US20100015144A1 - Methods for dosing an activin-actriia antagonist and monitoring of treated patients - Google Patents

Methods for dosing an activin-actriia antagonist and monitoring of treated patients Download PDF

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US20100015144A1
US20100015144A1 US12/459,202 US45920209A US2010015144A1 US 20100015144 A1 US20100015144 A1 US 20100015144A1 US 45920209 A US45920209 A US 45920209A US 2010015144 A1 US2010015144 A1 US 2010015144A1
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actriia
activin
patient
amino acid
polypeptide
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Matthew L. Sherman
Niels Borgstein
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Acceleron Pharma Inc
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Assigned to ACCELERON PHARMA, INC. reassignment ACCELERON PHARMA, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BORGSTEIN, NIELS, SHERMAN, MATTHEW L.
Publication of US20100015144A1 publication Critical patent/US20100015144A1/en
Priority to US15/005,762 priority patent/US20160279203A1/en
Priority to US16/821,593 priority patent/US20210038689A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1796—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
    • G01N33/721—Haemoglobin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/90—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving iron binding capacity of blood
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • TGF-beta The transforming growth factor-beta (TGF-beta) superfamily contains a variety of growth factors that share common sequence elements and structural motifs. These proteins are known to exert biological effects on a large variety of cell types in both vertebrates and invertebrates. Members of the superfamily perform important functions during embryonic development in pattern formation and tissue specification 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 general branches: the BMP/GDF and the TGF-beta/Activin/BMP10 branches, whose members have diverse, often complementary effects.
  • the Piedmontese and Belgian Blue cattle breeds carry a loss-of-function mutation in the GDF8 (also called myostatin) gene that causes a marked increase in muscle mass. Grobet et al., Nat Genet. 1997, 17(1):71-4. Furthermore, in humans, inactive alleles of GDF8 are associated with increased muscle mass and, reportedly, exceptional strength. Schuelke et al., N Engl J Med 2004, 350:2682-8.
  • Changes in muscle, bone, cartilage and other tissues may be achieved by agonizing or antagonizing signaling that is mediated by an appropriate TGF-beta family member. It is an object of the present disclosure to provide alternative methods for administering modulators of the TGF-beta superfamily to patients.
  • the disclosure relates to methods for administering activin antagonists, as well as ActRIIa antagonists (collectively, “activin-ActRIIa antagonists”), to patients in a manner that is appropriate given the effects that such antagonists can have on a variety of tissues, including red blood cells.
  • activin-ActRIIa antagonists can increase red blood cell and hemoglobin levels and also increase bone density. This dual effect has particular advantages in patients that have both anemia and bone loss, such as many cancer patients (where anemia and bone loss can be a consequence of the tumor or a consequence of irradiation or chemotherapy), patients with osteoporosis and patients with renal failure.
  • soluble form of ActRIIa acts as an inhibitor of activin and, when administered in vivo, increases red blood cell levels. While soluble ActRIIa may affect red blood cell levels through a mechanism other than activin antagonism, the disclosure nonetheless demonstrates that desirable therapeutic agents may be selected on the basis of activin antagonism or ActRIIa antagonism or both. Such agents are referred to collectively as activin-ActRIIa antagonists.
  • activin-ActRIIa antagonists also have a variety of other therapeutic uses including, for example, promoting bone growth, decreasing FSH levels, treating multiple myeloma and treating breast cancer.
  • an activin-ActRIIa antagonists when administering an activin-ActRIIa antagonists for promoting bone growth or treating breast cancer, it may be desirable to monitor the effects on red blood cells during administration of an activin-ActRIIa antagonists, or to determine or adjust the dosing of an activin-ActRIIa antagonists, in order to reduce undesired effects on red blood cells. For example, excessive increases in red blood cell levels, hemoglobin levels, or hematocrit levels may cause increases in blood pressure or other undesirable side effects. It may also be desirable to restrict dosing of activin-ActRIIa antagonists to patients who have appropriate hematologic parameters.
  • hemoglobin level below normal (e.g., below 12 g/dL, below 11 g/dL, below 10 g/dL or below 9 g/dL or lower).
  • the disclosure provides methods for managing a patient that has been treated with, or is a candidate to be treated with, an activin-ActRIIa antagonist, including, for example, activin-binding ActRIIa polypeptides, anti-activin antibodies, anti-ActRIIa antibodies, activin- or ActRIIa-targeted small molecules and aptamers, and nucleic acids that decrease expression of activin or ActRIIa, by monitoring in the patient one or more hematologic parameters that correlate with an increase in red blood cell levels, such as, for example, red blood cell levels, blood pressure, or iron stores.
  • an activin-ActRIIa antagonist including, for example, activin-binding ActRIIa polypeptides, anti-activin antibodies, anti-ActRIIa antibodies, activin- or ActRIIa-targeted small molecules and aptamers, and nucleic acids that decrease expression of activin or ActRIIa, by monitoring in the patient one or more hematologic parameters that correlate with an increase in red blood cell levels, such as
  • the disclosure provides polypeptides comprising a soluble, activin-binding ActRIIa polypeptide that binds to activin.
  • ActRIIa polypeptides may be formulated as a pharmaceutical preparation comprising the activin-binding ActRIIa polypeptide and a pharmaceutically acceptable carrier.
  • the activin-binding ActRIIa polypeptide may bind to activin with a KD less than 1 micromolar or less than 100, 10 or 1 nanomolar.
  • the activin-binding ActRIIa polypeptide selectively binds activin versus GDF11 and/or GDF8, and optionally with a KD that is at least 10-fold, 20-fold or 50-fold lower with respect to activin than with respect to GDF11 and/or GDF8. While not wishing to be bound to a particular mechanism of action, it is expected that this degree of selectivity for activin inhibition over GDF11/GDF8 inhibition in ActRIIa-Fc accounts for effects on bone or erythropoiesis without a consistently measurable effect on muscle. In many embodiments, an ActRIIa polypeptide will be selected for causing less than 15%, less than 10% or less than 5% increase in muscle at doses that achieve desirable effects on red blood cell levels.
  • the composition may be at least 95% pure, with respect to other polypeptide components, as assessed by size exclusion chromatography, and optionally, the composition is at least 98% pure.
  • An activin-binding ActRIIa polypeptide for use in such a preparation may be any of those disclosed herein, such as a polypeptide having (i.e. comprising) an amino acid sequence selected from SEQ ID NOs: 2, 3, 7, 12 or 13, or having (i.e. comprising) an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97% or 99% identical to an amino acid sequence selected from SEQ ID NOs: 2, 3, 7, 12 or 13.
  • An activin-binding ActRIIa polypeptide may include a functional fragment of a natural ActRIIa polypeptide, such as one comprising at least 10, 20 or 30 amino acids of a sequence selected from SEQ ID NOs: 1-3 or a sequence of SEQ ID NO: 2, lacking the C-terminal 10 to 15 amino acids (the “tail”).
  • a soluble, activin-binding ActRIIa polypeptide may include one or more alterations in the amino acid sequence (e.g., in the ligand-binding domain) relative to a naturally occurring ActRIIa polypeptide.
  • altered ActRIIa polypeptides are provided in WO 2006/012627, pp. 59-60 and pp. 55-58, respectively, which is incorporated by reference herein, and throughout U.S. patent application Ser. No. 12/012,652, incorporated by reference herein.
  • the alteration in the amino acid sequence may, for example, alter glycosylation of the polypeptide when produced in a mammalian, insect or other eukaryotic cell or alter proteolytic cleavage of the polypeptide relative to the naturally occurring ActRIIa polypeptide.
  • An activin-binding ActRIIa polypeptide may be a fusion protein that has, as one domain, an ActRIIa polypeptide, (e.g., a ligand-binding portion of an ActRIIa) and one or more additional domains that provide a desirable property, such as improved pharmacokinetics, easier purification, targeting to particular tissues, etc.
  • 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 protein complexes, multimerization of the fusion protein, and/or purification.
  • An activin-binding ActRIIa fusion protein may include an immunoglobulin Fc domain (wild-type or mutant) or a serum albumin or other polypeptide portion that provides desirable properties such as improved pharmacokinetics, improved solubility or improved stability.
  • an ActRIIa-Fc fusion comprises a relatively unstructured linker positioned between the Fc domain and the extracellular ActRIIa domain.
  • This unstructured linker may be an artificial sequence of 1, 2, 3, 4 or 5 amino acids or a length of between 5 and 15, 20, 30, 50 or more amino acids that are relatively free of secondary structure, or a mixture of both.
  • a linker may be rich in glycine and proline residues and may, for example, contain a single sequence of threonine/serine and glycines or repeating sequences of threonine/serine and glycines (e.g., TG 4 (SEQ ID NO: 15) or SG 4 (SEQ ID NO: 16) singlets or repeats).
  • a fusion protein may include a purification subsequence, such as an epitope tag, a FLAG tag, a polyhistidine sequence, and a GST fusion.
  • a soluble ActRIIa polypeptide includes one or more modified amino acid residues selected from: a glycosylated amino acid, a PEGylated amino acid, a famesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, and an amino acid conjugated to an organic derivatizing agent.
  • a pharmaceutical preparation may also include one or more additional compounds such as a compound that is used to treat a bone disorder or a compound that is used to treat anemia.
  • a pharmaceutical preparation is substantially pyrogen free.
  • an ActRIIa protein be expressed in a mammalian cell line that mediates suitably natural glycosylation of the ActRIIa protein so as to diminish the likelihood of an unfavorable immune response in a patient.
  • Human and CHO cell lines have been used successfully, and it is expected that other common mammalian expression systems will be useful.
  • ActRIIa proteins designated ActRIIa-Fc have desirable properties, including selective binding to activin versus GDF8 and/or GDF11, high affinity ligand binding and serum half life greater than two weeks in animal models and in human patients.
  • the invention provides ActRIIa-Fc polypeptides and pharmaceutical preparations comprising such polypeptides and a pharmaceutically acceptable excipient.
  • the disclosure provides nucleic acids encoding a soluble activin-binding ActRIIa polypeptide.
  • An isolated polynucleotide may comprise a coding sequence for a soluble, activin-binding ActRIIa polypeptide, such as described above.
  • an isolated nucleic acid may include a sequence coding for an extracellular domain (e.g., ligand-binding domain) of an ActRIIa and a sequence that would code for part or all of the transmembrane domain and/or the cytoplasmic domain of an ActRIIa, but for a stop codon positioned within the transmembrane domain or the cytoplasmic domain, or positioned between the extracellular domain and the transmembrane domain or cytoplasmic domain.
  • an extracellular domain e.g., ligand-binding domain
  • an isolated polynucleotide may comprise a full-length ActRIIa polynucleotide sequence such as SEQ ID NO: 4 or a partially truncated version of ActRIIa, such as a nucleic acid comprising the nucleic acid sequence of SEQ ID NO:5, which corresponds to the extracellular domain of ActRIIa.
  • An isolated polynucleotide may further comprise a transcription termination codon at least six hundred nucleotides before the 3′-terminus or otherwise positioned such that translation of the polynucleotide gives rise to an extracellular domain optionally fused to a truncated portion of a full-length ActRIIa.
  • a preferred nucleic acid sequence for ActRIIa is SEQ ID NO:14.
  • Nucleic acids disclosed herein may be operably linked to a promoter for expression, and the disclosure provides cells transformed with such recombinant polynucleotides.
  • the cell is a mammalian cell such as a CHO cell.
  • the disclosure provides methods for making a soluble, activin-binding ActRIIa polypeptide.
  • a method may include expressing any of the nucleic acids (e.g., SEQ ID NO: 4, 5 or 14) disclosed herein in a suitable cell, such as a Chinese hamster ovary (CHO) cell or human cell.
  • a suitable cell such as a Chinese hamster ovary (CHO) cell or human cell.
  • Such a method may comprise: a) culturing a cell under conditions suitable for expression of the soluble ActRIIa polypeptide, wherein said cell is transformed with a soluble ActRIIa expression construct; and b) recovering the soluble ActRIIa polypeptide so expressed.
  • Soluble ActRIIa polypeptides may be recovered as crude, partially purified or highly purified fractions.
  • Purification may be achieved by a series of purification steps, including, for example, one, two or three or more of the following, in any order: protein A chromatography, anion exchange chromatography (e.g., Q sepharose), hydrophobic interaction chromatography (e.g., phenylsepharose), size exclusion chromatography, and cation exchange chromatography.
  • Soluble ActRIIa polypeptides may be formulated in liquid or solid (e.g., lyophilized) forms.
  • the disclosure provides a method for dosing a patient with an activin-ActRIIa antagonist, comprising dosing the patient in amounts and at intervals selected so as to reduce the risk of causing a rise in hemoglobin levels greater than 0.5 g/dL, 1 g/dl or 1.5 g/dL in two weeks.
  • the disclosure provides a method for administering an ActRIIa-Fc fusion protein to a patient, comprising administering the ActRIIa fusion protein no more frequently than once per 60 days, once per 90 days, or once per 120 days.
  • the patient may be a patient in need of bone growth or a patient suffering from or at risk for developing breast cancer or multiple myeloma, or a patient in need of having decreased FSH.
  • FIG. 1 shows the purification of ActRIIa-hFc expressed in CHO cells.
  • the protein purifies as a single, well-defined peak as visualized by sizing column (left panel) and Coomassie stained SDS-PAGE (right panel) (left lane: molecular weight standards; right lane: ActRIIa-hFc).
  • FIG. 2 shows the binding of ActRIIa-hFc to activin and GDF-11, as measured by BiaCoreTM assay.
  • FIG. 3 shows the effects of ActRIIa-hFc on red blood cell counts in female non-human primates.
  • Female cynomolgus monkeys (four groups of five monkeys each) were treated with placebo or 1 mg/kg, 10 mg/kg or 30 mg/kg of ActRIIa-hFc on day 0, day 7, day 14 and day 21.
  • FIG. 3A shows red blood cell (RBC) counts.
  • FIG. 3B shows hemoglobin levels. Statistical significance is relative to baseline for each treatment group. At day 57, two monkeys remained in each group.
  • FIG. 4 shows the effects of ActRIIa-hFc on red blood cell counts in male non-human primates.
  • Male cynomolgus monkeys (four groups of five monkeys each) were treated with placebo or 1 mg/kg, 10 mg/kg or 30 mg/kg of ActRIIa-hFc on day 0, day 7, day 14 and day 21.
  • FIG. 4A shows red blood cell (RBC) counts.
  • FIG. 4B shows hemoglobin levels. Statistical significance is relative to baseline for each treatment group. At day 57, two monkeys remained in each group.
  • FIG. 5 shows the effects of ActRIIa-hFc on reticulocyte counts in female non-human primates. Cynomolgus monkeys (four groups of five monkeys each) were treated with placebo or 1 mg/kg, 10 mg/kg or 30 mg/kg of ActRIIa-hFc on day 0, day 7, day 14 and day 21.
  • FIG. 5A shows absolute reticulocyte counts.
  • FIG. 5B shows the percentage of reticulocytes relative to RBCs. Statistical significance is relative to baseline for each group. At day 57, two monkeys remained in each group.
  • FIG. 6 shows the effects of ActRIIa-hFc on reticulocyte counts in female non-human primates. Cynomolgus monkeys (four groups of five monkeys each) were treated with placebo or 1 mg/kg, 10 mg/kg or 30 mg/kg of ActRIIa-hFc on day 0, day 7, day 14 and day 21.
  • FIG. 6A shows absolute reticulocyte counts.
  • FIG. 6B shows the percentage of reticulocytes relative to RBCs. Statistical significance is relative to baseline for each group. At day 57, two monkeys remained in each group.
  • FIG. 7 shows results from the human clinical trial described in Example 5, where the area-under-curve (AUC) and administered dose of ActRIIa-hFc have a linear correlation, regardless of whether ActRIIa-hFc was administered intravenously (IV) or subcutaneously (SC).
  • AUC area-under-curve
  • IV intravenously
  • SC subcutaneously
  • FIG. 8 shows a comparison of serum levels of ActRIIa-hFc in patients administered IV or SC.
  • FIG. 9 shows bone alkaline phosphatase (BAP) levels in response to different dose levels of ActRIIa-hFc.
  • BAP is a marker for anabolic bone growth.
  • FIG. 10 depicts the median change from baseline of hematocrit levels from the human clinical trial described in Example 5. ActRIIa-hFc was administered intravenously (IV) at the indicated dosage.
  • FIG. 11 depicts the median change from baseline of hemoglobin levels from the human clinical trial described in Example 5. ActRIIa-hFc was administered intravenously (IV) at the indicated dosage.
  • FIG. 12 depicts the median change from baseline of RBC (red blood cell) count from the human clinical trial described in Example 5. ActRIIa-hFc was administered intravenously (IV) at the indicated dosage.
  • FIG. 13 depicts the median change from baseline of reticulocyte count from the human clinical trial described in Example 5. ActRIIa-hFc was administered intravenously (IV) at the indicated dosage.
  • FIG. 14 shows an alignment of human ActRIIA and ActRIIB with the residues that are deduced herein, based on composite analysis of multiple ActRIIB and ActRIIA crystal structures to directly contact ligand (the ligand binding pocket) indicated with boxes.
  • FIG. 15 shows the effect of ActRIIA-mFc on hematocrit in a mouse model of chemotherapy-induced anemia. Data are means ⁇ SEM. *, P ⁇ 0.05 vs. vehicle at same time point. A single dose of ActRIIA-mFc before chemotherapy prevented the decline in hematocrit level otherwise observed after administration of the chemotherapeutic paclitaxel.
  • FIG. 16 shows the dose-dependent effect of ActRIIA-mFc on hematocrit in a mouse model of chemotherapy-induced anemia. Data are means ⁇ SEM. **, P ⁇ 0.01; ***, P ⁇ 0.001 vs. vehicle at same time point. Two weeks after paclitaxel administration, ActRIIA-mFc treatment increased hematocrit level as a function of dose number.
  • FIG. 17 shows the effect of ActRIIA-mFc on hematocrit in a partially nephrectomized (NEPHX) mouse model of chronic kidney disease. Data are means ⁇ SEM. *, P ⁇ 0.05 vs. vehicle at same time point. ActRIIA-mFc treatment prevented the decline in hematocrit level otherwise observed at 4 weeks and produced a beneficial trend in hematocrit at 8 weeks.
  • TGF-beta The transforming growth factor-beta (TGF-beta) superfamily contains a variety of growth factors that share common sequence elements and structural motifs. These proteins are known to exert biological effects on a large variety of cell types in both vertebrates and invertebrates. Members of the superfamily perform important functions during embryonic development in pattern formation and tissue specification 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 general branches: the BMP/GDF and the TGF-beta/Activin/BMP10 branches, whose members have diverse, often complementary effects.
  • the Piedmontese and Belgian Blue cattle breeds carry a loss-of-function mutation in the GDF8 (also called myostatin) gene that causes a marked increase in muscle mass. Grobet et al., Nat Genet. 1997, 17(1):71-4. Furthermore, in humans, inactive alleles of GDF8 are associated with increased muscle mass and, reportedly, exceptional strength. Schuelke et al., N Engl J Med 2004, 350:2682-8.
  • Activins are dimeric polypeptide growth factors that belong to the TGF-beta superfamily. There are three principal activin forms (A, B, and AB) that are homo/heterodimers of two closely related ⁇ subunits ( ⁇ A ⁇ A , ⁇ B ⁇ B , and ⁇ A ⁇ B , respectively).
  • the human genome also encodes an activin C and an activin E, which are primarily expressed in the liver, and heterodimeric forms containing ⁇ C or ⁇ E are also known.
  • activins are unique and multifunctional factors that can stimulate hormone production in ovarian and placental cells, support neuronal cell survival, influence cell-cycle progress positively or negatively depending on cell type, and induce mesodermal differentiation at least in amphibian embryos (DePaolo et al., 1991, Proc Soc Ep Biol Med. 198:500-512; Dyson et al., 1997, Curr Biol. 7:81-84; Woodruff, 1998, Biochem Pharmacol. 55:953-963).
  • erythroid differentiation factor isolated from the stimulated human monocytic leukemic cells was found to be identical to activin A (Murata et al., 1988, PNAS, 85:2434). It has been suggested that activin A promotes erythropoiesis in the bone marrow. In several tissues, activin signaling is antagonized by its related heterodimer, 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.
  • FSH follicle-stimulating hormone
  • Other proteins that may regulate activin bioactivity and/or bind to activin include follistatin (FS), follistatin-related protein (FSRP) and ⁇ 2 -macroglobulin.
  • TGF- ⁇ signals are mediated by heteromeric complexes of type I and type II serine/threonine kinase receptors, which phosphorylate and activate downstream Smad proteins upon ligand stimulation (Massagué, 2000, Nat. Rev. Mol. Cell Biol. 1:169-178).
  • type I and type II receptors are transmembrane proteins, composed of a ligand-binding extracellular domain with cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine specificity.
  • Type I receptors are essential for signaling; and type II receptors are required for binding ligands and for expression of type I receptors.
  • Type I and II activin receptors form a stable complex after ligand binding, resulting in phosphorylation of type I receptors by type II receptors.
  • ActRIIa and ActRIIb Two related type II receptors (ActRII), ActRIIa and ActRIIb, have been identified as the type II receptors for activins (Mathews and Vale, 1991, Cell 65:973-982; Attisano et al., 1992, Cell 68: 97-108). Besides activins, ActRIIa and ActRIIb can biochemically interact with several other TGF- ⁇ family proteins, including BMP7, Nodal, GDF8, and GDF11 (Yamashita et al., 1995, J. Cell Biol. 130:217-226; Lee and McPherron, 2001, Proc. Natl. Acad. Sci. 98:9306-9311; Yeo and Whitman, 2001, Mol.
  • ALK4 is the primary type I receptor for activins, particularly for activin A, and ALK-7 may serve as a receptor for activins as well, particularly for activin B.
  • sActRIIa a soluble ActRIIa polypeptide
  • GDF8 or GDF11 TGF-beta family members
  • hematopoiesis is a complex process, regulated by a variety of factors, including erythropoietin, G-CSF and iron homeostasis.
  • the terms “increase red blood cell levels” and “promote red blood cell formation” refer to clinically observable metrics, such as hematocrit, red blood cell counts and hemoglobin measurements, and are intended to be neutral as to the mechanism by which such changes occur.
  • activin-ActRIIa antagonists are useful for a variety of therapeutic applications, including, for example, promoting bone growth (see PCT Publication No. WO2007/062188, which is hereby incorporated by reference in its entirety), and treating breast cancer (see PCT Application No. PCT/US2008/001429, which is hereby incorporated by reference in its entirety).
  • promoting bone growth see PCT Publication No. WO2007/062188, which is hereby incorporated by reference in its entirety
  • treating breast cancer see PCT Application No. PCT/US2008/001429, which is hereby incorporated by reference in its entirety.
  • a dual effect on blood cells and bone or other tissue will be desirable, but it is generally recognized that pharmaceutically promoted increases in red blood cells, even up to a level that is typically considered normal, can have adverse effects on patients, and thus are often monitored or managed with care.
  • appropriate dosing including amounts and frequency of administration
  • therapeutic progress and effects on one or more hematologic parameters over time may be useful in managing patients being dosed with an activin-ActRIIa antagonist by facilitating patient care, determining appropriate maintenance dosing (both amounts and frequency), etc.
  • Activin-ActRIIa antagonists include, for example, activin-binding soluble ActRIIa polypeptides, antibodies that bind to activin (particularly the activin A or B subunits, also referred to as ⁇ A or ⁇ B ) and disrupt ActRIIa binding, antibodies that bind to ActRIIa and disrupt activin binding, non-antibody proteins selected for activin or ActRIIa binding (see e.g., WO/2002/088171, WO/2006/055689, and WO/2002/032925 for examples of such proteins and methods for design and selection of same), randomized peptides selected for activin or ActRIIa binding, often affixed to an Fc domain.
  • activin binders that block the type I (e.g., a soluble type I activin receptor) and type II (e.g., a soluble type II activin receptor) binding sites, respectively, may be linked together to create a bifunctional binding molecule.
  • Nucleic acid aptamers, small molecules and other agents that inhibit the activin-ActRIIa signaling axis are included as activin-ActRIIa antagonists.
  • activin-ActRIIa 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, FLRG, activin C, alpha(2)-macroglobulin, and an M108A (methionine to alanine change at position 108) mutant activin A.
  • inhibin i.e., inhibin alpha subunit
  • follistatin e.g., follistatin-288 and follistatin-315
  • FSRP follistatin-288 and follistatin-315
  • FLRG activin C
  • alpha(2)-macroglobulin e.g., follistatin-288 and follistatin-315
  • M108A methionine to alanine change at position 108 mutant activin A.
  • nucleic acids such as antisense molecules, siRNAs or ribozymes that inhibit activin A, B, C or E, or, particularly, ActRIIa expression, can be used as activin-ActRIIa antagonists.
  • the activin-ActRIIa antagonist to be used may exhibit selectivity for inhibiting activin-mediated signaling versus other members of the TGF-beta family, and particularly with respect to GDF8 and GDF11.
  • “About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values.
  • the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.
  • the methods of the invention may include steps of comparing sequences to each other, including wild-type sequence to one or more mutants (sequence variants).
  • Such comparisons typically comprise alignments of polymer sequences, e.g., using sequence alignment programs and/or algorithms that are well known in the art (for example, BLAST, FASTA and MEGALIGN, to name a few).
  • sequence alignment programs and/or algorithms that are well known in the art (for example, BLAST, FASTA and MEGALIGN, to name a few).
  • sequence alignment programs and/or algorithms that are well known in the art (for example, BLAST, FASTA and MEGALIGN, to name a few).
  • sequence similarity in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin.
  • homologous when modified with an adverb such as “highly,” may refer to sequence similarity and may or may not relate to a common evolutionary origin.
  • the present invention relates to ActRIIa polypeptides.
  • ActRIIa refers to a family of activin receptor type IIa (ActRIIa) proteins from any species and variants derived from such ActRIIa proteins by mutagenesis or other modification. Reference to ActRIIa herein is understood to be a reference to any one of the currently identified forms.
  • ActRIIa Members of the ActRIIa family are generally transmembrane proteins, composed of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine kinase activity.
  • ActRIIa polypeptide includes polypeptides comprising any naturally occurring polypeptide of an ActRIIa family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity. See, for example, WO/2006/012627.
  • ActRIIa polypeptides include polypeptides derived from the sequence of any known ActRIIa having a sequence at least about 80% identical to the sequence of an ActRIIa polypeptide, and optionally at least 85%, 90%, 95%, 97%, 99% or greater identity.
  • an ActRIIapolypeptide of the invention may bind to and inhibit the function of an ActRIIa protein and/or activin.
  • An ActRIIa polypeptide may be selected for activity in promoting red blood cell formation in vivo.
  • Examples of ActRIIa polypeptides include human ActRIIa precursor polypeptide (SEQ ID NO: 1) and soluble human ActRIIa polypeptides (e.g., SEQ ID NOs: 2, 3, 7 and 12).
  • the human ActRIIa precursor protein sequence is as follows:
  • the signal peptide is single underlined; the extracellular domain is in bold and the potential N-linked glycosylation sites are double underlined.
  • the human ActRIIa soluble (extracellular), processed polypeptide sequence is as follows:
  • the C-terminal “tail” of the extracellular domain is underlined.
  • the sequence with the “tail” deleted is as follows:
  • nucleic acid sequence encoding human ActRIIa precursor protein is as follows (nucleotides 164-1705 of Genbank entry NM — 001616):
  • the nucleic acid sequence encoding a human ActRIIa soluble (extracellular) polypeptide is as follows:
  • the invention relates to soluble ActRIIa polypeptides.
  • soluble ActRIIa polypeptide generally refers to polypeptides comprising an extracellular domain of an ActRIIa protein.
  • soluble ActRIIa polypeptide includes any naturally occurring extracellular domain of an ActRIIa protein as well as any variants thereof (including mutants, fragments and peptidomimetic forms).
  • An activin-binding ActRIIa polypeptide is one that retains the ability to bind to activin, including, for example, activin AA, AB, BB, or forms that include a C or E subunit.
  • an activin-binding ActRIIa polypeptide will bind to activin AA with a dissociation constant of 1 nM or less.
  • the extracellular domain of an ActRIIa protein binds to activin and is generally soluble in physiological conditions, and thus can be termed a soluble, activin-binding ActRIIa polypeptide.
  • Examples of soluble, activin-binding ActRIIa polypeptides include the soluble polypeptides illustrated in SEQ ID NOs: 2, 3, 7, 12 and 13. SEQ ID NO:7 is referred to as ActRIIa-hFc, and is described further in the Examples.
  • soluble, activin-binding ActRIIa polypeptides comprise a signal sequence in addition to the extracellular domain of an ActRIIa protein, for example, the honey bee mellitin leader sequence (SEQ ID NO: 8), the tissue plaminogen activator (TPA) leader (SEQ ID NO: 9) or the native ActRIIa leader (SEQ ID NO: 10).
  • the ActRIIa-hFc polypeptide illustrated in SEQ ID NO:13 uses a TPA leader.
  • a general formula for an active ActRIIa variant protein is one that comprises amino acids 12-82 of SEQ ID NO: 2, respectively, but optionally beginning at a position ranging from 1-5 or 3-5 and ending at a position ranging from 110-116 or 110-115, respectively, and comprising no more than 1, 2, 5, 10 or 15 conservative amino acid changes in the ligand binding pocket, and zero, one or more non-conservative alterations at positions 40, 53, 55, 74, 79 and/or 82 in the ligand binding pocket.
  • Such a protein may comprise an amino acid sequence that retains greater than 80%, 90%, 95% or 99% sequence identity to the sequence of amino acids 29-109 of SEQ ID NO: 2.
  • Functionally active fragments of ActRIIa polypeptides can be obtained by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding an ActRIIa polypeptide.
  • 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 can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments that can function as antagonists (inhibitors) of ActRIIa protein or signaling mediated by activin.
  • a functional variant of the ActRIIa polypeptides can be obtained by screening libraries of modified polypeptides recombinantly produced from the corresponding mutagenized nucleic acids encoding an ActRIIa polypeptide. The variants can be produced and tested to identify those that can function as antagonists (inhibitors) of ActRIIa protein or signaling mediated by activin.
  • a functional variant of the ActRIIa polypeptides comprises an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NOs: 2 or 3. In certain cases, the functional variant has an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 2 or 3.
  • Functional variants may be generated by modifying the structure of an ActRIIa polypeptide for such purposes as enhancing therapeutic efficacy, or stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo).
  • modified ActRIIa polypeptides when selected to retain activin binding, are considered functional equivalents of the naturally-occurring ActRIIa polypeptides.
  • Modified ActRIIa polypeptides can also be produced, for instance, by amino acid substitution, deletion, or addition.
  • the present invention contemplates specific mutations of the ActRIIa polypeptides so as to alter the glycosylation of the polypeptide.
  • Such mutations may be selected so as to introduce or eliminate one or more glycosylation sites, such as O-linked or N-linked glycosylation sites.
  • Asparagine-linked glycosylation recognition sites generally comprise a tripeptide sequence, asparagine-X-threonine or asparagine-X-serine (where “X” is any amino acid) which is specifically recognized by appropriate cellular glycosylation enzymes.
  • the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the wild-type ActRIIa polypeptide (for O-linked glycosylation sites).
  • a variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a glycosylation recognition site (and/or amino acid deletion at the second position) results in non-glycosylation at the modified tripeptide sequence.
  • Another means of increasing the number of carbohydrate moieties on an ActRIIa polypeptide is by chemical or enzymatic coupling of glycosides to the ActRIIa polypeptide.
  • the sugar(s) may be attached to (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups such as those of cysteine; (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan; or (f) the amide group of glutamine. Removal of one or more carbohydrate moieties present on an ActRIIa polypeptide may be accomplished chemically and/or enzymatically.
  • Chemical deglycosylation may involve, for example, exposure of the ActRIIa polypeptide to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the amino acid sequence intact.
  • Enzymatic cleavage of carbohydrate moieties on ActRIIa polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al. (1987) Meth. Enzymol. 138:350.
  • ActRIIa polypeptide may be adjusted, as appropriate, depending on the type of expression system used, as mammalian, yeast, insect and plant cells may all introduce differing glycosylation patterns that can be affected by the amino acid sequence of the peptide.
  • ActRIIa proteins for use in humans may be expressed in a mammalian cell line that provides proper glycosylation, such as HEK293 or CHO cell lines, although other mammalian expression cell lines are expected to be useful as well.
  • Other non-mammalian cell lines may be used (e.g., yeast, E. coli, insect cells), and in some cases, such cell lines may be engineered to include enzymes that confer mammalian-type glycosylation patterns on the expressed proteins.
  • This disclosure further contemplates a method of generating mutants, particularly sets of combinatorial mutants of an ActRIIa polypeptide, as well as truncation mutants; pools of combinatorial mutants are especially useful for identifying functional variant sequences.
  • the purpose of screening such combinatorial libraries may be to generate, for example, ActRIIa polypeptide variants which bind to activin or other ligands.
  • a variety of screening assays are provided below, and such assays may be used to evaluate variants.
  • an ActRIIa polypeptide variant may be screened for ability to bind to an ActRIIa ligand, to prevent binding of an ActRIIa ligand to an ActRIIa polypeptide or to interfere with signaling caused by an ActRIIa ligand.
  • an ActRIIa polypeptide or its variants may also be tested in a cell-based or in vivo assay. For example, the effect of an ActRIIa polypeptide variant on the expression of genes involved in hematopoiesis may be assessed. This may, as needed, be performed in the presence of one or more recombinant ActRIIa ligand proteins (e.g., activin), and cells may be transfected so as to produce an ActRIIa polypeptide and/or variants thereof, and optionally, an ActRIIa ligand. Likewise, an ActRIIa polypeptide may be administered to a mouse or other animal, and one or more blood measurements, such as an RBC count, hemoglobin, or reticulocyte count may be assessed.
  • ActRIIa polypeptide may be administered to a mouse or other animal, and one or more blood measurements, such as an RBC count, hemoglobin, or reticulocyte count may be assessed.
  • Combinatorially-derived variants can be generated which have a selective or generally increased potency relative to a naturally occurring ActRIIa polypeptide.
  • mutagenesis can give rise to variants which have intracellular half-lives dramatically different than the corresponding a wild-type ActRIIa polypeptide.
  • the altered protein can be rendered either more stable or less stable to proteolytic degradation or other cellular processes which result in destruction of, or otherwise inactivation of a native ActRIIa polypeptide.
  • Such variants, and the genes which encode them can be utilized to alter ActRIIa polypeptide levels by modulating the half-life of the ActRIIa polypeptides.
  • a short half-life can give rise to more transient biological effects and, when part of an inducible expression system, can allow tighter control of recombinant ActRIIa polypeptide levels within the cell.
  • mutations may be made in the linker (if any) and/or the Fc portion to alter the half-life of the protein.
  • a combinatorial library may be produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential ActRIIa polypeptide sequences.
  • a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential ActRIIa polypeptide nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display).
  • the library of potential homologs can be generated from a degenerate oligonucleotide sequence.
  • Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes can then be ligated into an appropriate vector for expression.
  • the synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, S A (1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. A G Walton, Amsterdam: Elsevier pp 273-289; Itakura et al., (1984) Annu. Rev.
  • ActRIIa polypeptide variants can be generated and isolated from a library by screening using, for example, alanine scanning mutagenesis and the like (Ruf et al., (1994) Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al., (1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol. Chem.
  • a wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations and truncations, and, for that matter, for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of ActRIIa polypeptides.
  • the most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected.
  • Preferred assays include activin binding assays and activin-mediated cell signaling assays.
  • the ActRIIa polypeptides of the invention may further comprise post-translational modifications in addition to any that are naturally present in the ActRIIa polypeptides.
  • modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • the modified ActRIIa polypeptides may contain non-amino acid elements, such as polyethylene glycols, lipids, poly- or mono-saccharide, and phosphates. Effects of such non-amino acid elements on the functionality of an ActRIIa polypeptide may be tested as described herein for other ActRIIa polypeptide variants.
  • an ActRIIa polypeptide When an ActRIIa polypeptide is produced in cells by cleaving a nascent form of the ActRIIa polypeptide, post-translational processing may also be important for correct folding and/or function of the protein.
  • Different cells such as CHO, HeLa, MDCK, 293, W138, NIH-3T3 or HEK293 have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the ActRIIa polypeptides.
  • fusion proteins having at least a portion of the ActRIIa polypeptides and one or more fusion domains.
  • fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), or human serum albumin.
  • a fusion domain may be selected so as to confer a desired property. For example, some fusion domains are particularly useful for isolation of the fusion proteins by affinity chromatography.
  • matrices for affinity chromatography such as glutathione-, amylase-, and nickel- or cobalt-conjugated resins are used.
  • Many of such matrices are available in “kit” form, such as the Pharmacia GST purification system and the QIAexpress system (Qiagen) useful with (HIS 6 ) fusion partners.
  • a fusion domain may be selected so as to facilitate detection of the ActRIIa polypeptides. Examples of such detection domains include the various fluorescent proteins (e.g., GFP) as well as “epitope tags,” which are usually short peptide sequences for which a specific antibody is available.
  • fusion domains have a protease cleavage site, such as for Factor Xa or Thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby liberate the recombinant proteins therefrom. The liberated proteins can then be isolated from the fusion domain by subsequent chromatographic separation.
  • an ActRIIa polypeptide is fused with a domain that stabilizes the ActRIIa polypeptide in vivo (a “stabilizer” domain).
  • stabilizing is meant anything that increases serum half life, regardless of whether this is because of decreased destruction, decreased clearance by the kidney, or other pharmacokinetic effect. Fusions with the Fc portion of an immunoglobulin are known to confer desirable pharmacokinetic properties on a wide range of proteins. Constant domains from an immunoglobulin, particularly an IgG heavy chain, may also be used as stabilizing domains. Likewise, fusions to human serum albumin can confer desirable properties. Other types of fusion domains that may be selected include multimerizing (e.g., dimerizing, tetramerizing) domains and functional domains (that confer an additional biological function, such as further stimulation of bone growth).
  • the present invention provides a fusion protein comprising a soluble extracellular domain of ActRIIa fused to an Fc domain.
  • a fusion protein comprising a soluble extracellular domain of ActRIIa fused to an Fc domain.
  • An example of an IgG1 Fc domain is shown below (SEQ ID NO: 6).
  • the Fc domain has one or more mutations at residues such as Asp-265, lysine 322, and Asn-434.
  • the mutant Fc domain having one or more of these mutations e.g., Asp-265 mutation
  • the mutant Fc domain having one or more of these mutations has increased ability of binding to the MHC class I-related Fc-receptor (FcRN) relative to a wildtype Fc domain.
  • Fc domains from IgG2, IgG3 and IgG4 may also be used.
  • an ActRIIa polypeptide may be placed C-terminal to a heterologous domain, or, alternatively, a heterologous domain may be placed C-terminal to an ActRIIA polypeptide.
  • the ActRIIa polypeptide domain and the heterologous domain need not be adjacent in a fusion protein, and additional domains or amino acid sequences may be included C- or N-terminal to either domain or between the domains.
  • the ActRIIaa polypeptides of the present invention contain one or more modifications that are capable of stabilizing the ActRIIa polypeptides.
  • modifications enhance the in vitro half life of the ActRIIa polypeptides, enhance circulatory half life of the ActRIIa polypeptides or reducing proteolytic degradation of the ActRIIa polypeptides.
  • stabilizing modifications include, but are not limited to, fusion proteins (including, for example, fusion proteins comprising an ActRIIa polypeptide and a stabilizer domain), modifications of a glycosylation site (including, for example, addition of a glycosylation site to an ActRIIa polypeptide), and modifications of carbohydrate moiety (including, for example, removal of carbohydrate moieties from an ActRIIa polypeptide).
  • fusion proteins including, for example, fusion proteins comprising an ActRIIa polypeptide and a stabilizer domain
  • modifications of a glycosylation site including, for example, addition of a glycosylation site to an ActRIIa polypeptide
  • carbohydrate moiety including, for example, removal of carbohydrate moieties from an ActRIIa polypeptide.
  • stabilizer domain not only refers to a fusion domain (e.g., Fc) as in the case of fusion proteins, but also includes nonproteinaceous modifications such as a carbohydrate moiety, or nonproteinace
  • the present invention makes available isolated and/or purified forms of the ActRIIa polypeptides, which are isolated from, or otherwise substantially free of, other proteins.
  • ActRIIa polypeptides will generally be produced by expression from recombinant nucleic acids.
  • the invention provides isolated and/or recombinant nucleic acids encoding any of the ActRIIa polypeptides (e.g., soluble ActRIIa polypeptides), including fragments, functional variants and fusion proteins disclosed herein.
  • SEQ ID NO: 4 encodes the naturally occurring human ActRIIa precursor polypeptide
  • SEQ ID NO: 5 encodes the processed extracellular domain of ActRIIa.
  • 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 for making ActRIIa polypeptides or as direct therapeutic agents (e.g., in a gene therapy approach).
  • the subject nucleic acids encoding ActRIIa polypeptides are further understood to include nucleic acids that are variants of SEQ ID NO: 4 or 5.
  • the invention provides isolated or recombinant nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 4 or 5.
  • nucleic acid sequences complementary to SEQ ID NOs: 4 or 5, and variants of SEQ ID NOs: 4 or 5 are also within the scope of this invention.
  • the nucleic acid sequences of the invention can be isolated, recombinant, and/or fused with a heterologous nucleotide sequence, or in a DNA library.
  • nucleic acids of the invention also include nucleotide sequences, and the ActRIIa polypeptides encoded by such nucleic acids, that hybridize under highly stringent conditions to the nucleotide sequence designated in SEQ ID NOs: 4 or 5, complement sequence of SEQ ID NOs: 4 or 5 or fragments thereof.
  • appropriate stringency conditions which promote DNA hybridization can be varied.
  • appropriate stringency conditions which promote DNA hybridization can be varied. For example, one could perform the hybridization at 6.0 ⁇ sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2.0 ⁇ SSC at 50° C.
  • SSC sodium chloride/sodium citrate
  • the salt concentration in the wash step can be selected from a low stringency of about 2.0 ⁇ SSC at 50° C. to a high stringency of about 0.2 ⁇ SSC at 50° C.
  • the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C., to high stringency conditions at about 65° C. Both temperature and salt may be varied, or temperature or salt concentration may be held constant while the other variable is changed.
  • the invention provides nucleic acids which hybridize under low stringency conditions of 6 ⁇ SSC at room temperature followed by a wash at 2 ⁇ SSC at room temperature.
  • Isolated nucleic acids which differ from the nucleic acids as set forth in SEQ ID NOs: 4 or 5 due to degeneracy in the genetic code are also within the scope of the invention.
  • a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in “silent” mutations which do not affect the amino acid sequence of the protein.
  • CAU and CAC are synonyms for histidine
  • nucleotides up to about 3-5% of the nucleotides
  • nucleic acids encoding a particular protein may exist among individuals of a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention.
  • the recombinant nucleic acids of the invention may be operably linked to one or more regulatory nucleotide sequences in an expression construct.
  • Regulatory nucleotide sequences will generally be appropriate to the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.
  • said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are contemplated by the invention.
  • the promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter.
  • An expression construct may be present in a cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome.
  • the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.
  • the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding an ActRIIa polypeptide and operably linked to at least one regulatory sequence.
  • Regulatory sequences are art-recognized and are selected to direct expression of the ActRIIa polypeptide.
  • the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990). For instance, any of a wide variety of expression control sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding an ActRIIa polypeptide.
  • Such useful expression control sequences include, for example, the early and late promoters of SV40, tet promoter, adenovirus or cytomegalovirus immediate early promoter, RSV promoters, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast ⁇ -mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered.
  • a recombinant nucleic acid of the invention can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian), or both.
  • Expression vehicles for production of a recombinant ActRIIa polypeptide include plasmids and other vectors.
  • suitable vectors include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.
  • Some mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells.
  • the pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells.
  • vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells.
  • bacterial plasmids such as pBR322
  • derivatives of viruses such as the 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.
  • BBV-1 bovine papilloma virus
  • pHEBo Epstein-Barr virus
  • pREP-derived and p205 Epstein-Barr virus
  • examples of other viral (including retroviral) expression systems can be found below in the description of gene therapy delivery systems.
  • the various methods employed in the preparation of the plasmids and in transformation of host organisms are well known in the art.
  • baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUWI), and pBlueBac-derived vectors (such as the ⁇ -gal containing pBlueBac III).
  • pVL-derived vectors such as pVL1392, pVL1393 and pVL941
  • pAcUW-derived vectors such as pAcUWI
  • pBlueBac-derived vectors such as the ⁇ -gal containing pBlueBac III.
  • a vector will be designed for production of the subject ActRIIa polypeptides in CHO cells, such as a Pcmv-Script vector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen, Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wis.).
  • the subject gene constructs can be used to cause expression of the subject ActRIIa polypeptides in cells propagated in culture, e.g., to produce proteins, including fusion proteins or variant proteins, for purification.
  • This disclosure also pertains to a host cell transfected with a recombinant gene including a coding sequence (e.g., SEQ ID NO: 4 or 5) for one or more of the subject ActRIIa polypeptides.
  • the host cell may be any prokaryotic or eukaryotic cell.
  • an ActRIIa polypeptide of the invention may be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.
  • the present invention further pertains to methods of producing the subject ActRIIa polypeptides.
  • a host cell transfected with an expression vector encoding an ActRIIa polypeptide can be cultured under appropriate conditions to allow expression of the ActRIIa polypeptide to occur.
  • the ActRIIa polypeptide may be secreted and isolated from a mixture of cells and medium containing the ActRIIa polypeptide.
  • the ActRIIa polypeptide may be retained cytoplasmically or in a membrane fraction and the cells harvested, lysed and the protein isolated.
  • a cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art.
  • the subject ActRIIa polypeptides can be isolated from cell culture medium, host cells, or both, using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, immunoaffinity purification with antibodies specific for particular epitopes of the ActRIIa polypeptides and affinity purification with an agent that binds to a domain fused to the ActRIIa polypeptide (e.g., a protein A column may be used to purify an ActRIIa-Fc fusion).
  • the ActRIIa polypeptide is a fusion protein containing a domain which facilitates its purification.
  • purification is achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography.
  • the purification could be completed with viral filtration and buffer exchange.
  • ActRIIa-hFc protein was purified to a purity of >98% as determined by size exclusion chromatography and >95% as determined by SDS PAGE. This level of purity was sufficient to achieve desirable results in mice, rats, non-human primates and humans.
  • a fusion gene coding for a purification leader sequence such as a poly-(His)/enterokinase cleavage site sequence at the N-terminus of the desired portion of the recombinant ActRIIa polypeptide, can allow purification of the expressed fusion protein by affinity chromatography using a Ni 2+ metal resin.
  • the purification leader sequence can then be subsequently removed by treatment with enterokinase to provide the purified ActRIIa polypeptide (e.g., see Hochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al., PNAS USA 88:8972).
  • fusion genes are well known. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).
  • activin-ActRIIa signaling can be used to increase red blood cell or hemoglobin levels.
  • soluble ActRIIa polypeptides, and particularly ActRIIa-Fc are preferred antagonists, and although such antagonists may affect red blood cell levels through a mechanism other than activin antagonism (e.g., activin inhibition may be an indicator of the tendency of an agent to inhibit the activities of a spectrum of molecules, including, perhaps, other members of the TGF-beta superfamily, and such collective inhibition may lead to the desired effect on hematopoiesis), other types of activin-ActRIIa antagonists are expected to be useful, including anti-activin (e.g., activin ⁇ A , ⁇ B , ⁇ C and ⁇ E ) antibodies, anti-ActRIIa antibodies, antisense, RNAi or ribozyme nucleic acids that inhibit the production of ActRIIa, and other inhibitors of activin or ActRIIa, particularly those that disrupt activ
  • anti-activin e.g
  • an antibody that is specifically reactive with an ActRIIa polypeptide e.g., a soluble ActRIIa polypeptide
  • an antibody that is specifically reactive with an activin ⁇ A , ⁇ B , ⁇ C or ⁇ E polypeptide, or any heterodimer thereof, and which disrupts ActRIIa binding may be used as an antagonist.
  • anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (see, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)).
  • a mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the activin or ActRIIa polypeptide, an antigenic fragment which is capable of eliciting an antibody response, or a fusion protein.
  • Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art.
  • An immunogenic portion of an ActRIIa or activin polypeptide can be administered in the presence of adjuvant.
  • the progress of immunization can be monitored by detection of antibody titers in plasma or serum.
  • Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.
  • antibody-producing cells can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells.
  • Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with an activin or ActRIIa polypeptide and monoclonal antibodies isolated from a culture comprising such hybridoma cells.
  • antibody as used herein is intended to include whole antibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), and includes fragments or domains of immunoglobulins which are reactive with a selected antigen.
  • Antibodies can be fragmented using conventional techniques and the fragments screened for utility and/or interaction with a specific epitope of interest.
  • the term includes segments of proteolytically-cleaved or recombinantly-prepared portions of an antibody molecule that are capable of selectively reacting with a certain protein.
  • Non-limiting examples of such proteolytic and/or recombinant fragments include Fab, F(ab′)2, Fab′, Fv, and single chain antibodies (scFv) containing a V[L] and/or V[H] domain joined by a peptide linker.
  • the scFv's may be covalently or non-covalently linked to form antibodies having two or more binding sites.
  • the term antibody also includes polyclonal, monoclonal, or other purified preparations of antibodies and recombinant antibodies.
  • recombinant antibody means an antibody, or antigen binding domain of an immunoglobulin, expressed from a nucleic acid that has been constructed using the techniques of molecular biology, such as a humanized antibody or a fully human antibody developed from a single chain antibody. Single domain and single chain antibodies are also included within the term “recombinant antibody”.
  • an antibody of the invention is a monoclonal antibody
  • the invention makes available methods for generating novel antibodies.
  • a method for generating a monoclonal antibody that binds specifically to an ActRIIa polypeptide or activin polypeptide may comprise administering to a mouse an amount of an immunogenic composition comprising the antigen polypeptide effective to stimulate a detectable immune response, obtaining antibody-producing cells (e.g., cells from the spleen) from the mouse and fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody-producing hybridomas to identify a hybridoma that produces a monocolonal antibody that binds specifically to the antigen.
  • antibody-producing cells e.g., cells from the spleen
  • a hybridoma can be propagated in a cell culture, optionally in culture conditions where the hybridoma-derived cells produce the monoclonal antibody that binds specifically to the antigen.
  • the monoclonal antibody may be purified from the cell culture.
  • the adjective “specifically reactive with” as used in reference to an antibody is intended to mean, as is generally understood in the art, that the antibody is sufficiently selective between the antigen of interest (e.g., an activin or ActRIIa polypeptide) and other antigens that are not of interest that the antibody is useful for, at minimum, detecting the presence of the antigen of interest in a particular type of biological sample. In certain methods employing the antibody, such as therapeutic applications, a higher degree of specificity in binding may be desirable. Monoclonal antibodies generally have a greater tendency (as compared to polyclonal antibodies) to discriminate effectively between the desired antigens and cross-reacting polypeptides. One characteristic that influences the specificity of an antibody:antigen interaction is the affinity of the antibody for the antigen. Although the desired specificity may be reached with a range of different affinities, generally preferred antibodies will have an affinity (a dissociation constant) of about 10 ⁇ 6 , 10 ⁇ 7 , 10 ⁇ 8 , 10 ⁇ 9 M or less.
  • the techniques used to screen antibodies in order to identify a desirable antibody may influence the properties of the antibody obtained. For example, if an antibody is to be used for binding an antigen in solution, it may be desirable to test solution binding.
  • a variety of different techniques are available for testing interaction between antibodies and antigens to identify particularly desirable antibodies. Such techniques include ELISAs, surface plasmon resonance binding assays (e.g., the BiacoreTM binding assay, Biacore AB, Uppsala, Sweden), sandwich assays (e.g., the paramagnetic bead system of IGEN International, Inc., Gaithersburg, Md.), western blots, immunoprecipitation assays, and immunohistochemistry.
  • a nucleic acid compound may be single or double stranded.
  • a double stranded compound may also include regions of overhang or non-complementarity, where one or the other of the strands is single stranded.
  • a single stranded compound may include regions of self-complementarity, meaning that the compound forms a so-called “hairpin” or “stem-loop” structure, with a region of double helical structure.
  • a nucleic acid compound may comprise a nucleotide sequence that is complementary to a region consisting 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 ActRIIa nucleic acid sequence or activin ⁇ A , ⁇ B , ⁇ C , or ⁇ E nucleic acid sequence.
  • the region of complementarity will preferably be at least 8 nucleotides, and optionally about 18 to 35 nucleotides.
  • a region of complementarity may fall within an intron, a coding sequence or a noncoding sequence of the target transcript, such as the coding sequence portion.
  • a nucleic acid compound will have a length of about 8 to about 500 nucleotides or base pairs in length, and optionally the length will be about 14 to about 50 nucleotides.
  • a nucleic acid may be a DNA (particularly for use as an antisense), RNA or RNA:DNA hybrid. Any one strand may include a mixture of DNA and RNA, as well as modified forms that cannot readily be classified as either DNA or RNA.
  • a double stranded compound may be DNA:DNA, DNA:RNA or RNA:RNA, and any one strand may also include a mixture of DNA and RNA, as well as modified forms that cannot readily be classified as either DNA or RNA.
  • a nucleic acid compound may include any of a variety of modifications, including one or modifications to the backbone (the sugar-phosphate portion in a natural nucleic acid, including intemucleotide linkages) or the base portion (the purine or pyrimidine portion of a natural nucleic acid).
  • An antisense nucleic acid compound will preferably have a length of about 15 to about 30 nucleotides and will often contain one or more modifications to improve characteristics such as stability in the serum, in a cell or in a place where the compound is likely to be delivered, such as the stomach in the case of orally delivered compounds and the lung for inhaled compounds.
  • the strand complementary to the target transcript will generally be RNA or modifications thereof.
  • the other strand may be RNA, DNA or any other variation.
  • the duplex portion of double stranded or single stranded “hairpin” RNAi construct will generally have a length of 18 to 40 nucleotides in length and optionally about 21 to 23 nucleotides in length, so long as it serves as a Dicer substrate.
  • Catalytic or enzymatic nucleic acids may be ribozymes or DNA enzymes and may also contain modified forms.
  • Nucleic acid compounds may inhibit expression of the target by about 50%, 75%, 90% or more when contacted with cells under physiological conditions and at a concentration where a nonsense or sense control has little or no effect. Preferred concentrations for testing the effect of nucleic acid compounds are 1, 5 and 10 micromolar. Nucleic acid compounds may also be tested for effects on, for example, red blood cell levels.
  • an activin-ActRIIa antagonist may be a follistatin polypeptide that antagonizes activin bioactivity and/or binds to activin.
  • follistatin polypeptide includes polypeptides comprising any naturally occurring polypeptide of follistatin as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity, and further includes any functional monomer or multimer of follistatin. Variants of follistatin polypeptides that retain activin binding properties can be identified based on previous studies involving follistatin and activin interactions.
  • WO2008/030367 discloses specific follistatin domains (“FSDs”) that are shown to be important for activin binding.
  • FSDs specific follistatin domains
  • SEQ ID NOs: 19-21 the N-terminus follistatin domain (“FSND” SEQ ID NO: 19), FSD2 (SEQ ID NO: 20), and to a lesser extent FSD1 (SEQ ID NO: 21) represent exemplary domains within follistatin important for activin binding.
  • methods for making and testing libraries of polypeptides are described above in the context of ActRIIa polypeptides and such methods also pertain to making and testing variants of follistatin.
  • Follistatin polypeptides include polypeptides derived from the sequence of any known follistatin having a sequence at least about 80% identical to the sequence of a follistatin polypeptide, and optionally at least 85%, 90%, 95%, 97%, 99% or greater identity.
  • follistatin polypeptides include the mature follistatin polypeptide or shorter isoforms or other variants of the human follistatin precursor polypeptide (SEQ ID NO: 17) as described, for example, in WO2005/025601.
  • the human follistatin precursor polypeptide isoform FST344 is as follows:
  • the human follistatin precursor polypeptide isoform FST317 is as follows:
  • N-terminus follistatin domain (FSND) sequence is as follows:
  • the FSD1 and FSD2 sequences are as follows:
  • an activin-ActRIIa antagonist may be a follistatin-like related gene (FLRG) that antagonizes activin bioactivity and/or binds to activin.
  • FLRG polypeptide includes polypeptides comprising any naturally occurring polypeptide of FLRG as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity. Variants of FLRG polypeptides that retain activin binding properties can be identified using routine methods to assay FLRG and activin interactions. See, for example, U.S. Pat. No. 6,537,966.
  • FLRG polypeptides include polypeptides derived from the sequence of any known FLRG having a sequence at least about 80% identical to the sequence of an FLRG polypeptide, and optionally at least 85%, 90%, 95%, 97%, 99% or greater identity.
  • the human FLRG precursor polypeptide is as follows:
  • MRPGAPGPLWPLPWGALAWAVGFVSS MGSGNPAPGGVCWLQQGQEATCSL VLQTDVTRAECCASGNIDTAWSNLTHPGNKINLLGFLGLVHCLPCKDSCD GVECGPGKACRMLGGRPRCECAPDCSGLPARLQVCGSDGATYRDECELPA ARCRGHPDLSVMYRGRCRKSCEHVVCPRPQSCVVDQTGSAHCVVCRAAPC VPSSPGQELCGNNNVTYISSCHMRQATCFLGRSIGVRHAGSCAGTPEEPP GGESAEEEENFV (SEQ ID NO: 22; NP_005851) The signal peptide is single underlined.
  • functional variants or modified forms of the follistatin polypeptides and FLRG polypeptides include fusion protein having at least a portion of the follistatin polypeptides or FLRG polypeptides and one or more fusion domains, such as, for example, domains that facilitate isolation, detection, stabilization or multimerization of the polypeptide. Suitable fusion domains are discussed in detail above with reference to the ActRIIa polypeptides.
  • an activin-ActRIIa antagonist is a fusion protein comprising an activin binding portion of a follistaton polypeptide fused to an Fc domain.
  • an activin-ActRIIa antagonist is a fusion protein comprising an activin binding portion of an FLRG polypeptide fused to an Fc domain.
  • Follistatin and FLRG have been shown in the literature, and by the applicants with respect to FLRG, to have affinities for Activin A in the picomolar range, indicating that these agents will inhibit activin A signaling to a similar degree as ActRIIa-Fc.
  • the present invention provides methods for managing a patient that has been treated with, or is a candidate to be treated with, an activin-ActRIIa antagonist by measuring one or more hematologic parameters in the patient.
  • the hematologic parameters may be used to evaluate appropriate dosing for a patient who is a candidate to be treated with an activin-ActRIIa anatagonist, to monitor the hematologic parameters during treatment with an activin-ActRIIa antagonist, to evaluate whether to adjust the dosage during treatment with an activin-ActRIIa antagonist, and/or to evaluate an appropriate maintenance dose of an activin-ActRIIa antagonist. If one or more of the hematologic parameters are outside the normal level, dosing with the activin-ActRIIa antagonist may be reduced, delayed or terminated.
  • Hematologic parameters that may be measured in accordance with the methods provided herein include, for example, red blood cell levels, blood pressure, iron stores, and other agents found in bodily fluids that correlate with increased red blood cell levels, using art recognized methods. Such parameters may be determined using a blood sample from a patient. Increases in red blood cell levels, hemoglobin levels, and/or hematocrit levels may cause increases in blood pressure.
  • Red blood cell levels may be determined, for example, by determining a red blood cell count, by measuring hemoglobin levels or by measuring hematocrit levels.
  • a red blood cell count may be determined using a commercially available Coulter Counter.
  • the normal range for a red blood cell count is between 4.2-5.9 million cells/cm, although individual variations should be taken into account.
  • Hemoglobin levels may be determined by lysing the red blood cells, converting the hemoglobin into cyanomethemoglobin and measuring the amount of hemoglobin with a calorimeter.
  • the normal ranges for hemoglobin are 14-18 gm/dl for adult males and 12-16 gm-dl for adult females, although individual variations should be taken into account.
  • Hematocrit or packed cell volume (PCV) refers to the ratio of the volume of red blood cells to the volume of whole blood. Hematocrit may be determined, for example, by centrifugation of a blood sample followed by analysis of the layers produced. Normal ranges for hematocrit are approximately 41-51% for men and 35-45% for women, although individual variations should be taken into account.
  • Blood pressure including systolic blood pressure, diastolic blood pressure, or mean arterial blood pressure, may be determined using art recognized methods. Blood pressure is most commonly measured using a sphygmomanometer. Typical values for a resting, healthy adult human are approximately 120 mmHg systolic and 80 mmHg diastolic, although individual variations should be taken into account. Individuals suffering from hypertension typically have a blood pressure ⁇ 140 mmHg systolic and ⁇ 90 diastolic blood pressure. Individuals having a level above normal but less than 140/90 mmHg are generally referred to as prehypertensive. Additional methods for measuring blood pressure may be found in Pickering et al., Hypertension 45: 142-161 (2005).
  • Iron stores may be measured using a variety of art recognized techniques including, for example, by determining levels of one or more of the following: serum ferritin (SF), transferrin saturation (TSAT), total iron binding capacity, hemoglobin concentration, zinc protoporphyrin, mean cell volume (MCV), or transferrin receptor in serum.
  • Serum ferritin levels may be determined, for example, using an immunoassay such as an enzyme-linked immunosorbent assay (ELISA) or immunoturbidometry. In normal patients, serum ferritin levels range from 13 to 220 ng/mL, although individual variations should be taken into account.
  • Transferrin saturation levels represent the proportion of transferrin bound to iron and may be determined by dividing serum iron by total iron biding capacity (TIBC).
  • transferrin saturation levels range from 20% to 40%, although individual variations should be taken into account.
  • Serum iron may be determiend using colorimetry and is expressed as ug/dl or umol/l.
  • Total iron binding capacity reflects the total capacity of circulating transferrin bound to iron and may be determined using a colorimetric assay to determine the amount of iron that can be bound to unsaturated transferrin in vitro. The normal range of total iron binding capacity is about 250-450 ug/dl, although individual variations should be taken into account. Additional information about measuring iron stores may be found in the World Health Organization report entitled Assessing the Iron Status of Populations dated April 2004 and in Yamanishi et al., Clinical Chemistry 48: 1565-1570 (2002).
  • hematologic parameters are outside the normal range, or on the high side of normal, in a patient who is a candidate to be treated with an activin-ActRIIa antagonist then onset of administration of the activin-ActRIIa antagonist may be delayed until the hematologic parameters have returned to a normal or acceptable level either naturally or via therapeutic intervention.
  • a candidate patient is hypertensive or prehypertensive, then the patient may be treated with a blood pressure lowering agent in order to reduce the patient's blood pressure.
  • Any blood pressure lowering agent appropriate for the individual patient's condition may be used including, for example, diuretics, adrenergic inhibitors (including alpha blockers and beta blockers), vasodilators, calcium channel blockers, angiotensin-converting enzyme (ACE) inhibitors, or angiotensin II receptor blockers.
  • Blood pressure may alternatively be treated using a diet and exercise regimen.
  • the patient may be treated with an appropriate regimen of diet and/or iron supplements until the patient's iron stores have returned to a normal or acceptable level.
  • administration of the activin-ActRIIa antagonist may be delayed until the levels have returned to a normal or acceptable level.
  • the dosage amount or frequency of dosing of the activin-ActRIIa antagonist may be set at an amount that would reduce the risk of an unacceptable increase in the hematologic parameters arising upon administration of the activin-ActRIIa antagonist.
  • a therapeutic regimen may be developed for the patient that combines an activin-ActRIIa antagonist with a therapeutic agent that addresses the undesirable level of the hematologic parameter.
  • a therapeutic regimen involving administration of an activin-ActRIIa antagonist and a blood pressure lowering agent may be designed.
  • a therapeutic regimen of an activin-ActRIIa antagonist and iron supplementation may be developed.
  • baseline parameter(s) for one or more hematologic parameters may be established for a patient who is a candidate to be treated with an activin-ActRIIa antagonists and an appropriate dosing regimen establish for that patient based on the baseline value(s).
  • established baseline parameters based on a patient's medical history could be used to inform an appropriate activin-ActRIIa antagonist dosing regimen for a patient. For example, if a healthy patient has an established baseline blood pressure reading that is above the defined normal range it may not be necessary to bring the patient's blood pressure into the range that is considered normal for the general population prior to treatment with the activin-ActRIIa antagonist.
  • a patient's baseline values for one or more hematologic parameters prior to treatment with an activin-ActRIIa antagonist may also be used as the relevant comparative values for monitoring any changes to the hematologic parameters during treatment with the activin-ActRIIa antagonist.
  • one or more hematologic parameters are measured in patients who are being treated with an activin-ActRIIa antagonist.
  • the hematologic parameters may be used to monitor the patient during treatment and permit adjustment or termination of the dosing with the activin-ActRIIa antagonist or additional dosing with another therapeutic agent. For example, if administration of an activin-ActRIIa antagonist results in an increase in blood pressure, red blood cell level, or hemoglobin level, or a reduction in iron stores, then the dose of the activin-ActRIIa antagonists may be reduced in amount or frequency in order to decrease the effects of the activin-ActRIIa antagonist on the one or more hematologic parameters.
  • the dosing of the activin-ActRIIa antagonist may be terminated either temporarily, until the hematologic parameter(s) return to an acceptable level, or permanently. Similarly, if one or more hematologic parameters are not brought within an acceptable range after reducing the dose or frequency of administration of the activin-ActRIIa antagonist then the dosing may be terminated.
  • the patient may be dosed with an additional therapeutic agent that addresses the undesirable level in the hematologic parameter(s), such as, for example, a blood pressure lowering agent or an iron supplement.
  • an additional therapeutic agent that addresses the undesirable level in the hematologic parameter(s), such as, for example, a blood pressure lowering agent or an iron supplement.
  • dosing with the activin-ActRIIa antagonist may continue at the same level and a blood pressure lowering agent is added to the treatment regimen, dosing with the activin-ActRIIa antagonist may be reduce (e.g., in amount and/or frequency) and a blood pressure lowering agent is added to the treatment regimen, or dosing with the activin-ActRIIa antagonist may be terminated and the patient may be treated with a blood pressure lowering agent.
  • a patient being treated with an activin-ActRIIa antagonist or a patient who is a candidate for treatment with an activin-ActRIIa antagonist has one or more of the following: a hemoglobin level greater than 12 g/dl, a hemoglobin level greater than 15 g/dl, a blood pressure ⁇ 120/80 mmHg, a blood pressure ⁇ 140/90 mmHg, a transferrin saturation level less than 20%, and/or a ferritin level less than 100 ng/ml, then dosing with the activin-ActRIIa antagonist is reduced, delayed or terminated.
  • a therapeutic agent that addresses the undesired level of one or more hematologic parameters (such as a blood pressure lowering agent or an iron supplement) may be administered to the patient.
  • the present invention provides a method for dosing a patient with an activin-ActRIIa antagonist by administering to the patient an activin-ActRIIa antagonist in an amount and at a frequency which reduces the risk of causing a rise in hemoglobin levels greater than 1 g/dl over a two week period.
  • the methods may comprise measuring one or more hematologic parameters either before beginning administration of the activin-ActRIIa antagonist and/or during administration of the activin-ActRIIa antagonist.
  • the initial dose of the activin-ActRIIa antagonist may be set so as to reduce the risk of causing a rise in hemoglobin levels greater than 1 g/dl over a two week period.
  • the dose may be adjusted over time to in order to maintain a reduced risk of causing a rise in hemoglobin levels greater than 1 g/dl in two weeks.
  • the present invention provides a method for administering an ActRIIa-Fc fusion protein to a patient by administering the ActRIIa fusion protein no more frequently than once per 60 days, once per 90 days, or once per 120 days, or 1-6 times per year, 2-6 times per year, 1-5 times per year, 2-5 times per year, 1-4 times per year, 2-4 times per year, 1-3 times per year, or 2-3 times per year.
  • increases in red blood cell levels arising from administration peak around 60 days after administration. At about 90 days after administration, a significant reduction in red blood cell levels is seen and after about 120 days red blood cell levels return to the baseline level.
  • the activin-ActRIIa antagonist is being administered for purposes other than increasing red blood cell levels, it may be desirable to administer subsequent doses of the activin-ActRIIa antagonist after the peak increase in red blood cell levels from the previous dose, or even after red blood cell levels have returned to normal.
  • the invention provides methods for determining dosing and monitoring therapeutic progress with activin-ActRIIa antagonist treatment in patients in which the activin-ActRIIa antagonist is being administered to increase red blood cell levels.
  • the methods involve determining one or more hematologic parameters either prior to beginning dosing with the activin-ActRIIa antagonist and/or during treatment with the activin-ActRIIa antagonist.
  • one or more hematologic parameters may be determined in a patient who is a candidate for administration of an activin-ActRIIa antagonist for increasing blood cell levels to facilitate determination of dosage amount and frequency.
  • One or more hematologic parameters may also be determined in a patient being treated with an activin-ActRIIa antagonist for purposes of increasing red blood cell levels in order to monitor progress of the treatment, facilitate dosing adjustments, and to determine maintenance dosing levels, etc.
  • one or more hematologic parameters may be measured at various time points and at varying frequencies as needed for an individual patient based on various factors such as a patient's baseline levels, responsiveness to treatment with an activin-ActRIIa antagonist, general health, age, sex, weight, etc. Measuring of one or more hematologic parameters may be carried out before and/or during treatment with an activin-ActRIIa antagonist. If conducting multiple measurements of hematologic parameters at various time points, the same set of hematologic parameter(s) need not be measured at each time point. Similarly, the same test for an individual parameter need not be used at each time point.
  • hematologic parameters and tests for such parameters may be chosen for an individual taking into account factors specific to the given individual. Testing of hematologic parameters may occur as frequently as needed for a given individual, such as, for example, once per day, once per week, once per every two weeks, once per month, once per each 2 month period, once per each 3 month period, once per each 6 month period, or once per year. In addition, the frequency of testing may vary over time.
  • the frequency of testing may be reduced to, for example, once per month, once per every two months, once per every three months, once per every six months, or once per year.
  • an activin-ActRIIa antagonist e.g., either amount or frequency of administration
  • patients being treated with an activin-ActRIIa antagonist may be mammals such as rodents and primates, and particularly human patients.
  • patients being treated with an activin-ActRIIa antagonist, or candidate patients to be treated with an activin-ActRIIa antagonist are patients in need of bone and/or cartilage formation, prevention of bone loss, increased bone mineralization or prevention of bone demineralization, such as patients with low bone density, decreased bone strength, or bone damage due to breakage, loss or demineralization.
  • the patients or candidate patients may be patients suffering from, or at risk for developing, osteoporosis (including secondary osteoporosis), hyperparathyroidism, Cushing's disease, Paget's disease, thyrotoxicosis, chronic diarrheal state or malabsorption, renal tubular acidosis, or anorexia nervosa.
  • Osteoporosis resulting from drugs or another medical condition is known as secondary osteoporosis.
  • Medications that can cause secondary osteoporosis include, for example, corticosteroids, methotrexate (Rheumatrex, Immunex, Folex PFS), cyclosporine (Sandimmune, Neoral), luteinizing hormone-releasing hormone agonists (Lupron, Zoladex), and heparin (Calciparine, Liquaemin).
  • CTIBL cancer therapy induced bone loss
  • patients being treated with an activin-ActRIIa antagonist, or candidate patients to be treated with an activin-ActRIIa antagonist are patients suffering from or at high risk for developing breast cancer.
  • a woman with a high risk for developing breast cancer is a woman whose risk factors confer a greater probability of developing the disease compared to the general population or the population of women within a certain age group.
  • risk factors include age, family history or genetic makeup, lifestyle habits such as exercise and diet, exposure to radiation or other cancer-causing agents, age at the time the first child was born, genetic changes, and weight gain after menopause.
  • Exemplary patients include, for example, patients that have mutations in the BRCA1/2 genes or other genes shown to predispose women to breast and ovarian cancer are also included. Patients also include individuals with solid tumors, metastatic cancer, precancerous lesions of the breast, benign lesions of the breast, or with any abnormal proliferative lesions including typical hyperplasia, atypical hyperplasia, and noninvasive or in situ carcinoma. Patients also include those with both hormone-dependent or hormone-responsive cancers (e.g., estrogen receptor positive cancers) and hormone-independent cancers (e.g., estrogen receptor negative or estrogen receptor mutant cancers).
  • hormone-dependent or hormone-responsive cancers e.g., estrogen receptor positive cancers
  • hormone-independent cancers e.g., estrogen receptor negative or estrogen receptor mutant cancers.
  • cancers in which growth factors or oncogenes are activated e.g., breast cancers in which c-erbB-2 (also known as HER-2/Neu) tyrosine kinase is expressed
  • c-erbB-2 also known as HER-2/Neu tyrosine kinase is expressed
  • patients being treated with an activin-ActRIIa antagonist, or candidate patients to be treated with an activin-ActRIIa antagonist are patients with undesirably low red blood cell or hemoglobin levels, such as patients having an anemia, and those that are at risk for developing undesirably low red blood cell or hemoglobin levels, such as those patients that are about to undergo major surgery or other procedures that may result in substantial blood loss, such as having blood drawn and stored for a later transfusion.
  • Patients and candidate patients may also include those patients in need of an increase in red blood cells and/or hemoglobin levels that do not respond well to Epo.
  • anemia When observing hemoglobin levels in humans, a level of less than normal for the appropriate age and gender category may be indicative of anemia, although individual variations are taken into account.
  • Potential causes of anemia include blood-loss, nutritional deficits, medication reaction, various problems with the bone marrow and many diseases. More particularly, anemia has been associated with a variety of disorders that include, for example, chronic renal failure, myelodysplastic syndrome, rheumatoid arthritis, bone marrow transplantation.
  • Anemia may also be associated with the following conditions: solid tumors (e.g. breast cancer, lung cancer, colon cancer); tumors of the lymphatic system (e.g.
  • chronic lymphocyte leukemia non-Hodgkins and Hodgkins lymphomas
  • tumors of the hematopoietic system eg. leukemia, myelodysplastic syndrome, multiple myeloma
  • radiation therapy e.g. platinum containing regimens
  • inflammatory and autoimmune diseases including, but not limited to, rheumatoid arthritis, other inflammatory arthritides, systemic lupus erythematosis (SLE), acute or chronic skin diseases (e.g. psoriasis), inflammatory bowel disease (e.g.
  • a therapeutic that “prevents” a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.
  • treating includes prophylaxis of the named condition or amelioration or elimination of the condition once it has been established. In either case, prevention or treatment may be discerned in the diagnosis provided by a physician or other health care provider and the intended result of administration of the therapeutic agent.
  • activin-ActRIIa antagonists e.g., ActRIIa polypeptides
  • a pharmaceutically acceptable carrier e.g., an ActRIIa polypeptide can be administered alone or as a component of a pharmaceutical formulation (therapeutic composition).
  • the subject compounds may be formulated for administration in any convenient way for use in human or veterinary medicine.
  • the therapeutic method of the invention includes administering the composition systemically, or locally as an implant or device.
  • the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form.
  • Therapeutically useful agents other than the activin-ActRIIa antagonists which may also optionally be included in the composition as described above, may be administered simultaneously or sequentially with the subject compounds (e.g., ActRIIa polypeptides) in the methods of the invention.
  • activin-ActRIaI antagonists will be administered parenterally.
  • Pharmaceutical compositions suitable for parenteral administration may comprise one or more ActRIIa polypeptides in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as 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.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, 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.
  • compositions of the present invention may include a matrix capable of delivering one or more therapeutic compounds (e.g., ActRIIa polypeptides) to a target tissue site (e.g., bone marrow), providing a structure for the developing tissue and optimally capable of being resorbed into the body.
  • the matrix may provide slow release of the ActRIIa polypeptides.
  • Such matrices may be formed of materials presently in use for other implanted medical applications.
  • matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties.
  • Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid and polyanhydrides.
  • Other potential materials are biodegradable and biologically well defined, such as bone or dermal collagen.
  • Further matrices are comprised of pure proteins or extracellular matrix components.
  • Other potential matrices are non-biodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics.
  • Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate.
  • the bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.
  • methods of the invention can be administered for orally, e.g., in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a 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 mouth washes and the like, each containing a predetermined amount of an agent as an active ingredient.
  • An agent may also be administered as a bolus, electuary or paste.
  • one or more therapeutic compounds of the present invention may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, 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, such as
  • 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 such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as 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.
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents,
  • Suspensions in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • compositions of the invention may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.
  • the dosage regimen will be determined by the attending physician considering various factors which modify the action of the subject compounds of the invention (e.g., ActRIIa polypeptides).
  • the various factors include, but are not limited to, the patient's red blood cell count, hemoglobin level or other diagnostic assessments, the desired target red blood cell count, the patient's age, sex, and diet, the severity of any disease that may be contributing to a depressed red blood cell level, time of administration, and other clinical factors.
  • the addition of other known growth factors to the final composition may also affect the dosage. Progress can be monitored by periodic assessment of red blood cell and hemoglobin levels, as well as assessments of reticulocyte levels and other indicators of the hematopoietic process.
  • a dosing scheme may be designed to deliver a serum concentration of between about 100 and 1000 ng/ml over a period of about 20 to 30 days.
  • a dosing scheme may be designed to deliver a dosing scheme of between about 100 and 1000 ng/ml with a dosing frequency that occurs less than once every 60 days, once every 90 days, or once every 120 days.
  • serum levels of 200 ng/ml may be achieved with a single dose of 0.1 mg/kg or greater and serum levels of 1000 ng/ml may be achieved with a single dose of 0.3 mg/kg or greater.
  • the observed serum half-life of the molecule is between about 20 and 30 days, substantially longer than most Fc fusion proteins, and thus a sustained effective serum level may be achieved, for example, by dosing with about 0.05 to 0.5 mg/kg on a weekly or biweekly basis, or higher doses may be used with longer intervals between dosings. For example, doses of 0.1 to 1 mg/kg might be used on a monthly or bimonthly basis.
  • the present invention also provides gene therapy for the in vivo production of ActRIIa polypeptides.
  • Such therapy would achieve its therapeutic effect by introduction of the ActRIIa polynucleotide sequences into cells or tissues having the disorders as listed above.
  • Delivery of ActRIIa polynucleotide sequences can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system.
  • Preferred for therapeutic delivery of ActRIIa polynucleotide sequences is the use of targeted liposomes.
  • RNA virus such as a retrovirus
  • the retroviral vector may be a derivative of a murine or avian retrovirus.
  • retroviral vectors in 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).
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • RSV Rous Sarcoma Virus
  • Retroviral vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated.
  • Retroviral vectors can be made target-specific by attaching, for example, a sugar, a glycolipid, or a protein. Preferred targeting is accomplished by using an antibody.
  • specific polynucleotide sequences can be inserted into the retroviral genome or attached to a viral envelope to allow target specific delivery of the retroviral vector containing the ActRIIa polynucleotide.
  • tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.
  • colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • the preferred colloidal system of this invention is a liposome.
  • Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (see e.g., Fraley, et al., Trends Biochem. Sci., 6:77, 1981).
  • compositions of the liposome is usually a combination of phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used.
  • the physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.
  • lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.
  • Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.
  • the targeting of liposomes is also possible based on, for example, organ-specificity, cell-specificity, and organelle-specificity and is known in the art.
  • Applicants constructed a soluble ActRIIa fusion protein that has the extracellular domain of human ActRIIa fused to a human or mouse Fc domain with a minimal linker in between.
  • the constructs are referred to as ActRIIa-hFc and ActRIIa-mFc, respectively.
  • ActRIIa-hFc is shown below as purified from CHO cell lines (SEQ ID NO: 7):
  • the ActRIIa-hFc and ActRIIa-mFc proteins were expressed in CHO cell lines. Three different leader sequences were considered:
  • the selected form employs the TPA leader and has the following unprocessed amino acid sequence:
  • This polypeptide is encoded by the following nucleic acid sequence:
  • both ActRIIa-hFc and ActRIIa-mFc were remarkably amenable to recombinant expression.
  • the protein was purified as a single, well-defined peak of protein.
  • N-terminal sequencing revealed a single sequence of -ILGRSTQE (SEQ ID NO: 11).
  • Purification could be achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography.
  • the purification could be completed with viral filtration and buffer exchange.
  • the ActRIIa-hFc protein was purified to a purity of >98% as determined by size exclusion chromatography and >95% as determined by SDS PAGE.
  • ActRIIa-hFc and ActRIIa-mFc showed a high affinity for ligands, particularly activin A.
  • GDF-11 or Activin A (“ActA”) were immobilized on a Biacore CM5 chip using standard amine coupling procedure.
  • ActA Activin A
  • ActA Activin A
  • ActIa-hFc and ActRIIa-mFc proteins were loaded onto the system, and binding was measured.
  • ActRIIa-hFc bound to activin with a dissociation constant (KD) of 5 ⁇ 10 ⁇ 12 , and the protein bound to GDF11 with a KD of 9.96 ⁇ 10 ⁇ 9 . See FIG. 2 .
  • ActRIIa-mFc behaved similarly.
  • the ActRIIa-hFc was very stable in pharmacokinetic studies. Rats were dosed with 1 mg/kg, 3 mg/kg or 10 mg/kg of ActRIIa-hFc protein and plasma levels of the protein were measured at 24, 48, 72, 144 and 168 hours. In a separate study, rats were dosed at 1 mg/kg, 10 mg/kg or 30 mg/kg.
  • ActRIIa-hFc had an 11-14 day serum half life and circulating levels of the drug were quite high after two weeks (11 ⁇ g/ml, 110 ⁇ g/ml or 304 ⁇ g/ml for initial administrations of 1 mg/kg, 10 mg/kg or 30 mg/kg, respectively.)
  • the plasma half life was substantially greater than 14 days and circulating levels of the drug were 25 ⁇ g/ml, 304 ⁇ g/ml or 1440 ⁇ g/ml for initial administrations of 1 mg/kg, 10 mg/kg or 30 mg/kg, respectively.
  • Preliminary results in humans suggests that the serum half life is between about 20 and 30 days.
  • ActRIIa-hFc fusion protein was expressed in stably transfected CHO-DUKX B11 cells from a pAID4 vector (SV40 ori/enhancer, CMV promoter), using a tissue plasminogen leader sequence of SEQ ID NO:9.
  • the protein purified as described above in Example 1, had a sequence of SEQ ID NO:7.
  • the Fc portion is a human IgG1 Fc sequence, as shown in SEQ ID NO:7.
  • Sialic acid analysis showed that the protein contained, on average, between about 1.5 and 2.5 moles of sialic acid per molecule of ActRIIa-hFc fusion protein.
  • This purified protein showed a remarkably long serum half-life in all animals tested, including a half-life of 25-32 days in human patients (see Example 6, below). Additionally, the CHO cell expressed material has a higher affinity for activin B ligand than that reported for an ActRIIa-hFc fusion protein expressed in human 293 cells (del Re et al., J Biol Chem. Dec. 17, 2004;279(51):53126-35.) Additionally, the use of the tPa leader sequence provided greater production than other leader sequences and, unlike ActRIIa-Fc expressed with a native leader, provided a highly pure N-terminal sequence. Use of the native leader sequence resulted in two major species of ActRIIa-Fc, each having a different N-terminal sequence.
  • the study employed four groups of five male and five female cynomolgus monkeys each, with three per sex per group scheduled for termination on Day 29, and two per sex per group scheduled for termination on Day 57.
  • Each animal was administered the vehicle (Group I) or ActRIIa-Fc at doses of 1, 10, or 30 mg/kg (Groups 2, 3 and 4, respectively) via intravenous (IV) injection on Days 1, 8, 15 and 22.
  • the dose volume was maintained at 3 mL/kg.
  • Various measures of red blood cell levels were assessed two days prior to the first administration and at days 15, 29 and 57 (for the remaining two animals) after the first administration.
  • the ActRIIa-hFc caused statistically significant increases in mean red blood cell parameters (red blood cell count [RBC], hemoglobin [HGB], and hematocrit [HCT]) for males and females, at all dose levels and time points throughout the study, with accompanying elevations in absolute and relative reticulocyte counts (ARTC; RTC). See FIGS. 3-6 .
  • red blood cell counts and hemoglobin levels are roughly equivalent in magnitude to effects reported with erythropoietin.
  • the onset of these effects is more rapid with ActRIIa-Fc than with erythropoietin.
  • ActRIIa-hFc Increases Red Blood Cell Levels and Markers of Bone Formation in Human Patients
  • the ActRIIa-hFc fusion protein described in Example 1 was administered to human patients in a randomized, double-blind, placebo-controlled study that was conducted to evaluate, primarily, the safety of the protein in healthy, postmenopausal women. Forty-eight subjects were randomized in cohorts of 6 to receive either a single dose of ActRIIa-hFc or placebo (5 active:1 placebo). Dose levels ranged from 0.01 to 3.0 mg/kg intravenously (IV) and 0.03 to 0.1 mg/kg subcutaneously (SC). All subjects were followed for 120 days. In addition to pharmacokinetic (PK) analyses, the biologic activity of ActRIIa-hFc was also assessed by measurement of biochemical markers of bone formation and resorption, and FSH levels.
  • PK pharmacokinetic
  • hemoglobin and RBC numbers were examined in detail for all subjects over the course of the study and compared to the baseline levels. Platelet counts were compared over the same time as the control. There were no clinically significant changes from the baseline values over time for the platelet counts.
  • PK analysis of ActRIIa-hFc displayed a linear profile with dose, and a mean half-life of approximately 25-32 days.
  • the area-under-curve (AUC) for ActRIIa-hFc was linearly related to dose, and the absorption after SC dosing was essentially complete (see FIGS. 7 and 8 ).
  • ActRIIa-hFc caused a rapid, sustained dose-dependent increase in serum levels of bone-specific alkaline phosphatase (BAP), which is a marker for anabolic bone growth, and a dose-dependent decrease in C-terminal type 1 collagen telopeptide and tartrate-resistant acid phosphatase 5b levels, which are markers for bone resorption.
  • BAP bone-specific alkaline phosphatase
  • Other markers, such as P1NP showed inconclusive results.
  • BAP levels showed near saturating effects at the highest dosage of drug, indicating that half-maximal effects on this anabolic bone biomarker could be achieved at a dosage of 0.3 mg/kg, with increases ranging up to 3 mg/kg.
  • the EC50 is 51,465 (day*ng/ml). See FIG. 9 . These bone biomarker changes were sustained for approximately 120 days at the highest dose levels tested. There was also a dose-dependent decrease in serum FSH levels consistent with inhibition of activin.
  • ActRIIa-hFc showed a dose-dependent effect on red blood cell counts and reticulocyte counts, and a dose-dependent effect on markers of bone formation.
  • a clinical study was designed to treat patients with multiple doses of ActRIIa-hFc, at dose levels of 0.1 mg/kg, 0.3 mg/kg and 1.0 mg/kg, with dosing every thirty days.
  • Normal healthy patients in the trial exhibited an increase in hemoglobin and hematocrit that is consistent with the increases seen in the Phase I clinical trial reported in Example 4, except that, in some instances, the hemoglobin and hematocrit were elevated beyond the normal range.
  • An anemic patient with hemoglobin of approximately 7.5 also received two doses at the 1 mg/kg level, resulting in a hemoglobin level of approximately 10.5 after two months.
  • the patient's anemia was a microcytic anemia, thought to be caused by chronic iron deficiency.
  • the compound treated group experienced a statistically significant increase in the nucleated cell number before red blood cell lysis and in the mature erythroid progenitor (CFU-E) colony number per dish, frequency and total progenitor number per spleen.
  • CFU-GM myeloid
  • BFU-E immature erythroid
  • mice Sixteen BDF1 female mice 6-8 weeks of age were used in the study. Eight mice were injected subcutaneously with test compound ActRIIa-mFc at days 1 and 3 at a dose of 10 mg/kg and eight mice were injected subcutaneously with vehicle control, phosphate buffered saline (PBS), at a volume of 100 ⁇ L per mouse. All mice were sacrificed 8 days after first injection in accordance with the relevant Animal Care Guidelines. Peripheral blood (PB) samples from individual animals were collected by cardiac puncture and used for complete blood counts and differential (CBC/Diff). Femurs and spleens were harvested from each mouse.
  • PB peripheral blood
  • PB from each mouse was collected via cardiac puncture and placed into the appropriate microtainer tubes. Samples were sent to CLV for analysis on a CellDyn 3500 counter.
  • Clonogenic progenitors of the myeloid, erythroid and lymphoid lineages were assessed using the in vitro methylcellulose-based media systems described below.
  • Clonogenic progenitors of the mature erythroid (CFU-E) lineages were cultured in MethoCultTM 3334, a methylcellulose-based medium containing recombinant human (rh) Erythropoietin (3 U/mL).
  • Clonogenic progenitors of the lymphoid (CFU-pre-B) lineage were cultured in MethoCult® 3630, a methylcellulose-based medium containing rh Interleukin 7 (10 ng/mL).
  • Clonogenic progenitors of the granulocyte-monocyte (CFU-GM), erythroid (BFU-E) and multipotential (CFU-GEMM) lineages were cultured in MethoCultTM 3434, a methylcellulose-based medium containing recombinant murine (rm) Stem Cell Factor (50 ng/mL), rh Interleukin 6 (10 ng/mL), rm Interleukin 3 (10 ng/mL) and rh Erythropoietin (3 U/mL).
  • MethoCultTM 3434 a methylcellulose-based medium containing recombinant murine (rm) Stem Cell Factor (50 ng/mL), rh Interleukin 6 (10 ng/mL), rm Interleukin 3 (10 ng/mL) and rh Erythropoietin (3 U/mL).
  • Mouse femurs and spleens were processed by standard protocols. Briefly, bone marrow was obtained by flushing the femoral cavity with Iscove's Modified Dulbecco's Media containing 2% fetal bovine serum (IMDM 2% FBS) using a 21 gauge needle and 1 cc syringe. Spleen cells were obtained by crushing spleens through a 70 ⁇ M filter and rinsing the filter with IMDM 2% FBS. Nucleated cell counts in 3% glacial acetic acid were then performed on the single cells suspensions using a Neubauer counting chamber so that the total cells per organ could be calculated. To remove contaminating red blood cells, total spleen cells were then diluted with 3 times the volume of ammonium chloride lysis buffer and incubated on ice 10 minutes. The cells were then washed and resuspended in
  • IMDM 2% FBS and a second cell count were performed to determine the cell concentration of cells after lysis.
  • Bone marrow cells were plated at 1 ⁇ 10 5 cells per dish in MethoCultTM 3334 to assess mature erythroid progenitors, 2 ⁇ 10 5 cells per dish in MethoCultTM 3630 to assess lymphoid progenitors and 3 ⁇ 10 4 cells per dish in MethoCultTM 3434 to assess immature erythroid and myeloid progenitors.
  • Spleen cells were plated at 4 ⁇ 10 5 cells per dish in MethoCultTM 3334 to assess mature erythroid progenitors, 4 ⁇ 10 5 cells per dish in MethoCultTM 3630 to assess lymphoid progenitors and 2 ⁇ 10 5 cells per dish in MethoCultTM 3434 to assess immature erythroid and myeloid progenitors.
  • Cultures plated in triplicate dishes were incubated at 37° C., 5% CO2 until colony enumeration and evaluation was performed by trained personnel.
  • Mature erythroid progenitors were cultured for 2 days, lymphoid progenitors were cultured for 7 days and mature erythroid and myeloid progenitors were cultured for 12 days.
  • the mean ⁇ 1 standard deviation was calculated for the triplicate cultures of the clonogenic assays and for the control and treatment groups for all data sets.
  • CFC colony forming cells
  • mice with ActRIIa-mFc resulted in significant increases in a number of hematopoietic parameters.
  • peripheral blood a significant increase in the red blood cell and hemoglobin content was seen in compound treated mice.
  • the compound treated group experienced a statistically significant increase in the nucleated cell number before red blood cell lysis and in the mature erythroid progenitor (CFU-E) colony number per dish, frequency and total progenitor number per spleen.
  • CFU-GM myeloid
  • BFU-E immature erythroid
  • total progenitor number per spleen an increase was seen in the number of myeloid (CFU-GM), immature erythroid (BFU-E) and total progenitor number per spleen.
  • ActRIIa variants that may be used according to the methods described herein are described in the International Patent Application published as WO2006/012627 (see e.g., pp. 55-58), incorporated herein by reference in its entirety.
  • An alternative construct may have a deletion of the C-terminal tail (the final 15 amino acids of the extracellular domain of ActRIIa. The sequence for such a construct is presented below (Fc portion underlined)(SEQ ID NO: 12):
  • ActRIIA-mFc 10 mg/kg, s.c.
  • vehicle (phosphate-buffered saline) 3 days before a single dose of the chemotherapeutic paclitaxel (20 mg/kg, i.p.).
  • ActRIIA-mFc prevented the decline in hematocrit level otherwise observed after paclitaxel ( FIG. 15 ), and similar effects were observed for hemoglobin concentration and RBC count.
  • female C57BL/6 mice underwent a partial surgical nephrectomy, with removal of approximately five-sixths of total kidney volume, to reduce production of erythropoietin.
  • Mice were given a 4-week recovery period with a high-fat diet to further promote renal deficiency and were then treated twice-weekly with ActRIIA-mFc (10 mg/kg, s.c.) or vehicle (PBS) for a total of 8 weeks.
  • soluble ActRIIA-Fc fusion proteins can be used as antagonists of signaling by TGF-family ligands to increase circulating levels of red blood cells, and thereby, to treat hypoproliferative anemias resulting from chronic diseases such as cancer and renal disease, and potentially other inflammatory or infectious diseases as well.
  • effects of ACE-011 on anemia in human patients are typically robust compared to the more modest effects in rodents.

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