A variety of suitable expression vector types and suitable regulatory sequences for a variety of host cells are known in the art. The one or more regulatory nucleotide sequences can generally include, but are not limited to, a promoter sequence, a leader or signal sequence, a ribosome binding site, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters known in the art are within the contemplation of the present invention. The promoter may be a naturally occurring promoter, or a hybrid promoter incorporating elements of more than one promoter. The expression construct may be present in the cell episomally (e.g., on a plasmid), or the expression construct may be inserted into the chromosome. In a preferred embodiment, the expression vector comprises a selectable marker gene, thereby allowing selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.

In certain aspects of the invention, the subject nucleic acid(s) is provided in an expression vector comprising a nucleotide sequence encoding an ActRII polypeptide operably linked to at least one regulatory sequence. Regulatory sequences are known in the art and may be selected to direct expression of the ActRII polypeptides. Thus, the term regulatory sequence includes promoters, enhancers and other expression control elements. Exemplary regulatory sequences are described in Goeddel; gene expression technology: MethodsinEnzymology, academic Press, san Diego, Calif. (1990). For example, any of a variety of expression control sequences that control the expression of a DNA sequence when operably linked to the DNA sequence may be used in these vectors to express a DNA sequence encoding an ActRII polypeptide. Such useful expression control sequences include, for example, the early and late promoters of SV40, the tet promoter, the adenovirus and cytomegalovirus immediate early promoters, the RSV promoter, the lac system, the trp system, the TAC or TRC system, the T7 promoter for expression directed by T7RNA polymerase, the major operator and promoter regions of bacteriophage lambda, the control regions of fd coat protein, the promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatases, e.g., Pho5, the promoters of yeast α -mating factor, the polyhedrin promoter of baculovirus system, and other sequences that control gene expression of prokaryotic or eukaryotic cells or viruses thereof, and various combinations thereof. It will be appreciated that the design of the expression vector may depend on factors such as the choice of host cell to be transformed and/or the type of protein that is desired to be expressed. In addition, the copy number of the vector, the ability to control the copy number, and the expression of any other proteins encoded by the vector (e.g., antibiotic markers) should also be considered.

The recombinant nucleic acids of the invention may be produced by ligating the cloned genes or portions thereof into vectors suitable for expression in prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian), or both. Expression vectors for the production of recombinant ActRII polypeptides include plasmids and other vectors. For example, suitable vectors include the following types of plasmids: pBR 322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids that can be expressed in prokaryotic cells (e.g., E.coli).

Some mammalian expression vectors contain both prokaryotic sequences that facilitate propagation of the vector in bacteria and one or more eukaryotic transcription units that are expressed 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 cell expression vectors suitable for transfection of eukaryotic cells. These vectors are modified in part with sequences from bacterial plasmids (e.g., pBR322) to facilitate replication and drug tolerance selection in prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as bovine papilloma virus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found in the description of gene therapy delivery systems below. Various methods for the preparation of plasmids and transformation of host organisms are known in the art. Other suitable prokaryotic and eukaryotic cell expression systems and general recombination procedures can be found in molecular cloning, in laboratory Manual, 3rd Ed, ed.Sambrook, FritschandManiatis (Cold spring harbor laboratory Press, 2001). In some cases, it may be desirable to express the recombinant polypeptide using a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (e.g., pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (e.g., pAcUW1), and pBlueBac-derived vectors (e.g., beta-gal containing pBlueBacIII).

In a preferred embodiment, vectors designed to produce the subject ActRII polypeptides in CHO cells, such as the Pcmv-Script vector (Stratagene, LaJolla, Calif), the pcDNA4 vector (Invitrogen, Carlsbad, Calif.), and the pCI-neo vector (Promega, Madison, Wisc), will be used. Obviously, the subject genetic constructs may be used to elicit expression of the subject ActRII polypeptides in cells propagated in culture, e.g., to produce proteins, including fusion proteins or variant proteins, for purification.

The disclosure also relates to transfecting a host cell with a recombinant gene comprising a coding sequence for one or more of the subject ActRII polypeptides (e.g., seq id nos:4, 5, 18, or 19). The host cell may be a prokaryotic or eukaryotic cell. For example, an ActRII polypeptide of the invention can be expressed in a bacterial cell, such as e.coli, an insect cell (e.g., using a baculovirus expression system), a yeast, or a mammalian cell. Other suitable host cells are known to those skilled in the art.

Accordingly, the present invention further relates to methods of producing the subject ActRII polypeptides. For example, a host cell transfected with an expression vector encoding an ActRIIa or ActRIIb polypeptide may be cultured under appropriate conditions for expression of the multiple actriii peptides. The ActRII polypeptides may be secreted and isolated from a mixture of the cell and a medium containing the ActRII polypeptides. Alternatively, the ActRII polypeptides may be retained within the cytoplasm or in a membrane fraction, and the cells harvested, lysed, and the proteins isolated. The cell culture comprises host cells, culture medium and other by-products. Suitable media for cell culture are well known in the art. The subject ActRII polypeptides can be isolated from the cell culture medium, the host cell, or both using techniques known in the art for purifying proteins, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, immunoaffinity purification by antibodies specific for particular epitopes of the ActRII protein, and affinity purification by agents that bind to a domain fused to the ActRII polypeptide (e.g., a protein a column can be used to purify ActRIIa-Fc or ActRIIb-Fc fusions). In a preferred embodiment, the ActRII polypeptide is a fusion protein that includes a domain that facilitates purification thereof. In a preferred embodiment, purification may be achieved by a series of column chromatography steps, including, for example, the following three or more steps in any order: protein a chromatography, Q sepharose chromatography, phenyl sepharose chromatography, size exclusion chromatography, and cation exchange chromatography. This purification can be accomplished by virus filtration and buffer exchange. As shown herein, ActRIIa-hFc proteins were purified to > 98% purity as measured by size exclusion chromatography and > 95% purity as measured by SDSPAGE. This level of purity is sufficient to achieve the desired results in mice, rats and non-human primates.

In another embodiment, a fusion gene encoding a purification leader sequence, such as a poly- (His)/enterokinase cleavage site sequence N-terminal to a desired portion of a recombinant ActRII polypeptide, may allow for the use of Ni via affinity chromatography²⁺Metal resin to purify the expressed fusion protein. The purified leader sequence can then be removed by treatment with enterokinase to provide a purified ActRII polypeptide (see, e.g., Hochuli et al, (1987) J. Chromatography411: 177; and Janknecht et al, PNAS USA88: 8972).

Techniques for preparing fusion genes are well known. Basically, various DNA fragments encoding different polypeptide sequences are ligated according to conventional techniques, ligated using blunt or staggered ends to provide appropriate ends for restriction enzyme digestion, sticky ends are filled in as needed, alkaline phosphatase treated to avoid unnecessary ligation, and then enzymatically ligated. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that produce a complementary overhang between two consecutive gene fragments that can then anneal to form a chimeric gene sequence (see, e.g., CurrentProtocol molecular biology, eds. Ausubel et al, John Wiley & Sons: 1992).

4.Alternativin and ActRII antagonists

The data presented herein demonstrate that antagonists of activin-ActRII signaling can be used to increase red blood cell or hemoglobin levels. Although soluble ActRIIa and ActRIIb polypeptides, particularly ActRIIa-Fc and ActRIIb-Fc, are preferred antagonists, and although such antagonists may affect red blood cell levels by mechanisms other than activin antagonism (e.g., activin inhibition may be an indication of the propensity of an agent to inhibit the activity of a range of molecules, potentially including other members of the TGF- β superfamily, and such collective inhibition may result in the desired effect of hematopoiesis), other types of activin-ActRII antagonists are expected to be useful, including anti-activin (e.g., activin β a, β B, β C, and β E) antibodies, anti-ActRIIa antibodies, anti-ActRIIb antibodies, antisense, RNAi, or ribozyme nucleic acids that inhibit ActRIIa and/or ActRIIb production, as well as other activin, ActRIIb, or ActRIIa inhibitors, particularly those that disrupt activin-ActRIIa and/or activin-ActRIIb binding.

Antibodies that specifically react with an ActRII polypeptide (e.g., a soluble ActRIIa or ActRIIb polypeptide) and compete with the ActRII polypeptide for binding to a ligand or inhibit ActRII-mediated signaling can be used as antagonists of ActRII polypeptide activity. Likewise, antibodies that specifically react with an activin β a, β B, β C, or β E polypeptide or any heterodimer thereof and disrupt ActRIIa and/or ActRIIb binding may be used as antagonists.

Using immunogens derived from ActRIIa polypeptides, ActRIIb polypeptides, or activin polypeptides, anti-protein/anti-peptide antisera or monoclonal Antibodies can be prepared by standard methods (see, e.g., Antibodies: Antibodies administered. by Harlowland Lane (ColdSpringHarborPress: 1988)). Mammals such as mice, hamsters, or rabbits may be immunized with activin, an immunogenic form of an ActRIIa or ActRIIb polypeptide, an antigenic fragment or fusion protein capable of eliciting an antibody response. Techniques for conferring immunogenicity on a protein or peptide include conjugation to a carrier or other techniques known in the art. An immunogenic portion of an ActRII or activin polypeptide may be administered in the presence of an adjuvant. The progress of the immunization can be monitored by detecting antibody titers in plasma or serum. Standard ELISA or other immunoassays can assess antibody levels with immunogen as the antigen.

After immunizing an animal with an antigenic preparation of activin, ActRIIa, or ActRIIb polypeptide, antisera may be obtained and, if desired, polyclonal antibodies may be isolated from the sera. For the production of monoclonal antibodies, antibody-producing cells (lymphocytes) can be collected from the immunized animal and fused with immortal cells such as myeloma cells by standard somatic cell fusion procedures to obtain hybridoma cells. Such techniques are well known in the art and include, for example, the generation of human monoclonal antibodies by hybridoma technology (originally developed: Kohlerand Milstein, (1975) Nature, 256: 495-. Hybridoma cells that produce antibodies specifically reactive with activin, ActRIIa, or ActRIIb polypeptides can be obtained by immunochemical screening and monoclonal antibodies isolated from cultures containing such hybridoma cells.

The term "antibody" as used herein is intended to include whole antibodies, such as any antibody isotype (IgG, IgA, IgM, IgE, etc.), and includes fragments or domains of immunoglobulins that react with selected antigens. Antibodies can be fragmented by conventional techniques and the fragments screened for utility and/or interaction with a specific epitope of interest. Thus, the term includes proteolytic cleavage fragments or recombinantly produced portions of antibody molecules that are capable of selectively reacting with a particular protein. Non-limiting examples of such proteolytic and/or recombinant fragments include Fab, F (ab ')2, Fab', Fv, and single chain antibodies (scFv) comprising VL and/or VH domains linked by a peptide linker. The scfvs may be linked, covalently or non-covalently, to form an antibody having two or more binding sites. The term antibody also includes polyclonal antibodies, monoclonal antibodies, or other purified preparations of antibodies and recombinant antibodies. The term "recombinant antibody" refers to an antibody or antigen-binding domain of an immunoglobulin expressed from a nucleic acid constructed by molecular biological techniques, such as a humanized antibody or a fully human antibody developed from a single chain antibody. Single domain and single chain antibodies are generally included within the term "recombinant antibody".

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

The adjective "specifically reacts with", when referring to an antibody, as is generally understood in the art, means that the antibody has sufficient selectivity between the target antigen (e.g., activin, ActRIIa, or ActRIIb polypeptide) and other non-target antigens such that the antibody is at least useful for detecting the presence of the target antigen in a particular type of biological sample. In certain methods of using the antibodies (e.g., therapeutic applications), a higher degree of binding specificity may be required. Monoclonal antibodies generally haveA stronger tendency to effectively distinguish between the antigen of interest and the cross-reactive polypeptide (compared to polyclonal antibodies). Influence on antibodies: one characteristic of antigen-interaction specificity is the affinity of the antibody for the antigen. Although the desired specificity can be achieved through a range of different affinities, it is generally preferred that the antibody have about 10^-6、10^-7、10^-8、10^-9M or less affinity (dissociation constant).

In addition, the techniques used to screen antibodies to identify desired antibodies can also affect the properties of the resulting antibodies. For example, if an antibody is used to bind an antigen in solution, it may be necessary to test for solution binding. There are a number of different techniques for testing the interaction between an antibody and an antigen to identify a particular antibody of interest. These techniques include ELISA, surface plasmon resonance binding assays (e.g., Biacore)^TMBinding assays, BiacoreAB, Uppsala, sweden), sandwich assays (e.g., paramagnetic bead systems, igen international, inc., Gaithersburg, Maryland), western blots, immunoprecipitation assays, and immunohistochemistry.

Examples of classes of nucleic acid compounds that are activin or ActRII antagonists include antisense nucleic acids, RNAi constructs, and catalytic nucleic acid constructs. The nucleic acid compound may be single-stranded or double-stranded. The double stranded compound may also comprise a dangling or non-complementary region, wherein one strand is single stranded. A single-stranded compound may comprise a self-complementary region, meaning that the compound forms a so-called "hairpin" or "stem-loop" structure in a double-stranded helical structural region. The nucleic acid compound can comprise a nucleotide sequence that is complementary to a region of no more than 1000, no more than 500, no more than 250, no more than 100, or no more than 50, 35, 25, 22, 20, 18, or 15 nucleotides of the full-length ActRII nucleic acid sequence or activin β a, β B, β C, or β E nucleic acid sequence. The complementary region is preferably at least 8 nucleotides, and optionally about 18 to 35 nucleotides. The complementary region may be in an intron, the latter being the coding or non-coding sequence of the transcript of interest, e.g., a portion of the coding sequence. Generally, the nucleic acid compound can have a length of about 8 to about 500 nucleotides or base pairs, and optionally the length is about 14 to about 50 nucleotides. The nucleic acid may be DNA (particularly when used as antisense), RNA or RNA: DNA hybrids. Either strand may include a mixture of DNA and RNA as well as modified forms that cannot be simply classified as DNA or RNA. Likewise, the double-stranded compound may be DNA, RNA or RNA, and any strand may also comprise a mixture of DNA and RNA and modified forms that cannot be simply classified as DNA or RNA. The nucleic acid compound can comprise any of a variety of modifications, including one or more modifications to the backbone (sugar-phosphate moieties in a natural nucleic acid, including internucleotide linkages) or base moieties (purine or pyrimidine moieties of a natural nucleic acid). The antisense nucleic acid compound will preferably have a length of about 15 to about 30 nucleotides and will typically comprise one or more modifications to improve its characteristics, such as stability in serum, in cells, or at a site where the compound is likely to be delivered (e.g., stomach when the compound is delivered orally and lung when the compound is inhaled). In the case of RNAi constructs, the strand complementary to the transcript of interest is typically RNA or a modified version thereof. The other strand may be RNA, DNA or any other variant. The duplex portion of a double-stranded or single-stranded "hairpin" RNAi construct typically has a length of 18-40 amino acids, optionally a length of about 21-23 nucleotides, so long as it serves as a Dicer substrate. The catalytic or enzymatic nucleic acid may be a ribozyme or a dnase, and may further comprise a modified form. The nucleic acid compound can inhibit expression of the target by about 50%, 75%, 90% or more under physiological conditions and in contact with the cell at a concentration where there is little or no effect of nonsense or sense control. Preferred concentrations for testing the effect of the nucleic acid compound are 1, 5 and 10 micromolar. Nucleic acid compounds can also be tested for their effect on, for example, red blood cell levels.

5.Screening assays

In certain aspects, the invention relates to the use of ActRII polypeptides (e.g., soluble ActRIIa or ActRIIb polypeptides) and activin polypeptides to identify compounds (agents) that are agonists or antagonists of the activin-ActRIIa and/or activin-ActRIIb signaling pathway. Compounds identified by this assay can be tested to assess their ability to modulate red blood cell, hemoglobin, and/or reticulocyte levels in vivo or in vitro. These compounds can be tested, for example, in animal models.

There are a variety of methods to screen for therapeutic agents that increase red blood cell or hemoglobin levels by targeting activin and ActRII signaling. In certain embodiments, high throughput screening of compounds can be performed to identify agents that perturb activin or ActRII-mediated effects in selected cell lines. In certain embodiments, the assay may be performed to screen for and identify compounds that specifically inhibit or reduce binding of an ActRIIa or ActRIIb polypeptide to activin. Alternatively, the assay may be used to identify compounds that increase the binding of an ActRIIa or ActRIIb polypeptide to activin. In further embodiments, compounds may be identified by their ability to interact with activin, an ActRIIb polypeptide, or an ActRIIa polypeptide.

A variety of assay formats may suffice, and assays not explicitly described herein will be understood by one of ordinary skill in the art in light of this disclosure. As described herein, the test compounds (agents) of the present invention can be generated by any combination of chemical methods. Alternatively, the subject compound may be a naturally occurring biomolecule synthesized in vivo or in vitro. The compounds (agents) to be tested for their ability to act as tissue growth regulators may be produced, for example, by bacteria, yeast, plants or other organisms (e.g., natural products), by chemical means (e.g., small molecules, including peptidomimetics) or by recombination. Test compounds contemplated by the present invention include non-peptidyl organic molecules, peptides, polypeptides, peptidomimetics, saccharides, hormones, and nucleic acid molecules. In particular embodiments, the test agent is a small organic molecule having a molecular weight of less than about 2000 daltons.

The test compounds of the invention may be provided as individual, discrete entities, or in more complex libraries, such as by combinatorial chemistry. These libraries may contain, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers, and other classes of organic compounds. The test compound may be present in the test system (particularly in the initial screening step) in isolated form or as a mixture of compounds. Alternatively, the compound may optionally be derivatized with other compounds and have a derivatizing group that facilitates separation of the compound. Non-limiting examples of derivatizing groups include biotin, fluorescein, digoxigenin, green fluorescent protein, isotopes, polyhistidines, magnetic beads, Glutathione S Transferase (GST), light activated cross-linkers, or any combination thereof.

High throughput assays are required in many drug screening programs to test libraries of compounds and natural extracts to maximize the number of compounds under investigation over a given time frame. Assays performed in cell-free systems (e.g., by purification or semi-purification of proteins) are often preferred as "primary" screens by which rapid development and relatively simple detection of target molecule modification mediated by test compounds can be performed. Furthermore, the cytotoxic or biocompatible effects of test compounds can often be neglected in vitro systems, allowing assays to focus primarily on the effect of the drug on molecular targets, as demonstrated by engineering the binding affinity between an ActRIIa polypeptide and activin and/or between an ActRIIb polypeptide and activin.

For illustrative purposes only, in an exemplary screening assay of the invention, the target compound is contacted with an isolated and purified ActRIIa polypeptide (which is generally capable of binding activin). A composition containing an ActRIIa ligand is then added to the mixture of the compound and ActRIIa polypeptide. The detection and quantification of the ActRIIa/activin complex provides a means for determining the potency of a compound to inhibit (or promote) complex formation between an ActRIIa polypeptide and activin. The efficacy of the compound can be assessed by generating a dose-response curve from data obtained for various concentrations of the test compound. In addition, control assays may also be performed to provide a comparative baseline. For example, in a control assay, isolated and purified activin is added to a composition containing the ActRIIa polypeptide and formation of an ActRIIa/activin complex is quantified in the absence of the test compound. It should be understood that in general the order in which the reactants are mixed may vary, and may be mixed simultaneously. In addition, cellular extracts and lysates can be substituted for purified proteins to provide suitable cell-free assay systems. Compounds that affect ActRIIb signaling may be identified in a similar manner using an ActRIIb polypeptide and an ActRIIb ligand.

Complex formation between an ActRII polypeptide and activin can be detected by a variety of techniques. For example, a detectably labeled protein such as a radiolabeled protein (e.g.,³²P、³⁵S、¹⁴c or³H) Fluorescently labeled (e.g., FITC) or enzymatically labeled ActRIIa or ActRIIb polypeptides or activin, and modulation of complex formation is quantified by immunoassay or by chromatographic detection.

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 degree of interaction between an ActRII polypeptide and its binding protein. In addition, other detection modes, such as detection based on optical waveguides (PCT publication WO96/26432 and U.S. Pat. No. 5,677,196), Surface Plasmon Resonance (SPR), surface charge sensors, and surface force sensors, may be applicable to various embodiments of the present invention.

In addition, the present disclosure contemplates the use of a mutual capture assay (also referred to as a "two-hybrid assay") to identify agents that disrupt or promote the interaction between an ActRII polypeptide and its binding protein. See, for example, U.S. patent 5,283,317; zervos et al (1993) Cell72: 223-232; madura et al (1993) JBiolchem268: 12046-12054; bartel et al (1993) Biotechniques14: 920-924; and Iwabuchi et al (1993) Oncogene8: 16993-. In particular embodiments, the present disclosure contemplates the use of a reverse two-hybrid system to identify compounds (e.g., small molecules or peptides) that isolate the interaction between an ActRII polypeptide and its binding protein. See, for example, Vidal and Legrain, (1999) nucleic acids 27: 919-29; vidal and Legrain, (1999) trends Biotechnol17: 374-81; and U.S. patent 5,525,490; 5,955,280, respectively; and 5,965,368.

In certain embodiments, a subject compound may be identified by its ability to interact with an ActRII or activin polypeptide of the invention. The interaction between a compound and an ActRIIa, ActRIIb, or activin polypeptide may be covalent or non-covalent. For example, such interactions can be identified at the protein level by in vitro biochemical methods including photocrosslinking, radiolabeled ligand binding, and affinity chromatography (JakobyWB et al, 1974, Methodsinenzymology46: 1). In certain instances, the compound may be screened by a mechanism-based assay (e.g., an assay that detects a compound that binds to an activin or ActRII polypeptide). This may include solid or liquid phase binding events. Alternatively, the gene encoding an activin or ActRII polypeptide may be transfected into the cell by a reporter system (e.g., β -galactosidase, luciferase, or green fluorescent protein), and the library optionally screened by high throughput screening or individual members of the library screened. Other mechanism-based binding assays may be used, for example, binding assays that detect changes in free energy. Binding assays can be performed with the target immobilized to a microwell, bead or chip or captured by an immobilized antibody or resolved by capillary electrophoresis. The binding compound can generally be detected by colorimetric or fluorescent or surface plasmon resonance methods.

6.Exemplary therapeutic uses

In certain embodiments, activin-ActRII antagonists of the invention (e.g., ActRIIa or ActRIIb polypeptides) are useful for increasing red blood cell levels in mammals (e.g., rodents and primates, particularly in human patients). In certain embodiments, the present invention provides methods of treating or preventing anemia in an individual by administering to the individual in need thereof a therapeutically effective amount of an activin-ActRIIa antagonist (e.g., an ActRIIa polypeptide) or a therapeutically effective amount of an activin-ActRIIb antagonist (e.g., an ActRIIb polypeptide). In certain embodiments, the present invention provides methods of promoting erythropoiesis in an individual by administering to the individual a therapeutically effective amount of an activin-ActRII antagonist, particularly an ActRII polypeptide. These methods are useful for the therapeutic or prophylactic treatment of mammals, particularly humans.

As used herein, "preventing" treatment of a disease or disorder refers to a compound that, in a statistical sample, reduces the occurrence of the disease or disorder in a treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disease or disorder relative to an untreated sample. The term "treating" as used herein includes preventing the specified condition or ameliorating or eliminating the condition after its formation. In either case, the prevention or treatment can be differentiated from the diagnosis provided by the physician or other health care provider or the expected result of the administration of the therapeutic agent.

As shown herein, activin-ActRIIa antagonists and activin-ActRIIb antagonists are useful for increasing red blood cell, hemoglobin, or reticulocyte levels in healthy individuals, and these antagonists are useful in selected patient populations. Examples of suitable patient populations include patients with undesirably low red blood cell or hemoglobin levels, such as patients with anemia, and patients at risk of developing undesirably low red blood cell or hemoglobin levels, such as patients who are about to undergo major surgery or other procedures that can result in significant blood loss. In one embodiment, a patient with sufficient red blood cell levels may be treated with an activin-ActRIIa antagonist to increase red blood cell levels, and then blood is withdrawn and stored for later transfusion. In one embodiment, a patient with sufficient red blood cell levels may be treated with an activin-ActRIIb antagonist to increase red blood cell levels, and then blood is withdrawn and stored for later transfusion.

activin-ActRII antagonists disclosed herein, particularly ActRIIa-Fc and ActRIIb proteins, may be used to increase red blood cell levels in patients with anemia. When hemoglobin levels are observed in patients, levels lower than the normal values of the appropriate age and gender groups can be used as indicators of anemia, although individual differences are considered. For example, a hemoglobin level of 12g/dl is generally considered to be the lower limit of normal in the average adult population. Potential causes include blood loss, nutritional deficiencies, drug reactions, various problems associated with bone marrow and various diseases. More specifically, anemia is associated with a variety of diseases, including, for example, chronic renal failure, myelodysplastic syndrome, rheumatoid arthritis, and bone marrow transplantation. Anemia may also be associated with the following conditions: solid tumors (e.g., breast, lung, colon); tumors of the lymphatic system (e.g., chronic lymphocytic leukemia, non-hodgkin's and hodgkin's lymphomas); hematopoietic tumors (e.g., leukemia, myelodysplastic syndrome, multiple myeloma); radiotherapy; chemotherapy (e.g., platinum-containing regimens); inflammatory and autoimmune diseases, including, but not limited to, rheumatoid arthritis, other inflammatory arthritis, Systemic Lupus Erythematosus (SLE), acute or chronic skin diseases (e.g., psoriasis), inflammatory bowel diseases (e.g., Crohn's disease and ulcerative colitis); acute or chronic kidney disease or failure, including primary or congenital conditions; acute or chronic liver disease; acute or chronic bleeding; a condition in which red blood cells cannot be infused due to the patient's hetero-or auto-antibodies and/or due to religious reasons (e.g., certain yeres and witnesses); infections (e.g., malaria, osteomyelitis); hemoglobinopathies, including, for example, sickle cell disease, thalassemia; drug use or abuse, e.g., ethanol misuse; pediatric patients with anemia due to any cause of blood transfusion avoidance; and anemia-suffering elderly patients or cardiopulmonary ailments who are unable to receive blood transfusions due to fear of circulatory overload.

Although lower target levels may cause fewer cardiovascular side effects, patients may be treated with a regimen aimed at restoring the patient to a target hemoglobin level, typically between about 10g/dl and about 12.5g/dl, typically about 11.0g/dl (see also Jacobs et al (2000) nephroldialTransplant15, 15-19). Alternatively, hematocrit levels (the percentage of cells by volume of the blood sample) may be used as a measure of the red blood cell disorder. For hematocrit levels of healthy individuals, the range is 41-51% for adult males and 35-45% for adult females. The target hematocrit level is typically about 30-33%. In addition, hemoglobin/hematocrit levels may vary from individual to individual. Thus, optimally, the target hemoglobin/hematocrit level can be individualized for each patient.

The rapid effect of activin-ActRIIa antagonists disclosed herein on the level of erythrocytes suggests that these agents may act differently from the mechanism of Epo. Thus, these antagonists can be used to increase red blood cell and hemoglobin levels in patients who do not respond well to Epo. For example, an activin-ActRIIa antagonist may be beneficial for patients who are administered a normal to increased (>300 IU/kg/week) dose of Epo that fails to raise hemoglobin levels to target levels. Patients with an insufficient Epo response were found in all types of anemia, while more non-responders were found particularly frequently in cancer patients and end stage renal disease patients. An insufficient response by Epo can be either constitutive (i.e., found when first treated with Epo) or acquired (e.g., found when treatment with Epo is repeated).

activin-ActRII antagonists may also be used to treat patients susceptible to side effects of Epo. The major side effects of Epo are an excessive increase in hematocrit or hemoglobin levels and erythrocytosis. Elevated hematocrit levels can lead to hypertension (and more particularly, exacerbation of hypertension) and vascular thrombosis. Other side effects of Epo, partly related to hypertension, have been reported as headache, influenza-like syndrome, shunt obstruction, myocardial infarction and cerebral convulsions due to thrombosis, hypertensive encephalopathy, and erythrocytic dysplasia (Singiberti (1994). J. ClinInvestig72(suppl6), S36-S43; Horl et al (2000) Nephroll DialTransplant15(suppl4), 51-56; Delanaty et al (1997) Neurology49, 686-.

7.Pharmaceutical composition

In certain embodiments, an activin-ActRII antagonist (e.g., an ActRIIa and ActRIIb polypeptide) of the invention may be formulated with a pharmaceutically acceptable carrier. For example, an ActRII polypeptide may be administered alone or as a component of a pharmaceutical formulation (therapeutic composition). The subject compounds may be formulated for administration as human or veterinary drugs in any convenient manner.

In certain embodiments, the therapeutic methods of the invention comprise systemic or local administration of the composition as a graft or device. The therapeutic compositions useful in the present invention, when administered, are, of course, in a pyrogen-free, physiologically acceptable form. Therapeutically useful agents other than activin-ActRII antagonists may also optionally be included in the compositions described above and may be administered concurrently or sequentially with the subject compounds (e.g., ActRIIa and ActRIIb polypeptides) in the methods of the invention.

activin-ActRII antagonists are typically administered parenterally. Pharmaceutical compositions suitable for parenteral administration may include one or more ActRII polypeptides in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that may be reconstituted into sterile injectable solutions or dispersions prior to use, which may include 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 nonaqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained in a variety of ways, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

In addition, the composition may be encapsulated or injected in a form that can be delivered to a target tissue site (e.g., bone marrow). In certain embodiments, the compositions of the present invention may comprise a matrix capable of delivering one or more therapeutic compounds (e.g., ActRIIa or ActRIIb polypeptides) to a target tissue site (e.g., bone marrow) to provide structure to developing tissue (developingtisue) and optimally capable of being resorbed by the body. For example, the matrix may provide for slow release of an ActRII polypeptide. Such matrices may be formed from materials currently employed in other implanted medical applications.

The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, decorative appearance and interfacial properties. The particular application of the subject compositions will define the appropriate formulation. Potential matrices for the composition may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, and polyanhydrides. Other potential materials are biodegradable and biocomponent defined, such as bone or dermal collagen. Other matrices are composed of pure proteins or extracellular matrix components. Other potential matrices are non-biodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminate or other ceramics. The matrix may be composed of a combination of any of the above material types, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. Bioceramics can modify composition, for example to calcium-aluminate-phosphate and by processing to change pore size, particle shape and biodegradability.

In certain embodiments, the methods of the present invention may be administered in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, typically sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base such as gelatin and glycerin or sucrose and acacia) and/or as a mouthwash, each containing a predetermined amount of the agent as the active ingredient. The therapeutic agent can also be administered as a pill, 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 admixed with one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binding agents, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, for example, cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; and (10) a colorant. For capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using lactose or milk lactose, as well as high molecular weight polyethylene glycols and the like as excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, the oral compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, odorant, and preservative agents.

In addition to the active compounds, suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methylhydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof, may be included in the suspension.

The compositions of the present invention may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol sorbic acid, and the like). It may also be desirable to include isotonic agents, for example, sugars, sodium chloride and the like in the compositions. In addition, sustained absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption (e.g., aluminum monostearate and gelatin).

It is understood that the dosing regimen may be determined by the attending physician in light of various factors that alter the action of the compounds of the subject invention (e.g., ActRIIa and ActRIIb polypeptides). Such factors include, but are not limited to, the number of red blood cells in the patient, the hemoglobin level or other diagnostic assessment, the desired target number of red blood cells, the age, sex, and diet of the patient, the severity of any disease that may result in a decrease in red blood cell levels, the time of administration, and other clinical factors. The addition of other known growth factors to the final composition may also affect the dosage. Progression can be monitored by periodic assessment of red blood cell and hemoglobin levels, as well as assessment of reticulocyte levels and other indicators of hematopoietic processes.

Experiments with primates and humans have demonstrated that the effect of the compound on red blood cell levels is measurable when ActRIIa-Fc is administered at intervals and amounts sufficient to achieve serum concentrations of about 100ng/ml or greater over a period of at least about 20-30 days. Doses that achieve serum levels of 200ng/ml, 500ng/ml, 1000ng/ml or higher over a period of at least 20-30 days may also be used. While substantial effects on bone are observed at serum levels of about 200ng/ml, substantial effects begin at about 1000ng/ml or more for a period of at least about 20-30 days. Thus, if one wants to achieve an effect on red blood cells while having a minor effect on bone, the dosing regimen can be designed to deliver a serum concentration of between 100 and 1000ng/ml over a period of about 20-30 days. For humans, a serum level of 200ng/ml may be achieved by a single dose of 0.1mg/kg or higher, while a serum level of 1000ng/ml may be achieved by a single dose of 0.3mg/kg or higher. The serum half-life observed for this molecule is between about 20-30 days, much longer than most Fc fusion proteins, and thus sustained effective serum levels can be achieved by, for example, administering about 0.05-0.5mg/kg weekly or biweekly, or higher doses can be used with longer dosing intervals. For example, a dosage of 0.1-1mg/kg is used monthly or bimonthly.

In certain embodiments, the present disclosure also provides gene therapy for the in vivo production of ActRII polypeptides. Such treatment may be effected by introducing an ActRIIa or ActRIIb polynucleotide sequence into a cell or tissue having the above-listed diseases. The delivery of an ActRII polynucleotide sequence may be accomplished using a recombinant expression vector (e.g., a chimeric virus or a colloidal dispersion system). Therapeutic delivery of ActRII polynucleotide sequences is preferably performed using targeted liposomes.

Various viral vectors taught herein that may be used in gene therapy include adenovirus, herpes virus, vaccinia or RNA viruses such as retrovirus. The retroviral vector may be a derivative of a murine or avian retrovirus. Examples of retroviral vectors into which a single foreign gene can be inserted include, but are not limited to: moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). Some other retroviral vectors can integrate multiple genes. All of these vectors can deliver or integrate a selectable marker gene so that transduced cells can be identified and generated. Retroviral vectors can have targeting specificity by linking, for example, a sugar, glycolipid, or protein. Targeting is preferably achieved by using antibodies. One skilled in the art will recognize that a particular polynucleotide sequence may be inserted into the retroviral genome or linked to the viral envelope to allow for targeted specific delivery of a retroviral vector comprising the ActRII polynucleotide.

Alternatively, tissue culture cells can be directly transfected by conventional calcium phosphate transfection using plasmids encoding the retroviral structural genes gag, pol and env. 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 ActRII polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes). The preferred colloidal system of the present invention is a liposome. Liposomes are artificial membrane vesicles that can be used as delivery vehicles in vitro and in vivo. RNA, DNA, and intact virus particles can be encapsulated inside the aqueous phase and delivered to the cells in a biologically active form (see, e.g., franley et al, trends biochem. sci., 6:77, 1981). Methods for efficient gene delivery using liposome carriers are known in the art, see, e.g., Mannino et al, Biotechniques, 6:682, 1988. Liposomes are generally composed of a combination of phospholipids, often in combination with steroids (particularly cholesterol). Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.

Examples of lipids that can be used to generate liposomes 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.

Examples

The present invention will now be generally described, and can be readily understood by reference to the following examples, which are included merely for purposes of illustration of certain embodiments and embodiments of the present invention, and are not intended to limit the invention.

Example 1: ActRIIa-Fc fusion polypeptides

Applicants constructed soluble ActRIIa fusion proteins with a human ActRIIa extracellular domain linked to a human or mouse Fc domain by a minimal linker. This construct was called ActRIIa-hFc and ActRIIa-mFc, respectively.

ActRIIa-hFc purified from the CHO cell line is shown below (SEQ ID NO: 7):

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

ActRIIa-hFc and ActRIIa-mFc proteins were expressed in CHO cell lines. Different leader sequences may be considered:

(i) bee Melittin (IIBML): MKFLVNVALVFMVVYISYIYA (SEQIDNO:8)

(ii) Tissue Plasminogen Activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO:9)

(iii) Nature: MGAAAKLAFAVFLISCSSGA (SEQ ID NO: 10).

The selected format employed a TPA leader sequence and had the following unprocessed amino acid sequence:

MDAMKRGLCCVLLLCGAVFVSPGAAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQIDNO:13)

the polypeptide is encoded by the following nucleic acid sequence:

ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCGGCGCCGCTATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATTGAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTCTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCGGAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCCACCGGTGGTGGAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGTCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGAATTC(SEQIDNO:14)

both ActRIIa-hFc and ActRIIa-mFc were highly amenable to recombinant expression. As shown in fig. 1, the protein was purified as a single protein with a good peak profile. N-terminal sequencing showed the individual sequence-ILGRSTQE (SEQ ID NO: 11). Purification can be achieved by a series of column chromatography steps, including, for example, the following three or more steps in any order: protein a chromatography, Q sepharose chromatography, phenyl sepharose chromatography, size exclusion chromatography, and cation exchange chromatography. This purification can be accomplished by virus filtration and buffer exchange. The ActRIIa-hFc protein was purified to > 98% purity as determined by size exclusion chromatography and > 95% purity as determined by SDSPAGE.

ActRIIa-hFc and ActRIIa-mFc showed high affinity for ligands, particularly activin A. GDF-11 or activin A ("ActA") was immobilized on a BiacoreCM5 chip by standard amine coupling procedures. Loading ActRIIa-hFc and ActRIIa-mFc proteinsTo the system and binding is measured. ActRIIa-hFc at 5x10¹²Dissociation constant (K) of_D) Binding to activin, whereas the protein was expressed at 9.96X10^-9K of_DBinds to GDF 11. See fig. 2. ActRIIa-mFc behaved similarly.

ActRIIa-hFc was very stable in pharmacokinetic studies. Rats were administered a 1mg/kg, 3mg/kg, or 10mg/kg dose of the ActRIIa-hFc protein and plasma levels of the protein were measured at 24, 48, 72, 144, and 168 hours. In another study, rats were given a dose of 1mg/kg, 10mg/kg or 30 mg/kg. ActRIIa-hFc had a serum half-life of 11-14 days in rats, and circulating levels of this drug were fairly high after two weeks (11. mu.g/ml, 110. mu.g/ml or 304. mu.g/ml after initial administration of 1mg/kg, 10mg/kg or 30mg/kg, respectively). In cynomolgus monkeys, the plasma half-life was much greater than 14 days, and the circulating levels of the drug after initial administration of 1mg/kg, 10mg/kg or 30mg/kg were 25. mu.g/ml, 304. mu.g/ml or 1440. mu.g/ml, respectively.

Example 2: ActRIIa-hFc increases erythrocyte levels in non-human primates

The study was divided into four groups of five male and five female cynomolgus monkeys, with three of each sex in each group being scheduled to be stopped on day 29 and two of each sex in each group being scheduled to be stopped on day 57. Each animal was administered vehicle (group 1) or ActRIIa-Fc at a dose of 1, 10 or 30mg/kg by Intravenous (IV) injection on days 1, 8, 15 and 22 (groups 2, 3 and 4). The dose volume was maintained at 3 mL/kg. Red blood cell levels were measured 2 days before the first administration and 15, 29 and 57 days after the first administration (for the remaining two animals).

ActRIIa-hFc induced statistically significant increases in mean red blood cell parameters (red blood cell count [ RBC ], hemoglobin [ HGB ], hematocrit [ HCT ]) in males and females at all dose levels and time points throughout the study, with increases in absolute and relative reticulocyte counts (ARTC; RTC). See fig. 3-6.

Statistical significance was calculated for each treatment group relative to the baseline mean of that treatment group.

It is clear that the magnitude of the increase in red blood cell number and hemoglobin level is approximately equal to the effect reported for erythropoietin. The onset of these effects of ActRIIa-Fc was more rapid than that of erythropoietin.

Similar results were observed in rats and mice.

Example 3: ActRIIa-hFc increases red blood cell levels in human patients

The fusion protein described in example 1 was administered to human patients in a randomized, double-blind, placebo-controlled study, which was used primarily to assess the safety of the protein in healthy, postmenopausal women. 48 subjects were randomized into 6 groups to receive a single dose of ActRIIa-hFc or placebo (5 active: 1 placebo). The dosage level ranges from 0.01-3.0mg/kg for Intravenous (IV) and 0.03-0.1mg/kg for Subcutaneous (SC). All subjects were followed for 120 days. In addition to Pharmacokinetic (PK) analysis, ActRIIa-hFc biological activity was also assessed by measuring biomarkers of bone formation and resorption as well as FSH levels.

To look for potential changes, hemoglobin and RBC counts for each subject were examined in detail during the study and compared to baseline levels. Platelet counts were compared to controls at the same time. The platelet count did not change clinically significant to the baseline values over time.

PK analysis of ActRIIa-hFc showed a dose-linear curve with an average half-life of about 25-32 days. The area under the curve (AUC) of ActRIIa-hFc correlated linearly with dose, and absorption was essentially complete after SC administration (see fig. 7 and 8). These data show that SC is an ideal route of administration as it provides equivalent bioavailability and serum half-life for the drug, while avoiding the peak serum concentration of the drug that occurs within the first few days of IV administration (see fig. 8). ActRIIa-hFc caused a rapid, sustained dose-dependent increase in serum levels of bone-specific alkaline phosphatase (BAP), a marker for synthetic bone growth, and a dose-dependent decrease in C-terminal type 1 collagen telopeptide and anti-tartrate acid phosphatase 5b levels, a marker for bone resorption. Other markers (e.g., P1NP) show inconclusive results. BAP levels show near saturation effects at the highest drug dose, indicating that a half-maximal effect on this synthetic bone marker can be achieved at a dose of 0.3mg/kg, which can rise up to 3 mg/kg. EC50 was calculated to be 51,465 (days × ng/ml) from the relationship between the pharmacodynamic action of the drug and AUC. See fig. 9. At the highest dose level tested, changes in these bone markers lasted about 120 days. Consistent with the inhibition of activin, there is also a dose-dependent decrease in serum FSH levels.

Overall, there was only minimal non-drug related hemoglobin reduction in the first week of the study, which may be associated with the phlebotomy bleeding study of the 0.01 and 0.03mg/kg groups, whether IV or SC administration. The 0.1mg/kgSC and IV hemoglobin results remained stable or showed a slight increase on days 8-15. At the 0.3mg/kgIV dose level, a significant rise in HGB levels was seen as early as day 2, with a peak typically occurring at days 15-29, which did not occur in placebo subjects. At this point, the change did not reach statistical significance.

Overall, ActRIIa-hFc showed a dose-dependent effect on red blood cell number and reticulocyte number. A summary of hematological changes is seen in figures 10-13.

Example 4: alternative ActRIIa-Fc proteins

A variety of ActRIIa variants that can be used with reference to the methods described herein are described in International patent application published as WO2006/012627 (see, e.g., pages 55-58), the entire contents of which are incorporated herein by reference. Alternative constructs may delete the C-terminal tail (the last 15 amino acids of the ActRIIa extracellular domain). The sequence of this construct is shown below (the Fc portion is underlined) (SEQ ID NO: 12):

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMTGGGTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK

example 5: ActRIIB-Fc fusion polypeptides

Applicants constructed soluble ActRIIb fusion proteins with a human ActRIIb extracellular domain linked to a human Fc domain. The co-crystal structure of activin and extracellular ActRIIb did not show any effect of the last (C-terminal) 15 amino acids of the extracellular domain (referred to herein as the "tail") in ligand binding. The sequence was not resolved from the crystal structure (resolve), suggesting that these residues are present in flexible loops (flexleboops) that are not uniformly packed in the crystal. Thompson et al EMBOJ.2003Apr1; 22(7):1555-66. This sequence is poorly conserved between ActRIIb and ActRIIa. Thus, these residues are deleted in the basic, or background, ActRIIb-Fc fusion construct. In addition, position 64 in the background form is occupied by alanine, which is generally considered to be the "wild-type" form, despite the naturally occurring a64R allele. Thus, background ActRIIB-Fc fusions have the following sequence (with the Fc portion underlined) (SEQ ID NO: 20):

SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGGTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK

surprisingly, it has been found that this C-terminal tail enhances activin binding to GDF-11, and thus the preferred form of ActRIIb-Fc has the following sequence (with the Fc portion underlined) (SEQ ID NO: 21):

SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

a variety of ActRIIb variants that can be used with reference to the methods described herein are described in International patent application published as WO2006/012627 (see, e.g., pages 59-60), the entire contents of which are incorporated herein by reference.

Example 6: ActRIIb-hFc stimulation of erythropoiesis in non-human primates

ActRIIb-hfc (iggl) was administered to male and female cynomolgus monkeys once a week over 1 month by subcutaneous injection. 48 cynomolgus monkeys (24/sex) were assigned to one of four treatment groups (6 animals/sex/group) and injected subcutaneously with vehicle or 3, 10 or 30mg/kg of ActRIIb-hFc once a week for 4 weeks (5 doses total). Parameters evaluated included general clinical pathology (hematology, clinical chemistry, coagulation and urinalysis). ActRIIb-hFc caused a statistically significant increase in mean absolute reticulocyte values in treated animals by day 15. By day 36, ActRIIb-hFc caused a number of hematological changes, including rising mean absolute reticulocyte and erythrocyte distribution width values and decreasing mean cellular hemoglobin concentrations. All treatment groups and both sexes were affected. These effects are consistent with a positive effect of ActRIIb-hFc on immature reticulocyte release from bone marrow. This effect was reversed after the drug had cleared from the treated animals (by day 56 of the study). Therefore, we conclude that ActRIIb-hFc stimulates erythropoiesis.

Incorporation by reference

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 matter have been discussed, the foregoing description is intended to be illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims that follow. The full scope of the invention should be determined with reference to the claims, along with their full scope of equivalents, and the specification and modifications.

Claims

1. Use of a polypeptide in the manufacture of a medicament for treating myelodysplastic syndrome, treating hemoglobinopathy, treating thalassemia, treating sickle cell disease, increasing red blood cell levels, or increasing hemoglobin levels in a patient in need thereof, wherein the polypeptide is selected from the group consisting of:

a) a polypeptide comprising an amino acid sequence having at least 90% identity to seq id No. 2;

b) a polypeptide comprising an amino acid sequence having at least 90% identity to seq id No. 3;

c) a polypeptide comprising an amino acid sequence having at least 90% identity to seq id No. 7;

d) polypeptide comprising an amino acid sequence having at least 90% identity to seq id No. 12

e) A polypeptide comprising an amino acid sequence having at least 90% identity to seq id No. 16;

f) a polypeptide comprising an amino acid sequence having at least 90% identity to seq id No. 17;

g) a polypeptide comprising an amino acid sequence having at least 90% identity to seq id No. 20; and

h) a polypeptide comprising an amino acid sequence having at least 90% identity to seq id No. 21.

2. A polypeptide for use in treating myelodysplastic syndrome, treating hemoglobinopathy, treating thalassemia, treating sickle cell disease, increasing red blood cell levels, or increasing hemoglobin levels in a patient in need thereof, wherein the polypeptide is selected from the group consisting of: :

3. The use or polypeptide of claim 1 or 2, wherein the polypeptide binds activin and/or GDF 11.

4. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises an amino acid sequence having at least 90% identity with seq id No. 3.

5. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises an amino acid sequence having at least 95% identity with seq id No. 3.

6. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises the amino acid sequence of seq id No. 3.

7. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises an amino acid sequence having at least 90% identity to seq id No. 2.

8. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises an amino acid sequence having at least 95% identity to seq id No. 2.

9. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises the amino acid sequence of seq id No. 2.

10. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises an amino acid sequence having at least 90% identity with seq id No. 17.

11. The use according to claim 1, wherein the polypeptide comprises an amino acid sequence having at least 95% identity to seq id No. 17.

12. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises the amino acid sequence of seq id No. 17.

13. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises an amino acid sequence having at least 90% identity with seq id No. 16.

14. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises an amino acid sequence having at least 95% identity with seq id No. 16.

15. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises the amino acid sequence of seq id No. 16.

16. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises an amino acid sequence having at least 90% identity with seq id No. 7.

17. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises an amino acid sequence having at least 95% identity with seq id No. 7.

18. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises the amino acid sequence of seq id No. 7.

19. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises an amino acid sequence having at least 90% identity with seq id No. 12.

20. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises an amino acid sequence having at least 95% identity with seq id No. 12.

21. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises the amino acid sequence of seq id No. 12.

22. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises an amino acid sequence having at least 90% identity with seq id No. 20.

23. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises an amino acid sequence having at least 95% identity with seq id No. 20.

24. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises the amino acid sequence of seq id No. 20.

25. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises an amino acid sequence having at least 90% identity with seq id No. 21.

26. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises an amino acid sequence having at least 95% identity with seq id No. 21.

27. The use or polypeptide according to claim 1 or 2, wherein the polypeptide comprises the amino acid sequence of seq id No. 21.

28. The use or polypeptide of claim 1 or 2, wherein the polypeptide binds activin.

29. The use of claim 28, wherein the polypeptide binds to activin a.

30. The use of claim 28, wherein the polypeptide binds to activin B.

31. The use or polypeptide of claim 1 or 2, wherein the polypeptide binds GDF 11.

32. The use or polypeptide of claim 1 or 2, wherein the polypeptide binds activin and GDF 11.

33. The use or polypeptide of claim 1 or 2, wherein the polypeptide has one or more of the following characteristics:

i) at least 10^-7K of M_DBinding an ActRII ligand; and

ii) inhibiting ActRII signaling in the cell.

34. The use or polypeptide according to claim 1 or 2, wherein the polypeptide is a fusion protein comprising an immunoglobulin Fc domain.

35. The use of claim 34, wherein the immunoglobulin Fc domain is an IgG1Fc domain.

36. The use or polypeptide of claim 1 or 2, wherein the polypeptide comprises one or more modified amino acid residues selected from the group consisting of: glycosylated amino acids, pegylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, and amino acids conjugated to a lipid moiety.

37. The use of claim 34, wherein the medicament is for administration such that a serum concentration of at least 100ng/ml of fusion protein is achieved in the patient for a period of about 20 to 30 days.

38. The use of claim 34, wherein the medicament is for administration such that a serum concentration in the range of 100ng/ml to 1000ng/ml of fusion protein is achieved in the patient.

39. The use of claim 34, wherein the fusion protein has a serum half-life of between 15 and 30 days.

40. The use of claim 34, wherein the medicament is for administration to the patient at a frequency of no more than once per week.

41. The use of claim 34, wherein the medicament is for administration to the patient at a frequency of no more than once a month.

42. The use or polypeptide of any one of claims 1-41 for the preparation of a medicament for the treatment of myelodysplastic syndrome.

43. The use or polypeptide of any one of claims 1-41 for the preparation of a medicament for the treatment of hemoglobinopathies.

44. The use or polypeptide of claim 43, wherein the hemoglobinopathy is a sickle cell disease.

45. The use or polypeptide of claim 43, wherein the hemoglobinopathy is thalassemia.

46. Use or polypeptide according to any one of claims 1 to 42 for the preparation of a medicament for increasing red blood cell levels.

47. Use or polypeptide according to any one of claims 1 to 42 for the preparation of a medicament for increasing hemoglobin levels.

48. Use of an activin-ActRII antagonist in the manufacture of a medicament for treating a myelodysplastic syndrome, treating a hemoglobinopathy, treating a thalassemia, treating a sickle cell disease, elevating a level of red blood cells, or elevating a level of hemoglobin in a patient in need thereof, wherein the activin-ActRII antagonist is selected from the group consisting of:

a) an activin-binding antibody;

b) an antibody that binds ActRII; and

c) follistatin; and

d) a statin.

49. An antagonist for use in treating myelodysplastic syndrome, treating hemoglobinopathy, treating thalassemia, treating sickle cell disease, increasing red blood cell levels, or increasing hemoglobin levels in a patient in need thereof, wherein said antagonist is selected from the group consisting of:

a) an activin-binding antibody;

b) an antibody that binds ActRII; and

c) follistatin; and

d) a statin.

50. The use or antagonist of claim 49, wherein the activin-ActRII antagonist is an antibody that disrupts activin-ActRIIa binding.

51. The use or antagonist of claim 49, wherein the activin-ActRII antagonist is an antibody that binds activin A.

52. The use or antagonist of claim 49, wherein the activin-ActRII antagonist is an antibody that binds activin B.

53. The use or antagonist of claim 49, wherein the activin-ActRII antagonist is follistatin.

54. The use or antagonist of claim 53, wherein the activin-ActRII antagonist is follistatin-288.

55. The use or antagonist of claim 53, wherein the activin-ActRII antagonist is follistatin-315.

56. The use or antagonist of claim 49, wherein the activin-ActRII antagonist is a statin.

57. The use or antagonist of claim 49, wherein the polypeptide has one or more of the following characteristics:

i) at least 10^-7K of M_DBinding an ActRII ligand; and

ii) inhibiting ActRII signaling in the cell.

58. The use or antagonist of claim 49, wherein the medicament is for administration such that a serum concentration of at least 100ng/ml of the activin-ActRII antagonist is achieved in the patient for a period of from about 20 to 30 days.

59. The use or antagonist of claim 49, wherein the medicament is for administration such that a serum concentration in the range of 100ng/ml to 1000ng/ml of the activin-ActRII antagonist is achieved in the patient.

60. The use or antagonist of claim 49, wherein the fusion protein has a serum half-life of between 15 and 30 days.

61. The use or antagonist of claim 49, wherein the medicament is for administration to the patient at a frequency of no more than once per week.

62. The use or antagonist of claim 49, wherein the medicament is for administration to the patient at a frequency of no more than once a month.

63. The use or antagonist of any one of claims 49-61 for the preparation of a medicament for the treatment of myelodysplastic syndrome.

64. The use or antagonist of any one of claims 49-61 for the preparation of a medicament for the treatment of hemoglobinopathies.

65. The use or antagonist of claim 64 wherein the hemoglobinopathy is a sickle cell disease.

66. The use or antagonist of claim 64, wherein the hemoglobinopathy is thalassemia.

67. The use or antagonist of any one of claims 49-61 for the preparation of a medicament for increasing red blood cell levels.

68. The use or antagonist of any one of claims 49-61 for the preparation of a medicament for increasing hemoglobin levels.